Ionizable lipids and methods of manufacture and use thereof

This application claims the benefit of U.S. provisional application No. 63/091,616, which was filed on Oct. 14, 2020; U.S. provisional application No. 63/179,885, which was filed on Apr. 26, 2021; U.S. provisional application No. 63/091,603, which was filed on Oct. 14, 2020; and U.S. provisional application No. 63/179,872, which was filed on Apr. 26, 2021, each of which is incorporate herein by reference in its entirety.

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Dec. 21, 2021, is named AEXR-001-02US-343269-2014_SL.txt and is 1,460 bytes in size.

The invention encompasses novel ionizable lipids compounds and their use in lipid nanoparticles delivery systems that are useful in the delivery of nucleic acids to a mammalian subject that can be included for use, for example, as cancer vaccines, delivering gene editing therapies, in the delivery of nucleic acid (e.g., mRNA) encoding antibodies, vaccines for infectious disease, and protein replacement therapeutics. Additionally, the invention encompasses compositions and therapeutics comprising ionizable lipids in the lipid nanoparticles and the use of the composition and therapeutics for the preparation of a pharmaceutical composition, especially a vaccine (e.g., for use in the prophylaxis or treatment of infectious diseases, tumor or cancer diseases, rare diseases, allergies, or autoimmune diseases). The invention also encompasses methods of treatment or prophylaxis of the aforementioned diseases.

Gene therapy and genetic vaccination belong to the most promising and quickly developing methods of modern medicine. They may provide highly specific and individual options for therapy of a large variety of diseases. As a fundamental biological concept, the cellular machinery exploits mRNA as a transient carrier of information for synthesizing genetically encoded proteins. Hence, from a theoretical standpoint, mRNA should be capable of replacing DNA or recombinant proteins for therapeutic purposes. For example, RNA-interference (RNAi) agents such as small-interfering RNA (siRNA) and micro-RNA (miRNA) have strong potential as therapeutic agents for the treatment of a broad range of diseases such as malignancies, infections, autoimmune diseases and neurological diseases that are associated with undesirable gene expression.

For systemic administration of these poorly permeable and easily degradable macromolecules, a safe and efficient delivery platform is highly desirable. Because of high biocompatibility, biodegradability and a solid track record for clinical use, nanocarriers made of lipids and/or phospholipids have been commonly employed to facilitate RNA delivery (e.g. liposomes, lipid nanoparticles, and lipid nanoemulsions).

Genetic vaccination evokes a desired immune response to selected antigens, such as characteristic components of bacterial surfaces, viral particles, tumor antigens or the like. Genetic vaccines (i.e., vaccines for genetic vaccination) typically composed of genetically engineered nucleic acid molecules which allow expression of peptide or protein (i.e., antigen) fragments characteristic for a pathogen or a tumor antigen in vivo. Genetic vaccines are expressed upon administration to a patient after uptake by target cells. Expression of the administered nucleic acids results in production of the encoded proteins. In the event these proteins are recognized as foreign by the patient's immune system, an immune response is triggered.

DNA, as well as RNA, may be used as nucleic acid molecules for administration in the context of genetic vaccination. DNA is known to be relatively stable and easy to handle. However, the use of DNA bears the risk of undesired insertion of the administered DNA-fragments into the patient's genome potentially resulting in mutagenic events such as in loss of function of the impaired genes. As a further risk, the undesired generation of anti-DNA antibodies may emerge. Another drawback is the limited expression level of the encoded peptide or protein that is achievable upon DNA administration because the DNA must enter the nucleus in order to be transcribed before the resulting mRNA can be translated. Among other reasons, the expression level of the administered DNA will be dependent on the presence of specific transcription factors which regulate DNA transcription. In the absence of such factors, DNA transcription will not yield satisfying amounts of RNA. As a result, the level of translated peptide or protein obtained is limited.

By using RNA instead of DNA for gene therapy and genetic vaccination, the risk of undesired genomic integration and generation of anti-DNA antibodies is minimized or avoided. However, RNA is considered to be a rather unstable molecular species which may readily be degraded by ubiquitous RNAses. There is a need in the art for providing an efficient method for mRNA administration (e.g., for vaccination), which allows eliciting an adaptive immune response, wherein the administration is not severely impaired by early degradation of the antigen or by an inefficient translation of the mRNA due to inefficient release of the mRNA in the cell. Furthermore, there is a need to decrease the dose of mRNA vaccines to decrease potential safety concerns and to make the vaccines affordable for the third world.

There are many challenges associated with the delivery of nucleic acids to affect a desired response in a biological system. Nucleic acid based therapeutics, such as vaccines, have enormous potential, but there remains a need for more effective delivery of nucleic acids to appropriate sites within a cell, tissue, or organism in order to realize this potential. However, three problems currently face the use of oligonucleotides in therapeutic contexts. First, free RNAs are susceptible to nuclease digestion in plasma. Second, free RNAs have limited ability to gain access to the intracellular compartment where the relevant translation machinery resides. Third, only a small fraction of internalized oligonucleotide is released into the cytoplasm to become bioactive. Lipid nanoparticles (LNP) formed from the Ionizable Lipids of the Invention (as defined herein) combined with other lipid components, such as neutral lipids, steroids, PEG, PEGylated lipids, and oligonucleotides have been used to block degradation of the RNAs in plasma and facilitate the cellular uptake of the oligonucleotides. They may if correctly designed release therapeutic levels of oligonucleotide into the cytoplasm.

Localized delivery results in the systemic presence of RNAs resulting in unwanted side effects and reduction in efficacy. Vaccines are typically administered via intramuscular (IM) injection, and LNP requirements for delivery to dendritic cells via the IM route are different than intravenous (IV) delivery to hepatocytes. Hepatocyte targeting is due to the LNP associating with ApoE that targets LNP uptake to hepatocyte LDL receptors while ApoE may not have the same role in IM administration. A second targeting mechanism for LNPs is their net charge. Negatively charged LNPs target the spleen upon IV administration while positively charged LNPs target the lungs and near neutral LNPs target the liver. No published study has evaluated the influence of LNP charge in IM administration.

There remains a need for improved ionizable lipids and lipid nanoparticles for the delivery of oligonucleotides.

The Ionizable Lipids of the Invention (as defined herein) included in the lipid nanoparticles provide optimal drug:lipid ratios, protect the nucleic acid from degradation, and clearance in serum, are suitable for systemic or local delivery, and provide intracellular delivery of the nucleic acid. In addition, Ionizable Lipids of the Invention included in the lipid-nucleic acid particles are well-tolerated and provide an adequate therapeutic index, such that patient treatment at an effective dose of the nucleic acid is not associated with unacceptable toxicity and/or risk to the patient. The present invention provides these and related advantages.

The instant invention provides the surprising and unexpected discovery of Ionizable Lipids of the Invention (as defined below) for use in lipid nanoparticles (LNP) that provide an increase in potency of nucleic acid delivery (e.g., mRNA) for use in, for example, vaccine candidates that greatly facilitate the ability to meet three criteria for a successful mRNA vaccine—(1) lasting protection, (2) large volumes manufactured per year, and (3) a worldwide population with access and willingness to be vaccinated. Increased potency afforded by the Ionizable Lipids of the Invention reduces the required dose and adverse reactions while maintaining efficacy and increasing the ability to vaccinate globally by reducing cost and increasing manufacturing capacity. In certain embodiments, increased potency at these same doses increases protection.

The inventors have surprising and unexpectedly discovered a new class of ionizable lipids that can be used in LNPs that increase potency—meaning mRNA expression, immunogenicity and protection (e.g., when used in delivery of a vaccine) through the design of the lipid nanoparticle by two means:

1) improved ionizable lipid design and performance, and 2) production of more efficient lipid nanoparticles through control of LNP assembly.

In certain embodiments, the measure of potency increase is the reduction in dose the LNPs of the invention will achieve to obtain the same level of protection against viral challenge as a standard LNP representing those used in previous clinical trials.

In certain embodiments, the present invention encompasses a novel approach to the design of the ionizable lipid focusing on three structural features that are known to control delivery efficiency: (1) ionization in the endosomal pH range, (2) net charge at physiological pH, and (3) lipid tail conformation related to branching and saturation/unsaturation.

The invention also encompasses in silico analysis of candidates for synthesis, which is capable of eliminating undesirable candidates based on inappropriate predicted properties. The invention encompasses novel synthetic methods to make ionizable lipids. In other embodiments, the novel methods adhere to the design strategy by characterizing molecular ionization of the Ionizable Lipids of the Invention using NMR of water-soluble analogs and by measuring zeta potentials of LNPs over a pH range (3-10). In certain embodiments, the approach to candidate evaluation and elucidating structure-function relationships (SFRs) is comprehensive, including, but not limited to, in vitro and in vivo evaluation of translation and toxicity, in vitro assessment of cell uptake, endosomal release and innate immune sensor activation, in vivo characterization of distribution and cell trafficking, immunogenicity, and the use of current and evolving rodent and non-rodent animal models in viral challenge studies.

In certain embodiments, increased in vivo expression (e.g., >5×) of mRNA LNPs due to increased mixing concentration of the lipids and mRNA is achieved during assembly. In certain embodiments, key technical difficulties and high costs associated with systematically examining these parameters is overcome. By generalizing this finding to an array of ionizable lipids, the instant invention could transform the industrial production of more potent LNPs.

In certain embodiments, the invention encompasses the design and synthesis of potent ionizable lipids for intramuscular delivery and focused administration to a desired cell, tissue, or organ of mRNA vaccines

In another embodiment, the invention encompasses ionizable lipids that increase potency and reduce dose (e.g., by at least 2×, 3×, 4×, 5×, 10×, 20×, 50×, or 100×) compared to previous delivery methods, while maintaining similar expression, immunogenicity and protection against viral challenge.

In another embodiment, the invention encompasses optimization of ionization properties of multivalent headgroups of the Ionizable Lipids of the Invention that produce both a positively charged LNP at neutral pH to limit systemic dissemination and increase endosomal ionization that increases vaccine potency, a highly branched and degradable lipid tails that can further increase potency through endosomal release, and the charge and structure of the ionizable lipid can influence LNP adjuvanticity.

In certain embodiments, the LNPs of the invention increase potency to limit adverse events, reduce manufacturing cost, and increase worldwide capacity to vaccinate.

In other embodiments, the invention encompasses the design and synthesis of ionizable lipids and LNPs for assessments of physicochemical and biological properties to identify more potent systems and underlying structure-function relationship (SFRs).

In another embodiment, the invention encompasses a method of significantly accelerating design of Ionizable Lipids of the Invention by the prediction of the pKa of ionizable lipids using software including ACDLabs Percepta.

In other embodiments, the predicted aqueous pKas for lipids are all significantly higher than that measured by TNS. In certain embodiments, a drop in pKa (e.g., 1-3 point drop) from an aqueous environment to the LNP environment has not been previously acknowledged in the LNP literature but is known for basic amino acids in lipid membranes. Without being bound by theory, a reasonable explanation can be found due to the higher solvation energy of protons in the lipid phase compared to the aqueous phase and electrostatic repulsion of protons from the cationic LNP, which can combine to reduce pKa by 1-3 points from aqueous to lipid phases. In certain embodiments, this capability is exploited in order to select specific headgroups and carbon spacers by examining pKa tables of potential candidates and selecting only those with appropriate pKa values. The majority of candidates can be accurately eliminated in this manner, generating enormous savings in synthesis and testing time and expense.

In certain embodiments, the invention encompasses optimized Ionizable Lipids of the Invention used in a lipid nanoparticle (LNP) formulation. Exemplary LNPs comprise an Ionizable Lipid of the Invention, a second lipid, a steroid, a polymer conjugated lipid, and a therapeutic agent, or a pharmaceutically acceptable salt thereof, encapsulated within or associated with the LNP.

In certain embodiments, the inventors have unexpectedly discovered that characteristics of the optimized formulations of the Ionizable Lipids of the Invention provide important improvements for LNP properties related to delivery of the therapeutic agent (e.g., increased stability and enhanced delivery).

In certain embodiments, the Ionizable Lipids of the Invention included in the lipid nanoparticles are used to deliver nucleic acids such as small interfering, antisense, micro- and/or messenger RNA.

Accordingly, in one embodiment is provided a LNP comprising:

i) an Ionizable Lipid of the Invention; ii) a second lipid, for example, a neutral or zwitterionic lipid; iii) a steroid; iv) a polymer conjugated lipid; and v) a therapeutic agent (e.g., DNA or RNA), or a pharmaceutically acceptable salt thereof, encapsulated within or associated with the lipid nanoparticle.

Another embodiment provides an LNP comprising:

i) from 0.1 to 75 mol percent of an Ionizable Lipid of the Invention having an effective LNP pKa greater than 5.5; ii) from 0 to 50 mol percent of a neutral or zwitterionic lipid; iii) from 0 to 50 mol percent of an anionic lipid; iv) from 10 to 55 mol percent of a steroid; v) from 0.1 to 10 mol percent of a polymer conjugated lipid; and vi) a therapeutic agent, or a pharmaceutically acceptable salt thereof, encapsulated within or associated with the lipid nanoparticle, wherein the mol percent is determined based on total mol of lipids present in the lipid nanoparticle.

Another embodiment provides a lipid nanoparticle comprising:

i) an Ionizable Lipid of the Invention having an effective pKa; ii) a neutral lipid; iii) a steroid; iv) a polymer conjugated lipid; and v) a therapeutic agent, or a pharmaceutically acceptable salt or prodrug thereof, encapsulated within or associated with the lipid nanoparticle.

In other embodiments, the invention encompasses pharmaceutical compositions comprising the Ionizable Lipids of the Invention for use in lipid nanoparticles and methods for use of the same for treatment of various diseases or conditions, such as those caused by infectious entities, cancer, and/or insufficiency of a protein, are also provided.

In other embodiments, the present invention provides a method for the targeted administration (e.g., to a particular tissue or organ) of a therapeutic agent to a patient in need thereof, the method comprising administering the Ionizable Lipids of the Invention or pharmaceutical composition comprising the same, to the subject.

In certain specific embodiments, the inventions encompass a compound of the formula V:

wherein each R 1 and each R 2 is independently selected from the group consisting of H, an optionally substituted C 1 -C 22 alkyl, optionally substituted C 2 -C 22 alkenyl, optionally substituted C 2 -C 22 alkynyl, optionally substituted C 3 -C 6 cycloalkyl, optionally substituted C 4 -C 6 heterocycloalkyl, optionally substituted C 4 -C 6 alkylcycloalkyl, optionally substituted C 4 -C 6 aryl, optionally substituted C 3 -C 6 heteroaryl, optionally substituted C 4 -C 8 aryloxy, optionally substituted C 7 -C 10 arylalkyl; optionally substituted C 5 -C 10 heteroarylalkyl group, optionally substituted amine; or R 1 and R 2 can together form a 3-7 membered heterocycloalkyl or heteroaryl ring,

wherein each R 3 , R 4 , R 13 and R 14 is independently selected from the group consisting of an optionally substituted C 1 -C 22 alkyl, optionally substituted C 2 -C 22 alkenyl, optionally substituted C 2 -C 22 alkynyl;

wherein each R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 15 , and R 16 is independently selected from the group consisting of H, OH, halo, phenyl, benzyl, optionally substituted C 1 -C 22 alkyl, optionally substituted C 2 -C 22 alkenyl, optionally substituted C 2 -C 22 alkynyl;

wherein each of w, x, y, and z is independently an integer from 0-10;

wherein each Q is independently an atom selected from O, NH, NR 1 , and S;

wherein each of m is an integer from 0 to 8, preferably 0, 1, or 2; and

wherein each of L 1 and L 2 is independently selected from the group consisting of —C(═O)—; OC(═O)—; —OC(═O)O—; —C(═O)O—; —C(═O)O(CR 5 R 6 R 7 ); —NH—C(═O)—; —C(═O)NH—; —SO—; —SO 2 —; —SO 3 —; —NSO 2 —; —SO 2 N—; —NH((C 1 -C 8 )alkyl); —N((C 1 -C 8 )alkyl) 2 ; —NH((C 6 )aryl); —N((C 6 )aryl) 2 ; —NHC(═O)NH—; —NHC(═O)O—; —OC(═O)NH—; —NHC(═O)NR 1 —; —NHC(═O)O—; —OC(═O)NR 1 —; —C(═O)R 1 —; —CO((C 1 -C 8 )alkyl); —CO((C 6 )aryl); —CO 2 ((C 1 -C 8 )alkyl); —CO 2 ((C 6 )aryl); —SO 2 ((C 1 -C 8 )alkyl); and —SO 2 ((C 6 )aryl).

A compound of Formula III:

wherein each R 1′ , R 1 , R 2 , R 11 , and R 12 is independently selected from the group consisting of H, an optionally substituted C 1 -C 22 alkyl, optionally substituted C 2 -C 22 alkenyl, optionally substituted C 2 -C 22 alkynyl, optionally substituted C 3 -C 6 cycloalkyl, optionally substituted C 4 -C 6 heterocycloalkyl, optionally substituted C 4 -C 6 alkylcycloalkyl, optionally substituted C 4 -C 6 aryl, optionally substituted C 3 -C 6 heteroaryl, optionally substituted C 4 -C 8 aryloxy, optionally substituted C 7 -C 10 arylalkyl; optionally substituted C 5 -C 10 heteroarylalkyl group, optionally substituted amine; or R 1 and R 2 can together form cycloalkyl or heterocycloalkyl ring, wherein if Q is S or O the R 1 attached to the S or O is an electron pair;

wherein each R 3 and R 4 is independently selected from the group consisting of an optionally substituted C 1 -C 22 alkyl, optionally substituted C 2 -C 22 alkenyl, optionally substituted C 2 -C 22 alkynyl;

wherein each R 5 , R 6 , R 7 , R 8 , R 9 , and R 10 is independently selected from the group consisting of H, OH, halo, phenyl, benzyl, optionally substituted C 1 -C22 alkyl, optionally substituted C 2 -C 22 alkenyl, optionally substituted C 2 -C 22 alkynyl,

wherein each of x, y, and z is independently an integer from 0-10;

wherein G and Q are each independently an atom selected from CH, 0, N, and S;

wherein each of m and n is an integer from 0-8; and

wherein each of L 1 and L 2 is independently selected from the group consisting of —C(═O)—; OC(═O)—; —OC(═O)O—; —C(═O)O—; —C(═O)O(CR 1 R 2 R 3 ); —NH—C(═O)—; —C(═O)NH—; —SO—; —SO 2 —; —SO 3 —; —NSO 2 —; —SO 2 N—; —NH((C 1 -C 8 )alkyl); —N((C 1 -C 8 )alkyl) 2 ; —NH((C 6 )aryl); —N((C 6 )aryl) 2 ; —NHC(═O)NH—; —NHC(═O)O—; —OC(═O)NH—; —NHC(═O)NR 1 —; —NHC(═O)O—; —OC(═O)NR 1 —; —C(═O)R 1 —; —CO((C 1 -C 8 )alkyl); —CO((C 6 )aryl); —CO 2 ((C 1 -C 8 )alkyl); —CO 2 ((C 6 )aryl); —SO 2 ((C 1 -C 8 )alkyl); and —SO 2 ((C 6 )aryl).

In certain preferred embodiments, the compound has the following structure:

In certain preferred embodiments, the compound has the following structure:

In certain preferred embodiments, the compound has the following structure:

In certain preferred embodiments, the compound has the following structure:

In certain preferred embodiments, the compound has the following structure:

In certain preferred embodiments, the compound has the following structure:

In certain preferred embodiments, the compound has the following structure:

In certain preferred embodiments, the compound has the following structure:

In certain preferred embodiments, the compound has the following structure:

In certain preferred embodiments, the compound has the following structure:

In certain preferred embodiments, the compound has the following structure:

In other embodiments, the invention encompasses a lipid nanoparticle comprising:

i) a compound encompassed by the structures of Formula I, II, III, IV, V, VI, or VII; ii) a second lipid, for example, a neutral or zwitterionic lipid; iii) a steroid; iv) a polymer conjugated lipid; and v) a therapeutic agent, for example, mRNA, or a pharmaceutically acceptable salt thereof, encapsulated within or associated with the lipid nanoparticle.

In other embodiments, the invention encompasses a lipid nanoparticle comprising:

i) a compound encompassed by the structures of Formula I, II, III, IV, V, VI, or VII; ii) a second lipid, for example, a neutral or zwitterionic lipid; iii) a steroid; iv) a polymer conjugated lipid; and v) a therapeutic agent, or a pharmaceutically acceptable salt thereof, encapsulated within or associated with the lipid nanoparticle.

In other embodiments, the invention encompasses a method of targeted delivery a nucleic acid to a subject comprising administering to said subject a lipid nanoparticle comprising:

i) a compound encompassed by the structures of Formula I, II, III, IV, V, VI, or VII; ii) a second lipid, for example, a neutral or zwitterionic lipid; iii) a steroid; iv) a polymer conjugated lipid; and v) a therapeutic agent, for example, mRNA or siRNA, or a pharmaceutically acceptable salt thereof, encapsulated within or associated with the lipid nanoparticle.

In other embodiments, the invention encompasses a method of targeted delivery a nucleic acid to a subject comprising administering to said subject a lipid nanoparticle comprising:

i) a compound encompassed by the structures of Formula I, II, III, IV, V, VI, or VII; ii) a second lipid, for example, a neutral lipid; iii) a steroid; iv) a polymer conjugated lipid; and v) a therapeutic agent, or a pharmaceutically acceptable salt thereof, encapsulated within or associated with the lipid nanoparticle.

In other embodiments, the invention encompasses a lipid nanoparticle comprising:

i) an Ionizable Lipid of the Invention encompassed by the structures of Formula I, II, III, IV, V, VI, or VII; ii) a second lipid, for example, a neutral or zwitterionic lipid; iii) a steroid; iv) a polymer conjugated lipid; and v) a therapeutic agent, or a pharmaceutically acceptable salt thereof, encapsulated within or associated with the lipid nanoparticle.

In other embodiments, the invention encompasses a lipid nanoparticle comprising:

i) a compound of structure:

ii) a second lipid, for example, a neutral or zwitterionic lipid;

iii) a steroid;

iv) a polymer conjugated lipid; and

v) a therapeutic agent, or a pharmaceutically acceptable salt thereof, encapsulated within or associated with the lipid nanoparticle.

In other embodiments, the invention encompasses a method of delivering a nucleic acid to a subject comprising administering to said subject a lipid nanoparticle comprising:

i) an Ionizable Lipid of the Invention encompassed by the structures of Formula I, II, III, IV, V, VI, or VII; ii) a second lipid, for example, a neutral or zwitterionic lipid; iii) a steroid; iv) a polymer conjugated lipid; and v) a therapeutic agent, or a pharmaceutically acceptable salt thereof, encapsulated within or associated with the lipid nanoparticle.

In other embodiments, the invention encompasses a method of delivering a nucleic acid to a subject comprising administering to said subject a lipid nanoparticle comprising:

i) an Ionizable Lipid of the Invention encompassed by the structures of Formula I, II, III, IV, V, VI, or VII, for example, a compound of formula;

ii) a second lipid, for example, a neutral or zwitterionic lipid;

iii) a steroid;

iv) a polymer conjugated lipid; and

v) a therapeutic agent, or a pharmaceutically acceptable salt thereof, encapsulated within or associated with the lipid nanoparticle.

In other embodiments, the invention encompasses a method of delivering a therapeutic agent comprising a therapeutic protein, a vaccine, a gene editing RNA to a subject comprising administering to said subject a lipid nanoparticle comprising:

i) an Ionizable Lipid of the Invention encompassed by the structures of Formula I, II, III, IV, V, VI, or VII, for example, a compound of structure:

ii) a second lipid, for example, a neutral or zwitterionic lipid;

iii) a steroid;

iv) a polymer conjugated lipid; and

v) a therapeutic agent, or a pharmaceutically acceptable salt thereof, encapsulated within or associated with the lipid nanoparticle.

FIG. 1 illustrates an exemplary approach to enhancing and improving mRNA potency and delivery using the Ionizable Lipids of the Invention included in Lipid Nanoparticles.

FIG. 2 A illustrates a graph of the aqueous phase pKa of a custom-synthesized water-soluble head group of MC3 titrated from pH 7 to ph 12 and the chemical shift if the dimethylamine protons measured to fit the Henderson-Hasselbach equation (HH) to obtain a pKa of 9.45 similar to the 9.4 predicted by ACDLabs Percepta. FIG. 2 B illustrates a graph of the zeta potential of 5 for Azane Diyl Diester (ADDE) LNPs and 2 LNPs made with MC3 and KC2 standards was measured from pH 3 to pH 10. FIG. 2 C illustrates LNP pKas measured using traditional TNS method fit to HH.

FIG. 3 A illustrates Firefly Luciferase Assay for mRNA Delivery Efficiency. FIG. 3 B illustrates in vivo Translation of mRNA Fluc at 4 hours post-injection in BALB/c mice. FIG. 3 C illustrates cryoTEM images of empty LNPs.

FIG. 4 illustrates robust binding antibody responses exhibited by exemplary first generation ADDE ionizable lipids of the invention. FIG. 4 A illustrates SARS-CoV-2 S-specific IgG by ELISA. FIG. 4 B illustrates neutralizing antibodies against VSVDG-RFP SARS-CoV-2 pseudovirus.

FIG. 5 illustrates cryoTEM images of empty LNPs. KC2 LNPs without mRNA were mixed and ejected into pH 4 buffer showing small electron lucent structures vs electron dense at pH 7.

FIG. 6 A illustrates the increase in LNP potency when the concentration of lipids and mRNA are increased during microfluidic mixing. FIG. 6 B illustrates in vivo targeting of LNP's of the invention.

FIG. 7 illustrates a graph of Firefly Luciferase Assay for mRNA Delivery Efficiency as exemplified in Example 1A.

FIG. 8 illustrates a graph of Ribogreen Assay for mRNA Encapsulation Efficiency as exemplified in Example 1B.

FIG. 9 illustrates a graph of Dynamic Light Scattering for LNP Size (white dots are PDI right y axis) as exemplified in Example 1C.

FIG. 10 illustrates a graph of Toxicity Assay based on Presto Blue HS viability reagent as exemplified in Example 1D.

FIG. 11 illustrates a graph of Firefly Luciferase Assay for mRNA Delivery Efficiency as exemplified in Example 2A.

FIG. 12 illustrates a graph of Ribogreen Assay for mRNA Encapsulation Efficiency as exemplified in Example 2B.

FIG. 13 illustrates a graph of Dynamic Light Scattering for LNP Size (white dots are PDI right y axis) as exemplified in Example 2C.

FIG. 14 illustrates a graph of Firefly Luciferase Assay for mRNA Delivery Efficiency as exemplified in Example 3A.

FIG. 15 illustrates a graph of Ribogreen Assay for mRNA Encapsulation Efficiency as exemplified in Example 3B.

FIG. 16 illustrates a graph of Dynamic Light Scattering for LNP Size (white dots are PDI right y axis) as exemplified in Example 3C.

FIG. 17 illustrates a graph of Toxicity Assay based on Presto Blue HS viability reagent as exemplified in Example 3D.

FIG. 18 illustrates a graph of Firefly Luciferase Assay for mRNA Delivery Efficiency as exemplified in Example 4A.

FIG. 19 illustrates a graph of Ribogreen Assay for mRNA Encapsulation Efficiency as exemplified in Example 4B.

FIG. 20 illustrates a graph of Dynamic Light Scattering for LNP Size (white dots are PDI right y axis) as exemplified in Example 4C.

FIG. 21 illustrates a graph of Firefly Luciferase Assay for mRNA Delivery Efficiency as exemplified in Example 5A.

FIG. 22 illustrates a graph of Ribogreen Assay for mRNA Encapsulation Efficiency as exemplified in Example 5B.

FIG. 23 illustrates a graph of Dynamic Light Scattering for LNP Size (white dots are PDI right y axis) as exemplified in Example 5C.

FIG. 24 illustrates a graph of In vivo Translation of mRNA Fluc at 4 hours post-injection in BALB/c mice as exemplified in Example 5D.

FIG. 25 illustrates a graph of Firefly Luciferase Assay for mRNA Delivery Efficiency as exemplified in Example 6A.

FIG. 26 illustrates a graph of Ribogreen Assay for mRNA Encapsulation Efficiency as exemplified in Example 6B.

FIG. 27 illustrates a graph of Dynamic Light Scattering for LNP Size (white dots are PDI right y axis) as exemplified in Example 6C.

FIG. 28 illustrates a graph of Firefly Luciferase Assay for mRNA Delivery Efficiency as exemplified in Example 7A.

FIG. 29 illustrates a graph of Ribogreen Assay for mRNA Encapsulation Efficiency as exemplified in Example 7B.

FIG. 30 illustrates a graph of Dynamic Light Scattering for LNP Size (white dots are PDI right y axis) as exemplified in Example 7C.

FIG. 31 illustrates graphs of molecular and LNP characterization of ionization and charge. FIG. 31 A illustrates the aqueous phase pKa of a custom-synthesized water-soluble headgroup of MC3 titrated from pH 7 to pH 12 and the chemical shift of the DMA protons measured to fit the Henderson-Hasselbach (HH) equation. FIG. 31 B illustrates the zeta potential of 5 ADDE LNPs and 2 LNPs made with MC3 and KC2 standards measured from pH 3 to pH 10. FIG. 31 C illustrates LNP pKa's measured using traditional fluorescence enhancement of TNS due to binding to LNP fit to the HH equation.

FIG. 32 illustrates luciferase reported expression in vitro and in vivo. FIG. 32 A illustrates Fluc mRNA delivered in 5 ADDE LNPs and MC3 and KC2 LNPs to HEK 293 cells in serum containing media at doses from 25-200 ng per well containing 12 k cells. FIG. 32 B illustrates 2.5 μg of Fluc mRNA delivered by IM injections in BALB/c mice (MC3 and KC2 containing LNPs display off target systemic expression in liver whiled DL-ADDE LNPs target expression to muscle and draining lymph nodes (see ex vivo). FIG. 32 C illustrates KC2 containing LNPs imaged using CryoTEM showing spherical LNPs with diameters corresponding to that measured by DLS number size of 65 nm.

FIG. 33 A illustrates ADDE ionizable lipids eliciting robust binding antibody responses in BALB/c mice (n=5/group) immunized at weeks 0 and 3 with 0.3 mg (green) and 10 mg (blue) of S-2P mRNA-encoded immunogen in 4 different LNPs and assessed for SARS-CoV-2 S-specific IgG by ELISA. FIG. 33 B illustrates neutralizing antibodies against VSVΔG-RFP SARS-CoV-2 pseudovirus.

FIG. 34 illustrates a graph of Ribogreen Assay for mRNA Encapsulation Efficiency as exemplified in Example 8A.

FIG. 35 illustrates a graph of Dynamic Light Scattering for LNP Size (white dots are PDI right y axis) as exemplified in Example 8B.

FIG. 36 illustrates a graph of Zeta Potential for LNP charge, pKa and Pi as exemplified in Example 8C.

FIG. 37 illustrates a graph of TNS Assay for LNP pKa as exemplified in Example 8D.

FIG. 38 illustrates a graph of Ribogreen Assay for mRNA Encapsulation Efficiency as exemplified in Example 9A.

FIG. 39 illustrates a graph of Dynamic Light Scattering for LNP Size (white dots are PDI right y axis) as exemplified in Example 9B.

FIG. 40 illustrates in vivo vivo Firefly Luciferase expression in IM administration of 5 ug of encapsulated mRNA in mice in intramuscular (I.M.) injections. FIG. 40 A illustrates 4 hour in-vivo imaging (left group 1, right group 2) from left to right (MC3/DL-ADDE-C2C2-4Me-PipZ/BODD-ADDE-C2C4-4Me-PipZ). FIG. 40 B illustrates the presence in muscle and liver illustrating systemic distribution and local concentration of from left to right (MC3/DL-ADDE-C2C2-4Me-PipZ/BODD-ADDE-C2C4-4Me-PipZ) after intramuscular administration. FIG. 40 C illustrates 24 hour in-vivo imaging (left group 1, right group 2) from left to right (MC3/DL-ADDE-C2C2-4Me-PipZ/BODD-ADDE-C2C4-4Me-PipZ). FIG. 40 D illustrates the presence in muscle and liver illustrating systemic distribution and local concentration of from left to right (MC3/DL-ADDE-C2C2-4Me-PipZ/BODD-ADDE-C2C4-4Me-PipZ) after intramuscular administration. FIG. 40 E illustrates the distribution of samples of MC3/DL-ADDE-C2C2-4Me-PipZ/BODD-ADDE-C2C4-4Me-PipZ after intramuscular administration. FIG. 40 F illustrates the distribution of samples of MC3 after intramuscular administration. FIG. 40 G illustrates the distribution of samples of DL-ADDE-C2C2-4Me-PipZ after intramuscular administration. FIG. 40 H illustrates the distribution of samples of BODD-ADDE-C2C4-4Me-PipZ after intramuscular administration.

FIG. 41 illustrates a graph of Firefly Luciferase Assay for mRNA Delivery Efficiency as exemplified in Example 10A.

FIG. 42 illustrates a graph of Ribogreen Assay for mRNA Encapsulation Efficiency as exemplified in Example 10B.

FIG. 43 illustrates a graph of Dynamic Light Scattering for LNP Size (white dots are PDI right y axis) as exemplified in Example 10C.

FIG. 44 illustrates a graph of Firefly Luciferase Assay for mRNA Delivery Efficiency as exemplified in Example 11A.

FIG. 45 illustrates a graph of Ribogreen Assay for mRNA Encapsulation Efficiency as exemplified in Example 11B.

FIG. 46 illustrates a graph of Dynamic Light Scattering for LNP Size (white dots are PDI right y axis) as exemplified in Example 11C.

FIG. 47 illustrates graphs of Stability Assay based on in vitro potency using Firefly Luciferase Assays as exemplified in Example 12B.

FIG. 48 illustrates a graph of Dynamic Light Scattering for LNP Size (white dots are PDI right y axis) as exemplified in Example 12C.

FIG. 49 illustrates a graph of Firefly Luciferase Assay for mRNA Delivery Efficiency as exemplified in Example 13A.

FIG. 50 illustrates a graph of Ribogreen Assay for mRNA Encapsulation Efficiency as exemplified in Example 13B.

FIG. 51 illustrates a graph of Dynamic Light Scattering for LNP Size (white dots are PDI right y axis) as exemplified in Example 13C.

FIG. 52 illustrates a graph of Firefly Luciferase Assay for mRNA Delivery Efficiency as exemplified in Example 14A.

FIG. 53 illustrates a graph of Ribogreen Assay for mRNA Encapsulation Efficiency as exemplified in Example 14B.

FIG. 54 illustrates a graph of Dynamic Light Scattering for LNP Size (white dots are PDI right y axis) as exemplified in Example 14C.

FIG. 55 illustrates a graph of Ribogreen Assay for mRNA Encapsulation Efficiency as exemplified 6 in Example 15A.

FIG. 56 illustrates a graph of Dynamic Light Scattering for LNP Size (white dots are PDI right y axis) as exemplified in Example 15B

FIGS. 57 A- 57 I illustrate exemplary ionizable lipid formulation BODD C2C4 PipZ at high 1.5 mg/ml mRNA mixing concentration for Rapid Microfluidic Mixing showing high delivery efficiency and potency in vivo versus reference lipid MC3 at standard mixing concentration 0.2 mg/ml.

FIG. 58 illustrates In vivo immunogenicity Endpoint ELISA Anti-RBD titers as exemplified in Example 16A— FIG. 58 A (PREBOOST), FIG. 58 B (POSTBOOST).

FIG. 59 illustrates In vivo immunogenicity FRNT50 titer for Psuedoneutralisation assay as exemplified in Example 16B.

FIG. 60 illustrates In vivo protection against viral challenge—Survival proportion, Weight and Temperature in Challenge model as exemplified in Example 17A; FIG. 60 A (SURVIVAL PROPORTIONS: MC3-SR); FIG. 60 B (SURVIVAL PROPORTIONS: BODD C2C4 PipZ-HO)

FIG. 61 A- 61 D illustrate In vivo weight and temperature in Challenge model as exemplified in Example 17B.

FIG. 62 illustrates a graph of Ribogreen Assay for mRNA Encapsulation Efficiency as exemplified in Example 18A.

FIG. 63 illustrates a graph of Dynamic Light Scattering for LNP Size (white dots are PDI right y axis) as exemplified in Example 18B.

FIG. 64 A- 64 H illustrate In vivo Firefly Luciferase expression in IM administration as exemplified in Example 18C. FIG. 64 I-J illustrate in vivo screening of multiple 2 nd generation Ionizable Lipids of the Invention for IM ( FIG. 64 I ) and IV ( FIG. 64 J ) administration that are superior to an MC3 standard.

FIG. 65 and FIG. 66 illustrates Firefly Luciferase Assay for mRNA Delivery Efficiency as exemplified in Example 19A.

FIG. 67 illustrates a graph of Ribogreen Assay for mRNA Encapsulation Efficiency as exemplified in Example 19B.

FIG. 68 illustrates a graph of Dynamic Light Scattering for LNP Size (white dots are PDI right y axis) as exemplified in Example 19C.

FIG. 69 illustrates Firefly Luciferase Assay for mRNA Delivery Efficiency as exemplified in Example 20A.

FIG. 70 illustrates a graph of Ribogreen Assay for mRNA Encapsulation Efficiency as exemplified in Example 20B.

FIG. 71 illustrates a graph of Dynamic Light Scattering for LNP Size (white dots are PDI right y axis) as exemplified in Example 20C.

FIG. 72 illustrates Firefly Luciferase Assay for mRNA Delivery Efficiency as exemplified in Example 21A.

FIG. 73 illustrates a graph of Ribogreen Assay for mRNA Encapsulation Efficiency as exemplified in Example 21B.

FIG. 74 illustrates a graph of Dynamic Light Scattering for LNP Size (white dots are PDI right y axis) as exemplified in Example 21C.

FIG. 75 and FIG. 76 illustrates the results of In vivo Firefly Luciferase expression of the Injection site in IM administration as exemplified in Example 22A.

FIGS. 77 and 78 illustrates the results of Ex vivo Firefly Luciferase expression of the Injection site in IM administration as exemplified in Example 22B.

FIG. 79 and FIG. 80 illustrates the results of in vivo Firefly Luciferase expression of the Injection site in IM administration as exemplified in Example 23A.

FIG. 81 , FIG. 82 , and FIG. 83 illustrates the results of ex vivo Firefly Luciferase expression of the Injection site in IM administration as exemplified in Example 23B.

FIG. 84 A - FIG. 84 F illustrates the results of multiprotic C24 ionizable lipid produces multistage protonation in the LNP and greater protonation in the endosomal pH range than the MC3 LNP and comparative data between MC3 LNP and C24 LNP.

FIG. 85 A- 85 C illustrates the effects of structural properties of C24 and MC3 lipid nanoparticles. FIG. 85 A- 85 C illustrates empty LNPs.

FIG. 86 A - FIG. 86 F illustrates the results of luciferase expression after intramuscular (IM) administration in Balb/c mice shows significantly higher on-target and lower off-target mRNA expression with the C24 LNP than for MC3.

FIG. 87 A - FIG. 87 E illustrates the results of C24 LNPs generate 10 fold higher binding and pseudoneutralizing antibody titers than MC3 LNPs in immunogenicity studies with Balb/c mice.

FIG. 88 A - FIG. 88 I illustrates C24 LNPs, which are protective against lethal SARS-CoV-2 challenge at low doses of the S2P immunogen and completely eliminate lung infection. FIG. 88 B - FIG. 88 I illustrate comparative data for MC3 and C24 LNPs.

FIG. 89 A - FIG. 891 illustrates local injection site inflammation is lower for C24 than MC3 mRNA LNPs.

FIG. 90 A - FIG. 90 F illustrates the bioactivity and mRNA integrity of C24 and MC3 LNPs, which are stable at 4° C. but decline at higher temperatures over 2 weeks.

FIG. 91 illustrates weight and temperature effects in K18-hACE2 mice subject to lethal challenge on Day 0 after a Prime-Boost vaccination with C24 and MC3 LNPs containing the mRNA-encoded S2P immunogen at doses ranging from 0.1 to 1.0 μg.

FIG. 92 illustrates LNP potency and endosomal protonation increase when the concentrations of lipids and mRNA are increased during microfluidic mixing. FIG. 92 A ) KC2 LNPs assembled at higher concentrations produced higher Fluc expression in vitro at the same doses of 25-200 ng per well containing 12 k HEK293 cells. FIG. 92 B ) LNPs produced at higher mixing concentrations (total lipid concentration in mM at mixing is shown above the animal), and diluted to a constant 5 μg dose in 50 μL for IM injection, are more potent (color bar is Radiance in 107 p/sec/cm2/sr). FIG. 92 C ) Zeta potential measurements reveal a greater increase in protonation when pH drops from 7.4 to 5 for the LNP prepared by high concentration mixing, suggesting greater endosomal release.

FIG. 93 illustrates determinants of LNP performance that will be measured, modeled and related to delivery efficiency, targeting, toxicity and reactogenicity in vitro and in animal models.

FIG. 94 illustrates exemplary BODD PipZ types of ionizable lipids. 12 members of this family were synthesized with different carbon spacers to generate ionizable lipids with molecular macro-ionization constants (shown on the Ns above) that span a 2 point range to characterize resulting LNP charge and endosomal protonation and relate these features to delivery efficiency and targeting.

FIG. 95 illustrates negatively charged LNP reduces liver expression and increases expression in spleen. The anionic lipid 18-1PA was added to the lipid mix prior to mRNA LNP assembly at 15% and 30% of total lipids. Adding 18-1PA at 15% total lipids reduced liver expression of FLuc and increased spleen expression 2.5× upon IV administration.

FIG. 96 illustrates assembly of Fluc LNPs at standard concentration (0.2 mg/ml mRNA in A,B) and high concentration (1.5 mg/ml mRNA in C,D) was imaged after ejection in ethanol/water at 1:2 v/v (A,C) or after dialysis in PBS to pH 7.4 (B, D). High potency mRNA LNPs produced at high mixing concentrations displayed greater levels of fused structures immediately upon mixing (C vs A) which were transformed to more homogeneous LNPs after dialysis.

V.1. Definitions

Unless defined otherwise, or unless the specific context requires otherwise, all technical terms used herein have the same meaning as is commonly understood by a person skilled in the relevant technical field.

Percentages in the context of numbers should be understood as relative to the total number of the respective items. In other cases, and unless the context dictates otherwise, percentages should be understood as percentages by weight (wt.-%) or percentages by mole (mol.-%).

The singular forms “a”, “an” and “the” should be understood as to include plural references unless the context clearly dictates otherwise. The expressions “an embodiment,” “a specific embodiment,” “one embodiment” and the like mean that a particular feature, property or characteristic, or a particular group or combination of features, properties or characteristics, as referred to in combination with the respective expression, is present in at least one of the embodiments of the invention. The occurrence of these expressions in various places throughout this description do not necessarily refer to the same embodiment. Moreover, the particular features, properties or characteristics may be combined in any suitable manner in one or more embodiments.

As used herein, the “adaptive immune system” is composed of highly specialized, systemic cells and processes that eliminate or prevent pathogenic growth and replication. The adaptive immune response provides the vertebrate immune system with the ability to recognize and remember specific pathogens (e.g., to generate immunity), and to mount stronger attacks each time the pathogen is encountered. The system is highly adaptable because of somatic hypermutation (a process of increased frequency of somatic mutations), and V(D)J recombination (an irreversible genetic recombination of antigen receptor gene segments). This mechanism allows a small number of genes to generate a vast number of different antigen receptors, which are then uniquely expressed on each individual lymphocyte. Because the gene rearrangement leads to an irreversible change in the DNA of each cell, all of the progeny (offspring) of that cell will then inherit genes encoding the same receptor specificity, including the Memory B cells and Memory T cells that are the keys to long-lived specific immunity. Immune network theory is a theory of how the adaptive immune system works, that is based on interactions between the variable regions of the receptors of T cells, B cells and of molecules made by T cells and B cells that have variable regions. As used herein, the term “adaptive immune response” is typically understood to be antigen-specific. Antigen specificity allows for the generation of responses that are tailored to specific antigens, pathogens or pathogen-infected cells. The ability to mount these tailored responses is maintained in the body by “memory cells.” Should a pathogen infect the body more than once, these specific memory cells are used to quickly eliminate it. In this context, the first step of an adaptive immune response is the activation of naive antigen-specific T cells or different immune cells able to induce an antigen-specific immune response by antigen-presenting cells. This occurs in the lymphoid tissues and organs through which naive T cells are constantly passing. Cell types that can serve as antigen-presenting cells are, inter alia, dendritic cells, macrophages, and B cells. Each of these cells has a distinct function in eliciting immune responses. Dendritic cells take up antigens by phagocytosis and macropinocytosis and are stimulated by contact with for example a foreign antigen to migrate to the local lymphoid tissue, where they differentiate into mature dendritic cells. Macrophages ingest particulate antigens such as bacteria and are induced by infectious agents or other appropriate stimuli to express MHC molecules. The unique ability of B cells to bind and internalize soluble protein antigens via their receptors may also be important to induce T cells. Presenting the antigen on MHC molecules leads to activation of T cells, which induces their proliferation and differentiation into armed effector T cells. The most important function of effector T cells is the killing of infected cells by CD8+ cytotoxic T cells and the activation of macrophages by Th1 cells, which together make up cell-mediated immunity, and the activation of B cells by both Th2 and Th1 cells to produce different classes of antibody, thus driving the humoral immune response. T cells recognize an antigen by their T cell receptors which do not recognize and bind antigen directly, but instead recognize short peptide fragments e.g. of pathogen-derived protein antigens, which are bound to MHC molecules on the surfaces of other cells.

As used herein, the term “adjuvant or an adjuvant component” in the broadest sense is typically a (e.g., pharmacological or immunological) agent or composition that may modify, e.g. enhance, the efficacy of other agents, such as a drug or vaccine. Conventionally, the term refers in the context of the invention to a compound or composition that serves as a carrier or auxiliary substance for immunogens and/or other pharmaceutically active compounds. It is to be interpreted in a broad sense and refers to a broad spectrum of substances that are able to increase the immunogenicity of antigens incorporated into or co-administered with an adjuvant in question. In the context of the invention an adjuvant will preferably enhance the specific immunogenic effect of the active agents of the present invention. Typically, “adjuvant” or “adjuvant component” has the same meaning and can be used mutually. Adjuvants may be divided, e.g., into immuno potentiators, antigenic delivery systems or even combinations thereof. As used herein, the term “adjuvant” is typically understood not to comprise agents which confer immunity by themselves. An adjuvant assists the immune system unspecifically to enhance the antigen-specific immune response by e.g. promoting presentation of an antigen to the immune system or induction of an unspecific innate immune response. Furthermore, an adjuvant may preferably e.g. modulate the antigen-specific immune response by e.g. shifting the dominating Th2-based antigen specific response to a more Th1-based antigen specific response or vice versa. Accordingly, an adjuvant may favourably modulate cytokine expression/secretion, antigen presentation, type of immune response etc.

As used herein, the term “alkyl group” means a saturated, monovalent unbranched or branched (substituted or unsubstituted) hydrocarbon chain. Examples of alkyl groups include, but are not limited to, (C 1 -C 22 )alkyl groups, such as methyl, ethyl, propyl, isopropyl, 2-methyl-1-propyl, 2 methyl 2-propyl, 2-methyl-1-butyl, 3-methyl-1-butyl, 2 methyl-3-butyl, 2,2 dimethyl 1-propyl, 2-methyl-1-pentyl, 3 methyl-1-pentyl, 4 methyl-1-pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4 methyl 2 pentyl, 2,2 dimethyl 1 butyl, 3,3-dimethyl-1-butyl, 2-ethyl-1-butyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, neopentyl, and hexyl, and longer alkyl groups, such as heptyl, octyl, decyl, dodecyl, etc. An alkyl group can be unsubstituted or substituted with one or two suitable substituents.

As used herein, the term an “alkenyl group” means a monovalent unbranched or branched hydrocarbon chain having one or more double bonds therein. The double bond of an alkenyl group can be unconjugated or conjugated to another unsaturated group. Suitable alkenyl groups include, but are not limited to (C 2 -C 22 )alkenyl groups, such as vinyl, allyl, butenyl, pentenyl, hexenyl, butadienyl, pentadienyl, hexadienyl, 2-ethylhexenyl, 2-propyl-2-butenyl, 4-(2-methyl-3-butene)-pentenyl. An alkenyl group can be unsubstituted or substituted with one or two suitable substituents.

As used herein, the term “alkoxy group” means an —O-alkyl group, wherein alkyl is as defined above. An alkoxy group can be unsubstituted or substituted with one or two suitable substituents. Preferably, the alkyl chain of an alkyloxy group is from 1 to 6 carbon atoms in length, referred to herein as “(C 1 -C 22 )alkoxy”.

As used herein, the term “alkoxycarbonyl” group means a monovalent group of the formula —C(O)-alkoxy. Preferably, the hydrocarbon chain of an alkoxycarbonyl group is from 1 to 22 carbon atoms in length.

As used herein, the term an “alkynyl group” means monovalent unbranched or branched hydrocarbon chain having one or more triple bonds therein. The triple bond of an alkynyl group can be unconjugated or conjugated to another unsaturated group. Suitable alkynyl groups include, but are not limited to, (C 2 -C 6 )alkynyl groups, such as ethynyl, propynyl, butynyl, pentynyl, hexynyl, methylpropynyl, 4-methyl-1-butynyl, 4-propyl-2-pentynyl, and 4-butyl-2-hexynyl. An alkynyl group can be unsubstituted or substituted with one or two suitable substituents.

As used herein, in the context of the present invention “antigen” refers typically to a substance which may be recognized by the immune system, preferably by the adaptive immune system, and is capable of triggering an antigen-specific immune response (e.g., by formation of antibodies and/or antigen-specific T cells as part of an adaptive immune response). Typically, an antigen may be or may comprise a peptide or protein which may be presented by the MHC to T-cells. In the sense of the present invention an antigen may be the product of translation of a provided nucleic acid molecule, preferably an mRNA as defined herein. In this context, also fragments, variants and derivatives of peptides and proteins comprising at least one epitope are understood as antigen.

As used herein, the term “antigen-providing mRNA” in the context of the invention may typically be an mRNA, having at least one open reading frame that can be translated by a cell or an organism provided with that mRNA. The product of this translation is a peptide or protein that may act as an antigen, preferably as an immunogen. The product may also be a fusion protein composed of more than one immunogen (e.g., a fusion protein that consists of two or more epitopes, peptides or proteins derived from the same or different virus-proteins), wherein the epitopes, peptides or proteins may be linked by linker sequences.

As used herein, the term “artificial mRNA (sequence)” may typically be understood to be an mRNA molecule, that does not occur naturally. In other words, an artificial mRNA molecule may be understood as a non-natural mRNA molecule. Such mRNA molecule may be non-natural due to its individual sequence (which does not occur naturally) and/or due to other modifications, for example, structural modifications of nucleotides which do not occur naturally. Typically, artificial mRNA molecules may be designed and/or generated by genetic engineering methods to correspond to a desired artificial sequence of nucleotides (heterologous sequence). In this context an artificial sequence is usually a sequence that may not occur naturally, i.e. it differs from the wild type sequence by at least one nucleotide. The term “wild type” may be understood as a sequence occurring in nature. Further, the term “artificial nucleic acid molecule” is not restricted to mean “one single molecule” but is, typically, understood to comprise an ensemble of identical molecules. Accordingly, it may relate to a plurality of identical molecules contained in an aliquot.

As used herein, the term an “aryl group” means a monocyclic or polycyclic-aromatic radical comprising carbon and hydrogen atoms. Examples of suitable aryl groups include, but are not limited to, phenyl, tolyl, anthacenyl, fluorenyl, indenyl, azulenyl, and naphthyl, as well as benzo-fused carbocyclic moieties such as 5,6,7,8-tetrahydronaphthyl. An aryl group can be unsubstituted or substituted with one or two suitable substituents. Preferably, the aryl group is a monocyclic ring, wherein the ring comprises 6 carbon atoms, referred to herein as “(C 6 )aryl”.

As used herein, the term “aryloxy group” means an —O-aryl group, wherein aryl is as defined above. An aryloxy group can be unsubstituted or substituted with one or two suitable substituents. Preferably, the aryl ring of an aryloxy group is a monocyclic ring, wherein the ring comprises 6 carbon atoms, referred to herein as “(C 6 )aryloxy”.

As used herein, the term “B cell epitopes” are typically fragments located on the outer surface of (native) protein or peptide antigens as defined herein, preferably having 5 to 15 amino acids, more preferably having 5 to 12 amino acids, even more preferably having 6 to 9 amino acids, which may be recognized by antibodies, i.e. in their native form. Such epitopes of proteins or peptides may furthermore be selected from any of the herein mentioned variants of such proteins or peptides. In this context antigenic determinants can be conformational or discontinuous epitopes which are composed of segments of the proteins or peptides as defined herein that are discontinuous in the amino acid sequence of the proteins or peptides as defined herein but are brought together in the three-dimensional structure or continuous or linear epitopes which are composed of a single polypeptide chain.

As used herein, the term “benzyl” means —CH 2 -phenyl.

As used herein, the term “Bi-/multicistronic mRNA” is mRNA that typically may have two (bicistronic) or more (multicistronic) open reading frames (ORF) (coding regions or coding sequences). An open reading frame in this context is a sequence of several nucleotide triplets (codons) that can be translated into a peptide or protein. Translation of such an mRNA yields two (bicistronic) or more (multicistronic) distinct translation products (provided the ORFs are not identical). For expression in eukaryotes such mRNAs may for example comprise an internal ribosomal entry site (IRES) sequence.

As used herein, the term “CAP analogue” refers to a non-polymerizable di-nucleotide that has CAP functionality in that it facilitates translation or localization, and/or prevents degradation of the RNA molecule when incorporated at the 5′-end of the RNA molecule. Non-polymerizable means that the CAP analogue will be incorporated only at the 5′-terminus because it does not have a 5′ triphosphate and therefore cannot be extended in the 3′-direction by a template-dependent RNA polymerase. In certain embodiments, the 5′-cap analogue utilizes Trilink Biotechnologies CleanCap Technology—https://www.trilinkbiotech.com/cleancap. As used herein, the term “CAP analogues” include, but are not limited to, a chemical structure selected from the group consisting of m7GpppG, m7GpppA, m7GpppC; unmethylated CAP analogues (e.g., GpppG); dimethylated CAP analogue (e.g., m2,7GpppG), trimethylated CAP analogue (e.g., m2,2,7GpppG), dimethylated symmetrical CAP analogues (e.g., m7Gpppm7G), or anti reverse CAP analogues (e.g., ARCA; m7,2′OmeGpppG, m7,2′dGpppG, m7,3′OmeGpppG, m7,3′dGpppG and their tetraphosphate derivatives) (Stepinski et al., 2001. RNA 7(10):1486-95). Further CAP analogues have been described previously (U.S. Pat. No. 7,074,596, WO2008/016473, WO2008/157688, WO2009/149253, WO2011/015347, and WO2013/059475), which are incorporated herein in their entirety. The synthesis of N7-(4-chlorophenoxyethyl) substituted dinucleotide CAP analogues has been described recently (Kore et al. (2013) Bioorg. Med. Chem. 21(15): 4570-4), which is incorporated herein in their entirety. As used herein, the term “5′-CAP structure” is typically a modified nucleotide (CAP analogue), particularly a guanine nucleotide, added to the 5′-end of an mRNA molecule. Preferably, the 5′-CAP is added using a 5′-5′-triphosphate linkage (also named m7GpppN). Further examples of 5′-CAP structures include glyceryl, inverted deoxy abasic residue (moiety), 4′,5′ methylene nucleotide, 1-(beta-D-erythrofuranosyl) nucleotide, 4′-thio nucleotide, carbocyclic nucleotide, 1,5-anhydrohexitol nucleotide, L-nucleotides, alpha-nucleotide, modified base nucleotide, threo-pentofuranosyl nucleotide, acyclic 3′,4′-seco nucleotide, acyclic 3,4-dihydroxybutyl nucleotide, acyclic 3,5 dihydroxypentyl nucleotide, 3′-3′-inverted nucleotide moiety, 3′-3′-inverted abasic moiety, 3′-2′-inverted nucleotide moiety, 3′-2′-inverted abasic moiety, 1,4-butanediol phosphate, 3′-phosphoramidate, hexylphosphate, aminohexyl phosphate, 3′-phosphate, 3′phosphorothioate, phosphorodithioate, or bridging or non-bridging methylphosphonate moiety. These modified 5′-CAP structures may be used in the context of the present invention to modify the mRNA sequence of the inventive composition. Further modified 5′-CAP structures which may be used in the context of the present invention are CAP1 (additional methylation of the ribose of the adjacent nucleotide of m7GpppN), CAP2 (additional methylation of the ribose of the 2nd nucleotide downstream of the m7GpppN), CAP3 (additional methylation of the ribose of the 3rd nucleotide downstream of the m7GpppN), CAP4 (additional methylation of the ribose of the 4th nucleotide downstream of the m7GpppN), ARCA (anti-reverse CAP analogue), modified ARCA (e.g. phosphothioate modified ARCA), inosine, N1-methyl-guanosine, 2′-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine. In the context of the present invention, a 5′-CAP structure may also be formed in chemical RNA synthesis or RNA in vitro transcription (co-transcriptional capping) using cCAP analogues, or a CAP structure may be formed in vitro using capping enzymes (e.g., commercially available capping kits).

As used herein, a “carbamoyl” group means the radical —C(O)N(R′) 2 , wherein R′ is chosen from the group consisting of hydrogen, alkyl, and aryl.

As used herein, a “carbonyl” group is a divalent group of the formula —C═(O)—.

As used herein, the term “carrier” in the context of the invention may typically be a compound that facilitates transport and/or complexation of another compound. Said carrier may form a complex with said other compound. A polymeric carrier is a carrier that is formed of a polymer.

Unless a different meaning is clear from the specific context, the term “cationic” means that the respective structure bears a positive charge, either permanently, or not permanently but in response to certain conditions such as pH. Thus, the term “cationic” covers both “permanently cationic” and “cationisable”. As used herein, “permanently cationic” means that the respective compound, or group or atom, is positively charged at any pH value or hydrogen ion activity of its environment. Typically, the positive charge is results from the presence of a quaternary nitrogen atom. Where a compound carries a plurality of such positive charges, it may be referred to as permanently polycationic, which is a subcategory of permanently cationic.

As used herein, the term “Cellular immunity/cellular immune response” relates typically to the activation of macrophages, natural killer cells (NK), antigen-specific cytotoxic T-lymphocytes, and the release of various cytokines in response to an antigen. In a more general way, cellular immunity is not related to antibodies but to the activation of cells of the immune system. A cellular immune response is characterized e.g., by activating antigen-specific cytotoxic T-lymphocytes that are able to induce apoptosis in body cells displaying epitopes of an antigen on their surface, such as virus-infected cells, cells with intracellular bacteria, and cancer cells displaying tumor antigens; activating macrophages and natural killer cells, enabling them to destroy pathogens; and stimulating cells to secrete a variety of cytokines that influence the function of other cells involved in adaptive immune responses and innate immune responses.

In the context of the invention, a “composition” refers to any type of composition in which the specified ingredients may be incorporated, optionally along with any further constituents, usually with at least one pharmaceutically acceptable carrier or excipient. Thus, the composition may be a dry composition such as a powder or granules, or a solid unit such as a lyophilised form or a tablet. Alternatively, the composition may be in liquid form, and each constituent may be independently incorporated in dissolved or dispersed (e.g. suspended or emulsified) form. In one of the preferred embodiments, the composition is formulated as a sterile solid composition, such as a powder or lyophilised form for reconstitution with an aqueous liquid carrier. Such formulation is also preferred for those versions of the composition which comprise a nucleic acid cargo as described in further detail below.

As used herein, a “compound” means a chemical substance (e.g., an Ionizable Lipid of the Invention), which is a material consisting of molecules having essentially the same chemical structure and properties. For a small molecular compound, the molecules are typically identical with respect to their atomic composition and structural configuration. For a macromolecular or polymeric compound, the molecules of a compound are highly similar but not all of them are necessarily identical. For example, a segment of a polymer that is designated to consist of 50 monomeric units may also contain individual molecules with e.g. 48 or 53 monomeric units.

Unless the context indicates or requires otherwise, the words “comprise”, “comprises” and “comprising” and similar expressions are to be construed in an open and inclusive sense, as “including, but not limited to” in this description and in the claims.

As used herein, the term “cycloalkyl group” means a monocyclic or polycyclic saturated ring comprising carbon and hydrogen atoms and having no carbon-carbon multiple bonds. Examples of cycloalkyl groups include, but are not limited to, (C 3 -C 7 )cycloalkyl groups, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl, and saturated cyclic and bicyclic terpenes. A cycloalkyl group can be unsubstituted or substituted by one or two suitable substituents. Preferably, the cycloalkyl group is a monocyclic ring or bicyclic ring.

As used herein, the term “derivative of a peptide or protein” is typically understood to be a molecule that is derived from another molecule, such as said peptide or protein. A “derivative” of a peptide or protein also encompasses fusions comprising a peptide or protein used in the present invention. For example, the fusion comprises a label, such as, for example, an epitope, e.g., a FLAG epitope or a V5 epitope. For example, the epitope is a FLAG epitope. Such a tag is useful for, for example, purifying the fusion protein.

As used herein, the term “epitope (also called “antigen determinant”): refers to T cell epitopes or parts of the proteins in the context of the invention may comprise fragments preferably having a length of about 6 to about 20 or even more amino acids, e.g. fragments as processed and presented by MHC class I molecules, preferably having a length of about 8 to about 10 amino acids, e.g. 8, 9, or 10, (or even 11, or 12 amino acids), or fragments as processed and presented by MHC class II molecules, preferably having a length of about 13 or more amino acids, e.g. 13, 14, 15, 16, 17, 18, 19, 20 or even more amino acids, wherein these fragments may be selected from any part of the amino acid sequence. These fragments are typically recognized by T cells in form of a complex consisting of the peptide fragment and an MHC molecule.

As used herein, the term “fragments” of proteins or peptides in the context of the present invention may, typically, comprise a sequence of a protein or peptide as defined herein, which is, with regard to its amino acid sequence (or its encoded nucleic acid molecule), N-terminally and/or C-terminally truncated compared to the amino acid sequence of the original (native) protein (or its encoded nucleic acid molecule). Such truncation may thus occur either on the amino acid level or correspondingly on the nucleic acid level. A sequence identity with respect to such a fragment as defined herein may therefore preferably refer to the entire protein or peptide as defined herein or to the entire (coding) nucleic acid molecule of such a protein or peptide. In this context a fragment of a protein may typically comprise an amino acid sequence having a sequence identity of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably of at least 70%, more preferably of at least 80%, even more preferably at least 85%, even more preferably of at least 90% and most preferably of at least 95% or even 97%, with an amino acid sequence of the respective naturally occurring fill-length protein.

As used herein, the term “fragments of proteins or peptides” in the context of the present invention may furthermore comprise a sequence of a protein or peptide as defined herein, which has a length of for example at least 5 amino acids, preferably a length of at least 6 amino acids, preferably at least 7 amino acids, more preferably at least 8 amino acids, even more preferably at least 9 amino acids; even more preferably at least 10 amino acids; even more preferably at least 11 amino acids; even more preferably at least 12 amino acids; even more preferably at least 13 amino acids; even more preferably at least 14 amino acids; even more preferably at least 15 amino acids; even more preferably at least 16 amino acids; even more preferably at least 17 amino acids; even more preferably at least 18 amino acids; even more preferably at least 19 amino acids; even more preferably at least 20 amino acids; even more preferably at least 25 amino acids; even more preferably at least 30 amino acids; even more preferably at least 35 amino acids; even more preferably at least 50 amino acids; or most preferably at least 100 amino acids. For example such fragment may have a length of about 6 to about 20 or even more amino acids, e.g. fragments as processed and presented by MHC class I molecules, preferably having a length of about 8 to about 10 amino acids, e.g. 8, 9, or 10, (or even 6, 7, 11, or 12 amino acids), or fragments as processed and presented by MHC class II molecules, preferably having a length of about 13 or more amino acids, e.g. 13, 14, 15, 16, 17, 18, 19, 20 or even more amino acids, wherein these fragments may be selected from any part of the amino acid sequence. These fragments are typically recognized by T-cells in form of a complex consisting of the peptide fragment and an MHC molecule, i.e. the fragments are typically not recognized in their native form. Fragments of proteins or peptides may comprise at least one epitope of those proteins or peptides. Furthermore also domains of a protein, like the extracellular domain, the intracellular domain or the transmembrane domain and shortened or truncated versions of a protein may be understood to comprise a fragment of a protein.

As used herein, the term “full-length protein” as used herein typically refers to a protein that substantially comprises the entire amino acid sequence of the naturally occurring protein. Nevertheless, substitutions of amino acids e.g. due to mutation in the protein are also encompassed in the term full-length protein.

As used herein, “halogen” means fluorine, chlorine, bromine, or iodine. Accordingly, the meaning of the terms “halo” and “Hal” encompass fluoro, chloro, bromo, and iodo.

As used herein, the term an “heteroaryl group” means a monocyclic- or polycyclic aromatic ring comprising carbon atoms, hydrogen atoms, and one or more heteroatoms, preferably 1 to 3 heteroatoms, independently selected from nitrogen, oxygen, and sulfur. Illustrative examples of heteroaryl groups include, but are not limited to, pyridinyl, pyridazinyl, pyrimidinyl, pyrazyl, triazinyl, pyrrolyl, pyrazolyl, imidazolyl, (1,2,3)- and (1,2,4)-triazolyl, pyrazinyl, pyrimidinyl, tetrazolyl, furyl, thiophenyl, isoxazolyl, thiazolyl, furyl, phenyl, isoxazolyl, and oxazolyl. A heteroaryl group can be unsubstituted or substituted with one or two suitable substituents. Preferably, a heteroaryl group is a monocyclic ring, wherein the ring comprises 2 to 5 carbon atoms and 1 to 3 heteroatoms, referred to herein as “(C 2 -C 5 )heteroaryl”.

