Provided are methods and compositions for the treating a patient with a urea cycle disorder. Methods and compositions are also provided for modulating genes encoding enzymes that participate in the urea cycle by altering gene signaling networks.

Description

62 parts

›RELATED APPLICATION DATA

This application is a continuation application of International Application No. PCT/US2020/027693, filed Apr. 10, 2020, which claims the benefit of U.S. Provisional Application No. 62/832,187, filed Apr. 10, 2019, which is hereby incorporated in its entirety by reference.

›SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Dec. 14, 2020, is named CTC-017WOC1.txt and is 1,309,331 bytes in size.

›BACKGROUND

Urea cycle disorders are a group of genetic disorders caused by defects in the metabolism of waste nitrogen via the urea cycle. The urea cycle is a cycle of biochemical reactions that produces urea from ammonia, a product of protein catabolism. The urea cycle mainly occurs in the mitochondria of liver cells. The urea produced by the liver enters the bloodstream where it travels to the kidneys and is ultimately excreted in urine. Genetic defects in any of the enzymes or transporters in the urea cycle can cause hyperammonemia (elevated blood ammonia), or the buildup of a cycle intermediate. Ammonia then reaches the brain through the blood, where it can cause cerebral edema, seizures, coma, long term disabilities in survivors, and/or death.

The onset and severity of urea cycle disorders is highly variable. It is influenced by the position of the defective protein in the cycle and the severity of the defect. Mutations that lead to severe deficiency or total absence of activity of any of the enzymes can result in the accumulation of ammonia and other precursor metabolites during the first few days of life. Because the urea cycle is the principal clearance system for ammonia, complete disruption of this pathway results in the rapid accumulation of ammonia and development of related symptoms. Mild to moderate mutations represent a broad spectrum of enzyme function, providing some ability to detoxify ammonia, and result in mild to moderate urea cycle disorders.

According to National Urea Cycle Disorders Foundation, the incidence of urea cycle disorders is estimated to be 1 in 8,500 live birth in the United States. The estimated incidence of individual urea cycle disorder varies from less than 1:2,000,000 to about 1:56,500 (See N A Mew et al., Urea Cycle Disorders Overview, 2015). They occur in both children and adults. These disorders are most often diagnosed in infancy, but some children do not develop symptoms until early childhood. Newborns with severe urea cycle disorders become catastrophically ill within 36-48 hours of life. In children with mild or moderate urea cycle disorders, symptoms may be seen as early as one year of age. Early symptoms include disliking meat or other high-protein foods, inconsolable crying, failure to thrive, mental confusion, and hyperactive behavior. Symptoms can progress to frequent episodes of vomiting, lethargy, delirium, and coma. Some individuals with mild urea cycle defects are diagnosed in adulthood. Ammonia accumulation may be triggered by illness or stress (e.g., viral infection, surgery, prolonged fasting, excessive exercising, and excessive dieting), resulting in multiple mild elevations of plasma ammonia concentration. Without proper diagnosis and treatment, these individuals are at risk for permanent brain damage, coma, and death.

Treatment for urea cycle disorders is a lifelong process. Symptoms are usually managed by using a combination of strategies including diet restriction, amino acid supplements, medications, dialysis, and/or hemofiltration. Dietary management is key to restricting the level of ammonia produced in the body. A careful balance of dietary protein, carbohydrates and fats is necessary to lower protein intake, while providing adequate calories for energy needs, as well as adequate essential amino acids for cell growth and development. Depending on the type of urea cycle disorder, amino acid supplements such as arginine or citrulline may be added to the diet. Sodium phenylbutyrate (BUPHENYL®), glycerol phenylbutyrate (RAVICTI®) and sodium benzoate are FDA approved drugs for the treatment of urea cycle disorders. They function as nitrogen binding agents to allow the kidneys to excrete excess nitrogen in place of urea. Dialysis and/or hemofiltration are used to quickly reduce plasma ammonia concentration to normal physiological level. When other treatment and management options fail, or for neonatal onset CPS1 and OTC deficiency, liver transplant is an option. Although the transplant alternative has been proven to be effective, the cost of the surgery, shortage of donors, and possible side effects of immunosuppressants can be difficult to overcome.

Therefore, there is a high unmet need for developing effective therapeutics for the treatment of urea cycle disorders. Provided herein are compositions and methods for the treatment of urea cycle disorders in humans.

›SUMMARY

Provided herein are methods for increasing OTC gene expression in a cell harboring an OTC mutation associated with a partial reduction of OTC function, comprising: contacting the cell with an effective amount of a compound that increases CPS1 expression, wherein the compound inhibits a target selected from the group consisting of JAK1, JAK2, JAK3, HSP90, and IRF9.

In some embodiments, the cell is a hepatocyte.

In some embodiments, the target is JAK1, JAK2 or JAK3.

In some embodiments, the target is JAK1.

In some embodiments, the compound is selected from the group consisting of tofacitinib, momelotinib, baricitinib, upadacitinib, oclacitinib maleate, GLPG0634, GLPG0634 analog, LYS2784544, ruxolitinib, itacitinib, AZD1480, CP690550 citrate, cerdulatinib, decernotinib, peficitinib, and PF-06263276.

In some embodiments, the compound is tofacitinib.

In some embodiments, the target is HSP90

In some embodiments, the compound is selected from the group consisting of EC144, 17-AAG, BIIB021, HSP-990, retaspimycin hydrochloride (HCl), PF-0492911, luminespib, alvespimycin, and alvespimycin hydrochloride (HCl).

In some embodiments, the compound is EC144.

In some embodiments, compound is an siRNA compound that inhibits a target selected from the group consisting of JAK1, JAK2, JAK3, HSP90, and IRF9.

In some embodiments, the compound is an siRNA compound that inhibits IRF9.

In some embodiments, the OTC mutation is selected from the group consisting of the mutations appearing in Table 40 that are associated with non-zero percent enzyme activity.

In another aspect, provided herein are methods for increasing OTC expression in a human subject harboring an OTC mutation associated with a partial reduction of OTC function, comprising: administering to the subject an effective amount of a compound that increases CPS1 expression, wherein the compound inhibits a target selected from the group consisting of JAK1, JAK2, JAK3, HSP90, and IRF9.

In some embodiments, the subject is human.

In some embodiments, the target is JAK1, JAK2 or JAK3.

In some embodiments, the target is JAK1.

In some embodiments, the compound is tofacitinib.

In some embodiments, the target is HSP90

In some embodiments, the compound is EC144.

In some embodiments, compound is an siRNA compound that inhibits a target selected from the group consisting of JAK1, JAK2, JAK3, HSP90, and IRF9.

In some embodiments, the compound is an siRNA compound that inhibits IRF9.

In some embodiments, the OTC mutation is selected from the group consisting of the mutations appearing in Table 40 that are associated with non-zero percent enzyme activity.

In another aspect, provided herein are methods for increasing ureagenesis in a subject comprising any method disclosed herein.

In some embodiments, the compound increases a serum urea level in the subject as compared to administration of a control compound.

In some embodiments, the serum urea level is determined by a colorimetric chemical assay or photometric assay.

In some embodiments, the assay is performed on an AutoAnalyzer.

In some embodiments, the serum urea level is increased by about at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.

In some embodiments, the serum urea level is increased by about at least 10%.

A method for increasing ureagenesis in a cell comprising any method disclosed herein.

In some embodiments, the compound increases a urea level in the cell as compared to the urea level in an untreated cell.

In some embodiments, the urea level is determined by a colorimetric chemical assay or photometric assay.

In some embodiments, the assay is performed on an AutoAnalyzer.

In some embodiments, the serum urea level is increased by about at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.

In some embodiments, the serum urea level is increased by about at least 10%.

›BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the packaging of chromosomes in a nucleus, the localized topological domains into which chromosomes are organized, insulated neighborhoods in TADs and finally an example of an arrangement of a signaling center(s) around a particular disease gene.

FIG. 2A and FIG. 2B illustrate a linear and 3D arrangement of the CTCF boundaries of an insulated neighborhood.

FIG. 3A and FIG. 3B illustrate tandem insulated neighborhoods and gene loops formed in such insulated neighborhoods.

FIG. 4 illustrates the concept of an insulated neighborhood contained within a larger insulated neighborhood and the signaling which may occur in each.

FIG. 5 illustrates the components of a signaling center; including transcriptional factors, signaling proteins, and/or chromatin regulators.

FIG. 6 shows OTC gene expression after treatment with the indicated compounds.

FIG. 7 shows ASS1 gene expression after treatment with the indicated compounds.

FIG. 8 shows ASS1 gene expression after treatment with the indicated compounds.

FIG. 9 shows ARG1 gene expression after treatment with the indicated compounds.

FIG. 10 shows ARG1 gene expression after treatment with the indicated compounds.

FIG. 11 shows CPS1 gene expression after treatment with the indicated compounds.

FIG. 12 shows CPS1 gene expression after treatment with the indicated compounds.

FIG. 13 shows NAGS gene expression after treatment with the indicated compounds.

FIG. 14 shows NAGS gene expression after treatment with the indicated compounds.

FIG. 15 shows SLC25A13 gene expression after treatment with the indicated compounds.

FIG. 16A and FIG. 16B show SLC25A15 gene expression after treatment with the indicated compounds.

FIG. 17 shows ASL gene expression after treatment with the indicated compounds.

FIG. 18 shows OTC gene expression after treatment with the indicated compounds.

FIG. 19 shows CPS1 gene expression after treatment with the indicated compounds.

FIG. 20 shows OTC and CPS1 gene expression compared to each other after treatment with the indicated JAK inhibitors that target JAK1. Compounds that increased CPS1 expression also increased OTC expression.

FIG. 21A shows a dose dependent increase in CPS1 expression in hepatocytes in a Donor A cell line after treatment with the indicated compounds. FIG. 21B shows a dose dependent increase in CPS1 expression in hepatocytes in a Donor B cell line after treatment with the indicated compounds. FIG. 21C shows a dose dependent increase in OTC expression in hepatocytes in a Donor A cell line after treatment with the indicated compounds. FIG. 21D shows a dose dependent increase in OTC expression in hepatocytes in a Donor B cell line after treatment with the indicated compounds.

FIG. 22A shows that JAK1 siRNA knockdown increased OTC and CPS1 mRNA expression. FIG. 22B shows that JAK1 knockdown after FANA (2′-deoxy-2′-fluoro-beta-D-arabinonucleic acid antisense) RNA treatment increased OTC and CPS1 mRNA expression. FIG. 22C shows that IFNγ, a JAK activator, decreases OTC expression in hepatocytes. FIG. 22D shows that STAT5B siRNA knockdown increased OTC expression.

FIG. 23 shows opening of a novel chromatin domain upstream of the OTC gene after treatment with the indicated JAK inhibitors.

FIG. 24A shows CPS1 expression after treatment with CPS1 siRNA. FIG. 24B shows CPS1 expression after treatment with JAK1 FANA, or both JAK1 FANA and CPS1 siRNA. FIG. 24C shows OTC expression after treatment with CPS1 siRNA. FIG. 24D shows OTC expression after treatment with JAK1 FANA, or both JAK1 FANA and CPS1 siRNA.

FIG. 25A shows an increase in CPS1 gene expression in vivo after treatment with the indicated JAK1 inhibitors. FIG. 25B shows an increase in OTC gene expression in vivo after treatment with tofacitinib. FIG. 25C shows a decrease in SOCS1 gene expression after treatment with the indicated JAK1 inhibitors.

FIG. 26A shows that siRNA knockdown of JAK1 and JAK2 has an additive effect in increasing OTC expression, as compared to over JAK1 only siRNA knockdown. FIG. 26B shows that siRNA knockdown of JAK1 and JAK2 has an additive effect in increasing CPS1 expression, as compared to over JAK1 only siRNA knockdown.

FIG. 27A shows JAK1 expression after knockdown of the indicated target genes with siRNA. FIG. 27B shows the target gene expression after treatment with siRNA. FIG. 27C shows OTC expression after knockdown of the indicated target genes with siRNA. FIG. 27D shows CPS1 expression after knockdown of the indicated target genes with siRNA.

FIG. 28A shows CPS1 gene expression in Donor A cell line after treatment with the indicated compounds. FIG. 28B shows CPS1 gene expression in Donor B cell line after treatment with the indicated compounds. FIG. 28C shows OTC gene expression in Donor A cell line after treatment with the indicated compounds. FIG. 28D shows OTC gene expression in Donor B cell line after treatment with the indicated compounds.

FIG. 29A shows HPS90 inhibitors, but not HP70 inhibitors, increase OTC expression.

FIG. 29B shows HPS90 inhibitors, but not HP70 inhibitors, increase CPS1 expression.

FIG. 30 shows opening of a novel chromatin domain and increased accessibility of an open chromatin domain upstream of the OTC gene after treatment with the indicated HSP90 inhibitors.

FIG. 31A shows HSP90 inhibitor BIIB021 increases ureagenesis in hepatocytes. FIG. 31B shows HSP90 inhibitor BIIB021 increases expression of multiple urea cycle genes.

FIG. 32 shows in vivo gene expression of the indicated urea cycle genes after treatment with EC144 or control vehicle (V) at various time points post administration.

FIG. 33A shows OTC gene expression after treatment with EC144. FIG. 33B shows CPS1 gene expression after treatment with EC144. FIG. 33C shows serum urea levels in vivo after treatment with an HSP90 inhibitor.

›DETAILED DESCRIPTION · 1 of 28

I. Introduction

Provided herein are compositions and methods for the treatment of urea cycle disorders in mammalian subjects, particularly in human subjects. In particular, provided herein are compounds and related use for the modulation of at least one gene encoding a protein (e.g., an enzyme or a transporter) involved in the urea cycle.

Binding Sites for Signaling Molecules

A series of consensus binding sites, or binding motifs for binding sites, for signaling molecules has been identified by the present inventors. These consensus sequences reflect binding sites along a chromosome, gene, or polynucleotide for signaling molecules or for complexes which include one or more signaling molecules. These sites are provided by Table 11 of U.S. 62/501,795, which is hereby incorporated by reference in its entirety, and is reproduced below as Table 34 of the instant specification.