As used herein, the term “heterocycloalkyl group” means a monocyclic or polycyclic ring comprising carbon and hydrogen atoms and at least one heteroatom, preferably, 1 to 3 heteroatoms selected from nitrogen, oxygen, and sulfur, and having no unsaturation. Examples of heterocycloalkyl groups include pyrrolidinyl, pyrrolidino, piperidinyl, piperidino, piperazinyl, piperazino, morpholinyl, morpholino, thiomorpholinyl, dioxolanyl, thiomorpholino, and pyranyl. A heterocycloalkyl group can be unsubstituted or substituted with one or two suitable substituents. Preferably, the heterocycloalkyl group is a monocyclic or bicyclic ring, more preferably, a monocyclic ring, wherein the ring comprises from 3 to 6 carbon atoms and from 1 to 3 heteroatoms, referred to herein as (C 1 -C 6 )heterocycloalkyl.

As used herein, the terms “heterocyclic radical” or “heterocyclic ring” mean a heterocycloalkyl group or a heteroaryl group.

As used herein, the term “humoral immunity” refers typically to antibody production and the accessory processes that may accompany it. A humoral immune response may be typically characterized, e.g., by Th2 activation and cytokine production, germinal center formation and isotype switching, affinity maturation and memory cell generation. Humoral immunity also typically may refer to the effector functions of antibodies, which include pathogen and toxin neutralization, classical complement activation, and opsonin promotion of phagocytosis and pathogen elimination.

As used herein, the term “hydrate” means a compound of the invention or a salt thereof, that further includes a stoichiometric or non-stoichiometric amount of water bound by non-covalent intermolecular forces. The term hydrate includes solvates, which are stoichiometric or non-stoichiometric amounts of a solvent bound by non-covalent intermolecular forces. Preferred solvents are volatile, non-toxic, and/or acceptable for administration to humans in trace amounts.

As used herein, the term “hydrocarbyl” group means a monovalent group selected from (C 1 -C 22 )alkyl, (C 2 -C 22 )alkenyl, and (C 2 -C 5 )alkynyl, optionally substituted with one or two suitable substituents. Preferably, the hydrocarbon chain of a hydrocarbyl group is from 1 to 22 carbon atoms in length, referred to herein in certain instances as “(C 1 -C 22 )hydrocarbyl”.

As used herein, the term “identity of a sequence” means the percentage to which two sequences are identical, e.g. nucleic acid sequences or amino acid sequences as defined herein, preferably the amino acid sequences encoded by a nucleic acid sequence of the polymeric carrier as defined herein or the amino acid sequences themselves, the sequences can be aligned in order to be subsequently compared to one another. Therefore, e.g. a position of a first sequence may be compared with the corresponding position of the second sequence. If a position in the first sequence is occupied by the same component (residue) as is the case at a position in the second sequence, the two sequences are identical at this position. If this is not the case, the sequences differ at this position. If insertions occur in the second sequence in comparison to the first sequence, gaps can be inserted into the first sequence to allow a further alignment. If deletions occur in the second sequence in comparison to the first sequence, gaps can be inserted into the second sequence to allow a further alignment. The percentage to which two sequences are identical is then a function of the number of identical positions divided by the total number of positions including those positions which are only occupied in one sequence. The percentage to which two sequences are identical can be determined using a mathematical algorithm. A preferred, but not limiting, example of a mathematical algorithm which can be used is the algorithm of Karlin et al. (1993), PNAS USA, 90:5873-5877 or Altschul et al. (1997), Nucleic Acids Res., 25:3389-3402. Such an algorithm is integrated in the BLAST program. Sequences which are identical to the sequences of the present invention to a certain extent can be identified by this program.

As used herein, the term “immune system” may protect organisms from infection. If a pathogen breaks through a physical barrier of an organism and enters this organism, the innate immune system provides an immediate, but non-specific response. If pathogens evade this innate response, vertebrates possess a second layer of protection, the adaptive immune system. Here, the immune system adapts its response during an infection to improve its recognition of the pathogen. This improved response is then retained after the pathogen has been eliminated, in the form of an immunological memory, and allows the adaptive immune system to mount faster and stronger attacks each time this pathogen is encountered. According to this, the immune system comprises the innate and the adaptive immune system. Each of these two parts contains so called humoral and cellular components.

As used herein, the term “an immune response” may typically either be a specific reaction of the adaptive immune system to a particular antigen (so called specific or adaptive immune response) or an unspecific reaction of the innate immune system (so called unspecific or innate immune response). The invention relates to the core to specific reactions (adaptive immune responses) of the adaptive immune system. Particularly, it relates to adaptive immune responses to infections by viruses like e.g. Influenza viruses. However, this specific response can be supported by an additional unspecific reaction (innate immune response). Therefore, the invention also relates to a compound for simultaneous stimulation of the innate and the adaptive immune system to evoke an efficient adaptive immune response.

As used herein, the term “immunostimulatory RNA (isRNA)” in the context of the invention may typically be an RNA that is able to induce an innate immune response itself. It usually does not have an open reading frame and thus does not provide a peptide-antigen or immunogen but elicits an innate immune response e.g. by binding to a specific kind of Toll-like-receptor (TLR) or other suitable receptors. However, of course also mRNAs having an open reading frame and coding for a peptide/protein (e.g. an antigenic function) may induce an innate immune response.

As used herein, the term “influenza pandemic or pandemic flu” can occur when a non-human (novel) influenza virus gains the ability for efficient and sustained human-to-human transmission and then spreads globally. Influenza viruses that have the potential to cause a pandemic are referred to as “influenza viruses with pandemic potential” or “pandemic influenza virus”. Examples of influenza viruses with pandemic potential include avian influenza A (H5N1) and avian influenza A (H7N9), which are two different “bird flu” viruses. These are non-human viruses (i.e., they are novel among humans and circulate in birds in parts of the world) so there is little to no immunity against these viruses among people. Human infections with these viruses have occurred rarely, but if either of these viruses was to change in such a way that it was able to infect humans easily and spread easily from person to person, an influenza pandemic could result.

As used herein, the term “innate immune system” also known as non-specific immune system, comprises the cells and mechanisms that defend the host from infection by other organisms in a non-specific manner. This means that the cells of the innate system recognize and respond to pathogens in a generic way, but unlike the adaptive immune system, it does not confer long-lasting or protective immunity to the host. The innate immune system may be e.g. activated by ligands of pathogen-associated molecular patterns (PAMP) receptors, e.g. Toll-like receptors (TLRs) or other auxiliary substances such as lipopolysaccharides, TNF-alpha, CD40 ligand, or cytokines, monokines, lymphokines, interleukins or chemokines, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IFN-alpha, IFN-beta, IFN-gamma, GM-CSF, G-CSF, M-CSF, LT-beta, TNF-alpha, growth factors, and hGH, a ligand of human Toll-like receptor TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, a ligand of murine Toll-like receptor TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12 or TLR13, a ligand of a NOD-like receptor, a ligand of a RIG-I like receptor, an immunostimulatory nucleic acid, an immunostimulatory RNA (isRNA), a CpG-DNA, an antibacterial agent, or an anti-viral agent. Typically a response of the innate immune system includes recruiting immune cells to sites of infection, through the production of chemical factors, including specialized chemical mediators, called cytokines; activation of the complement cascade; identification and removal of foreign substances present in organs, tissues, the blood and lymph, by specialized white blood cells; activation of the adaptive immune system through a process known as antigen presentation; and/or acting as a physical and chemical barrier to infectious agents.

As used herein, the term “ionizable” (e.g., as used in the term ‘an Ionizable Lipid of the Invention’) means that a compound, or group or atom, is charged at a certain pH and uncharged at another pH of its environment. Also in non-aqueous environments where no pH value can be determined, a ionizable compound, group or atom is positively charged at a high hydrogen ion concentration and negatively or uncharged at a low concentration or activity of hydrogen ions. It depends on the individual properties of the ionizable or polyionizable compound, in particular the pKa of the respective ionizable group or atom, at which pH or hydrogen ion concentration it is charged or uncharged. In diluted aqueous environments, the fraction of ionizable compounds, groups or atoms bearing a charge may be estimated using the so-called Henderson-Hasselbalch equation, which is well-known to a person skilled in the art. For example, in some embodiments, if a compound or moiety is ionizable, it is preferred that it is positively charged at a pH value of about 1 to 9, preferably 4 to 9, 5 to 8 or even 6 to 8, more preferably of a pH value of or below 9, of or below 8, of or below 7, most preferably at physiological pH values, e.g. about 7.3 to 7.4, i.e. under physiological conditions, particularly under physiological salt conditions of the cell in vivo. In other embodiments, the ionizable compound or moiety is predominantly neutral at physiological pH values, e.g. about 7.0-7.4, but becomes positively charged at lower pH values. In some embodiments, the preferred range of pKa for the cationisable compound or moiety is about 5 to about 7.

As used herein, the phrase “Ionizable Lipids of the Invention” means compounds disclosed herein including the ionizable lipid nanoparticle compounds disclosed herein. Particular compounds of the invention are compounds of formulas I, II, III, IV, V, VI, VII, VIII, or IX and pharmaceutically acceptable salts, hydrates, enantiomers, diastereomer, racemates or mixtures of stereoisomers thereof. Thus, “Ionizable Lipids of the invention” collectively means compound of formulas I, II, III, IV, V, VI, VII, VIII, or IX and pharmaceutically acceptable salts, hydrates, enantiomers, diastereomer, racemates or mixtures of stereoisomers thereof. The Ionizable Lipids of the invention are identified herein by their chemical structure and/or chemical name. Where a compound is referred to by both a chemical structure and a chemical name, and the chemical structure and chemical name conflict, the chemical structure is to be accorded more weight. The term “Ionizable Lipid of the Invention” typically refers to a molecule capable of being charged, which is, for example, positively charged (cationic) at a pH value of typically about 1 to 9. In some embodiments, the cationic component/compound is preferably charged at a pH value of or below 9 (e.g. 5 to 9), of or below 8 (e.g. 5 to 8), of or below 7 (e.g. 5 to 7), most preferably at physiological pH values, e.g. about 7.3 to 7.4, and endosomal pH values, e.g. about 7 to 5. Accordingly, a cationic peptide, protein, polysaccharide, lipid or polymer according to one embodiment of the present invention is, in certain embodiments, positively charged under physiological conditions, particularly under physiological salt conditions of the cell in vivo. In another preferred embodiment, the lipid nanoparticle, the cationic peptide, protein, polysaccharide, lipid or polymer according to the present invention is uncharged, has a neutral charge or is respectively electrically neutral under physiological conditions, particularly under physiological salt conditions of the cell in vivo. A cationic peptide or protein preferably contains a larger number of cationic amino acids, e.g. a larger number of Arg, His, Lys or Orn than other amino acid residues (in particular more cationic amino acids than anionic amino acid residues like Asp or Glu) or contains blocks predominantly formed by cationic amino acid residues. The expression “cationic” may also refer to “polycationic” components/compounds. The cationic component/compound may also refer to a cationic lipid capable of being positively charged. Exemplary cationic lipids include one or more amine group(s) which bear the positive charge. Preferred cationic lipids are ionizable such that they can exist in a positively charged or neutral form depending on pH. The ionization of the cationic lipid affects the surface charge of a lipid nanoparticle (LNP) under different pH conditions. This charge state can influence plasma protein absorption, blood clearance and tissue distribution (Semple, S. C., et al., Adv. Drug Deliv Rev 32:3-17 (1998)) as well as the ability to form non-bilayer structures (Hafez, I. M., et al., Gene Ther 8:1188-1196 (2001)) critical to the intracellular delivery of nucleic acids. As described elsewhere, the pKa of formulated cationic lipids is correlated with the effectiveness of LNPs for delivery of nucleic acids (see Jayaraman et al, Angewandte Chemie, International Edition (2012), 51(34), 8529-8533; Semple et al, Nature Biotechnology 28, 172-176 (2010)). In some embodiments of the present invention, the preferred range of pKa is about 5 to about 7. In preferred embodiments, the Ionizable Lipids of the Invention have a neutral charge with a slight positive or negative charge near pH 7. The Ionizable Lipids of the Invention can contain one or more chiral centers and/or double bonds and, therefore, exist as stereoisomers, such as double-bond isomers (i.e., geometric isomers), enantiomers, or diastereomers. According to the invention, the chemical structures depicted herein, and therefore the Ionizable Lipids of the Invention, encompass all of the corresponding compounds' enantiomers and stereoisomers, that is, both the stereomerically pure form (e.g., geometrically pure, enantiomerically pure, or diastereomerically pure) and enantiomeric and stereoisomeric mixtures. An Ionizable Lipids of the Invention is considered optically active or enantiomerically enriched (i.e., substantially the R-form or substantially the S-form) with respect to a chiral center when the compound is about 90% ee (enantiomeric excess) or greater, preferably, equal to or greater than 95% ee with respect to a particular chiral center. An Ionizable Lipids of the Invention is considered to be in enantiomerically-enriched form when the compound has an enantiomeric excess of greater than about 1% ee, preferably greater than about 5% ee, more preferably, greater than about 10% ee with respect to a particular chiral center. An Ionizable Lipids of the Invention is considered diastereomerically pure with respect to multiple chiral centers when the compound is about 90% de (diastereomeric excess) or greater, preferably, equal to or greater than 95% de with respect to a particular chiral center. An Ionizable Lipids of the Invention is considered to be in diastereomerically-enriched form when the compound has an diastereomeric excess of greater than about 1% de, preferably greater than about 5% de, more preferably, greater than about 10% de with respect to a particular chiral center. As used herein, a racemic mixture means about 50% of one enantiomer and about 50% of is corresponding enantiomer relative to all chiral centers in the molecule. Thus, the invention encompasses all enantiomerically-pure, enantiomerically-enriched, diastereomerically pure, diastereomerically enriched, and racemic mixtures of compounds of Formulas I through V. Enantiomeric and diastereomeric mixtures can be resolved into their component enantiomers or stereoisomers by well-known methods, such as chiral-phase gas chromatography, chiral-phase high performance liquid chromatography, crystallizing the compound as a chiral salt complex, or crystallizing the compound in a chiral solvent. Enantiomers and diastereomers can also be obtained from diastereomerically- or enantiomerically-pure intermediates, reagents, and catalysts by well-known asymmetric synthetic methods. The Ionizable Lipids of the Invention are defined herein by their chemical structures and/or chemical names. Where a compound is referred to by both a chemical structure and a chemical name, and the chemical structure and chemical name conflict, the chemical structure is determinative of the compound's identity. When administered to a patient, e.g., to an animal for veterinary use or for improvement of livestock, or to a human for clinical use, the Ionizable Lipids of the Invention are administered in isolated form or as the isolated form in a pharmaceutical composition. As used herein, “isolated” means that the compounds of the invention are separated from other components of either (a) a natural source, such as a plant or cell, preferably bacterial culture, or (b) a synthetic organic chemical reaction mixture. Preferably, via conventional techniques, the Ionizable Lipids of the Invention are purified. As used herein, “purified” means that when isolated, the isolate contains at least 95%, preferably at least 98%, of a single hydroxy compound of the invention by weight of the isolate.

As used herein, the term “jet injection”, as used herein, refers to a needle-free injection method, wherein a fluid containing at least one inventive mRNA sequence and, optionally, further suitable excipients is forced through an orifice, thus generating an ultra-fine liquid stream of high pressure that is capable of penetrating mammalian skin and, depending on the injection settings, subcutaneous tissue or muscle tissue. In principle, the liquid stream forms a hole in the skin, through which the liquid stream is pushed into the target tissue. Preferably, jet injection is used for intradermal, subcutaneous or intramuscular injection of the mRNA sequence according to the invention. In a preferred embodiment, jet injection is used for intramuscular injection of the mRNA sequence according to the invention. In a further preferred embodiment, jet injection is used for intradermal injection of the mRNA sequence according to the invention.

As used herein the term “lipidoid compound,” also simply referred to as lipidoid, is a lipid-like compound, i.e. an amphiphilic compound with lipid-like physical properties. In the context of the present invention the term lipid is considered to encompass lipidoids.

As used herein, the term “microneedle injection” refers to microscopic applicators used to deliver vaccines or other drugs across various barriers: while transdermal application is the most popular use of microneedles, intraocular and intracochlear microneedle drug delivery systems are emerging. Microneedles are constructed through various methods usually involving photolithographic processes or micromolding. These methods involve etching microscopic structure into resin or silicon in order to cast microneedles. Microneedles are made from a variety of material ranging from silicon, titanium, stainless steel, and polymers. Some microneedles are made of a drug to be delivered to the body but are shaped into a needle so they will penetrate the skin. The microneedles range in size, shape, and function but are all used as an alternative to other delivery methods like the conventional hypodermic needle or other injection apparatus.

As used herein, the term “monocistronic mRNA” may typically be an mRNA, that comprises only one open reading frame (coding sequence or coding region). An open reading frame in this context is a sequence of several nucleotide triplets (codons) that can be translated into a peptide or protein.

As used herein, the term “nucleic acid” means any DNA- or RNA-molecule. The term may be used for a polynucleotide and/or oligonucleotide. Wherever herein reference is made to a nucleic acid or nucleic acid sequence encoding a particular protein and/or peptide, said nucleic acid or nucleic acid sequence, respectively, preferably also comprises regulatory sequences allowing in a suitable host, e.g. a human being, its expression, i.e. transcription and/or translation of the nucleic acid sequence encoding the particular protein or peptide.

As used herein, the term “nucleoside modification” in the context of the present invention the term nucleoside modification refers to mRNA molecules or compounds comprising nucleosides, which are not usually part of mRNA, preferably non-natural nucleosides. In particular, the term preferably refers to mRNA nucleosides other than adenine, guanine, cytosine, uracil and in some cases thymine.

As used herein, the term “peptide” is an oligomer or polymer of at least two amino acid monomers. Usually the monomers are linked by peptide bonds. The term “peptide” does not limit the length of the polymer chain of amino acids. In some embodiments of the present invention a peptide may for example contain less than 50 monomer units. Longer peptides are also called polypeptides, typically having 50 to 600 monomeric units, more specifically 50 to 300 monomeric units.

As described herein “nucleoside” is defined as a compound containing a five-carbon sugar molecule (a pentose or ribose) or derivative thereof, and an organic base, purine or pyrimidine, or a derivative thereof. As described herein, “nucleotide” is defined as a nucleoside consisting of a phosphate group. In some embodiments, the chemical modifications can include an amino group, a thiol group, an alkyl group, or a halo group. In some embodiments, modified nucleosides include pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine. In some embodiments, modified nucleosides include 5-aza-cytidine, pseudoisocytidme, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, and 4-methoxy-1-methyl-pseudoisocytidine. In other embodiments, modified nucleosides include 2-aminopurine, 2, 6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2, 6-diaminopurine, 7-deaza-8-aza-2, 6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine. In some embodiments, modified nucleosides include inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.

The phrase “pharmaceutically acceptable salt(s),” as used herein includes, but is not limited to, salts of acidic or basic groups that may be present in the compounds of the invention. Compounds that are basic in nature are capable of forming a wide variety of salts with various inorganic and organic acids. The acids that may be used to prepare pharmaceutically acceptable acid addition salts of such basic compounds are those that form non-toxic acid addition salts, i.e., salts containing pharmacologically acceptable anions, including but not limited to sulfuric, citric, maleic, acetic, oxalic, hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate and pamoate (i.e., 1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts. Compounds of the invention that include an amino moiety also can form pharmaceutically acceptable salts with various amino acids, in addition to the acids mentioned above. Compounds of the invention that are acidic in nature are capable of forming base salts with various pharmacologically acceptable cations. Examples of such salts include alkali metal or alkaline earth metal salts and, particularly, calcium, magnesium, sodium lithium, zinc, potassium, and iron salts.

As used herein, the term “pharmaceutically effective amount” in the context of the invention is typically understood to be an amount that is sufficient to induce an immune response.

As used herein, the term “phenyl” means —C 6 H 5 . A phenyl group can be unsubstituted or substituted with one or two suitable substituents, wherein the substituent replaces an H of the phenyl group. As used herein, “Ph,” represents a phenyl group or a substituted phenyl group.

As used herein, the prefix “poly-” refers to a plurality of atoms or groups having the respective property in a compound. If put in parenthesis, the presence of a plurality is optional. For example, (poly)cationic means cationic and/or polycationic. However, the absence of the prefix should not be interpreted such as to exclude a plurality. For example, a polycationic compound is also a cationic compound and may be referred to as such.

As used herein, the term “poly-A-tail” also called “3′-poly(A) tail or poly(A) sequence” is typically a long sequence of adenosine nucleotides of up to about 400 adenosine nucleotides, e.g. from about 25 to about 400, preferably from about 50 to about 400, more preferably from about 50 to about 300, even more preferably from about 50 to about 250, most preferably from about 60 to about 250 adenosine nucleotides, added to the 3′-end of a RNA. Moreover, poly(A) sequences, or poly(A) tails may be generated in vitro by enzymatic polyadenylation of the RNA, e.g. using Poly(A)polymerases derived from E. coli or yeast.

As used herein, the term “polyadenylation” is typically understood to be the addition of a poly(A) sequence to a nucleic acid molecule, such as an RNA molecule, e.g. to a premature mRNA. Polyadenylation may be induced by a so called polyadenylation signal. This signal is preferably located within a stretch of nucleotides at the 3′-end of a nucleic acid molecule, such as an RNA molecule, to be polyadenylated. A polyadenylation signal typically comprises a hexamer consisting of adenine and uracil/thymine nucleotides, preferably the hexamer sequence AAUAAA. Other sequences, preferably hexamer sequences, are also conceivable. Polyadenylation typically occurs during processing of a pre-mRNA (also called premature-mRNA). Typically, RNA maturation (from pre-mRNA to mature mRNA) comprises the step of polyadenylation.

As used herein, the term “Poly (C) sequence” is typically a long sequence of cytosine nucleotides, typically about 10 to about 200 cytosine nucleotides, preferably about 10 to about 100 cytosine nucleotides, more preferably about 10 to about 70 cytosine nucleotides or even more preferably about 20 to about 50 or even about 20 to about 30 cytosine nucleotides. A poly(C) sequence may preferably be located 3′ of the coding region comprised by a nucleic acid.

As used herein, the term “protein” typically consists of one or more peptides and/or polypeptides folded into a 3-dimensional form, facilitating a biological function.

As used herein, the terms “Pyr” “Pyrd” and “PyrD” are used interchangeably and refer to a pyridine or pyridyl substituent.

As used herein, RNA encompasses messenger RNA and modified messenger RNA, as well as coding (cRNA) and noncoding RNA (ncRNA) including housekeeping ncRNAs (transfer RNA (i.e., tRNA) and ribosomalRNA (i.e, rRNA)) and regulatory ncRNAs, which are further classified according to their size including long ncRNAs (lncRNA) having at least 200 nucleotides and small ncRNAs have fewer than 200 nucleotides including micro RNA (miRNA), small nucleolar RNA (snoRNA), small nuclear RNA (snRNA), small-interfering RNA (siRNA), and PIWI-interacting RNA (piRNA).

As used herein, the terms “RNA in vitro transcription” or “in vitro transcription” relate to a process wherein RNA is synthesized in a cell-free system (in vitro). DNA, particularly plasmid DNA, is used as template for the generation of RNA transcripts. RNA may be obtained by DNA-dependent in vitro transcription of an appropriate DNA template, which according to the present invention is preferably a linearized plasmid DNA template. The promoter for controlling in vitro transcription can be any promoter for any DNA-dependent RNA polymerase. Particular examples of DNA-dependent RNA polymerases are the T7, T3, and SP6 RNA polymerases. A DNA template for in vitro RNA transcription may be obtained by cloning of a nucleic acid, in particular cDNA corresponding to the respective RNA to be in vitro transcribed, and introducing it into an appropriate vector for in vitro transcription, for example into plasmid DNA. In a preferred embodiment of the present invention the DNA template is linearized with a suitable restriction enzyme before it is transcribed in vitro. The cDNA may be obtained by reverse transcription of mRNA or chemical synthesis. Moreover, the DNA template for in vitro RNA synthesis may also be obtained by gene synthesis. As used herein, the methods for in vitro transcription are known in the art (see, e.g., Geall et al. (2013) Semin. Immunol. 25(2): 152-159; Brunelle et al. (2013) Methods Enzymol. 530:101-14). Reagents used in said method typically include:

1) a linearized DNA template with a promoter sequence that has a high binding affinity for its respective RNA polymerase such as bacteriophage-encoded RNA polymerases; 2) ribonucleoside triphosphates (NTPs) for the four bases (adenine, cytosine, guanine and uracil); 3) optionally a CAP analogue as defined above (e.g. m7G(5′)ppp(5′)G (m7G) or 10.1126/scitranslmed.aav5701): RNAs were capped using the m7G capping kit with 2′-O-methyltransferase (ScriptCap, CELLSCRIPT) to obtain cap1); 4) a DNA-dependent RNA polymerase capable of binding to the promoter sequence within the linearized DNA template (e.g. T7, T3 or SP6 RNA polymerase); 5) optionally a ribonuclease (RNase) inhibitor to inactivate any contaminating RNase; 6) optionally a pyrophosphatase to degrade pyrophosphate, which may inhibit transcription; 7) MgCl 2 , which supplies Mg2+ ions as a co-factor for the polymerase; 8) a buffer to maintain a suitable pH value, which can also contain antioxidants (e.g. DTT), and/or polyamines such as spermidine at optimal concentrations.

As used herein, the term “stabilized nucleic acid, preferably mRNA” typically, exhibits a modification increasing resistance to in vivo degradation (e.g. degradation by an exo- or endo-nuclease) and/or ex vivo degradation (e.g. by the manufacturing process prior to vaccine administration, e.g. in the course of the preparation of the vaccine solution to be administered). Stabilization of RNA can, e.g., be achieved by providing a 5′-CAP-Structure, a Poly-A-Tail, or any other UTR-modification. It can also be achieved by chemical modification or modification of the G/C-content of the nucleic acid. Various other methods are known in the art and conceivable in the context of the invention.

As used herein, a composition that “substantially” comprises a compound means that the composition contains more than about 80% by weight, more preferably more than about 90% by weight, even more preferably more than about 95% by weight, and most preferably more than about 97% by weight of the compound.

As used herein, a reaction that is “substantially complete” means that the reaction contains more than about 80% by weight of the desired product, more preferably more than about 90% by weight of the desired product, even more preferably more than about 95% by weight of the desired product, and most preferably more than about 97% by weight of the desired product.

As used herein, a composition that is “substantially free” of a compound means that the composition contains less than about 20% by weight, more preferably less than about 10% by weight, even more preferably less than about 5% by weight, and most preferably less than about 3% by weight of the compound.

As used herein, a “substituted” or “substituent” each means a group that does not nullify the synthetic or pharmaceutical utility of the compounds of the invention or the intermediates useful for preparing them. Examples of suitable substituents include, but are not limited to: —NH 2 ; CN; halo, heterocycloalkyl; heterocycloaryl; (C 1 -C 22 )alkyl; (C 2 -C 22 )alkenyl; (C 2 -C 22 )alkynyl; (C 6 )aryl; (C 2 -C 5 )heteroaryl; (C 3 -C 7 )cycloalkyl; (C 1 -C 8 )alkoxy; (C 6 )aryloxy; —CN; —OH; oxo; halo, —CO 2 H; —NH 2 ; —NH((C 1 -C 22 )alkyl); —N((C 1 -C 22 )alkyl) 2 ; —NH((C 6 )aryl); —N((C 6 )aryl) 2 ; —CHO; —CO((C 1 -C 22 )alkyl); —CO((C 6 )aryl); —CO 2 ((C 1 -C 22 )alkyl); —CO 2 ((C 6 )aryl); —SO 2 ((C 1 -C 22 )alkyl); and —SO 2 ((C 6 )aryl) or any of the groups identified herein that can act as a suitable substituent. One of skill in the art can readily choose a suitable substituent based on the stability and pharmacological and synthetic activity of the compound of the invention.

As used herein, the term “5′-terminal oligopyrimidine tract (TOP)” is typically a stretch of pyrimidine nucleotides located at the 5′-terminal region of a nucleic acid molecule, such as the 5′-terminal region of certain mRNA molecules or the 5′-terminal region of a functional entity, e.g. the transcribed region, of certain genes. The sequence starts with a cytidine, which usually corresponds to the transcriptional start site, and is followed by a stretch of usually about 3 to 30 pyrimidine nucleotides. For example, the TOP may comprise 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or even more nucleotides. The pyrimidine stretch and thus the 5′-TOP ends one nucleotide 5′ to the first purine nucleotide located downstream of the TOP. Messenger RNA that contains a 5′-terminal oligopyrimidine tract is often referred to as TOP mRNA. Accordingly, genes that provide such messenger RNAs are referred to as TOP genes. TOP sequences have, for example, been found in genes and mRNAs encoding peptide elongation factors and ribosomal proteins.

As used herein, the term “TOP motif” is a nucleic acid sequence which corresponds to a 5′-TOP as defined above. Thus, a TOP motif in the context of the present invention is preferably a stretch of pyrimidine nucleotides having a length of 3-30 nucleotides. Preferably, the TOP motif consists of at least 3 pyrimidine nucleotides, preferably at least 4 pyrimidine nucleotides, preferably at least 5 pyrimidine nucleotides, more preferably at least 6 nucleotides, more preferably at least 7 nucleotides, most preferably at least 8 pyrimidine nucleotides, wherein the stretch of pyrimidine nucleotides preferably starts at its 5′-end with a cytosine nucleotide. In TOP genes and TOP mRNAs, the TOP motif preferably starts at its 5′-end with the transcriptional start site and ends one nucleotide 5′ to the first purine residue in said gene or mRNA. A TOP motif in the sense of the present invention is preferably located at the 5′-end of a sequence which represents a 5′-UTR or at the 5′-end of a sequence which codes for a 5′-UTR. Thus, preferably, a stretch of 3 or more pyrimidine nucleotides is called “TOP motif” in the sense of the present invention if this stretch is located at the 5′end of a respective sequence, such as the inventive mRNA, the 5′-UTR element of the inventive mRNA, or the nucleic acid sequence which is derived from the 5′-UTR of a TOP gene as described herein. In other words, a stretch of 3 or more pyrimidine nucleotides which is not located at the 5′-end of a 5′-UTR or a 5′-UTR element but anywhere within a 5′-UTR or a 5′-UTR element is preferably not referred to as “TOP motif”.

As used herein, the term “TOP genes” are typically characterized by the presence of a 5′-terminal oligopyrimidine tract. Furthermore, most TOP genes are characterized by a growth-associated translational regulation. However, also TOP genes with a tissue specific translational regulation are known. As defined above, the 5′-UTR of a TOP gene corresponds to the sequence of a 5′-UTR of a mature mRNA derived from a TOP gene, which preferably extends from the nucleotide located 3′ to the 5′-CAP to the nucleotide located 5′ to the start codon. A 5′-UTR of a TOP gene typically does not comprise any start codons, preferably no upstream AUGs (uAUGs) or upstream open reading frames (uORFs). Therein, upstream AUGs and upstream open reading frames are typically understood to be AUGs and open reading frames that occur 5′ of the start codon (AUG) of the open reading frame that should be translated. The 5′-UTRs of TOP genes are generally rather short. The lengths of 5′-UTRs of TOP genes may vary between 20 nucleotides up to 500 nucleotides, and are typically less than about 200 nucleotides, preferably less than about 150 nucleotides, more preferably less than about 100 nucleotides. Exemplary 5′-UTRs of TOP genes in the sense of the present invention are the nucleic acid sequences extending from the nucleotide at position 5 to the nucleotide located immediately 5′ to the start codon (e.g. the ATG) in the sequences according to SEQ ID NOs: 1-1363, SEQ ID NO: 1395, SEQ ID NO: 1421 and SEQ ID NO: 1422 of international patent application WO2013/143700 or homologs or variants thereof, whose disclosure is incorporated herewith by reference. In this context a particularly preferred fragment of a 5′-UTR of a TOP gene is a 5′-UTR of a TOP gene lacking the 5′-TOP motif. The term “5′-UTR of a TOP gene” preferably refers to the 5′-UTR of a naturally occurring TOP gene.

As used herein, the “3′-untranslated region (3′-UTR)” is typically the part of an mRNA which is located between the protein coding region (i.e. the open reading frame) and the poly(A) sequence of the mRNA. A 3′-UTR of the mRNA is not translated into an amino acid sequence. The 3′-UTR sequence is generally encoded by the gene which is transcribed into the respective mRNA during the gene expression process. The genomic sequence is first transcribed into pre-mature mRNA, which comprises optional introns. The pre-mature mRNA is then further processed into mature mRNA in a maturation process. This maturation process comprises the steps of 5′-Capping, splicing the pre-mature mRNA to excise optional introns and modifications of the 3′-end, such as polyadenylation of the 3′-end of the pre-mature mRNA and optional endo- or exonuclease cleavages etc. In the context of the present invention, a 3′-UTR corresponds to the sequence of a mature mRNA which is located 3′ to the stop codon of the protein coding region, preferably immediately 3′ to the stop codon of the protein coding region, and which extends to the 5′-side of the poly(A) sequence, preferably to the nucleotide immediately 5′ to the poly(A) sequence. The term “corresponds to” means that the 3′-UTR sequence may be an RNA sequence, such as in the mRNA sequence used for defining the 3′-UTR sequence, or a DNA sequence which corresponds to such RNA sequence. In the context of the present invention, the term “a 3′-UTR of a gene”, such as “a 3′-UTR of an albumin gene”, is the sequence which corresponds to the 3′-UTR of the mature mRNA derived from this gene, i.e. the mRNA obtained by transcription of the gene and maturation of the pre-mature mRNA. The term “3′-UTR of a gene” encompasses the DNA sequence and the RNA sequence of the 3′-UTR.

As used herein, the term “5′-untranslated region (5′-UTR)” is typically understood to be a particular section of messenger RNA (mRNA). It is located 5′ of the open reading frame of the mRNA. Typically, the 5′-UTR starts with the transcriptional start site and ends one nucleotide before the start codon of the open reading frame. The 5′-UTR may comprise elements for controlling gene expression, also called regulatory elements. Such regulatory elements may be, for example, ribosomal binding sites or a 5′-Terminal Oligopyrimidine Tract. The 5′-UTR may be posttranscriptionally modified, for example by addition of a 5′-CAP. In the context of the present invention, a 5′-UTR corresponds to the sequence of a mature mRNA which is located between the 5′-CAP and the start codon. Preferably, the 5′-UTR corresponds to the sequence which extends from a nucleotide located 3′ to the 5′-CAP, preferably from the nucleotide located immediately 3′ to the 5′-CAP, to a nucleotide located 5′ to the start codon of the protein coding region, preferably to the nucleotide located immediately 5′ to the start codon of the protein coding region. The nucleotide located immediately 3′ to the 5′-CAP of a mature mRNA typically corresponds to the transcriptional start site. The term “corresponds to” means that the 5′-UTR sequence may be an RNA sequence, such as in the mRNA sequence used for defining the 5′-UTR sequence, or a DNA sequence which corresponds to such RNA sequence. In the context of the present invention, the term “a 5′-UTR of a gene”, such as “a 5′-UTR of a TOP gene”, is the sequence which corresponds to the 5′-UTR of the mature mRNA derived from this gene, i.e. the mRNA obtained by transcription of the gene and maturation of the pre-mature mRNA. The term “5′-UTR of a gene” encompasses the DNA sequence and the RNA sequence of the 5′-UTR.

As used herein, the term “vaccine” is typically understood to be a prophylactic or therapeutic material providing at least one antigen or antigenic function. The antigen or antigenic function may stimulate the body's adaptive immune system to provide an adaptive immune response. As used herein, the term “vaccine for pandemic influenza/flu or pandemic influenza/flu vaccine” refers to a vaccine directed against a pandemic influenza virus is called herein as a vaccine for pandemic influenza/flu or pandemic influenza/flu vaccine. As used herein, the term “vaccine for seasonal influenza/flu or seasonal influenza/flu vaccine” refers to a vaccine directed against the seasonal occurring influenza viruses in a flu season is termed herein “vaccine for seasonal influenza/flu or seasonal influenza/flu vaccine”.

As used herein, the term “variants” of proteins or peptides as defined in the context of the present invention may be generated, having an amino acid sequence which differs from the original sequence in one or more mutation(s), such as one or more substituted, inserted and/or deleted amino acid(s). Preferably, these fragments and/or variants have the same biological function or specific activity compared to the full-length native protein, e.g. its specific antigenic property. “Variants” of proteins or peptides as defined in the context of the present invention may comprise conservative amino acid substitution(s) compared to their native, i.e. non-mutated physiological, sequence. Those amino acid sequences as well as their encoding nucleotide sequences in particular fall under the term variants as defined herein. Substitutions in which amino acids, which originate from the same class, are exchanged for one another are called conservative substitutions. In particular, these are amino acids having aliphatic side chains, positively or negatively charged side chains, aromatic groups in the side chains or amino acids, the side chains of which can enter into hydrogen bridges, e.g. side chains which have a hydroxyl function. This means that e.g. an amino acid having a polar side chain is replaced by another amino acid having a likewise polar side chain, or, for example, an amino acid characterized by a hydrophobic side chain is substituted by another amino acid having a likewise hydrophobic side chain (e.g. serine (threonine) by threonine (serine) or leucine (isoleucine) by isoleucine (leucine)). Insertions and substitutions are possible, in particular, at those sequence positions which cause no modification to the three-dimensional structure or do not affect the binding region. Modifications to a three-dimensional structure by insertion(s) or deletion(s) can easily be determined e.g. using CD spectra (circular dichroism spectra) (Urry, 1985, Absorption, Circular Dichroism and ORD of Polypeptides, in: Modern Physical Methods in Biochemistry, Neuberger et al. (ed.), Elsevier, Amsterdam).

As used herein, the term “variant” of a protein or peptide may have at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% amino acid identity over a stretch of 10, 20, 30, 50, 75 or 100 amino acids of such protein or peptide. Furthermore, variants of proteins or peptides as defined herein, which may be encoded by a nucleic acid molecule, may also comprise those sequences, wherein nucleotides of the encoding nucleic acid sequence are exchanged according to the degeneration of the genetic code, without leading to an alteration of the respective amino acid sequence of the protein or peptide, i.e. the amino acid sequence or at least part thereof may not differ from the original sequence in one or more mutation(s) within the above meaning.

As used herein, the term “vehicle” means an agent, e.g. a carrier, that may typically be used within a pharmaceutical composition or vaccine for facilitating administering of the components of the pharmaceutical composition or vaccine to an individual.

V.2. Exemplary Ionizable Lipids of the Invention

The inventors have discovered a new class of ionizable lipids, the Ionizable Lipids of the Invention, that were synthesized including two or more ionizable amine head groups, two or more degradable linker groups, and two or more saturated or unsaturated or branched alkyl tails.

In its broadest embodiment, the Ionizable Lipids of the Invention have a structure of Formula I:

Head-Spacer-Linker-Spacer-Tail

Formula I

In certain embodiments, the spacer is optional and is a substituted or unsubstituted C 1 -C 22 alkyl group. In preferred embodiments, each spacer is independently a substituted or unsubstituted C 1 carbon, a substituted or unsubstituted C 2 carbon, a substituted or unsubstituted C 3 carbon, a substituted or unsubstituted C4 carbon, a substituted or unsubstituted C 5 carbon, a substituted or unsubstituted C 6 carbon, a substituted or unsubstituted C7 carbon, a substituted or unsubstituted C 8 carbon, a substituted or unsubstituted C9 carbon, a substituted or unsubstituted C 10 carbon, a substituted or unsubstituted C11 carbon, a substituted or unsubstituted C 12 carbon, a substituted or unsubstituted C 13 carbon, a substituted or unsubstituted C 14 carbon, a substituted or unsubstituted Cis carbon, a substituted or unsubstituted C 16 carbon, a substituted or unsubstituted C 17 carbon, a substituted or unsubstituted Cis carbon, a substituted or unsubstituted C 19 carbon, a substituted or unsubstituted C 20 carbon, a substituted or unsubstituted C 21 carbon, or a substituted or unsubstituted C 22 carbon.

In certain embodiments, the Ionizable Lipids of the Invention include candidate headgroup structures for DL=DiLinoleic Acid, ADDE=Azane Diyl DiEthyl, Cx/Cy=carbon spacers, DMA=DiMethylAmine, Pyr=Pyridine, PipZ=PiperaZine, PipD=PiperiDine, DIPA=DiIsopropylamine, DM=DiMethyl, BOD=BisOctylDecyl headgroups and Percepta predictions of pKa.

In certain embodiments exemplary, non-limited headgroups for each of Dimethyl Amine (Table 2), Piperdine (Table 3), Pyrrolidine (Table 4), and Piperazine (Table 5) are illustrated below. In certain embodiments, the pKas of the two amines can be tuned to achieve specific values to facilitate delivery to a specific tissue or organ. Percepta predictions for pKa are shown in black for the classic algorithm and in red for the Galas algorithm. Distancing the amines from each other brings the two pKas closer due to reduced electrostatic repulsion between the charged sites. Distancing the internal amine from the ester bond raises its pKa since the proton-binding free electron pair of the amine is further removed from the electron withdrawing ester. Choosing headgroup structures with appropriate pKas are expected to achieve both a slight positive charge or neutral LNP for IM localization and strong increasing positive charge below pH 7 for endolysosomal release capacity.

In certain embodiments, additional bivalent headgroups can be chosen along with Dimethylamine, Pyrrolidine, and Piperazine illustrated in Tables 2, 4, and 5, where carbon spacers can be changed to produce specific molecular pKas. In addition to specific ionization properties, some headgroups may have increased immune-adjuvanticity.

In certain embodiments, the Ionizable Lipids of the Invention include one or more linker groups, preferably 2, 3, 4, 5, 6, 7, 8, 9, or 10 linker groups.

Exemplary linkers are illustrated in Tables 7A-7E below.

In certain embodiments, the shape of the alkyl tails of the Ionizable Lipid of the Invention is preferably cone-shaped and diverge in order to promote endolysosomal release by creating nonbilayer inverted hexagonal or other structures. Exemplary alkyl tails are illustrated in Table 8A, 8B, and 8C. In certain embodiments, this can be achieved by adding saturated bonds or branching. In other embodiments, adding ester bonds can increase tolerability through more rapid degradation.

Exemplary non-limiting examples of Ionizable Lipids of the Invention with Dimethylamine, Pyrrolidine, and Piperazine headgroups, ester linker, C4 spacers, and alkyl/alkylene tails are illustrated in Table 9 and can have pKas that permit a positively charged LNP at pH 7.4 for IM localization and a second internal amine with a pKa near 6.5 which can further ionize during endosomal acidification and provide release.

In another embodiment, the invention encompasses Ionizable Lipids of the Invention of Formula II:

wherein each R 1 and R 2 is independently selected from the group consisting of H, an optionally substituted C 1 -C 22 alkyl, optionally substituted C 2 -C 22 alkenyl, optionally substituted C 2 -C 22 alkynyl, optionally substituted C 3 -C 6 cycloalkyl, optionally substituted C 4 -C 6 heterocycloalkyl, optionally substituted C 4 -C 6 alkylcycloalkyl, optionally substituted C 4 -C 6 aryl, optionally substituted C 3 -C 6 heteroaryl, optionally substituted C 4 -C 8 aryloxy, optionally substituted C 7 -C 10 arylalkyl or optionally substituted C 5 -C 10 heteroarylalkyl group, or R 1 and R 2 can together form a 3-7 membered heterocycle or heteroaryl ring;

wherein each R 3 and R 4 is independently selected from the group consisting of an optionally substituted C 1 -C 22 alkyl, optionally substituted C 2 -C 22 alkenyl, optionally substituted C 2 -C 22 alkynyl, or wherein in R 1 and R 2 together comprise a 3-7 membered heterocycle or heteroaromatic ring;

wherein each R 5 , R 6 , R 7 , R 8 , R 9 , and R 10 is independently selected from the group consisting of H, OH, halo, phenyl, benzyl, optionally substituted C 1 -C 22 alkyl, optionally substituted C 2 -C 22 alkenyl, optionally substituted C 2 -C 22 alkynyl,

wherein each of x, y, and z is independently an integer from 0-10; and

wherein each of L 1 and L 2 is independently selected from the group consisting of —C(═O)—; OC(═O)—; —OC(═O)O—; —C(═O)O—; —C(═O)O(CR 5 R 6 R 7 ) m ; —NH—C(═O)—; —C(═O)NH—; —SO—; —SO 2 —; —SO 3 —; —NSO 2 —; —SO 2 N—; —NH((C 1 -C 8 )alkyl); —N((C 1 -C 8 )alkyl) 2 ; —NH((C 6 )aryl); —N((C 6 )aryl) 2 ; —NHC(═O)NH—; —NHC(═O)O—; —OC(═O)NH—; —NHC(═O)NR 1 —; —NHC(═O)O—; —OC(═O)NR 1 —; dioxolopyrrolidine-dione; —C(═O)R 1 —; —CO((C 1 -C 22 )alkyl); —CO((C 6 )aryl); —CO 2 ((C 1 -C 22 )alkyl); —CO 2 ((C 6 )aryl); —SO 2 ((C 1 -C 22 )alkyl); and —SO 2 ((C 6 )aryl).

The Ionizable Lipids of the Invention can optionally include carbon spacers between the first nitrogen and the second nitrogen (i.e., CR 5 R 6 in Formula II above) and between second nitrogen and the ester linker (i.e., CR 7 R 8 and CR 9 R 10 in Formula II above). In certain embodiments, tails are symmetric including, for example, DiLinoleic Acid (DL) and the branched BisOctylDecyl (BOD).

In certain embodiments, R 1 is H.

In certain embodiments, R 1 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 1 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 1 is substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 1 is substituted or unsubstituted C 3 -C 6 cycloalkyl.

In certain embodiments, R 1 is substituted or unsubstituted C 4 -C 6 heterocycloalkyl.

In certain embodiments, R 1 is substituted or unsubstituted C 4 -C 6 alkylcycloalkyl.

In certain embodiments, R 1 is substituted or unsubstituted C 4 -C 6 aryl.

In certain embodiments, R 1 is substituted or unsubstituted C 3 -C 6 heteroaryl.

In certain embodiments, R 1 is substituted or unsubstituted C 4 -C 8 aryloxy.

In certain embodiments, R 1 is substituted or unsubstituted C 7 -C 10 arylalkyl.

In certain embodiments, R 1 is substituted or unsubstituted C 5 -C 10 heteroarylalkyl group.

In certain embodiments, R 2 is H.

In certain embodiments, R 2 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 2 is substituted or unsubstituted C 2 -C 22 alkenyl

In certain embodiments, R 2 is substituted or unsubstituted C 2 -C 22 alkynyl

In certain embodiments, R 2 is substituted or unsubstituted C 3 -C 6 cycloalkyl.

In certain embodiments, R 2 is substituted or unsubstituted C 4 -C 6 heterocycloalkyl.

In certain embodiments, R 2 is substituted or unsubstituted C 4 -C 6 alkylcycloalkyl.

In certain embodiments, R 2 is substituted or unsubstituted C 4 -C 6 aryl.

In certain embodiments, R 2 is substituted or unsubstituted C 3 -C 6 heteroaryl.

In certain embodiments, R 2 is substituted or unsubstituted C 4 -C 8 aryloxy.

In certain embodiments, R 2 is substituted or unsubstituted C 7 -C 10 arylalkyl.

In certain embodiments, R 2 is substituted or unsubstituted C 5 -C 10 heteroarylalkyl group.

In certain embodiments, R 3 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 3 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 3 is substituted or unsubstituted C 2 -C 22 alkynyl.

In a preferred embodiment, R 3 is substituted or unsubstituted —C(═O)O—C 1 -C 22 alkyl.

In a preferred embodiment, R 3 is substituted or unsubstituted —C(═O)O—C 1 -C 22 alkenyl.

In a preferred embodiment, R 3 is substituted or unsubstituted —C(═O)O—C 1 -C 22 alkynyl.

In certain embodiments, R 4 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 4 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 4 is substituted or unsubstituted C 2 -C 22 alkynyl.

In a preferred embodiment, R 4 is substituted or unsubstituted —C(═O)O—C 1 -C 22 alkyl.

In a preferred embodiment, R 4 is substituted or unsubstituted —C(═O)O—C 1 -C 22 alkenyl.

In a preferred embodiment, R 4 is substituted or unsubstituted —C(═O)O—C 1 -C 22 alkynyl.

In certain preferred embodiments, the substitution on R 3 and/or R 4 includes a terminal dioxolane group.

In certain embodiments, R 5 is H, OH, halo, phenyl, benzyl, substituted or unsubstituted C 1 -C 22 alkyl, substituted or unsubstituted C 2 -C 22 alkenyl; or substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 5 is H.

In certain embodiments, R 5 is OH.

In certain embodiments, R 5 is halo.

In certain embodiments, R 5 is phenyl.

In certain embodiments, R 5 is benzyl.

In certain embodiments, R 5 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 5 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 5 is substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 6 is H, OH, halo, phenyl, benzyl, substituted or unsubstituted C 1 -C 22 alkyl, substituted or unsubstituted C 2 -C 22 alkenyl; or substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 6 is H.

In certain embodiments, R 6 is OH.

In certain embodiments, R 6 is halo.

In certain embodiments, R 6 is phenyl.

In certain embodiments, R 6 is benzyl.

In certain embodiments, R 6 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 6 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 6 is substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 7 is H, OH, halo, phenyl, benzyl, substituted or unsubstituted C 1 -C 22 alkyl, substituted or unsubstituted C 2 -C 22 alkenyl; or substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 7 is H.

In certain embodiments, R 7 is OH.

In certain embodiments, R 7 is halo.

In certain embodiments, R 7 is phenyl.

In certain embodiments, R 7 is benzyl.

In certain embodiments, R 7 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 7 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 7 is substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 8 is H, OH, halo, phenyl, benzyl, substituted or unsubstituted C 1 -C 22 alkyl, substituted or unsubstituted C 2 -C 22 alkenyl; or substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 8 is H.

In certain embodiments, R 8 is OH.

In certain embodiments, R 8 is halo.

In certain embodiments, R 8 is phenyl.

In certain embodiments, R 8 is benzyl.

In certain embodiments, R 8 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 8 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 8 is substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 9 is H, OH, halo, phenyl, benzyl, substituted or unsubstituted C 1 -C 22 alkyl, substituted or unsubstituted C 2 -C 22 alkenyl; or substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 9 is H.

In certain embodiments, R 9 is OH.

In certain embodiments, R 9 is halo.

In certain embodiments, R 9 is phenyl.

In certain embodiments, R 9 is benzyl.

In certain embodiments, R 9 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 9 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 9 is substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 10 is H, OH, halo, phenyl, benzyl, substituted or unsubstituted C 1 -C 22 alkyl, substituted or unsubstituted C 2 -C 22 alkenyl; or substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 10 is H.

In certain embodiments, R 10 is OH.

In certain embodiments, R 10 is halo.

In certain embodiments, R 10 is phenyl.

In certain embodiments, R 10 is benzyl.

In certain embodiments, R 10 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 10 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 10 is substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, x is 0.

In certain embodiments, x is 1.

In certain embodiments, x is 2.

In certain embodiments, x is 3.

In certain embodiments, x is 4.

In certain embodiments, x is 5.

In certain embodiments, x is 6.

In certain embodiments, x is 7.

In certain embodiments, x is 8.

In certain embodiments, x is 9.

In certain embodiments, x is 10.

In certain embodiments, x is 11.

In certain embodiments, x is 12.

In certain embodiments, x is 13.

In certain embodiments, x is 14.

In certain embodiments, x is 15.

In certain embodiments, x is 16.

In certain embodiments, x is 17.

In certain embodiments, x is 18.

In certain embodiments, x is 19.

In certain embodiments, x is 20.

In certain embodiments, y is 0.

In certain embodiments, y is 1.

In certain embodiments, y is 2.

In certain embodiments, y is 3.

In certain embodiments, y is 4.

In certain embodiments, y is 5.

In certain embodiments, y is 6.

In certain embodiments, y is 7.

In certain embodiments, y is 8.

In certain embodiments, y is 9.

In certain embodiments, y is 10.

In certain embodiments, y is 11.

In certain embodiments, y is 12.

In certain embodiments, y is 13.

In certain embodiments, y is 14.

In certain embodiments, y is 15.

In certain embodiments, y is 16.

In certain embodiments, y is 17.

In certain embodiments, y is 18.

In certain embodiments, y is 19.

In certain embodiments, y is 20.

In certain embodiments, z is 0.

In certain embodiments, z is 1.

In certain embodiments, z is 2.

In certain embodiments, z is 3.

In certain embodiments, z is 4.

In certain embodiments, z is 5.

In certain embodiments, z is 6.

In certain embodiments, z is 7.

In certain embodiments, z is 8.

In certain embodiments, z is 9.

In certain embodiments, z is 10.

In certain embodiments, z is 11.

In certain embodiments, z is 12.

In certain embodiments, z is 13.

In certain embodiments, z is 14.

In certain embodiments, z is 15.

In certain embodiments, z is 16.

In certain embodiments, z is 17.

In certain embodiments, z is 18.

In certain embodiments, z is 19.

In certain embodiments, z is 20.

In certain embodiments L 1 is a bond.

In certain embodiments, L 1 is —C(═O)—.

In certain embodiments, L 1 is —OC(═O)O—.

In certain embodiments, L 1 is —NH—C(═O)—.

In certain embodiments, L 1 is —SO—.

In certain embodiments, L 1 is —SO 2 —.

In certain embodiments, L 1 is OC(═O).

In certain embodiments, L 1 is —C(═O)O—.

In certain embodiments, L 1 is —C(═O)NH—.

In certain embodiments, L 1 is —SO 3 —.

In certain embodiments, L 1 is —NSO 2 —.

In certain embodiments, L 1 is —SO 2 N.

In certain embodiments, L 1 is —NH((C 1 -C 22 )alkyl).

In certain embodiments, L 1 is —N((C 1 -C 8 )alkyl) 2 .

In certain embodiments, L 1 is —NH((C 6 )aryl).

In certain embodiments, L 1 is —N((C 6 )aryl) 2 .

In certain embodiments, L 1 is dioxolopyrrolidine-dione.

In certain embodiments, L 1 is —C(═O)R 1 —.

In certain embodiments, L 1 is —CO((C 1 -C 22 )alkyl).

In certain embodiments, L 1 is —CO((C 6 )aryl).

In certain embodiments, L 1 is —CO 2 ((C 1 -C 22 )alkyl).

In certain embodiments, L 1 is —CO 2 ((C 6 )aryl).

In certain embodiments, L 1 is —SO 2 ((C 1 -C 22 )alkyl).

In certain embodiments, L 1 is —SO 2 ((C 6 )aryl).

In certain embodiments L 2 is a bond.

In certain embodiments, L 2 is —C(═O)—.

In certain embodiments, L 2 is —OC(═O)O—.

In certain embodiments, L 2 is —OC(═O)O(CR 1 R 2 R 3 ).

In certain embodiments, L 2 is —NH—C(═O)—.

In certain embodiments, L 2 is —SO—.

In certain embodiments, L 2 is —SO 2 —.

In certain embodiments, L 2 is OC(═O).

In certain embodiments, L 2 is —C(═O)O—.

In certain embodiments, L 2 is —C(═O)NH—.

In certain embodiments, L 2 is —SO 3 —.

In certain embodiments, L 2 is —NSO 2 —.

In certain embodiments, L 2 is —SO 2 N.

In certain embodiments, L 2 is —NH((C 1 -C 22 )alkyl).

In certain embodiments, L 2 is —N((C 1 -C 8 )alkyl) 2 .

In certain embodiments, L 2 is —NH((C 6 )aryl).

In certain embodiments, L 2 is —N((C 6 )aryl) 2 .

In certain embodiments, L 2 is dioxolopyrrolidine-dione.

In certain embodiments, L 2 is —C(═O)R 1 —.

In certain embodiments, L 2 is —CO((C 1 -C 22 )alkyl).

In certain embodiments, L 2 is —CO((C 6 )aryl).

In certain embodiments, L 2 is —CO 2 ((C 1 -C 22 )alkyl).

In certain embodiments, L 2 is —CO 2 ((C 6 )aryl).

In certain embodiments, L 2 is —SO 2 ((C 1 -C 22 )alkyl).

In certain embodiments, L 2 is —SO 2 ((C 6 )aryl).

In another embodiment, the invention encompasses Ionizable Lipids of the Invention of Formula III:

wherein each R 1′ , R 1 , R 2 , R 11 , and R 12 is independently selected from the group consisting of H, an optionally substituted C 1 -C 12 alkyl, optionally substituted C 2 -C 12 alkenyl, optionally substituted C 2 -C 12 alkynyl, optionally substituted C 3 -C 6 cycloalkyl, optionally substituted C 4 -C 6 heterocycloalkyl, optionally substituted C 4 -C 6 alkylcycloalkyl, optionally substituted C 4 -C 6 aryl, optionally substituted C 3 -C 6 heteroaryl, optionally substituted C 4 -C 8 aryloxy, optionally substituted C 7 -C 10 arylalkyl; optionally substituted C 5 -C 10 heteroarylalkyl group, optionally substituted amine; or R 1 and R 2 can together form cycloalkyl or heterocycloalkyl ring, wherein if Q is S or O the R 1 attached to the S or O is an electron pair;

wherein each R 3 and R 4 is independently selected from the group consisting of an optionally substituted C 1 -C 22 alkyl, optionally substituted C 2 -C 22 alkenyl, optionally substituted C 2 -C 22 alkynyl;

wherein each R 5 , R 6 , R 7 , R 8 , R 9 , and R 10 is independently selected from the group consisting of H, OH, halo, phenyl, benzyl, optionally substituted C 1 -C 22 alkyl, optionally substituted C 2 -C 22 alkenyl, optionally substituted C 2 -C 22 alkynyl,

wherein each of x, y, and z is independently an integer from 0-20;

wherein G and Q are each independently an atom selected from CH, 0, N, and S;

wherein each of m and n is an integer from 0-4; and

wherein each of L 1 and L 2 is independently selected from the group consisting of —C(═O)—; OC(═O)—; —OC(═O)O—; —C(═O)O—; —C(═O)O(CR 5 R 6 R 7 ) m ; —NH—C(═O)—; —C(═O)NH—; —SO—; —SO 2 —; —SO 3 —; —NSO 2 —; —SO 2 N—; —NH((C 1 -C 8 )alkyl); —N((C 1 -C 8 )alkyl) 2 ; —NH((C 6 )aryl); —N((C 6 )aryl) 2 ; —NHC(═O)NH—; —NHC(═O)O—; —OC(═O)NH—; —NHC(═O)NR 1 —; —NHC(═O)O—; —OC(═O)NR 1 —; —C(═O)R 1 —; —CO((C 1 -C 8 )alkyl); —CO((C 6 )aryl); —CO 2 ((C 1 -C 8 )alkyl); —CO 2 ((C 6 )aryl); —SO 2 ((C 1 -C 8 )alkyl); —SO 2 ((C 6 )aryl); and a disulfide bond.

In certain embodiments, R 1 is H.

In certain embodiments, R 1 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 1 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 1 is substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 1 is substituted or unsubstituted C 3 -C 6 cycloalkyl.

In certain embodiments, R 1 is substituted or unsubstituted C 4 -C 6 heterocycloalkyl.

In certain embodiments, R 1 is substituted or unsubstituted C 4 -C 6 alkylcycloalkyl.

In certain embodiments, R 1 is substituted or unsubstituted C 4 -C 6 aryl.

In certain embodiments, R 1 is substituted or unsubstituted C 3 -C 6 heteroaryl.

In certain embodiments, R 1 is substituted or unsubstituted C 4 -C 8 aryloxy.

In certain embodiments, R 1 is substituted or unsubstituted C 7 -C 10 arylalkyl.

In certain embodiments, R 1 is substituted or unsubstituted C 5 -C 10 heteroarylalkyl group.

In certain embodiments, R 2 is H.

In certain embodiments, R 2 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 2 is substituted or unsubstituted C 2 -C 22 alkenyl

In certain embodiments, R 2 is substituted or unsubstituted C 2 -C 22 alkynyl

In certain embodiments, R 2 is substituted or unsubstituted C 3 -C 6 cycloalkyl.

In certain embodiments, R 2 is substituted or unsubstituted C 4 -C 6 heterocycloalkyl.

In certain embodiments, R 2 is substituted or unsubstituted C 4 -C 6 alkylcycloalkyl.

In certain embodiments, R 2 is substituted or unsubstituted C 4 -C 6 aryl.

In certain embodiments, R 2 is substituted or unsubstituted C 3 -C 6 heteroaryl.

In certain embodiments, R 2 is substituted or unsubstituted C 4 -C 8 aryloxy.

In certain embodiments, R 2 is substituted or unsubstituted C 7 -C 10 arylalkyl.

In certain embodiments, R 2 is substituted or unsubstituted C 5 -C 10 heteroarylalkyl group.

In certain embodiments, R 3 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 3 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 3 is substituted or unsubstituted C 2 -C 22 alkynyl.

In a preferred embodiment, R 3 is substituted or unsubstituted —C(═O)O—C 1 -C 22 alkyl.

In a preferred embodiment, R 3 is substituted or unsubstituted —C(═O)O—C 1 -C 22 alkenyl.

In a preferred embodiment, R 3 is substituted or unsubstituted —C(═O)O—C 1 -C 22 alkynyl.

In certain embodiments, R 4 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 4 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 4 is substituted or unsubstituted C 2 -C 22 alkynyl.

In a preferred embodiment, R 4 is substituted or unsubstituted —C(═O)O—C 1 -C 22 alkyl.

In a preferred embodiment, R 4 is substituted or unsubstituted —C(═O)O—C 1 -C 22 alkenyl.

In a preferred embodiment, R 4 is substituted or unsubstituted —C(═O)O—C 1 -C 22 alkynyl.

In certain embodiments, R 5 is H, OH, halo, phenyl, benzyl, substituted or unsubstituted C 1 -C 22 alkyl, substituted or unsubstituted C 2 -C 22 alkenyl; or substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 5 is H.

In certain embodiments, R 5 is OH.

In certain embodiments, R 5 is halo.

In certain embodiments, R 5 is phenyl.

In certain embodiments, R 5 is benzyl.

In certain embodiments, R 5 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 5 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 5 is substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 6 is H, OH, halo, phenyl, benzyl, substituted or unsubstituted C 1 -C 22 alkyl, substituted or unsubstituted C 2 -C 22 alkenyl; or substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 6 is H.

In certain embodiments, R 6 is OH.

In certain embodiments, R 6 is halo.

In certain embodiments, R 6 is phenyl.

In certain embodiments, R 6 is benzyl.

In certain embodiments, R 6 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 6 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 6 is substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 7 is H, OH, halo, phenyl, benzyl, substituted or unsubstituted C 1 -C 22 alkyl, substituted or unsubstituted C 2 -C 22 alkenyl; or substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 7 is H.

In certain embodiments, R 7 is OH.

In certain embodiments, R 7 is halo.

In certain embodiments, R 7 is phenyl.

In certain embodiments, R 7 is benzyl.

In certain embodiments, R 7 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 7 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 7 is substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 8 is H, OH, halo, phenyl, benzyl, substituted or unsubstituted C 1 -C 22 alkyl, substituted or unsubstituted C 2 -C 22 alkenyl; or substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 8 is H.

In certain embodiments, R 8 is OH.

In certain embodiments, R 8 is halo.

In certain embodiments, R 8 is phenyl.

In certain embodiments, R 8 is benzyl.

In certain embodiments, R 8 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 8 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 8 is substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 9 is H, OH, halo, phenyl, benzyl, substituted or unsubstituted C 1 -C 22 alkyl, substituted or unsubstituted C 2 -C 22 alkenyl; or substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 9 is H.

In certain embodiments, R 9 is OH.

In certain embodiments, R 9 is halo.

In certain embodiments, R 9 is phenyl.

In certain embodiments, R 9 is benzyl.

In certain embodiments, R 9 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 9 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 9 is substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 10 is H, OH, halo, phenyl, benzyl, substituted or unsubstituted C 1 -C 22 alkyl, substituted or unsubstituted C 2 -C 22 alkenyl; or substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 10 is H.

In certain embodiments, R 10 is OH.

In certain embodiments, R 10 is halo.

In certain embodiments, R 10 is phenyl.

In certain embodiments, R 10 is benzyl.

In certain embodiments, R 10 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 10 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 10 is substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, x is 0.

In certain embodiments, x is 1.

In certain embodiments, x is 2.

In certain embodiments, x is 3.

In certain embodiments, x is 4.

In certain embodiments, x is 5.

In certain embodiments, x is 6.

In certain embodiments, x is 7.

In certain embodiments, x is 8.

In certain embodiments, x is 9.

In certain embodiments, x is 10.

In certain embodiments, x is 11.

In certain embodiments, x is 12.

In certain embodiments, x is 13.

In certain embodiments, x is 14.

In certain embodiments, x is 15.

In certain embodiments, x is 16.

In certain embodiments, x is 17.

In certain embodiments, x is 18.

In certain embodiments, x is 19.

In certain embodiments, x is 20.

In certain embodiments, y is 0.

In certain embodiments, y is 1.

In certain embodiments, y is 2.

In certain embodiments, y is 3.

In certain embodiments, y is 4.

In certain embodiments, y is 5.

In certain embodiments, y is 6.

In certain embodiments, y is 7.

In certain embodiments, y is 8.

In certain embodiments, y is 9.

In certain embodiments, y is 10.

In certain embodiments, y is 11.

In certain embodiments, y is 12.

In certain embodiments, y is 13.

In certain embodiments, y is 14.

In certain embodiments, y is 15.

In certain embodiments, y is 16.

In certain embodiments, y is 17.

In certain embodiments, y is 18.

In certain embodiments, y is 19.

In certain embodiments, y is 20.

In certain embodiments, z is 0.

In certain embodiments, z is 1.

In certain embodiments, z is 2.

In certain embodiments, z is 3.

In certain embodiments, z is 4.

In certain embodiments, z is 5.

In certain embodiments, z is 6.

In certain embodiments, z is 7.

In certain embodiments, z is 8.

In certain embodiments, z is 9.