In some embodiments, binding sites are associated with more than one signaling molecule or complex of molecules. Further, non-limiting examples of such motifs or sites are also provided in Table 12 of U.S. 62/501,795, which is hereby incorporated by reference in its entirety, and is reproduced below as Table 35 of the instant specification.

It has further been determined that certain patterns are found in the binding motifs. A list of such patterns for complexes is provided by Tables 13 and 14 of U.S. 62/501,795, which is hereby incorporated by reference in its entirety, and for single molecules is provided in Tables 15 and 16 of U.S. 62/501,795, which is hereby incorporated by reference in its entirety. Each of these are reproduced below, respectively, as Tables 36-39 of the instant specification.

In the Motif Tables 13-16 of U.S. 62/501,795 which is hereby incorporated by reference in its entirety, certain designators are used according to the IUPAC nucleotide code. This code is shown in Table 17 of U.S. 62/501,795, which is hereby incorporated by reference in its entirety, and is reproduced below as Table 40 of the instant specification.

Table 18 of U.S. 62/501,795, which is hereby incorporated by reference in its entirety, provides a list of signaling molecules including those which act as transcription factors (TF) and/or chromatin remodeling factors (CR) that function in various cellular signaling pathways. The methods described herein may be used to inhibit or activate the expression of one or more signaling molecules associated with the regulatory sequence region of the primary neighborhood gene encoded within an insulated neighborhood. The methods may thus alter the signaling signature of one or more primary neighborhood genes which are differentially expressed upon treatment with the therapeutic agent compared to an untreated control.

Various embodiments of the transcripts encoding the signaling proteins of Table 18 of U.S. 62/501,795, which is hereby incorporated by reference in its entirety, contain internal stop codons. These internal stop codons result in translation of multiple polypeptides. In one embodiment, a polypeptide that is a fragment of the signaling proteins taught in Table 18 of U.S. 62/501,795 may have signaling properties. As a non-limiting example, the polypeptide may be a fragment such as SEQ ID NO: 9 and SEQ ID NO: 10 from SEQ ID NO: 11. As a non-limiting example, the polypeptide may be a fragment such as SEQ ID NO: 12 and SEQ ID NO: 13 from SEQ ID NO: 14. As a non-limiting example, the polypeptide may be a fragment such as SEQ ID NO: 15 and SEQ ID NO: 16 from SEQ ID NO: 17. As a non-limiting example, the polypeptide may be a fragment such as SEQ ID NO: 18-19 from SEQ ID NO: 20. As a non-limiting example, the polypeptide may be a fragment such as SEQ ID NO: 21 and SEQ ID NO: 22 from SEQ ID NO: 23. As a non-limiting example, the polypeptide may be a fragment such as SEQ ID NO: 24 and SEQ ID NO: 25 from SEQ ID NO: 26. As a non-limiting example, the polypeptide may be a fragment such as SEQ ID NO: 27 and SEQ ID NO: 28 from SEQ ID NO: 29. As a non-limiting example, the polypeptide may be a fragment such as SEQ ID NO: 30 and SEQ ID NO: 31 from SEQ ID NO: 32. As a non-limiting example, the polypeptide may be a fragment such as SEQ ID NO: 33-37 from SEQ ID NO: 38. Table 18 of U.S. 62/501,795 is reproduced below as Table 41 of the instant specification.

In some embodiments, at least one compound selected from Tables 19-21, of U.S. 62/501,795, which are hereby incorporated by reference in their entirety, and Tables 22-26 and 28 of U.S. 62/501,795, which are hereby incorporated by reference in their entirety, may be used to modulate RNAs derived from regulatory sequence regions to alter or elucidate the gene signaling networks of the present disclosure.

II. Urea Cycle Disorders and Related Genes

Compositions and methods described herein may be used to treat one or more urea cycle disorders. As used herein, the term “urea cycle disorder” refers to any disorder that is caused by a defect or malfunction in the urea cycle. The urea cycle is a cycle of biochemical reactions that produces urea from ammonia, a product of protein catabolism. It is composed of 5 key enzymes including carbamoyl phosphate synthetase 1 (CPS1), ornithine transcarbamylase (OTC), argininosuccinate synthetase (ASS1), argininosuccinate lyase (ASL), and arginase 1 (ARG1), but also requires other enzymes, such as N-acetylglutamate synthetase (NAGS), and mitochondrial amino acid transporters, such as ornithine translocase (ORNT1) and citrin.

As used herein, a “urea cycle-related gene” refers to a gene whose gene product (e.g., RNA or protein) is involved in the urea cycle. Urea cycle-related genes include, but are not limited to, CPS1 (encoding CPS1), OTC (encoding OTC), ASS1 (encoding ASS1), NAGS (encoding NAGS), ARG1 (encoding ARG1), SLC25A15 (encoding ORNT1), and SLC25A13 (encoding citrin). Mutations in the urea cycle-related genes or their regulatory regions may lead to production of dysfunctional proteins and disruption of the urea cycle. In some cases, patients with a urea cycle disorder may carry a single functional allele and a mutated allele of a urea cycle-related gene. This results in the production of insufficient amount of the functional protein. The phenomenon is known as “haploinsufficiency.”

›DETAILED DESCRIPTION · 2 of 28

The urea cycle mainly occurs in the mitochondria of liver cells. The urea produced by the liver enters the bloodstream where it travels to the kidneys and is ultimately excreted in urine. Genetic defects in any of the enzymes or transporters in the urea cycle can cause hyperammonemia (elevated blood ammonia), or the buildup of a cycle intermediate. Ammonia then reaches the brain through the blood, where it can cause cerebral edema, seizures, coma, long term disabilities in survivors, and/or death.

The onset and severity of urea cycle disorders is highly variable. It is influenced by the position of the defective protein in the cycle and the severity of the defect. Mutations that lead to severe deficiency or total absence of activity of any of the first four enzymes in the pathway (CPS1, OTC, ASS1, and ASL) or the cofactor producer (NAGS) can result in the accumulation of ammonia and other precursor metabolites during the first few days of life. Because the urea cycle is the principal clearance system for ammonia, complete disruption of this pathway results in the rapid accumulation of ammonia and development of related symptoms. Mild to moderate mutations represent a broad spectrum of enzyme function, providing some ability to detoxify ammonia, and result in mild to moderate urea cycle disorders.

According to National Urea Cycle Disorders Foundation, the incidence of urea cycle disorders is estimated to be 1 in 8,500 live birth in the United States. The estimated incidence of individual urea cycle disorder varies from less than 1:2,000,000 to about 1:56,500 (See NA Mew et al., Urea Cycle Disorders Overview, 2015, which is incorporated by reference in its entirety). They occur in both children and adults. These disorders are most often diagnosed in infancy, but some children do not develop symptoms until early childhood. Newborns with severe urea cycle disorders become catastrophically ill within 36-48 hours of life. In children with mild or moderate urea cycle disorders, symptoms may be seen as early as one year of age. Early symptoms include disliking meat or other high-protein foods, inconsolable crying, failure to thrive, mental confusion or hyperactive behavior. Symptoms can progress to frequent episodes of vomiting, lethargy, delirium, and coma. Some individuals with mild urea cycle defects are diagnosed in adulthood. Ammonia accumulation may be triggered by illness or stress (e.g., viral infection, surgery, prolonged fasting, excessive exercising, and excessive dieting), resulting in multiple mild elevations of plasma ammonia concentration. Without proper diagnosis and treatment, these individuals are at risk for permanent brain damage, coma, and death.

Specific types of urea cycle disorder include, but are not limited to, Phosphate Synthetase 1 (CPS1) deficiency, Ornithine Transcarbamylase (OTC) deficiency, Argininosuccinate Synthetase (ASS1) deficiency, Argininosuccinate Lyase (ASL) deficiency, Arginase-1 (ARG1) deficiency, N-Acetylglutamate Synthetase (NAGS) deficiency, Ornithine translocase (ORNT1) deficiency, and Citrin deficiency. Any one or more of these disorders may be treated or targeted by the compositions and methods described herein.

Carbamoyl Phosphate Synthetase 1 (CPS1) Deficiency

In some embodiments, methods and compositions provided herein may be used to treat Carbamoyl Phosphate Synthetase 1 (CPS1) deficiency. CPS1 deficiency (MIM #237300) is an autosomal recessive disorder caused by mutations in the CPS1 gene. CPS1 catalyzes the synthesis of carbamoyl phosphate from ammonia and bicarbonate. CPS1 deficiency is the most severe type of the urea cycle disorders. Approximately 10 mutations that cause CPS1 deficiency have been identified in the CPS1 gene. Individuals with complete CPS1 deficiency rapidly develop hyperammonemia in the newborn period. Children who are successfully rescued from crisis are chronically at risk for repeated episodes of hyperammonemia.

In some embodiments, methods provided herein involve modulating the expression of the CPS1 gene. CPS1 may also be referred to as Carbamoyl-Phosphate Synthase 1, Mitochondrial; Carbamoyl-Phosphate Synthase (Ammonia); EC 6.3.4.16; Carbamoyl-Phosphate Synthase [Ammonia], Mitochondrial; Carbamoyl-Phosphate Synthetase I; Carbamoylphosphate Synthetase I; CPSase I; CPSASE1; and PHN. The CPS1 gene has a cytogenetic location of 2q34 and the genomic coordinate are on Chromosome 2 on the forward strand at position 210,477,682-210,679,107. LANCL1-AS1 (ENSG00000234281) and LANCL1 (ENSG00000115365) are the genes upstream of CPS1 and LOC107985978 is the gene downstream of CPS1. CPS1-IT1 (ENSG00000280837) is a gene located within CPS1 on the forward strand. The CPS1 gene has a NCBI gene ID of 1373, Uniprot ID of P31327 and Ensembl Gene ID of ENSG00000021826. The genomic sequence of CPS1 is shown as in SEQ ID NO: 1.

›DETAILED DESCRIPTION · 3 of 28

Ornithine Transcarbamylase (OTC) Deficiency

In some embodiments, methods and compositions provided herein may be used to treat Ornithine Transcarbamylase (OTC) deficiency. OTC deficiency (MIM #311250) is an X-linked genetic disorder caused by mutations in the OTC gene. OTC catalyzes the reaction between carbamoyl phosphate and ornithine to form citrulline and phosphate. More than 500 OTC gene mutations have been identified in people with OTC deficiency. The severe, early-onset form of the disorder, caused by complete absence of OTC activity, usually affects males. This form is as severe as CPS1 deficiency. The later-onset form of the disorder occurs in both males and females. These individuals develop hyperammonemia during their lifetime and many require chronic medical management for hyperammonemia.

In some embodiments, methods provided herein involve modulating the expression of the OTC gene. OTC may also be referred to as Ornithine Carbamoyltransferase; Ornithine Transcarbamylase; EC 2.1.3.3; OTCase, Ornithine Carbamoyltransferase, Mitochondrial; EC 2.1.3; and OCTD. The OTC gene has a cytogenetic location of Xp11.4 and the genomic coordinate are on Chromosome X on the forward strand at position 38,352,545-38,421,450. RPGR (ENSG00000 156313) is the gene upstream of OTC and LOC392442 is the gene downstream of OTC. TDGF1P1 (ENSG00000227988) is the gene located within OTC on the reverse strand. The OTC gene has a NCBI gene ID of 5009, Uniprot ID of P00480 and Ensembl Gene ID of ENSG00000036473. The genomic sequence of OTC is shown as in SEQ ID NO: 2.

Argininosuccinate Synthetase (ASS1) Deficiency

In some embodiments, methods and compositions provided herein may be used to treat Argininosuccinate Synthetase (ASS1) deficiency. ASS1 deficiency (MIM #215700), also known as Citrullinemia type I, is an autosomal recessive disorder caused by mutations in the ASS1 gene. ASS1 catalyzes the synthesis of argininosuccinate from citrulline and aspartate. About 118 mutations that cause ASS1 deficiency have been identified in the ASS1 gene. The early onset form of this disorder can also be quite severe. The symptoms associated with hyperammonemia are life-threatening in many cases. Affected individuals are able to incorporate some waste nitrogen into urea cycle intermediates, which makes treatment slightly easier than in the other urea cycle disorders.

In some embodiments, methods provided herein involve modulating the expression of the ASS1 gene. ASS1 may also be referred to as EC 6.3.4.5, Argininosuccinate Synthase, ASS, Argininosuccinic Acid Synthetase 1, Argininosuccinate Synthetase 1, Argininosuccinate Synthetase, Citrulline-Aspartate Ligase, and CTLN1. The ASS1 gene has a cytogenetic location of 9q34.11 and the genomic coordinate are on Chromosome 9 on the forward strand at position 130,444,929-130,501,274. HMCN2 (ENSG00000148357) and LOC107987134 are the genes upstream of ASS1, and FUBP3 (ENSG00000107164) and LOC100272217 are the genes downstream of ASS1. LOC105376294 is the gene that overlaps with the 3′ region of ASS1 on the reverse strand. The ASS1 gene has a NCBI gene ID of 445, Uniprot ID of P00966 and Ensembl Gene ID of ENSG00000130707. The genomic sequence of ASS1 is shown as in SEQ ID NO: 3.

Argininosuccinate Lyase (ASL) Deficiency

In some embodiments, methods and compositions provided herein may be used to treat Argininosuccinate Lyase (ASL) deficiency. ASL deficiency (MIM #207900) is an autosomal recessive disorder caused by mutations in the ASL gene. ASL cleaves argininosuccinic acid to produce arginine and fumarate in the fourth step of the urea cycle. More than 30 different mutations in the ASL gene have been identified worldwide. This disorder has a severe neonatal onset form and a late onset form. The severe neonatal onset form is indistinguishable from that of other urea cycle disorders. The late onset form ranges from episodic hyperammonemia triggered by acute infection or stress to cognitive impairment, behavioral abnormalities, and/or learning disabilities in the absence of any documented episodes of hyperammonemia.

In some embodiments, methods provided herein involve modulating the expression of the ASL gene. ASL may also be referred to as Argininosuccinase, EC 4.3.2.1, ASAL, and Argininosuccinase. The ASL gene has a cytogenetic location of 7q11.21 and the genomic coordinate are on Chromosome 7 on the forward strand at position 66,075,798-66,093,558. LOC644667 is the gene upstream of ASL and CRCP (ENSG00000241258) is the genes downstream of ASL. The ASL gene has a NCBI gene ID of 435, Uniprot ID of P04424 and Ensembl Gene ID of ENSG00000126522. The genomic sequence of ASL is shown as in SEQ ID NO: 4.