In certain embodiments, z is 10.

In certain embodiments, z is 11.

In certain embodiments, z is 12.

In certain embodiments, z is 13.

In certain embodiments, z is 14.

In certain embodiments, z is 15.

In certain embodiments, z is 16.

In certain embodiments, z is 17.

In certain embodiments, z is 18.

In certain embodiments, z is 19.

In certain embodiments, z is 20.

In certain embodiments L 1 is a bond.

In certain embodiments, L 1 is —C(═O)—.

In certain embodiments, L 1 is —OC(═O)O—.

In certain embodiments, L 1 is —NH—C(═O)—.

In certain embodiments, L 1 is —SO—.

In certain embodiments, L 1 is —SO 2 —.

In certain embodiments, L 1 is OC(═O).

In certain embodiments, L 1 is —C(═O)O—.

In certain embodiments, L 1 is —C(═O)NH—.

In certain embodiments, L 1 is —SO 3 —.

In certain embodiments, L 1 is —NSO 2 —.

In certain embodiments, L 1 is —SO 2 N.

In certain embodiments, L 1 is —NH((C 1 -C 22 )alkyl).

In certain embodiments, L 1 is —N((C 1 -C 8 )alkyl) 2 .

In certain embodiments, L 1 is —NH((C 6 )aryl).

In certain embodiments, L 1 is —N((C 6 )aryl) 2 .

In certain embodiments, L 1 is dioxolopyrrolidine-dione.

In certain embodiments, L 1 is —C(═O)R 1 —.

In certain embodiments, L 1 is —CO((C 1 -C 22 )alkyl).

In certain embodiments, L 1 is —CO((C 6 )aryl).

In certain embodiments, L 1 is —CO 2 ((C 1 -C 22 )alkyl).

In certain embodiments, L 1 is —CO 2 ((C 6 )aryl).

In certain embodiments, L 1 is —C(═O)O(R 1 R 2 R 3 )

In certain embodiments, L 1 is —SO 2 ((C 1 -C 22 )alkyl).

In certain embodiments, L 1 is —SO 2 ((C 6 )aryl).

In certain embodiments L 2 is a bond.

In certain embodiments, L 2 is —C(═O)—.

In certain embodiments, L 2 is —OC(═O)O—.

In certain embodiments, L 2 is —NH—C(═O)—.

In certain embodiments, L 2 is —SO—.

In certain embodiments, L 2 is —SO 2 —.

In certain embodiments, L 2 is OC(═O).

In certain embodiments, L 2 is —C(═O)O—.

In certain embodiments, L 2 is —C(═O)NH—.

In certain embodiments, L 2 is —SO 3 —.

In certain embodiments, L 2 is —NSO 2 —.

In certain embodiments, L 2 is —SO 2 N.

In certain embodiments, L 2 is —NH((C 1 -C 22 )alkyl).

In certain embodiments, L 2 is —N((C 1 -C 8 )alkyl) 2 .

In certain embodiments, L 2 is —NH((C 6 )aryl).

In certain embodiments, L 2 is —N((C 6 )aryl) 2 .

In certain embodiments, L 2 is dioxolopyrrolidine-dione.

In certain embodiments, L 2 is —C(═O)R 1 —.

In certain embodiments, L 2 is —CO((C 1 -C 22 )alkyl).

In certain embodiments, L 2 is —CO((C 6 )aryl).

In certain embodiments, L 2 is —CO 2 ((C 1 -C 22 )alkyl).

In certain embodiments, L 2 is —CO 2 ((C 6 )aryl).

In certain embodiments, L 2 is —SO 2 ((C 1 -C 22 )alkyl).

In certain embodiments, L 2 is —SO 2 ((C 6 )aryl).

In certain embodiments, Q is CH.

In certain embodiments, Q is O.

In certain embodiments, Q is S.

In certain embodiments, Q is N.

In certain embodiments, G is CH.

In certain embodiments, G is O.

In certain embodiments, G is S.

In certain embodiments, G is N.

In another embodiment, the invention encompasses Ionizable Lipids of the Invention of Formula IV:

wherein R 1 is selected from the group consisting of H, an optionally substituted C 1 -C 12 alkyl, optionally substituted C 2 -C 12 alkenyl, optionally substituted C 2 -C 12 alkynyl, optionally substituted C 3 -C 6 cycloalkyl, optionally substituted C 4 -C 6 heterocycloalkyl, optionally substituted C 4 -C 6 alkylcycloalkyl, optionally substituted C 4 -C 6 aryl, optionally substituted C 3 -C 6 heteroaryl, optionally substituted C 4 -C 8 aryloxy, optionally substituted C 7 -C 10 arylalkyl, and optionally substituted C 5 -C 10 heteroarylalkyl group;

wherein each R 3 and R 4 is independently selected from the group consisting of an optionally substituted C 1 -C 22 alkyl, optionally substituted C 2 -C 22 alkenyl, optionally substituted C 2 -C 22 alkynyl; wherein each R 5 , R 6 , R 7 , R 8 , R 9 , and R 10 is independently selected from the group consisting of H, OH, halo, phenyl, benzyl, optionally substituted C 1 -C 22 alkyl, optionally substituted C 2 -C 22 alkenyl, optionally substituted C 2 -C 22 alkynyl, wherein each of x, y, and z is independently an integer from 0-10; and wherein each of L 1 and L 2 is independently selected from the group consisting of —C(═O)—; OC(═O)—; —OC(═O)O—; —C(═O)O—; —C(═O)O(CR 5 R 6 R 7 ); —NH—C(═O)—; —C(═O)NH—; —SO—; —SO 2 —; —SO 3 —; —NSO 2 —; —SO 2 N—; —NH((C 1 -C 8 )alkyl); —N((C 1 -C 8 )alkyl) 2 ; —NH((C 6 )aryl); —N((C 6 )aryl) 2 ; —NHC(═O)NH—; —NHC(═O)O—; —OC(═O)NH—; —NHC(═O)NR 1 —; —NHC(═O)O—; —OC(═O)NR 1 —; —C(═O)R 1 —; —CO((C 1 -C 8 )alkyl); —CO((C 6 )aryl); —CO 2 ((C 1 -C 8 )alkyl); —CO 2 ((C 6 )aryl); —SO 2 ((C 1 -C 8 )alkyl); and —SO 2 ((C 6 )aryl).

In certain embodiments, R 1 is H.

In certain embodiments, R 1 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 1 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 1 is substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 1 is substituted or unsubstituted C 3 -C 6 cycloalkyl.

In certain embodiments, R 1 is substituted or unsubstituted C 4 -C 6 heterocycloalkyl.

In certain embodiments, R 1 is substituted or unsubstituted C 4 -C 6 alkylcycloalkyl.

In certain embodiments, R 1 is substituted or unsubstituted C 4 -C 6 aryl.

In certain embodiments, R 1 is substituted or unsubstituted C 3 -C 6 heteroaryl.

In certain embodiments, R 1 is substituted or unsubstituted C 4 -C 8 aryloxy.

In certain embodiments, R 1 is substituted or unsubstituted C 7 -C 10 arylalkyl.

In certain embodiments, R 1 is substituted or unsubstituted C 5 -C 10 heteroarylalkyl group.

In certain embodiments, R 3 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 3 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 3 is substituted or unsubstituted C 2 -C 22 alkynyl.

In a preferred embodiment, R 3 is substituted or unsubstituted —C(═O)O—C 1 -C 22 alkyl.

In a preferred embodiment, R 3 is substituted or unsubstituted —C(═O)O—C 1 -C 22 alkenyl.

In a preferred embodiment, R 3 is substituted or unsubstituted —C(═O)O—C 1 -C 22 alkynyl.

In certain embodiments, R 4 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 4 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 4 is substituted or unsubstituted C 2 -C 22 alkynyl.

In a preferred embodiment, R 4 is substituted or unsubstituted —C(═O)O—C 1 -C 22 alkyl.

In a preferred embodiment, R 4 is substituted or unsubstituted —C(═O)O—C 1 -C 22 alkenyl.

In a preferred embodiment, R 4 is substituted or unsubstituted —C(═O)O—C 1 -C 22 alkynyl.

In certain embodiments, R 5 is H, OH, halo, phenyl, benzyl, substituted or unsubstituted C 1 -C 22 alkyl, substituted or unsubstituted C 2 -C 22 alkenyl; or substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 5 is H.

In certain embodiments, R 5 is OH.

In certain embodiments, R 5 is halo.

In certain embodiments, R 5 is phenyl.

In certain embodiments, R 5 is benzyl.

In certain embodiments, R 5 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 5 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 5 is substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 6 is H, OH, halo, phenyl, benzyl, substituted or unsubstituted C 1 -C 22 alkyl, substituted or unsubstituted C 2 -C 22 alkenyl; or substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 6 is H.

In certain embodiments, R 6 is OH.

In certain embodiments, R 6 is halo.

In certain embodiments, R 6 is phenyl.

In certain embodiments, R 6 is benzyl.

In certain embodiments, R 6 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 6 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 6 is substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 7 is H, OH, halo, phenyl, benzyl, substituted or unsubstituted C 1 -C 22 alkyl, substituted or unsubstituted C 2 -C 22 alkenyl; or substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 7 is H.

In certain embodiments, R 7 is OH.

In certain embodiments, R 7 is halo.

In certain embodiments, R 7 is phenyl.

In certain embodiments, R 7 is benzyl.

In certain embodiments, R 7 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 7 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 7 is substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 8 is H, OH, halo, phenyl, benzyl, substituted or unsubstituted C 1 -C 22 alkyl, substituted or unsubstituted C 2 -C 22 alkenyl; or substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 8 is H.

In certain embodiments, R 8 is OH.

In certain embodiments, R 8 is halo.

In certain embodiments, R 8 is phenyl.

In certain embodiments, R 8 is benzyl.

In certain embodiments, R 8 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 8 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 8 is substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 9 is H, OH, halo, phenyl, benzyl, substituted or unsubstituted C 1 -C 22 alkyl, substituted or unsubstituted C 2 -C 22 alkenyl; or substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 9 is H.

In certain embodiments, R 9 is OH.

In certain embodiments, R 9 is halo.

In certain embodiments, R 9 is phenyl.

In certain embodiments, R 9 is benzyl.

In certain embodiments, R 9 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 9 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 9 is substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 10 is H, OH, halo, phenyl, benzyl, substituted or unsubstituted C 1 -C 22 alkyl, substituted or unsubstituted C 2 -C 22 alkenyl; or substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 10 is H.

In certain embodiments, R 10 is OH.

In certain embodiments, R 10 is halo.

In certain embodiments, R 10 is phenyl.

In certain embodiments, R 10 is benzyl.

In certain embodiments, R 10 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 10 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 10 is substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, x is 0.

In certain embodiments, x is 1.

In certain embodiments, x is 2.

In certain embodiments, x is 3.

In certain embodiments, x is 4.

In certain embodiments, x is 5.

In certain embodiments, x is 6.

In certain embodiments, x is 7.

In certain embodiments, x is 8.

In certain embodiments, x is 9.

In certain embodiments, x is 10.

In certain embodiments, x is 11.

In certain embodiments, x is 12.

In certain embodiments, x is 13.

In certain embodiments, x is 14.

In certain embodiments, x is 15.

In certain embodiments, x is 16.

In certain embodiments, x is 17.

In certain embodiments, x is 18.

In certain embodiments, x is 19.

In certain embodiments, x is 20.

In certain embodiments, y is 0.

In certain embodiments, y is 1.

In certain embodiments, y is 2.

In certain embodiments, y is 3.

In certain embodiments, y is 4.

In certain embodiments, y is 5.

In certain embodiments, y is 6.

In certain embodiments, y is 7.

In certain embodiments, y is 8.

In certain embodiments, y is 9.

In certain embodiments, y is 10.

In certain embodiments, y is 11.

In certain embodiments, y is 12.

In certain embodiments, y is 13.

In certain embodiments, y is 14.

In certain embodiments, y is 15.

In certain embodiments, y is 16.

In certain embodiments, y is 17.

In certain embodiments, y is 18.

In certain embodiments, y is 19.

In certain embodiments, y is 20.

In certain embodiments, z is 0.

In certain embodiments, z is 1.

In certain embodiments, z is 2.

In certain embodiments, z is 3.

In certain embodiments, z is 4.

In certain embodiments, z is 5.

In certain embodiments, z is 6.

In certain embodiments, z is 7.

In certain embodiments, z is 8.

In certain embodiments, z is 9.

In certain embodiments, z is 10.

In certain embodiments, z is 11.

In certain embodiments, z is 12.

In certain embodiments, z is 13.

In certain embodiments, z is 14.

In certain embodiments, z is 15.

In certain embodiments, z is 16.

In certain embodiments, z is 17.

In certain embodiments, z is 18.

In certain embodiments, z is 19.

In certain embodiments, z is 20.

In certain embodiments L 1 is a bond.

In certain embodiments, L 1 is —C(═O)—.

In certain embodiments, L 1 is —OC(═O)O—.

In certain embodiments, L 1 is —NH—C(═O)—.

In certain embodiments, L 1 is —SO—.

In certain embodiments, L 1 is —SO 2 —.

In certain embodiments, L 1 is OC(═O).

In certain embodiments, L 1 is —C(═O)O—.

In certain embodiments, L 1 is —C(═O)NH—.

In certain embodiments, L 1 is —SO 3 —.

In certain embodiments, L 1 is —NSO 2 —.

In certain embodiments, L 1 is —SO 2 N.

In certain embodiments, L 1 is —NH((C 1 -C 22 )alkyl).

In certain embodiments, L 1 is —N((C 1 -C 8 )alkyl) 2 .

In certain embodiments, L 1 is —NH((C 6 )aryl).

In certain embodiments, L 1 is —N((C 6 )aryl) 2 .

In certain embodiments, L 1 is dioxolopyrrolidine-dione.

In certain embodiments, L 1 is —C(═O)R 1 —.

In certain embodiments, L 1 is —CO((C 1 -C 22 )alkyl).

In certain embodiments, L 1 is —CO((C 6 )aryl).

In certain embodiments, L 1 is —CO 2 ((C 1 -C 22 )alkyl).

In certain embodiments, L 1 is —CO 2 ((C 6 )aryl).

In certain embodiments, L 1 is —C(═O)O(CR 1 R 2 R 3 )

In certain embodiments, L 1 is —SO 2 ((C 1 -C 22 )alkyl).

In certain embodiments, L 1 is —SO 2 ((C 6 )aryl).

In certain embodiments L 2 is a bond.

In certain embodiments, L 2 is —C(═O)—.

In certain embodiments, L 2 is —OC(═O)O—.

In certain embodiments, L 2 is —NH—C(═O)—.

In certain embodiments, L 2 is —SO—.

In certain embodiments, L 2 is —SO 2 —.

In certain embodiments, L 2 is OC(═O).

In certain embodiments, L 2 is —C(═O)O—.

In certain embodiments, L 2 is —C(═O)NH—.

In certain embodiments, L 2 is —SO 3 —.

In certain embodiments, L 2 is —NSO 2 —.

In certain embodiments, L 2 is —SO 2 N.

In certain embodiments, L 2 is —NH((C 1 -C 22 )alkyl).

In certain embodiments, L 2 is —N((C 1 -C 8 )alkyl) 2 .

In certain embodiments, L 2 is —NH((C 6 )aryl).

In certain embodiments, L 2 is —N((C 6 )aryl) 2 .

In certain embodiments, L 2 is dioxolopyrrolidine-dione.

In certain embodiments, L 2 is —C(═O)R 1 —.

In certain embodiments, L 2 is —CO((C 1 -C 22 )alkyl).

In certain embodiments, L 2 is —CO((C 6 )aryl).

In certain embodiments, L 2 is —CO 2 ((C 1 -C 22 )alkyl).

In certain embodiments, L 2 is —CO 2 ((C 6 )aryl).

In certain embodiments, L 2 is —SO 2 ((C 1 -C 22 )alkyl).

In certain embodiments, L 2 is —SO 2 ((C 6 )aryl).

In another embodiment, the invention encompasses Ionizable Lipids of the Invention of Formula V:

wherein each R 1 and each R 2 is independently selected from the group consisting of H, an optionally substituted C 1 -C 12 alkyl, optionally substituted C 2 -C 12 alkenyl, optionally substituted C 2 -C 12 alkynyl, optionally substituted C 3 -C 6 cycloalkyl, optionally substituted C 4 -C 6 heterocycloalkyl, optionally substituted C 4 -C 6 alkylcycloalkyl, optionally substituted C 4 -C 6 aryl, optionally substituted C 3 -C 6 heteroaryl, optionally substituted C 4 -C 8 aryloxy, optionally substituted C 7 -C 10 arylalkyl; optionally substituted C 5 -C 10 heteroarylalkyl group, optionally substituted amine; or R 1 and R 2 can together form a 3-7 membered heterocycle or heteroaryl ring;

wherein each R 3 , R 4 , R 3 , and R 14 is independently selected from the group consisting of an optionally substituted C 1 -C 22 alkyl, optionally substituted C 2 -C 22 alkenyl, optionally substituted C 2 -C 22 alkynyl;

wherein each R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 15 , and R 16 is independently selected from the group consisting of H, OH, halo, phenyl, benzyl, optionally substituted C 1 -C 22 alkyl, optionally substituted C 2 -C 22 alkenyl, optionally substituted C 2 -C 22 alkynyl,

wherein each of w, x, y, and z is independently an integer from 0-10;

wherein each Q is independently an atom selected from 0, NH, S, or a disulfide bond;

wherein each of m is an integer from 0-4, preferably 0, 1, or 2; and

wherein each of L 1 and L 2 is independently selected from the group consisting of —C(═O)—; OC(═O)—; —OC(═O)O—; —C(═O)O—; —C(═O)O(CR 6 R 7 ) m ; —NH—C(═O)—; —C(═O)NH—; —SO—; —SO 2 —; —SO 3 —; —NSO 2 —; —SO 2 N—; —NH((C 1 -C 8 )alkyl); —N((C 1 -C 8 )alkyl) 2 ; —NH((C 6 )aryl); —N((C 6 )aryl) 2 ; —NHC(═O)NH—; —NHC(═O)O—; —OC(═O)NH—; —NHC(═O)NR 1 —; —NHC(═O)O—; —OC(═O)NR 1 —; —C(═O)R 1 —; —CO((C 1 -C 8 )alkyl); —CO((C 6 )aryl); —CO 2 ((C 1 -C 8 )alkyl); —CO 2 ((C 6 )aryl); —SO 2 ((C 1 -C 8 )alkyl); and —SO 2 ((C 6 )aryl).

In certain embodiments, R 1 is H.

In certain embodiments, R 1 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 1 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 1 is substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 1 is substituted or unsubstituted C 3 -C 6 cycloalkyl.

In certain embodiments, R 1 is substituted or unsubstituted C 4 -C 6 heterocycloalkyl.

In certain embodiments, R 1 is substituted or unsubstituted C 4 -C 6 alkylcycloalkyl.

In certain embodiments, R 1 is substituted or unsubstituted C 4 -C 6 aryl.

In certain embodiments, R 1 is substituted or unsubstituted C 3 -C 6 heteroaryl.

In certain embodiments, R 1 is substituted or unsubstituted C 4 -C 8 aryloxy.

In certain embodiments, R 1 is substituted or unsubstituted C 7 -C 10 arylalkyl.

In certain embodiments, R 1 is substituted or unsubstituted C 5 -C 10 heteroarylalkyl group.

In certain embodiments, R 2 is H.

In certain embodiments, R 2 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 2 is substituted or unsubstituted C 2 -C 22 alkenyl

In certain embodiments, R 2 is substituted or unsubstituted C 2 -C 22 alkynyl

In certain embodiments, R 2 is substituted or unsubstituted C 3 -C 6 cycloalkyl.

In certain embodiments, R 2 is substituted or unsubstituted C 4 -C 6 heterocycloalkyl.

In certain embodiments, R 2 is substituted or unsubstituted C 4 -C 6 alkylcycloalkyl.

In certain embodiments, R 2 is substituted or unsubstituted C 4 -C 6 aryl.

In certain embodiments, R 2 is substituted or unsubstituted C 3 -C 6 heteroaryl.

In certain embodiments, R 2 is substituted or unsubstituted C 4 -C 8 aryloxy.

In certain embodiments, R 2 is substituted or unsubstituted C 7 -C 10 arylalkyl.

In certain embodiments, R 2 is substituted or unsubstituted C 5 -C 10 heteroarylalkyl group.

In certain embodiments, R 3 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 3 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 3 is substituted or unsubstituted C 2 -C 22 alkynyl.

In a preferred embodiment, R 3 is substituted or unsubstituted —C(═O)O—C 1 -C 22 alkyl.

In a preferred embodiment, R 3 is substituted or unsubstituted —C(═O)O—C 1 -C 22 alkenyl.

In a preferred embodiment, R 3 is substituted or unsubstituted —C(═O)O—C 1 -C 22 alkynyl.

In certain embodiments, R 4 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 4 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 4 is substituted or unsubstituted C 2 -C 22 alkynyl.

In a preferred embodiment, R 4 is substituted or unsubstituted —C(═O)O—C 1 -C 22 alkyl.

In a preferred embodiment, R 4 is substituted or unsubstituted —C(═O)O—C 1 -C 22 alkenyl.

In a preferred embodiment, R 4 is substituted or unsubstituted —C(═O)O—C 1 -C 22 alkynyl.

In certain embodiments, each R 5 is independently H, OH, halo, phenyl, benzyl, substituted or unsubstituted C 1 -C 22 alkyl, substituted or unsubstituted C 2 -C 22 alkenyl; or substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 5 is H.

In certain embodiments, R 5 is OH.

In certain embodiments, R 5 is halo.

In certain embodiments, R 5 is phenyl.

In certain embodiments, R 5 is benzyl.

In certain embodiments, R 5 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 5 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 5 is substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, each R 6 is independently H, OH, halo, phenyl, benzyl, substituted or unsubstituted C 1 -C 22 alkyl, substituted or unsubstituted C 2 -C 22 alkenyl; or substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 6 is H.

In certain embodiments, R 6 is OH.

In certain embodiments, R 6 is halo.

In certain embodiments, R 6 is phenyl.

In certain embodiments, R 6 is benzyl.

In certain embodiments, R 6 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 6 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 6 is substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 7 is H, OH, halo, phenyl, benzyl, substituted or unsubstituted C 1 -C 22 alkyl, substituted or unsubstituted C 2 -C 22 alkenyl; or substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 7 is H.

In certain embodiments, R 7 is OH.

In certain embodiments, R 7 is halo.

In certain embodiments, R 7 is phenyl.

In certain embodiments, R 7 is benzyl.

In certain embodiments, R 7 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 7 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 7 is substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 8 is H, OH, halo, phenyl, benzyl, substituted or unsubstituted C 1 -C 22 alkyl, substituted or unsubstituted C 2 -C 22 alkenyl; or substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 8 is H.

In certain embodiments, R 8 is OH.

In certain embodiments, R 8 is halo.

In certain embodiments, R 8 is phenyl.

In certain embodiments, R 8 is benzyl.

In certain embodiments, R 8 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 8 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 8 is substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 9 is H, OH, halo, phenyl, benzyl, substituted or unsubstituted C 1 -C 22 alkyl, substituted or unsubstituted C 2 -C 22 alkenyl; or substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 9 is H.

In certain embodiments, R 9 is OH.

In certain embodiments, R 9 is halo.

In certain embodiments, R 9 is phenyl.

In certain embodiments, R 9 is benzyl.

In certain embodiments, R 9 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 9 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 9 is substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 10 is H, OH, halo, phenyl, benzyl, substituted or unsubstituted C 1 -C 22 alkyl, substituted or unsubstituted C 2 -C 22 alkenyl; or substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 10 is H.

In certain embodiments, R 10 is OH.

In certain embodiments, R 10 is halo.

In certain embodiments, R 10 is phenyl.

In certain embodiments, R 10 is benzyl.

In certain embodiments, R 10 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 10 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 10 is substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, w is 0.

In certain embodiments, w is 1.

In certain embodiments, w is 2.

In certain embodiments, w is 3.

In certain embodiments, w is 4.

In certain embodiments, w is 5.

In certain embodiments, w is 6.

In certain embodiments, w is 7.

In certain embodiments, w is 8.

In certain embodiments, w is 9.

In certain embodiments, w is 10.

In certain embodiments, w is 11.

In certain embodiments, w is 12.

In certain embodiments, w is 13.

In certain embodiments, w is 14.

In certain embodiments, w is 15.

In certain embodiments, w is 16.

In certain embodiments, w is 17.

In certain embodiments, w is 18.

In certain embodiments, w is 19.

In certain embodiments, w is 20.

In certain embodiments, x is 0.

In certain embodiments, x is 1.

In certain embodiments, x is 2.

In certain embodiments, x is 3.

In certain embodiments, x is 4.

In certain embodiments, x is 5.

In certain embodiments, x is 6.

In certain embodiments, x is 7.

In certain embodiments, x is 8.

In certain embodiments, x is 9.

In certain embodiments, x is 10.

In certain embodiments, x is 11.

In certain embodiments, x is 12.

In certain embodiments, x is 13.

In certain embodiments, x is 14.

In certain embodiments, x is 15.

In certain embodiments, x is 16.

In certain embodiments, x is 17.

In certain embodiments, x is 18.

In certain embodiments, x is 19.

In certain embodiments, x is 20.

In certain embodiments, y is 0.

In certain embodiments, y is 1.

In certain embodiments, y is 2.

In certain embodiments, y is 3.

In certain embodiments, y is 4.

In certain embodiments, y is 5.

In certain embodiments, y is 6.

In certain embodiments, y is 7.

In certain embodiments, y is 8.

In certain embodiments, y is 9.

In certain embodiments, y is 10.

In certain embodiments, y is 11.

In certain embodiments, y is 12.

In certain embodiments, y is 13.

In certain embodiments, y is 14.

In certain embodiments, y is 15.

In certain embodiments, y is 16.

In certain embodiments, y is 17.

In certain embodiments, y is 18.

In certain embodiments, y is 19.

In certain embodiments, y is 20.

In certain embodiments, z is 0.

In certain embodiments, z is 1.

In certain embodiments, z is 2.

In certain embodiments, z is 3.

In certain embodiments, z is 4.

In certain embodiments, z is 5.

In certain embodiments, z is 6.

In certain embodiments, z is 7.

In certain embodiments, z is 8.

In certain embodiments, z is 9.

In certain embodiments, z is 10.

In certain embodiments, z is 11.

In certain embodiments, z is 12.

In certain embodiments, z is 13.

In certain embodiments, z is 14.

In certain embodiments, z is 15.

In certain embodiments, z is 16.

In certain embodiments, z is 17.

In certain embodiments, z is 18.

In certain embodiments, z is 19.

In certain embodiments, z is 20.

In certain embodiments L 1 is a bond.

In certain embodiments, L 1 is —C(═O)—.

In certain embodiments, L 1 is —OC(═O)O—.

In certain embodiments, L 1 is —NH—C(═O)—.

In certain embodiments, L 1 is —SO—.

In certain embodiments, L 1 is —SO 2 —.

In certain embodiments, L 1 is OC(═O).

In certain embodiments, L 1 is —C(═O)O—.

In certain embodiments, L 1 is —C(═O)NH—.

In certain embodiments, L 1 is —SO 3 —.

In certain embodiments, L 1 is —NSO 2 —.

In certain embodiments, L 1 is —SO 2 N.

In certain embodiments, L 1 is —NH((C 1 -C 22 )alkyl).

In certain embodiments, L 1 is —N((C 1 -C 8 )alkyl) 2 .

In certain embodiments, L 1 is —NH((C 6 )aryl).

In certain embodiments, L 1 is —N((C 6 )aryl) 2 .

In certain embodiments, L 1 is dioxolopyrrolidine-dione.

In certain embodiments, L 1 is —C(═O)R 1 —.

In certain embodiments, L 1 is —CO((C 1 -C 22 )alkyl).

In certain embodiments, L 1 is —CO((C 6 )aryl).

In certain embodiments, L 1 is —CO 2 ((C 1 -C 22 )alkyl).

In certain embodiments, L 1 is —CO 2 ((C 6 )aryl).

In certain embodiments, L 1 is —C(═O)O(CR 1 R 2 R 3 )

In certain embodiments, L 1 is —SO 2 ((C 1 -C 22 )alkyl).

In certain embodiments, L 1 is —SO 2 ((C 6 )aryl).

In certain embodiments L 2 is a bond.

In certain embodiments, L 2 is —C(═O)—.

In certain embodiments, L 2 is —OC(═O)O—.

In certain embodiments, L 2 is —NH—C(═O)—.

In certain embodiments, L 2 is —SO—.

In certain embodiments, L 2 is —SO 2 —.

In certain embodiments, L 2 is OC(═O).

In certain embodiments, L 2 is —C(═O)O—.

In certain embodiments, L 2 is —C(═O)NH—.

In certain embodiments, L 2 is —SO 3 —.

In certain embodiments, L 2 is —NSO 2 —.

In certain embodiments, L 2 is —SO 2 N.

In certain embodiments, L 2 is —NH((C 1 -C 22 )alkyl).

In certain embodiments, L 2 is —N((C 1 -C 8 )alkyl) 2 .

In certain embodiments, L 2 is —NH((C 6 )aryl).

In certain embodiments, L 2 is —N((C 6 )aryl) 2 .

In certain embodiments, L 2 is dioxolopyrrolidine-dione.

In certain embodiments, L 2 is —C(═O)R 1 —.

In certain embodiments, L 2 is —CO((C 1 -C 22 )alkyl).

In certain embodiments, L 2 is —CO((C 6 )aryl).

In certain embodiments, L 2 is —CO 2 ((C 1 -C 22 )alkyl).

In certain embodiments, L 2 is —CO 2 ((C 6 )aryl).

In certain embodiments, L 2 is —SO 2 ((C 1 -C 22 )alkyl).

In certain embodiments, L 2 is —SO 2 ((C 6 )aryl).

In certain embodiments, Q is CH.

In certain embodiments, Q is O.

In certain embodiments, Q is S.

In certain embodiments, Q is NH.

In certain embodiments, Q is a disulfide bond.

In certain embodiments, m is 0.

In certain embodiments, m is 1.

In certain embodiments, m is 2.

In certain embodiments, m is 3.

In certain embodiments, m is 4.

In certain embodiments, m is 5.

In certain embodiments, m is 6.

In certain embodiments, m is 7.

In certain embodiments, m is 8.

In certain embodiments, m is 9.

In certain embodiments, m is 10.

In certain embodiments, m is 11.

In certain embodiments, m is 12.

In certain embodiments, m is 13.

In certain embodiments, m is 14.

In certain embodiments, m is 15.

In certain embodiments, m is 16.

In certain embodiments, m is 17.

In certain embodiments, m is 18.

In certain embodiments, m is 19.

In certain embodiments, m is 20.

In another embodiment, the invention encompasses Ionizable Lipids of the Invention of Formula VI:

wherein each R 1 and each R 2 is independently selected from the group consisting of H, an optionally substituted C 1 -C 12 alkyl, optionally substituted C 2 -C 12 alkenyl, optionally substituted C 2 -C 12 alkynyl, optionally substituted C 3 -C 6 cycloalkyl, optionally substituted C 4 -C 6 heterocycloalkyl, optionally substituted C 4 -C 6 alkylcycloalkyl, optionally substituted C 4 -C 6 aryl, optionally substituted C 3 -C 6 heteroaryl, optionally substituted C 4 -C 8 aryloxy, optionally substituted C 7 -C 10 arylalkyl; optionally substituted C 5 -C 10 heteroarylalkyl group, optionally substituted amine; or R 1 and R 2 can together form a 3-7 membered heterocycle or heteroaryl ring;

wherein each R 3 , R 4 , R 23 and R 24 is independently selected from the group consisting of an optionally substituted C 1 -C 22 alkyl, optionally substituted C 2 -C 22 alkenyl, optionally substituted C 2 -C 22 alkynyl;

wherein each R 5 , R 6 , R 7 , R 8 , R 11 , R 12 , R 13 , R 17 , R 18 , R 34 , R 35 , R 36 is independently selected from the group consisting of H, OH, halo, phenyl, benzyl, optionally substituted C 1 -C 22 alkyl, optionally substituted C 2 -C 22 alkenyl, optionally substituted C 2 -C 22 alkynyl,

wherein each of u, v, w, x, y, and z is independently an integer from 0-20;

wherein each Q is independently an atom selected from 0, NH, S, or a disulfide bond;

wherein each of m is an integer from 0-4, preferably 0, 1, or 2; and

wherein each of L 1 and L 2 is independently selected from the group consisting of —C(═O)—; OC(═O)—; —OC(═O)O—; —C(═O)O—; —C(═O)O(CR 6 R 7 ) m ; —NH—C(═O)—; —C(═O)NH—; —SO—; —SO 2 —; —SO 3 —; —NSO 2 —; —SO 2 N—; —NH((C 1 -C 8 )alkyl); —N((C 1 -C 8 )alkyl) 2 ; —NH((C 6 )aryl); —N((C 6 )aryl) 2 ; —NHC(═O)NH—; —NHC(═O)O—; —OC(═O)NH—; —NHC(═O)NR 1 —; —NHC(═O)O—; —OC(═O)NR 1 —; —C(═O)R 1 —; —CO((C 1 -C 8 )alkyl); —CO((C 6 )aryl); —CO 2 ((C 1 -C 8 )alkyl); —CO 2 ((C 6 )aryl); —SO 2 ((C 1 -C 8 )alkyl); and —SO 2 ((C 6 )aryl).

In certain embodiments, R 1 is H.

In certain embodiments, R 1 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 1 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 1 is substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 1 is substituted or unsubstituted C 3 -C 6 cycloalkyl.

In certain embodiments, R 1 is substituted or unsubstituted C 4 -C 6 heterocycloalkyl.

In certain embodiments, R 1 is substituted or unsubstituted C 4 -C 6 alkylcycloalkyl.

In certain embodiments, R 1 is substituted or unsubstituted C 4 -C 6 aryl.

In certain embodiments, R 1 is substituted or unsubstituted C 3 -C 6 heteroaryl.

In certain embodiments, R 1 is substituted or unsubstituted C 4 -C 8 aryloxy.

In certain embodiments, R 1 is substituted or unsubstituted C 7 -C 10 arylalkyl.

In certain embodiments, R 1 is substituted or unsubstituted C 5 -C 10 heteroarylalkyl group.

In certain embodiments, R 2 is H.

In certain embodiments, R 2 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 2 is substituted or unsubstituted C 2 -C 22 alkenyl

In certain embodiments, R 2 is substituted or unsubstituted C 2 -C 22 alkynyl

In certain embodiments, R 2 is substituted or unsubstituted C 3 -C 6 cycloalkyl.

In certain embodiments, R 2 is substituted or unsubstituted C 4 -C 6 heterocycloalkyl.

In certain embodiments, R 2 is substituted or unsubstituted C 4 -C 6 alkylcycloalkyl.

In certain embodiments, R 2 is substituted or unsubstituted C 4 -C 6 aryl.

In certain embodiments, R 2 is substituted or unsubstituted C 3 -C 6 heteroaryl.

In certain embodiments, R 2 is substituted or unsubstituted C 4 -C 8 aryloxy.

In certain embodiments, R 2 is substituted or unsubstituted C 7 -C 10 arylalkyl.

In certain embodiments, R 2 is substituted or unsubstituted C 5 -C 10 heteroarylalkyl group.

In certain embodiments, R 3 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 3 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 3 is substituted or unsubstituted C 2 -C 22 alkynyl.

In a preferred embodiment, R 3 is substituted or unsubstituted —C(═O)O—C 1 -C 22 alkyl.

In a preferred embodiment, R 3 is substituted or unsubstituted —C(═O)O—C 1 -C 22 alkenyl.

In a preferred embodiment, R 3 is substituted or unsubstituted —C(═O)O—C 1 -C 22 alkynyl.

In certain embodiments, R 4 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 4 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 4 is substituted or unsubstituted C 2 -C 22 alkynyl.

In a preferred embodiment, R 4 is substituted or unsubstituted —C(═O)O—C 1 -C 22 alkyl.

In a preferred embodiment, R 4 is substituted or unsubstituted —C(═O)O—C 1 -C 22 alkenyl.

In a preferred embodiment, R 4 is substituted or unsubstituted —C(═O)O—C 1 -C 22 alkynyl.

In certain embodiments, each R 5 is independently H, OH, halo, phenyl, benzyl, substituted or unsubstituted C 1 -C 22 alkyl, substituted or unsubstituted C 2 -C 22 alkenyl; or substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 5 is H.

In certain embodiments, R 5 is OH.

In certain embodiments, R 5 is halo.

In certain embodiments, R 5 is phenyl.

In certain embodiments, R 5 is benzyl.

In certain embodiments, R 5 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 5 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 5 is substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, each R 6 is independently H, OH, halo, phenyl, benzyl, substituted or unsubstituted C 1 -C 22 alkyl, substituted or unsubstituted C 2 -C 22 alkenyl; or substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 6 is H.

In certain embodiments, R 6 is OH.

In certain embodiments, R 6 is halo.

In certain embodiments, R 6 is phenyl.

In certain embodiments, R 6 is benzyl.

In certain embodiments, R 6 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 6 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 6 is substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 7 is H, OH, halo, phenyl, benzyl, substituted or unsubstituted C 1 -C 22 alkyl, substituted or unsubstituted C 2 -C 22 alkenyl; or substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 7 is H.

In certain embodiments, R 7 is OH.

In certain embodiments, R 7 is halo.

In certain embodiments, R 7 is phenyl.

In certain embodiments, R 7 is benzyl.

In certain embodiments, R 7 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 7 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 7 is substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 8 is H, OH, halo, phenyl, benzyl, substituted or unsubstituted C 1 -C 22 alkyl, substituted or unsubstituted C 2 -C 22 alkenyl; or substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 8 is H.

In certain embodiments, R 8 is OH.

In certain embodiments, R 8 is halo.

In certain embodiments, R 8 is phenyl.

In certain embodiments, R 8 is benzyl.

In certain embodiments, R 8 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 8 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 8 is substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 9 is H, OH, halo, phenyl, benzyl, substituted or unsubstituted C 1 -C 22 alkyl, substituted or unsubstituted C 2 -C 22 alkenyl; or substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 9 is H.

In certain embodiments, R 9 is OH.

In certain embodiments, R 9 is halo.

In certain embodiments, R 9 is phenyl.

In certain embodiments, R 9 is benzyl.

In certain embodiments, R 9 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 9 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 9 is substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 10 is H, OH, halo, phenyl, benzyl, substituted or unsubstituted C 1 -C 22 alkyl, substituted or unsubstituted C 2 -C 22 alkenyl; or substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 10 is H.

In certain embodiments, R 10 is OH.

In certain embodiments, R 10 is halo.

In certain embodiments, R 10 is phenyl.

In certain embodiments, R 10 is benzyl.

In certain embodiments, R 10 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 10 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 10 is substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 11 is H, OH, halo, phenyl, benzyl, substituted or unsubstituted C 1 -C 22 alkyl, substituted or unsubstituted C 2 -C 22 alkenyl; or substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 11 is H.

In certain embodiments, R 11 is OH.

In certain embodiments, R 11 is halo.

In certain embodiments, R 11 is phenyl.

In certain embodiments, R 11 is benzyl.

In certain embodiments, R 11 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 11 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 11 is substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 12 is H, OH, halo, phenyl, benzyl, substituted or unsubstituted C 1 -C 22 alkyl, substituted or unsubstituted C 2 -C 22 alkenyl; or substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 12 is H.

In certain embodiments, R 12 is OH.

In certain embodiments, R 12 is halo.

In certain embodiments, R 12 is phenyl.

In certain embodiments, R 12 is benzyl.

In certain embodiments, R 12 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 12 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 12 is substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 13 is H, OH, halo, phenyl, benzyl, substituted or unsubstituted C 1 -C 22 alkyl, substituted or unsubstituted C 2 -C 22 alkenyl; or substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 13 is H.

In certain embodiments, R 13 is OH.

In certain embodiments, R 13 is halo.

In certain embodiments, R 13 is phenyl.

In certain embodiments, R 13 is benzyl.

In certain embodiments, R 13 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 13 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 13 is substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 14 is H, OH, halo, phenyl, benzyl, substituted or unsubstituted C 1 -C 22 alkyl, substituted or unsubstituted C 2 -C 22 alkenyl; or substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 14 is H.

In certain embodiments, R 14 is OH.

In certain embodiments, R 14 is halo.

In certain embodiments, R 14 is phenyl.

In certain embodiments, R 14 is benzyl.

In certain embodiments, R 14 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 14 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 14 is substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 15 is H, OH, halo, phenyl, benzyl, substituted or unsubstituted C 1 -C 22 alkyl, substituted or unsubstituted C 2 -C 22 alkenyl; or substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 15 is H.

In certain embodiments, R 15 is OH.

In certain embodiments, R 15 is halo.

In certain embodiments, R 15 is phenyl.

In certain embodiments, R 15 is benzyl.

In certain embodiments, R 15 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 15 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 15 is substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 16 is H, OH, halo, phenyl, benzyl, substituted or unsubstituted C 1 -C 22 alkyl, substituted or unsubstituted C 2 -C 22 alkenyl; or substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 16 is H.

In certain embodiments, R 16 is OH.

In certain embodiments, R 16 is halo.

In certain embodiments, R 16 is phenyl.

In certain embodiments, R 16 is benzyl.

In certain embodiments, R 16 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 16 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 16 is substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, u is 0.

In certain embodiments, u is 1.

In certain embodiments, u is 2.

In certain embodiments, u is 3.

In certain embodiments, u is 4.

In certain embodiments, u is 5.

In certain embodiments, u is 6.

In certain embodiments, u is 7.

In certain embodiments, u is 8.

In certain embodiments, u is 9.

In certain embodiments, u is 10.

In certain embodiments, u is 11.

In certain embodiments, u is 12.

In certain embodiments, u is 13.

In certain embodiments, u is 14.

In certain embodiments, u is 15.

In certain embodiments, u is 16.

In certain embodiments, u is 17.

In certain embodiments, u is 18.

In certain embodiments, u is 19.

In certain embodiments, u is 20.

In certain embodiments, v is 0.

In certain embodiments, v is 1.

In certain embodiments, v is 2.

In certain embodiments, v is 3.

In certain embodiments, v is 4.

In certain embodiments, v is 5.

In certain embodiments, v is 6.

In certain embodiments, v is 7.

In certain embodiments, v is 8.

In certain embodiments, v is 9.

In certain embodiments, v is 10.

In certain embodiments, v is 11.

In certain embodiments, v is 12.

In certain embodiments, v is 13.

In certain embodiments, v is 14.

In certain embodiments, v is 15.

In certain embodiments, v is 16.

In certain embodiments, v is 17.

In certain embodiments, v is 18.

In certain embodiments, v is 19.

In certain embodiments, v is 20.

In certain embodiments, w is 0.

In certain embodiments, w is 1.

In certain embodiments, w is 2.

In certain embodiments, w is 3.

In certain embodiments, w is 4.

In certain embodiments, w is 5.

In certain embodiments, w is 6.

In certain embodiments, w is 7.

In certain embodiments, w is 8.

In certain embodiments, w is 9.

In certain embodiments, w is 10.

In certain embodiments, w is 11.

In certain embodiments, w is 12.

In certain embodiments, w is 13.

In certain embodiments, w is 14.

In certain embodiments, w is 15.

In certain embodiments, w is 16.

In certain embodiments, w is 17.

In certain embodiments, w is 18.

In certain embodiments, w is 19.

In certain embodiments, w is 20.

In certain embodiments, x is 0.

In certain embodiments, x is 1.

In certain embodiments, x is 2.

In certain embodiments, x is 3.

In certain embodiments, x is 4.

In certain embodiments, x is 5.

In certain embodiments, x is 6.

In certain embodiments, x is 7.

In certain embodiments, x is 8.

In certain embodiments, x is 9.

In certain embodiments, x is 10.

In certain embodiments, x is 11.

In certain embodiments, x is 12.

In certain embodiments, x is 13.

In certain embodiments, x is 14.

In certain embodiments, x is 15.

In certain embodiments, x is 16.

In certain embodiments, x is 17.

In certain embodiments, x is 18.

In certain embodiments, x is 19.

In certain embodiments, x is 20.

In certain embodiments, y is 0.

In certain embodiments, y is 1.

In certain embodiments, y is 2.

In certain embodiments, y is 3.

In certain embodiments, y is 4.

In certain embodiments, y is 5.

In certain embodiments, y is 6.

In certain embodiments, y is 7.

In certain embodiments, y is 8.

In certain embodiments, y is 9.

In certain embodiments, y is 10.

In certain embodiments, y is 11.

In certain embodiments, y is 12.

In certain embodiments, y is 13.

In certain embodiments, y is 14.

In certain embodiments, y is 15.

In certain embodiments, y is 16.

In certain embodiments, y is 17.

In certain embodiments, y is 18.

In certain embodiments, y is 19.

In certain embodiments, y is 20.

In certain embodiments, z is 0.

In certain embodiments, z is 1.

In certain embodiments, z is 2.

In certain embodiments, z is 3.

In certain embodiments, z is 4.

In certain embodiments, z is 5.

In certain embodiments, z is 6.

In certain embodiments, z is 7.

In certain embodiments, z is 8.

In certain embodiments, z is 9.

In certain embodiments, z is 10.

In certain embodiments, z is 11.

In certain embodiments, z is 12.

In certain embodiments, z is 13.

In certain embodiments, z is 14.

In certain embodiments, z is 15.

In certain embodiments, z is 16.

In certain embodiments, z is 17.

In certain embodiments, z is 18.

In certain embodiments, z is 19.

In certain embodiments, z is 20.

In certain embodiments L 1 is a bond.

In certain embodiments, L 1 is —C(═O)—.

In certain embodiments, L 1 is —OC(═O)O—.

In certain embodiments, L 1 is —NH—C(═O)—.

In certain embodiments, L 1 is —SO—.

In certain embodiments, L 1 is —SO 2 —.

In certain embodiments, L 1 is OC(═O).

In certain embodiments, L 1 is —C(═O)O—.

In certain embodiments, L 1 is —C(═O)NH—.

In certain embodiments, L 1 is —SO 3 —.

In certain embodiments, L 1 is —NSO 2 —.

In certain embodiments, L 1 is —SO 2 N.

In certain embodiments, L 1 is —NH((C 1 -C 22 )alkyl).

In certain embodiments, L 1 is —N((C 1 -C 8 )alkyl) 2 .

In certain embodiments, L 1 is —NH((C 6 )aryl).

In certain embodiments, L 1 is —N((C 6 )aryl) 2 .

In certain embodiments, L 1 is dioxolopyrrolidine-dione.

In certain embodiments, L 1 is —C(═O)R 1 —.

In certain embodiments, L 1 is —CO((C 1 -C 22 )alkyl).

In certain embodiments, L 1 is —CO((C 6 )aryl).

In certain embodiments, L 1 is —CO 2 ((C 1 -C 22 )alkyl).

In certain embodiments, L 1 is —CO 2 ((C 6 )aryl).

In certain embodiments, L 1 is —C(═O)O(CR 1 R 2 R 3 )

In certain embodiments, L 1 is —SO 2 ((C 1 -C 22 )alkyl).

In certain embodiments, L 1 is —SO 2 ((C 6 )aryl).

In certain embodiments L 2 is a bond.

In certain embodiments, L 2 is —C(═O)—.

In certain embodiments, L 2 is —OC(═O)O—.

In certain embodiments, L 2 is —NH—C(═O)—.

In certain embodiments, L 2 is —SO—.

In certain embodiments, L 2 is —SO 2 —.

In certain embodiments, L 2 is OC(═O).

In certain embodiments, L 2 is —C(═O)O—.

In certain embodiments, L 2 is —C(═O)NH—.

In certain embodiments, L 2 is —SO 3 —.

In certain embodiments, L 2 is —NSO 2 —.

In certain embodiments, L 2 is —SO 2 N.

In certain embodiments, L 2 is —NH((C 1 -C 22 )alkyl).

In certain embodiments, L 2 is —N((C 1 -C 8 )alkyl) 2 .

In certain embodiments, L 2 is —NH((C 6 )aryl).

In certain embodiments, L 2 is —N((C 6 )aryl) 2 .

In certain embodiments, L 2 is dioxolopyrrolidine-dione.

In certain embodiments, L 2 is —C(═O)R 1 —.

In certain embodiments, L 2 is —CO((C 1 -C 22 )alkyl).

In certain embodiments, L 2 is —CO((C 6 )aryl).

In certain embodiments, L 2 is —CO 2 ((C 1 -C 22 )alkyl).

In certain embodiments, L 2 is —CO 2 ((C 6 )aryl).

In certain embodiments, L 2 is —SO 2 ((C 1 -C 22 )alkyl).

In certain embodiments, L 2 is —SO 2 ((C 6 )aryl).

In certain embodiments, Q is CH.

In certain embodiments, Q is O.

In certain embodiments, Q is S.

In certain embodiments, Q is NH.

In certain embodiments, Q is a disulfide bond.

In certain embodiments, m is 0.

In certain embodiments, m is 1.

In certain embodiments, m is 2.

In certain embodiments, m is 3.

In certain embodiments, m is 4.

In certain embodiments, m is 5.

In certain embodiments, m is 6.

In certain embodiments, m is 7.

In certain embodiments, m is 8.

In certain embodiments, m is 9.

In certain embodiments, m is 10.

In certain embodiments, m is 11.

In certain embodiments, m is 12.

In certain embodiments, m is 13.

In certain embodiments, m is 14.

In certain embodiments, m is 15.

In certain embodiments, m is 16.

In certain embodiments, m is 17.

In certain embodiments, m is 18.

In certain embodiments, m is 19.

In certain embodiments, m is 20.

In another embodiment, the invention encompasses Ionizable Lipids of the Invention of Formula VII:

wherein each R 1 and each R 2 is independently selected from the group consisting of H, an optionally substituted C 1 -C 12 alkyl, optionally substituted C 2 -C 12 alkenyl, optionally substituted C 2 -C 12 alkynyl, optionally substituted C 3 -C 6 cycloalkyl, optionally substituted C 4 -C 6 heterocycloalkyl, optionally substituted C 4 -C 6 alkylcycloalkyl, optionally substituted C 4 -C 6 aryl, optionally substituted C 3 -C 6 heteroaryl, optionally substituted C 4 -C 8 aryloxy, optionally substituted C 7 -C 10 arylalkyl; optionally substituted C 5 -C 10 heteroarylalkyl group, optionally substituted amine; or R 1 and R 2 can together form a 3-7 membered heterocycloalkyl or heteroaryl ring;

wherein each R 5 , R 6 , R 5′ , R 6′ , R 5″ , and R 6″ is independently selected from the group consisting of H, OH, halo, phenyl, benzyl, optionally substituted C 1 -C 22 alkyl, optionally substituted C 2 -C 22 alkenyl, optionally substituted C 2 -C 22 alkynyl,

wherein each R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , and R 16 is independently selected from the group consisting of H, OH, halo, phenyl, benzyl, optionally substituted C 1 -C 22 alkyl, optionally substituted C 2 -C 22 alkenyl, optionally substituted C 2 -C 22 alkynyl,

wherein each of u, v, w, y, and z is independently an integer from 0-20;

wherein each Q is independently an atom selected from 0, NH, S, or a disulfide bond; and

wherein each of L 1 and L 2 is independently selected from the group consisting of —C(═O)—; OC(═O)—; —OC(═O)O—; —C(═O)O—; —C(═O)O(CR 5 R 6 R 7 ) m ; —NH—C(═O)—; —C(═O)NH—; —SO—; —SO 2 —; —SO 3 —; —NSO 2 —; —SO 2 N—; —NH((C 1 -C 8 )alkyl); —N((C 1 -C 8 )alkyl) 2 ; —NH((C 6 )aryl); —N((C 6 )aryl) 2 ; —NHC(═O)NH—; —NHC(═O)O—; —OC(═O)NH—; —NHC(═O)NR 1 —; —NHC(═O)O—; —OC(═O)NR 1 —; —C(═O)R 1 —; —CO((C 1 -C 8 )alkyl); —CO((C 6 )aryl); —CO 2 ((C 1 -C 8 )alkyl); —CO 2 ((C 6 )aryl); —SO 2 ((C 1 -C 8 )alkyl); and —SO 2 ((C 6 )aryl).

In certain embodiments, R 1 is H.

In certain embodiments, R 1 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 1 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 1 is substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 1 is substituted or unsubstituted C 3 -C 6 cycloalkyl.

In certain embodiments, R 1 is substituted or unsubstituted C 4 -C 6 heterocycloalkyl.

In certain embodiments, R 1 is substituted or unsubstituted C 4 -C 6 alkylcycloalkyl.

In certain embodiments, R 1 is substituted or unsubstituted C 4 -C 6 aryl.

In certain embodiments, R 1 is substituted or unsubstituted C 3 -C 6 heteroaryl.

In certain embodiments, R 1 is substituted or unsubstituted C 4 -C 8 aryloxy.

In certain embodiments, R 1 is substituted or unsubstituted C 7 -C 10 arylalkyl.

In certain embodiments, R 1 is substituted or unsubstituted C 5 -C 10 heteroarylalkyl group.

In certain embodiments, R 2 is H.

In certain embodiments, R 2 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 2 is substituted or unsubstituted C 2 -C 22 alkenyl

In certain embodiments, R 2 is substituted or unsubstituted C 2 -C 22 alkynyl

In certain embodiments, R 2 is substituted or unsubstituted C 3 -C 6 cycloalkyl.

In certain embodiments, R 2 is substituted or unsubstituted C 4 -C 6 heterocycloalkyl.

In certain embodiments, R 2 is substituted or unsubstituted C 4 -C 6 alkylcycloalkyl.

In certain embodiments, R 2 is substituted or unsubstituted C 4 -C 6 aryl.

In certain embodiments, R 2 is substituted or unsubstituted C 3 -C 6 heteroaryl.

In certain embodiments, R 2 is substituted or unsubstituted C 4 -C 8 aryloxy.

In certain embodiments, R 2 is substituted or unsubstituted C 7 -C 10 arylalkyl.

In certain embodiments, R 2 is substituted or unsubstituted C 5 -C 10 heteroarylalkyl group.

In certain embodiments, each R 5 is independently H, OH, halo, phenyl, benzyl, substituted or unsubstituted C 1 -C 22 alkyl, substituted or unsubstituted C 2 -C 22 alkenyl; or substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 5 is H.

In certain embodiments, R 5 is OH.

In certain embodiments, R 5 is halo.

In certain embodiments, R 5 is phenyl.

In certain embodiments, R 5 is benzyl.

In certain embodiments, R 5 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 5 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 5 is substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, each R 6 is independently H, OH, halo, phenyl, benzyl, substituted or unsubstituted C 1 -C 22 alkyl, substituted or unsubstituted C 2 -C 22 alkenyl; or substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 6 is H.

In certain embodiments, R 6 is OH.

In certain embodiments, R 6 is halo.

In certain embodiments, R 6 is phenyl.

In certain embodiments, R 6 is benzyl.

In certain embodiments, R 6 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 6 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 6 is substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 7 is H, OH, halo, phenyl, benzyl, substituted or unsubstituted C 1 -C 22 alkyl, substituted or unsubstituted C 2 -C 22 alkenyl; or substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 7 is H.

In certain embodiments, R 7 is OH.

In certain embodiments, R 7 is halo.

In certain embodiments, R 7 is phenyl.

In certain embodiments, R 7 is benzyl.

In certain embodiments, R 7 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 7 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 7 is substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 8 is H, OH, halo, phenyl, benzyl, substituted or unsubstituted C 1 -C 22 alkyl, substituted or unsubstituted C 2 -C 22 alkenyl; or substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 8 is H.

In certain embodiments, R 8 is OH.

In certain embodiments, R 8 is halo.

In certain embodiments, R 8 is phenyl.

In certain embodiments, R 8 is benzyl.

In certain embodiments, R 8 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 8 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 8 is substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 9 is H, OH, halo, phenyl, benzyl, substituted or unsubstituted C 1 -C 22 alkyl, substituted or unsubstituted C 2 -C 22 alkenyl; or substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 9 is H.

In certain embodiments, R 9 is OH.

In certain embodiments, R 9 is halo.

In certain embodiments, R 9 is phenyl.

In certain embodiments, R 9 is benzyl.

In certain embodiments, R 9 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 9 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 9 is substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 10 is H, OH, halo, phenyl, benzyl, substituted or unsubstituted C 1 -C 22 alkyl, substituted or unsubstituted C 2 -C 22 alkenyl; or substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 10 is H.

In certain embodiments, R 10 is OH.

In certain embodiments, R 10 is halo.

In certain embodiments, R 10 is phenyl.

In certain embodiments, R 10 is benzyl.

In certain embodiments, R 10 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 10 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 10 is substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 11 is H, OH, halo, phenyl, benzyl, substituted or unsubstituted C 1 -C 22 alkyl, substituted or unsubstituted C 2 -C 22 alkenyl; or substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 11 is H.

In certain embodiments, R 11 is OH.

In certain embodiments, R 11 is halo.

In certain embodiments, R 11 is phenyl.

In certain embodiments, R 11 is benzyl.

In certain embodiments, R 11 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 11 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 11 is substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 11 is H, OH, halo, phenyl, benzyl, substituted or unsubstituted C 1 -C 22 alkyl, substituted or unsubstituted C 2 -C 22 alkenyl; or substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 12 is H.

In certain embodiments, R 12 is OH.

In certain embodiments, R 12 is halo.

In certain embodiments, R 12 is phenyl.

In certain embodiments, R 12 is benzyl.

In certain embodiments, R 12 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 12 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 12 is substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 13 is H, OH, halo, phenyl, benzyl, substituted or unsubstituted C 1 -C 22 alkyl, substituted or unsubstituted C 2 -C 22 alkenyl; or substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 13 is H.

In certain embodiments, R 13 is OH.

In certain embodiments, R 13 is halo.

In certain embodiments, R 13 is phenyl.

In certain embodiments, R 13 is benzyl.

In certain embodiments, R 13 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 3 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 13 is substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 14 is H, OH, halo, phenyl, benzyl, substituted or unsubstituted C 1 -C 22 alkyl, substituted or unsubstituted C 2 -C 22 alkenyl; or substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 14 is H.

In certain embodiments, R 14 is OH.

In certain embodiments, R 14 is halo.

In certain embodiments, R 14 is phenyl.

In certain embodiments, R 14 is benzyl.

In certain embodiments, R 14 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 14 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 14 is substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 15 is H, OH, halo, phenyl, benzyl, substituted or unsubstituted C 1 -C 22 alkyl, substituted or unsubstituted C 2 -C 22 alkenyl; or substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 15 is H.

In certain embodiments, R 15 is OH.

In certain embodiments, R 15 is halo.

In certain embodiments, R 15 is phenyl.

In certain embodiments, R 15 is benzyl.

In certain embodiments, R 15 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 15 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 15 is substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 16 is H, OH, halo, phenyl, benzyl, substituted or unsubstituted C 1 -C 22 alkyl, substituted or unsubstituted C 2 -C 22 alkenyl; or substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 16 is H.

In certain embodiments, R 16 is OH.

In certain embodiments, R 16 is halo.

In certain embodiments, R 16 is phenyl.

In certain embodiments, R 16 is benzyl.

In certain embodiments, R 16 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 16 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 16 is substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, u is 0.

In certain embodiments, u is 1.

In certain embodiments, u is 2.

In certain embodiments, u is 3.

In certain embodiments, u is 4.

In certain embodiments, u is 5.

In certain embodiments, u is 6.

In certain embodiments, u is 7.

In certain embodiments, u is 8.

In certain embodiments, u is 9.

In certain embodiments, u is 10.

In certain embodiments, u is 11.

In certain embodiments, u is 12.

In certain embodiments, u is 13.

In certain embodiments, u is 14.

In certain embodiments, u is 15.

In certain embodiments, u is 16.

In certain embodiments, u is 17.

In certain embodiments, u is 18.

In certain embodiments, u is 19.

In certain embodiments, u is 20.

In certain embodiments, v is 0.

In certain embodiments, v is 1.

In certain embodiments, v is 2.

In certain embodiments, v is 3.

In certain embodiments, v is 4.

In certain embodiments, v is 5.

In certain embodiments, v is 6.

In certain embodiments, v is 7.

In certain embodiments, v is 8.

In certain embodiments, v is 9.

In certain embodiments, v is 10.

In certain embodiments, v is 11.

In certain embodiments, v is 12.

In certain embodiments, v is 13.

In certain embodiments, v is 14.

In certain embodiments, v is 15.

In certain embodiments, v is 16.

In certain embodiments, v is 17.

In certain embodiments, v is 18.

In certain embodiments, v is 19.

In certain embodiments, v is 20.

In certain embodiments, w is 0.

In certain embodiments, w is 1.

In certain embodiments, w is 2.

In certain embodiments, w is 3.

In certain embodiments, w is 4.

In certain embodiments, w is 5.

In certain embodiments, w is 6.

In certain embodiments, w is 7.

In certain embodiments, w is 8.

In certain embodiments, w is 9.

In certain embodiments, w is 10.

In certain embodiments, w is 11.

In certain embodiments, w is 12.

In certain embodiments, w is 13.

In certain embodiments, w is 14.

In certain embodiments, w is 15.

In certain embodiments, w is 16.

In certain embodiments, w is 17.

In certain embodiments, w is 18.

In certain embodiments, w is 19.

In certain embodiments, w is 20.

In certain embodiments, y is 0.

In certain embodiments, y is 1.

In certain embodiments, y is 2.

In certain embodiments, y is 3.

In certain embodiments, y is 4.

In certain embodiments, y is 5.

In certain embodiments, y is 6.

In certain embodiments, y is 7.

In certain embodiments, y is 8.

In certain embodiments, y is 9.

In certain embodiments, y is 10.

In certain embodiments, y is 11.

In certain embodiments, y is 12.

In certain embodiments, y is 13.

In certain embodiments, y is 14.

In certain embodiments, y is 15.

In certain embodiments, y is 16.

In certain embodiments, y is 17.

In certain embodiments, y is 18.

In certain embodiments, y is 19.

In certain embodiments, y is 20.

In certain embodiments, z is 0.

In certain embodiments, z is 1.

In certain embodiments, z is 2.

In certain embodiments, z is 3.

In certain embodiments, z is 4.

In certain embodiments, z is 5.

In certain embodiments, z is 6.

In certain embodiments, z is 7.

In certain embodiments, z is 8.

In certain embodiments, z is 9.

In certain embodiments, z is 10.

In certain embodiments, z is 11.

In certain embodiments, z is 12.

In certain embodiments, z is 13.

In certain embodiments, z is 14.

In certain embodiments, z is 15.

In certain embodiments, z is 16.

In certain embodiments, z is 17.

In certain embodiments, z is 18.

In certain embodiments, z is 19.

In certain embodiments, z is 20.

In certain embodiments L 1 is a bond.

In certain embodiments, L 1 is —C(═O)—.

In certain embodiments, L 1 is —OC(═O)O—.

In certain embodiments, L 1 is —NH—C(═O)—.

In certain embodiments, L 1 is —SO—.

In certain embodiments, L 1 is —SO 2 —.

In certain embodiments, L 1 is OC(═O).

In certain embodiments, L 1 is —C(═O)O—.

In certain embodiments, L 1 is —C(═O)NH—.

In certain embodiments, L 1 is —SO 3 —.

In certain embodiments, L 1 is —NSO 2 —.

In certain embodiments, L 1 is —SO 2 N.

In certain embodiments, L 1 is —NH((C 1 -C 22 )alkyl).

In certain embodiments, L 1 is —N((C 1 -C 8 )alkyl) 2 .

In certain embodiments, L 1 is —NH((C 6 )aryl).

In certain embodiments, L 1 is —N((C 6 )aryl) 2 .

In certain embodiments, L 1 is dioxolopyrrolidine-dione.

In certain embodiments, L 1 is —C(═O)R 1 —.

In certain embodiments, L 1 is —CO((C 1 -C 22 )alkyl).

In certain embodiments, L 1 is —CO((C 6 )aryl).

In certain embodiments, L 1 is —CO 2 ((C 1 -C 22 )alkyl).

In certain embodiments, L 1 is —CO 2 ((C 6 )aryl).

In certain embodiments, L 1 is —SO 2 ((C 1 -C 22 )alkyl).

In certain embodiments, L 1 is —SO 2 ((C 6 )aryl).

In certain embodiments L 2 is a bond.

In certain embodiments, L 2 is —C(═O)—.

In certain embodiments, L 2 is —OC(═O)O—.

In certain embodiments, L 2 is —NH—C(═O)—.

In certain embodiments, L 2 is —SO—.

In certain embodiments, L 2 is —SO 2 —.

In certain embodiments, L 2 is OC(═O).

In certain embodiments, L 2 is —C(═O)O—.

In certain embodiments, L 2 is —C(═O)NH—.

In certain embodiments, L 2 is —SO 3 —.

In certain embodiments, L 2 is —NSO 2 —.

In certain embodiments, L 2 is —SO 2 N.

In certain embodiments, L 2 is —NH((C 1 -C 22 )alkyl).

In certain embodiments, L 2 is —N((C 1 -C 8 )alkyl) 2 .

In certain embodiments, L 2 is —NH((C 6 )aryl).

In certain embodiments, L 2 is —N((C 6 )aryl) 2 .

In certain embodiments, L 2 is dioxolopyrrolidine-dione.

In certain embodiments, L 2 is —C(═O)R 1 —.

In certain embodiments, L 2 is —CO((C 1 -C 22 )alkyl).

In certain embodiments, L 2 is —CO((C 6 )aryl).

In certain embodiments, L 2 is —CO 2 ((C 1 -C 22 )alkyl).

In certain embodiments, L 2 is —CO 2 ((C 6 )aryl).

In certain embodiments, L 2 is —CO 2 (CR 1 R 2 R 3 ).

In certain embodiments, L 2 is —SO 2 ((C 1 -C 22 )alkyl).

In certain embodiments, L 2 is —SO 2 ((C 6 )aryl).

In certain embodiments, Q is CH.

In certain embodiments, Q is O.

In certain embodiments, Q is S.

In certain embodiments, Q is NH.

In certain embodiments, Q is a disulfide bond.

In certain embodiments, m is 0.

In certain embodiments, m is 1.

In certain embodiments, m is 2.

In certain embodiments, m is 3.

In certain embodiments, m is 4.

In certain embodiments, m is 5.

In certain embodiments, m is 6.

In certain embodiments, m is 7.

In certain embodiments, m is 8.

In certain embodiments, m is 9.

In certain embodiments, m is 10.

In certain embodiments, m is 11.

In certain embodiments, m is 12.

In certain embodiments, m is 13.

In certain embodiments, m is 14.

In certain embodiments, m is 15.

In certain embodiments, m is 16.

In certain embodiments, m is 17.

In certain embodiments, m is 18.

In certain embodiments, m is 19.

In certain embodiments, m is 20.

In another embodiment, the invention encompasses Ionizable Lipids of the Invention of Formula VIII:

wherein

R 1 and R 2 are each independently selected from the group consisting of H, an optionally substituted C 1 -C 22 alkyl, optionally substituted C 2 -C 22 alkenyl, optionally substituted C 2 -C 22 alkynyl, optionally substituted C 3 -C 6 cycloalkyl, optionally substituted C 4 -C 6 heterocycloalkyl, optionally substituted C 4 -C 6 alkylcycloalkyl, optionally substituted C 4 -C 6 aryl, optionally substituted C 3 -C 6 heteroaryl, optionally substituted C 4 -C 8 aryloxy, optionally substituted C 7 -C 10 arylalkyl or optionally substituted C 5 -C 10 heteroarylalkyl group;

R 3 and R 4 are each independently optionally substituted C 10 -C 22 alkyl, optionally substituted C 10 -C 22 alkenyl, optionally substituted C 10 -C 22 alkynyl, or R 1 and R 2 can together form a 3-7 membered heterocycle or heteroaryl ring

X is OH, or NR 1 R 2 ; and

Z is an integer from 0 to 5.

In certain embodiments, the ionizable lipid of Formula VIII is selected from the group consisting of:

In certain embodiments, the ionizable lipid of Formula VIII is selected from the group consisting of:

In certain embodiments, the ionizable lipid of Formula VIII is selected from the group consisting of:

In certain embodiments, the ionizable lipid of Formula VIII is selected from the group consisting of:

In certain embodiments, the ionizable lipid of Formula VIII is selected from the group consisting of:

In certain embodiments, the ionizable lipid of Formula VIII is selected from the group consisting of:

In certain embodiments, the ionizable lipid of Formula VIII is selected from the group consisting of:

In certain embodiments, the ionizable lipid of Formula VIII is selected from the group consisting of:

In another embodiment, the invention encompasses Ionizable Lipids of the Invention of Formula IX:

wherein each R 1 and each R 2 is independently selected from the group consisting of H, an electron pair, an optionally substituted C 1 -C 12 alkyl, optionally substituted C 2 -C 12 alkenyl, optionally substituted C 2 -C 12 alkynyl, optionally substituted C 3 -C 6 cycloalkyl, optionally substituted C 4 -C 6 heterocycloalkyl, optionally substituted C 4 -C 6 alkylcycloalkyl, optionally substituted C 4 -C 6 aryl, optionally substituted C 3 -C 6 heteroaryl, optionally substituted C 4 -C 8 aryloxy, optionally substituted C 7 -C 10 arylalkyl; optionally substituted C 5 -C 10 heteroarylalkyl group, optionally substituted amine; or R 1 and R 2 can together form a 3-7 membered heterocycle or heteroaryl ring;

wherein each R 5 , R 6 , R 5′ , R 6′ , R 5″ , and R 6″ is independently selected from the group consisting of H, OH, halo, phenyl, benzyl, optionally substituted C 1 -C 22 alkyl, optionally substituted C 2 -C 22 alkenyl, optionally substituted C 2 -C 22 alkynyl,

wherein each R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 3 , R 14 , R 5 , and R 16 is independently selected from the group consisting of H, OH, halo, phenyl, benzyl, optionally substituted C 1 -C 22 alkyl, optionally substituted C 2 -C 22 alkenyl, optionally substituted C 2 -C 22 alkynyl,

wherein each of u, v, w, y, and z is independently an integer from 0-20;

wherein X is O, S, or N; and

wherein each of L 1 and L 2 is independently selected from the group consisting of —C(═O)—; OC(═O)—; —OC(═O)O—; —C(═O)O—; —C(═O)O(CR 5 R 6 R 7 ) m ; —NH—C(═O)—; —C(═O)NH—; —SO—; —SO 2 —; —SO 3 —; —NSO 2 —; —SO 2 N—; —NH((C 1 -C 8 )alkyl); —N((C 1 -C 8 )alkyl) 2 ; —NH((C 6 )aryl); —N((C 6 )aryl) 2 ; —NHC(═O)NH—; —NHC(═O)O—; —OC(═O)NH—; —NHC(═O)NR 1 —; —NHC(═O)O—; —OC(═O)NR 1 —; —C(═O)R 1 —; —CO((C 1 -C 8 )alkyl); —CO((C 6 )aryl); —CO 2 ((C 1 -C 8 )alkyl); —CO 2 ((C 6 )aryl); —SO 2 ((C 1 -C 8 )alkyl); and —SO 2 ((C 6 )aryl).

In certain embodiments, R 1 is H.

In certain embodiments, R 1 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 1 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 1 is substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 1 is substituted or unsubstituted C 3 -C 6 cycloalkyl.

In certain embodiments, R 1 is substituted or unsubstituted C 4 -C 6 heterocycloalkyl.

In certain embodiments, R 1 is substituted or unsubstituted C 4 -C 6 alkylcycloalkyl.

In certain embodiments, R 1 is substituted or unsubstituted C 4 -C 6 aryl.

In certain embodiments, R 1 is substituted or unsubstituted C 3 -C 6 heteroaryl.

In certain embodiments, R 1 is substituted or unsubstituted C 4 -C 8 aryloxy.

In certain embodiments, R 1 is substituted or unsubstituted C 7 -C 10 arylalkyl.

In certain embodiments, R 1 is substituted or unsubstituted C 5 -C 10 heteroarylalkyl group.

In certain embodiments, R 2 is H.

In certain embodiments, R 2 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 2 is substituted or unsubstituted C 2 -C 22 alkenyl

In certain embodiments, R 2 is substituted or unsubstituted C 2 -C 22 alkynyl

In certain embodiments, R 2 is substituted or unsubstituted C 3 -C 6 cycloalkyl.

In certain embodiments, R 2 is substituted or unsubstituted C 4 -C 6 heterocycloalkyl.

In certain embodiments, R 2 is substituted or unsubstituted C 4 -C 6 alkylcycloalkyl.

In certain embodiments, R 2 is substituted or unsubstituted C 4 -C 6 aryl.

In certain embodiments, R 2 is substituted or unsubstituted C 3 -C 6 heteroaryl.

In certain embodiments, R 2 is substituted or unsubstituted C 4 -C 8 aryloxy.

In certain embodiments, R 2 is substituted or unsubstituted C 7 -C 10 arylalkyl.

In certain embodiments, R 2 is substituted or unsubstituted C 5 -C 10 heteroarylalkyl group.

In certain embodiments, each R 5 is independently H, OH, halo, phenyl, benzyl, substituted or unsubstituted C 1 -C 22 alkyl, substituted or unsubstituted C 2 -C 22 alkenyl; or substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 5 is H.

In certain embodiments, R 5 is OH.

In certain embodiments, R 5 is halo.

In certain embodiments, R 5 is phenyl.

In certain embodiments, R 5 is benzyl.

In certain embodiments, R 5 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 5 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 5 is substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, each R 6 is independently H, OH, halo, phenyl, benzyl, substituted or unsubstituted C 1 -C 22 alkyl, substituted or unsubstituted C 2 -C 22 alkenyl; or substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 6 is H.

In certain embodiments, R 6 is OH.

In certain embodiments, R 6 is halo.

In certain embodiments, R 6 is phenyl.

In certain embodiments, R 6 is benzyl.

In certain embodiments, R 6 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 6 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 6 is substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 7 is H, OH, halo, phenyl, benzyl, substituted or unsubstituted C 1 -C 22 alkyl, substituted or unsubstituted C 2 -C 22 alkenyl; or substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 7 is H.

In certain embodiments, R 7 is OH.

In certain embodiments, R 7 is halo.

In certain embodiments, R 7 is phenyl.

In certain embodiments, R 7 is benzyl.

In certain embodiments, R 7 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 7 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 7 is substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 8 is H, OH, halo, phenyl, benzyl, substituted or unsubstituted C 1 -C 22 alkyl, substituted or unsubstituted C 2 -C 22 alkenyl; or substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 8 is H.

In certain embodiments, R 8 is OH.

In certain embodiments, R 8 is halo.

In certain embodiments, R 8 is phenyl.

In certain embodiments, R 8 is benzyl.

In certain embodiments, R 8 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 8 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 8 is substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 9 is H, OH, halo, phenyl, benzyl, substituted or unsubstituted C 1 -C 22 alkyl, substituted or unsubstituted C 2 -C 22 alkenyl; or substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 9 is H.

In certain embodiments, R 9 is OH.

In certain embodiments, R 9 is halo.

In certain embodiments, R 9 is phenyl.

In certain embodiments, R 9 is benzyl.

In certain embodiments, R 9 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 9 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 9 is substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 10 is H, OH, halo, phenyl, benzyl, substituted or unsubstituted C 1 -C 22 alkyl, substituted or unsubstituted C 2 -C 22 alkenyl; or substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 10 is H.

In certain embodiments, R 10 is OH.

In certain embodiments, R 10 is halo.

In certain embodiments, R 10 is phenyl.

In certain embodiments, R 10 is benzyl.

In certain embodiments, R 10 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 10 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 10 is substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 11 is H, OH, halo, phenyl, benzyl, substituted or unsubstituted C 1 -C 22 alkyl, substituted or unsubstituted C 2 -C 22 alkenyl; or substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 11 is H.

In certain embodiments, R 11 is OH.

In certain embodiments, R 11 is halo.

In certain embodiments, R 11 is phenyl.

In certain embodiments, R 11 is benzyl.

In certain embodiments, R 11 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 11 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 11 is substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 11 is H, OH, halo, phenyl, benzyl, substituted or unsubstituted C 1 -C 22 alkyl, substituted or unsubstituted C 2 -C 22 alkenyl; or substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 12 is H.

In certain embodiments, R 12 is OH.

In certain embodiments, R 12 is halo.

In certain embodiments, R 12 is phenyl.

In certain embodiments, R 2 is benzyl.

In certain embodiments, R 12 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 12 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 12 is substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 13 is H, OH, halo, phenyl, benzyl, substituted or unsubstituted C 1 -C 22 alkyl, substituted or unsubstituted C 2 -C 22 alkenyl; or substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 13 is H.

In certain embodiments, R 13 is OH.

In certain embodiments, R 13 is halo.

In certain embodiments, R 13 is phenyl.

In certain embodiments, R 13 is benzyl.

In certain embodiments, R 13 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 13 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 13 is substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 14 is H, OH, halo, phenyl, benzyl, substituted or unsubstituted C 1 -C 22 alkyl, substituted or unsubstituted C 2 -C 22 alkenyl; or substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 14 is H.

In certain embodiments, R 14 is OH.

In certain embodiments, R 14 is halo.

In certain embodiments, R 14 is phenyl.

In certain embodiments, R 14 is benzyl.

In certain embodiments, R 14 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 14 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 14 is substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 15 is H, OH, halo, phenyl, benzyl, substituted or unsubstituted C 1 -C 22 alkyl, substituted or unsubstituted C 2 -C 22 alkenyl; or substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 15 is H.

In certain embodiments, R 15 is OH.

In certain embodiments, R 15 is halo.

In certain embodiments, R 15 is phenyl.

In certain embodiments, R 15 is benzyl.

In certain embodiments, R 15 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 15 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 15 is substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 16 is H, OH, halo, phenyl, benzyl, substituted or unsubstituted C 1 -C 22 alkyl, substituted or unsubstituted C 2 -C 22 alkenyl; or substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, R 16 is H.

In certain embodiments, R 16 is OH.

In certain embodiments, R 16 is halo.

In certain embodiments, R 16 is phenyl.

In certain embodiments, R 16 is benzyl.

In certain embodiments, R 16 is substituted or unsubstituted C 1 -C 22 alkyl.

In certain embodiments, R 16 is substituted or unsubstituted C 2 -C 22 alkenyl.

In certain embodiments, R 16 is substituted or unsubstituted C 2 -C 22 alkynyl.

In certain embodiments, u is 0.

In certain embodiments, u is 1.

In certain embodiments, u is 2.

In certain embodiments, u is 3.

In certain embodiments, u is 4.

In certain embodiments, u is 5.

In certain embodiments, u is 6.

In certain embodiments, u is 7.

In certain embodiments, u is 8.

In certain embodiments, u is 9.

In certain embodiments, u is 10.

In certain embodiments, u is 11.

In certain embodiments, u is 12.

In certain embodiments, u is 13.

In certain embodiments, u is 14.

In certain embodiments, u is 15.

In certain embodiments, u is 16.

In certain embodiments, u is 17.

In certain embodiments, u is 18.

In certain embodiments, u is 19.

In certain embodiments, u is 20.

In certain embodiments, v is 0.

In certain embodiments, v is 1.

In certain embodiments, v is 2.

In certain embodiments, v is 3.

In certain embodiments, v is 4.

In certain embodiments, v is 5.

In certain embodiments, v is 6.

In certain embodiments, v is 7.

In certain embodiments, v is 8.

In certain embodiments, v is 9.

In certain embodiments, v is 10.

In certain embodiments, v is 11.

In certain embodiments, v is 12.

In certain embodiments, v is 13.

In certain embodiments, v is 14.

In certain embodiments, v is 15.

In certain embodiments, v is 16.

In certain embodiments, v is 17.

In certain embodiments, v is 18.

In certain embodiments, v is 19.

In certain embodiments, v is 20.

In certain embodiments, w is 0.

In certain embodiments, w is 1.

In certain embodiments, w is 2.

In certain embodiments, w is 3.

In certain embodiments, w is 4.

In certain embodiments, w is 5.

In certain embodiments, w is 6.

In certain embodiments, w is 7.

In certain embodiments, w is 8.

In certain embodiments, w is 9.

In certain embodiments, w is 10.

In certain embodiments, w is 11.

In certain embodiments, w is 12.

In certain embodiments, w is 13.

In certain embodiments, w is 14.

In certain embodiments, w is 15.

In certain embodiments, w is 16.

In certain embodiments, w is 17.

In certain embodiments, w is 18.

In certain embodiments, w is 19.

In certain embodiments, w is 20.

In certain embodiments, y is 0.

In certain embodiments, y is 1.

In certain embodiments, y is 2.

In certain embodiments, y is 3.

In certain embodiments, y is 4.

In certain embodiments, y is 5.

In certain embodiments, y is 6.

In certain embodiments, y is 7.

In certain embodiments, y is 8.

In certain embodiments, y is 9.

In certain embodiments, y is 10.

In certain embodiments, y is 11.

In certain embodiments, y is 12.

In certain embodiments, y is 13.

In certain embodiments, y is 14.

In certain embodiments, y is 15.

In certain embodiments, y is 16.

In certain embodiments, y is 17.

In certain embodiments, y is 18.

In certain embodiments, y is 19.

In certain embodiments, y is 20.

In certain embodiments, z is 0.

In certain embodiments, z is 1.

In certain embodiments, z is 2.

In certain embodiments, z is 3.

In certain embodiments, z is 4.

In certain embodiments, z is 5.

In certain embodiments, z is 6.

In certain embodiments, z is 7.

In certain embodiments, z is 8.

In certain embodiments, z is 9.

In certain embodiments, z is 10.

In certain embodiments, z is 11.

In certain embodiments, z is 12.

In certain embodiments, z is 13.

In certain embodiments, z is 14.

In certain embodiments, z is 15.

In certain embodiments, z is 16.

In certain embodiments, z is 17.

In certain embodiments, z is 18.

In certain embodiments, z is 19.

In certain embodiments, z is 20.

In certain embodiments L 1 is a bond.

In certain embodiments, L 1 is —C(═O)—.

In certain embodiments, L 1 is —OC(═O)O—.

In certain embodiments, L 1 is —NH—C(═O)—.

In certain embodiments, L 1 is —SO—.

In certain embodiments, L 1 is —SO 2 —.

In certain embodiments, L 1 is OC(═O).

In certain embodiments, L 1 is —C(═O)O—.

In certain embodiments, L 1 is —C(═O)NH—.

In certain embodiments, L 1 is —SO 3 —.

In certain embodiments, L 1 is —NSO 2 —.

In certain embodiments, L 1 is —SO 2 N.

In certain embodiments, L 1 is —NH((C 1 -C 22 )alkyl).

In certain embodiments, L 1 is —N((C 1 -C 8 )alkyl) 2 .

In certain embodiments, L 1 is —NH((C 6 )aryl).

In certain embodiments, L 1 is —N((C 6 )aryl) 2 .

In certain embodiments, L 1 is dioxolopyrrolidine-dione.

In certain embodiments, L 1 is —C(═O)R 1 —.

In certain embodiments, L 1 is —CO((C 1 -C 22 )alkyl).

In certain embodiments, L 1 is —CO((C 6 )aryl).

In certain embodiments, L 1 is —CO 2 ((C 1 -C 22 )alkyl).

In certain embodiments, L 1 is —CO 2 ((C 6 )aryl).

In certain embodiments, L 1 is —SO 2 ((C 1 -C 22 )alkyl).

In certain embodiments, L 1 is —SO 2 ((C 6 )aryl).

In certain embodiments L 2 is a bond.

In certain embodiments, L 2 is —C(═O)—.

In certain embodiments, L 2 is —OC(═O)O—.

In certain embodiments, L 2 is —NH—C(═O)—.

In certain embodiments, L 2 is —SO—.

In certain embodiments, L 2 is —SO 2 —.

In certain embodiments, L 2 is OC(═O).

In certain embodiments, L 2 is —C(═O)O—.

In certain embodiments, L 2 is —C(═O)NH—.

In certain embodiments, L 2 is —SO 3 —.

In certain embodiments, L 2 is —NSO 2 —.

In certain embodiments, L 2 is —SO 2 N.

In certain embodiments, L 2 is —NH((C 1 -C 22 )alkyl).

In certain embodiments, L 2 is —N((C 1 -C 8 )alkyl) 2 .

In certain embodiments, L 2 is —NH((C 6 )aryl).

In certain embodiments, L 2 is —N((C 6 )aryl) 2 .

In certain embodiments, L 2 is dioxolopyrrolidine-dione.

In certain embodiments, L 2 is —C(═O)R 1 —.

In certain embodiments, L 2 is —CO((C 1 -C 22 )alkyl).

In certain embodiments, L 2 is —CO((C 6 )aryl).

In certain embodiments, L 2 is —CO 2 ((C 1 -C 22 )alkyl).

In certain embodiments, L 2 is —CO 2 ((C 6 )aryl).

In certain embodiments, L 2 is —CO 2 (CR 1 R 2 R 3 ).

In certain embodiments, L 2 is —SO 2 ((C 1 -C 22 )alkyl).

In certain embodiments, L 2 is —SO 2 ((C 6 )aryl).

In certain embodiments, Q is CH.

In certain embodiments, X is O.

In certain embodiments, X is S.

In certain embodiments, X is N.

In certain embodiments, m is 0.

In certain embodiments, m is 1.

In certain embodiments, m is 2.

In certain embodiments, m is 3.

In certain embodiments, m is 4.

In certain embodiments, m is 5.

In certain embodiments, m is 6.

In certain embodiments, m is 7.

In certain embodiments, m is 8.

In certain embodiments, m is 9.

In certain embodiments, m is 10.

In certain embodiments, m is 11.

In certain embodiments, m is 12.

In certain embodiments, m is 13.

In certain embodiments, m is 14.

In certain embodiments, m is 15.

In certain embodiments, m is 16.

In certain embodiments, m is 17.

In certain embodiments, m is 18.

In certain embodiments, m is 19.

In certain embodiments, m is 20.

V.3. Illustrative Ionizable Lipids of the Invention

Illustrative, non-limiting examples of Ionizable Lipids of the Invention are illustrated in Table 10 (the column with the heading “Method” refers to the method of synthesizing the particular compound as set for below in section V.4. Methods Of Making The Ionizable Lipids Of The Invention).

In another embodiment of the invention, adjusting the pKa of the Ionizable Lipids of the Invention can direct the administration of the lipid nanoparticle-nucleic acid complex (LNP-NA) to a particular tissue or organ. The inventors surprisingly found that the design of the Ionizable Lipids of the Invention can be significantly accelerated using the prediction of the pKa of ionizable lipids using software, for example, ACDLabs Percepta. The inventors found that the predicted aqueous pKas for ionizable lipids are all significantly higher than that measured by TNS. In certain embodiments, a drop in pKa from an aqueous environment to the LNP environment has not been previously acknowledged in the literature. Without being bound by theory, a reasonable explanation can be found due to the higher solvation energy of protons in the lipid phase compared with the aqueous phase and electrostatic repulsion of protons from the Ionizable Lipids of the Invention in the LNP, which can combine to reduce pKa by 1-3 points from aqueous to lipid phases. In certain embodiments, this predictive capability can be used in order to select specific headgroups and carbon spacers by examining pKa tables of potential candidates and selecting only those with appropriate pKa values. Candidates can be accurately eliminated in this manner, generating enormous savings in synthesis and testing time and expense.

In certain embodiments, the pKa of the Ionizable Lipids of the Invention included in the LNP is measured by fluorescent enhancement of an anionic lipophilic dye TNS binding to the LNP containing the ionizable lipid. Prior studies have shown that the TNS pKa needs to be near 6.5 for efficient delivery to liver upon IV administration. The pKa in the aqueous phase can be predicted by software such ACDLabs Percepta, resulting in pKas that are higher than the TNS pKa. Given the importance of the LNP pKa in determining their efficiency, we developed new methods (zeta potential, 1 H NMR, modeling) to link the intrinsic pKa of the ionizable lipid to LNP ionization properties, in order to rationally design effective systems for mRNA delivery.

In certain embodiments, the invention encompasses a new class of ionizable lipids (the Ionizable Lipids of the Invention) synthesized bearing two ionizable amines and two degradable ester linkers to linoleic acid tails or branched tails. DL=DiLinoleic Acid, ADDE=Azane Diyl DiEthyl, Cx/Cy=carbon spacers, DMA=DiMethylAmine, Pyr=Pyridine, PipZ=PiperaZine, PipD=PiperiDine, DIPA=DiIsopropylamine, DM=DiMethyl, BOD=BisOctylDecyl. Most ADDE ionizable lipids successfully transfected HEK 293 cells in vitro, with the exception of 4 listed below that were not active, possibly due to the bulkiness of their headgroups and restricted conformations.

In certain embodiments, additional ionizable lipids were synthesized bearing one ionizable amine and two linoleic acid tails linked to headgroups such as Isopentylamine (IP), Ethylalcohol (EA), Propyl alcohol (PA) via degradable esters as illustrated in Table 13A.

In certain embodiments, a second class of lipids contains one ionizable groups (DMA) and two different linkers: degradable ester and non-degradable OctaSulfonAmido (OSA). Additionally, these lipids contain either a linear (Linoyl) or branched tail (Octadecanyl) as illustrated in Table 13B.

In certain embodiments, a third class of ionizable lipids with one ionizable amine and one linoleic acid (L) tail both linked to the headgroup such as (Aminoethane (AE), Dimethylamine, and Aminoethyldisulfonyl (AEDS)) via non degradable amides as illustrated in Table 13C.

In certain embodiments, exemplary Ionizable Lipids of the Invention including alkyl lipids are illustrated with predicted pKa from ACD Classic and ACD Galas including the Method of synthesis are illustrated in Table 13D.

V.4. Illustrative Methods of Making the Ionizable Lipids of the Invention

The Ionizable Lipids of the Invention can be synthesized by various routes using commercially available reagents. The below methods provide illustrative techniques to make the Ionizable Lipids of the Invention.

Illustrative embodiments of the Ionizable Lipids of the Invention with two degradable esters (e.g., compound A-7) can be prepared according to general reaction Scheme 1 (“Method A”), wherein R 1 is a saturated or unsaturated C 1 -C 18 alkyl with linear chains or saturated/unsaturated chains and bilayer tails (symmetric and unsymmetrical), n is an integer from 1 to 6. Referring to the General Reaction Scheme 1, compounds of structure A-1 can be purchased from commercial sources or prepared according to the methods familiar to one of the ordinary skill in the art. A mixture of A-1 in solvent ethanol is treated with catalytic amount of sulfuric acid to give the ester A-2, then A-2 is treated with LAH (e.g., lithium aluminium hydride) in anhydrous THF for 9-10 h to obtain alcohol derivative A-3. The purified A-3 is reacted with acryloyl chloride derivatives (where Ra=CH 3 , C 2 H 5 and/or isopropyl groups) A-4, a base (e.g., triethyl amine) in dichloromethane for 4-5 hours to get substituted acylation products A-5. A mixture of the acylated product and head groups (R1) N,N-dimethyldiamine A-6 is heated at a temperature and time sufficient to produce A-7 after any necessarily workup and or purification step.

Illustrative embodiments of the Ionizable Lipids of the Invention with carbon spacers increased between the linker and internal amine (e.g., compound B-3) can be prepared according to general reaction Scheme 2 (“Method B”), wherein R 1 is a saturated or unsaturated C 1 -C 18 alkyl with linear chains or saturated/unsaturated with bilayer tails (symmetric and unsymmetrical), n is an integer from 1 to 6. Referring to the General Reaction Scheme 2, compounds of structure A-2 are prepared according to the methods familiar to one of the ordinary skill in art. A mixture of B-1 and A-2 in methylene dichloride as the solvent is treated with a catalytic amount of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC·HCl), hydroxybenzotriazole (HOBt) with a base such as triethyl amine to give the ester derivative B-2, then the mixture of B-2 and A-6 is treated with base (e.g., N,N-diisopropylethyl amine or 1,8-diazabicyclo[5.4.0]undec-7-ene) in anhydrous THF heated at a temperature and time sufficient 2-5 days to produce B-3 after any necessarily workup and or purification step.

Illustrative embodiments of the Ionizable Lipids of the Invention (symmetrical with 4 ester groups) (e.g., compound C-4) can be prepared according to general reaction Scheme 3 (“Method C”), wherein R 1 is a saturated or unsaturated C 1 -C 18 alkyl with linear chains or saturated/unsaturated with bilayer tails (symmetric and unsymmetrical), where m is an integer from −1 to 2, n is an integer from 1 to 6. Referring to the General Reaction Scheme 3, compounds of structure A-2 are prepared according to the methods familiar to one of the ordinary skill in the art. A mixture of C-1 and A-2 are treated with solvent free conditions in Schlenk tube purged with nitrogen gas (maintained under anhydrous conditions) with a catalytic amount of the organocatalyst 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD) heated at a temperature to afford ester derivative C-2. The purified C-2 is mixed with acryloyl chloride A-4 using methylene chloride as a solvent in the presence of triethylamine. Then the reaction is allowed to stir at room temperature until the consumption of starting materials to obtain C-3. A mixture of the acylated product and head groups C-3 and N,N-dimethyldiamine A-6 is heated at a temperature and time sufficient to produce C-4 after any necessarily workup and or purification step.

Illustrative embodiments of the Ionizable Lipids of the Invention (unsymmetrical with 4 ester groups) (e.g., compound C-7) can be prepared according to general reaction Scheme 4 (“Method-C1”), wherein R 1 is a saturated or unsaturated C 1 -C 18 is alkyl with linear chains or saturated/unsaturated with bilayer tails (symmetric and unsymmetrical), where m is an integer from −1 to 2, n is an integer from 1 to 6. Referring to the General Reaction Scheme 4, compounds of structure A-2 are prepared according to the methods familiar to one of the ordinary skill in the art. A mixture of C-1 and A-2 are treated with solvent free conditions in a Schlenk tube purged with nitrogen gas (maintained under anhydrous conditions) with a catalytic amount of the organocatalyst 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD) heated at a temperature to afford ester derivative C-2. The purified C-2 is mixed with acryloyl chloride A-4 using methylene chloride as a solvent in the presence of triethylamine. The reaction is allowed to stir at room temperature until the consumption of stating materials to obtain C-3. A mixture of the acylated C-3 product and head groups N,N-dimethyldiamine A-6 in equal amounts are reacted with heating at a temperature and time sufficient to produce C-5. Further purified C-5 is treated with C-6 (bilayered tail) in the same equivalents and heated at a temperature and time sufficient to produce C-7 after any necessarily workup and or purification step.

Illustrative embodiments of the Ionizable Lipids of the Invention with six degradable esters (e.g., compound D-8) can be prepared according to general reaction Scheme 5 (“Method D”), wherein R 1 is a saturated or unsaturated C 1 -C 18 alkyl with linear chains or saturated/unsaturated with bilayer tails (symmetric and unsymmetrical), n is an integer from 1 to 6, R 2 is alkyl, cyclic, heterocyclic substituents and R 4 is ester derivative with an alkyl chain. Referring to the General Reaction Scheme 5, compounds of structure B-1, A-1, A-2, A-5 and A-6 can be purchased from commercial sources or prepared according to the methods familiar to one of the ordinary skill in the art. Esterification of B-1 in presence of ethanol using a catalytic amount of sulfuric acid obtained D-1. Further, the alkyl bromide is converted to the corresponding alkyl iodide D-2 with a solution of sodium iodide in acetone by the classic Finkelstein reaction. A mixture of D-2 and D-3 is reacted with sodium ethoxide (NaOEt) in ethanol at reflux, followed by acidification to low pH to obtain D-4. In Step 4, esterification was done in the presence of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC·HCl) and a catalytic amount of 4-dimethylaminopyridine (4-DMAP) in methylene dichloride for overnight to obtain D-5. Further D-5 is reduced to the corresponding alcohol in the presence of sodium borohydride (NaBH 4 ) to get D-6. Purified D-6 is reacted with acryloyl chloride derivatives (where Ra=CH 3 , C 2 H 5 and/or isopropyl groups) A-5 in the presence of a base (e.g., triethylamine) in tetrahydrofuran solvent for 4-5 hours to get the substituted acylation products D-7. A mixture of the acylated product D-7 and head groups (R1) N,N-dimethyldiamine A-6 is heated at a temperature and time sufficient to produce D-8 after any necessarily workup and or purification step.

Illustrative embodiments of the Ionizable Lipids of the Invention with degradable and non-degradable esters (e.g., compounds E, F and G) can be prepared according to general reaction Scheme 6 (“Method E”), wherein R 1 is a saturated or unsaturated C 1 -C 18 alkyl with linear chains or saturated/unsaturated with bilayer tails (symmetric and unsymmetrical), n is an integer from 1 to 6, R 2 is alkyl, cyclic, heterocyclic substituents. Referring to the General Reaction Scheme 6, compounds of structure E-1, A-4, E-2, A-5 and A-6 can be purchased from commercial sources or prepared according to the methods familiar to one of the ordinary skill in the art. E-1 is reacted with acryloyl chloride A-4 in methylene chloride in the presence of the reagent triethylamine to obtain E-2. In Step-2, A-6 is reacted with A-5 to obtain the monoalkylated product in situ. Further addition of A-5 dropwise under solvent free conditions is followed by heating for a time sufficient to produce E-3 after any necessarily workup and or purification step. In step 3, A-6 is reacted with 2 equivalents of acryloylamide under a nitrogen atmosphere under solvent free conditions followed by heating at a temperature and time sufficient to produce compound F after any necessarily workup and or purification step. In Step 4 A-6 is reacted with one equivalent of E-2 to obtain mono alkylated product and it further reacted with alkyl sulfonyl chloride to get nondegradable linkers derivatives G after any necessarily workup and or purification step.

Illustrative embodiments of the Ionizable Lipids of the Invention with spiro dioxolane derivatives (e.g., compound H) is prepared according to general reaction Scheme 7 (“Method H”), wherein R 1 is a saturated or unsaturated C 1 -C 18 alkyl with linear chains or saturated/unsaturated with bilayer tails (symmetric and unsymmetrical), n is an integer from 1 to 6, R 2 is alkyl, cyclic, heterocyclic substituents. Referring to the General Reaction Scheme 5, compounds of structure A-6, H-1, H-5 and necessary catalysts can be purchased from commercial sources or prepared according to the methods familiar to one of the ordinary skill in the art. Maleic anhydride reacts with 3-(dimethylamino)-1-propylamine (DMAPA) to yield 3-(N,N-dimethylamino)propyl maleamic acid H-2. The reaction was carried out in chloroform by addition of the amine to maleic anhydride at room temperature and subsequent heating to 60° C. After purification, H-2 is reacted with sodium acetate in acetic anhydride and heated at a temperature and time sufficient to produce G-3 after any necessarily workup and or purification step. In step 3, a hydroxylation reaction is performed using osmium tetroxide in anhydrous THF overnight to obtain HA. A mixture of H-4 and H-5 are dissolved in toluene solvent and the round bottom flask is equipped with Dean-Stark apparatus and the reaction is subsequently heated to reflux for 8 h and the obtained water in the Dean-Stark apparatus is collected in a separate flask as the reaction continues until the consumption of starting materials to obtain H-6 after any necessarily workup and or purification step.

Illustrative embodiments of the Ionizable Lipids of the Invention with spiro dioxolane derivatives (e.g., compound I) is prepared according to general reaction Scheme 8 (“Method I”), wherein R 1 is a saturated or unsaturated C 1 -C 18 alkyl with linear chains or saturated/unsaturated with bilayer tails (symmetric and unsymmetrical), n is an integer from 1 to 6, R 2 is alkyl, cyclic, heterocyclic substituents. Referring to the General Reaction Scheme 8, compounds of structure I-1, I-5 and necessary catalysts can be purchased from commercial sources or prepared according to the methods familiar to one of the ordinary skill in the art. A mixture of I-1, 1,2,5-pentanetriol and pyridinium p-toluenesulfonate in toluene is refluxed under nitrogen overnight with a Dean-Stark apparatus to remove water and the reaction is allowed to reflux until complete consumption of reactants to obtain I-2. Esterification was done in step 2, I-2 was reacted with different alkyl saturated/unsaturated linear or bilayer acids with reagents like EDC·HCl and 4-DMAP in methylene dichloride for overnight to obtain I-3. Deprotection of the amine affords the I-4 derivative. In step 3, I-4 and I-5 are reacted in the presence of EDC·HCl, HOBt and DMAP or triethylamine. All these mixtures are dissolved in dimethylformamide and allowed to stir at room temperature until the consumption of starting materials to obtain I-6 after any necessarily workup and or purification step.

Illustrative embodiments of a lipid with two degradable esters (e.g., compound A-5) can be prepared according to general reaction Scheme 9, wherein R 1 is a saturated or unsaturated C 1 -C 18 alkyl with linear chains or saturated/unsaturated with bilayer tails (symmetric and unsymmetrical), n is an integer from 1 to 6. Referring to the General Reaction Scheme 9, compounds of structure A-1 are purchased from commercial sources and/or prepared according to the methods familiar to one of the ordinary skill in art. A-1 is reacted with A-2 acryloyl chloride derivatives (where Ra=CH 3 , C 2 H 5 and/or isopropyl groups) with catalytic amount of base (e.g., triethyl amine) in methylene chloride solvent for 4-5 hours to get substituted acylation products A-3. A mixture of the acylated product and head groups (R1) N,N-dimethyldiamine and or primary amines substituents such as N-alkyl, acyclic, cyclic, heterocyclic, aromatic amines) A-4 is heated at a temperature and time sufficient to produce A-5 after any necessary workup and or purification step.

Illustrative embodiments of the lipids with carbon spacers increased between the linker and internal amine (e.g., compound B-2) can be prepared according to general reaction Scheme 10, wherein R 1 and R 2 is a saturated or unsaturated C 1 -C 18 alkyl with linear chains or saturated/unsaturated with bilayer tails (symmetric and unsymmetrical), n is an integer from 1 to 6. Referring to the General Reaction Scheme 10, compounds of structure B-2 are prepared according to the methods familiar to one of the ordinary skill in art. Mixtures of B-1 and B-2 in methylene dichloride as the solvent is treated with stoichiometric amounts of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride(EDC·HCl) or DCC Dicyclohexyl carbodimide, hydroxybenzotriazole (HOBt) with a base such as triethyl amine to give the ester derivative B-3, then the mixture of B-3 and A-4 is treated with base (e.g., N,N-diisopropylethyl amine) in anhydrous acetonitrile solvent heated at a 67° C. temperature for 16 h, to produce B-4 after any necessarily workup and or purification.

Illustrative embodiments of the lipid with degradable/non degradable (e.g., compound C-2) can be prepared according to general reaction Scheme 11, wherein R Tail is a saturated or unsaturated C 1 -C 18 alkyl with linear chains or saturated/unsaturated with bilayer tails (symmetric and unsymmetrical), n is an integer from 1 to 8. Referring to the General Reaction Scheme 11, compounds of structure A-4 purchased from commercial sources and or prepared according to the methods familiar to one of the ordinary skill in art. A-4 is reacted with C-1 with catalytic amount of base (e.g., potassium carbonate, N,N-diisopropyl ethylamine, potassium iodide) in acetonitrile solvent for 16 hours at 67° C. temperature to produce C-2 after any necessarily workup and or purification step.

Illustrative embodiments of the lipid with degradable/non degradable (e.g., compound D-4) can be prepared according to general reaction Scheme 12, wherein R Tail is a saturated or unsaturated C 1 -C 18 alkyl with linear chains or saturated/unsaturated with bilayer tails (symmetric and unsymmetrical), n is an integer from 1 to 12. Referring to the General Reaction Scheme 12, Compounds of structure A-4 purchased from commercial sources and or prepared according to the methods familiar to one of the ordinary skill in art. Excess equivalents of A-4 is reacted with one equivalents of C-1 with catalytic amount of base (e.g., potassium carbonate, N,N-diisopropyl ethylamine, potassium iodide) in acetonitrile solvent for 16 hours at 67° C. temperature to produce D-1 after any necessarily workup and or purification step. Then D-1 is reacted with D-2 same as step-1 to produce D-3. React A-3 with D-3 under solvent free condition to produce D-4 for procedure follow scheme-1. Moreover, N, N-alkylation is done by reacting with B-3 and D-3 in the same procedure as in general reaction scheme 10.

Illustrative embodiments of the lipid with degradable/non degradable (e.g., compound E-2) can be prepared according to general reaction Scheme 13, wherein R Tail is a saturated or unsaturated C 1 -C 18 alkyl with linear chains or saturated/unsaturated with bilayer tails (symmetric and unsymmetrical), n is an integer from 1 to 12. Referring to the General Reaction Scheme 13, compounds of structure A-4 and E-1 purchased from commercial sources and or prepared according to the methods familiar to one of the ordinary skill in art. Excess equivalents of E-1 is reacted with one equivalents of A-4 with stoichiometric amount of sodium triacetoxyborohydride in methylene chloride solvent for 4-5 hours at room temperature to produce E-2 after any necessarily workup and or purification step.

Illustrative embodiments of the lipid with degradable/non degradable (e.g., compound F-2) can be prepared according to general reaction Scheme 14, wherein R Tail is a saturated or unsaturated C 1 -C 18 alkyl with linear chains or saturated/unsaturated with bilayer tails (symmetric and unsymmetrical), n is an integer from 1 to 14. Referring to the General Reaction Scheme 14, compounds of structures A-4 and F-1 purchased from commercial sources and or prepared according to the methods familiar to one of the ordinary skill in art. Excess equivalents of F-1 is reacted with one equivalents of A-4 with catalytic amount of tetra butyl ammonium fluoride in tetrahydrofuran solvent for 3 days at 80° C. to produce F-2 after any necessarily workup and or purification step.

Illustrative embodiments of the lipid with six degradable esters (e.g., compound D-8) can be prepared according to general reaction Scheme 15, wherein RTail is a saturated or unsaturated C 1 -C 18 alkyl with linear chains or saturated/unsaturated with bilayer tails (symmetric and unsymmetrical), n is an integer from 1 to 8, RHead is alkyl, cyclic, heterocyclic substituents. Referring to the General Reaction Scheme 7, compounds of structure B-1 and A-4 can be purchased from commercial sources or prepared according to the methods familiar to one of the ordinary skill in the art. Esterification of B-1 in presence of ethanol using a catalytic amount of sulfuric acid obtained G-1. Further, the alkyl bromide is converted to the corresponding alkyl iodide G-2 with a solution of sodium iodide in acetone by the classic Finkelstein reaction. A mixture of G-2 and G-3 is reacted with sodium ethoxide (NaOEt) in ethanol at reflux, followed by acidification to low pH to obtain GA. In Step 4, Fischer esterification was done in the presence of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC·HCl) and a catalytic amount of 4-dimethylaminopyridine (4-DMAP) in methylene dichloride for overnight to obtain G-5. Further G-5 is reduced to the corresponding reductive amination in the presence of sodium borohydride (NaBH4) to get G-6. Purified G-6 is reacted with acyl or sulfonyl chloride derivatives G-8 in the presence of a base (e.g., triethylamine) in tetrahydrofuran solvent for 4-5 hours to get the substituted acylation products G-9 at a temperature and time sufficient to produce D-8 after any necessarily workup and or purification step.

Illustrative embodiments of the lipids with carbon spacers increased between the linker and internal amine (e.g., compound H-4) can be prepared according to general reaction Scheme 16, wherein R1 and R2 is a saturated or unsaturated C 1 -C 18 alkyl with linear chains or saturated/unsaturated with bilayer tails (symmetric and unsymmetrical), n and n1 are integer's from 1 to 6. Referring to the General Reaction Scheme 16, compounds of structure HA are prepared according to the methods familiar to one of the ordinary skill in art. Mixtures of B-1 and B-2 in methylene dichloride as the solvent is treated with stoichiometric amounts of N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride(EDC·HCl) or DCC Dicyclohexyl carbodimide, hydroxybenzotriazole (HOBt) with a base such as triethyl amine to give the ester derivative B-3. On the other hand, protection of amine H-1 was done according to previous methods to produce H-1, then the mixture of B-3 and H-1 is treated with base (e.g., N,N-diisopropylethyl amine) in anhydrous acetonitrile solvent heated at a 67 C temperature for 16 h, to produce H-3, then deprotection is done in presence of acid media then followed by N-alkyation to produce H-4 after any necessarily workup and or purification.

V.5. Targeted Delivery of the LNPs Including the Ionizable Lipids of the Invention

In certain embodiments, it was surprisingly found that the Ionizable Lipids of the Invention when used in vaccines for intramuscular (IM) injection, the LNP requirements for delivery to dendritic cells via the IM route are different than intravenous (IV) delivery to hepatocytes. In certain embodiments, hepatocyte targeting is due to the LNP associating with ApoE that targets LNP uptake to hepatocyte LDL receptors while ApoE may not have the same role in IM administration. In certain embodiments, a second targeting mechanism for LNPs is their net charge. In certain embodiments, negatively charged LNPs target the spleen upon IV administration while positively charged LNPs target the lungs and near neutral LNPs target the liver. The inventors have surprisingly discovered, however, a significant charge effect based on the finding that previously known LNPs rapidly disseminate and express systemically upon IM administration, while the Ionizable Lipids of the Invention utilized in LNP can be tuned to have a slight positive charge or be near neutral at physiological pH and do not disseminate and are expressed locally in muscle and draining lymph nodes to generate a robust immune response through targeted delivery. In certain embodiments, localization of expression can increase potency and reduce systemic adverse events by avoiding off-target expression.

In certain embodiments, protonation of the Ionizable Lipids of the Invention and branched lipid tails promote endosomal release, and LNP charge influences targeting. In other embodiments, the Ionizable Lipids of the Invention influence LNP structure on its adjuvanticity. In certain embodiments, an asymmetric ionizable lipid LNP acted as a strong Th2-biased adjuvant when delivered with protein subunit antigens.

In certain embodiments, the variety of these LNP driven adjuvant effects motivates the identification of structure function relationships (SFRs) controlling these properties which may involve the cyclic vs linear nature of the headgroups.

Without being bound by theory, the invention encompasses Ionizable Lipids of the Invention and potent LNPs, for example, for use in mRNA vaccines, based on the understanding of SFRs connecting LNP ionization, charge and structure to delivery efficiency, targeting, and adjuvanticity for intramuscular administration with potential application to other sites of delivery.

In certain embodiments, pKas were obtained from ACDLabs Percepta for the ionizable lipid, and by zeta potential and TNS for LNPs containing the ionizable lipids. In other embodiments, LNP isoelectric pI was obtained from the zeta potential, and LNP diameter is number-average from DLS. Average mRNA copies per LNP was calculated using a molecular volume model and the DLS diameter and is proportional to LNP volume.

In certain embodiments, the invention encompasses Ionizable Lipids of the Invention encompassed by the structure of Formula I:

Head-Spacer-Linker-Spacer-Tail   Formula I

wherein the pKa can be adjusted to effectuate targeted delivery to a specific tissue or organ of the body. One of ordinary skill will recognize that the Ionizable Lipids of the Invention of Formulas I-IX and specific embodiments thereof can be used for targeted delivery of LNPs.

In certain embodiments, the Ionizable Lipids of the Invention can be tuned to have a slight positive charge or be near neutral at physiological pH, so they do not systemically disseminate and are expressed locally, for example, in muscle and draining lymph nodes to generate a robust immune response through targeted delivery. In certain embodiments, localization of expression can increase potency and reduce systemic adverse events by avoiding off-target expression.

In certain embodiments, protonation and utilization of branched lipid tails in the Ionizable Lipids of the Invention for use in LNPs promote endosomal release, and LNP charge influencing targeting—a third SFR is the influence of LNP structure on its adjuvanticity. In certain embodiments, asymmetric Ionizable Lipids of the Invention in the LNP act as a strong Th2-biased adjuvant when delivered with protein subunit antigens and the LNP mRNA vaccines drive a Tfh-biased response that stimulates the proliferation of Tfh and germinal center B cells and a potent long-lived neutralizing antibody response.

In certain embodiments, the Ionizable Lipids of the Invention are designed with three structural features that are known to control delivery efficiency—ionization in the endosomal pH range, net charge at physiological pH, and lipid tail conformation related to branching and saturation/unsaturation. In certain embodiments, the approach to candidate evaluation and elucidating structure-function relationships (SFRs) includes in vitro and in vivo evaluation of translation and toxicity, in vitro assessment of cell uptake, endosomal release and innate immune sensor activation, in vivo characterization of distribution and cell trafficking, immunogenicity, and the use of current and evolving rodent and non-rodent animal models in viral challenge studies.

In certain embodiments, the Ionizable Lipids of the Invention possess increased in vivo expression (>5×) of mRNA LNPs due to increased mixing concentration of the lipids and mRNA during assembly.

In certain embodiments, the targeted delivery of LNPs including the Ionizable Lipids of the Invention involves: 1) Optimization of ionization properties of multivalent headgroups that can produce both a slightly positive or near neutral LNP at physiological pH to limit systemic dissemination and increase endosomal ionization that increases vaccine potency. In other embodiments, the targeted delivery of LNPs including Ionizable Lipids of the Invention involves highly branched and degradable lipid tails that can further increase potency through endosomal release. In other embodiments, the targeted delivery of LNPs including Ionizable Lipids of the Invention involves the charge and structure of the Ionizable Lipids of the Invention can influence LNP adjuvanticity.

In certain embodiments, the LNP potency is increased to limit adverse events, reduce manufacturing cost, and increase ability to vaccinate large population groups.

In certain embodiments, the invention encompasses a LNP delivery system that is superior to the current LNPs with protection at lower dose, lower reactogenicity, lower systemic distribution and greater stability in storage than the current LNPs in vaccines. In certain embodiments, first generation C24 LNPs were superior to MC3, the standard reference LNP in the field, in terms of all of these properties, and exceeded the published neutralizing titers of the vaccine in their preclinical study. In certain embodiments, head-to-head comparisons with the disclosed vaccines formulations using our latest second generation LNPs that are improvements over C24. In certain embodiments, the invention encompasses mechanisms-of-action to identify cell types and cell reactions involved in the vaccine response. In certain embodiments, by combining charge-mediated targeting to eliminate off-target liver expression and increase spleen targeting, with ligand-mediated targeting for specific cell uptake in T cells, we will precisely target delivery to splenic T cells. In certain embodiments, the systematic screening of a diverse and unique library of highly potent ionizable lipids allows identification of several other achievable cell-specific targets that will be optimized through charge- and ligand-mediated targeting combined with formulation and manufacturing process parameters.

In certain embodiments, the invention encompasses using 3 mRNA-encoded reporters, luciferase, mCherry and Cre-recombinase and one mRNA-encoded immunogen, the S2P SARS-Cov-2 immunogen. In certain embodiments, these constructs are sufficient to develop general principles and models of expression, targeting and toxicity of mRNA lipid nanoparticles. In certain embodiments, substitution of other RNA sequences, whether small (siRNA) or large (self-amplifying RNA, multivalent vaccines, gene-editing designs) will require relatively minor or moderate modifications to LNP formulations and manufacturing.

In certain embodiments, the invention encompasses methods to identify LNP features that predict the targeting performance attributes and provide methods to measure them. In certain embodiments, the improved manufacturing processes permit the identification of appropriate in process controls (IPCs) to ensure consistent manufacturing of high potency LNPs. In certain embodiments, the methods could be implemented on various systems requiring targeted delivery of a therapeutic or prophylactic agent.

In certain embodiments, the invention encompasses a novel library of ionizable lipids with systematic changes in structure and theoretical ionization properties. In certain embodiments, a series of project modules described herein allows the acquisition of the data sets that will permit the predictive models to relate performance determining properties of the LNPs to molecular, formulation and manufacturing parameters (multiple models for each arrow on the three far left boxes below).

In certain embodiments, some of the models are statistical involving machine learning approaches while some will be mechanistic. In certain embodiments, the performance of LNPs that have been characterized for features currently understood to influence performance (in middle box above and predicted as described above). In certain embodiments, the measured in vitro and in vivo performance include expression efficiency, organ- and cell-targeting, toxicity, degradability and immunogenicity (far right box above).

In certain embodiments, in vitro cell culture based characterization and in vivo characterization of performance in animal models predict LNP performance from LNP characteristics (arrow from central box to far right box above).

In certain embodiments, the determinants of mRNA-LNP expression and targeting in vivo include, but are not limited to LNP net charge (NCLNP), surface charge (SCLNP), endosomal protonation (EPLNP), size (DLNP), ionizable lipid membrane-disrupting ability (MDIL), ionizable lipid RNA-release ability (RRIL), targeting ligand density (ρL) and binding affinity (KL). In certain embodiments, these parameters are in turn determined by ionizable lipid ionization and structural properties as well as by formulation parameters (e.g., lipid types and mole ratios), manufacturing processes (e.g., concentrations, buffer, pH, solvent ratios, flow rates) and ligand conjugation. In certain embodiments, the models predict NCLNP, SCLNP, EPLNP, DLNP, MDIL, RRIL, ρL and KL from ionizable lipid properties, formulation parameters and manufacturing process parameters and a model to predict mRNA stability in LNPs.

In one embodiment, LNP net charge (NCLNP), surface charge (SCLNP), and endosomal protonation (EPLNP) prediction are derived from ionizable lipid structure and ionization properties. In one embodiment, the invention encompasses a method to theoretically predict LNP ionization properties from the molecular ionization constants of monoprotic ionizable lipids and extended to multiprotic ionizable lipids that have greater potential to control both endosomal protonation and net charge of the LNP.

In one embodiment, the structure of lipid tails is important for the delivery, release and expression efficiency of mRNA LNPs and is thought to be due to their endosomal membrane disrupting and RNA release abilities.

In certain embodiments, the invention encompasses methods to assess mRNA cleavage during storage in LNPs. In one embodiment, the ionizable lipid influences the rate of cleavage through the pH dependence of the kinetics of this reaction.

In one embodiment, LNP net charge (NCLNP), surface charge (SCLNP), endosomal protonation (EPLNP), size (DLNP), ionizable lipid membrane disrupting ability (MDIL) and RNA release ability (RRIL) prediction is obtained from LNP formulation. In one embodiment, LNP charge can be modified by changing formulation ratios, adding a 5th lipid and by designing specific ionization properties into the ionizable lipid. In one embodiment, different PEG lipid structures can also influence expression and targeting.

In one embodiment, methods of preparing the lipid solutions and mRNA solutions prior to mixing and self-assembly form LNPs that are more efficient delivery vehicles according to reporter expression in vitro and in vivo. In one embodiment, these novel solutions have higher concentrations of the lipids and mRNA during self-assembly and also specific methods for protonating the ionizable lipid. In one embodiment, a statistical machine learning model will relate manufacturing parameters to LNP characteristics.

In one embodiment, conjugated targeting ligands directly to intact LNPs show large increases in cell-specific targeting.

In other embodiments, for certain LNPs, ApoE adsorption is necessary for the cellular uptake of LNPs. In certain embodiments, decreased activity of LNPs with higher PEG-lipid content in hepatocytes resulted in reduced association of ApoE with LNPs. In certain embodiments, LNPs with 5% PEG-lipid have a lower binding affinity with ApoE compared to LNPs with 1.5% PEG-lipid. In certain embodiments, by introducing unique targeting ligands to the highly PEGylated LNPs, ApoE-mediated cellular uptake can be blocked and selective delivery of LNPs can be achieved.

In certain embodiments, mannose-conjugated nanoparticles have been investigated in various targeted delivery studies, and these nanoparticles have been proven to be safe. To introduce mannose moieties on the surface of LNPs, mannose-PEG lipid is used.

V.6. Lipid Nanoparticles Containing Ionizable Lipids of the Invention

In certain embodiments, the invention encompasses Lipid Nanoparticles including one or more Ionizable Lipids of the Invention. In certain embodiments, the LNPs include a second lipid. In certain embodiments, the LNP's include a steroid. In other embodiments, the LNP's include a pegylated lipid. In other embodiments, the LNP's include a nucleic acid, preferably mRNA.

As used herein the term “lipid nanoparticle,” also referred to as LNP, refers to a particle having at least one dimension on the order of nanometers (e.g., 1-1,000 nm) which includes Ionizable Lipid of the Invention, for example an Ionizable Lipid of the Invention encompassed by Formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), or (IX) or a pharmaceutically acceptable salt thereof. In some embodiments, such lipid nanoparticles comprise, for example an Ionizable Lipid of the Invention encompassed by Formula (I), (II), (III), (IV), (V), (VI), (VII), (VIII), or (IX) and one or more excipients selected from neutral lipids, charged lipids, steroids and polymer conjugated lipids (e.g., a pegylated lipid). In some embodiments, the nucleic acid, preferably mRNA, or a portion thereof, is encapsulated in the lipid portion of the lipid nanoparticle or an aqueous space enveloped by some or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects induced by the mechanisms of the host organism or cells e.g. an adverse immune response. In some embodiments, the mRNA or a portion thereof is associated with the lipid nanoparticles.

In the context of the present invention, lipid nanoparticles are not restricted to any particular morphology, and should be interpreted as to include any morphology generated when an Ionizable Lipid of the Invention encompassed by Formula (I), (II), (III) (IV), (V), (VI), (VII), (VIII), or (IX) and optionally one or more further lipids are combined, e.g. in an aqueous environment and/or in the presence of a nucleic acid compound. For example, a liposome, a lipid complex, a lipoplex and the like are within the scope of a lipid nanoparticle.

In various embodiments, the lipid nanoparticles have a mean diameter of from about 30 nm to about 150 nm, from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, from about 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm, and are substantially non-toxic. In certain embodiments, the mRNA, when present in the lipid nanoparticles, is resistant in aqueous solution to degradation with a nuclease. As used herein, the mean diameter may be represented by the number-weighted average as determined by dynamic light scattering.

An LNP may comprise any Ionizable Lipid of the Invention encompassed by Formula (I), (II), (III) (IV), (V), (VI), (VII), (VIII), and (IX) capable of forming a particle to which the one or more nucleic acid molecules are attached, or in which the one or more nucleic acid molecules are encapsulated. The term “lipid” refers to a group of organic compounds that are derivatives of fatty acids (e.g., esters) and are generally characterized by being insoluble in water but soluble in many organic solvents. Lipids are usually divided in at least three classes: (1) “simple lipids” which include fats and oils as well as waxes; (2) “compound lipids” which include phospholipids and glycolipids; and (3) “derived lipids” such as steroids.

In one embodiment, the mRNA-comprising LNP comprises one or more Ionizable Lipids of the Invention encompassed by Formula (I), (II), (III) (IV), (V), (VI), (VII), (VIII), or (IX) as defined herein, and one or more stabilizing lipids. Stabilizing lipids include neutral lipids and pegylated lipids.

As mentioned, the LNP comprises for example an Ionizable Lipid of the Invention encompassed by Formula (I), (II), (III) (IV), (V), (VI), (VII), (VIII), or (IX). In certain embodiments, the Ionizable Lipids of the Invention are preferably cationisable, (i.e., it becomes protonated as the pH is lowered below the pKa of the ionizable group of the lipid), but is progressively more neutral at higher pH values. In certain embodiments, when positively charged, the lipid is then able to associate with negatively charged nucleic acids. In certain embodiments, the Ionizable Lipid of the Invention comprises a zwitterionic lipid that assumes a positive charge on pH decrease. The LNP may comprise any lipid capable of forming a particle to which the one or more nucleic acid molecules are attached, or in which the one or more nucleic acid molecules are encapsulated.

In certain embodiments, the LNP may comprise a further cationic or cationisable lipid, (i.e., any of a number of lipid species which carry a net positive charge at a selective pH) such as physiological pH. Examples of such lipids include, but are not limited to, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC); N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA); N,N-distearyl-N,N-dimethylammonium bromide (DDAB); N-(2,3dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP); 3-(N—(N′,N′dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N-(1-(2,3-dioleoyloxy)propyl)N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoracetate (DOSPA), dioctadecylamidoglycyl carboxyspermine (DOGS), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), N,N-dimethyl-2,3-dioleoyloxy)propylamine (DODMA), and N-(1,2dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide (DMRIE).

Additionally, a number of commercial preparations of lipids are available which can be used in the present invention. These include, for example, LIPOFECTIN® (commercially available cationic liposomes comprising DOTMA and 1,2-dioleoyl-sn-3phosphoethanolamine (DOPE), from GIBCO/BRL, Grand Island, N.Y.); LIPOFECTAMINE® (commercially available cationic liposomes comprising N-(1-(2,3dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA) and (DOPE), from GIBCO/BRL); and TRANSFECTAM® (commercially available cationic lipids comprising dioctadecylamidoglycyl carboxyspermine (DOGS) in ethanol from Promega Corp., Madison, Wis.). The following lipids are cationic and have a positive charge at below physiological pH: DODAP, DODMA, DMDMA, 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA).

In further embodiment, the further lipid is an amino lipid. Suitable amino lipids useful in the invention include those described in WO2012/016184, incorporated herein by reference in its entirety. Representative amino lipids include, but are not limited to, 1,2-dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyoxy-3morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyloxy-3dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), 3-(N,Ndilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), and 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA).

In certain embodiments, the LNP comprises one or more additional lipids which stabilize the formation of particles during their formation.

Suitable stabilizing lipids include neutral lipids and anionic lipids. The term “neutral lipid” refers to any one of a number of lipid species that exist in either an uncharged or neutral zwitterionic form at physiological pH. Representative neutral lipids include diacylphosphatidylcholines, diacylphosphatidylethanolamines, ceramides, sphingomyelins, dihydro sphingomyelins, cephalins, and cerebrosides.

Exemplary neutral lipids include, for example, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-lcarboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearioyl-2-oleoylphosphatidyethanol amine (SOPE), and 1,2-dielaidoyl-sn-glycero-3-phophoethanolamine (transDOPE). In one embodiment, the neutral lipid is 1,2-distearoyl-sn-glycero-3phosphocholine (DSPC).

In some embodiments, the LNPs comprise a neutral lipid selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM. In various embodiments, the molar ratio of the Ionizable Lipid of the Invention (e.g., lipid encompassed by the structure of Formula (I), (II) (III), (IV), (V), (VI), or (VII)) to the neutral lipid ranges from about 2:1 to about 8:1. In certain embodiments, the molar ration is 2:1, 2.5:1, 3:1, 3.5:1, 4:1; 4.5:1, 5:1, 5.5:1, 6:1, 6.5:1, 7:1; 7.5:1; 8:1; 8.5:1, 9:1, 9.5:1; 10:1; 11:1; 15:1; 20:1; 30:1; 50:1, and all molar ratios included within these ranges.

In various embodiments, the LNPs further comprise a steroid or steroid analogue.

In certain embodiments, the steroid or steroid analogue is cholesterol. In some of these embodiments, the molar ratio of the Ionizable Lipids of the Invention (e.g., lipids encompassed by the structure of Formula (I), (II), (III), (IV), (V), (VI), or (VII)) to steroid ranges from about 5:1 to 1:1. In certain embodiments, the molar ration is 2:1, 2.5:1, 3:1, 3.5:1, 4:1; 4.5:1, 5:1, 5.5:1, 6:1, 6.5:1, 7:1; 7.5:1; 8:1; 8.5:1, 9:1, 9.5:1; 10:1; 11:1; 15:1; 20:1; 30:1; 50:1, and all molar ratios included within these ranges.

The term “anionic lipid” refers to any lipid that is negatively charged at physiological pH. These lipids include phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, Ndodecanoylphosphatidylethanolamines, N-succinylphosphatidylethanolamines, Nglutarylphosphatidylethanolamines, lysylphosphatidylglycerols, palmitoyloleyolphosphatidylglycerol (POPG), and other anionic modifying groups joined to neutral lipids.

In certain embodiments, the LNP comprises glycolipids (e.g., monosialoganglioside GM1).

In some embodiments, the LNPs comprise a polymer conjugated lipid. The term “polymer conjugated lipid” refers to a molecule comprising both a lipid portion and a polymer portion. An example of a polymer conjugated lipid is a pegylated lipid. The term “pegylated lipid” refers to a molecule comprising both a lipid portion and a polyethylene glycol portion. Pegylated lipids are known in the art and include 1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-s-DMG) and the like.

In certain embodiments, the LNP comprises an additional, stabilizing-lipid which is a polyethylene glycol-lipid (pegylated lipid). Suitable polyethylene glycollipids include PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramides (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols. Representative polyethylene glycol-lipids include PEG-c-DOMG, PEG-c-DMA, and PEG-s-DMG. In one embodiment, the polyethylene glycol-lipid is N-[(methoxy poly(ethylene glycol)2000)carbamyl]-1,2-dimyristyloxlpropyl-3-amine (PEG-c-DMA). In one embodiment, the polyethylene glycol-lipid is PEG-c-DOMG). In other embodiments, the LNPs comprise a pegylated diacylglycerol (PEG-DAG) such as 1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG), a pegylated phosphatidylethanoloamine (PEG-PE), a PEG succinate diacylglycerol (PEG-S-DAG) such as 4-O-(2′,3′-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), a pegylated ceramide (PEG-cer), or a PEG dialkoxypropylcarbamate such as o-methoxy(polyethoxy)ethyl-N-(2,3di(tetradecanoxy)propyl)carbamate or 2,3-di(tetradecanoxy)propyl-N-(ω-methoxy(polyethoxy)ethyl)carbamate. In various embodiments, the molar ratio of the cationic lipid to the pegylated lipid ranges from about 400:1 to about 10:1.

As mentioned, the mRNA comprising lipid nanoparticle may comprise a pegylated lipid having the following structure:

or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein:

R 8 and R 9 are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds; and w has mean value ranging from 30 to 60.

In some of the foregoing embodiments of the pegylated lipid, R 8 and R 9 are not both n-octadecyl when w is 42. In some other embodiments, R 8 and R 9 are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 18 carbon atoms. In some embodiments, R 8 and R 9 are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 12 to 16 carbon atoms. In some embodiments, R 8 and R 9 are each independently a straight or branched, saturated or unsaturated alkyl chain containing 12 carbon atoms. In some embodiments, R 8 and R 9 are each independently a straight or branched, saturated or unsaturated alkyl chain containing 14 carbon atoms. In other embodiments, R 8 and R 9 are each independently a straight or branched, saturated or unsaturated alkyl chain containing 16 carbon atoms. In still more embodiments, R 8 and R 9 are each independently a straight or branched, saturated or unsaturated alkyl chain containing 18 carbon atoms. In still other embodiments, R 8 is a straight or branched, saturated or unsaturated alkyl chain containing 12 carbon atoms and R 9 is a straight or branched, saturated or unsaturated alkyl chain containing 14 carbon atoms.

In various embodiments, w spans a range that is selected such that the PEG portion has an average molecular weight of about 400 to about 6000 g/mol. In some embodiments, the average w is about 50.

In certain embodiments, the PEG lipid is present in the LNP in an amount from about 1 to about 10 mole percent, relative to the total lipid content of the nanoparticle. In one embodiment, the PEG lipid is present in the LNP in an amount from about 1 to about 5 mole percent. In one embodiment, the PEG lipid is present in the LNP in about 1 mole percent or about 1.5 mole percent.

In certain embodiments, the LNP comprises one or more targeting moieties which are capable of targeting the LNP to a cell or cell population. For example, in one embodiment, the targeting moiety is a ligand which directs the LNP to a receptor found on a cell surface.

In certain embodiments, the LNP comprises one or more internalization domains. For example, in one embodiment, the LNP comprises one or more domains which bind to a cell to induce the internalization of the LNP. For example, in one embodiment, the one or more internalization domains bind to a receptor found on a cell surface to induce receptor-mediated uptake of the LNP. In certain embodiments, the LNP is capable of binding a biomolecule in vivo, where the LNP-bound biomolecule can then be recognized by a cell-surface receptor to induce internalization. For example, in one embodiment, the LNP binds systemic ApoE, which leads to the uptake of the LNP and associated cargo.