N-Acetylglutamate Synthetase (NAGS) Deficiency

In some embodiments, methods and compositions provided herein may be used to treat N-Acetylglutamate Synthetase (NAGS) deficiency. NAGS deficiency (MIM #237310) is an autosomal recessive disorder caused by mutations in the NAGS gene. NAGS catalyzes the production of N-Acetylglutamate (NAG) from glutamate and acetyl-CoA. NAG is a cofactor of CPS1. Approximately 12 mutations in the NAGS gene have been identified in people with NAGS deficiency. Symptoms of NAGS deficiency mimic those of CPS1 deficiency, as CPS1 is rendered inactive in the absence of NAG.

In some embodiments, methods provided herein involve modulating the expression of the NAGS gene. NAGS may also be referred to as Amino-Acid Acetyltransferase, N-Acetylglutamate Synthase, Mitochondrial, EC 2.3.1.1, AGAS, and ARGA. The NAGS gene has a cytogenetic location of 17q21.31 and the genomic coordinate are on Chromosome 17 on the forward strand at position 44,004,546-44,009,063. PPY (ENSG00000108849) is the gene upstream of NAGS and TMEM101 (ENSG00000091947) is the genes downstream of NAGS. PYY (ENSG00000131096) is a gene that overlaps with NAGS on the reserve strand. The NAGS gene has a NCBI gene ID of 162417, Uniprot ID of Q8N159 and Ensembl Gene ID of ENSG00000161653. The genomic sequence of NAGS is shown as in SEQ ID NO: 5.

›DETAILED DESCRIPTION · 4 of 28

Arginase-1 (ARG1) Deficiency

In some embodiments, methods and compositions provided herein may be used to treat Arginase-1 (ARG1) deficiency. ARG1 deficiency (MIM #207800) is an autosomal recessive disorder caused by mutations in the ARG1 gene. ARG1 catalyzes the hydrolysis of arginine to ornithine and urea, which is the final step in the urea cycle. More than 40 mutations have been found in the ARG1 gene that cause partial or complete loss of enzyme function. Defects in ARG1 cause hyperargininemia, a more subtle disorder involving neurologic symptoms. Arginase deficiency usually becomes evident by about the age of 3. It most often appears as stiffness, especially in the legs, caused by abnormal tensing of the muscles (spasticity). Other symptoms may include slower than normal growth, developmental delay and eventual loss of developmental milestones, intellectual disability, seizures, tremor, and difficulty with balance and coordination (ataxia). Occasionally, high protein meals or stress caused by illness or periods without food (fasting) may cause ammonia to accumulate more quickly in the blood. This rapid increase in ammonia may lead to episodes of irritability, refusal to eat, and vomiting. In some affected individuals, signs and symptoms of arginase deficiency may be less severe, and may not appear until later in life. Hyperammonemia is rare or usually not severe in Arginase deficiency. Arginase deficiency is a very rare disorder; it has been estimated to occur once in every 300,000 to 1,000,000 individuals.

In some embodiments, methods provided herein involve modulating the expression of the ARG1 gene. ARG1 may also be referred to as Liver-Type Arginase; Type I Arginase; Arginase, liver; and EC 3.5.3.1. The ARG1 gene has a cytogenetic location of 6q23.2 and the genomic coordinate are on Chromosome 6 on the forward strand at position 131,573,144-131,584,332. RPL21P67 (ENSG00000219776) is the gene upstream of ARG1 and ENPP3 (ENSG00000154269) is the genes downstream of ARG1 MED23 (ENSG00000112282) is a gene that overlaps with ARG1 on the reserve strand. The ARG1 gene has a NCBI gene ID of 383, Uniprot ID of P05089 and Ensembl Gene ID of ENSG00000118520. The genomic sequence of ARG1 is shown as in SEQ ID NO: 6.

Ornithine Translocase (ORNT1) Deficiency

In some embodiments, methods and compositions provided herein may be used to treat Ornithine translocase (ORNT1) deficiency. ORNT1 deficiency (MIM #238970), also known as the hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome, is an autosomal recessive disorder caused by mutations in the SLC25A15 gene. ORNT1 is a transporter protein that transports ornithine across the inner mitochondrial membrane to the mitochondrial matrix, where it participates in the urea cycle. Failure to transport ornithine results in an interruption of the urea cycle and the accumulation of ammonia. Approximately 17 mutations in the SLC25A15 gene have been identified in individuals with ORNT1 deficiency, including F188delta, E180K, T32R, Q89X, G27R, G190D, R275Q, and a 13q14 microdeletion, etc (Camacho et al., Nat Genet. 1999 June; 22(2):151-8; Camacho et al., Pediatric Research (2006) 60, 423-429; Salvi et al., Human Mutation, Mutation in Brief #457 (2001), which are hereby incorporated by reference in their entirety). Affected newborns typically present with lethargy, muscular hypotonia, and seizures. If untreated, death occurs within the first few days. The majority of survivors have pyramidal tract signs, with spastic paraparesis (Lemay et al., J Pediatr. 1992 November; 121(5 Pt 1): 725-30, Salvi et al., Neurology. 2001; 57(5):911, which are hereby incorporated by reference in their entirety). Most have myoclonic seizures, ataxia, and mental retardation. A milder form of the disorder has been reported in adults, who become symptomatic following protein-rich meals.

In some embodiments, methods provided herein involve modulating the expression of the SLC25A15 gene. SLC25A15 may also be referred to as Solute Carrier Family 25 Member 15, Solute Carrier Family 25 (Mitochondrial Carrier; Ornithine Transporter) Member 15, Ornithine Transporter 1, ORNT1, Mitochondrial Ornithine Transporter 1, D135327, ORC1, and HHH. SLC25A15 has a cytogenetic location of 13q14.11 and the genomic coordinate are on Chromosome 13 on the forward strand at position 40,789,412-40,810,111. MRPS31 (ENSG00000102738) is the gene upstream of SLC25A15 and MIR621 (ENSG00000207652) is the genes downstream of SLC25A15. TPTE2P5 (ENSG00000168852) is a gene that overlaps with SLC25A15 on the reserve strand. SLC25A15 has a NCBI gene ID of 10166, Uniprot ID of Q9Y619 and Ensembl Gene ID of ENSG00000102743. The genomic sequence of SLC25A15 is shown as in SEQ ID NO: 7.