Other exemplary LNPs and their manufacture are described in the art, for example in U.S. Patent Application Publication No. US20120276209, Semple et al., 2010, Nat Biotechnol., 28(2):172-176; Akinc et al., 2010, Mol Ther., 18(7): 1357-1364; Basha et al., 2011, Mol Ther, 19(12): 2186-2200; Leung et al., 2012, J Phys Chem C Nanomater Interfaces, 116(34): 18440-18450; Lee et al., 2012, Int J Cancer., 131(5): E781-90; Belliveau et al., 2012, Mol Ther nucleic Acids, 1: e37; Jayaraman et al., 2012, Angew Chem Int Ed Engl., 51(34): 8529-8533; Mui et al., 2013, Mol Ther Nucleic Acids. 2, e139; Maier et al., 2013, Mol Ther., 21(8): 1570-1578; and Tam et al., 2013, Nanomedicine, 9(5): 665-74, each of which are incorporated by reference in their entirety.

In preferred embodiments, the lipid nanoparticles have a mean diameter of from about 30 nm to about 150 nm, from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, from about 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm, and are substantially non-toxic. As mentioned, the mean diameter may correspond to the number-weighted average as determined by dynamic light scattering.

In another preferred embodiment of the invention the lipid nanoparticles have a hydrodynamic diameter in the range from about 50 nm to about 300 nm, or from about 60 nm to about 250 nm, from about 60 nm to about 150 nm, or from about 60 nm to about 120 nm, respectively.

In certain embodiments, the mRNA, when present in the lipid nanoparticles, is resistant in aqueous solution to degradation with a nuclease.

The total amount of mRNA in the lipid nanoparticles varies and may be defined depending on the mRNA to total lipid w/w ratio. In certain embodiments, the nanoparticle ratio is from 2 to 10. In one embodiment of the invention, the mRNA to total lipid ratio is less than 0.06 w/w, preferably between 0.03 and 0.04 w/w.

In some embodiments, the LNPs comprise an Ionizable Lipid of the Invention of encompassed by any of the structures of Formula (I), (II), (III), (IV), (V), (VI), or (VII); a mRNA compound as defined above, a neutral lipid, a steroid and a pegylated lipid.

In certain embodiments, the LNP comprises one or more targeting moieties which are capable of targeting the LNP to a cell or cell population. For example, in one embodiment, the targeting moiety is a ligand which directs the LNP to a receptor found on a cell surface.

In certain embodiments, the LNP comprises one or more internalization domains. For example, in one embodiment, the LNP comprises one or more domains which bind to a cell to induce the internalization of the LNP. For example, in one embodiment, the one or more internalization domains bind to a receptor found on a cell surface to induce receptor-mediated uptake of the LNP. In certain embodiments, the LNP is capable of binding a biomolecule in vivo, where the LNP-bound biomolecule can then be recognized by a cell-surface receptor to induce internalization. For example, in one embodiment, the LNP binds systemic ApoE, which leads to the uptake of the LNP and associated cargo.

In particular the invention relates to the following non-limiting specific embodiments.

In a preferred embodiment, the invention relates to a mRNA comprising lipid nanoparticle comprising a Ionizable Lipid of the Invention according to formula (I), (II), (II), (IV), (V), (VI), or (VII) as defined above and a mRNA compound comprising a mRNA sequence encoding at least one antigenic peptide or protein, and DSPC, cholesterol and a PEG lipid at a ratio of about 50:10:38.5:1.

In a specific preferred embodiment the invention relates to a mRNA comprising lipid nanoparticle, comprising a Ionizable Lipid of the Invention, a PEG-lipid, a mRNA compound comprising a mRNA sequence encoding at least one antigenic peptide or protein, a steroid and a neutral lipid, wherein preferably, at a ratio of about 50:10:38.5:1.5 that encapsulates unmodified, 1-methylpseudouridine modified or codon-optimized mRNA. Preferably the antigenic peptide or protein is derived from pathogenic antigens, tumor antigens, allergenic antigens or autoimmune self-antigens or a fragment or variant thereof.

In certain embodiments, the invention encompasses a 50-100 nm diameter LNP including the Ionizable Lipids of the Invention composed of the nucleic acid and 4 lipids: an Ionizable Lipids of the Invention with an amine group (50%), a second lipid (e.g., 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)) (10%), a steroid (e.g., cholesterol) (38.5%), and a pegylated lipid (e.g., 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000)) (1.5%) (mole ratios in parentheses).

In certain embodiments, the amount of the Ionizable Lipid of the Invention included in the LNP is 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4. 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4. 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.10, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, 12, 12.1, 12.2, 12.3, 12.4. 12.5, 12.6, 12.7, 12.8, 12.9, 13, 13.1, 13.2, 13.3, 13.4, 13.5, 13.6, 13.7, 13.8, 13.9, 14, 14.1, 14.2, 14.3, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9, 15, 15.1, 15.2, 15.3, 15.4, 15.5, 15.6, 15.7, 15.8, 15.9, 16, 16.1, 16.2, 16.3, 16.4. 16.5, 16.6, 16.7, 16.8, 16.9, 17, 17.1, 17.2, 17.3, 17.4, 17.5, 17.6, 17.7, 17.8, 17.9, 18, 18.1, 18.2, 18.3, 18.4, 18.5, 18.6, 18.7, 18.8, 18.9, 19, 19.1, 19.2, 19.3, 19.4, 19.5, 19.6, 19.7, 19.8, 19.9, 20, 21.1, 21.2, 21.3, 21.4, 21.5, 21.6, 21.7, 21.8, 21.9, 30, 31, 31.1, 31.2, 31.3, 31.4, 31.5, 31.6, 31.7, 31.8, 31.9, 32, 32.1, 32.2, 32.3, 32.4. 32.5, 32.6, 32.7, 32.8, 32.9, 33, 33.1, 33.2, 33.3, 33.4, 33.5, 33.6, 33.7, 33.8, 33.9, 34, 34.1, 34.2, 34.3, 34.4, 34.5, 34.6, 34.7, 34.8, 34.9, 35, 35.1, 35.2, 35.3, 35.4, 35.5, 35.6, 35.7, 35.8, 35.9, 36, 36.1, 36.2, 36.3, 36.4. 36.5, 36.6, 36.7, 36.8, 36.9, 37, 37.1, 37.2, 37.3, 37.4, 37.5, 37.6, 37.7, 37.8, 37.9, 38, 38.1, 38.2, 38.3, 38.4, 38.5, 38.6, 38.7, 38.8, 38.9, 39, 39.1, 39.2, 39.3, 39.4, 39.5, 39.6, 39.7, 39.8, 39.9, 40, 40.1, 40.2, 40.3, 40.4, 40.5, 40.6, 40.7, 40.8, 40.9, 41, 41.1, 41.2, 41.3, 41.4, 41.5, 41.6, 41.7, 41.8, 41.9, 42, 42.1, 42.2, 42.3, 42.4. 42.5, 42.6, 42.7, 42.8, 42.9, 43, 43.1, 43.2, 43.3, 43.4, 43.5, 43.6, 43.7, 43.8, 43.9, 44, 44.1, 44.2, 44.3, 44.4, 44.5, 44.6, 44.7, 44.8, 44.9, 45, 45.1, 45.2, 45.3, 45.4, 45.5, 45.6, 45.7, 45.8, 45.9, 46, 46.1, 46.2, 46.3, 46.4. 46.5, 46.6, 46.7, 46.8, 46.9, 47, 47.1, 47.2, 47.3, 47.4, 47.5, 47.6, 47.7, 47.8, 47.9, 48, 48.1, 48.2, 48.3, 48.4, 48.5, 48.6, 48.7, 48.8, 48.9, 49, 49.1, 49.2, 49.3, 49.4, 49.5, 49.6, 49.7, 49.8, 49.9, 50, 55, 60, 65, 70, or 75 molar percent.

In certain embodiments, the amount of the second lipid included in the LNP is 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4. 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4. 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.10, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, 12, 12.1, 12.2, 12.3, 12.4. 12.5, 12.6, 12.7, 12.8, 12.9, 13, 13.1, 13.2, 13.3, 13.4, 13.5, 13.6, 13.7, 13.8, 13.9, 14, 14.1, 14.2, 14.3, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9, 15, 15.1, 15.2, 15.3, 15.4, 15.5, 15.6, 15.7, 15.8, 15.9, 16, 16.1, 16.2, 16.3, 16.4. 16.5, 16.6, 16.7, 16.8, 16.9, 17, 17.1, 17.2, 17.3, 17.4, 17.5, 17.6, 17.7, 17.8, 17.9, 18, 18.1, 18.2, 18.3, 18.4, 18.5, 18.6, 18.7, 18.8, 18.9, 19, 19.1, 19.2, 19.3, 19.4, 19.5, 19.6, 19.7, 19.8, 19.9, 20, 21.1, 21.2, 21.3, 21.4, 21.5, 21.6, 21.7, 21.8, 21.9, 30, 31, 31.1, 31.2, 31.3, 31.4, 31.5, 31.6, 31.7, 31.8, 31.9, 32, 32.1, 32.2, 32.3, 32.4. 32.5, 32.6, 32.7, 32.8, 32.9, 33, 33.1, 33.2, 33.3, 33.4, 33.5, 33.6, 33.7, 33.8, 33.9, 34, 34.1, 34.2, 34.3, 34.4, 34.5, 34.6, 34.7, 34.8, 34.9, 35, 35.1, 35.2, 35.3, 35.4, 35.5, 35.6, 35.7, 35.8, 35.9, 36, 36.1, 36.2, 36.3, 36.4. 36.5, 36.6, 36.7, 36.8, 36.9, 37, 37.1, 37.2, 37.3, 37.4, 37.5, 37.6, 37.7, 37.8, 37.9, 38, 38.1, 38.2, 38.3, 38.4, 38.5, 38.6, 38.7, 38.8, 38.9, 39, 39.1, 39.2, 39.3, 39.4, 39.5, 39.6, 39.7, 39.8, 39.9, or 40 mole percent.

In certain embodiments, the amount of the steroid included in the LNP is 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4. 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4. 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.10, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, 12, 12.1, 12.2, 12.3, 12.4. 12.5, 12.6, 12.7, 12.8, 12.9, 13, 13.1, 13.2, 13.3, 13.4, 13.5, 13.6, 13.7, 13.8, 13.9, 14, 14.1, 14.2, 14.3, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9, 15, 15.1, 15.2, 15.3, 15.4, 15.5, 15.6, 15.7, 15.8, 15.9, 16, 16.1, 16.2, 16.3, 16.4. 16.5, 16.6, 16.7, 16.8, 16.9, 17, 17.1, 17.2, 17.3, 17.4, 17.5, 17.6, 17.7, 17.8, 17.9, 18, 18.1, 18.2, 18.3, 18.4, 18.5, 18.6, 18.7, 18.8, 18.9, 19, 19.1, 19.2, 19.3, 19.4, 19.5, 19.6, 19.7, 19.8, 19.9, 20, 21.1, 21.2, 21.3, 21.4, 21.5, 21.6, 21.7, 21.8, 21.9, 30, 31, 31.1, 31.2, 31.3, 31.4, 31.5, 31.6, 31.7, 31.8, 31.9, 32, 32.1, 32.2, 32.3, 32.4. 32.5, 32.6, 32.7, 32.8, 32.9, 33, 33.1, 33.2, 33.3, 33.4, 33.5, 33.6, 33.7, 33.8, 33.9, 34, 34.1, 34.2, 34.3, 34.4, 34.5, 34.6, 34.7, 34.8, 34.9, 35, 35.1, 35.2, 35.3, 35.4, 35.5, 35.6, 35.7, 35.8, 35.9, 36, 36.1, 36.2, 36.3, 36.4. 36.5, 36.6, 36.7, 36.8, 36.9, 37, 37.1, 37.2, 37.3, 37.4, 37.5, 37.6, 37.7, 37.8, 37.9, 38, 38.1, 38.2, 38.3, 38.4, 38.5, 38.6, 38.7, 38.8, 38.9, 39, 39.1, 39.2, 39.3, 39.4, 39.5, 39.6, 39.7, 39.8, 39.9, 40, 40.1, 40.2, 40.3, 40.4, 40.5, 40.6, 40.7, 40.8, 40.9, 41, 41.1, 41.2, 41.3, 41.4, 41.5, 41.6, 41.7, 41.8, 41.9, 42, 42.1, 42.2, 42.3, 42.4. 42.5, 42.6, 42.7, 42.8, 42.9, 43, 43.1, 43.2, 43.3, 43.4, 43.5, 43.6, 43.7, 43.8, 43.9, 44, 44.1, 44.2, 44.3, 44.4, 44.5, 44.6, 44.7, 44.8, 44.9, 45, 45.1, 45.2, 45.3, 45.4, 45.5, 45.6, 45.7, 45.8, 45.9, 46, 46.1, 46.2, 46.3, 46.4. 46.5, 46.6, 46.7, 46.8, 46.9, 47, 47.1, 47.2, 47.3, 47.4, 47.5, 47.6, 47.7, 47.8, 47.9, 48, 48.1, 48.2, 48.3, 48.4, 48.5, 48.6, 48.7, 48.8, 48.9, 49, 49.1, 49.2, 49.3, 49.4, 49.5, 49.6, 49.7, 49.8, 49.9, 50, 55, 60, 65, 70, or 75 mole percent.

In certain embodiments, the amount of the pegylated lipid included in the LNP is 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4. 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4. 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.10, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, 12, 12.1, 12.2, 12.3, 12.4. 12.5, 12.6, 12.7, 12.8, 12.9, 13, 13.1, 13.2, 13.3, 13.4, 13.5, 13.6, 13.7, 13.8, 13.9, 14, 14.1, 14.2, 14.3, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9, 15, 15.1, 15.2, 15.3, 15.4, 15.5, 15.6, 15.7, 15.8, 15.9, 16, 16.1, 16.2, 16.3, 16.4. 16.5, 16.6, 16.7, 16.8, 16.9, 17, 17.1, 17.2, 17.3, 17.4, 17.5, 17.6, 17.7, 17.8, 17.9, 18, 18.1, 18.2, 18.3, 18.4, 18.5, 18.6, 18.7, 18.8, 18.9, 19, 19.1, 19.2, 19.3, 19.4, 19.5, 19.6, 19.7, 19.8, 19.9, or 20 mole percent.

In certain embodiments, the hydrophilic DMG-PEG forms the shell of the LNP. The amount of nucleic acid is represented by the NP mole ratio of the amine on the ionizable lipid to the phosphate groups on the nucleic acid backbone and is typically 3-6. In certain embodiments, the pKa of the LNP to be in the 6-7 range corresponding to the pH in the early endosome. A link between the pKa of the ionizable lipid in the LNP and gene silencing efficiency showed an LNP pKa in the range 6-7 produced more silencing for the ionizable lipid DLinDMA and was associated with promoting lipid structures that could disrupt the membrane of the endosome. The pKa of the LNP was measured using the pH-dependence of fluorescence enhancement of the anionic dye TNS. In certain embodiments, the pKa-dependent endosomal release mechanism was described as an ion-pairing event between the cationic protonated ionizable lipid and anionic endosomal phospholipids where a divergent cone-shape lipid tail of the ionizable lipid facilitates membrane rupture.

In particular preferred embodiments, the lipid nanoparticle includes a mRNA included in the lipid nanoparticle, wherein preferably the antigenic peptide or protein is derived from hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), matrix protein 1 (M1), matrix protein 2 (M2), non-structural protein 1 (NS1), non-structural protein 2 (NS2), nuclear export protein (NEP), polymerase acidic protein (PA), polymerase basic protein PB1, PB1-F2, or polymerase basic protein 2 (PB2) of an influenza virus or a fragment or variant thereof. More preferably the antigenic peptide or protein is derived from hemagglutinin (HA) or neuraminidase (NA) of an influenza virus or a fragment or variant thereof. Even more preferably the antigenic peptide or protein is at least one full-length protein of hemagglutinin (HA) and/or at least one full-length protein of neuraminidase (NA) of an influenza virus or a variant thereof. In a further preferred embodiment the influenza virus is selected from an influenza A, B or C virus. In a particularly preferred embodiment the influenza A virus is selected from an influenza virus characterized by a hemagglutinin (HA) selected from the group consisting of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17 and H18 and/or the influenza A virus is selected from an influenza virus characterized by a neuraminidase (NA) selected from the group consisting of N1, N2, N3, N4, N5, N6, N7, N8, N9, N10, and N11. Preferably, the influenza A virus is selected from the group consisting of H1N1, H1N2, H2N2, H3N1, H3N2, H3N8, H5N1, H5N2, H5N3, H5N8, H5N9, H7N1, H7N2, H7N3, H7N4, H7N7, H7N9, H9N2, and H10N7, preferably from H1N1, H3N2, H5N1. Most preferably, the mRNA sequence comprises at least one coding region encoding at least one antigenic peptide or protein derived from hemagglutinin (HA) of an influenza virus or a fragment or variant thereof and at least one antigenic peptide or protein derived from neuraminidase (NA) of an influenza virus or a fragment or variant thereof. In a specifically preferred embodiment the mRNA sequence comprises at least one coding region encoding at least one antigenic peptide or protein derived from hemagglutinin (HA) and/or at least one antigenic peptide or protein derived from neuraminidase (NA) of an influenza A virus selected from the group consisting of H1N1, H1N2, H2N2, H3N1, H3N2, H3N8, H5N1, H5N2, H5N3, H5N8, H5N9, H7N1, H7N2, H7N3, H7N4, H7N7, H7N9, H9N2, and H10N7, preferably from H1N1, H3N2, H5N1 or a fragment or variant thereof.

V.7. Methods of Making the LNPs of the Invention

The invention further relates to a method of preparing lipid nanoparticles comprising the steps of:

(i) combining (a) an Ionizable Lipid of the Invention as defined above or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, (b) a PEG lipid; (c) at least one mRNA compound comprising an mRNA sequence encoding at least one antigenic peptide or protein; and (d) optionally a steroid; and (e) optionally a neutral lipid; (ii) solubilizing the Ionizable lipid and/or the PEG lipid and optionally the neutral lipid and/or the steroid or a steroid derivative in ethanol; (iii) mixing the ethanolic lipid solution with an aqueous solution comprising the mRNA polynucleotide (iv) removing the ethanol to form lipid nanoparticles encapsulating or associating with the mRNA polynucleotide; and optionally; (v) pH adjustment through dilution, dialysis, or filtration; (vi) separating or purifying the lipid nanoparticles.

The ethanol may be removed by any suitable method which does not negatively affect the lipids or the forming lipid nanoparticles. In one embodiment of the invention the ethanol is removed by dialysis. In an alternative embodiment the ethanol is removed by diafiltration.

Separation and optimal purification of the lipid nanoparticles might also be performed by any suitable method. Preferably the lipid nanoparticles are filtrated, more preferably the lipid nanoparticles are separated or purified by filtration through a sterile filter.

V.8. Pharmaceutical Compositions of the Invention

The invention also encompasses pharmaceutical compositions and/or vaccines comprising an Ionizable Lipid of the Invention and a nucleic acid.

In a preferred embodiment, the pharmaceutical composition or the vaccine according to the invention comprising mRNA comprises lipid nanoparticles, which have a molar ratio of approximately 50:10:38.5:1.5, preferably 47.5:10:40.8:1.7 or more preferably 47.4:10:40.9:1.7 (i.e., proportion (mol %) of Ionizable Lipid of the Invention, DSPC, cholesterol and PEG-lipid; solubilized in ethanol). In another embodiment, the molar ratio is 48:12:38:2.

The invention further relates to a pharmaceutical composition comprising at least one lipid nanoparticle according to the present invention. In certain embodiments, the lipid nanoparticle comprises an mRNA compound comprising a sequence encoding at least one antigenic peptide or protein as defined herein.

In one embodiment of the invention the mRNA sequence encodes one antigenic peptide or protein. In an alternative embodiment of the invention the mRNA sequence encodes more than one antigenic peptide or protein.

In one embodiment of the invention, the pharmaceutical composition comprises a lipid nanoparticle according to the invention, wherein the lipid nanoparticle comprises more than one mRNA compounds, which each comprise a different mRNA sequence encoding an antigenic peptide or protein.

In an alternative embodiment of the invention the pharmaceutical composition comprises a second lipid nanoparticle, wherein the mRNA compound comprised by the second lipid nanoparticle is different from the mRNA compound comprised by the first lipid nanoparticle.

In a further aspect, the present invention concerns a composition comprising mRNA comprising lipid nanoparticles wherein the mRNA comprises an mRNA sequence comprising at least one coding region as defined herein and a pharmaceutically acceptable carrier. The composition according to the invention is preferably provided as a pharmaceutical composition or as a vaccine.

According to a preferred embodiment, the pharmaceutical composition or the vaccine according to the invention comprises mRNA comprising lipid nanoparticles comprising at least one mRNA comprising at least one mRNA sequence as defined above, wherein the at least one coding region of the at least one mRNA sequence encodes at least one antigenic peptide or protein preferably derived from a protein of an influenza virus or Rabies virus, preferably any one of the hemagglutinin (HA) or neuraminidase (NA) proteins or glycoproteins, or a fragment or variant of any one of these proteins.

Preferably, the pharmaceutical composition or the vaccine according to the invention comprises mRNA comprising lipid nanoparticles comprising at least one mRNA comprising at least one mRNA sequence as defined above, wherein the at least one coding sequence of the at least one mRNA sequence comprises or consists of a nucleic acid sequence encoding at least one antigenic peptide or protein preferably derived from a protein of an influenza virus, preferably any one of the hemagglutinin (HA) or neuraminidase (NA) proteins, or a fragment or variant thereof, wherein the protein derived from a protein of an influenza virus preferably comprises or consists of any one of the amino acid sequences or a fragment or variant of any one of these sequences. Alternatively, the antigenic peptide or protein is derived from a Rabies virus, preferably from glycoprotein of a Rabies virus, or a fragment or variant of any one of these sequences.

Preferably, the pharmaceutical composition or the vaccine according to the invention comprises mRNA comprising lipid nanoparticles comprising at least one mRNA comprising at least one mRNA sequence as defined above, wherein the at least one coding sequence of the mRNA sequence comprises or consists of a nucleic acid sequence encoding at least one antigenic peptide or protein derived from a protein of an influenza virus or Rabies virus, or a fragment or variant thereof, wherein the antigenic peptide or protein derived from a protein of an influenza virus or Rabies virus.

The pharmaceutical composition or vaccine according to the invention may thus comprise mRNA comprising lipid nanoparticles comprising at least one mRNA comprising at least one mRNA sequence comprising at least one coding region, encoding at least one antigenic peptide or protein, preferably derived from a protein of an influenza virus or Rabies virus or a fragment or variant thereof, wherein the at least one coding region of the at least one mRNA sequence encodes one specific antigenic peptide or protein e.g. derived from a protein of an influenza virus defined herein or a fragment or a variant thereof.

Alternatively, the pharmaceutical composition or vaccine of the present invention may comprise mRNA comprising lipid nanoparticles comprising at least one mRNA compound comprising at least one mRNA sequence according to the invention, wherein the at least one mRNA sequence encodes at least two, three, four, five, six, seven, eight, nine, ten, eleven or twelve distinct antigenic peptides or proteins e.g. derived from a protein of an influenza virus as defined herein or a fragment or variant thereof.

In this context it is particularly preferred that the at least one mRNA compound comprised in the pharmaceutical composition or vaccine is a bi- or multicistronic mRNA, which encodes the at least two, three, four, five, six, seven, eight, nine, ten, eleven or twelve distinct antigenic peptides or proteins e.g. derived from a protein of an influenza virus. Mixtures between these embodiments are also envisaged, such as compositions comprising more than one mRNA sequence, wherein at least one mRNA sequence may be monocistronic, while at least one other mRNA sequence may be bi- or multicistronic.

The pharmaceutical composition or vaccine according to the present invention, preferably the at least one coding sequence of the mRNA sequence comprised therein, may thus comprise any combination of the nucleic acid sequences as defined herein.

Preferably, the pharmaceutical composition or vaccine comprises mRNA comprising lipid nanoparticle comprising a plurality or more than one of the mRNA sequences according to the invention, wherein each mRNA sequence comprises at least one coding region encoding at least one antigenic peptide or protein derived from a protein of an influenza virus or a fragment or variant thereof.

In a particularly preferred embodiment the composition comprises at least 2, 3, 4, 5, 6, 7, 6, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, or 100 different mRNA sequences each encoding at least one antigenic peptide or protein, for example, derived from a protein of an influenza virus or a fragment or variant thereof as defined above, for example, derived from hemagglutinin (HA) or neuraminidase (NA) of an influenza virus or a fragment or variant thereof.

In another embodiment the composition comprises 4 different mRNA sequences each encoding at least one antigenic peptide or protein preferably derived from a protein of an influenza virus or a fragment or variant thereof as defined above, for example, derived from hemagglutinin (HA) or neuraminidase (NA) of an influenza virus or a fragment or variant thereof.

In this context it is particularly preferred that each mRNA sequence encodes at least one different antigenic peptide or protein derived from proteins of the same pathogen, e.g. influenza virus, wherein it is particularly preferred that the antigenic peptide or protein is derived from different proteins of the same pathogen, e.g. influenza virus. Preferably the composition comprises at least two mRNA sequences, wherein at least one mRNA sequence encodes at least one antigenic peptide or protein derived from hemagglutinin (HA) of the influenza virus and at least one mRNA sequence encodes at least one antigenic peptide or protein derived from neuraminidase (NA) of the same influenza virus.

In another preferred embodiment each mRNA sequence encodes at least one different antigenic peptide or protein derived from proteins of different pathogens, e.g. influenza viruses. Preferably each mRNA sequence encodes at least one antigenic peptide or protein derived from hemagglutinin (HA) and/or neuraminidase (NA) of different influenza viruses.

Preferably, the pharmaceutical composition or vaccine according to the invention comprises a plurality of mRNA sequences each encoding at least one antigenic peptide or protein derived from hemagglutinin (HA) and/or neuraminidase (NA) of an influenza virus, wherein at least one antigenic peptide or protein derived from hemagglutinin (HA) and/or neuraminidase (NA) of 2, 3, 4, 5, 6, 7, 6, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, or 100 different influenza viruses are encoded by the plurality of mRNA sequences.

In this context it is particularly preferred that the pharmaceutical composition or vaccine comprises at least one mRNA comprising lipid nanoparticle comprising a mRNA compound comprising a mRNA sequence encoding at least one antigenic peptide or protein derived from a protein of influenza A virus H1, preferably hemagglutinin (HA) and/or neuraminidase (NA), at least one mRNA sequence encoding at least one antigenic peptide or protein derived from a protein of influenza A virus H3, preferably hemagglutinin (HA) and/or neuraminidase (NA), at least one mRNA sequence encoding at least one antigenic peptide or protein derived from a protein of influenza A virus H5, preferably hemagglutinin (HA) and/or neuraminidase (NA), and optionally at least one mRNA sequence encoding at least one antigenic peptide or protein derived from a protein of influenza A virus H7, preferably hemagglutinin (HA) and/or neuraminidase (NA), and/or optionally at least one mRNA sequence encoding at least one antigenic peptide or protein derived from a protein of influenza A virus H9, preferably hemagglutinin (HA) and/or neuraminidase (NA).

Preferably, the pharmaceutical composition or vaccine comprises at least one mRNA comprising lipid nanoparticle comprising a mRNA compound comprising a mRNA sequence encoding at least one antigenic peptide or protein derived from hemagglutinin (HA) and/or at least one mRNA sequence encoding at least one antigenic peptide or protein derived from neuraminidase (NA) of influenza A virus H1, at least one mRNA sequence encoding at least one antigenic peptide or protein derived from hemagglutinin (HA) and/or at least one mRNA sequence encoding at least one antigenic peptide or protein derived from neuraminidase (NA) of influenza A virus H3, at least one mRNA sequence encoding at least one antigenic peptide or protein derived from hemagglutinin (HA) and/or at least one mRNA sequence encoding at least one antigenic peptide or protein derived from neuraminidase (NA) of influenza A virus H5, and optionally at least one mRNA sequence encoding at least one antigenic peptide or protein derived from preferably hemagglutinin (HA) and/or at least one mRNA sequence encoding at least one antigenic peptide or protein derived from neuraminidase (NA) of influenza A virus H7, and/or optionally at least one mRNA sequence encoding at least one antigenic peptide or protein derived from, preferably hemagglutinin (HA) and/or at least one mRNA sequence encoding at least one antigenic peptide or protein derived from neuraminidase (NA) of influenza A virus H9.

In a specific embodiment the pharmaceutical composition or vaccine comprises at least one mRNA comprising lipid nanoparticle comprising an mRNA compound comprising a mRNA sequence encoding at least one antigenic peptide or protein derived from hemagglutinin (HA) and/or at least one mRNA sequence encoding at least one antigenic peptide or protein derived from neuraminidase (NA) of influenza A virus H1N1, at least one mRNA sequence encoding at least one antigenic peptide or protein derived from hemagglutinin (HA) and/or at least one mRNA sequence encoding at least one antigenic peptide or protein derived from neuraminidase (NA) of influenza A virus H3N2, at least one mRNA sequence encoding at least one antigenic peptide or protein derived from hemagglutinin (HA) and/or at least one mRNA sequence encoding at least one antigenic peptide or protein derived from neuraminidase (NA) of influenza A virus H5N1.

Additionally, the pharmaceutical composition or vaccine preferably further comprises at least one mRNA comprising lipid nanoparticle comprising a mRNA compound comprising a mRNA sequence encoding at least one antigenic peptide or protein derived from hemagglutinin (HA) and/or at least one mRNA sequence encoding at least one antigenic peptide or protein derived from neuraminidase (NA) of at least one influenza B virus, encapsulated or associated with mRNA comprising lipid nanoparticles according to the invention.

V.9. Nucleic Acids for Use in the LNPs of the Invention

The present invention is based on the surprising finding that a nucleic acid (e.g., m-RNA) encoding at least one antigenic peptide or protein included in lipid nanoparticles (LNPs) induces very efficiently antigen-specific immune responses against the encoded antigenic peptide or protein at a low dosage and dosing regimen, which do not require frequent administration.

Another advantages of the inventive mRNA encoding at least one antigenic peptide or protein comprised in lipid nanoparticles (LNPs) are: induction of a strong humoral immune response; induction of b-cell memory; faster onset of immune protection; longevity of the induced immune responses; induction of broad cellular t-cell responses; induction of a (local and transient) pro-inflammatory environment; no induction of systemic cytokine or chemokine response; well tolerability, no side-effects, non-toxic; advantageous stability characteristics; formulation compatible with many different antigens: larger antigen cocktails feasible based on the same (production) technology; no vector immunity, i.e. technology can be used to vaccinate the same subject multiple times against multiple (different) antigens; speed, adaptability, simplicity and scalability of production.

In particular, the invention relates to lipid nanoparticles and uses thereof. In certain embodiments, the lipid nanoparticles comprise at least:

(i) an Ionizable Lipid of the Invention and/or a PEG-lipid; and (ii) an mRNA compound comprising an mRNA sequence encoding an antigenic peptide or protein.

In certain embodiments, the lipid nanoparticle comprising mRNA may comprise further compounds, such as one or more neutral lipids, steroids and combinations of said compounds. Suitable compounds will be described in detail below.

In certain embodiments, the mRNA compound comprising an mRNA sequence encoding an antigenic peptide or protein may be a mRNA molecule. In one embodiment of the invention, the mRNA compound is a natural and non-modified mRNA. Within the context of the present invention, natural and non-modified mRNA encompasses mRNA generated in vitro, without chemical modifications or changes in the sequence.

In an alternative embodiment of the invention, the mRNA compound comprises an artificial mRNA. In the context of the present invention artificial mRNA encompasses mRNA with chemical modifications, sequence modifications or non-natural sequences.

In a preferred embodiment of the invention, the mRNA compound does not comprise nucleoside modifications, in particular no base modifications. In a further embodiment, the mRNA compound does not comprise 1-methylpseudouridine modifications. In one preferred embodiment, the mRNA comprises only the naturally existing nucleosides. In a further preferred embodiment, the mRNA compound does not comprise any chemical modification and optionally comprises sequence modifications. In a further preferred embodiment of the invention the mRNA compound only comprises the naturally existing nucleosides adenine, uracil, guanine and cytosine.

According to certain embodiments of the present invention, the mRNA sequence is mono-, bi-, or multicistronic, preferably as defined herein. The coding sequences in a bi- or multicistronic mRNA preferably encode distinct peptides or proteins as defined herein or a fragment or variant thereof. Preferably, the coding sequences encoding two or more peptides or proteins may be separated in the bi- or multicistronic mRNA by at least one IRES (internal ribosomal entry site) sequence, as defined below. Thus, the term “encoding two or more peptides or proteins” may mean, without being limited thereto, that the bi- or even multicistronic mRNA, may encode e.g. at least two, three, four, five, six or more (preferably different) peptides or proteins or their fragments or variants within the definitions provided herein. More preferably, without being limited thereto, the bi- or even multicistronic mRNA, may encode, for example, at least two, three, four, five, six or more (preferably different) peptides or proteins as defined herein or their fragments or variants as defined herein.

In this context, a so-called IRES (internal ribosomal entry site) sequence as defined above can function as a sole ribosome binding site, but it can also serve to provide a bi- or even multicistronic mRNA as defined above, which encodes several peptides or proteins which are to be translated by the ribosomes independently of one another. Examples of IRES sequences, which can be used according to the invention, are those from picornaviruses (e.g. FMDV), pestiviruses (CFFV), polioviruses (PV), encephalomyocarditis viruses (ECMV), foot and mouth disease viruses (FMDV), hepatitis C viruses (HCV), classical swine fever viruses (CSFV), mouse leukoma virus (MLV), simian immunodeficiency viruses (SIV) or cricket paralysis viruses (CrPV). The TEV, tobacco etch virus 5′ UTR we use is an IRES.

According to a further embodiment the at least one coding region of the mRNA sequence according to the invention may encode at least two, three, four, five, six, seven, eight and more peptides or proteins (or fragments and derivatives thereof) as defined herein linked with or without an amino acid linker sequence, wherein said linker sequence can comprise rigid linkers, flexible linkers, cleavable linkers (e.g., self-cleaving peptides) or a combination thereof. Therein, the peptides or proteins may be identical or different or a combination thereof. Particular peptide or protein combinations can be encoded by said mRNA encoding at least two peptides or proteins as explained herein (also referred to herein as “multi-antigen-constructs/mRNA”).

In a particular aspect of the invention, the lipid nanoparticles comprise an mRNA compound, comprising an mRNA sequence encoding an antigenic peptide or protein, or a fragment, variant or derivative thereof.

These antigenic peptides or proteins may be derived from pathogenic antigens, tumor antigens, allergenic antigens or autoimmune self-antigens, preferably as defined herein. In the context of the present invention, antigenic peptides or proteins preferably exclude luciferases.

V.9.1. Pathogenic Antigens

Such pathogenic antigens are derived from pathogenic organisms, in particular bacterial, viral or protozoological (multicellular) pathogenic organisms, which evoke an immunological reaction by subject, in particular a mammalian subject, more particularly a human. More specifically, pathogenic antigens are preferably surface antigens, e.g. proteins (or fragments of proteins, e.g. the exterior portion of a surface antigen) located at the surface of the virus or the bacterial or protozoological organism.

Pathogenic antigens are peptide or protein antigens preferably derived from a pathogen associated with infectious disease which are preferably selected from antigens derived from the pathogens Acinetobacter baumannii, Anaplasma genus, Anaplasma phagocytophilum, Ancylostoma braziliense, Ancylostoma duodenale, Arcanobacterium haemolyticum, Ascaris lumbricoides, Aspergillus genus, Astroviridae, Babesia genus, Bacillus anthracis, Bacillus cereus, Bartonella henselae , BK virus, Blastocystis hominis, Blastomyces dermatitidis, Bordetella pertussis, Borrelia burgdorferi, Borrelia genus, Borrelia spp, Brucella genus, Brugia malayi , Bunyaviridae family, Burkholderia cepacia and other Burkholderia species, Burkholderia mallei, Burkholderia pseudomallei , Caliciviridae family, Campylobacter genus, Candida albicans, Candida spp, Chlamydia trachomatis, Chlamydophila pneumoniae, Chlamydophila psittaci , CD prion, Clonorchis sinensis, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium perfringens, Clostridium spp, Clostridium tetani, Coccidioides spp, coronaviruses, Corynebacterium diphtheriae, Coxiella burnetii , Crimean-Congo hemorrhagic fever virus, Cryptococcus neoformans, Cryptosporidium genus, Cytomegalovirus (CMV), Dengue viruses (DEN-1, DEN-2, DEN-3 and DEN-4), Dientamoeba fragilis , Ebolavirus (EBOV), Echinococcus genus, Ehrlichia chaffeensis, Ehrlichia ewingii, Ehrlichia genus, Entamoeba histolytica, Enterococcus genus, Enterovirus genus, Enteroviruses, mainly Coxsackie A virus and Enterovirus 71 (EV71), Epidermophyton spp, Epstein-Barr Virus (EBV), Escherichia coli O157:H7, 0111 and 0104:H4, Fasciola hepatica and Fasciola gigantica , FFI prion, Filarioidea superfamily, Flaviviruses, Francisella tularensis, Fusobacterium genus, Geotrichum candidum, Giardia intestinalis, Gnathostoma spp, GSS prion, Guanarito virus, Haemophilus ducreyi, Haemophilus influenzae, Helicobacter pylori , Henipavirus (Hendra virus Nipah virus), Hepatitis A Virus, Hepatitis B Virus (HBV), Hepatitis C Virus (HCV), Hepatitis D Virus, Hepatitis E Virus, Herpes simplex virus 1 and 2 (HSV-1 and HSV-2), Histoplasma capsulatum , HIV (Human immunodeficiency virus), Hortaea werneckii , Human bocavirus (HBoV), Human herpesvirus 6 (HHV-6) and Human herpesvirus 7 (HHV-7), Human metapneumovirus (hMPV), Human papillomavirus (HPV), Human parainfluenza viruses (HPIV), Japanese encephalitis virus, JC virus, Junin virus, Kingella kingae, Klebsiella granulomatis , Kuru prion, Lassa virus, Legionella pneumophila, Leishmania genus, Leptospira genus, Listeria monocytogenes , Lymphocytic choriomeningitis virus (LCMV), Machupo virus, Malassezia spp, Marburg virus, Measles virus, Metagonimus yokagawai, Microsporidia phylum, Molluscum contagiosum virus (MCV), Mumps virus, Mycobacterium leprae and Mycobacterium lepromatosis, Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycoplasma pneumoniae, Naegleria fowleri, Necator americanus, Neisseria gonorrhoeae, Neisseria meningitidis, Nocardia asteroides, Nocardia spp, Onchocerca volvulus , Orientia tsutsugamushi, Orthomyxoviridae family (Influenza), Paracoccidioides brasiliensis, Paragonimus spp, Paragonimus westermani , Parvovirus B19, Pasteurella genus, Plasmodium genus, Pneumocystis jirovecii , Poliovirus, Rabies virus, Respiratory syncytial virus (RSV), Rhinovirus, rhinoviruses, Rickettsia akari, Rickettsia genus, Rickettsia prowazekii, Rickettsia rickettsii, Rickettsia typhi , Rift Valley fever virus, Rotavirus, Rubella virus, Sabia virus, Salmonella genus, Sarcoptes scabiei , SARS coronavirus, Schistosoma genus, Shigella genus, Sin Nombre virus, Hantavirus, Sporothrix schenckii, Staphylococcus genus, Staphylococcus genus, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Strongyloides stercoralis, Taenia genus, Taenia solium , Tick-borne encephalitis virus (TBEV), Toxocara canis or Toxocara cati, Toxoplasma gondii, Treponema pallidum, Trichinella spiralis, Trichomonas vaginalis, Trichophyton spp, Trichuris trichiura, Trypanosoma brucei, Trypanosoma cruzi, Ureaplasma urealyticum , Varicella zoster virus (VZV), Varicella zoster virus (VZV), Variola major or Variola minor, vCJD prion, Venezuelan equine encephalitis virus, Vibrio cholerae , West Nile virus, Western equine encephalitis virus, Wuchereria bancrofti , Yellow fever virus, Yersinia enterocolitica, Yersinia pestis , and Yersinia pseudotuberculosis.

In this context particularly preferred are antigens from the pathogens selected from Influenza virus, SARS-COV-2, respiratory syncytial virus (RSV), Herpes simplex virus (HSV), human Papilloma virus (HPV), Human immunodeficiency virus (HIV), Plasmodium, Clostridium difficile, Staphylococcus aureus , Dengue virus, Chlamydia trachomatis, Brucella , Cytomegalovirus (CMV), Hepatitis B virus (HBV), Mycobacterium tuberculosis , Rabies virus, Crimean-Congo Hemorrhagic Fever virus and Yellow Fever Virus.

Furthermore, the pathogenic antigen (antigen derived from a pathogen associated with infectious disease) may be preferably selected from the following antigens: Outer membrane protein A OmpA, biofilm associated protein Bap, transport protein MucK ( Acinetobacter baumannii, Acinetobacter infections)); variable surface glycoprotein VSG, microtubule-associated protein MAPP15, trans-sialidase TSA ( Trypanosoma brucei , African sleeping sickness (African trypanosomiasis)); HIV p24 antigen, HIV envelope proteins (Gp120, Gp41, Gp160), polyprotein GAG, negative factor protein Nef, trans-activator of transcription Tat (HIV (Human immunodeficiency virus), AIDS (Acquired immunodeficiency syndrome)); galactose-inhibitable adherence protein GIAP, 29 kDa antigen Eh29, Gal/GalNAc lectin, protein CRT, 125 kDa immunodominant antigen, protein M17, adhesin ADH112, protein STIRP ( Entamoeba histolytica , Amoebiasis); Major surface proteins 1-5 (MSP1a, MSP1b, MSP2, MSP3, MSP4, MSP5), type IV secretion system proteins (VirB2, VirB7, VirB11, VirD4) ( Anaplasma genus, Anaplasmosis); protective Antigen PA, edema factor EF, lethal factor LF, the S-layer homology proteins SLH ( Bacillus anthracis , Anthrax); acranolysin, phospholipase D, collagen-binding protein CbpA ( Arcanobacterium haemolyticum, Arcanobacterium haemolyticum infection); nucleocapsid protein NP, glycoprotein precursor GPC, glycoprotein GPI, glycoprotein GP2 (Junin virus, Argentine hemorrhagic fever); chitin-protein layer proteins, 14 kDa surface antigen A14, major sperm protein MSP, MSP polymerization-organizing protein MPOP, MSP fiber protein 2 MFP2, MSP polymerization-activating kinase MPAK, ABA-1-like protein ALB, protein ABA-1, cuticulin CUT-1 ( Ascaris lumbricoides , Ascariasis); 41 kDa allergen Asp v13, allergen Asp f3, major conidial surface protein rodlet A, protease Pepip, GPI-anchored protein Gellp, GPI-anchored protein Crflp ( Aspergillus genus, Aspergillosis); family VP26 protein, VP29 protein (Astroviridae, Astrovirus infection); Rhoptry-associated protein 1 RAP-1, merozoite surface antigens MSA-1, MSA-2 (a1, a2, b, c), 12D3, 11C5, 21B4, P29, variant erythrocyte surface antigen VESA1, Apical Membrane Antigen 1 AMA-1 ( Babesia genus, Babesiosis); hemolysin, enterotoxin C, PXO1-51, glycolate oxidase, ABC-transporter, penicillin-binding protein, zinc transporter family protein, pseudouridine synthase Rsu, plasmid replication protein RepX, oligoendopeptidase F, prophage membrane protein, protein HemK, flagellar antigen H, 28.5-kDa cell surface antigen ( Bacillus cereus, Bacillus cereus infection); large T antigen LT, small T antigen, capsid protein VP1, capsid protein VP2 (BK virus, BK virus infection); 29 kDa-protein, caspase-3-like antigens, glycoproteins ( Blastocystis hominis, Blastocystis hominis infection); yeast surface adhesin WI-1 ( Blastomyces dermatitidis , Blastomycosis); nucleoprotein N, polymerase L, matrix protein Z, glycoprotein GP (Machupo virus, Bolivian hemorrhagic fever); outer surface protein A OspA, outer surface protein OspB, outer surface protein OspC, decorin binding protein A DbpA, decorin binding protein B DbpB, flagellar filament 41 kDa core protein Fla, basic membrane protein A precursor BmpA (Immunodominant antigen P39), outer surface 22 kDa lipoprotein precursor (antigen IPLA7), variable surface lipoprotein vlsE ( Borrelia genus, Borrelia infection); Botulinum neurotoxins BoNT/A1, BoNT/A2, BoNT/A3, BoNT/B, BoNT/C, BoNT/D, BoNT/E, BoNT/F, BoNT/G, recombinant botulinum toxin F He domain FHc ( Clostridium botulinum , Botulism (and Infant botulism)); nucleocapsid, glycoprotein precursor (Sabia virus, Brazilian hemorrhagic fever); copper/Zinc superoxide dismutase SodC, bacterioferritin Bfr, 50S ribosomal protein RplL, OmpA-like transmembrane domain-containing protein Omp31, immunogenic 39-kDa protein M5 P39, zinc ABC transporter periplasmic zinc-binding protein znuA, periplasmic immunogenic protein Bp26, 30S ribosomal protein S12 RpsL, glyceraldehyde-3-phosphate dehydrogenase Gap, 25 kDa outer-membrane immunogenic protein precursor Omp25, invasion protein B lalB, trigger factor Tig, molecular chaperone DnaK, putative peptidyl-prolyl cis-trans isomerase SurA, lipoprotein Omp19, outer membrane protein MotY Omp16, conserved outer membrane protein D15, malate dehydrogenase Mdh, component of the Type-IV secretion system (T4SS) VirJ, lipoprotein of unknown function BAB1_0187 ( Brucella genus, Brucellosis); members of the ABC transporter family (LoiC, OppA, and PotF), putative lipoprotein releasing system transmembrane protein LolC/E, flagellin FliC, Burkholderia intracellular motility A BimA, bacterial Elongation factor-Tu EF-Tu, 17 kDa OmpA-like protein, boaA coding protein, boaB coding protein ( Burkholderia cepacia and other Burkholderia species, Burkholderia infection); mycolyl-transferase Ag85A, heat-shock protein Hsp65, protein TB10.4, 19 kDa antigen, protein PstS3, heat-shock protein Hsp70 ( Mycobacterium ulcerans , Buruli ulcer); norovirus major and minor viral capsid proteins VP1 and VP2, genome polyprotein, Sapoviurus capsid protein VP1, protein Vp3, geome polyprotein (Caliciviridae family, Calicivirus infection (Norovirus and Sapovirus)); major outer membrane protein PorA, flagellin FlaA, surface antigen CjaA, fibronectin binding protein CadF, aspartate/glutamate-binding ABC transporter protein PeblA, protein FspAI, protein FspA2 ( Campylobacter genus, Campylobacteriosis); glycolytic enzyme enolase, secreted aspartyl proteinases SAP1-10, glycophosphatidylinositol (GPI)-linked cell wall protein, protein Hyrl, complement receptor 3-related protein CR3-RP, adhesin Als3p, heat shock protein 90 kDa hsp90, cell surface hydrophobicity protein CSH (usually Candida albicans and other Candida species, Candidiasis); 17-kDa antigen, protein P26, trimeric autotransporter adhesins TAAs, Bartonella adhesin A BadA, variably expressed outer-membrane proteins Vomps, protein Pap3, protein HbpA, envelope-associated protease HtrA, protein OMP89, protein GroEL, protein LalB, protein OMP43, dihydrolipoamide succinyltransferase SucB ( Bartonella henselae , Cat-scratch disease); amastigote surface protein-2, amastigote-specific surface protein SSP4, cruzipain, trans-sialidase TS, trypomastigote surface glycoprotein TSA-1, complement regulatory protein CRP-10, protein G4, protein G2, paraxonemal rod protein PAR2, paraflagellar rod component Par1, mucin-Associated Surface Proteins MPSP ( Trypanosoma cruzi , Chagas Disease (American trypanosomiasis)); envelope glycoproteins (gB, gC, gE, gH, gI, gK, gL) (Varicella zoster virus (VZV), Chickenpox); major outer membrane protein MOMP, probable outer membrane protein PMPC, outer membrane complex protein B OmcB, heat shock proteins Hsp60 HSP10, protein IncA , proteins from the type III secretion system, ribonucleotide reductase small chain protein NrdB, plasmid protein Pgp3, chlamydial outer protein N CopN, antigen CT521, antigen CT425, antigen CT043, antigen TC0052, antigen TC0189, antigen TC0582, antigen TC0660, antigen TC0726, antigen TC0816, antigen TC0828 ( Chlamydia trachomatis, Chlamydia ); low calcium response protein E LCrE, chlamydial outer protein N CopN, serine/threonine-protein kinase PknD, acyl-carrier-protein S-malonyltransferase FabD, single-stranded DNA-binding protein Ssb, major outer membrane protein MOMP, outer membrane protein 2 Omp2, polymorphic membrane protein family (Pmp1, Pmp2, Pmp3, Pmp4, Pmp5, Pmp6, Pmp7, Pmp8, Pmp9, Pmp10, Pmp11, Pmp12, Pmp13, Pmp14, Pmp15, Pmp16, Pmp17, Pmp18, Pmp19, Pmp20, Pmp21) ( Chlamydophila pneumoniae, Chlamydophila pneumoniae infection); cholera toxin B CTB, toxin coregulated pilin A TcpA, toxin coregulated pilin TcpF, toxin co-regulated pilus biosynthesis ptrotein F TcpF, cholera enterotoxin subunit A, cholera enterotoxin subunit B, Heat-stable enterotoxin ST, mannose-sensitive hemagglutinin MSHA, outer membrane protein U Porin ompU, Poring B protein, polymorphic membrane protein-D ( Vibrio cholerae , Cholera); propionyl-CoA carboxylase PCC, 14-3-3 protein, prohibitin, cysteine proteases, glutathione transferases, gelsolin, cathepsin L proteinase CatL, Tegumental Protein 20.8 kDa TP20.8, tegumental protein 31.8 kDa TP31.8, lysophosphatidic acid phosphatase LPAP, ( Clonorchis sinensis , Clonorchiasis); surface layer proteins SLPs, glutamate dehydrogenase antigen GDH, toxin A, toxin B, cysteine protease Cwp84, cysteine protease Cwp13, cysteine protease Cwp19, Cell Wall Protein CwpV, flagellar protein FliC, flagellar protein FIiD, Zmp-1, CdeM, CdeC ( Clostridium difficile, Clostridium difficile infection); rhinoviruses: capsid proteins VP1, VP2, VP3, VP4; coronaviruses: sprike proteins S, envelope proteins E, membrane proteins M, nucleocapsid proteins N (usually rhinoviruses and coronaviruses, Common cold (Acute viral rhinopharyngitis; Acute coryza)); prion protein Prp (CJD prion, Creutzfeldt-Jakob disease (CJD)); envelope protein Gc, envelope protein Gn, nucleocapsid proteins (Crimean-Congo hemorrhagic fever virus, Crimean-Congo hemorrhagic fever (CCHF)); virulence-associated DEAD-box RNA helicase VAD1, galactoxylomannan-protein GalXM, glucuronoxylomannan GXM, mannoprotein MP ( Cryptococcus neoformans , Cryptococcosis); acidic ribosomal protein P2 CpP2, mucin antigens MucI, Muc2, Muc3 Muc4, Muc5, Muc6, Muc7, surface adherence protein CP20, surface adherence protein CP23, surface protein CP12, surface protein CP21, surface protein CP40, surface protein CP60, surface protein CP15, surface-associated glycopeptides gp40, surface-associated glycopeptides gp15, oocyst wall protein AB, profilin PRF, apyrase ( Cryptosporidium genus, Cryptosporidiosis); fatty acid and retinol binding protein-1 FAR-1, tissue inhibitor of metalloproteinase TIMP (TMP), cysteine proteinase ACEY-1, cysteine proteinase ACCP-1, surface antigen Ac-16, secreted protein 2 ASP-2, metalloprotease 1 MTP-1, aspartyl protease inhibitor API-1, surface-associated antigen SAA-1, adult-specific secreted factor Xa serine protease inhibitor anticoagulant AP, cathepsin D-like aspartic protease ARR-1 (usually Ancylostoma braziliense ; multiple other parasites, Cutaneous larva migrans (CLM)); cathepsin L-like proteases, 53/25-kDa antigen, 8 kDa family members, cysticercus protein with a marginal trypsin-like activity TsAg5, oncosphere protein TSOL18, oncosphere protein TSOL45-1A, lactate dehydrogenase A LDHA, lactate dehydrogenase B LDHB ( Taenia solium , Cysticercosis); pp65 antigen, membrane protein pp15, capsid-proximal tegument protein pp150, protein M45, DNA polymerase UL54, helicase UL105, glycoprotein gM, glycoprotein gN, glcoprotein H, glycoprotein B gB, protein UL83, protein UL94, protein UL99 (Cytomegalovirus (CMV), Cytomegalovirus infection); capsid protein C, premembrane protein prM, membrane protein M, envelope protein E (domain I, domain IL, domain II), protein NS1, protein NS2A, protein NS2B, protein NS3, protein NS4A, protein 2K, protein NS4B, protein NS5 (Dengue viruses (DEN-1, DEN-2, DEN-3 and DEN-4)-Flaviviruses, Dengue fever); 39 kDa protein ( Dientamoeba fragilis , Dientamoebiasis); diphtheria toxin precursor Tox, diphteria toxin DT, pilin-specific sortase SrtA, shaft pilin protein SpaA, tip pilin protein SpaC, minor pilin protein SpaB, surface-associated protein DIP1281 ( Corynebacterium diphtheriae , Diphtheria); glycoprotein GP, nucleoprotein NP, minor matrix protein VP24, major matrix protein VP40, transcription activator VP30, polymerase cofactor VP35, RNA polymerase L (Ebolavirus (EBOV), Ebola hemorrhagic fever); prion protein (vCOD prion, Variant Creutzfeldt-Jakob disease (vCOD, nvCOD)); UvrABC system protein B, protein Flp1, protein Flp2, protein Flp3, protein TadA, hemoglobin receptor HgbA, outer membrane protein TdhA, protein CpsRA, regulator CpxR, protein SapA, 18 kDa antigen, outer membrane protein NcaA, protein LspA, protein LspAl, protein LspA2, protein LspB, outer membrane component DsrA, lectin DltA, lipoprotein Hip, major outer membrane protein OMP, outer membrane protein OmpA2 ( Haemophilus ducreyi , Chancroid); aspartyl protease 1 Pep1, phospholipase B PLB, alpha-mannosidase 1 AMN1, glucanosyltransferase GEL1, urease URE, peroxisomal matrix protein Pmpl, proline-rich antigen Pra, humal T-cell reactive protein TcrP ( Coccidioides immitis and Coccidioides posadasii, Coccidioidomycosis); allergen Tri r 2, heat shock protein 60 Hsp60, fungal actin Act, antigen Tri r2, antigen Tri r4, antigen Tri t1, protein IV, glycerol-3-phosphate dehydrogenase Gpdl, osmosensor HwSholA, osmosensor HwSholB, histidine kinase HwHhk7B, allergen Mala s 1, allergen Mala s 11, thioredoxin Trx Mala s 13, allergen Mala f, allergen Mala s (usually Trichophyton spp, Epidermophyton spp., Malassezia spp., Hortaea werneckii , Dermatophytosis); protein EG95, protein EG10, protein EG18, protein EgA31, protein EM18, antigen EPC1, antigen B, antigen 5, protein P29, protein 14-3-3, 8-kDa protein, myophilin, heat shock protein 20 HSP20, glycoprotein GP-89, fatty acid binding protein FAPB ( Echinococcus genus, Echinococcosis); major surface protein 2 MSP2, major surface protein 4 MSP4, MSP variant SGV1, MSP variant SGV2, outer membrane protein OMP, outer membrane protein 19 OMP-19, major antigenic protein MAP1, major antigenic protein MAP1-2, major antigenic protein MAPiB, major antigenic protein MAP1-3, Erum2510 coding protein, protein GroEL, protein GroES, 30-kDA major outer membrane proteins, GE 100-kDa protein, GE 130-kDa protein, GE 160-kDa protein ( Ehrlichia genus, Ehrlichiosis); secreted antigen SagA, sagA-like proteins SalA and SalB, collagen adhesin Scm, surface proteins Fmsl (EbpA(fm), Fms5 (EbpB(fm), Fms9 (EpbC(fm) and Fms10, protein EbpC(fm), 96 kDa immunoprotective glycoprotein G1 ( Enterococcus genus, Enterococcus infection); genome polyprotein, polymerase 3D, viral capsid protein VP1, viral capsid protein VP2, viral capsid protein VP3, viral capsid protein VP4, protease 2A, protease 3C ( Enterovirus genus, Enterovirus infection); outer membrane proteins OM, 60 kDa outer membrane protein, cell surface antigen OmpA, cell surface antigen OmpB (sca5), 134 kDa outer membrane protein, 31 kDa outer membrane protein, 29.5 kDa outer membrane protein, cell surface protein SCA4, cell surface protein Adr1 (RP827), cell surface protein Adr2 (RP828), cell surface protein SCA1, Invasion protein invA, cell division protein fts, secretion proteins sec 0family, virulence proteins virB, tlyA, tlyC, parvulin-like protein Pip, preprotein translocase SecA, 120-kDa surface protein antigen SPA, 138 kD complex antigen, major 100-kD protein (protein I), intracytoplasmic protein D, protective surface protein antigen SPA ( Rickettsia prowazekii , Epidemic typhus); Epstein-Barr nuclear antigens (EBNA-1, EBNA-2, EBNA-3A, EBNA-3B, EBNA-3C, EBNA-leader protein (EBNA-LP)), latent membrane proteins (LMP-1, LMP-2A, LMP-2B), early antigen EBV-EA, membrane antigen EBV-MA, viral capsid antigen EBV-VCA, alkaline nuclease EBV-AN, glycoprotein H, glycoprotein gp350, glycoprotein gp110, glycoprotein gp42, glycoprotein gHgL, glycoprotein gB (Epstein-Barr Virus (EBV), Epstein-Barr Virus Infectious Mononucleosis); capsid protein VP2, capsid protein VP1, major protein NS1 (Parvovirus B19, Erythema infectiosum (Fifth disease)); pp65 antigen, glycoprotein 105, major capsid protein, envelope glycoprotein H, protein U51 (Human herpesvirus 6 (HHV-6) and Human herpesvirus 7 (HHV-7), Exanthem subitum); thioredoxin-glutathione reductase TGR, cathepsins L1 and L2, Kunitz-type protein KTM, leucine aminopeptidase LAP, cysteine proteinase Fas2, saposin-like protein-2 SAP-2, thioredoxin peroxidases TPx, Prx-1, Prx-2, cathepsin I cysteine proteinase CL3, protease cathepsin L CL1, phosphoglycerate kinase PGK, 27-kDa secretory protein, 60 kDa protein HSP35alpha, glutathione transferase GST, 28.5 kDa tegumental antigen 28.5 kDa TA, cathepsin B3 protease CatB3, Type I cystatin stefin-1, cathepsin L5, cathepsin Llg and cathepsin B, fatty acid binding protein FABP, leucine aminopeptidases LAP ( Fasciola hepatica and Fasciola gigantica , Fasciolosis); prion protein (FFI prion, Fatal familial insomnia (FFI)); venom allergen homolog-like protein VAL-1, abundant larval transcript ALT-1, abundant larval transcript ALT-2, thioredoxin peroxidase TPX, vespid allergen homologue VAH, thiordoxin peroxidase 2 TPX-2, antigenic protein SXP (peptides N, N1, N2, and N3), activation associated protein-1 ASP-1, Thioredoxin TRX, transglutaminase BmTGA, glutathione-S-transferases GST, myosin, vespid allergen homologue VAH, 175 kDa collagenase, glyceraldehyde-3-phosphate dehydrogenase GAPDH, cuticular collagen Col-4, secreted larval acidic proteins SLAPs, chitinase CHI-1, maltose binding protein MBP, glycolytic enzyme fructose-1,6-bisphosphate aldolase Fba, tropomyosin TMY-1, nematode specific gene product OvB20, onchocystatin CPI-2, Cox-2 (Filarioidea superfamily, Filariasis); phospholipase C PLC, heat-labile enterotoxin B, Iota toxin component Ib, protein CPE1281, pyruvate ferredoxin oxidoreductase, elongation factor G EF-G, perfringolysin O Pfo, glyceraldehyde-3-phosphate dehydrogenase GapC, Fructose-bisphosphate aldolase Alf2, Clostridium perfringens enterotoxin CPE, alpha toxin AT, alpha toxoid ATd, epsilon-toxoid ETd, protein HP, large cytotoxin TpeL, endo-beta-N-acetylglucosaminidase Naglu, phosphoglyceromutase Pgm ( Clostridium perfringens , Food poisoning by Clostridium perfringens ); leukotoxin IktA, adhesion FadA, outer membrane protein RadD, high-molecular weight arginine-binding protein ( Fusobacterium genus, Fusobacterium infection); phospholipase C PLC, heat-labile enterotoxin B, Iota toxin component Ib, protein CPE1281, pyruvate ferredoxin oxidoreductase, elongation factor G EF-G, perfringolysin O Pfo, glyceraldehyde-3-phosphate dehydrogenase GapC, fructose-bisphosphate aldolase Alf2, Clostridium perfringens enterotoxin CPE, alpha toxin AT, alpha toxoid ATd, epsilon-toxoid ETd, protein HP, large cytotoxin TpeL, endo-beta-N-acetylglucosaminidase Naglu, phosphoglyceromutase Pgm (usually Clostridium perfringens ; other Clostridium species, Gas gangrene (Clostridial myonecrosis)); lipase A, lipase B, peroxidase DecI ( Geotrichum candidum , Geotrichosis); prion protein (GSS prion, Gerstmann-Striussler-Scheinker syndrome (GSS)); cyst wall proteins CWP1, CWP2, CWP3, variant surface protein VSP, VSP1, VSP2, VSP3, VSP4, VSP5, VSP6, 56 kDa antigen, pyruvate ferredoxin oxidoreductase PFOR, alcohol dehydrogenase E ADHE, alpha-giardin, alpha8-giardin, alpha1-guiardin, beta-giardin, cystein proteases, glutathione-S-transferase GST, arginine deiminase ADI, fructose-1,6-bisphosphat aldolase FBA, Giardia trophozoite antigens GTA (GTA1, GTA2), ornithine carboxyl transferase OCT, striated fiber-asseblin-like protein SALP, uridine phosphoryl-like protein UPL, alpha-tubulin, beta-tubulin ( Giardia intestinalis , Giardiasis); members of the ABC transporter family (LolC, OppA, and PotF), putative lipoprotein releasing system transmembrane protein LolC/E, flagellin FliC, Burkholderia intracellular motility A BimA, bacterial Elongation factor-Tu EF-Tu, 17 kDa OmpA-like protein, boaA coding protein ( Burkholderia mallei , Glanders); cyclophilin CyP, 24 kDa third-stage larvae protien GS24, excretion-secretion products ESPs (40, 80, 120 and 208 kDa) ( Gnathostoma spinigerum and Gnathostoma hispidum , Gnathostomiasis); pilin proteins, minor pilin-associated subunit pilC, major pilin subunit and variants pilE, pilS, phase variation protein porA, Porin B PorB, protein TraD, Neisserial outer membrane antigen H.8, 70 kDa antigen, major outer membrane protein PI, outer membrane proteins PIA and PIB, W antigen, surface protein A NspA, transferrin binding protein TbpA, transferrin binding protein TbpB, PBP2, mtrR coding protein, ponA coding protein, membrane permease FbpBC, FbpABC protein system, LbpAB proteins, outer membrane protein Opa, outer membrane transporter FetA, iron-repressed regulator MpeR ( Neisseria gonorrhoeae , Gonorrhea); outer membrane protein A OmpA, outer membrane protein C OmpC, outer membrane protein K17 OmpK17 ( Klebsiella granulomatis , Granuloma inguinale (Donovanosis)); fibronectin-binding protein Sfb, fibronectin/fibrinogen-binding protein FBP54, fibronectin-binding protein FbaA, M protein type 1 Emm1, M protein type 6 Emm6, immunoglobulin-binding protein 35 Sib35, Surface protein R28 Spr28, superoxide dismutase SOD, C5a peptidase ScpA, antigen I/II AgI/II, adhesin AspA, G-related alpha2-macroglobulin-binding protein GRAB, surface fibrillar protein M5 ( Streptococcus pyogenes , Group A streptococcal infection); C protein P3 antigen, arginine deiminase proteins, adhesin BibA, 105 kDA protein BPS, surface antigens c, surface antigens R, surface antigens X, trypsin-resistant protein R1, trypsin-resistant protein R3, trypsin-resistant protein R4, surface immunogenic protein Sip, surface protein Rib, Leucine-rich repeats protein LrrG, serine-rich repeat protein Srr-2, C protein alpha-antigen Bca, Beta antigen Bag, surface antigen Epsilon, alpha-like protein ALP1, alpha-like protein ALP5 surface antigen delta, alpha-like protein ALP2, alpha-like protein ALP3, alpha-like protein ALP4, Cbeta protein Bac ( Streptococcus agalactiae , Group B streptococcal infection); transferrin-binding protein 2 Tbp2, phosphatase P4, outer membrane protein P6, peptidoglycan-associated lipoprotein Pal, protein D, protein E, adherence and penetration protein Hap, outer membrane protein 26 Omp26, outer membrane protein P5 (Fimbrin), outer membrane protein D15, outer membrane protein OmpP2, 5′-nucleotidase NucA, outer membrane protein P1, outer membrane protein P2, outer membrane lipoprotein Pcp, Lipoprotein E, outer membrane protein P4, fuculokinase FucK, [Cu,Zn]-superoxide dismutase SodC, protease HtrA, protein 0145, alpha-galactosylceramide ( Haemophilus influenzae, Haemophilus influenzae infection); polymerase 3D, viral capsid protein VP1, viral capsid protein VP2, viral capsid protein VP3, viral capsid protein VP4, protease 2A, protease 3C (Enteroviruses, mainly Coxsackie A virus and Enterovirus 71 (EV71), Hand, foot and mouth disease (HFMD)); RNA polymerase L, protein L, glycoprotein Gn, glycoprotein Gc, nucleocapsid protein S, envelope glycoprotein G1, nucleoprotein NP, protein N, polyprotein M (Sin Nombre virus, Hantavirus, Hantavirus Pulmonary Syndrome (HPS)); heat shock protein HspA, heat shock protein HspB, citrate synthase GItA, protein UreB, heat shock protein Hsp60, neutrophil-activating protein NAP, catalase KatA, vacuolating cytotoxin VacA, urease alpha UreA, urease beta Ureb, protein Cpn10, protein groES, heat shock protein Hsp10, protein MopB, cytotoxicity-associated 10 kDa protein CAG, 36 kDa antigen, beta-lactamase HcpA, Beta-lactamase HcpB ( Helicobacter pylori, Helicobacter pylori infection); integral membrane proteins, aggregation-prone proteins, 0-antigen, toxin-antigens Stx2B, toxin-antigen StxlB, adhesion-antigen fragment Int28, protein EspA, protein EspB, Intimin, protein Tir, protein IntC300, protein Eae ( Escherichia coli O157:H7, 0111 and 0104:H4, Hemolytic-uremic syndrome (HUS)); RNA polymerase L, protein L, glycoprotein Gn, glycoprotein Gc, nucleocapsid protein S, envelope glycoprotein G1, nucleoprotein NP, protein N, polyprotein M (Bunyaviridae family, Hemorrhagic fever with renal syndrome (HFRS)); glycoprotein G, matrix protein M, nucleoprotein N, fusion protein F, polymerase L, protein W, proteinC, phosphoprotein p, non-structural protein V (Henipavirus (Hendra virus Nipah virus), Henipavirus infections); polyprotein, glycoproten Gp2, hepatitis A surface antigen HBAg, protein 2A, virus protein VP1, virus protein VP2, virus protein VP3, virus protein VP4, protein P1B, protein P2A, protein P3AB, protein P3D (Hepatitis A Virus, Hepatitis A); hepatitis B surface antigen HBsAg, Hepatitis B core antigen HbcAg, polymerase, protein Hbx, preS2 middle surface protein, surface protein L, large S protein, virus protein VP1, virus protein VP2, virus protein VP3, virus protein VP4 (Hepatitis B Virus (HBV), Hepatitis B); envelope glycoprotein E1 gp32 gp35, envelope glycoprotein E2 NS1 gp68 gp70, capsid protein C, core protein Core, polyprotein, virus protein VP1, virus protein VP2, virus protein VP3, virus protein VP4, antigen G, protein NS3, protein NS5A, (Hepatitis C Virus, Hepatitis C); virus protein VP1, virus protein VP2, virus protein VP3, virus protein VP4, large hepatitis delta antigen, small hepatitis delta antigen (Hepatitis D Virus, Hepatitis D); virus protein VP1, virus protein VP2, virus protein VP3, virus protein VP4, capsid protein E2 (Hepatitis E Virus, Hepatitis E); glycoprotein L UL1, uracil-DNA glycosylase UL2, protein UL3, protein UL4, DNA replication protein UL5, portal protein UL6, virion maturation protein UL7, DNA helicase UL8, replication origin-binding protein UL9, glycoprotein M UL10, protein UL11, alkaline exonuclease UL12, serine-threonine protein kinase UL13, tegument protein UL14, terminase UL15, tegument protein UL16, protein UL17, capsid protein VP23 UL18, major capsid protein VP5 UL19, membrane protein UL20, tegument protein UL21, Glycoprotein H (UL22), Thymidine Kinase UL23, protein UL24, protein UL25, capsid protein P40 (UL26, VP24, VP22A), glycoprotein B (UL27), ICP18.5 protein (UL28), major DNA-binding protein ICP8 (UL29), DNA polymerase UL30, nuclear matrix protein UL31, envelope glycoprotein UL32, protein UL33, inner nuclear membrane protein UL34, capsid protein VP26 (UL35), large tegument protein UL36, capsid assembly protein UL37, VP19C protein (UL38), ribonucleotide reductase (Large subunit) UL39, ribonucleotide reductase (Small subunit) UL40, tegument protein/virion host shutoff VHS protein (UL41), DNA polymerase processivity factor UL42, membrane protein UL43, glycoprotein C (UL44), membrane protein UL45, tegument proteins VP11/12 (UL46), tegument protein VP13/14 (UL47), virion maturation protein VP16 (UL48, Alpha-TIF), envelope protein UL49, dUTP diphosphatase UL50, tegument protein UL51, DNA helicase/primase complex protein UL52, glycoprotein K (UL53), transcriptional regulation protein IE63 (ICP27, UL54), protein UL55, protein UL56, viral replication protein ICP22 (IE68, US1), protein US2, serine/threonine-protein kinase US3, glycoprotein G (US4), glycoprotein J (US5), glycoprotein D (US6), glycoprotein I (US7), glycoprotein E (US8), tegument protein US9, capsid/tegument protein US10, Vmw21 protein (US11), ICP47 protein (IE12, US12), major transcriptional activator ICP4 (IE175, RS1), E3 ubiquitin ligase ICPO (IE110), latency-related protein 1 LRP1, latency-related protein 2 LRP2, neurovirulence factor RL 1 (ICP34.5), latency-associated transcript LAT (Herpes simplex virus 1 and 2 (HSV-1 and HSV-2), Herpes simplex); heat shock protein Hsp60, cell surface protein HIC, dipeptidyl peptidase type IV DppIV, M antigen, 70 kDa protein, 17 kDa histone-like protein ( Histoplasma capsulatum , Histoplasmosis); fatty acid and retinol binding protein-1 FAR-1, tissue inhibitor of metalloproteinase TIMP (TMP), cysteine proteinase ACEY-1, cysteine proteinase ACCP-1, surface antigen Ac-16, secreted protein 2 ASP-2, metalloprotease 1 MTP-1, aspartyl protease inhibitor API-1, surface-associated antigen SAA-1, surface-associated antigen SAA-2, adult-specific secreted factor Xa, serine protease inhibitor anticoagulant AP, cathepsin D-like aspartic protease ARR-1, glutathione S-transferase GST, aspartic protease APR-1, acetylcholinesterase AChE ( Ancylostoma duodenale and Necator americanus , Hookworm infection); protein NS1, protein NP1, protein VPI, protein VP2, protein VP3 (Human bocavirus (HBoV), Human bocavirus infection); major surface protein 2 MSP2, major surface protein 4 MSP4, MSP variant SGV1, MSP variant SGV2, outer membrane protein OMP, outer membrane protein 19 OMP-19, major antigenic protein MAP1, major antigenic protein MAP1-2, major antigenic protein MAPIB, major antigenic protein MAP1-3, Erum2510 coding protein, protein GroEL, protein GroES, 30-kDA major outer membrane proteins, GE 100-kDa protein, GE 130-kDa protein, GE 160-kDa protein ( Ehrlichia ewingii, Human ewingii ehrlichiosis); major surface proteins 1-5 (MSP1a, MSP1b, MSP2, MSP3, MSP4, MSP5), type IV secreotion system proteins VirB2, VirB7, VirB11, VirD4 ( Anaplasma phagocytophilum , Human granulocytic anaplasmosis (HGA)); protein NS1, small hydrophobic protein NS2, SH protein, fusion protein F, glycoprotein G, matrix protein M, matrix protein M2-1, matrix protein M2-2, phosphoprotein P, nucleoprotein N, polymerase L (Human metapneumovirus (hMPV), Human metapneumovirus infection); major surface protein 2 MSP2, major surface protein 4 MSP4, MSP variant SGV1, MSP variant SGV2, outer membrane protein OMP, outer membrane protein 19 OMP-19, major antigenic protein MAP1, major antigenic protein MAP1-2, major antigenic protein MAP1B, major antigenic protein MAP1-3, Erum2510 coding protein, protein GroEL, protein GroES, 30-kDA major outer membrane proteins, GE 100-kDa protein, GE 130-kDa protein, GE 160-kDa protein ( Ehrlichia chaffeensis , Human monocytic ehrlichiosis); replication protein E1, regulatory protein E2, protein E3, protein E4, protein E5, protein E6, protein E7, protein E8, major capsid protein L1, minor capsid protein L2 (Human papillomavirus (HPV), Human papillomavirus (HPV) infection); fusion protein F, hemagglutinin-neuramidase HN, glycoprotein G, matrix protein M, phosphoprotein P, nucleoprotein N, polymerase L (Human parainfluenza viruses (HPIV), Human parainfluenza virus infection); Hemagglutinin (HA), Neuraminidase (NA), Nucleoprotein (NP), M1 protein, M2 protein, NS1 protein, NS2 protein (NEP protein: nuclear export protein), PA protein, PB1 protein (polymerase basic 1 protein), PB1-F2 protein and PB2 protein (Orthomyxoviridae family, Influenza virus (flu)); genome polyprotein, protein E, protein M, capsid protein C (Japanese encephalitis virus, Japanese encephalitis); RTX toxin, type IV pili, major pilus subunit PilA, regulatory transcription factors PilS and PilR, protein sigma54, outer membrane proteins (Kingella kingae, Kingella kingae infection); prion protein (Kuru prion, Kuru); nucleoprotein N, polymerase L, matrix protein Z, glycoprotein GP (Lassa virus, Lassa fever); peptidoglycan-associated lipoprotein PAL, 60 kDa chaperonin Cpn60 (groEL, HspB), type IV pilin PilE, outer membrane protein MIP, major outer membrane protein MompS, zinc metalloproteinase MSP ( Legionella pneumophila , Legionellosis (Legionnaires' disease, Pontiac fever)); P4 nuclease, protein WD, ribonucleotide reductase M2, surface membrane glycoprotein Pg46, cysteine proteinase CP, glucose-regulated protein 78 GRP-78, stage-specific S antigen-like protein A2, ATPase F1, beta-tubulin, heat shock protein 70 Hsp70, KMP-11, glycoprotein GP63, protein BT1, nucleoside hydrolase NH, cell surface protein B1, ribosomal protein P1-like protein P1, sterol 24-c-methyltransferase SMT, LACK protein, histone H1, SPB1 protein, thiol specific antioxidant TSA, protein antigen STIl, signal peptidase SP, histone H2B, surface antigen PSA-2, cystein proteinase b Cpb ( Leishmania genus, Leishmaniasis); major membrane protein I, serine-rich antigen-45 kDa, 10 kDa caperonin GroES, HSP kDa antigen, amino-oxononanoate synthase AONS, protein recombinase A RecA, Acetyl-/propionyl-coenzyme A carboxylase alpha, alanine racemase, 60 kDa chaperonin 2, ESAT-6-like protein EcxB (L-ESAT-6), protein Lsr2, protein ML0276, Heparin-binding hemagglutinin HBHA, heat-shock protein 65 Hsp65, mycPI or ML0041 coding protein, htrA2 or ML0176 coding protein, htrA4 or ML2659 coding protein, gcp or ML0379 coding protein, clpC or ML0235 coding protein ( Mycobacterium leprae and Mycobacterium lepromatosis, Leprosy); outer membrane protein LipL32, membrane protein LIC10258, membrane protein LP30, membrane protein LIC12238, Ompa-like protein Lsa66, surface protein LigA, surface protein LigB, major outer membrane protein OmpL1, outer membrane protein LipL41, protein LigAni, surface protein LcpA, adhesion protein LipL53, outer membrane protein UpL32, surface protein Lsa63, flagellin FlaB1, membran lipoprotein LipL21, membrane protein pL40, leptospiral surface adhesin Lsa27, outer membrane protein OmpL36, outer membrane protein OmpL37, outer membrane protein OmpL47, outer membrane protein OmpL54, acyltransferase LpxA (Leptospira genus, Leptospirosis); listeriolysin O precursor My (LLO), invasion-associated protein Iap (P60), Listeriolysin regulatory protein PrfA, Zinc metalloproteinase Mpl, Phosphatidylinositol-specific phospholipase C PLC (PicA, PlcB), O-acetyltransferase Oat, ABC-transporter permease Im.G_1771, adhesion protein LAP, LAP receptor Hsp60, adhesin LapB, haemolysin listeriolysin O LLO, protein ActA, Internalin A InlA, protein InlB ( Listeria monocytogenes , Listeriosis); outer surface protein A OspA, outer surface protein OspB, outer surface protein OspC, decorin binding protein A DbpA, decorin binding protein B DbpB, flagellar filament 41 kDa core protein Fla, basic membrane protein A BmpA (Immunodominant antigen P39), outer surface 22 kDa lipoprotein precursor (antigen IPLA7), variable surface lipoprotein vlsE (usually Borrelia burgdorferi and other Borrelia species, Lyme disease (Lyme borreliosis)); venom allergen homolog-like protein VAL-1, abundant larval transcript ALT-1, abundant larval transcript ALT-2, thioredoxin peroxidase TPX, vespid allergen homologue VAH, thiordoxin peroxidase 2 TPX-2, antigenic protein SXP (peptides N, N1, N2, and N3), activation associated protein-1 ASP-1, thioredoxin TRX, transglutaminase BmTGA, glutathione-S-transferases GST, myosin, vespid allergen homologue VAH, 175 kDa collagenase, glyceraldehyde-3-phosphate dehydrogenase GAPDH, cuticular collagen Col-4, Secreted Larval Acidic Proteins SLAPs, chitinase CHI-1, maltose binding protein MBP, glycolytic enzyme fructose-1,6-bisphosphate aldolase Fba, tropomyosin TMY-1, nematode specific gene product OvB20, onchocystatin CPI-2, protein Cox-2 ( Wuchereria bancrofti and Brugia malayi , Lymphatic filariasis (Elephantiasis)); glycoprotein GP, matrix protein Z, polymerase L, nucleoprotein N (Lymphocytic choriomeningitis virus (LCMV), Lymphocytic choriomeningitis); thrombospondin-related anonymous protein TRAP, SSP2 Sporozoite surface protein 2, apical membrane antigen 1 AMA1, rhoptry membrane antigen RMA1, acidic basic repeat antigen ABRA, cell-traversal protein PF, protein Pvs25, merozoite surface protein 1 MSP-1, merozoite surface protein 2 MSP-2, ring-infected erythrocyte surface antigen RESALiver stage antigen 3 LSA-3, protein Eba-175, serine repeat antigen 5 SERA-5, circumsporozoite protein CS, merozoite surface protein 3 MSP3, merozoite surface protein 8 MSP8, enolase PF10, hepatocyte erythrocyte protein 17 kDa HEP17, erythrocyte membrane protein 1 EMP1, protein Kbetamerozoite surface protein 4/5 MSP 4/5, heat shock protein Hsp90, glutamate-rich protein GLURP, merozoite surface protein 4 MSP-4, protein STARP, circumsporozoite protein-related antigen precursor CRA ( Plasmodium genus, Malaria); nucleoprotein N, membrane-associated protein VP24, minor nucleoprotein VP30, polymerase cofactor VP35, polymerase L, matrix protein VP40, envelope glycoprotein GP (Marburg virus, Marburg hemorrhagic fever (MHF)); protein C, matrix protein M, phosphoprotein P, non-structural protein V, hemagglutinin glycoprotein H, polymerase L, nucleoprotein N, fusion protein F (Measles virus, Measles); members of the ABC transporter family (LolC, OppA, and PotF), putative lipoprotein releasing system transmembrane protein LolC/E, flagellin FliC, Burkholderia intracellular motility A BimA, bacterial Elongation factor-Tu EF-Tu, 17 kDa OmpA-like protein, boaA coding protein, boaB coding protein ( Burkholderia pseudomallei , Melioidosis (Whitmore's disease)); pilin proteins, minor pilin-associated subunit pilC, major pilin subunit and variants pilE, pilS, phase variation protein porA, Porin B PorB, protein TraD, Neisserial outer membrane antigen H.8, 70 kDa antigen, major outer membrane protein PI, outer membrane proteins PIA and PIB, W antigen, surface protein A NspA, transferrin binding protein TbpA, transferrin binding protein TbpB, PBP2, mtrR coding protein, ponA coding protein, membrane permease FbpBC, FbpABC protein system, LbpAB proteins, outer membrane protein Opa, outer membrane transporter FetA, iron-repressed regulator MpeR, factor H-binding protein fHbp, adhesin NadA, protein NhbA, repressor FarR ( Neisseria meningitidis , Meningococcal disease); 66 kDa protein, 22 kDa protein (usually Metagonimus yokagawai , Metagonimiasis); polar tube proteins (34, 75, and 170 kDa in Glugea, 35, 55 and 150 kDa in Encephalitozoon), kinesin-related protein, RNA polymerase II largest subunit, similar to integral membrane protein YIPA, anti-silencing protein 1, heat shock transcription factor HSF, protein kinase, thymidine kinase, NOP-2 like nucleolar protein (Microsporidia phylum, Microsporidiosis); CASP8 and FADD-like apoptosis regulator, Glutathione peroxidase GPX1, RNA helicase NPH-II NPH2, Poly(A) polymerase catalytic subunit PAPL, Major envelope protein P43K, early transcription factor 70 kDa subunit VETFS, early transcription factor 82 kDa subunit VETFL, metalloendopeptidase Gi-type, nucleoside triphosphatase I NPH1, replication protein A28-like MC134L, RNA polymease 7 kDa subunit RPO7 (Molluscum contagiosum virus (MCV), Molluscum contagiosum (MC)); matrix protein M, phosphoprotein PN, small hydrophobic protein SH, nucleoprotein N, protein V, fusion glycoprotein F, hemagglutinin-neuraminidase HN, RNA polymerase L (Mumps virus, Mumps); Outer membrane proteins OM, cell surface antigen OmpA, cell surface antigen OmpB (sca5), cell surface protein SCA4, cell surface protein SCA1, intracytoplasmic protein D, crystalline surface layer protein SLP, protective surface protein antigen SPA ( Rickettsia typhi , Murine typhus (Endemic typhus)); adhesin P1, adhesion P30, protein p116, protein P40, cytoskeletal protein HNMW1, cytoskeletal protein HMW2, cytoskeletal protein HMW3, MPN152 coding protein, MPN426 coding protein, MPN456 coding protein, MPN-500 coding protein ( Mycoplasma pneumoniae, Mycoplasma pneumonia); NocA, Iron dependent regulatory protein, VapA, VapD, VapF, VapG, caseinolytic protease, filament tip-associated 43-kDa protein, protein P24, protein P61, 15-kDa protein, 56-kDa protein (usually Nocardia asteroides and other Nocardia species, Nocardiosis); venom allergen homolog-like protein VAL-1, abundant larval transcript ALT-1, abundant larval transcript ALT-2, thioredoxin peroxidase TPX, vespid allergen homologue VAH, thiordoxin peroxidase 2 TPX-2, antigenic protein SXP (peptides N, N1, N2, and N3), activation associated protein-1 ASP-1, Thioredoxin TRX, transglutaminase BmTGA, glutathione-S-transferases GST, myosin, vespid allergen homologue VAH, 175 kDa collagenase, glyceraldehyde-3-phosphate dehydrogenase GAPDH, cuticular collagen Col-4, Secreted Larval Acidic Proteins SLAPs, chitinase CHI-1, maltose binding protein MBP, glycolytic enzyme fructose-1,6-bisphosphate aldolase Fba, tropomyosin TMY-1, nematode specific gene product OvB20, onchocystatin CPI-2, Cox-2 ( Onchocerca volvulus , Onchocerciasis (River blindness)); 43 kDa secreted glycoprotein, glycoprotein gp0, glycoprotein gp75, antigen Pb27, antigen Pb40, heat shock protein Hsp65, heat shock protein Hsp70, heat shock protein Hsp90, protein P10, triosephosphate isomerase TPI, N-acetyl-glucosamine-binding lectin Paracoccin, 28 kDa protein Pb28 ( Paracoccidioides brasiliensis , Paracoccidioidomycosis (South American blastomycosis)); 28-kDa cruzipain-like cystein protease Pw28CCP (usually Paragonimus westermani and other Paragonimus species, Paragonimiasis); outer membrane protein OmpH, outer membrane protein Omp28, protein PM1539, protein PM0355, protein PM1417, repair protein MutL, protein BcbC, prtein PM0305, formate dehydrogenase-N, protein PM0698, protein PM1422, DNA gyrase, lipoprotein PlpE, adhesive protein Cp39, heme aquisition system receptor HasR, 39 kDa capsular protein, iron-regulated OMP IROMP, outer membrane protein OmpA87, fimbrial protein Ptf, fimbrial subunit protein PtfA, transferrin binding protein Tbpl, esterase enzyme MesA, Pasteurella multocida toxin PMT, adhesive protein Cp39 ( Pasteurella genus, Pasteurellosis); “filamentous hemagglutinin FhaB, adenylate cyclase CyaA, pertussis toxin subunit 4 precursor PtxD, pertactin precursor Prn, toxin subunit 1 PtxA, protein Cpn60, protein brkA, pertussis toxin subunit 2 precursor PtxB, pertussis toxin subunit 3 precursor PtxC, pertussis toxin subunit 5 precursor PtxE, pertactin Prn, protein Fim2, protein Fim3; “( Bordetella pertussis , Pertussis (Whooping cough)); “F1 capsule antigen, virulence-associated V antigen, secreted effector protein LcrV, V antigen, outer membrane protease Pla, secreted effector protein YopD, putative secreted protein-tyrosine phosphatase YopH, needle complex major subunit YscF, protein kinase YopO, putative autotransporter protein YapF, inner membrane ABC-transporter YbtQ (Irp7), putative sugar binding protein YP00612, heat shock protein 90 HtpG, putative sulfatase protein YdeN, outer-membrane lipoprotein carrier protein LolA, secretion chaperone YerA, putative lipoprotein YP00420, hemolysin activator protein HpmB, pesticin/yersiniabactin outer membrane receptor Psn, secreted effector protein YopE, secreted effector protein YopF, secreted effector protein YopK, outer membrane protein YopN, outer membrane protein YopM, Coagulase/fibrinolysin precursor Pla; “( Yersinia pestis , Plague); protein PhpA, surface adhesin PsaA, pneumolysin Ply, ATP-dependent protease Clp, lipoate-protein ligase LplA, cell wall surface anchored protein psrP, sortase SrtA, glutamyl-tRNA synthetase GItX, choline binding protein A CbpA, pneumococcal surface protein A PspA, pneumococcal surface protein C PspC, 6-phosphogluconate dehydrogenase Gnd, iron-binding protein PiaA, Murein hydrolase LytB, proteon LytC, protease A1 ( Streptococcus pneumoniae , Pneumococcal infection); major surface protein B, kexin-like protease KEX1, protein A12, 55 kDa antigen P55, major surface glycoprotein Msg ( Pneumocystis jirovecii, Pneumocystis pneumonia (PCP)); genome polyprotein, polymerase 3D, viral capsid protein VP1, viral capsid protein VP2, viral capsid protein VP3, viral capsid protein VP4, protease 2A, protease 3C (Poliovirus, Poliomyelitis); protein Nfa1, exendin-3, secretory lipase, cathepsin B-like protease, cysteine protease, cathepsin, peroxiredoxin, protein Cry1Ac (usually Naegleria fowleri , Primary amoebic meningoencephalitis (PAM)); agnoprotein, large T antigen, small T antigen, major capsid protein VP1, minor capsid protein Vp2 (JC virus, Progressive multifocal leukoencephalopathy); low calcium response protein E LCrE, chlamydial outer protein N CopN, serine/threonine-protein kinase PknD, acyl-carrier-protein S-malonyltransferase FabD, single-stranded DNA-binding protein Ssb, major outer membrane protein MOMP, outer membrane protein 2 Omp2, polymorphic membrane protein family (Pmpl, Pmp2, Pmp3, Pmp4, Pmp5, Pmp6, Pmp7, Pmp8, Pmp9, Pmp10, Pmp11, Pmp12, Pmp13, Pmp14, Pmp15, Pmp16, Pmp17, Pmp18, Pmp19, Pmp20, Pmp21) ( Chlamydophila psittaci , Psittacosis); outer membrane protein P1, heat shock protein B HspB, peptide ABC transporter, GTP-binding protein, protein IcmB, ribonuclease R, phosphatas SixA, protein DsbD, outer membrane protein TolC, DNA-binding protein PhoB, ATPase DotB, heat shock protein B HspB, membrane protein Com1, 28 kDa protein, DNA-3-methyladenine glycosidase I, pouter membrane protein OmpH, outer membrane protein AdaA, glycine cleavage system T-protein ( Coxiella burnetii , Q fever); nucleoprotein N, large structural protein L, phophoprotein P, matrix protein M, glycoprotein G (Rabies virus, Rabies); fusionprotein F, nucleoprotein N, matrix protein M, matrix protein M2-1, matrix protein M2-2, phophoprotein P, small hydrophobic protein SH, major surface glycoprotein G, polymerase L, non-structural protein 1 NS1, non-structural protein 2 NS2 (Respiratory syncytial virus (RSV), Respiratory syncytial virus infection); genome polyprotein, polymerase 3D, viral capsid protein VP1, viral capsid protein VP2, viral capsid protein VP3, viral capsid protein VP4, protease 2A, protease 3C (Rhinovirus, Rhinovirus infection); outer membrane proteins OM, cell surface antigen OmpA, cell surface antigen OmpB (sca5), cell surface protein SCA4, cell surface protein SCA1, protein PS120, intracytoplasmic protein D, protective surface protein antigen SPA ( Rickettsia genus, Rickettsial infection); outer membrane proteins OM, cell surface antigen OmpA, cell surface antigen OmpB (sca5), cell surface protein SCA4, cell surface protein SCA1, intracytoplasmic protein D ( Rickettsia akari , Rickettsialpox); envelope glycoprotein GP, polymerase L, nucleoprotein N, non-structural protein NSS (Rift Valley fever virus, Rift Valley fever (RVF)); outer membrane proteins OM, cell surface antigen OmpA, cell surface antigen OmpB (sca5), cell surface protein SCA4, cell surface protein SCA1, intracytoplasmic protein D ( Rickettsia rickettsii , Rocky mountain spotted fever (RMSF)); non-structural protein 6 NS6, non-structural protein 2 NS2, intermediate capsid protein VP6, inner capsid protein VP2, non-structural protein 3 NS3, RNA-directed RNA polymerase L, protein VP3, non-structural protein 1 NS1, non-structural protein 5 NS5, outer capsid glycoprotein VP7, non-structural glycoprotein 4 NS4, outer capsid protein VP4; (Rotavirus, Rotavirus infection); polyprotein P200, glycoprotein E1, glycoprotein E2, protein NS2, capsid protein C (Rubella virus, Rubella); chaperonin GroEL (MopA), inositol phosphate phosphatase SopB, heat shock protein HslU, chaperone protein DnaJ, protein TviB, protein IroN, flagellin FliC, invasion protein SipC, glycoprotein gp43, outer membrane protein LamB, outer membrane protein PagC, outer membrane protein TolC, outer membrane protein NmpC, outer membrane protein FadL, transport protein SadA, transferase WgaP, effector proteins SifA, SteC, SseL, SseJ and SseF ( Salmonella genus, Salmonellosis ); “protein 14, non-structural protein NS7b, non-structural protein NS8a, protein 9b, protein 3a, nucleoprotein N, non-structural protein NS3b, non-structural protein NS6, protein 7a, non-structural protein NS8b, membrane protein M, envelope small membrane protein EsM, replicase polyprotein 1a, spike glycoprotein S, replicase polyprotein lab; SARS coronavirus, SARS (Severe Acute Respiratory Syndrome)); serin protease, Atypical Sarcoptes Antigen 1 ASA1, glutathione S-transferases GST, cystein protease, serine protease, apolipoprotein ( Sarcoptes scabiei , Scabies); glutathione S-transferases GST, paramyosin, hemoglbinase SM32, major egg antigen, 14 kDa fatty acid-binding protein Sm14, major larval surface antigen P37, 22.6 kDa tegumental antigen, calpain CANP, triphospate isomerase Tim, surface protein 9B, outer capsid protein VP2, 23 kDa integral membrane protein Sm23, Cu/Zn-superoxide dismutase, glycoprotein Gp, myosin ( Schistosoma genus, Schistosomiasis (Bilharziosis)); 60 kDa chaperonin, 56 kDa type-specific antigen, pyruvate phosphate dikinase, 4-hydroxybenzoate octaprenyltransferase (Orientia tsutsugamushi, Scrub typhus); dehydrogenase GuaB, invasion protein Spa32, invasin IpaA, invasin IpaB, invasin IpaC, invasin IpaD, invasin IpaH, invasin IpaJ ( Shigella genus, Shigellosis (Bacillary dysentery)); protein P53, virion protein US10 homolog, transcriptional regulator IE63, transcriptional transactivator IE62, protease P33, alpha trans-inducing factor 74 kDa protein, deoxyuridine 5′-triphosphate nucleotidohydrolase, transcriptional transactivator IE4, membrane protein UL43 homolog, nuclear phosphoprotein UL3 homolog, nuclear protein UL4 homolog, replication origin-binding protein, membrane protein 2, phosphoprotein 32, protein 57, DNA polymerase processivity factor, portal protein 54, DNA primase, tegument protein UL14 homolog, tegument protein UL21 homolog, tegument protein UL55 homolog, tripartite terminase subunit UL33 homolog, tripartite terminase subunit UL15 homolog, capsid-binding protein 44, virion-packaging protein 43 (Varicella zoster virus (VZV), Shingles (Herpes zoster)); truncated 3-beta hydroxy-5-ene steroid dehydrogenase homolog, virion membrane protein A13, protein A19, protein A31, truncated protein A35 homolog, protein A37.5 homolog, protein A47, protein A49, protein A51, semaphorin-like protein A43, serine proteinase inhibitor 1, serine proteinase inhibitor 2, serine proteinase inhibitor 3, protein A6, protein B15, protein C1, protein C5, protein C6, protein F7, protein F8, protein F9, protein F11, protein F14, protein F15, protein F16 (Variola major or Variola minor, Smallpox (Variola)); adhesin/glycoprotein gp70, proteases ( Sporothrix schenckii , Sporotrichosis); heme-iron binding protein IsdB, collagen adhesin Cna, clumping factor A ClfA, protein MecA, fibronectin-binding protein A FnbA, enterotoxin type A EntA, enterotoxin type B EntB, enterotoxin type C EntC1, enterotoxin type C EntC2, enterotoxin type D EntD, enterotoxin type E EntE, Toxic shock syndrome toxin-1 TSST-1, Staphylokinase, Penicillin binding protein 2a PBP2a (MecA), secretory antigen SssA ( Staphylococcus genus, Staphylococcal food poisoning); heme-iron binding protein IsdB, collagen adhesin Cna, clumping factor A ClfA, protein MecA, fibronectin-binding protein A FnbA, enterotoxin type A EntA, enterotoxin type B EntB, enterotoxin type C EntC1, enterotoxin type C EntC2, enterotoxin type D EntD, enterotoxin type E EntE, Toxic shock syndrome toxin-1 TSST-1, Staphylokinase, Penicillin binding protein 2a PBP2a (MecA), secretory antigen SssA ( Staphylococcus genus e.g. aureus , Staphylococcal infection); antigen Ss-IR, antigen NIE, strongylastacin, Na+-K+ ATPase Sseat-6, tropomysin SsTmy-1, protein LEC-5, 41 kDa aantigen P5, 41-kDa larval protein, 31-kDa larval protein, 28-kDa larval protein ( Strongyloides stercoralis , Strongyloidiasis); glycerophosphodiester phosphodiesterase GlpQ (Gpd), outer membrane protein TmpB, protein Tp92, antigen TpF1, repeat protein Tpr, repeat protein F TprF, repeat protein G TprG, repeat protein I TprI, repeat protein J TprJ, repeat protein K TprK, treponemal membrane protein A TmpA, lipoprotein, 15 kDa Tpp15, 47 kDa membrane antigen, miniferritin TpF1, adhesin Tp0751, lipoprotein TP0136, protein TpN17, protein TpN47, outer membrane protein TP0136, outer membrane protein TP0155, outer membrane protein TP0326, outer membrane protein TP0483, outer membrane protein TP0956 ( Treponema pallidum , Syphilis); Cathepsin L-like proteases, 53/25-kDa antigen, 8 kDa family members, cysticercus protein with a marginal trypsin-like activity TsAg5, oncosphere protein TSOL18, oncosphere protein TSOL45-1A, lactate dehydrogenase A LDHA, lactate dehydrogenase B LDHB ( Taenia genus, Taeniasis); tetanus toxin TetX, tetanus toxin C TTC, 140 kDa S layer protein, flavoprotein beta-subunit CT3, phospholipase (lecithinase), phosphocarrier protein HPr ( Clostridium tetani , Tetanus (Lockjaw)); genome polyprotein, protein E, protein M, capsid protein C (Tick-borne encephalitis virus (TBEV), Tick-borne encephalitis); 58-kDa antigen, 68-kDa antigens, Toxocara larvae excretory-secretory antigen TES, 32-kDa glycoprotein, glycoprotein TES-70, glycoprotein GP31, excretory-secretory antigen TcES-57, perienteric fluid antigen Pe, soluble extract antigens Ex, excretory/secretory larval antigens ES, antigen TES-120, polyprotein allergen TBA-1, cathepsin L-like cysteine protease c-cpl-1, 26-kDa protein ( Toxocara canis or Toxocara cati , Toxocariasis (Ocular Larva Migrans (OLM) and Visceral Larva Migrans (VLM))); microneme proteins (MIC1, MIC2, MIC3, MIC4, MIC5, MIC6, MIC7, MIC8), rhoptry protein Rop2, rhoptry proteins (Rop1, Rop2, Rop3, Rop4, Rop5, Rop6, Rop7, Rop16, Rjop17), protein SRi, surface antigen P22, major antigen p24, major surface antigen p30, dense granule proteins (GRA1, GRA2, GRA3, GRA4, GRA5, GRA6, GRA7, GRA8, GRA9, GRA10), 28 kDa antigen, surface antigen SAG1, SAG2 related antigen, nucleoside-triphosphatase 1, nucleoside-triphosphatase 2, protein Stt3, HesB-like domain-containing protein, rhomboid-like protease 5, toxomepsin 1 ( Toxoplasma gondii , Toxoplasmosis); 43 kDa secreted glycoprotein, 53 kDa secreted glycoprotein, paramyosin, antigen Ts21, antigen Ts87, antigen p46000, TSL-1 antigens, caveolin-1 CAV-1, 49 kDa newborn larva antigen, prosaposin homologue, serine protease, serine proteinase inhibitor, 45-kDa glycoprotein Gp45 ( Trichinella spiralis , Trichinellosis); Myb-like transcriptional factors (Myb1, Myb2, Myb3), adhesion protein AP23, adhesion protein AP33, adhesin protein AP33-3, adhesins AP51, adhesin AP65, adhesion protein AP65-1, alpha-actinin, kinesin-associated protein, teneurin, 62 kDa proteinase, subtilisin-like serine protease SUB1, cysteine proteinase gene 3 CP3, alpha-enolase Enol, cysteine proteinase CP30, heat shock proteins (Hsp70, Hsp60), immunogenic protein P270, ( Trichomonas vaginalis , Trichomoniasis); beta-tubulin, 47-kDa protein, secretory leucocyte-like proteinase-1 SLP-1, 50-kDa protein TT50, 17 kDa antigen, 43/47 kDa protein ( Trichuris trichiura , Trichuriasis (Whipworm infection)); protein ESAT-6 (EsxA), 10 kDa filtrate antigen EsxB, secreted antigen 85-B FBPB, fibronectin-binding protein A FbpA (Ag85A), serine protease PepA, PPE family protein PPE18, fibronectin-binding protein D FbpD, immunogenic protein MPT64, secreted protein MPT51, catalase-peroxidase-peroxynitritase T KATG, periplasmic phosphate-binding lipoprotein PSTS3 (PBP-3, Phos-1), iron-regulated heparin binding hemagglutinin Hbha, PPE family protein PPE14, PPE family protein PPE68, protein Mtb72F, protein Apa, immunogenic protein MPT63, periplasmic phosphate-binding lipoprotein PSTS1 (PBP-1), molecular chaperone DnaK, cell surface lipoprotein Mpt83, lipoprotein P23, phosphate transport system permease protein pstA, 14 kDa antigen, fibronectin-binding protein C FbpCI, Alanine dehydrogenase TB43, Glutamine synthetase 1, ESX-1 protein, protein CFP10, TB10.4 protein, protein MPT83, protein MTB12, protein MTB8, Rpf-like proteins, protein MTB32, protein MTB39, crystallin, heat-shock protein HSP65, protein PST-S(usually Mycobacterium tuberculosis , Tuberculosis); outer membrane protein FobA, outer membrane protein FobB, intracellular growth locus IgIC1, intracellular growth locus IgIC2, aminotransferase Wbt1, chaperonin GroEL, 17 kDa major membrane protein TUL4, lipoprotein LpnA, chitinase family 18 protein, isocitrate dehydrogenase, Nif3 family protein, type IV pili glycosylation protein, outer membrane protein tolC, FAD binding family protein, type IV pilin multimeric outer membrane protein, two component sensor protein KdpD, chaperone protein DnaK, protein ToIQ ( Francisella tularensis , Tularemia); “MB antigen, urease, protein GyrA, protein GyrB, protein ParC, protein ParE, lipid associated membrane proteins LAMP, thymidine kinase TK, phospholipase PL-A1, phospholipase PL-A2, phospholipase PL-C, surface-expressed 96-kDa antigen; “( Ureaplasma urealyticum, Ureaplasma urealyticum infection); non-structural polyprotein, structural polyprotein, capsid protein CP, protein E1, protein E2, protein E3, protease P1, protease P2, protease P3 (Venezuelan equine encephalitis virus, Venezuelan equine encephalitis); glycoprotein GP, matrix protein Z, polymerase L, nucleoprotein N (Guanarito virus, Venezuelan hemorrhagic fever); polyprotein, protein E, protein M, capsid protein C, protease NS3, protein NS1, protein NS2A, protein AS2B, protein NS4A, protein NS4B, protein NS5 (West Nile virus, West Nile Fever); capsid protein CP, protein E1, protein E2, protein E3, protease P2 (Western equine encephalitis virus, Western equine encephalitis); genome polyprotein, protein E, protein M, capsid protein C, protease NS3, protein NS1, protein NS2A, protein AS2B, protein NS4A, protein NS4B, protein NS5 (Yellow fever virus, Yellow fever); putative Yop targeting protein YobB, effector protein YopD, effector protein YopE, protein YopH, effector protein YopJ, protein translocation protein YopK, effector protein YopT, protein YpkA, flagellar biosyntheses protein FlhA, peptidase M48, potassium efflux system KefA, transcriptional regulatoer RovA, adhesin Ifp, translocator portein LcrV, protein PcrV, invasin Inv, outer membrane protein OmpF-like porin, adhesin YadA, protein kinase C, phospholipase C1, protein PsaA, mannosyltransferase-like protein WbyK, protein YscU, antigen YPMa ( Yersinia pseudotuberculosis, Yersinia pseudotuberculosis infection); effector protein YopB, 60 kDa chaperonin, protein WbcP, tyrosin-protein phosphatase YopH, protein YopQ, enterotoxin, Galactoside permease, reductaase NrdE, protein YasN, Invasin Inv, adhesin YadA, outer membrane porin F OmpF, protein UspAI, protein EibA, protein Hia, cell surface protein Ail, chaperone SycD, protein LcrD, protein LcrG, protein LcrV, protein SycE, protein YopE, regulator protein TyeA, protein YopM, protein YopN, protein YopO, protein YopT, protein YopD, protease ClpP, protein MyfA, protein FilA, and protein PsaA ( Yersinia enterocolitica , Yersiniosis).

The brackets in the preceding section indicate the particular pathogen or the family of pathogens of which the antigen(s) is/are derived and the infectious disease with which the pathogen is associated.

In a particularly preferred embodiment of the first aspect of the invention the mRNA compound comprises a mRNA sequence comprises a coding region, encoding at least one antigenic peptide or protein derived from hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), matrix protein 1 (M1), matrix protein 2 (M2), non-structural protein 1 (NS1), non-structural protein 2 (NS2), nuclear export protein (NEP), polymerase acidic protein (PA), polymerase basic protein PB1, PB1-F2, or polymerase basic protein 2 (PB2) of an influenza virus or a fragment or variant thereof.

In this context, the amino acid sequence of the at least one antigenic peptide or protein may be selected from any peptide or protein derived from hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), matrix protein 1 (M1), matrix protein 2 (M2), non-structural protein 1 (NS1), non-structural protein 2 (NS2), nuclear export protein (NEP), polymerase acidic protein (PA), polymerase basic protein PB1, PB1-F2, or polymerase basic protein 2 (PB2) of an influenza virus or a fragment or variant or from any synthetically engineered influenza virus peptide or protein.

In a preferred embodiment of the invention the coding region encodes at least one antigenic peptide or protein derived from hemagglutinin (HA) and/or neuraminidase (NA) of an influenza virus or a fragment or variant thereof. In this context the hemagglutinin (HA) and the neuraminidase (NA) may be chosen from the same influenza virus or from different influenza viruses.

In this context it is particularly preferred that the at least one coding region encodes at least one full-length protein of hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), matrix protein 1 (M1), matrix protein 2 (M2), non-structural protein 1 (NS1), non-structural protein 2 (NS2), nuclear export protein (NEP), polymerase acidic protein (PA), polymerase basic protein PB1, PB1-F2, or polymerase basic protein 2 (PB2) of an influenza virus or a variant thereof.

In particularly preferred embodiments the at least one coding region encodes at least one full-length protein of hemagglutinin (HA), and/or at least one full-length protein of neuraminidase (NA) of an influenza virus or a variant thereof.

The term “full-length protein” as used herein typically refers to a protein that substantially comprises the entire amino acid sequence of the naturally occurring protein.

In specific embodiments the influenza virus peptide or protein is derived from an influenza A, B or C virus (strain).

In specific embodiments the virus peptide or protein is derived from a coronavirus, for example, SARS-CoV-1, SARS-CoV-2, MERS, etc. (strain).

The influenza A virus may be selected from influenza A viruses characterized by a hemagglutinin (HA) selected from the group consisting of H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17 and H18. Preferably the influenza A virus is selected from an influenza virus characterized by a hemagglutinin (HA) selected from the group consisting of H1, H3, H5 or H9.

Furthermore, particularly preferred are influenza A viruses characterized by a neuraminidase (NA) selected from the group consisting of N1, N2, N3, N4, N5, N6, N7, N8, N9, N10, and N11. Most preferably the influenza A virus is characterized by a neuraminidase (NA) selected from the group consisting of N1, N2, and N8.

In particularly preferred embodiments the influenza A virus is selected from the group consisting of H1N1, H1N2, H2N2, H3N1, H3N2, H3N8, H5N1, H5N2, H5N3, H5N8, H5N9, H7N1, H7N2, H7N3, H7N4, H7N7, H7N9, H9N2, H10N8, and H10N7, preferably from H1N1, H3N2, H5N1, and H5N8.

In this context it is particularly preferred that the at least one coding region of the inventive mRNA sequence encodes at least one antigenic peptide or protein derived from hemagglutinin (HA) and/or at least one antigenic peptide or protein derived from neuraminidase (NA) of an influenza A virus selected from the group consisting of H1N1, H1N2, H2N2, H3N1, H3N2, H3N8, H5N1, H5N2, H5N3, H5N8, H5N9, H7N1, H7N2, H7N3, H7N4, H7N7, H7N9, H9N2, H10N8 and H10N7, preferably from H1N1, H3N2, H5N1, H5N8 or a fragment or variant thereof.

In the context of the present invention a fragment of a protein or a variant thereof encoded by the at least one coding region of the mRNA sequence according to the invention may typically comprise an amino acid sequence having a sequence identity of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably of at least 70%, more preferably of at least 80%, even more preferably at least 85%, even more preferably of at least 90% and most preferably of at least 95% or even 97%, with an amino acid sequence of the respective naturally occurring full-length protein or a variant thereof.

In specific embodiments the antigenic peptide or protein is derived from a hemagglutinin (HA) protein of an influenza A virus.

Furthermore, in this context the coding region encoding at least one antigenic peptide or protein derived from hemagglutinin (HA) and/or neuraminidase (NA) of an influenza virus or a fragment, variant or derivative thereof, may be selected from any nucleic acid sequence comprising a coding region encoding hemagglutinin (HA) or neuraminidase (NA) derived from any influenza virus isolate or a fragment or variant thereof.

In a particularly preferred embodiment of the first aspect of the invention the mRNA compound comprising an mRNA sequence comprises a coding region, encoding at least one antigenic peptide or protein derived from glycoprotein G of a Rabies virus or a fragment or variant thereof.

In this context, the amino acid sequence of the at least one antigenic peptide or protein may be selected from any peptide or protein derived from a glycoprotein of a Rabies virus or a fragment or variant or from any synthetically engineered Rabies virus peptide or protein.

In a preferred embodiment of the present invention the coding region encodes at least one antigenic peptide or protein derived from a glycoprotein of a Rabies virus or a fragment or variant thereof.

In this context it is particularly preferred that the at least one coding region encodes at least one full-length protein of a glycoprotein of a Rabies virus or a variant thereof.

As used herein, the term “full-length protein” preferably relates to the full-length sequence of protein indicated in the sequence listing of the present invention.

In this context it is further preferred that the at least one coding sequence of the mRNA sequence of the present invention encodes at least one antigenic peptide or protein which is derived from a glycoprotein of a Rabies virus, or a fragment or variant thereof.

Each glycoprotein of a Rabies virus is identified by the database accession number of the corresponding protein (see sequence listing numeric identifier <223> which indicates the Protein or Nucleic Acid Accession No. (GenBank)). If the respective Protein or Nucleic Acid Accession No. (GenBank) is searched further on in the sequence listing, the next SEQ ID NO: which show said Protein or Nucleic Acid Accession No. (GenBank) under numeric identifier <223> corresponding to the nucleic acid sequence of the wild type mRNA encoding said protein.

Furthermore, in this context the coding region encoding at least one antigenic peptide or protein derived from glycoprotein of a Rabies virus or a fragment, variant or derivative thereof, may be selected from any nucleic acid sequence comprising a coding region encoding glycoprotein derived from any Rabies virus isolate or a fragment or variant thereof.

Ebolaviruses and the genetically-related Marburgviruses are human pathogens that cause severe diseases. Ebolaviruses and Marburgviruses are filoviruses, which are enveloped viruses featuring a negative-stranded RNA genome. The family of Filoviridae comprises three genera: Ebolavirus, Marburgvirus and Cuevavirus. The genus of Cuevaviruses as well as Marburgviruses include only one species, i.e. Lloviu cuevavirus (Lloviu virus—LLOV) and Marburg marburgvirus, respectively, which is subdivided in Marburg virus (MARV) and Ravn virus (RAVV). The genus of Ebolaviruses comprises five known species, i.e. Bundibugyo ebolavirus (Bundibugyo virus—BDBV), Reston ebolavirus (Reston virus—RESTV), Sudan ebolavirus (Sudan virus—SUDV), Tai Forest ebolavirus (Tai Forest virus—TAFV) (=Côte d'Ivoire ebolavirus), and Zaire ebolavirus (Ebola virus—EBOV). While Cuevaviruses have been isolated from bats and their potential as a pathogen in humans remains unknown, both Ebolaviruses and Marburgviruses are human pathogens that cause Ebolavirus disease (EVD) and Marburgvirus disease, respectively, characterised by haemorrhagic fever and an extremely high mortality rate. In the context of the present invention, any virus, virus member, virus strain, virus type, virus sub-type, virus isolate, virus variant, or virus serotype or genetic reassortant of a virus belonging to or being related to or being derived from viruses of the families and genera listed above are considered to be a “Ebola virus”.

In a particularly preferred embodiment of the first aspect of the invention the mRNA compound comprising an mRNA sequence comprises a coding region, encoding at least one antigenic peptide or protein derived from the glycoprotein (GP) and/or the matrix protein 40 (VP40) and/or the nucleoprotein (NP) of a virus of the genus Ebolavirus or Marburgvirus or a fragment, variant or derivative thereof.

In this context, the amino acid sequence of the at least one antigenic peptide or protein may be selected from any peptide or protein derived from glycoprotein (GP) and/or the matrix protein 40 (VP40) and/or the nucleoprotein (NP) a glycoprotein of an Ebola virus or a fragment or variant or from any synthetically engineered Ebola virus peptide or protein.

In a preferred embodiment of the present invention the coding region encodes at least one antigenic peptide or protein derived from a glycoprotein of an Ebola virus or a fragment or variant thereof. In this context it is particularly preferred that the at least one coding region encodes at least one full-length protein of a glycoprotein of an Ebola virus or a variant thereof.

In particularly preferred embodiments the mRNA sequence comprises at least one coding region encoding glycoprotein of a Ebola virus. In particularly preferred embodiments the mRNA sequence comprises at least one coding region encoding VP40 of a Ebola virus. In particularly preferred embodiments the mRNA sequence comprises at least one coding region encoding NP of a Ebola virus.

In particularly preferred embodiments the mRNA sequence comprises at least one coding region encoding an antigenic peptide or protein of an Ebola virus comprising an RNA sequence selected from the following RNA sequences:

mRNA encoding GP protein of Ebola virus: SEQ ID NOs: 37-39 of the patent application WO2016097065, or fragments or variants of these sequences. In this context, SEQ ID NOs: 37-39 of WO2016097065 and the disclosure relating to SEQ ID NOs: 37-39 of WO2016097065 are incorporated herein by reference. mRNA encoding VP40 of Ebola virus: SEQ ID NOs: 40-42 of the patent application WO2016097065, or fragments or variants of these sequences. In this context, SEQ ID NOs: 40-42 of WO2016097065 and the disclosure relating to SEQ ID NOs: 40-42 of WO2016097065 are incorporated herein by reference. mRNA encoding NP of Ebola virus: SEQ ID NOs: 43-44 of the patent application WO2016097065, or fragments or variants of these sequences. In this context, SEQ ID NOs: 43-44 of WO2016097065 and the disclosure relating to SEQ ID NOs: 43-44 of WO2016097065 are incorporated herein by reference.

V.9.3. Tumor Antigens

Preferably, the at least one coding sequence of the mRNA compound comprising an mRNA sequence according to the invention encodes a tumor antigen, preferably as defined herein, or a fragment or variant thereof, wherein the tumor antigen is preferably selected from the group consisting of 1A01_HLA-A/m; 1A02; 5T4; ACRBP; AFP; AKAP4; alpha-actinin-_4/m; alpha-methylacyl-coenzyme_A_racemase; ANDR; ART-4; ARTC1/m; AURKB; B2MG; B3GN5; B4GN1; B7H4; BAGE-1; BASI; BCL-2; bcr/abl; beta-catenin/m; BING-4; BIRC7; BRCA1/m; BY55; calreticulin; CAMEL; CASP-8/m; CASPA; cathepsin_B; cathepsin_L; CD1A; CD1B; CD1C; CD1D; CD1E; CD20; CD22; CD276; CD33; CD3E; CD3Z; CD44_Isoform_1; CD44_Isoform_6; CD4; CD52; CD55; CD56; CD80; CD86; CD8A; CDC27/m; CDE30; CDK4/m; CDKN2A/m; CEA; CEAM6; CH3L2; CLCA2; CML28; CML66; COA-1/m; coactosin-like_protein; collagen_XXIII; COX-2; CP1B1; CSAG2; CT45A1; CT55; CT-_9/BRD6; CTAG2_Isoform_LAGE-1A; CTAG2_Isoform_LAGE-1B; CTCFL; Cten; cyclin_B1; cyclin_D1; cyp-B; DAM-10; DEPlA; E7; EF1A2; EFTUD2/m; EGFR; EGLN3; ELF2/m; EMMPRIN; EpCam; EphA2; EphA3; ErbB3; ERBB4; ERG; ETV6; EWS; EZH2; FABP7; FCGR3A_Version_1; FCGR3A_Version_2; FGF5; FGFR2; fibronectin; FOS; FOXP3; FUT1; G250; GAGE-1; GAGE-2; GAGE-3; GAGE-4; GAGE-5; GAGE-6; GAGE7b; GAGE-8_(GAGE-2D); GASR; GnT-V; GPC3; GPNMB/m; GRM3; HAGE; hepsin; Her2/neu; HLA-A2/m; homeobox_NKX3.1; HOM-TES-85; HPG1; HS71A; HS71B; HST-2; hTERT; iCE; IF2B3; IL10; IL-13Ra2; IL2-RA; IL2-RB; IL2-RG; IL-5; IMP3; ITA5; ITB1; ITB6; kallikrein-2; kallikrein-4; KI20A; KIAA0205; KIF2C; KK-LC-1; LDLR; LGMN; LIRB2; LY6K; MAGA5; MAGA8; MAGAB; MAGE-A10; MAGE-A12; MAGE-A1; MAGE-A2; MAGE-A3; MAGE-A4; MAGE-A6; MAGE-A9; MAGE-B10; MAGE-B16; MAGE-B17; MAGE-Bi; MAGE-B2; MAGE-B3; MAGE-B4; MAGE-B5; MAGE-B6; MAGE-C1; MAGE-C2; MAGE-C3; MAGE-D1; MAGE-D2; MAGE-D4; MAGE-_E1; MAGE-E1_(MAGE1); MAGE-E2; MAGE-F1; MAGE-H1; MAGEL2; mammaglobin_A; MART-1/melan-A; MART-2; MC1_R; M-CSF; mesothelin; MITF; MMP1_1; MMP7; MUC-1; MUM-1/m; MUM-2/m; MYCN; MYO1A; MYO1B; MYO1C; MYO1D; MYO1E; MYO1F; MYO1G; MYO1H; NA17; NA88-A; Neo-PAP; NFYC/m; NGEP; NPM; NRCAM; NSE; NUF2; NY-ESO-1; OA1; OGT; OS-9; osteocalcin; osteopontin; p53; PAGE-4; PAI-1; PAI-2; PAP; PATE; PAX3; PAX5; PD1L1; PDCD1; PDEF; PECA1; PGCB; PGFRB; Pim-1_-Kinase; Pin-1; PLAC1; PMEL; PML; POTEF; POTE; PRAME; PRDX5/m; PRM2; prostein; proteinase-3; PSA; PSB9; PSCA; PSGR; PSM; PTPRC; RAB8A; RAGE-1; RARA; RASH; RASK; RASN; RGS5; RHAMM/CD168; RHOC; RSSA; RU1; RU2; RUNX1; S-100; SAGE; SART-_1; SART-2; SART-3; SEPR; SERPINB5; SIA7F; SIA8A; SIAT9; SIRT2/m; SOX10; SP17; SPNXA; SPXN3; SSX-1; SSX-2; SSX3; SSX-4; ST1A1; STAG2; STAMP-1; STEAP-1; Survivin-2B; survivin; SYCP1; SYT-SSX-1; SYT-SSX-2; TARP; TCRg; TF2AA; TGFB1; TGFR2; TGM-4; TIE2; TKTL1; TPI/m; TRGV11; TRGV9; TRPC1; TRP-p8; TSG10; TSPY1; TVC_(TRGV3); TX101; tyrosinase; TYRP1; TYRP2; UPA; VEGFR1; WT1; and XAGE1.

Further antigens useful for the present invention are shown herein below (gene names followed by bracket with protein accession NOs):