Citrin Deficiency

In some embodiments, methods and compositions provided herein may be used to treat Citrin deficiency. Citrin deficiency (neonatal-onset MIM #605814 and adult-onset #603471), also known as Citrullinemia type II, is an autosomal recessive disorder caused by mutations in the SLC25A13 gene. Citrin is a transporter protein responsible for the transport of aspartate into the urea cycle. The loss of citrin blocks the aspartate transport and decrease the ability of ASS to produce argininosuccinate. More than 20 mutations in the SLC25A13 gene have been identified in people with adult-onset type II citrullinemia. It can manifest in newborns as neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD), in older children as failure to thrive and dyslipidemia caused by citrin deficiency (FTTDCD), and in adults as recurrent hyperammonemia with neuropsychiatric symptoms in citrullinemia type II (CTLN2). Citrin deficiency as a cause of neonatal intrahepatic cholestasis occurs almost exclusively in Asian infants (Yeh et al., J Pediatr 2006; 148:642; Zhang et al., Tohoku J Exp Med. 2014 August; 233(4):275-81; Tzun and Marques., EC Paediatrics 2.5 (2016); Lin et al., Sci Rep. 2016 Jul. 11; 6:29732; which are hereby incorporated by reference in their entirety). Adult-onset form of this disease is characterized by fatty liver, hyperammonemia, and neurological symptoms (Yasuda et a., Hum Genet 2000; 107:537; Lee et al., J Pediatr Gastroenterol Nutr 2010; 50:682, which are hereby incorporated by reference in their entirety).

›DETAILED DESCRIPTION · 5 of 28

In some embodiments, methods provided herein involve modulating the expression of the SLC25A13 gene. SLC25A13 may also be referred to as Solute Carrier Family 25 Member 13, Mitochondrial Aspartate Glutamate Carrier 2, Solute Carrier Family 25 (Aspartate/Glutamate Carrier) Member 13, ARALAR2, CITRIN, Calcium-Binding Mitochondrial Carrier Protein Aralar2, and CTLN2. SLC25A13 has a cytogenetic location of 7q21.3 and the genomic coordinate are on Chromosome 7 on the reverse strand at position 96,120,220-96,322,147. DYNC1I1 (ENSG00000158560) is the gene upstream of SLC25A13 and RNU6-532P (ENSG00000207045) is the gene downstream of SLC25A13. CYCSP18, MIR591 (ENSG00000208025), and RPL21P74 are genes located within SLC25A13. SLC25A13 has a NCBI gene ID of 10165, Uniprot ID of Q9UJS0 and Ensembl Gene ID of ENSG00000004864. The genomic sequence of SLC25A13 is shown as in SEQ ID NO: 8.

Ureagenesis

Provided herein are methods of increasing ureagenesis in a cell or subject, comprising administering to the subject or contacting the cell with an effective amount of a compound capable of modulating the expression of one or more genes selected from Carbamoyl Phosphate Synthetase 1 (CPS1), Ornithine Transcarbamylase (OTC), Argininosuccinate Synthetase 1 (ASS1), Argininosuccinate Lyase (ASL), N-Acetylglutamate Synthetase (NAGS), Arginase 1 (ARG1), Solute Carrier Family 25 Member 15 (SLC25A15), and Solute Carrier Family 25 Member 13 (SLC25A13). In some embodiments, the method comprises increasing OTC expression by administering a compound that increases CPS1 expression. In some embodiments, the compound modulates a target selected from JAK1, JAK2, JAK3, STAT5A, STAT5B, HSP90, or IRF9.

An increase in ureagenesis can result in an increase in a serum urea level in a subject, or a urea level in a cell. In some embodiments, the increase in urea level in a subject is as compared to administration of a control compound. In some embodiments, the increase in urea level in a subject is as compared to the urea level prior to administration of the compound that increases a urea cycle gene. In some embodiments, the increase in urea level in a cell is as compared to the urea level in an untreated cell.

In some embodiments, ureagenesis in a subject or cell is increased after administration of a JAK1, JAK2, JAK3, HSP90, MAPK, EGFR, FGFR, BRAF, RAF1, KDR, FLT1, TBK1, IKBKE, PRKAA1, PRKAA2, PRKAB1, BMPR1A, BMPR1B, JAK1, JAK2, JAK3, HSP90, MAPK, EGFR, FGFR, ABL, HER2, or HIF1a inhibitor. In some embodiments, ureagenesis in a subject or cell is increased after administration of a JAK1 inhibitor. In some embodiments, ureagenesis in a subject or cell is increased after administration of a JAK2 inhibitor. In some embodiments, ureagenesis in a subject or cell is increased after administration of a JAK3 inhibitor. In some embodiments, ureagenesis in a subject or cell is increased after administration of an HSP90 inhibitor. In some embodiments, ureagenesis in a subject or cell is increased after administration of a STAT5B inhibitor. In some embodiments, ureagenesis in a subject or cell is increased after administration of a STAT5A inhibitor. In some embodiments, ureagenesis in a subject or cell is increased after administration of an IRF9 inhibitor.

The methods described herein can increase the serum urea level in a subject by about at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more in the subject as compared to administration of a control compound. The methods described herein can increase the serum urea level in a subject by about at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more in the subject as compared to administration of a control compound as compared to the urea level prior to administration of the compound that increases a urea cycle gene. The methods described herein can increase the serum urea level in a cell, by about at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more in the cell as compared to the urea level in an untreated cell. In some embodiments, the urea level is increased by about at least 1%. In some embodiments, the urea level is increased by about at least 5%. In some embodiments, the urea level is increased by about at least 10%. In some embodiments, the urea level is increased by about at least 20%. In some embodiments, the urea level is increased by about at least 30%. In some embodiments, the urea level is increased by about at least 40%. In some embodiments, the urea level is increased by about at least 50%. In some embodiments, the urea level is increased by about at least 60%. In some embodiments, the urea level is increased by about at least 70%. In some embodiments, the urea level is increased by about at least 80%. In some embodiments, the urea level is increased by about at least 90%.

Urea levels in a subject or cell can be measured by any assay know in the art. For example, the blood urea nitrogen (BUN) is commonly used by health practitioners. In addition, commercially available colorimetric chemical assay kits that test urea levels in samples from multiple sources are available from Invitrogen (Blood Urea Nitrogen Assay Kit Cat #EIABUN), Abcam (Urea Assay Kit (ab83362)), BioAssays Systems (QuantiChrom™ Urea Assay Kit), and CellBioLabs (Urea Assay kit, STA-382). In addition, photometric assays that are processed by AutoAnalyzer machines can also be used to determine urea levels in samples. AutoAnalyzers are machines that can process and analyze samples using a flow technique called continuous flow analysis (CFA) or Segmented Flow Analysis (SFA). AutoAnalyzers are commercially available from a number of sources, including Beckman Coulter (Beckman Coulter AU analyzer), FIAlab Instruments, Inc (FIAlyzer-1000 and -2000), SEAL Analytical (AutoAnalyzer II/3 and QuAAtro CFA systems), Skalar Inc (SAN++ Continuous Flow Analyzers), and Astoria-Pacific International (Astoria Analyzer).

›DETAILED DESCRIPTION · 6 of 28

III. Compositions and Methods

Provided herein are compositions and methods for modulating the expression of one or more urea cycle-related genes to treat a urea cycle disorder. Any one or more of the compositions and methods described herein may be used to treat a urea cycle disorder in a subject.

The terms “subject” and “patient” are used interchangeably herein and refer to an animal to whom treatment with the compositions described herein is provided. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human being.

In some embodiments, subjects may have been diagnosed with or have symptoms for a urea cycle disorder, e.g., CPS1 deficiency, OTC deficiency, ASS1 Deficiency, ASL deficiency, NAGS deficiency, ARG1 deficiency, ORNT1 deficiency, and/or citrin deficiency. In other embodiments, subjects may be susceptible to or at risk for a urea cycle disorder, e.g., CPS1 deficiency, OTC deficiency, ASS1 Deficiency, ASL deficiency, NAGS deficiency, ARG1 deficiency, ORNT1 deficiency, and/or citrin deficiency.

In some embodiments, subjects may carry mutations within or near a urea cycle-related gene. In some embodiments, subjects may carry one or more mutations within or near the CPS1 gene. In some embodiments, subjects may carry one or more mutations within or near the OTC gene. In some embodiments, subjects may carry one or more mutations within or near the ASS1 gene. In some embodiments, subjects may carry one or more mutations within or near the ASL gene. In some embodiments, subjects may carry one or more mutations within or near the NAGS gene. In some embodiments, subjects may carry one or more mutations within or near the ARG1 gene. In some embodiments, subjects may carry one or more mutations within or near the SLC25A15 gene. In some embodiments, subjects may carry one or more mutations within or near the SLC25A13 gene. In some embodiment, subjects may carry one functional allele and one mutated allele of a urea cycle-related gene. In some embodiment, subjects may carry two mutated alleles of a urea cycle-related gene.

In some embodiments, subjects may have dysregulated expression of at least one urea cycle-related gene. In some embodiments, subjects may have a deficiency of at least one urea cycle-related protein. In some embodiments, subjects may have at least one urea cycle-related protein that is partially functional.

In some embodiments, compositions and methods provided herein may be used to increase the expression of a urea cycle-related gene in a cell or a subject. Changes in gene expression may be assessed at the RNA level or protein level by various techniques known in the art and described herein, such as RNA-seq, qRT-PCR, Western Blot, or enzyme-linked immunosorbent assay (ELISA). Changes in gene expression may be determined by dividing the level of target gene expression in the treated cell or subject by the level of expression in an untreated or control cell or subject. In some embodiments, compositions and methods provided herein cause an increase in the expression of a urea cycle-related gene by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 125%, at least about 150%, at least about 175%, at least about 200%, at least about 250%, at least about 300%, at least about 400%, at least about 500%, from about 25% to about 50%, from about 40% to about 60%, from about 50% to about 70%, from about 60% to about 80%, from about 80% to about 100%, from about 100% to about 125%, from about 100 to about 150%, from about 150% to about 200%, from about 200% to about 300%, from about 300% to about 400%, from about 400% to about 500%, or more than 500%. In some embodiments, compositions and methods provided herein cause a fold change in the expression of a urea cycle-related gene by about 2 fold, about 3 fold, about 4 fold, about 5 fold, about 6 fold, about 7 fold, about 8 fold, about 9 fold, about 10 fold, about 12 fold, about 15 fold, about 18 fold, about 20 fold, about 25 fold, or more than 30 fold.

In some embodiments, the increase in the expression of a urea cycle-related gene induced by compositions and methods provided herein may be sufficient to prevent or alleviate one or more signs or symptoms of a urea cycle disorder.

Small Molecules

In some embodiments, compounds used to modulate the expression of a urea cycle-related gene may include small molecules. As used herein, the term “small molecule” refers a low molecular weight drug, i.e. <5000 Daltons organic compound that may help regulate a biological process. In some embodiments, small molecule compounds described herein are applied to a genomic system to interfere with components (e.g., transcription factor, signaling proteins) of the gene signaling networks associated with one or more urea cycle-related genes, thereby modulating the expression of these genes. In some embodiments, small molecule compounds described herein are applied to a genomic system to alter the boundaries of an insulated neighborhood and/or disrupt signaling centers associated with one or more urea cycle-related genes, thereby modulating the expression of these genes.

A small molecule screen may be performed to identify small molecules that act through signaling centers of an insulated neighborhood to alter gene signaling networks which may modulate expression of a select group of urea cycle-related genes. For example, known signaling agonists/antagonists may be administered. Credible hits are identified and validated by the small molecules that are known to work through a signaling center and modulate expression of the target gene.

In some embodiments, small molecule compounds capable of modulating expression of one or more urea cycle-related genes include, but are not limited to, EC144, upadacitinib, oclacitinib maleate, tofacitinib, BIIB021, 17-AAG (Tanespimycin), Afatinib, Amlodipine Besylate, Amuvatinib, AZD2858, BAY 87-2243, BIRB 796, bms-986094 (inx-189), Bosutinib, Calcitriol, CD 2665, Ceritinib, CI-4AS-1, CO-1686 (Rociletinib), CP-673451, Crenolanib, Crizotinib, Darapladib, Dasatinib, Deoxycorticosterone, Echinomycin, Enzastaurin, Epinephrine, Erlotinib, EVP-6124 (hydrochloride) (encenicline), EW-7197, FRAX597, GDC-0879, G06983, GSK2334470, GZD824 Dimesylate, INNO-206 (aldoxorubicin), LDN193189, LDN-212854, Merestinib, MK-0752, Momelotinib, Oligomycin A, OSU-03012, Pacritinib (SB1518), PHA-665752, Phenformin, Phorbol 12,13-dibutyrate, PND-1186, prednisone, R788 (fostamatinib disodium hexahydrate), Rifampicin, Semaxinib, SIS3, SKL2001, SMI-4a, T0901317, TFP, Thalidomide, Tivozanib, TP-434 (Eravacycline), WYE-125132 (WYE-132), and Zibotentan, or derivatives or analogs thereof. Any one of these compounds or a combination thereof may be administered to a subject to treat a urea cycle disorder, such as CPS1 deficiency, OTC deficiency, ASS1 Deficiency, ASL deficiency, NAGS deficiency, ARG1 deficiency, ORNT1 deficiency, and/or citrin deficiency.