1A01_HLA-A/m (UniProtKB: P30443); 1A02 (UniProtKB: P01892); 5T4 (UniProtKB: Q13641); ACRBP (UniProtKB: Q8NEB7); AFP (UniProtKB: P02771); AKAP4 (UniProtKB: Q5JQC9); alpha-actinin-_4/m (UniProtKB: B4DSX0); alpha-actinin-4/m (UniProtKB: B4E337); alpha-actinin-_4/m (UniProtKB: 043707); alpha-methylacyl-coenzyme A_racemase (UniProtKB: A0A024RE16); alpha-methylacyl-coenzyme A_racemase (UniProtKB: A8KAC3); ANDR (UniProtKB: P10275); ART-4 (UniProtKB: Q9ULX3); ARTCi/m (UniProtKB: P52961); AURKB (UniProtKB: Q96GD4); B2MG (UniProtKB: P61769); B3GN5 (UniProtKB: Q9BYG0); B4GN1 (UniProtKB: Q00973); B7H4 (UniProtKB: Q7Z7D3); BAGE-1 (UniProtKB: Q13072); BASI (UniProtKB: P35613); BCL-2 (UniProtKB: A9QXG9); bcr/abl (UniProtKB: A9UEZ4); bcr/abl (UniProtKB: A9UEZ7); bcr/abl (UniProtKB: A9UEZ8); bcr/abl (UniProtKB: A9UEZ9); bcr/abl (UniProtKB: A9UF00); bcr/abl (UniProtKB: A9UF01); bcr/abl (UniProtKB: A9UF03); bcr/abl (UniProtKB: A9UF04); bcr/abl (UniProtKB: A9UF05); bcr/abl (UniProtKB: A9UF06); bcr/abl (UniProtKB: A9UF08); beta-catenin/m (UniProtKB: P35222); beta-catenin/m (UniProtKB: Q8WYA6); BING-4 (UniProtKB: 015213); BIRC7 (UniProtKB: Q96CA5); BRCA1/m (UniProtKB: A0A024R1V0); BRCA1/m (UniProtKB: A0A024R1V7); BRCA1/m (UniProtKB: A0A024RIZ8); BRCA1/m (UniProtKB: A0A068BFX7); BRCA1/m (UniProtKB: C6YB45); BRCA1/m (UniProtKB: C6YB47); BRCA1/m (UniProtKB: G3XAC3); BY55 (UniProtKB: 095971); calreticulin (UniProtKB: B4DHR1); calreticulin (UniProtKB: B4E2Y9); calreticulin (UniProtKB: P27797); calreticulin (UniProtKB: Q96L12); CAMEL (UniProtKB: 095987); CASP-8/m (UniProtKB: Q14790); CASPA (UniProtKB: Q92851-4); cathepsin_B (UniProtKB: A0A024R374); cathepsin_B (UniProtKB: P07858); cathepsin_L (UniProtKB: A0A024R276); cathepsin_L (UniProtKB: P07711); cathepsin_L (UniProtKB: Q9HBQ7); CD1A (UniProtKB: P06126); CD1B (UniProtKB: P29016); CD1C (UniProtKB: P29017); CD1D (UniProtKB: P15813); CD1E (UniProtKB: P15812); CD20 (UniProtKB: P11836); CD22 (UniProtKB: 060926); CD22 (UniProtKB: P20273); CD22 (UniProtKB: QOEAF5); CD276 (UniProtKB: Q5ZPR3); CD33 (UniProtKB: B4DF51); CD33 (UniProtKB: P20138); CD33 (UniProtKB: Q546G0); CD3E (UniProtKB: P07766); CD3Z (UniProtKB: P20963); CD44_Isoform_1 (UniProtKB: P16070); CD44_Isoform_6 (UniProtKB: P16070-6); CD4 (UniProtKB: P01730); CD52 (UniProtKB: P31358); CD52 (UniProtKB: Q6IBD0); CD52 (UniProtKB: V9HWN9); CD55 (UniProtKB: B1AP15); CD55 (UniProtKB: D3DT85); CD55 (UniProtKB: D3DT86); CD55 (UniProtKB: P08174); CD56 (UniProtKB: P13591); CD80 (UniProtKB: AONOP2); CD80 (UniProtKB: P33681); CD86 (UniProtKB: P42081); CD8A (UniProtKB: P01732); CDC27/m (UniProtKB: G5EA36); CDC27/m (UniProtKB: P30260); CDE30 (UniProtKB: P28908); CDK4/m (UniProtKB: A0A024RBB6); CDK4/m (UniProtKB: P11802); CDK4/m (UniProtKB: Q6LC83); CDK4/m (UniProtKB: Q96BE9); CDKN2A/m (UniProtKB: D1LYX3); CDKN2A/m (UniProtKB: G3XAG3); CDKN2A/m (UniProtKB: K7PML8); CDKN2A/m (UniProtKB: L8E941); CDKN2A/m (UniProtKB: Q8N726); CEA (RefSeq: NP_004354); CEAM6 (UniProtKB: P40199); CH3L2 (UniProtKB: Q15782); CLCA2 (UniProtKB: Q9UQC9); CML28 (UniProtKB: Q9NQT4); CML66 (UniProtKB: Q96RS6); COA-1/m (UniProtKB: Q5T124); coactosin-like_protein (UniProtKB: Q14019); collagen_XXIII (UniProtKB: L8EAS4); collagen_XXIII (UniProtKB: Q86Y22); COX-2 (UniProtKB: Q6ZYK7); CP1B1 (UniProtKB: Q16678); CSAG2 (UniProtKB: Q9Y5P2-2); CSAG2 (UniProtKB: Q9Y5P2); CT45A1 (UniProtKB: Q5HYN5); CT55 (UniProtKB: Q8WUE5); CT-_9/BRD6 (UniProtKB: Q58F21); CTAG2_Isoform_LAGE-1A (UniProtKB: 075638-2); CTAG2_Isoform_LAGE-1B (UniProtKB: 075638); CTCFL (UniProtKB: Q8NI51); Cten (UniProtKB: Q8IZW8); cyclin_B1 (UniProtKB: P14635); cyclin_D1 (UniProtKB: P24385); cyp-B (UniProtKB: P23284); DAM-10 (UniProtKB: P43366); DEPlA (UniProtKB: Q5TB30); E7 (UniProtKB: P03129); E7 (UniProtKB: P06788); E7 (UniProtKB: P17387); E7 (UniProtKB: P06429); E7 (UniProtKB: P27230); E7 (UniProtKB: P24837); E7 (UniProtKB: P21736); E7 (UniProtKB: P26558); E7 (UniProtKB: P36831); E7 (UniProtKB: P36833); E7 (UniProtKB: Q9QCZ1); E7 (UniProtKB: Q81965); E7 (UniProtKB: Q80956); EF1A2 (UniProtKB: Q05639); EFTUD2/m (UniProtKB: Q15029); EGFR (UniProtKB: A0A0B4J1Y5); EGFR (UniProtKB: E7BSV0); EGFR (UniProtKB: LOR6G1); EGFR (UniProtKB: P00533-2); EGFR (UniProtKB: P00533); EGFR (UniProtKB: Q147T7); EGFR (UniProtKB: Q504U8); EGFR (UniProtKB: Q8NDU8); EGLN3 (UniProtKB: Q9H6Z9); ELF2/m (UniProtKB: B7Z720); EMMPRIN (UniProtKB: Q54A51); EpCam (UniProtKB: P16422); EphA2 (UniProtKB: P29317); EphA3 (UniProtKB: P29320); EphA3 (UniProtKB: Q6P4R6); ErbB3 (UniProtKB: B3KWG5); ErbB3 (UniProtKB: B4DGQ7); ERBB4 (UniProtKB: Q15303); ERG (UniProtKB: P11308); ETV6 (UniProtKB: P41212); EWS (UniProtKB: Q01844); EZH2 (UniProtKB: F2YMM1); EZH2 (UniProtKB: G3XAL2); EZH2 (UniProtKB: LOR855); EZH2 (UniProtKB: Q15910); EZH2 (UniProtKB: S4S3R8); FABP7 (UniProtKB: 015540); FCGR3A_Version_1 (UniProtKB: P08637); FCGR3A_Version_2 (CCDS: CCDS1232.1); FGF5 (UniProtKB: P12034); FGF5 (UniProtKB: Q60518); FGFR2 (UniProtKB: P21802); fibronectin (UniProtKB: A0A024R5I6); fibronectin (UniProtKB: A0A024RB01); fibronectin (UniProtKB: A0A024RDT9); fibronectin (UniProtKB: A0A024RDV5); fibronectin (UniProtKB: A6NH44); fibronectin (UniProtKB: A8K6A5); fibronectin (UniProtKB: B2R627); fibronectin (UniProtKB: B3KXM5); fibronectin (UniProtKB: B4DIC5); fibronectin (UniProtKB: B4DN21); fibronectin (UniProtKB: B4DS98); fibronectin (UniProtKB: B4DTH2); fibronectin (UniProtKB: B4DTK1); fibronectin (UniProtKB: B4DU16); fibronectin (UniProtKB: B7Z3W5); fibronectin (UniProtKB: B7Z939); fibronectin (UniProtKB: G5E9X3); fibronectin (UniProtKB: Q9H382); FOS (UniProtKB: P01100); FOXP3 (UniProtKB: Q9BZS1); FUT1 (UniProtKB: P19526); G250 (UniProtKB: Q16790); GAGE-1 (Genbank: AAA82744); GAGE-2 (UniProtKB: Q6NT46); GAGE-3 (UniProtKB: Q13067); GAGE-4 (UniProtKB: Q13068); GAGE-5 (UniProtKB: Q13069); GAGE-6 (UniProtKB: Q13070); GAGE7b (UniProtKB: 076087); GAGE-8_(GAGE-2D) (UniProtKB: Q9UEU5); GASR (UniProtKB: P32239); GnT-V (UniProtKB: Q09328); GPC3 (UniProtKB: I6QTG3); GPC3 (UniProtKB: P51654); GPC3 (UniProtKB: Q8IYG2); GPNMB/m (UniProtKB: A0A024RA55); GPNMB/m (UniProtKB: Q14956); GPNMB/m (UniProtKB: Q8IXJ5); GPNMB/m (UniProtKB: Q96F58); GRM3 (UniProtKB: Q14832); HAGE (UniProtKB: Q9NXZ2); hepsin (UniProtKB: B2ZDQ2); hepsin (UniProtKB: P05981); Her2/neu (UniProtKB: B4DTR1); Her2/neu (UniProtKB: L8E8G2); Her2/neu (UniProtKB: P04626); Her2/neu (UniProtKB: Q9UK79); HLA-A2/m (UniProtKB: Q95387); HLA-A2/m (UniProtKB: Q9MYF8); homeobox_NKX3.1 (UniProtKB: Q99801); HOM-TES-85 (UniProtKB: B2RBQ6); HOM-TES-85 (UniProtKB: Q9P127); HPG1 (Pubmed: 12543784); HS71A (UniProtKB: PODMV8); HS71B (UniProtKB: PODMV9); HST-2 (UniProtKB: P10767); hTERT (UniProtKB: 094807); iCE (UniProtKB: 000748); IF2B3 (UniProtKB: 000425); I10 (UniProtKB: P22301); IL-13Ra2 (UniProtKB: Q14627); 1L2-RA (UniProtKB: P01589); IL2-RB (UniProtKB: P14784); IL2-RG (UniProtKB: P31785); IL-5 (UniProtKB: P05113); IMP3 (UniProtKB: Q9NV31); ITA5 (UniProtKB: P08648); ITB1 (UniProtKB: P05556); ITB6 (UniProtKB: P18564); kallikrein-2 (UniProtKB: A0A024R4J4); kallikrein-2 (UniProtKB: A0A024R4N3); kallikrein-2 (UniProtKB: BOAZU9); kallikrein-2 (UniProtKB: B4DU77); kallikrein-2 (UniProtKB: P20151); kallikrein-2 (UniProtKB: Q6T774); kallikrein-2 (UniProtKB: Q6T775); kallikrein-4 (UniProtKB: A0A0C4DFQ5); kallikrein-4 (UniProtKB: Q5BQA0); kallikrein-4 (UniProtKB: Q96PTO); kallikrein-4 (UniProtKB: Q96PT1); kallikrein-4 (UniProtKB: Q9Y5K2); KI20A (UniProtKB: 095235); KIAA0205 (UniProtKB: Q92604); KIF2C (UniProtKB: Q99661); KK-LC-1 (UniProtKB: Q5H943); LDLR (UniProtKB: P01130); LGMN (UniProtKB: Q99538); LIRB2 (UniProtKB: Q8N423); LY6K (UniProtKB: Q17RY6); MAGA5 (UniProtKB: P43359); MAGA8 (UniProtKB: P43361); MAGAB (UniProtKB: P43364); MAGE-A10 (UniProtKB: A0A024RC14); MAGE-A12 (UniProtKB: P43365); MAGE-A1 (UniProtKB: P43355); MAGE-A2 (UniProtKB: P43356); MAGE-A3 (UniProtKB: P43357); MAGE-A4 (UniProtKB: A0A024RC12); MAGE-A4 (UniProtKB: P43358); MAGE-A4 (UniProtKB: Q1RN33); MAGE-A6 (UniProtKB: A8K072); MAGE-A6 (UniProtKB: P43360); MAGE-A6 (UniProtKB: Q6FHI5); MAGE-A9 (UniProtKB: P43362); MAGE-B10 (UniProtKB: Q96LZ2); MAGE-B16 (UniProtKB: A2A368); MAGE-B17 (UniProtKB: A8MXT2); MAGE-B1 (UniProtKB: Q96TG1); MAGE-B2 (UniProtKB: 015479); MAGE-B3 (UniProtKB: 015480); MAGE-B4 (UniProtKB: 015481); MAGE-B5 (UniProtKB: Q9BZ81); MAGE-B6 (UniProtKB: Q8N7X4); MAGE-C1 (UniProtKB: 060732); MAGE-C2 (UniProtKB: Q9UBF1); MAGE-C3 (UniProtKB: Q8TD91); MAGE-D1 (UniProtKB: Q9Y5V3); MAGE-D2 (UniProtKB: Q9UNF1); MAGE-D4 (UniProtKB: Q96JG8); MAGE-E1 (UniProtKB: Q6IAI7); MAGE-E1_(MAGEl) (UniProtKB: Q9HCI5); MAGE-E2 (UniProtKB: Q8TD90); MAGE-F1 (UniProtKB: Q9HAY2); MAGE-H1 (UniProtKB: Q9H213); MAGEL2 (UniProtKB: Q9UJ55); mammaglobin_A (UniProtKB: Q13296); mammaglobin_A (UniProtKB: Q6NX70); MART-1/melan-A (UniProtKB: Q16655); MART-2 (UniProtKB: Q5VTY9); MC1_R (UniProtKB: Q01726); MC1_R (UniProtKB: Q1JUL4); MC1_R (UniProtKB: Q1JUL6); MC1_R (UniProtKB: QIJUL8); MC1_R (UniProtKB: Q1JUL9); MC1_R (UniProtKB: Q1JUM0); MC1_R (UniProtKB: Q1JUM2); MC1_R (UniProtKB: Q1JUM3); MC1_R (UniProtKB: Q1JUM4); MC1_R (UniProtKB: Q1JUM5); MC1_R (UniProtKB: Q6UR92); MC1_R (UniProtKB: Q6UR94); MC1_R (UniProtKB: Q6UR95); MC1_R (UniProtKB: Q6UR96); MC1_R (UniProtKB: Q6UR97); MC1_R (UniProtKB: Q6UR98); MC1_R (UniProtKB: Q6UR99); MC1_R (UniProtKB: Q6URA0); MC1_R (UniProtKB: Q86YW1); MC1_R (UniProtKB: V9Q5S2); MC1_R (UniProtKB: V9Q671); MC1_R (UniProtKB: V9Q783); MC1_R (UniProtKB: V9Q7F1); MC1_R (UniProtKB: V9Q8N1); MC1_R (UniProtKB: V9Q977); MC1_R (UniProtKB: V9Q9P5); MC1_R (UniProtKB: V9Q9R8); MC1_R (UniProtKB: V9QAE0); MC1_R (UniProtKB: V9QAR2); MC1_R (UniProtKB: V9QAW3); MC1_R (UniProtKB: V9QB02); MC1_R (UniProtKB: V9QB58); MC1_R (UniProtKB: V9QBY6); MC1_R (UniProtKB: V9QC17); MC1_R (UniProtKB: V9QC66); MC1_R (UniProtKB: V9QCQ4); MC1_R (UniProtKB: V9QDF4); MC1_R (UniProtKB: V9QDN7); MC1_R (UniProtKB: V9QDQ6); M-CSF (UniProtKB: P09603); mesothelin (UniProtKB: Q13421); MITF (UniProtKB: 075030-8); MITF (UniProtKB: 075030-9); MITF (UniProtKB: 075030); MMP1_1 (UniProtKB: B3KQS8); MMP7 (UniProtKB: P09237); MUC-1 (Genbank: AAA60019); MUM-1/m (RefSeq: NP_116242); MUM-2/m (UniProtKB: Q9Y5R8); MYCN (UniProtKB: P04198); MYO1A (UniProtKB: Q9UBC5); MYO1B (UniProtKB: 043795); MYO1C (UniProtKB: 000159); MYO1D (UniProtKB: 094832); MYO1E (UniProtKB: Q12965); MYO1F (UniProtKB: 000160); MYO1G (UniProtKB: BOI1T2); MYO1H (RefSeq: NP_001094891); NA17 (UniProtKB: Q3V5L5); NA88-A (Pubmed: 10790436); Neo-PAP (UniProtKB: Q9BWT3); NFYC/m (UniProtKB: Q13952); NGEP (UniProtKB: Q6IWH7); NPM (UniProtKB: P06748); NRCAM (UniProtKB: Q92823); NSE (UniProtKB: P09104); NUF2 (UniProtKB: Q9BZD4); NY-ESO-1 (UniProtKB: P78358); OA1 (UniProtKB: P51810); OGT (UniProtKB: 015294); OS-9 (UniProtKB: B4DH11); OS-9 (UniProtKB: B4E321); OS-9 (UniProtKB: B7Z8E7); OS-9 (UniProtKB: Q13438); osteocalcin (UniProtKB: P02818); osteopontin (UniProtKB: A0A024RDE2); osteopontin (UniProtKB: A0A024RDE6); osteopontin (UniProtKB: A0A024RDJ0); osteopontin (UniProtKB: B7Z351); osteopontin (UniProtKB: F2YQ21); osteopontin (UniProtKB: P10451); p53 (UniProtKB: P04637); PAGE-4 (UniProtKB: 060829); PAI-1 (UniProtKB: P05121); PAI-2 (UniProtKB: P05120); PAP (UniProtKB: Q06141); PAP (UniProtKB: Q53S56); PATE (UniProtKB: Q8WXA2); PAX3 (UniProtKB: P23760); PAX5 (UniProtKB: Q02548); PD1L1 (UniProtKB: Q9NZQ7); PDCD1 (UniProtKB: Q15116); PDEF (UniProtKB: 095238); PECA1 (UniProtKB: P16284); PGCB (UniProtKB: Q96GW7); PGFRB (UniProtKB: P09619); Pim-1_-Kinase (UniProtKB: A0A024RD25); Pin-1 (UniProtKB: 015428); Pin-1 (UniProtKB: Q13526); Pin-1 (UniProtKB: Q49AR7); PLAC1 (UniProtKB: Q9HBJ0); PMEL (UniProtKB: P40967); PML (UniProtKB: P29590); POTEF (UniProtKB: A5A3E0); POTE (UniProtKB: Q86YR6); PRAME (UniProtKB: A0A024R1E6); PRAME (UniProtKB: P78395); PRDX5/m (UniProtKB: P30044); PRM2 (UniProtKB: P04554); prostein (UniProtKB: Q96JT2); proteinase-3 (UniProtKB: D6CHE9); proteinase-3 (UniProtKB: P24158); PSA (UniProtKB: P55786); PSB9 (UniProtKB: P28065); PSCA (UniProtKB: D3DWI6); PSCA (UniProtKB: 043653); PSGR (UniProtKB: Q9H255); PSM (UniProtKB: Q04609); PTPRC (RefSeq: NP_002829); RAB8A (UniProtKB: P61006); RAGE-1 (UniProtKB: Q9UQ07); RARA (UniProtKB: P10276); RASH (UniProtKB: P01112); RASK (UniProtKB: P01116); RASN (UniProtKB: PO1111); RGS5 (UniProtKB: 015539); RHAMM/CD168 (UniProtKB: 075330); RHOC (UniProtKB: P08134); RSSA (UniProtKB: P08865); RU1 (UniProtKB: Q9UHJ3); RU2 (UniProtKB: Q9UHG0); RUNX1 (UniProtKB: Q01196); S-100 (UniProtKB: V9HW39); SAGE (UniProtKB: Q9NXZ1); SART-_1 (UniProtKB: 043290); SART-2 (UniProtKB: Q9UL01); SART-3 (UniProtKB: Q15020); SEPR (UniProtKB: Q12884); SERPINB5 (UniProtKB: P36952); SIA7F (UniProtKB: Q969X2); SIA8A (UniProtKB: Q92185); SIAT9 (UniProtKB: Q9UNP4); SIRT2/m (UniProtKB: A0A024ROG8); SIRT2/m (UniProtKB: Q8IXJ6); SOX10 (UniProtKB: P56693); SP17 (UniProtKB: Q15506); SPNXA (UniProtKB: Q9NS26); SPXN3 (UniProtKB: Q5MJ09); SSX-1 (UniProtKB: Q16384); SSX-2 (UniProtKB: Q16385); SSX3 (UniProtKB: Q99909); SSX-4 (UniProtKB: 060224); ST1A1 (UniProtKB: P50225); STAG2 (UniProtKB: Q8N3U4-2); STAMP-1 (UniProtKB: Q8NFT2); STEAP-1 (UniProtKB: A0A024RA63); STEAP-1 (UniProtKB: Q9UHE8); Survivin-2B (UniProtKB: 015392-2); survivin (UniProtKB: 015392); SYCP1 (UniProtKB: A0A024ROI2); SYCP1 (UniProtKB: B7ZLS9); SYCP1 (UniProtKB: Q15431); SYCP1 (UniProtKB: Q3MHC4); SYT-SSX-1 (UniProtKB: A4PIV7); SYT-SSX-1 (UniProtKB: A4PIV8); SYT-SSX-2 (UniProtKB: A4PIV9); SYT-SSX-2 (UniProtKB: A4PIW0); TARP (UniProtKB: Q0VGM3); TCRg (UniProtKB: A2JGV3); TF2AA (UniProtKB: P52655); TGFB1 (UniProtKB: P01137); TGFR2 (UniProtKB: P37173); TGM-4 (UniProtKB: B2R7D1); TIE2 (UniProtKB: Q02763); TKTL1 (UniProtKB: P51854); TPI/m (UniProtKB: P60174); TRGV11 (UniProtKB: Q99601); TRGV9 (UniProtKB: A4D1X2); TRGV9 (UniProtKB: Q99603); TRGV9 (UniProtKB: Q99604); TRPC1 (UniProtKB: P48995); TRP-p8 (UniProtKB: Q7Z2W7); TSG10 (UniProtKB: Q9BZW7); TSPY1 (UniProtKB: Q01534); TVC_(TRGV3) (Genbank: M13231.1); TX101 (UniProtKB: Q9BY14-2); tyrosinase (UniProtKB: A0A024DBG7); tyrosinase (UniProtKB: L8B082); tyrosinase (UniProtKB: L8B086); tyrosinase (UniProtKB: L8B0B9); tyrosinase (UniProtKB: 075767); tyrosinase (UniProtKB: P14679); tyrosinase (UniProtKB: U3M8NO); tyrosinase (UniProtKB: U3M9D5); tyrosinase (UniProtKB: U3M9J2); TYRP1 (UniProtKB: P17643); TYRP2 (UniProtKB: P40126); UPA (UniProtKB: Q96NZ9); VEGFR1 (UniProtKB: B5A924); WT1 (UniProtKB: A0A0H5AUY0); WT1 (UniProtKB: P19544); WT1 (UniProtKB: Q06250); XAGE1 (UniProtKB: Q9HD64).

V.9.4. Checkpoint Inhibitors

Negative regulatory T cell surface molecules were discovered, which are upregulated in activated T cells in order to dampen their activity, thus reducing the effectiveness of said activated T cells in the killing of tumor cells. These inhibitory molecules were termed negative co-stimulatory molecules due to their homology to the T cell co-stimulatory molecule CD28. These proteins, also referred to as immune checkpoint proteins, function in multiple pathways including the attenuation of early activation signals, competition for positive costimulation and direct inhibition of antigen presenting cells (Bour-Jordan et al., 2011. Immunol Rev. 241(1):180-205).

In the context of the present invention, a checkpoint modulator is typically a molecule, such as a protein (e.g. an antibody), a dominant negative receptor, a decoy receptor, or a ligand or a fragment or variant thereof, which modulates the function of an immune checkpoint protein, e.g. it inhibits or reduces the activity of checkpoint inhibitors (or inhibitory checkpoint molecules) or it stimulates or enhances the activity of checkpoint stimulators (or stimulatory checkpoint molecules). Therefore, checkpoint modulators as defined herein, influence the activity of checkpoint molecules.

In this context, inhibitory checkpoint molecules are defined as checkpoint inhibitors and can be used synonymously. In addition, stimulatory checkpoint molecules are defined as checkpoint stimulators and can be used synonymously.

Preferably, the checkpoint modulator is selected from agonistic antibodies, antagonistic antibodies, ligands, dominant negative receptors, and decoy receptors or combinations thereof.

Methods for generating and using mRNA-encoded antibodies are known in the art (e.g. WO2008/083949 or PCT/EP2017/060226).

Preferred inhibitory checkpoint molecules that may be inhibited by a checkpoint modulator in the context of the invention are PD-1, PD-L1, CTLA-4, PD-L2, LAG3, TIM3/HAVCR2, 2B4, A2aR, B7H3, B7H4, BTLA, CD30, CD160, CD155, GAL9, HVEM, IDO1, IDO2, KIR, LAIR1 and VISTA.

Preferred stimulatory checkpoint molecules that may be stimulated by a checkpoint modulator in the context of the invention are CD2, CD27, CD28, CD40, CD137, CD226, CD276, GITR, ICOS, OX40 and CD70.

According to a preferred embodiment, the pharmaceutical composition or vaccine comprising RNAs of the invention is for use as described herein, wherein the use comprises—as an additional pharmaceutically active ingredient—a checkpoint modulator selected from the group consisting of the checkpoint modulator is selected from the group consisting of a PD-1 inhibitor, a PD-L1 inhibitor, a PD-L2 inhibitor, a CTLA-4 inhibitor, a LAG3 inhibitor, a TIM3 inhibitor, a TIGIT-inhibitor, an OX40 stimulator, a 4-1BB stimulator, a CD40L stimulator, a CD28 stimulator and a GITR stimulator.

According to a preferred embodiment, the checkpoint modulator as used herein targets a member of the PD-1 pathway. Members of the PD-1 pathway are typically proteins, which are associated with PD-1 signaling. On the one hand, this group comprises proteins, which induce PD-1 signaling upstream of PD-1 as e.g. the ligands of PD-1, PD-L1 and PD-L2, and the signal transduction receptor PD-1. On the other hand, this group comprises signal transduction proteins downstream of PD-1 receptor. Particularly preferred as members of the PD-1 pathway in the context of the present invention are PD-1, PD-L1 and PD-L2.

In the context of the present invention, a PD-1 pathway antagonist (or PD-1 inhibitor) is preferably defined herein as a compound capable to impair the PD-1 pathway signaling, preferably signaling mediated by the PD-1 receptor. Therefore, the PD-1 pathway antagonist may be any antagonist directed against any member of the PD-1 pathway capable of antagonizing PD-1 pathway signaling.

In a preferred embodiment, the checkpoint modulator used herein is a PD-1 inhibitor or a PD-L1 inhibitor, wherein the PD-1 inhibitor is preferably an antagonistic antibody directed against PD-1 and the PD-L1 inhibitor is preferably an antagonistic antibody directed against PD-L1.

In this context, the antagonist may be an antagonistic antibody as defined herein, targeting any member of the PD-1 pathway, preferably an antagonistic antibody directed against PD-1 receptor, PD-L1 or PD-L2. Such an antagonistic antibody may also be encoded by a nucleic acid. Also, the PD-1 pathway antagonist may be a fragment of the PD-1 receptor blocking the activity of PD1 ligands. B7-1 or fragments thereof may act as PD1-antagonizing ligands as well. Additionally, a PD-1 pathway antagonist may be a protein comprising (or a nucleic acid coding for) an amino acid sequence capable of binding to PD-1 but preventing PD-1 signaling, e.g. by inhibiting PD-1 and B7-H1 or B7-DL interaction (WO 2014/127917; WO2012062218).

Particularly preferred are the anti-PD1 antibodies Nivolumab (MDX-1106/BMS-936558/ONO-4538), (Brahmer et al., 2010. J Clin Oncol. 28(19):3167-75; PMID: 20516446); Pidilizumab (CT-011), (Berger et al., 2008. Clin Cancer Res. 14(10):3044-51; PMID: 18483370); Pembrolizumab (MK-3475, SCH 900475); AMP-224, and MEDI0680 (AMP-514).

Particularly preferred are also the anti-PD-L1 antibodies MDX-1105/BMS-936559 (Brahmer et al. 2012. N Engl J Med. 366(26):2455-65; PMID: 22658128); atezolizumab (MPDL3280A/RG7446); durvalumab (MEDI4736); and avelumab (MSB0010718).

According to another embodiment, the checkpoint modulator used herein is an OX40 stimulator. OX40 is a member of the TNFR-superfamily of receptors, and is expressed on the surface of antigen-activated mammalian CD4+ and CD8+T lymphocytes. OX40 ligand (OX40L, also known as gp34, ACT-4-L, and CD252) is a protein that specifically interacts with the OX40 receptor. The term OX40L includes the entire OX40 ligand, soluble OX40 ligand, and fusion proteins comprising a functionally active portion of OX40 ligand covalently linked to a second moiety, e.g., a protein domain. Also included within the definition of OX40L are variants which vary in amino acid sequence from naturally occurring OX40L, but which retain the ability to specifically bind to the OX40 receptor. Further included within the definition of OX40L are variants thereof, which enhance the biological activity of OX40. An OX40 agonist is a molecule which induces or enhances the biological activity of OX40, e.g. signal transduction mediated by OX40. An OX40 agonist is preferably defined herein as a binding molecule capable of specific binding to OX40. Therefore, the OX40 agonist may be any agonist binding to OX40 and capable of stimulating OX40 signaling. In this context, the OX40 agonist may be an agonistic antibody binding to OX40.

OX40 agonists and anti-0X40 monoclonal antibodies are described in WO1995/021251, WO1995/012673 and WO1995/21915. Particularly preferred is the anti-0X40 antibody 9B12, a murine anti-0X40 monoclonal antibody directed against the extracellular domain of human OX40 (Weinberg et al., 2006. J. Immunother. 29(6):575-585).

In another embodiment, the checkpoint modulator as used herein is an antagonistic antibody is selected from the group consisting of anti-CTLA4, anti-PD1, anti-PD-L1, anti-Vista, anti-Tim-3, anti-TIGIT, anti-LAG-3, and anti-BTLA.

Preferably, an anti-CTLA4 antibody that may be used as a checkpoint modulator is directed against Cytotoxic T lymphocyte antigen-4 (CTLA-4). CTLA-4 is mainly expressed within the intracellular compartment of T cells. After a potent or long-lasting stimulus to a naïve T cell via the T cell receptor (TCR), CTLA-4 is transported to the cell surface and concentrated at the immunological synapse. CTLA-4 then competes with CD28 for CD80/CD86 and down-modulates TCR signaling via effects on Akt signaling. Thus CTLA-4 functions physiologically as a signal dampener (Weber, J. 2010. Semin. Oncol. 37(5):430-9).

In preferred embodiments, the pharmaceutical composition or vaccine comprising RNAs of the invention is for use as described herein, wherein the use comprises—as an additional pharmaceutically active ingredient—a CTLA4 antagonist, which is preferably an antagonistic antibody directed against CTLA4 (anti-CTLA4 antibody). The term ‘CTLA4 antagonist’ as used herein comprises any compound, such as an antibody, that antagonizes the physiological function of CTLA4. In the context of the present invention, the term ‘anti-CTLA4 antibody’ may refer to an antagonistic antibody directed against CTLA4 (or a functional fragment or variant of said antibody) or to a nucleic acid, preferably an RNA, encoding said antagonistic antibody (or a functional fragment thereof). A functional fragment or variant of an anti-CTLA4 antibody preferably acts as a CTLA4 antagonist. More preferably, the term ‘anti-CTLA4 antibody’ refers to a monoclonal antibody directed against CTLA4 (or a functional fragment or variant of such an antibody) or to a nucleic acid encoding a monoclonal antibody directed against CTLA4 (or a functional fragment or variant of such an antibody). The term ‘anti-CTLA4 antibody’ as used herein may refer to the heavy or light antibody chain, respectively, or also refer to both antibody chains (heavy and light chain), or to a fragment or variant of any one of these chains. Preferably, the fragment or variant of an anti-CTLA4 antibody as used herein is a functional fragment or variant, preferably as described herein.

Particularly preferred are the anti-CTLA-4 antibodies ipilimumab (Yervoy®), tremelimumab, and AGEN-1884. Further preferred anti-CTLA4 antibodies as used herein comprise BMS 734016; BMS-734016; BMS734016; MDX 010; MDX 101; MDX-010; MDX-101; MDX-CTLA-4; MDX-CTLA4; MDX010; Winglore; and Yervoy, or a functional fragment or variant of any one of these antibodies.

According to a further embodiment, the checkpoint modulator as used herein is at least one antibody described in Table 1 or a fragment or variant thereof

V.5. Combination Cancer Therapy

More preferably, the subject receiving the pharmaceutical composition or vaccine comprising RNAs of the invention, the combination thereof or the pharmaceutical composition or vaccine comprising said RNA(s) is a patient suffering from a tumor or cancer disease as described herein and who received or receives chemotherapy (e.g. first-line or second-line chemotherapy), radiotherapy, chemoradiation (combination of chemotherapy and radiotherapy), kinase inhibitors, antibody therapy and/or checkpoint modulators (e.g., CTLA4 inhibitors, PD1 pathway inhibitors), or a patient, who has achieved partial response or stable disease after having received one or more of the treatments specified above. More preferably, the subject is a patient suffering from a tumor or cancer disease as described herein and who received or receives a compound conventionally used in any of these diseases as described herein, more preferably a patient who receives or received a checkpoint modulator.

The following compounds are preferred compounds which preferably are used in standard therapies and can be applied in combination with the pharmaceutical compositions or vaccines comprising RNAs of the invention: Cetuximab (Erbitux), paclitaxel albumin bound (Abraxane), (gimeracil+oteracil+tegafur) (TS-1), Docetaxel (Docetaxel, Doxel, Taxotere, Docetaxel An, Docel, Nanoxel M, Tautax, Docetaxel-AS, Docetaxel-M, Qvidadotax, Relidoce, Taxelo, Oncodocel, Doxotel, Pacancer, Docetrust, Dodetax, Dodabur, Soulaxcin, Taxedol, Docefim, Docetaxel, Ribodocel, Critidoc, Asodoc, Chemodoc, Docelibbs, Docenat, Dincilezan, Dostradixinol, Docefrez, Camitotic, Oncotaxel, Somatixel, Belotaxel, Qvidadotax, Taxceus, Cetadocure, Docetaxel CT, Tevaxter, Docirena, Eurotere, Axtere, Celotax, Taxanit, Drobanos, Cetado, Doxocad, Taxceus, Egidox, Tedocad, Docecad, Docelex, Docetax, Docetaxel, Docetere, Dotax, Taxuba, Monotaxel, Taceedo, Detaxl, Docet, Docetaxel, Ferdotax, Wintaxel), (tegafur+uracil) (Uft, Uft E, Tefudex, Unitoral, Luporal, Tagracil), Fluorouracil (5-FU), (gimeracil+oteracil+tegafur) ODT (TS-1 Combination OD), bleomycin sulfate (Tecnomicina, Cinaleo, Bleomycin, Bloicin-S, Bonar, Bleocin, Bleomycin Sulfate, Bleo, Bleocel, Bleotex, Oncobleo, Bleonco, Bleosol, Lyoble, Bleomycin Sulfate, Blenamax, Bleomycin, Blenoxane, Bleomicina, Bleomycine Bellon, Bleoprim), carboplatin (Carboplatin, Platamine CS, Carbaccord, Carboplatina, Carboplatino, Paraplatin, Carbosin, Tecnocarb, Carbomerck, Paract, Carboplatine CTRS, Carboplatine Intsel Chimos, Carboplatin, Carbokem, Carbotinol, Fauldcarbo, Evocarb, Citoplatina, Platin), ciprofloxacin (Hypoflox, Ufexil), ciprofloxacin hydrochloride (Ciprofloxacin Pharma, Prodin, Ciproxin), cisplatin (Cisplatin, Stritin, Ifapla, Accocit, Unistin, Cancertin, Cisplan, Citoplax, Nuoxin, Placis, Cisplatino, Displanor, Randa, Cispla, Fauldcispla, Briplatin, Platinex, Platinol, Platinex, Riboplatin, Cisplatine, Platistine CS, Platosin, Accocit, Cisplatino) cyclophosphamide (Endoxan, Cyclophosphamide), doxifluridine (Doxifluridine, May Vladimir), doxorubicin (Doxorubicin Hydrochloride, Adriamycin RDF, Doxorubicin, Doxorubicin PFS), epirubicin, hydrochloride (Brecila, Cloridrato De Epirrubicina, Epirubicin, Farmorubicina, Nuovodox, Adnexa, 4-Eppedo, Favicin), fluorouracil (Agicil, Fluorouracil, Fauldfluor, Oncourcil, Flocil, 5 Flucel), folic acid+methotrexate (Truxofol), human adenovirus type 5 (recombinant) (Oncorine), hydroxyurea (Oxyrea, Durea, Myelostat, Riborea, Unidrea, Ondrea, Hydran, Leukocel, Hydroxyurea, Hydrea), ifosfamide (Holoxan, Ifosfamide EG), levamisole (Zirsol), methotrexate Methotrexate (Tratoben, Methotrexate, Fresexate, Neometho, Fauldmetro, Methotrexate Sodium, Methocel, Hytas, Methaccord, Methofill, Metotrexato, Traxacord, Plastomet, Tevatrex, Metrex, Caditrex, Carditrex, Vibzi, Imutrex, Biotrexate, Methorex, Mexate, Neotrexate, Oncotrex, Remtrex, Trixilem, Hi-Trex, Metorex, Trex, Unitrexate, Ebetrexac, Fauldexato, Lantarel, Maxtrex, Miantrex CS, Rheumatrex, Folex, Folex PFS, Abitrexate, Tevametho, Trexall, Emthexate, Abitrexate, Meadow), mitomycin (Mitomycin C, Mitomycin, Mitonco, Lyomit), nedaplatin (Jiebaishu, Aoxianda, Aqupla), nimesulide (Nimulid), nimotuzumab (Biomab EGFR, Laedemab), nitrofurantoin (Furatsilin), ofloxacin (Entof), paclitaxel (Paclitaxel, Taxol), peplomycin sulfate (Pepleo), picibanil (Picibanil), pirarubicin (Pirarubicin Hydrochloride, Therarubicin, Pinorubin), sodium glycididazole (CMNa), tegafur (Utefos, Icarus, Futraful, Tegafur Gimeracil Oteracil Potassium), temoporfin (Foscan), topotecan hydrochloride (Topotecan), ubenimex (Ubenimex), vinblastine sulfate (Vinblastine, Vblastin), vincristine sulfate (Vincristine, Vincristine Sulfate, Vincristin, Sutivin, vindesine sulfate (Eldisine), carboplatin (Carboplatine Qualimed, Carboplatine, Carboplatino, Carboplatin), cisplatin (Cisplatin), docetaxel (Kamdocon, Naltoxater, Docetaxel), fluorouracil (Fluorouracil, Fluorouracile, Fluorouracil), methotrexate (Methotrexate Sodium, Mexate, Mexate Aq, Biometrox, Medsatrexate, Otaxem), vincristine sulfate (Oncovin), fluorouracil, sunitinib malate, acitretin, fibrin sealant, cetuximab, cetuximab, erlotinib, cisplatin; docetaxel; fluorouracil, undisclosed anti-cancer drug, gefitinib, pravastatin sodium, sirolimus, undisclosed chemotherapy, cisplatin; docetaxel; fluorouracil, sirolimus, fluorouracil; undisclosed taxane, methyl aminolevulinate hydrochloride, cisplatin; docetaxel; fluorouracil, erlotinib hydrochloride, cetuximab, imiquimod, undisclosed Chinese herbal medicine, aspirin; enalapril maleate, undisclosed chemotherapy, cetuximab, (gimeracil+oteracil+tegafur); carboplatin; cisplatin, cisplatin; fluorouracil; nimotuzumab, carboplatin; paclitaxel albumin bound, cisplatin; nedaplatin, bleomycin, nedaplatin, cisplatin; paclitaxel, paclitaxel albumin bound, (gimeracil+oteracil+tegafur), bleomycin; undisclosed chemotherapy, apatinib; docetaxel, undisclosed immunomodulatory supplement, BCM-95, aminolevulinic acid hydrochloride, nedaplatin, cisplatin; palifermin, cetuximab, gefitinib, bevacizumab, belagenpumatucel-L, cisplatin; tirapazamine, cisplatin; tirapazamine, cisplatin; gemcitabine; paclitaxel; topotecan; vinorelbine, cisplatin; fluorouracil, panitumumab, carboplatin; docetaxel; gemcitabine hydrochloride; vinorelbine tartrate, amifostine; fluorouracil, cisplatin; fluorouracil, carboplatin; paclitaxel, tirapazamine, cisplatin; epoetin alfa, figitumumab, melphalan; tumor necrosis factor alf, cisplatin, cisplatin; fluorouracil, cisplatin; undisclosed chemotherapy, docetaxel, contusugene ladenovec, cisplatin; fluorouracil; paclitaxel, docetaxel, human papillomavirus [serotypes 16, 18] (bivalent) vaccine, isotretinoin, cisplatin; fluorouracil, misonidazole, paclitaxel, palifermin, endostatin, pilocarpine, cisplatin; docetaxel; filgrastim; fluorouracil; paclitaxel, cisplatin; docetaxel; filgrastim; fluorouracil; paclitaxel, cisplatin; irinotecan hydrochloride, cisplatin; gemcitabine, cisplatin; epirubicin; fluorouracil; undisclosed chemotherapy, methyl aminolevulinate hydrochloride, carboplatin; paclitaxel, carbogen; carbon dioxide; niacinamide, cisplatin; fluorouracil, talimogene laherparepvec, epoetin alfa, cisplatin; fluorouracil; panitumumab, cisplatin; fluorouracil, cisplatin; fluorouracil, aldesleukin, cisplatin; fluorouracil, cisplatin; paclitaxel, cisplatin; fluorouracil, fluorouracil; leucovorin; lobaplatin, cisplatin, cisplatin; ethyl mercaptan; ifosfamide; mesna; mitolactol, doxorubicin; levamisole, (tegafur+uracil), cisplatin; fluorouracil, cisplatin; vinorelbine, carboplatin; cisplatin; gemcitabine hydrochloride, Corynebacterium parvum ; doxorubicin, capecitabine; cisplatin; fluorouracil; paclitaxel, fluorouracil; leucovorin; methotrexate, rAd-p53, cetuximab; cisplatin; docetaxel, PV-10, methyl aminolevulinate hydrochloride, cisplatin; fluorouracil, paclitaxel; topotecan hydrochloride, carboplatin; cisplatin; paclitaxel, cisplatin; topotecan hydrochloride, cisplatin; etoposide, docetaxel; fluorouracil, aspirin, cisplatin; gemcitabine, Lactobacillus brevis CD2, cisplatin; docetaxel, fosbretabulin tromethamine, panitumumab, fluorouracil, paclitaxel, carboplatin; cisplatin; docetaxel; fluorouracil, fluorouracil, erlotinib hydrochloride, cisplatin; undisclosed chemotherapy; vinorelbine, (gimeracil+oteracil+tegafur); carboplatin, cetuximab, contusugene ladenovec, cetuximab, methyl aminolevulinate hydrochloride, cyclophosphamide, (gimeracil+oteracil+tegafur); cisplatin, paclitaxel albumin bound, carboplatin; paclitaxel, cisplatin; gemcitabine, capecitabine; cisplatin, docetaxel, Z-100, cisplatin; ifosfamide; paclitaxel, nimotuzumab, irinotecan hydrochloride, celecoxib; methotrexate, Nutrison, carboplatin; cisplatin; fluorouracil; paclitaxel, cisplatin; paclitaxel, cisplatin; docetaxel; vinorelbine, paclitaxel, (gimeracil+oteracil+tegafur); cisplatin, carboplatin; paclitaxel, methyl aminolevulinate hydrochloride, Aibin, cisplatin; fluorouracil, porfimer sodium, carboplatin; cisplatin; tocotrienol; vinorelbine, (gimeracil+oteracil+tegafur); cisplatin; paclitaxel, docetaxel, ipilimumab, cisplatin, VB-4847, celecoxib; thalidomide, cisplatin; epirubicin; fluorouracil, cisplatin; fluorouracil, fluorouracil, carboplatin; paclitaxel, cetuximab; cisplatin; docetaxel, autologous cytokine induced killer cells, cisplatin; docetaxel; fluorouracil, cisplatin; epirubicin; fluorouracil, tergenpumatucel-L, cetuximab; cisplatin; docetaxel, Elental, cisplatin; nimotuzumab; paclitaxel, eicosapentaenoic acid; undisclosed nutritional supplement, palbociclib, pembrolizumab (Keytruda), nimotuzumab, apatorsen and dacomitinib.

As used herein, the terms “tumor”, “cancer” or “cancer disease” refer to a malignant disease, which is preferably selected from the group consisting of Adenocystic carcinoma (Adenoid cystic carcinoma), Adrenocortical carcinoma, AIDS-related cancers, AIDS-related lymphoma, Anal cancer, Appendix cancer, Astrocytoma, Basal cell carcinoma, Bile duct cancer, Bladder cancer, Bone cancer, Osteosarcoma/Malignant fibrous histiocytoma, Brainstem glioma, Brain tumor, cerebellar astrocytoma, cerebral astrocytoma/malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumors, visual pathway and hypothalamic glioma, Breast cancer, Bronchial adenomas/carcinoids, Burkitt lymphoma, childhood Carcinoid tumor, gastrointestinal Carcinoid tumor, Carcinoma of unknown primary, primary Central nervous system lymphoma, childhood Cerebellar astrocytoma, childhood Cerebral astrocytoma/Malignant glioma, Cervical cancer, Childhood cancers, Chronic lymphocytic leukemia, Colon Cancer, Cutaneous T-cell lymphoma including Mycosis Fungoides and Sezary Syndrome, Desmoplastic small round cell tumor, Endometrial cancer, Ependymoma, Esophageal cancer, Ewing's sarcoma in the Ewing family of tumors, Childhood Extracranial germ cell tumor, Extragonadal Germ cell tumor, Extrahepatic bile duct cancer, Intraocular melanoma, Retinoblastoma, Gallbladder cancer, Gastric (Stomach) cancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal stromal tumor (GIST), extracranial, extragonadal, or ovarian Germ cell tumor, Gestational trophoblastic tumor, Glioma of the brain stem, Childhood Cerebral Astrocytoma, Childhood Visual Pathway and Hypothalamic Glioma, Gastric carcinoid, Hairy cell leukemia, Head and neck cancer, Heart cancer, Hepatocellular (liver) cancer, Hodgkin lymphoma, Human Papilloma Virus (HPV)-related cancer, Hypopharyngeal cancer, childhood Hypothalamic and visual pathway glioma, Intraocular Melanoma, Islet Cell Carcinoma (Endocrine Pancreas), Kaposi sarcoma, Kidney cancer (renal cell cancer), Laryngeal Cancer, Lip and Oral Cavity Cancer, Liposarcoma, Liver Cancer, Non-Small Cell Lung Cancer, Small Cell Lung Cancer, Lymphomas, AIDS-related Lymphoma, Burkitt Lymphoma, Hodgkin Lymphoma, Non-Hodgkin Lymphomas, Primary Central Nervous System Lymphoma, Malignant Fibrous Histiocytoma of Bone/Osteosarcoma, Childhood Medulloblastoma, Melanoma, Intraocular (Eye) Melanoma, Merkel Cell Carcinoma, Adult Malignant Mesothelioma, Childhood Mesothelioma, Head or Neck Cancer, Mouth Cancer, Childhood Multiple Endocrine Neoplasia Syndrome, Multiple Myeloma/Plasma Cell Neoplasm, Multiple Myeloma (Cancer of the Bone-Marrow), Nasal cavity and paranasal sinus cancer, Nasopharyngeal carcinoma, Neuroblastoma, Oral Cancer, Oropharyngeal cancer, Osteosarcoma/malignant fibrous histiocytoma of bone, Ovarian cancer, Ovarian epithelial cancer (Surface epithelial-stromal tumor), Ovarian germ cell tumor, Ovarian low malignant potential tumor, Pancreatic cancer, islet cell Pancreatic cancer, Paranasal sinus and nasal cavity cancer, Parathyroid cancer, Penile cancer, Pharyngeal cancer, Pheochromocytoma, Pineal astrocytoma, Pineal germinoma, childhood Pineoblastoma and supratentorial primitive neuroectodermal tumors, Pituitary adenoma, Plasma cell neoplasia/plasmocytoma/Multiple myeloma, Pleuropulmonary blastoma, Primary central nervous system lymphoma, Prostate cancer, Rectal cancer, Renal cell carcinoma (kidney cancer), Cancer of the Renal pelvis and ureter, Retinoblastoma, childhood Rhabdomyosarcoma, Salivary gland cancer, Sarcoma of the Ewing family of tumors, Kaposi Sarcoma, soft tissue Sarcoma, uterine Sarcoma, Skin cancer (nonmelanoma), Skin cancer (melanoma), Merkel cell Skin carcinoma, Small intestine cancer, Squamous cell carcinoma, metastatic Squamous neck cancer with occult primary, soft tissue sarcoma (STS), childhood Supratentorial primitive neuroectodermal tumor, Testicular cancer (seminoma and non-seminoma), Throat cancer, childhood Thymoma, Thymoma and Thymic carcinoma, Thyroid cancer, childhood Thyroid cancer, Transitional cell cancer of the renal pelvis and ureter, gestational Trophoblastic tumor, Urethral cancer, endometrial Uterine cancer, Uterine sarcoma, Vaginal cancer, childhood Visual pathway and hypothalamic glioma, Vulvar cancer, and childhood Wilms tumor (kidney cancer).

V.9.Allergenic Antigens

Antigens associated with allergy or allergic disease (allergens or allergenic antigens) are preferably derived from a source selected from the list consisting of:

Acarus spp (Aca s 1, Aca s 10, Aca s 10.0101, Aca s 13, Aca s 13.0101, Aca s 2, Aca s 3, Aca s 7, Aca s 8), Acanthocybium spp (Aca so 1), Acanthocheilonema spp (Aca v 3, Aca v 3.0101), Acetes spp (Ace ja 1), Actinidia spp (Act a 1, Act c 1, Act c 10, Act c 10.0101, Act c 2, Act c 4, Act c 5, Act c 5.0101, Act c 8, Act c 8.0101, Act c Chitinase, Act d 1, Act d 1.0101, Act d 10, Act d 10.0101, Act d 10.0201, Act d 11, Act d 11.0101, Act d 2, Act d 2.0101, Act d 3, Act d 3.0101, Act d 3.02, Act d 4, Act d 4.0101, Act d 5, Act d 5.0101, Act d 6, Act d 6.0101, Act d 7, Act d 7.0101, Act d 8, Act d 8.0101, Act d 9, Act d 9.0101, Act d Chitinase, Act e 1, Act e 5), Acyrthosiphon spp (Acy pi 7, Acy pi 7.0101, Acy pi 7.0102), Adenia spp (Ade v RIP), Aedes spp (Aed a 1, Aed a 1.0101, Aed a 2, Aed a 2.0101, Aed a 3, Aed a 3.0101, Aed a 4, Aed a 7, Aed a 7.0101, Aed a 7.0102, Aed a 7.0103, Aed a 7.0104, Aed a 7.0105, Aed a 7.0106, Aed a 7.0107, Aed a 7.0108, Aed a 7.0109, Aed a 7.0110, Aed a 7.0111, Aed al 1, Aed al 3, Aed al 37 kD, Aed v 37 kD, Aed v 63 kD), Aegilops spp (Aeg ta 28, Aeg ta alpha_Gliadin, Aeg um 28, Aeg un 28), Aethaloperca spp (Aet ro 1), Agropyron spp (Agr c 7), Agrostis spp (Agr ca 1, Agr ca 5, Agr g 1, Agr g 4, Agr s 5), Agrobacterium spp (Agr sp CP4 EPSPS), Ailuropoda spp (Ail me Phosvitin, Ail me TCTP), Aix spp (Aix ga 1, Aix sp 1), Aleuroglyphus spp (Ale o 1, Ale o 10, Ale o 10.0101, Ale o 10.0102, Ale o 13, Ale o 14, Ale o 2, Ale o 20, Ale o 3, Ale o 5, Ale o 7, Ale o 8, Ale o 9), Allium spp (All a 3, All a Alliin lyase, All c 3, All c 30 kD, All c 4, All c Alliin lyase, All p Alliin lyase, All s Alliin lyase), Alnus spp (Aln g 1, Aln g 1.0101, Aln g 1/Bet v 1/Cor a 1 TPC7, Aln g 1/Bet v 1/Cor a 1 TPC9, Aln g 2, Aln g 4, Aln g 4.0101), Alopochen spp (Alo ae 1), Alopecurus spp (Alo p 1, Alo p 5), Alternaria spp (Alt a 1, Alt a 1.0101, Alt a 1.0102, Alt a 10, Alt a 10.0101, Alt a 12, Alt a 12.0101, Alt a 13, Alt a 13.0101, Alt a 2, Alt a 3, Alt a 3.0101, Alt a 4, Alt a 4.0101, Alt a 5, Alt a 5.0101, Alt a 6, Alt a 6.0101, Alt a 7, Alt a 7.0101, Alt a 70 kD, Alt a 8, Alt a 8.0101, Alt a 9, Alt a MnSOD, Alt a NTF2, Alt a TCTP, Alt ar 1, Alt arg 1, Alt b 1, Alt bl 1, Alt br 1, Alt c 1, Alt ca 1, Alt ce 1, Alt ch 1, Alt ci 1, Alt co 1, Alt cr 1, Alt ct 1, Alt cu 1, Alt cy 1, Alt d 1, Alt du 1, Alt e 1, Alt et 1, Alt eu 1, Alt ga 1, Alt gr 1, Alt j 1, Alt I 1, Alt lo 1, Alt m 1, Alt me 1, Alt mi 1, Alt mo 1, Alt o 1, Alt p 1, Alt ph 1, Alt po 1, Alt ps 1, Alt r 1, Alt s 1, Alt se 1, Alt sm 1, Alt so 1, Alt su 1, Alt t 1, Alt te 1, Alt to 1), Amaranthus spp (Ama r 2, Ama r 2.0101, Ama v 2, Ama v 2.0101, Ama v 2.0201), Ambrosia spp (Amb a 1, Amb a 1.0101, Amb a 1.0201, Amb a 1.0202, Amb a 1.0301, Amb a 1.0302, Amb a 1.0303, Amb a 1.0304, Amb a 1.0305, Amb a 1.0401, Amb a 1.0402, Amb a 1.0501, Amb a 1.0502, Amb a 10, Amb a 10.0101, Amb a 3, Amb a 3.0101, Amb a 4, Amb a 4.0101, Amb a 5, Amb a 5.0101, Amb a 6, Amb a 6.0101, Amb a 7, Amb a 7.0101, Amb a 8, Amb a 8.0101, Amb a 8.0102, Amb a 9, Amb a 9.0101, Amb a 9.0102, Amb a CPI, Amb p 1, Amb p 5, Amb p 5.0101, Amb p 5.0201, Amb t 5, Amb t 5.0101, Amb t 8), Ammothea spp (Amm h 7, Amm h 7.0101), Anadara spp (Ana br 1), Ananas spp (Ana c 1, Ana c 1.0101, Ana c 2, Ana c 2.0101, Ana c 2.0101 (MUXF3)), Anas spp (Ana ca 1), Anarhichas spp (Ana I 1), Anacardium spp (Ana o 1, Ana o 1.0101, Ana o 1.0102, Ana o 2, Ana o 2.0101, Ana o 3, Ana o 3.0101), Anas spp (Ana p 1, Ana p 2, Ana p 3), Anguilla spp (Ang a 1, Ang j 1), Anisakis spp (Ani s 1, Ani s 1.0101, Ani s 10, Ani s 10.0101, Ani s 11, Ani s 11.0101, Ani s 12, Ani s 12.0101, Ani s 2, Ani s 2.0101, Ani s 24 kD, Ani s 3, Ani s 3.0101, Ani s 4, Ani s 4.0101, Ani s 5, Ani s 5.0101, Ani s 6, Ani s 6.0101, Ani s 7, Ani s 7.0101, Ani s 8, Ani s 8.0101, Ani s 9, Ani s 9.0101, Ani s CCOS3, Ani s Cytochrome B, Ani s FBPP, Ani s NADHDS4L, Ani s NARaS, Ani s PEPB, Ani s Troponin), Annona spp (Ann c Chitinase), Anopheles spp (Ano da 17, Ano da 17.0101, Ano da 27, Ano da 27.0101, Ano da 7, Ano da 7.0101, Ano g 7, Ano g 7.0101), Anser spp (Ans a 1, Ans a 2, Ans a 3, Ans in 1), Anthoxanthum spp (Ant o 1, Ant o 1.0101, Ant o 12, Ant o 13, Ant o 2, Ant o 4, Ant o 5, Ant o 6, Ant o 7), Apis spp (Api c 1, Api c 1.0101, Api c 10, Api c 2, Api c 4, Api d 1, Api d 1.0101, Api d 4, Api fl 4), Apium spp (Api g 1, Api g 1.0101, Api g 1.0201, Api g 2, Api g 2.0101, Api g 3, Api g 3.0101, Api g 4, Api g 4.0101, Api g 5, Api g 5.0101, Api g 6, Api g 6.0101), Apis spp (Api m 1, Api m 1.0101, Api m 10, Api m 10.0101, Api m 11, Api m 11.0101, Api m 11.0201, Api m 13 kD, Api m 2, Api m 2.0101, Api m 3, Api m 3.0101, Api m 4, Api m 4.0101, Api m 5, Api m 5.0101, Api m 6, Api m 6.0101, Api m 7, Api m 7.0101, Api m 8, Api m 8.0101, Api m 9, Api m 9.0101, Api m A1-A2, Api m A1-A2-A3, Api m Apalbumin 1, Api m Apalbumin 2, Api me 1, Api me 4), Arachis spp (Ara d 2, Ara d 6, Ara f 3, Ara f 4, Ara h 1, Ara h 1.0101, Ara h 10, Ara h 10.0101, Ara h 10.0102, Ara h 11, Ara h 11.0101, Ara h 2, Ara h 2.0101, Ara h 2.0102, Ara h 2.0201, Ara h 2.0202, Ara h 3, Ara h 3.0101, Ara h 4, Ara h 4.0101, Ara h 5, Ara h 5.0101, Ara h 6, Ara h 6.0101, Ara h 7, Ara h 7.0101, Ara h 7.0201, Ara h 7.0202, Ara h 8, Ara h 8.0101, Ara h 8.0201, Ara h 9, Ara h 9.0101, Ara h 9.0201, Ara h Agglutinin, Ara h Oleosin 18 kD, Ara i 2, Ara i 6), Arabidopsis spp (Ara t 3, Ara t 8, Ara t GLP), Archosargus spp (Arc pr 1), Archaeopotamobius spp (Arc s 8, Arc s 8.0101), Aequipecten spp (Arg i 1), Argas spp (Arg r 1, Arg r 1.0101), Ariopsis spp (Ari fe 1), Armoracia spp (Arm r HRP), Arrhenatherum spp (Arr e 1, Arr e 5), Artemisia spp (Art a 1, Art ap 1), Artemia spp (Art fr 1, Art fr 1.0101, Art fr 5, Art fr 5.0101), Arthrobacter spp (Art gl CO), Achorion spp (Art gy 7), Artocarpus spp (Art h 17 kD, Art h 4), Arthrospira spp (Art pl beta Phycocyanin), Artemisia spp (Art v 1, Art v 1.0101, Art v 1.0102, Art v 1.0103, Art v 1.0104, Art v 1.0105, Art v 1.0106, Art v 1.0107, Art v 2, Art v 2.0101, Art v 3, Art v 3.0101, Art v 3.0201, Art v 3.0202, Art v 3.0301, Art v 4, Art v 4.0101, Art v 4.0201, Art v 47 kD, Art v 5, Art v 5.0101, Art v 6, Art v 6.0101, Art v 60 kD), Arthroderma spp (Art va 4), Ascaris spp (Asc 1 3, Asc 1 3.0101, Asc 1 3.0102, Asc 134 kD, Asc s 1, Asc s 1.0101, Asc s 3, Asc s 3.0101, Asc s GST), Aspergillus spp (Asp aw Glucoamylase, Asp c 22, Asp f 1, Asp f 1.0101, Asp f 10, Asp f 10.0101, Asp f 11, Asp f 11.0101, Asp f 12, Asp f 12.0101, Asp f 13, Asp f 13.0101, Asp f 15, Asp f 15.0101, Asp f 16, Asp f 16.0101, Asp f 17, Asp f 17.0101, Asp f 18, Asp f 18.0101, Asp f 2, Asp f 2.0101, Asp f 22, Asp f 22.0101, Asp f 23, Asp f 23.0101, Asp f 27, Asp f 27.0101, Asp f 28, Asp f 28.0101, Asp f 29, Asp f 29.0101, Asp f 3, Asp f 3.0101, Asp f 34, Asp f 34.0101, Asp f 4, Asp f 4.0101, Asp f 5, Asp f 5.0101, Asp f 56 kD, Asp f 6, Asp f 6.0101, Asp f 7, Asp f 7.0101, Asp f 8, Asp f 8.0101, Asp f 99, Asp f 9.0101, Asp f AfCalAp, Asp f AT_V, Asp f Catalase, Asp f Chitosanase, Asp f CP, Asp f DPPV, Asp f FDH, Asp f gamma_Actin, Asp f Glucosidase, Asp f GPI, Asp f GST, Asp f GT, Asp f IAO, Asp f IPMI, Asp f LPL1, Asp f LPL3, Asp f Mannosidase, Asp f MDH, Asp f PL, Asp f PUP, Asp f RPS3, Asp f SXR, Asp fl 13, Asp fl 13.0101, Asp fl 18, Asp fl 2, Asp fl 21, Asp fl 3, Asp fl 4, Asp fl 7, Asp fl 8, Asp fl 9, Asp me Seaprose, Asp n 14, Asp n 14.0101, Asp n 18, Asp n 18.0101, Asp n 25, Asp n 25.0101, Asp n 30, Asp n Glucoamylase, Asp n Hemicellulase, Asp n Pectinase, Asp o 13, Asp o 13.0101, Asp o 21, Asp o 21.0101, Asp o 3, Asp o 4, Asp o 7, Asp o 8, Asp o Lactase, Asp o Lipase, Asp oc 13, Asp r 1, Asp sa AP, Asp sp Glucoamylase, Asp sp Glucoseoxidase, Asp sp PL, Asp sp PME, Asp sy 13, Asp v 13, Asp v 13.0101, Asp v Catalase A, Asp v Enolase, Asp v GAPDH, Asp v MDH, Asp v SXR), Asparagus spp (Aspa o 1, Aspa o 1.01, Aspa o 1.02, Aspa o 17 kD, Aspa o 4), Aspergillus spp (Aspe ni 2, Aspe ni 3, Aspe ni 4, Aspe ni 7, Aspe ni 8, Aspe ni 9), Avena spp (Ave s 1, Ave s 12, Ave s 13, Ave s 2, Ave s 4, Ave s 5, Ave s 7), Babylonia spp (Bab ja 1), Bacillus spp (Bac al Subtilisin, Bac cl Subtilisin, Bac I Subtilisin, Bac li aA, Bac li Subtilisin), Bactrocera spp (Bac ol 27, Bac ol 27.0101), Bacillus spp (Bac sp aA1, Bac sp aA3, Bac sp Decarboxylase, Bac st amyM, Bac su Subtilisin, Bac t CrylAb, Bac t Cry1Fa, Bac t Cry3Bbl, Bac t Cry9c), Bagre spp (Bag ma 1), Balistes spp (Bal ca 1), Balanus spp (Bal r 1, Bal r 1.0101), Beauveria spp (Bea b Aid, Bea b Enol, Bea b f2, Bea b Hex), Bertholletia spp (Ber e 1, Ber e 1.0101, Ber e 2, Ber e 2.0101), Beryx spp (Ber sp 1), Betula spp (Bet ab 1, Bet al 1, Bet ch 1, Bet co 1, Bet da 1, Bet gr 1, Bet hu 1, Bet le 1, Bet me 1, Bet n 1, Bet p 1, Bet pa 1, Bet po 1, Bet pu 1, Bet pu 2, Bet pu 4, Bet pu 6, Bet pu 7, Bet sc 1, Bet ut 1, Bet v 1, Bet v 1 B1-B1-B1, Bet v 1 fv Mal 4x, Bet v 1.0101, Bet v 1.0102, Bet v 1.0103, Bet v 1.0201, Bet v 1.0301, Bet v 1.0401, Bet v 1.0402, Bet v 1.0501, Bet v 1.0601, Bet v 1.0602, Bet v 1.0701, Bet v 1.0801, Bet v 1.0901, Bet v 1.1001, Bet v 1.1101, Bet v 1.1201, Bet v 1.1301, Bet v 1.1401, Bet v 1.1402, Bet v 1.1501, Bet v 1.1502, Bet v 1.1601, Bet v 1.1701, Bet v 1.1801, Bet v 1.1901, Bet v 1.2001, Bet v 1.2101, Bet v 1.2201, Bet v 1.2301, Bet v 1.2401, Bet v 1.2501, Bet v 1.2601, Bet v 1.2701, Bet v 1.2801, Bet v 1.2901, Bet v 1.3001, Bet v 1.3101, Bet v 2, Bet v 2.0101, Bet v 3, Bet v 3.0101, Bet v 4, Betv 4.0101, Bet v 6, Bet v 6.0101, Bet v 6.0102, Bet v 7, Bet v 7.0101, Bet v 8, Bet v Glucanase), Beta spp (Beta v 1, Beta v 1.0101, Beta v 2, Beta v 2.0101), Blattella spp (Bla g 1, Bla g 1.0101, Bla g 1.0102, Bla g 1.0103, Bla g 1.0201, Bla g 1.0202, Bla g 2, Bla g 2.0101, Bla g 2.0201, Bla g 36 kD, Bla g 4, Bla g 4.0101, Bla g 4.0201, Bla g 5, Bla g 5.0101, Bla g 5.0201, Bla g 6, Bla g 6.0101, Bla g 6.0201, Bla g 6.0301, Bla g 7, Bla g 7.0101, Bla g 8, Bla g 8.0101, Bla g 9, Bla g Enolase, Bla g GSTD1, Bla g RACK1, Bla g TPI, Bla g Trypsin, Bla g Vitellogenin), Blatta spp (Bla o 1, Bla o 7), Blomia spp (Blo t 1, Blo t 1.0101, Blo t 1.0201, Blo t 10, Blo t 10.0101, Blo t 10.0102, Blo t 11, Blo t 11.0101, Blo t 12, Blo t 12.0101, Blo t 12.0102, Blo t 13, Blo t 13.0101, Blo t 14, Blo t 15, Blo t 18, Blo t 19, Blo t 19.0101, Blo t 2, Blo t 2.0101, Blo t 2.0102, Blo t 2.0103, Blo t 20, Blo t 21, Blo t 21.0101, Blo t 3, Blo t 3.0101, Blo t 4, Blo t 4.0101, Blo t 5, Blo t 5.0101, Blo t 6, Blo t 6.0101, Blo t 7, Blo t 8, Blo t 9, Blo t HSP70), Bombus spp (Bom ar 4, Bom by 4, Bom p 1, Bom p 1.0101, Bom p 2, Bom p 3, Bom p 4, Bom p 4.0101, Bom t 1, Bom t 1.0101, Bom t 4, Bom t 4.0101), Bombyx spp (Bomb m 1, Bomb m 1.0101, Bomb m 7, Bomb m 7.0101, Bomb m 7.0102, Bomb m 7.0103, Bomb m 7.0104, Bomb m 7.0105, Bomb m 7.0106), Boophilus spp (Boo m 1, Boo m 7, Boo m 7.0101), Bos spp (Bos d 2, Bos d 2.0101, Bos d 2.0102, Bos d 2.0103, Bos d 3, Bos d 3.0101, Bos d 4, Bos d 4.0101, Bos d 5, Bos d 5.0101, Bos d 5.0102, Bos d 6, Bos d 6 (MDA), Bos d 6.0101, Bos d 7, Bos d 7.0101, Bos d 8, Bos d 8 alphaS1, Bos d 8 alphaS2, Bos d 8 beta, Bos d 8 kappa, Bos d alpha2I, Bos d alpha2I.0101, Bos d Chymosin, Bos d Fibrin, Bos d Gelatin, Bos d HG, Bos d Insulin, Bos d Lactoferrin, Bos d Lactoperoxidase, Bos d Myoglobin, Bos d OBP, Bos d OSCP, Bos d Phosvitin, Bos d PLA2, Bos d PRVB, Bos d Thrombin, Bos d TI, Bos gr ALA, Bos gr Myoglobin), Bothrops spp (Bot as 1, Bot at 1), Bouteloua spp (Bou g 1), Biting spp (Bov ov 1), Brama spp (Bra du 1), Brassica spp (Bra j 1, Bra j 1.0101, Bra n 1, Bra n 1.0101, Bra n 4, Bra n 7, Bra n 8, Bra n PG, Bran i 8, Bra o 3, Bra o 3.0101, Bra r 1, Bra r 1.0101, Bra r 2, Bra r 2.0101, Bra r 3, Bra r 4, Bra r 7), Bromus spp (Bro a 1, Bro a 4), Brosme spp (Bro br 1), Bromus spp (Bro i 1, Bro i 5, Bro i 7), Brugia spp (Bru m 3, Bru m 3.0101, Bru m Bm33), Bubalus spp (Bub b ALA, Bub b BLG, Bub b Casein, Bub b Casein alphaS1, Bub b Casein alphaS2, Bub b Casein beta, Bub b Casein kappa), Caenorhabditis spp (Cae b 3, Cae b 3.0101, Cae br 3, Cae br 3.0101, Cae e 3, Cae e 3.0101, Cae e 3.0102, Cae re 13, Cae re 13.0101), Cajanus spp (Caj c 1), Caligus spp (Cal cl 1, Cal cl 1.0101, Cal cl 1.0102), Calamus spp (Cal le 1), Callinectes spp (Cal s 2), Camelus spp (Cam d ALA, Cam d Casein, Cam d Casein alphaS1, Cam d Casein alphaS2, Cam d Casein beta, Cam d Casein kappa), Camponotus spp (Cam fl 7, Cam fl 7.0101), Canis spp (Can f 1, Can f 1.0101, Can f 2, Can f 2.0101, Can f 3, Can f 3.0101, Can f 4, Can f 4.0101, Can f 5, Can f 5.0101, Can f 6, Can f 6.0101, Can f Feld1-like, Can f Homs2-like, Can f Phosvitin, Can f TCTP), Canthidermis spp (Can ma 1), Cancer spp (Can mg 2, Can p 1), Cannabis spp (Can s 3), Candida spp (Cand a 1, Cand a 1.0101, Cand a 3, Cand a 3.0101, Cand a CAAP, Cand a CyP, Cand a Enolase, Cand a FPA, Cand a MnSOD, Cand a PGK, Cand b 2, Cand b 2.0101, Cand b FDH, Cand r Lipase), Capsicum spp (Cap a 1, Cap a 1.0101, Cap a 17 kD, Cap a 2, Cap a 2.0101, Cap a 30 kD, Cap a Glucanase, Cap ch 17 kD), Caprella spp (Cap e 1), Capra spp (Cap h ALA, Cap h BLG, Cap h Casein, Cap h Casein alphaS1, Cap h Casein alphaS2, Cap h Casein beta, Cap h Casein kappa, Cap h GSA), Capitulum spp (Cap m 1), Carassius spp (Car au 1), Carpinus spp (Car b 1, Car b 1.0101, Car b 1.0102, Car b 1.0103, Car b 1.0104, Car b 1.0105, Car b 1.0106, Car b 1.0107, Car b 1.0108, Car b 1.0109, Car b 1.0110, Car b 1.0111, Car b 1.0112, Car b 1.0113, Car b 1.0201, Car b 1.0301, Car b 1.0302, Car b 2, Car b 4), Caranx spp (Car cr 1), Carya spp (Car i 1, Car i 1.0101, Car i 2, Car i 4, Car i 4.0101), Carcinus spp (Car ma 2), Caryota spp (Car mi 2), Carica spp (Car p 1, Car p Chitinase, Car p Chymopapain, Car p Endoproteinase), Castanea spp (Cas c 24 kD, Cas s 1, Cas s 1.0101, Cas s 1.0102, Cas s 1.0103, Cas s 2, Cas s 5, Cas s 5.0101, Cas s 8, Cas s 8.0101, Cas s 9, Cas s 9.0101), Catharanthus spp (Cat r 1, Cat r 1.0101, Cat r 17 kD, Cat r 2), Caulolatilus spp (Cau ch 1), Cavia spp (Cav p 1, Cav p 1.0101, Cav p 2, Cav p 2.0101, Cav p 3, Cav p 3.0101, Cav p Gelatin, Cav p GSA), Centropristis spp (Cen s 1), Cephalopholis spp (Cep so 1), Charybdis spp (Cha f 1, Cha f 1.0101), Chaetodipterus spp (Cha fa 1), Chamaecyparis spp (Cha o 1, Cha o 1.0101, Cha o 2, Cha o 2.0101), Chenopodium spp (Che a 1, Che a 1.0101, Che a 2, Che a 2.0101, Che a 3, Che a 3.0101), Chironomus spp (Chi k 1, Chi k 10, Chi k 10.0101), Chinchilla spp (Chi 121 kD_a, Chi 121 kD_b), Chionoecetes spp (Chi o 1, Chi o 1.0101, Chi o 2, Chi o 4, Chi o 6, Chi o alpha_Actin, Chi o SERCA), Chironomus spp (Chi t 1, Chi t 1.0101, Chi t 1.0201, Chi t 2, Chi t 2.0101, Chi t 2.0102, Chi t 3, Chi t 3.0101, Chi t 4, Chi t 4.0101, Chi t 5, Chi t 5.0101, Chi t 6, Chi t 6.0101, Chi t 6.0201, Chi t 7, Chi t 7.0101, Chi t 8, Chi t 8.0101, Chi t 9, Chi t 9.0101), Chlamys spp (Chl n 1), Chloephaga spp (Chl pi 1), Chortoglyphus spp (Cho a 10), Chrysomela spp (Chr tr 7, Chr tr 7.0101), Cicer spp (Cic a 2S Albumin, Cic a Albumin), Cichorium spp (Cic i 1), Cimex spp (Cim I Nitrophorin), Citrus spp (Cit I 1, Cit 13, Cit I 3.0101), Citrullus spp (Cit la 2, Cit la MDH, Cit la TPI), Citrus spp (Cit r 3, Cit r 3.0101, Cit s 1, Cit s 1.0101, Cit s 2, Cit s 2.0101, Cit s 3, Cit s 3.0101, Cit s 3.0102, Cit s IFR), Cladosporium spp (Cla c 14, Cla c 14.0101, Cla c 9, Cla c 9.0101, Cla h 1, Cla h 10, Cla h 10.0101, Cla h 12, Cla h 12.0101, Cla h 2, Cla h 2.0101, Cla h 42 kD, Cla h 5, Cla h 5.0101, Cla h 6, Cla h 6.0101, Cla h 7, Cla h 7.0101, Cla h 8, Cla h 8 CSP, Cla h 8.0101, Cla h 9, Cla h 9.0101, Cla h abH, Cla h GST, Cla h HChl, Cla h HSP70, Cla h NTF2, Cla h TCTP), Clostridium spp (Clo hi Collagenase, Clo t Toxoid), Clupea spp (Clu h 1, Clu h 1.0101, Clu h 1.0201, Clu h 1.0301), Cocos spp (Coc n 2, Coc n 4, Coc n 5), Coccidioides spp (Coc po 8), Coffea spp (Cof a 1, Cof a 1.0101), Columba spp (Col I PSA), Coprinus spp (Cop c 1, Cop c 1.0101, Cop c 2, Cop c 2.0101, Cop c 3, Cop c 3.0101, Cop c 4, Cop c 5, Cop c 5.0101, Cop c 6, Cop c 7, Cop c 7.0101), Corylus spp (Cor a 1, Cor a 1.0101, Cor a 1.0102, Cor a 1.0103, Cor a 1.0104, Cor a 1.0201, Cor a 1.0301, Cor a 1.0401, Cor a 1.0402, Cor a 1.0403, Cor a 1.0404, Cor a 10, Cor a 10.0101, Cor a 11, Cor a 11.0101, Cor a 12, Cor a 12.0101, Cor a 13, Cor a 13.0101, Cor a 14, Cor a 14.0101, Cor a 2, Cor a 2.0101, Cor a 2.0102, Cor a 8, Cor a 8.0101, Cor a 9, Cor a 9.0101), Corynebacterium spp (Cor d Toxoid), Corylus spp (Cor he 1), Coryphaena spp (Cor hi 1), Coriandrum spp (Cor s 1, Cor s 11 kD, Cor s 2), Cotoneaster spp (Cot 13), Crangon spp (Cra c 1, Cra c 1.0101, Cra c 2, Cra c 2.0101, Cra c 4, Cra c 4.0101, Cra c 5, Cra c 5.0101, Cra c 6, Cra c 6.0101, Cra c 8, Cra c 8.0101), Crassostrea spp (Cra g 1), Cricetus spp (Cri c HSA), Crivellia spp (Cri pa 1), Crocus spp (Cro s 1, Cro s 1.0101, Cro s 2, Cro s 2.0101, Cro s 3, Cro s 3.01, Cro s 3.02), Cryptomeria spp (Cry j 1, Cry j 1.0101, Cry j 1.0102, Cry j 1.0103, Cry j 2, Cry j 2.0101, Cry j 2.0102, Cryj 3, Cryj 3.1, Cryj 3.2, Cryj 3.3, Cryj 3.4, Cryj 3.5, Cryj 3.6, Cryj 3.7, Cryj 3.8, Cryj 4, Cryj AP, Cry j Chitinase, Cry j CPA9, Cry j IFR, Cry j LTP, Cry j P1-P2), Cryphonectria spp (Cry p AP), Ctenocephalides spp (Cte f 1, Cte f 1.0101, Cte f 2, Cte f 2.0101, Cte f 3, Cte f 3.0101), Ctenopharyngodon spp (Cte id 1), Cucumis spp (Cuc m 1, Cuc m 1.0101, Cuc m 2, Cuc m 2.0101, Cuc m 3, Cuc m 3.0101, Cuc m Lec17, Cuc m MDH), Cucurbita spp (Cuc ma 18 kD, Cuc ma 2, Cuc p 2, Cuc p AscO), Cucumis spp (Cuc s 2), Culicoides spp (Cul n 1, Cul n 10, Cul n 11, Cul n 2, Cul n 3, Cul n 4, Cul n 5, Cul n 6, Cul n 7, Cul n 8, Cul n 9, Cul n HSP70), Culex spp (Cul q 28 kD, Cul q 35 kD, Cul q 7, Cul q 7.0101, Cul q 7.0102), Culicoides spp (Cul so 1), Cuminum spp (Cum c 1, Cum c 2), Cupressus spp (Cup a 1, Cup a 1.0101, Cup a 1.02, Cup a 2, Cup a 3, Cup a 4, Cup a 4.0101, Cup s 1, Cup s 1.0101, Cup s 1.0102, Cup s 1.0103, Cup s 1.0104, Cup s 1.0105, Cup s 3, Cup s 3.0101, Cup s 3.0102, Cup s 3.0103, Cup s 8), Cochliobolus spp (Cur I 1, Cur I 1.0101, Cur I 2, Cur I 2.0101, Cur I 3, Cur I 3.0101, Cur I 4, Cur I 4.0101, Cur I ADH, Cur I GST, Cur I MnSOD, Cur I Oryzin, Cur I Trx, Cur I ZPS1), Cyanochen spp (Cya cy 1), Cynoscion spp (Cyn ar 1), Cynosurus spp (Cyn cr 1, Cyn cr 5), Cynodon spp (Cyn d 1, Cyn d 1.0101, Cyn d 1.0102, Cyn d 1.0103, Cyn d 1.0104, Cyn d 1.0105, Cyn d 1.0106, Cyn d 1.0107, Cyn d 1.0201, Cyn d 1.0202, Cyn d 1.0203, Cyn d 1.0204, Cyn d 10, Cyn d 11, Cyn d 12, Cyn d 12.0101, Cyn d 13, Cyn d 15, Cyn d 15.0101, Cyn d 2, Cyn d 22, Cyn d 22.0101, Cyn d 23, Cyn d 23.0101, Cyn d 24, Cyn d 24.0101, Cyn d 4, Cyn d 5, Cyn d 6, Cyn d 7, Cyn d 7.0101), Cynoscion spp (Cyn ne 1), Cynomys spp (Cyn sp Lipocalin), Cyprinus spp (Cyp c 1, Cyp c 1.01, Cyp c 1.02), Daboia spp (Dab ru 1), Dactylis spp (Dac g 1, Dac g 1.01, Dac g 1.0101, Dac g 1.02, Dac g 12, Dac g 13, Dac g 2, Dac g 2.0101, Dac g 3, Dac g 3.0101, Dac g 4, Dac g 4.0101, Dac g 5, Dac g 5.0101, Dac g 7), Dama spp (Dam d CSA), Danio spp (Dan re 1, Dan re 2, Dan re alpha2I, Dan re CK), Dasyatis spp (Das ak 1, Das am 1, Das sa 1), Daucus spp (Dau c 1, Dau c 1.0101, Dau c 1.0102, Dau c 1.0103, Dau c 1.0104, Dau c 1.0105, Dau c 1.0201, Dau c 1.0301, Dau c 3, Dau c 4, Dau c 4.0101, Dau c CyP), Decapterus spp (Dec ru 1), Dendronephthya spp (Den n 1, Den n 1.0101), Dermatophagoides spp (Der f 1, Der f 1.0101, Der f 1.0102, Der f 1.0103, Der f 1.0104, Der f 1.0105, Der f 1.0106, Der f 1.0107, Der f 1.0108, Der f 1.0109, Der f 1.0110, Der f 10, Der f 10.0101, Der f 10.0102, Der f 11, Der f 11.0101, Der f 13, Der f 13.0101, Der f 14, Der f 14.0101, Der f 15, Der f 15.0101, Der f 16, Der f 16.0101, Der f 17, Der f 17.0101, Der f 18, Der f 18.0101, Der f 2, Der f 2.0101, Der f 2.0102, Der f 2.0103, Der f 2.0104, Der f 2.0105, Der f 2.0106, Der f 2.0107, Der f 2.0108, Der f 2.0109, Der f 2.0110, Der f 2.0111, Der f 2.0112, Der f 2.0113, Der f 2.0114, Der f 2.0115, Der f 2.0116, Der f 2.0117, Der f 20, Der f 21, Der f 22, Der f 22.0101, Der f 3, Der f 3.0101, Der f 4, Der f 5, Der f 6, Der f 6.0101, Der f 7, Der f 7.0101, Der f 8, Der f 9, Der f HSP70), Dermanyssus spp (Der g 10, Der g 10.0101), Dermatophagoides spp (Der m 1, Der m 1.0101, Der p 1, Der p 1.0101, Der p 1.0102, Der p 1.0103, Der p 1.0104, Der p 1.0105, Der p 1.0106, Der p 1.0107, Der p 1.0108, Der p 1.0109, Der p 1.0110, Der p 1.0111, Der p 1.0112, Der p 1.0113, Der p 1.0114, Der p 1.0115, Der p 1.0116, Der p 1.0117, Der p 1.0118, Der p 1.0119, Der p 1.0120, Der p 1.0121, Der p 1.0122, Der p 1.0123, Der p 1.0124, Der p 10, Der p 10.0101, Der p 10.0102, Der p 10.0103, Der p 11, Der p 11.0101, Der p 13, Der p 14, Der p 14.0101, Der p 15, Der p 18, Der p 2, Der p 2.0101, Der p 2.0102, Der p 2.0103, Der p 2.0104, Der p 2.0105, Der p 2.0106, Der p 2.0107, Der p 2.0108, Der p 2.0109, Der p 2.0110, Der p 2.0111, Der p 2.0112, Der p 2.0113, Der p 2.0114, Der p 2.0115, Der p 20, Der p 20.0101, Der p 21, Der p 21.0101, Der p 23, Der p 23.0101, Der p 3, Der p 3.0101, Der p 4, Der p 4.0101, Der p 5, Der p 5.0101, Der p 5.0102, Der p 6, Der p 6.0101, Der p 7, Der p 7.0101, Der p 8, Der p 8.0101, Der p 9, Der p 9.0101, Der p 9.0102, Der p P1-P2, Der p P2-P1, Der s 1, Der s 2, Der s 3), Dianthus spp (Dia c RIP), Dicranopteris spp (Dic I 2S Albumin), Diospyros spp (Dio k 17 kD, Dio k 4, Dio k IFR), Dioscorea spp (Dio p TSP), Diplodus spp (Dip ho 1), Distichlis spp (Dis s 1, Dis s 7), Ditrema spp (Dit te 1), Dolichovespula spp (Dol a 1, Dol a 2, Dol a 5, Dol a 5.0101), Dolichos spp (Dol b Agglutinin), Dolichovespula spp (Dol m 1, Dol m 1.0101, Dol m 1.02, Dol m 2, Dol m 2.0101, Dol m 5, Dol m 5.0101, Dol m 5.02), Drosophila spp (Dro an 7, Dro an 7.0101, Dro er 7, Dro er 7.0101, Dro er 7.0102, Dro gr 7, Dro gr 7.0101, Dro gr 7.0102, Dro m 7, Dro m 7.0101, Dro m 7.0102, Dro m 7.0103, Dro m 7.0104, Dro m 7.0105, Dro m 7.0106, Dro m 7.0107, Dro m 7.0108, Dro m 7.0109, Dro m 7.0110, Dro m 7.0111, Dro m 7.0112, Dro m 7.0113, Dro m 9, Dro m MnSOD, Dro mo 7, Dro mo 7.0101, Dro pp 7, Dro pp 7.0101, Dro se 7, Dro se 7.0101, Dro si 7, Dro si 7.0101, Dro si 7.0102, Dro vi 7, Dro vi 7.0101, Dro wi 7, Dro wi 7.0101, Dro y 7, Dro y 7.0101, Dro y 7.0102, Dro y 7.0103), Echium spp (Ech p Cytochrome C), Elaeis spp (Ela g 2, Ela g Bd31 kD), Elops spp (Elo sa 1), Embellisia spp (Emb a 1, Emb i 1, Emb nz 1, Emb t 1), Engraulis spp (Eng e 1), Enteroctopus spp (Ent d 1), Epinephelus spp (Epi bl 1, Epi co 1, Epi fl 1, Epi mc 1, Epi mo 1), Epicoccum spp (Epi p 1, Epi p 1.0101, Epi p 12 kD, Epi p GST), Epinephelus spp (Epi po 1, Epi un 1), Equisetum spp (Equ a 17 kD), Equus spp (Equ as 4, Equ as DSA, Equ bu 4, Equ c 1, Equ c 1.0101, Equ c 2, Equ c 2.0101, Equ c 2.0102, Equ c 3, Equ c 3.0101, Equ c 4, Equ c 4.0101, Equ c 5, Equ c 5.0101, Equ c ALA, Equ c BLG, Equ c Casein, Equ c Casein beta, Equ c Casein kappa, Equ c PRVB, Equ he 4, Equ z ZSA), Erimacrus spp (Eri i 1, Eri i 1.0101, Eri i 1.0102), Eriocheir spp (Eri s 1, Eri s 1.0101, Eri s 2), Erwinia spp (Erw ch Asparaginase), Escherichia spp (Esc c Asparaginase, Esc c beta GAL), Esox spp (Eso I 1), Euphausia spp (Eup p 1, Eup p 1.0101), Euphasia spp (Eup s 1, Eup s 1.0101), Euroglyphus spp (Eur m 1, Eur m 1.0101, Eur m 1.0102, Eur m 1.0103, Eur m 10, Eur m 14, Eur m 14.0101, Eur m 2, Eur m 2.0101, Eur m 2.0102, Eur m 3, Eur m 3.0101, Eur m 4, Eur m 4.0101), Evynnis spp (Evy j 1), Fagopyrum spp (Fag e 1, Fag e 1.0101, Fag e 10 kD, Fag e 19 kD, Fag e 2, Fag e 2.0101, Fag e TI), Fagus spp (Fag s 1, Fag s 1.0101, Fag s 2, Fag s 4), Fagopyrum spp (Fag t 1, Fag t 10 kD, Fag t 2, Fag t 2.0101), Felis spp (Fel d 1, Fel d 1.0101, Fel d 2, Fel d 2.0101, Fel d 3, Fel d 3.0101, Fel d 4, Fel d 4.0101, Fel d 5, Fel d 5.0101, Fel d 6, Fel d 6.0101, Fel d 7, Fel d 7.0101, Fel d 8, Fel d 8.0101, Fel d IgG), Fenneropenaeus spp (Fen c 1, Fen c 2, Fen me 1, Fen me 1.0101), Festuca spp (Fes e 1, Fes e 13, Fes e 4, Fes e 5, Fes e 7, Fes p 1, Fes p 13, Fes p 4, Fes p 4.0101, Fes p 5, Fes r 1, Fes r 5), Ficus spp (Fic c 17 kD, Fic c 4, Fic c Ficin), Foeniculum spp (Foe v 1, Foe v 2), Forsythia spp (For s 1), Forcipomyia spp (For t 1, For t 1.0101, For t 2, For t 2.0101, For t 7, For t FPA, For t Myosin, For t TPI), Fragaria spp (Fra a 1, Fra a 1.0101, Fra a 3, Fra a 3.0101, Fra a 3.0102, Fra a 3.0201, Fra a 3.0202, Fra a 3.0203, Fra a 3.0204, Fra a 3.0301, Fra a 4, Fra a 4.0101, Fra c 1), Fraxinus spp (Fra e 1, Fra e 1.0101, Fra e 1.0102, Fra e 1.0201, Fra e 12, Fra e 2, Fra e 3, Fra e 9), Fragaria spp (Fra v 1), Fusarium spp (Fus c 1, Fus c 1.0101, Fus c 2, Fus c 2.0101, Fus c 3, Fus s 1, Fus s 45 kD, Fus sp Lipase), Gadus spp (Gad c 1, Gad c 1.0101, Gad c APDH, Gad m 1, Gad m 1.0101, Gad m 1.0102, Gad m 1.0201, Gad m 1.0202, Gad m 45 kD, Gad m Gelatin, Gad ma 1), Gallus spp (Gal d 1, Gal d 1.0101, Gal d 2, Gal d 2.0101, Gal d 3, Gal d 3.0101, Gal d 4, Gal d 4.0101, Gal d 5, Gal d 5.0101, Gal d 6, Gal d 6.0101, Gal d Apo I, Gal d Apo VI, Gal d GPI, Gal d HG, Gal d IgY, Gal d L-PGDS, Gal d Ovomucin, Gal d Phosvitin, Gal d PRVB, Gal la 4), Galleria spp (Gal m 18 kD, Gal m 24 kD), Gallus spp (Gal so 4), Gammarus spp (Gam s TM), Gelonium spp (Gel m RIP), Geothelphusa spp (Geo de 1), Glossina spp (Glo m 5, Glo m 5.0101, Glo m 7, Glo m 7.0101, Glo m 7.0102, Glo m 7.0103), Glycine spp (Gly a Bd30K, Gly ar Bd30K, Gly ca Bd30K, Gly cl Bd30K, Gly cu Bd30K, Gly cy Bd30K), Glycyphagus spp (Gly d 10, Gly d 10.0101, Gly d 13, Gly d 2, Gly d 2.0101, Gly d 2.0201, Gly d 2.03, Gly d 2/Lep d 2 L1, Gly d 2/Lep d 2 L2, Gly d 2/Lep d 2 L3, Gly d 2/Lep d 2 L4, Gly d 2/Lep d 2 R1, Gly d 2/Lep d 2 R2, Gly d 2/Lep d 2 R3, Gly d 2/Lep d 2 R4, Gly d 2/Lep d 2 R5, Gly d 20, Gly d 3, Gly d 5, Gly d 5.01, Gly d 5.02, Gly d 7, Gly d 8), Glycine spp (Gly f Bd30K, Gly I Bd30K, Gly m 1, Gly m 1.0101, Gly m 1.0102, Gly m 2, Gly m 2.0101, Gly m 2S Albumin, Gly m 3, Gly m 3.0101, Gly m 3.0102, Gly m 39 kD, Gly m 4, Gly m 4.0101, Gly m 5, Gly m 5.0101, Gly m 5.0201, Gly m 5.0301, Gly m 5.0302, Gly m 50 kD, Gly m 6, Gly m 6.0101, Gly m 6.0201, Gly m 6.0301, Gly m 6.0401, Gly m 6.0501, Gly m 68 kD, Gly m Agglutinin, Gly m Bd28K, Gly m Bd30K, Gly m Bd60K, Gly m CPI, Gly m EAP, Gly m TI, Gly mi Bd30K, Gly s Bd30K, Gly t Bd30K, Gly to Bd30K), Gossypium spp (Gos h Vicilin), Haemophilus spp (Hae in P6), Haemaphysalis spp (Hae I 7, Hae I 7.0101, Hae q 7, Hae q 7.0101), Haliotis spp (Hal a 1, Hal d 1, Hal di 1, Hal di PM, Hal m 1, Hal m 1.0101, Hal r 1, Hal r 49 kD, Hal ru 1), Harmonia spp (Har a 1, Har a 1.0101, Har a 2, Har a 2.0101), Harpegnathos spp (Har sa 7, Har sa 7.0101, Har sa 7.0102), Helianthus spp (Hel a 1, Hel a 1.0101, Hel a 2, Hel a 2.0101, Hel a 2S Albumin, Hel a 3, Hel a 3.0101, Hel a 4), Helix spp (Hel ap 1, Hel as 1, Hel as 1.0101), Heligmosomoides spp (Hel p 3, Hel p 3.0101), Helianthus spp (Hel tu 1), Hemanthias spp (Hem le 1), Hemifusus spp (Hem t 1), Heterodera spp (Het g 3, Het g 3.0101), Hevea spp (Hev b 1, Hev b 1.0101, Hev b 10, Hev b 10.0101, Hev b 10.0102, Hev b 10.0103, Hev b 11, Hev b 11.0101, Hev b 11.0102, Hev b 12, Hev b 12.0101, Hev b 13, Hev b 13.0101, Hev b 14, Hev b 14.0101, Hev b 2, Hev b 2.0101, Hev b 3, Hev b 3.0101, Hev b 4, Hev b 4.0101, Hev b 5, Hev b 5.0101, Hev b 6, Hev b 6.01, Hev b 6.02, Hev b 6.0202, Hev b 6.03, Hev b 7, Hev b 7.01, Hev b 7.02, Hev b 7.D2, Hev b 7.S2, Hev b 8, Hev b 8.0101, Hev b 8.0102, Hev b 8.0201, Hev b 8.0202, Hev b 8.0203, Hev b 8.0204, Hev b 9, Hev b 9.0101, Hev b Citrate binding Protein, Hev b GAPDH, Hev b HSP80, Hev b IFR, Hev b Proteasome subunit, Hev b Rotamase, Hev b SPI, Hev b Trx, Hev b UDPGP), Hexagrammos spp (Hex ot 1), Hippoglossus spp (Hip h 1), Hippoglossoides spp (Hip pl 1), Hippoglossus spp (Hip st 1), Hirudo spp (Hir me Hirudin), Holcus spp (Hol I1, Hol 11.0101, Hol 11.0102, Hol 12, Hol 14, Hol 15, Hol I 5.0101, Hol I 5.0201), Holocnemus spp (Hol pl 9, Hol pl Hemocyanin), Homarus spp (Hom a 1, Hom a 1.0101, Hom a 1.0102, Hom a 1.0103, Hom a 3, Hom a 3.0101, Hom a 4, Hom a 6, Hom a 6.0101, Hom g 1, Hom g 2), Homo spp (Hom s 1, Hom s 1.0101, Hom s 2, Hom s 2.0101, Hom s 3, Hom s 3.0101, Hom s 4, Hom s 4.0101, Hom s 5, Hom s 5.0101, Hom s AAT, Hom s ACTH, Hom s Adalimumab, Hom s ALA, Hom s alpha_Actin, Hom s alpha-Galactosidase, Hom s APDH, Hom s Arylsulfatase B, Hom s Casein, Hom s CyP A, Hom s CyP B, Hom s CyP C, Hom s DSF70, Hom s DSG3, Hom s eIF6, Hom s Etanercept, Hom s Factor IX, Hom s Factor VII, Hom s Factor VIII, Hom s G-CSF, Hom s Glucocerebrosidase, Hom s Glucosidase, Hom s HLA-DR-alpha, Hom s HSA, Hom s Iduronidase, Hom s Idursulfase, Hom s IgA, Hom s Insulin, Hom s Lactoferrin, Hom s Laminin gamma_2, Hom s MnSOD, Hom s Oxytocin, Hom s P2, Hom s Phosvitin, Hom s Profilin, Hom s PSA, Hom s RP1, Hom s TCTP, Hom s TL, Hom s TPA, Hom s TPO, Hom s Transaldolase, Hom s Trx, Hom s Tubulin-alpha, Hom s/Mus m Basiliximab, Hom s/Mus m Cetuximab, Hom s/Mus m Cetuximab (Gal-Gal), Hom s/Mus m Infliximab, Hom s/Mus m Natalizumab, Hom s/Mus m Omalizumab, Hom s/Mus m Palivizumab, Hom s/Mus m Rituximab, Hom s/Mus m Tocilizumab, Hom s/Mus m Trastuzumab), Hoplostethus spp (Hop a 1), Hordeum spp (Hor v 1, Hor v 12, Hor v 12.0101, Hor v 13, Hor v 14, Hor v 15, Hor v 15.0101, Hor v 16, Hor v 16.0101, Hor v 17, Hor v 17.0101, Hor v 18 kD, Hor v 2, Hor v 21, Hor v 21.0101, Hor v 28, Hor v 33, Hor v 4, Hor v 5, Hor v 5.0101, Hor v BDAI, Hor v BTI), Humicola spp (Hum in Cellulase), Humulus spp (Hum j 1, Hum j 1.0101, Hum j 10 kD, Hum j 2), Huso spp (Hus h 1), Hylocereus spp (Hyl un LTP), Hymenocephalus spp (Hym st 1), Hyperoglyphe spp (Hyp by 1), Hypophthalmichthys spp (Hyp mo 1), Hypophthalmichthy spp (Hyp no 1), Ictalurus spp (Ict fu 1, Ict p 1), Imperata spp (Imp c 4, Imp c 5, Imp c VIIIel), Ixodes spp (Ixo r 2, Ixo sc 7, Ixo sc 7.0101), Jasus spp (Jas la 1, Jas la 1.0101, Jas la 1.0102), Juglans spp (Jug ca 1, Jug ca 2, Jug ci 1, Jug ci 2, Jug n 1, Jug n 1.0101, Jug n 2, Jug n 2.0101, Jug r 1, Jug r 1.0101, Jug r 2, Jug r 2.0101, Jug r 3, Jug r 3.0101, Jug r 4, Jug r 4.0101, Jug r 5), Juniperus spp (Jun a 1, Jun a 1.0101, Jun a 1.0102, Jun a 2, Jun a 2.0101, Jun a 3, Jun a 3.0101, Jun c 1, Jun o 1, Jun o 4, Jun o 4.0101, Jun r 3, Jun r 3.1, Jun r 3.2, Jun v 1, Jun v 1.0101, Jun v 1.0102, Jun v 3, Jun v 3.0101, Jun v 3.0102, Jun v 4), Katsuwonus spp (Kat p 1), Kyphosus spp (Kyp se 1), Lachnolaimus spp (Lac ma 1), Lachesis spp (Lac mu 1), Lactuca spp (Lac s 1, Lac s 1.0101), Lagocephalus spp (Lag la 1), Larus spp (Lar a 1, Lar a 2, Lar a 3), Larimichthys spp (Lar po 1), Lates spp (Lat c 1), Lateolabrax spp (Lat ja 1), Lathyrus spp (Lat oc Agglutinin), Leiostomus spp (Lei xa 1), Lens spp (Len c 1, Len c 1.0101, Len c 1.0102, Len c 1.0103, Len c 2, Len c 2.0101, Len c 3, Len c 3.0101, Len c Agglutinin), Leopardus spp (Leo p 1), Lepidoglyphus spp (Lep d 10, Lep d 10.0101, Lep d 12, Lep d 13, Lep d 13.0101, Lep d 2, Lep d 2.0101, Lep d 2.0102, Lep d 2.0201, Lep d 2.0202, Lep d 3, Lep d 39 kD, Lep d 5, Lep d 5.0101, Lep d 5.0102, Lep d 5.0103, Lep d 7, Lep d 7.0101, Lep d 8, Lep d alpha Tubulin), Lepomis spp (Lep gi 1), Leptomelanosoma spp (Lep i 1), Lepomis spp (Lep ma 1), Lepisma spp (Lep s 1, Lep s 1.0101, Lep s 1.0102), Lepeophtheirus spp (Lep sa 1, Lep sa 1.0101, Lep sa 1.0102, Lep sa 1.0103), Leptailurus spp (Lep se 1), Lepidorhombus spp (Lep w 1, Lep w 1.0101), Lethocerus spp (Let in 7, Let in 7.0101, Let in 7.0102), Leuciscus spp (Leu ce 1), Lewia spp (Lew in 1), Ligustrum spp (Lig v 1, Lig v 1.0101, Lig v 1.0102, Lig v 2), Lilium spp (Lil 12, Lil I PG), Limanda spp (Lim fe 1), Limnonectes spp (Lim m 1), Limulus spp (Lim p 1, Lim p 1.0101, Lim p 2, Lim p LPA), Liposcelis spp (Lip b 1, Lip b 1.0101), Litchi spp (Lit c 1, Lit c 1.0101, Lit c IFR, Lit c TPI), Lithobates spp (Lit ca 1), Litopenaeus spp (Lit se 1, Lit v 1, Lit v 1.0101, Lit v 2, Lit v 2.0101, Lit v 3, Lit v 3.0101, Lit v 4, Lit v 4.0101), Filiaria spp (Loa lo 3, Loa lo 3.0101), Lobotes spp (Lob su 1), Locusta spp (Loc m 7, Loc m 7.0101), Loligo spp (Lol b 1, Lol e 1), Lolium spp (Lol m 2, Lol m 5, Lol p 1, Lol p 1.0101, Lol p 1.0102, Lol p 1.0103, Lol p 10, Lol p 11, Lol p 11.0101, Lol p 12, Lol p 13, Lol p 2, Lol p 2.0101, Lol p 3, Lol p 3.0101, Lol p 4, Lol p 4.0101, Lol p 5, Lol p 5.0101, Lol p 5.0102, Lol p 7, Lol p CyP, Lol p FT, Lol p Legumin), Lonomia spp (Lon o 7, Lon o 7.0101), Lophodytes spp (Lop cu 1), Lophonetta spp (Lop sp 1), Lupinus spp (Lup a 1, Lup a alpha_Conglutin, Lup a delta_Conglutin, Lup a gamma_Conglutin, Lup an 1, Lup an 1.0101, Lup an alpha_Conglutin, Lup an delta_Conglutin, Lup an gamma_Conglutin, Lup 117 kD), Lutjanus spp (Lut a 1, Lut c 1, Lut cy 1, Lut gr 1, Lut gu 1, Lut jo 1), Lutraria spp (Lut p 1), Lutjanus spp (Lut pu 1, Lut sy 1), Lycopersicon spp (Lyc e 1, Lyc e 1.0101, Lyc e 11 S Globulin, Lyc e 2, Lyc e 2.0101, Lyc e 2.0102, Lyc e 3, Lyc e 3.0101, Lyc e 4, Lyc e 4.0101, Lyc e ARP60S, Lyc e Chitinase, Lyc e Glucanase, Lyc e Peroxidase, Lyc e PG, Lyc e PME, Lyc e PR23, Lyc e Vicilin), Maconellicoccus spp (Mac h 7, Mac h 7.0101), Macruronus spp (Mac ma 1, Mac n 1), Macdura spp (Mac po 17 kD), Macrobrachium spp (Mac ro 1, Mac ro 1.0101, Mac ro Hemocyanin), Macropus spp (Macr s Gelatin), Malus spp (Mal d 1, Mal d 1.0101, Mal d 1.0102, Mal d 1.0103, Mal d 1.0104, Mal d 1.0105, Mal d 1.0106, Mal d 1.0107, Mal d 1.0108, Mal d 1.0109, Mal d 1.0201, Mal d 1.0202, Mal d 1.0203, Mal d 1.0204, Mal d 1.0205, Mal d 1.0206, Mal d 1.0207, Mal d 1.0208, Mal d 1.0301, Mal d 1.0302, Mal d 1.0303, Mal d 1.0304, Mal d 1.0401, Mal d 1.0402, Mal d 1.0403, Mal d 2, Mal d 2.0101, Mal d 3, Mal d 3.0101, Mal d 3.0102, Mal d 3.0201, Mal d 3.0202, Mal d 3.0203, Mal d 4, Mal d 4.0101, Mal d 4.0102, Mal d 4.0201, Mal d 4.0202, Mal d 4.0301, Mal d 4.0302), Malpighia spp (Mal g 4, Mal g Hevein), Malus spp (Mal p 1), Malassezia spp ( Mala f 2, Mala f 2.0101, Mala f 3, Mala f 3.0101, Mala f 4, Mala f 4.0101, Mala g 10, Mala s 1, Mala s 1.0101, Mala s 10, Mala s 10.0101, Mala s 11, Mala s 11.0101, Mala s 12, Mala s 12.0101, Mala s 13, Mala s 13.0101, Mala s 5, Mala s 5.0101, Mala s 6, Mala s 6.0101, Mala s 7, Mala s 7.0101, Mala s 8, Mala s 8.0101, Mala s 9, Mala s 9.0101), Manihot spp (Man e 5, Man e 5.0101, Man e FPA, Man e GAPDH), Mangifera spp (Man i 1, Man i 14 kD, Man i 2, Man i 3, Man i 3.01, Man i 3.02, Man i Chitinase), Marsupenaeus spp (Mar j 1, Mar j 1.0101, Mar j 2, Mar j 4), Matricaria spp (Mat c 17 kD), Mecopoda spp (Mec e 7), Megalobrama spp (Meg am 2, Meg am CK), Megathura spp (Meg c Hemocyanin), Megalops spp (Meg sp 1), Melanogrammus spp (Mel a 1), Meleagris spp (Mel g 1, Mel g 2, Mel g 3, Mel g PRVB, Mel g TSA), Melicertus spp (Mel I 1), Menticirrhus spp (Men am 1), Mercurialis spp (Mer a 1, Mer a 1.0101), Merluccius spp (Mer ap 1, Mer au 1, Mer bi 1, Mer ca 1, Mer ga 1, Mer hu 1), Merlangius spp (Mer me 1), Merluccius spp (Mer mr 1, Mer pa 1, Mer po 1, Mer pr 1, Mer se 1), Meriones spp (Mer un 23 kD), Metarhizium spp (Met a 30), Metapenaeopsis spp (Met ba 1), Metapenaeus spp (Met e 1, Met e 1.0101, Met e 2), Metasequoia spp (Met gl 2), Metapenaeus spp (Met j 1, Met j 2), Metanephrops spp (Met ja 1), Metapenaeopsis spp (Met la 1), Metanephrops spp (Met t 2), Micromesistius spp (Mic po 1), Micropogonias spp (Mic un 1), Mimachlamys spp (Mim n 1), Momordica spp (Mom c RIP), Morus spp (Mor a 17 kD, Mor a 4), Morone spp (Mor am 1), Morus spp (Mor n 3, Mor n 3.0101), Morone spp (Mor sa 1, Mor sc 1), Mugil spp (Mug c 1), Muraenolepis spp (Mur mi 1), Musa spp (Mus a 1, Mus a 1.0101, Mus a 2, Mus a 2.0101, Mus a 3, Mus a 3.0101, Mus a 4, Mus a 4.0101, Mus a 5, Mus a 5.0101, Mus a 5.0102), Mus spp (Mus m 1, Musm 1.0101, Mus m 1.0102, Mus m 2, Mus m Gelatin, Mus m IgG, Mus m MSA, Mus m Muromonab, Mus m Phosvitin), Mustela spp (Mus p 17 kD), Musa spp (Mus xp 1, Mus xp 2, Mus xp 5), Mycteroperca spp (Myc bo 1, Myc mi 1, Myc ph 1), Myceliophthora spp (Myc sp Laccase), Myrmecia spp (Myr p 1, Myr p 1.0101, Myr p 2, Myr p 2.0101, Myr p 2.0102, Myr p 3, Myr p 3.0101), Mytilus spp (Myt e 1, Myt g 1, Myt g PM), Myzus spp (Myz p 7, Myz p 7.0101), Nemorhedus spp (Nae go Hya), Necator spp (Nec a Calreticulin), Nemipterus spp (Nem vi 1), Neosartorya spp (Neo fi 1, Neo fi 22), Neochen spp (Neo ju 1), Neoscona spp (Neo n 7, Neo n 7.0101), Nephelium spp (Nep I GAPDH), Nephrops spp (Nep n 1, Nep n DF9), Neptunea spp (Nep po 1, Nep po 1.0101), Nicotiana spp (Nic t 8, Nic t Osmotin, Nic t Villin), Nimbya spp (Nim c 1, Nim s 1), Nippostrongylus spp (Nip b Agl), Nycticebus spp (Nyc c 1), Octopus spp (Oct f 1, Oct I 1, Oct v 1, Oct v 1.0101, Oct v PM), Ocyurus spp (Ocy ch 1), Olea spp (Ole e 1, Ole e 1.0101, Ole e 1.0102, Ole e 1.0103, Ole e 1.0104, Ole e 1.0105, Ole e 1.0106, Ole e 1.0107, Ole e 10, Ole e 10.0101, Ole e 11, Ole e 11.0101, Ole e 11.0102, Ole e 12, Ole e 13, Ole e 2, Ole e 2.0101, Ole e 3, Ole e 3.0101, Ole e 36 kD, Ole e 4, Ole e 4.0101, Ole e 5, Ole e 5.0101, Ole e 6, Ole e 6.0101, Ole e 7, Ole e 7.0101, Ole e 8, Ole e 8.0101, Ole e 9, Ole e 9.0101), Ommastrephes spp (Omm b 1, Omm b 1.0101), Oncorhynchus spp (Onc ke 1, Onc ke 18 kD, Onc ke alpha2I, Onc ke Vitellogenin, Onc m 1, Onc m 1.0101, Onc m 1.0201, Onc m alpha2I, Onc m Protamine, Onc m Vitellogenin, Onc ma 1, Onc ma FPA, Onc ma FSA, Onc ma TPI, Onc n 1), Onchocerca spp (Onc o 3, Onc o 3.0101), Oncorhynchus spp (Onc ts 1), Onchocerca spp (Onc v 3, Onc v 3.0101), Oratosquilla spp (Ora o 1, Ora o 1.0101), Oreochromis spp (Ore a 1, Ore mo 1, Ore mo 2, Ore mo FPA, Ore mo SCAF7145, Ore ni 1, Ore ni 18 kD, Ore ni 45 kD), Ornithonyssus spp (Orn sy 10, Orn sy 10.0101, Orn sy 10.0102), Oryctolagus spp (Ory c 1, Ory c 1.0101, Ory c 2, Ory c Casein, Ory c Phosvitin, Ory c RSA), Oryza spp (Ory s 1, Ory s 1.0101, Ory s 11, Ory s 12, Ory s 12.0101, Ory s 13, Ory s 14, Ory s 17 kD, Ory s 19 kD, Ory s2, Ory s23, Ory s 3, Ory s 7, Ory s aA_TI, Ory s GLP52, Ory s GLP63, Ory s Glyoxalase I, Ory s NRA), Ostrya spp (Ost c 1, Ost c 1.0101), Ovis spp (Ovi a ALA, Ovi a BLG, Ovi a Casein, Ovi a Casein alphaSI, Ovi a Casein alphaS2, Ovi a Casein beta, Ovi a Casein kappa, Ovi a Phosvitin, Ovi a SSA), Pachycondyla spp (Pac c 3), Pagrus spp (Pag m 1, Pag pa 1), Pampus spp (Pam ar 1, Pam c 1), Pandalus spp (Pan b 1, Pan b 1.0101), Pangasius spp (Pan bo 1), Pandalus spp (Pan e 1, Pan e 1.0101, Pan e 4), Panulirus spp (Pan h 1, Pan by 1), Pangasius spp (Pan by 18 kD, Pan by 45 kD), Panulirus spp (Pan j 1), Panthera spp (Pan I 1, Pan o 1, Pan p 1), Panulirus spp (Pan s 1, Pan s 1.0101), Panthera spp (Pan t 1), Pan spp (Pan tr TCTP), Papaver spp (Pap s 17 kD, Pap s 2, Pap s 34 kD), Papilio spp (Pap xu 7, Pap xu 7.0101, Pap xu 7.0102), Paralichthys spp (Par a 1), Parasilurus spp (Par as 1, Par c 1), Paralithodes spp (Par c 1.0101, Par c 1.0102, Par f 1), Parthenium spp (Par h 1), Parietaria spp (Par j 1, Par j 1.0101, Par j 1.0102, Par j 1.0103, Par j 1.0201, Par j 2, Par j 2.0101, Par j 2.0102, Par j 3, Par j 3.0101, Par j 3.0102, Par j 4, Par j 4.0101, Par j J1-J2), Paralichthys spp (Par le 1), Parietaria spp (Par m 1, Par o 1, Par o 1.0101), Paralichthys spp (Par ol 1, Par of alpha2I), Parahucho spp (Par pe Vitellogenin), Passiflora spp (Pas e Chitinase, Pas e Hevein), Paspalum spp (Pas n 1, Pas n 1.0101, Pas n 13), Patinopecten spp (Pat y 1), Pediculus spp (Ped h 7, Ped h 7.0101), Penaeus spp (Pen a 1, Pen a 1.0101, Pen a 1.0102, Pen a 1.0102 (103-117), Pen a 1.0102 (109-123), Pen a 1.0102 (1-15), Pen a 1.0102 (115-129), Pen a 1.0102 (121-135), Pen a 1.0102 (127-141), Pen a 1.0102 (13-27), Pen a 1.0102 (133-147), Pen a 1.0102 (139-153), Pen a 1.0102 (145-159)), Farfantepenaeus spp (Pen a 1.0102 (151-165)), Penaeus spp (Pen a 1.0102 (157-171), Pen a 1.0102 (163-177), Pen a 1.0102 (169-183), Pen a 1.0102 (175-189), Pen a 1.0102 (181-195), Pen a 1.0102 (187-201), Pen a 1.0102 (193-207), Pen a 1.0102 (19-33), Pen a 1.0102 (199-213), Pen a 1.0102 (205-219), Pen a 1.0102 (211-225), Pen a 1.0102 (217-231), Pen a 1.0102 (223-237), Pen a 1.0102 (229-243)), Farfantepenaeus spp (Pen a 1.0102 (235-249)), Penaeus spp (Pen a 1.0102 (241-255), Pen a 1.0102 (247-261), Pen a 1.0102 (253-267), Pen a 1.0102 (25-39), Pen a 1.0102 (259-273), Pen a 1.0102 (265-279), Pen a 1.0102 (270-284), Pen a 1.0102 (31-45), Pen a 1.0102 (37-51), Pen a 1.0102 (43-57), Pen a 1.0102 (49-63)), Farfantepenaeus spp (Pen a 1.0102 (55-69)), Penaeus spp (Pen a 1.0102 (61-75), Pen a 1.0102 (67-81), Pen a 1.0102 (7-21), Pen a 1.0102 (73-87), Pen a 1.0102 (79-93), Pen a 1.0102 (85-99), Pen a 1.0102 (91-105), Pen a 1.0102 (97-111), Pen a 1.0103), Penicillium spp (Pen b 13, Pen b 13.0101, Pen b 26, Pen b 26.0101, Pen c 1, Pen c 13, Pen c 13.0101, Pen c 18, Pen c 19, Pen c 19.0101, Pen c 2, Pen c 22, Pen c 22.0101, Pen c 24, Pen c 24.0101, Pen c 3, Pen c 3.0101, Pen c 30, Pen c 30.0101, Pen c 32, Pen c 32.0101, Pen c MnSOD, Pen ch 13, Pen ch 13.0101, Pen ch 18, Pen ch 18.0101, Pen ch 20, Pen ch 20.0101, Pen ch 31, Pen ch 31.0101, Pen ch 33, Pen ch 33.0101, Pen ch 35, Pen ch 35.0101, Pen ch MnSOD), Penaeus spp (Pen i 1, Pen i 1.0101, Pen m 1, Penm 1.0101, Penm 1.0102, Pen m 2, Pen m 2.0101, Pen m 3, Pen m 3.0101, Pen m 4, Pen m 4.0101, Pen m 6, Pen m 6.0101), Penicillium spp (Pen o 18, Pen o 18.0101), Penaeus spp (Pena o 1, Pena o 1.0101), Periplaneta spp (Per a 1, Per a 1.0101, Per a 1.0102, Per a 1.0103, Per a 1.0104, Per a 1.0105, Per a 1.0201, Per a 10, Per a 10.0101, Per a 2, Per a 3, Per a 3.0101, Per a 3.0201, Per a 3.0202, Per a 3.0203, Per a 4, Per a 5, Per a 6, Per a 6.0101, Per a 7, Per a 7.0101, Per a 7.0102, Per a 7.0103, Per a 9, Per a 9.0101, Per a Cathepsin, Per a FABP, Per a Trypsin, Per f 1, Per f 7, Per f 7.0101), Perna spp (Per v 1), Persea spp (Pers a 1, Pers a 1.0101, Pers a 4), Petroselinum spp (Pet c 1, Pet c 2, Pet c 3), Phalaris spp (Pha a 1, Pha a 1.0101, Pha a 5, Pha a 5.0101, Pha a 5.02, Pha a 5.03, Pha a 5.04), Phaseolus spp (Pha v 3, Pha v 3.0101, Pha v 3.0201, Pha v aAI, Pha v aAI.0101, Pha v Chitinase, Pha v PHA, Pha v Phaseolin), Phleum spp (Phl p 1, Phl p 1.0101, Phi p 1.0102, Phi p 11, Phi p 11.0101, Phi p 12, Phi p 12.0101, Phi p 12.0102, Phi p 12.0103, Phi p 13, Phl p 13.0101, PhI p 2, Phi p 2.0101, Phi p 3, Phi p 3.0101, PhI p 3.0102, Phi p 4, Phi p 4.0101, Phi p 4.0102, Phl p 4.0201, Phl p 4.0202, Phl p 4.0203, Phl p 4.0204, Phl p 5, Phl p 5.0101, Phl p 5.0102, Phl p 5.0103, Phl p 5.0104, Phl p 5.0105, Phl p 5.0106, Phl p 5.0107, Phl p 5.0108, Phl p 5.0109, Phl p 5.0201, Phl p 5.0202, Phl p 5.0203, Phl p 5.0204, Phl p 5.0205, Phl p 5.0206, Phl p 5.0207, Phl p 6, Phl p 6.0101, Phl p 6.0102, Phl p 7, Phl p 7.0101, Phl p P1-P2-P5-P6, Phl p P2-P6, Phl p P5-P1, Phl p P6-P2), Phoenix spp (Pho d 2, Pho d 2.0101, Pho d 40 kD, Pho d 90 kD), Phodopus spp (Pho s 21 kD), Phoma spp (Pho t 1), Phragmites spp (Phr a 1, Phr a 12, Phr a 13, Phr a 4, Phr a 5), Phytolacca spp (Phy a RIP), Pimpinella spp (Pim a 1, Pim a 2), Pinna spp (Pin a 1), Piper spp (Pip n 14 kD, Pip n 28 kD), Pisum spp (Pis s 1, Pis s 1.0101, Pis s 1.0102, Pis s 2, Pis s 2.0101, Pis s 5, Pis s Agglutinin, Pis s Albumin), Pistacia spp (Pis v 1, Pis v 1.0101, Pis v 2, Pis v 2.0101, Pis v 2.0201, Pis v 3, Pis v 3.0101, Pis v 4, Pis v 4.0101, Pis v 5, Pis v 5.0101), Platanus spp (Pla a 1, Pla a 1.0101, Pla a 2, Pla a 2.0101, Pla a 3, Pla a 3.0101, Pla a 8), Platichthys spp (Pla f 1), Plantago spp (Pla I 1, Pla I 1.0101, Pla I 1.0102, Pla I 1.0103, Pla I Cytochrome C), Platanus spp (Pla oc 1, Pla or 1, Pla or 1.0101, Pla or 2, Pla or 2.0101, Pla or 3, Pla or 3.0101, Pla or 4, Pla or CyP, Pla r 1), Plectropomus spp (Pie ar 1), Pleospora spp (Pie h 1), Plectropomus spp (Pie le 1), Plodia spp (Plo i 1, Plo i 1.0101, Plo i 2, Plo i 2.0101), Poa spp (Poa p 1, Poa p 1.0101, Poa p 10, Poa p 12, Poa p 13, Poa p 2, Poa p 4, Poa p 5, Poa p 5.0101, Poa p 6, Poa p 7), Polistes spp (Pol a 1, Pol a 1.0101, Pol a 2, Pol a 2.0101, Pol a 5, Pol a 5.0101, Pol d 1, Pol d 1.0101, Pol d 1.0102, Pol d 1.0103, Pol d 1.0104, Pol d 4, Pol d 4.0101, Pol d 5, Pol d 5.0101, Pol e 1, Pol e 1.0101, Pol e 2, Pol e 4, Pol e 4.0101, Pol e 5, Pol e 5.0101, Pol f 5, Pol f 5.0101, Pol g 1, Pol g 1.0101, Pol g 2, Pol g 4, Pol g 5, Pol g 5.0101, Pol he MLT, Pol m 5, Pol m 5.0101), Polypedilum spp (Pol n 1), Pollicipes spp (Pol po 1), Pollachius spp (Pol vi 1), Polybia spp (Poly p 1, Poly p 1.0101, Poly p 2, Poly p 5, Poly s 5, Poly s 5.0101), Pomatomus spp (Pom sa 1), Pongo spp (Pon ab HSA), Pontastacus spp (Pon 14, Pon I 4.0101, Pon I 7, Pon I 7.0101), Portunus spp (Por s 1, Por s 1.0101, Por s 1.0102, Por tr 1, Por tr 1.0101), Protortonia spp (Pro ca 38 kD), Procumbarus spp (Pro cl 1, Pro cl 1.0101, Pro cl 21 kD), Prosopis spp (Pro j 20 kD), Prunus spp (Pru ar 1, Pru ar 1.0101, Pru ar 3, Pru ar 3.0101, Pru av 1, Pru av 1.0101, Pru av 1.0201, Pru av 1.0202, Pru av 1.0203, Pru av 2, Pru av 2.0101, Pru av 3, Pru av 3.0101, Pru av 4, Pru av 4.0101, Pru c 1, Pru d 1, Pru d 2, Pru d 3, Pru d 3.0101, Pru d 4, Pru du 1, Pru du 2, Pru du 2S Albumin, Pru du 3, Pru du 3.0101, Pru du 4, Pru du 4.0101, Pru du 4.0102, Pru du 5, Pru du 5.0101, Pru du 6, Pru du 6.0101, Pru du 6.0201, Pru du Conglutin, Pru p 1, Pru p 1.0101, Pru p 2, Pru p 2.0101, Pru p 2.0201, Pru p 2.0301, Pru p 3, Pru p 3.0101, Pru p 3.0102, Pru p 4, Pru p 4.0101, Pru p 4.0201, Pru sa 3), Psilocybe spp (Psi c 1, Psi c 1.0101, Psi c 2, Psi c 2.0101), Psoroptes spp (Pso o 1, Pso o 10, Pso o 10.0101, Pso o 11, Pso o 13, Pso o 14, Pso o 2, Pso o 21, Pso o 3, Pso o 5, Pso o 7), Puma spp (Pum c 1), Punica spp (Pun g 3), Pyrus spp (Pyr c 1, Pyr c 1.0101, Pyr c 3, Pyr c 3.0101, Pyr c 4, Pyr c 4.0101, Pyr c 5, Pyr c 5.0101, Pyr py 2), Quercus spp (Que a 1, Que a 1.0101, Que a 1.0201, Que a 1.0301, Que a 1.0401, Que a 2, Que a 4), Rachycentron spp (Rac ca 1), Rana spp (Ran e 1, Ran e 1.0101, Ran e 2, Ran e 2.0101), Ranina spp (Ran ra 1), Rangifer spp (Ran t BLG), Rattus spp (Rat n 1, Rat n 1.0101, Rat n Casein, Rat n Gelatin, Rat n IgG, Rat n Phosvitin, Rat n RSA, Rat n Transferrin), Rhizomucor spp (Rhi m AP), Rhizopus spp (Rhi nv Lipase, Rhi o Lipase), Rhomboplites spp (Rho au 1), Rhodotorula spp (Rho m 1, Rho m 1.0101, Rho m 2, Rho m 2.0101), Ricinus spp (Ric c 1, Ric c 1.0101, Ric c 2, Ric c 3, Ric c 8, Ric c RIP), Rivulus spp (Riv ma 1), Robinia spp (Rob p 2, Rob p 4, Rob p Glucanase), Rosa spp (Ros r 3), Roystonea spp (Roy e 2), Rubus spp (Rub i 1, Rub i 1.0101, Rub i 3, Rub i 3.0101, Rub i Chitinase, Rub i CyP), Saccharomyces spp (Sac c Carboxypeptidase Y, Sac c CyP, Sac c Enolase, Sac c Glucosidase, Sac c Invertase, Sac c MnSOD, Sac c P2, Sac c Profilin), Salvelinus spp (Sal f 1), Salsola spp (Sal k 1, Sal k 1.0101, Sal k 1.0201, Sal k 1.0301, Sal k 1.0302, Sal k 2, Sal k 2.0101, Sal k 3, Sal k 3.0101, Sal k 4, Sal k 4.0101, Sal k 4.0201, Sal k 5, Sal k 5.0101), Salvelinus spp (Sal le Vitellogenin), Salmo spp (Sal s 1, Sal s 1.0101, Sal s 1.0201, Sal s 2, Sal s 2.0101, Sal s Gelatin), Sambucus spp (Sam n 1), Sander spp (San lu 1), Saponaria spp (Sap o RIP), Sardinops spp (Sar m 1), Sarkidiornis spp (Sar ml 1), Sardina spp (Sar p 1), Sarcoptes spp (Sar s 1, Sar s 14, Sar s 3, Sar s GST, Sar s PM), Sardinops spp (Sar sa 1, Sar sa 1.0101), Schistosoma spp (Sch j GST, Sch j PM, Sch j Sj22, Sch j Sj67, Sch ma Sm20, Sch ma Sm21, Sch ma Sm22, Sch ma Sm31), Sciaenops spp (Sci oc 1), Scomber spp (Sco a 1), Scombermorus spp (Sco ca 1), Scomberomorus spp (Sco g 1), Scomber spp (Sco j 1, Sco ma 1, Sco s 1), Scolopendra spp (Sco y 7, Sco y 7.0101), Scylla spp (Scy o 1, Scy o 1.0101, Scy o 2, Scy pa 1, Scy pa 2, Scy s 1, Scy s 1.0101, Scy s 2), Sebastes spp (Seb fa 1, Seb in 1, Seb m 1, Seb m 1.0101, Seb m 1.0201), Secale spp (Sec c 1, Sec c 12, Sec c 13, Sec c 2, Sec c 20, Sec c 20.0101, Sec c 20.0201, Sec c 28, Sec c 3, Sec c 4, Sec c 4.0101, Sec c 4.0201, Sec c 5, Sec c 5.0101, Sec c aA_TI, Sec c aA_TI.0101), Senecio spp (Sen j MDH, Sen j PL), Sepia spp (Sep e 1, Sep e 1.0101), Sepioteuthis spp (Sep I 1, Sep 1 1.0101), Sepia spp (Sep m 1), Seriola spp (Ser d 1, Ser la 1), Sergestes spp (Ser lu 1), Seriola spp (Ser q 1, Ser ri 1), Sesamum spp (Ses i 1, Ses i 1.0101, Ses i 2, Ses i 2.0101, Ses i 3, Ses i 3.0101, Ses i 4, Ses i 4.0101, Ses i 5, Ses i 5.0101, Ses i 6, Ses i 6.0101, Ses i 7, Ses i 7.0101, Ses i 8), Shigella spp (Shi bo GST, Shi dy GST), Simulia spp (Sim vi 1, Sim vi 2, Sim vi 3, Sim vi 4, Sim vi 70 kD), Sinapis spp (Sin a 1, Sin a 1.0101, Sin a 1.0104, Sin a 1.0105, Sin a 1.0106, Sin a 1.0107, Sin a 1.0108, Sin a 2, Sin a 2.0101, Sin a 3, Sin a 3.0101, Sin a 4, Sin a 4.0101), Sinonovacula spp (Sin c 1, Sin c 1.0101), Solenopsis spp (Sol g 2, Sol g 2.0101, Sol g 3, Sol g 3.0101, Sol g 4, Sol g 4.0101, Sol g 4.0201, Sol i 1, Sol i 1.0101, Sol i 2, Sol i 2.0101, Sol i 3, Sol i 3.0101, Sol i 4, Sol i 4.0101), Solenocera spp (Sol me 1), Solenopsis spp (Sol r 1, Sol r 2, Sol r 2.0101, Sol r 3, Sol r 3.0101, Sol s 2, Sol s 2.0101, Sol s 3, Sol s 3.0101, Sol s 4), Solea spp (Sol so 1, Sol so TPI), Solanum spp (Sola t 1, Sola t 1.0101, Sola t 2, Sola t 2.0101, Sola t 3, Sola t 3.0101, Sola t 3.0102, Sola t 4, Sola t 4.0101, Sola t 8, Sola t Glucanase), Sorghum spp (Sor b 1, Sor h 1, Sor h 1.0101, Sor h 12, Sor h 7), Sparus spp (Spa a 1), Sphyrna spp (Sph ti 1), Spirulina spp (Spi mx beta Phycocyanin), Spinacia spp (Spi o 2, Spi o RuBisCO), Squilla spp (Squ ac 1, Squ ac 1.0101, Squ o 1, Squ o 1.0101), Staphylococcus spp (Sta a FBP, Sta a SEA, Sta a SEB, Sta a SEC, Sta a SED, Sta a SEE, Sta a TSST), Stachybotrys spp (Sta c 3, Sta c 3.0101, Sta c Cellulase, Sta c Hemolysin, Sta c SchS34, Sta c Stachyrase A), Stemphylium spp (Ste b 1, Ste c 1, Ste v 1), Stolephorus spp (Sto i 1), Struthio spp (Str c 1, Str c 2, Str c 3), Streptococcus spp (Str dy Streptokinase), Streptomyces spp (Str g Pronase), Streptococcus spp (Str pn PspC), Strongylocentrotus spp (Str pu 18 kD, Str pu Vitellogenin), Streptococcus spp (Str py SPEA, Str py SPEC, Str py Streptokinase), Strongyloides spp (Str st 45 kD), Streptomyces spp (Str v PAT), Styela spp (Sty p 1), Suidasia spp ( Sui m 1 , Sui m 13 , Sui m 2 , Sui m 3 , Sui m 5 , Sui m 5.01 , Sui m 5.02 , Sui m 5.03 , Sui m 6 , Sui m 7 , Sui m 8 , Sui m 9), Sus spp (Sus s ACTH, Sus s ALA, Sus s Amylase, Sus s BLG, Sus s Casein, Sus s Casein alphaS1, Sus s Casein alphaS2, Sus s Casein beta, Sus s Casein kappa, Sus s Gelatin, Sus s HG, Sus s Insulin, Sus s Lipase, Sus s Pepsin, Sus s Phosvitin, Sus s PRVB, Sus s PSA, Sus s TCTP), Syntelopodeuma spp (Syn y 7, Syn y 7.0101), Syringa spp (Syr v 1, Syr v 1.0101, Syr v 1.0102, Syr v 1.0103, Syr v 2, Syr v 3, Syr v 3.0101), Tabanus spp (Tab y 1, Tab y 1.0101, Tab y 2, Tab y 2.0101, Tab y 5, Tab y 5.0101), Tadorna spp (Tad ra 1), Talaromyces spp (Tal st 22, Tal st 3, Tal st 8), Taraxacum spp (Tar o 18 kD), Taxodium spp (Tax d 2), Tegenaria spp (Teg d Hemocyanin), Teladorsagia spp (Tel ci 3), Thaumetopoea spp (Tha p 1, Tha p 1.0101, Tha p 2, Tha p 2.0101), Theragra spp (The c 1), Thermomyces spp (The I Lipase, The sp Lipase, The sp Xylanase), Thunnus spp (Thu a 1, Thu a 1.0101, Thu a Collagen, Thu al 1, Thu at 1, Thu o 1, Thu o Collagen), Thuja spp (Thu oc 3, Thu p 1), Thunnus spp (Thu t 1, Thu to 1), Thyrsites spp (Thy at 1), Thyrophygus spp (Thy y 7, Thy y 7.0101), Todarodes spp (Tod p 1, Tod p 1.0101, Tod p 1.0102), Toxoptera spp (Tox c 7, Tox c 7.0101), Toxocara spp (Tox ca TES120, Tox ca TES26, Tox ca TES30), Toxoplasma spp (Tox g HSP70), Trachypenaeus spp (Tra c 1), Trachinotus spp (Tra ca 1), Trachurus spp (Tra j 1, Tra j Gelatin, Tra tr Gelatin), Triticum spp (Tri a 1, Tri a 10 kD, Tri a 12, Tri a 12.0101, Tri a 12.0102, Tri a 12.0103, Tri a 12.0104, Tri a 13, Tri a 14, Tri a 14.0101, Tri a 14.0201, Tri a 15, Tri a 15.0101, Tri a 18, Tri a 18.0101, Tri a 19, Tri a 19.0101, Tri a 2, Tri a 21, Tri a 21.0101, Tri a 23kd, Tri a 25, Tri a 25.0101, Tri a 26, Tri a 26.0101, Tri a 27, Tri a 27.0101, Tri a 28, Tri a 28.0101, Tri a 29, Tri a 29.0101, Tri a 29.0201, Tri a 3, Tri a 30, Tri a 30.0101, Tri a 31, Tri a 31.0101, Tri a 32, Tri a 32.0101, Tri a 33, Tri a 33.0101, Tri a 34, Tri a 34.0101, Tri a 35, Tri a 35.0101, Tri a 36, Tri a 36.0101, Tri a 37, Tri a 37.0101, Tri a 4, Tri a 4.0101, Tri a 4.0201, Tri a 5, Tri a 7, Tri a aA_SI, Tri a alpha_Gliadin, Tri a bA, Tri a Bd36K, Tri a beta Gliadin, Tri a Chitinase, Tri a CM16, Tri a DH, Tri a Endochitinase, Tri a gamma_Gliadin, Tri a Germin, Tri a Gliadin, Tri a GST, Tri a LMW Glu, Tri a LMW-GS B16, Tri a LMW-GS P42, Tri a LMW-GS P73, Tri a LTP2, Tri a omega2_Gliadin, Tri a Peroxidase, Tri a Peroxidase 1, Tri a SPI, Tri a TLP, Tri a Tritin, Tri a XI), Tritirachium spp (Tri al Proteinase K), Tribolium spp (Tri ca 17, Tri ca 17.0101, Tri ca 7, Tri ca 7.0101), Trichostrongylus spp (Tri co 3, Tri co 3.0101), Trichophyton spp (Tri eq 4), Trigonella spp (Tri fg 1, Tri fg 2, Tri fg 3, Tri fg 4), Trichosanthes spp (Tri k RIP), Trichiurus spp (Tri le 1), Triticum spp (Tri m Peroxidase), Trichophyton spp (Tri me 2, Tri me 4), Trisetum spp (Tri p 1, Tri p 5), Trichinella spp (Tri ps 3, Tri ps 3.0101), Trichophyton spp (Tri r 2, Tri r 2.0101, Tri r 4, Tri r 4.0101), Trichoderma spp (Tri rs Cellulase), Triticum spp (Tri s 14), Trichophyton spp (Tri sc 2, Tri sc 4, Tri so 2), Trichinella spp (Tri sp 3, Tri sp 3.0101, Tri sp 3.0102, Tri sp 3.0103, Tri sp 3.0104, Tri sp 3.0105, Tri sp 3.0106), Trichophyton spp (Tri t 1, Tri t 1.0101, Tri t 4, Tri t 4.0101), Triticum spp (Tri td 14, Tri td aA_TI), Trichoderma spp (Tri v Cellulase), Trichophyton spp (Tri ve 4), Triatoma spp (Tria p 1, Tria p 1.0101), Triplochiton spp (Trip s 1), Turbo spp (Tur c 1, Tur c PM), Tyrophagus spp (Tyr p 1, Tyr p 10, Tyr p 10.0101, Tyr p 10.0102, Tyr p 13, Tyr p 13.0101, Tyr p 2, Tyr p 2.0101, Tyr p 24, Tyr p 24.0101, Tyr p 3, Tyr p 3.0101, Tyr p 4, Tyr p 5, Tyr p 5.01, Tyr p 5.02, Tyr p 5.03, Tyr p 7, Tyr p alpha Tubulin), Ulocladium spp (Ulo a 1, Ulo at 1, Ulo b 1, Ulo c 1, Ulo co 1, Ulo cu 1, Ulo mu 1, Ulo ob 1, Ulo se 1, Ulo su 1, Ulo tu 1), Uncia spp (Unc u 1), Urophycis spp (Uro te 1), Vaccinium spp (Vac m 3), Varroa spp (Var j 13 kD), Venerupis spp (Ven ph 1, Ven ph 1.0101), Vespula spp (Ves f 1, Ves f 2, Ves f 5, Ves f 5.0101, Ves g 1, Ves g 2, Ves g 5, Ves g 5.0101, Ves m 1, Ves m 1.0101, Ves m 2, Ves m 2.0101, Ves m 5, Ves m 5.0101, Ves m MLT, Ves p 1, Ves p 2, Ves p 5, Ves p 5.0101, Ves s 1, Ves s 1.0101, Ves s 2, Ves s 5, Ves s 5.0101, Ves v 1, Ves v 1.0101, Ves v 2, Ves v 2.0101, Ves v 2.0201, Ves v 3, Ves v 3.0101, Ves v 5, Ves v 5.0101, Ves v 5-Pol a 5, Ves vi 5, Ves vi 5.0101), Vespa spp (Vesp c 1, Vesp c 1.0101, Vesp c 2, Vesp c 5, Vesp c 5.0101, Vesp c 5.0102, Vesp m 1, Vesp m 1.0101, Vesp m 5, Vesp m 5.0101, Vesp ma 1, Vesp ma 2, Vesp ma 5, Vesp ma MLT, Vesp v MLT), Vigna spp (Vig r 1, Vig r 1.0101, Vig r 17 kD, Vig r 5, Vig r 8S Globulin, Vig r Albumin, Vig r beta-Conglycinin), Vitis spp (Vit v 1, Vit v 1.0101, Vit v 4, Vit v 5, Vit v Glucanase, Vit v TLP), Xiphias spp (Xip g 1, Xip g 1.0101, Xip g 25 kD), Zea spp ( Zea m 1 , Zea m 1.0101 , Zea m 11 , Zea m 12 , Zea m 12.0101 , Zea m 12.0102 , Zea m 12.0103 , Zea m 12.0104 , Zea m 12.0105 , Zea m 13 , Zea m 14 , Zea m 14.0101 , Zea m 14.0102 , Zea m 2 , Zea m 20S, Zea m 22 , Zea m 25 , Zea m 25.0101 , Zea m 27 kD Zein, Zea m 3 , Zea m 4 , Zea m 5 , Zea m 50 kD Zein, Zea m 7 , Zea m Chitinase, Zea m G1 , Zea m G2 , Zea m PAO, Zea m Zm13), Zeus spp (Zeu fa 1), Ziziphus spp (Ziz m 1, Ziz m 1.0101), Zoarces spp (Zoa a ISP III), Zygophyllum spp (Zyg f 2).

In this context, the terms in brackets indicate the particular preferred allergenic antigens (allergens) from the particular source.

Most preferably the allergenic antigen is preferably derived from a source (e.g. a plant (e.g. grass or a tree), a natural product (e.g. milk, nuts etc.), a fungal source (e.g. Aspergillus ) or a bacterial source or from an animal source or animal poison (e.g. cat, dog, venom of bees etc.), preferably selected from the list consisting of grass pollen (e.g. pollen of rye), tree pollen (e.g. pollen of hazel, birch, alder, ash), flower pollen, herb pollen (e.g. pollen of mugwort), dust mite (e.g. Der f 1, Der p 1, Eur m 1, Der m 1 Der f 2, Der p 2, Eur m 2, Tyr p 2, Lep d 2), mold (e.g. allergens of Acremonium, Aspergillus, Cladosporium, Fusarium, Mucor, Penicillium, Rhizopus, Stachybotrys, Trichoderma , or Alternaria ), animals (e.g Fel d1, Fel d 2, Fel d3, or Fel d4 of cats), food (e.g. allergens of fish (e.g. bass, cod, flounder), seafood (e.g. crab, lobster, shrimps), egg, wheat, nuts (e.g. peanuts, almonds, cashews, walnuts), soya, milk, etc.) or insect venom (e.g. allergens from the venom of wasps, bees, hornets, ants, mosquitoes, or ticks).

Autoimmune Self-Antigens

Autoimmune self-antigens, i.e. antigens associated with autoimmune disease or autoantigens, may be associated with an autoimmune disease affecting at least one or more of the following organ systems: the circulatory system, the digestive system, the endocrine system, the excretory system, the immune system, the integumentary system, the muscular system, the nervous system, the reproductive system, the respiratory system, the skeletal system, preferably with the cardiovascular system, the neuroendocrine system, the musculoskeletal system or gastrointestinal system. Therein the circulatory system is the organ system which enables pumping and channeling blood to and from the body and lungs with heart, blood and blood vessels. The digestive system enables digestion and processing food with salivary glands, esophagus, stomach, liver, gallbladder, pancreas, intestines, colon, rect