›DETAILED DESCRIPTION · 7 of 28

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include 17-AAG (Tanespimycin), or a derivative or an analog thereof. 17-AAG (Tanespimycin), also known as NSC 330507 or CP 127374, is a potent HSP90 inhibitor with half-maximal inhibitory concentration (IC 50 ) of 5 nM, a 100-fold higher binding affinity for HSP90 derived from tumor cells than HSP90 from normal cells.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include Afatinib, or a derivative or an analog thereof. Afatinib, also known as BIBW2992, irreversibly inhibits epidermal growth factor receptor (EGFR)/HER2 including EGFR (wildtype), EGFR (L858R), EGFR (L858R/T790M) and HER2 with IC 50 of 0.5 nM, 0.4 nM, 10 nM and 14 nM, respectively. It is 100-fold more active against Gefitinib-resistant L858R-T790M EGFR mutant.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include Amlodipine Besylate, or a derivative or an analog thereof. Amlodipine, also known as Norvasc, is a long-acting calcium channel blocker with an IC 50 of 1.9 nM.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include Amuvatinib, or a derivative or an analog thereof. Amuvatinib, also known as MP-470, is a potent and multi-targeted inhibitor of c-Kit, PDGFRα and FLT3 with IC 50 of 10 nM, 40 nM and 81 nM, respectively.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include AZD2858, or a derivative or an analog thereof. AZD2858 is a selective GSK-3 inhibitor with an IC 50 of 68 nM. It activates Wnt signaling and increases bone mass in rats.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include BAY 87-2243, or a derivative or an analog thereof. BAY 87-2243 is a potent and selective hypoxia-inducible factor-1 (HIF-1) inhibitor.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include BIRB 796, or a derivative or an analog thereof. BIRB 796, also known as Doramapimod, is a highly selective p38α MAPK inhibitor with dissociation constant (Kd) of 0.1 nM, 330-fold greater selectivity versus JNK2. It shows weak inhibition for c-RAF, Fyn and Lck and insignificant inhibition of ERK-1, SYK, IKK2, ZAP-70, EGFR, HER2, PKA, PKC, and PKCα/β/γ.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include bms-986094 (inx-189), or a derivative or an analog thereof. Bms-986094, also known as INX-08189, INX-189, or IDX-189, is a prodrug of a guanosine nucleotide analogue (2′-C-methylguanosine). Bms-986094 is an RNA-directed RNA polymerase (NS5B) inhibitor originally developed by Inhibitex (acquired by Bristol-Myers Squibb in 2012). It was in phase II clinical trials for the treatment of hepatitis C virus infection. However, the study was discontinued due to unexpected cardiac and renal adverse events.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include Bosutinib, or a derivative or an analog thereof. Bosutinib, also known as SKI-606, is a novel, dual Src/Abl inhibitor with IC 50 of 1.2 nM and 1 nM, respectively.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include Calcitriol, or a derivative or an analog thereof. Calcitriol, also known as 1,25-Dihydroxyvitamin D3 or Rocaltrol, is the hormonally active form of vitamin D, Calcitriol is the active metabolite of vitamin D3 that activates the vitamin D receptor.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include CD 2665, or a derivative or an analog thereof. CD 2665 is a selective RARβγ antagonist with Kd values of 110 nM, 306 nM, and >1000 nM for RARγ, RARβ, and RARα, respectively. It blocks retinoic acid-induced apoptosis ex vivo.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include Ceritinib, or a derivative or an analog thereof. Ceritinib, also known as LDK378, is potent inhibitor against ALK with IC 50 of 0.2 nM, exhibiting 40- and 35-fold selectivity against IGF-1R and InsR, respectively.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include CI-4AS-1, or a derivative or an analog thereof. CI-4AS-1 is a potent steroidal androgen receptor agonist (IC 50 =12 nM). It mimics the action of 5α-dihydrotestosterone (DHT). It transactivates the mouse mammary tumor virus (MMTV) promoter; represses MMP1 promoter activity. It also inhibits 5α-reductase type I and II with IC 50 values of 6 nM and 10 nM, respectively.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include CO-1686 (Rociletinib), or a derivative or an analog thereof. CO-1686, also known as Rociletinib, is a novel, irreversible and orally delivered kinase inhibitor that specifically targets the mutant forms of EGFR including T790M (IC 50 =21 nM).

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include CP-673451, or a derivative or an analog thereof. CP 673451 is a selective inhibitor of PDGFRα/β with IC 50 of 10 nM/1 nM, exhibiting >450-fold selectivity over other angiogenic receptors. CP 673451 also has antiangiogenic and antitumor activity.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include Crenolanib, or a derivative or an analog thereof. Crenolanib, also known as CP-868596, is a potent and selective inhibitor of PDGFRα/β with Kd of 2.1 nM/3.2 nM. It also potently inhibits FLT3 and is sensitive to D842V mutation not V561D mutation. It is >100-fold more selective for PDGFR than c-Kit, VEGFR-2, TIE-2, FGFR-2, EGFR, erbB2, and Src.

›DETAILED DESCRIPTION · 8 of 28

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include Crizotinib, or a derivative or an analog thereof. Crizotinib, also known as PF-2341066, is a potent inhibitor of c-Met and ALK with IC 50 of 11 nM and 24 nM, respectively.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include Darapladib, or a derivative or an analog thereof. Darapladib is a selective and orally active inhibitor of lipoprotein-associated phospholipase A2 (Lp-PLA2) with IC 50 of 270 pM. Lp-PLA2 may link lipid metabolism with inflammation, leading to the increased stability of atherosclerotic plaques present in the major arteries. Darapladib is being studied as a possible add-on treatment for atherosclerosis.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include Dasatinib, or a derivative or an analog thereof. Dasatinib is a novel, potent and multi-targeted inhibitor that targets Abl, Src, and c-Kit, with IC50 of <1 nM, 0.8 nM, and 79 nM, respectively.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include Deoxycorticosterone, or a derivative or an analog thereof. Deoxycorticosterone acetate is a steroid hormone used for intramuscular injection for replacement therapy of the adrenocortical steroid. 11β-hydroxylation of deoxycorticosterone leads to corticosterone.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include Echinomycin, or a derivative or an analog thereof. Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that controls genes involved in glycolysis, angiogenesis, migration, and invasion. Echinomycin is a cell-permeable inhibitor of HIF-1-mediated gene transcription. It acts by intercalating into DNA in a sequence-specific manner, blocking the binding of either HIF-1a or HIF-10 to the hypoxia-responsive element. Echinomycin reversibly inhibits hypoxia-induced HIF-1 transcription activity in U215 cells with a half maximal effective concentration (EC 50 ) value of 1.2 nM. It inhibits hypoxia-induced expression of vascular endothelial growth factor, blocking angiogenesis and altering excitatory synaptic transmission in hippocampal neurons. Echinomycin also impairs expression of survivin, enhancing the sensitivity of multiple myeloma cells to melphalan.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include Enzastaurin, or a derivative or an analog thereof. Enzastaurin, also known as LY317615, is a potent PKCβ selective inhibitor with IC 50 of 6 nM, exhibiting 6- to 20-fold selectivity against PKCα, PKCγ and PKCε.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include Epinephrine, or a derivative or an analog thereof. Epinephrine HCl is a hormone and a neurotransmitter.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include Erlotinib, or a derivative or an analog thereof. Erlotinib is an EGFR inhibitor with IC 50 of 2 nM, >1000-fold more sensitive for EGFR than human c-Src or v-Abl.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include EVP-6124 (hydrochloride) (encenicline), or a derivative or an analog thereof. EVP-6124 hydrochloride, also known as encenicline, is a novel partial agonist of α7 neuronal nicotinic acetylcholine receptors (nAChRs). EVP-6124 shows selectivity for α7 nAChRs and does not activate or inhibit heteromeric α4β2 nAChRs.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include EW-7197. EW-7197 is a highly potent, selective, and orally bioavailable TGF-β receptor ALK4/ALK5 inhibitor with IC 50 of 13 nM and 11 nM, respectively.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include FRAX597, or a derivative or an analog thereof. FRAX597 is a potent, ATP-competitive inhibitor of group I PAKs with IC 50 of 8 nM, 13 nM, and 19 nM for PAK1, PAK2, and PAK3, respectively.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include GDC-0879, or a derivative or an analog thereof. GDC-0879 is a novel, potent, and selective B-Raf inhibitor with IC 50 of 0.13 nM with activity against c-Raf as well.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include G06983, or a derivative or an analog thereof. G06983 is a pan-PKC inhibitor against for PKCα, PKCβ, PKCγ and PKCδ with IC 50 of 7 nM, 7 nM, 6 nM and 10 nM, respectively. It is less potent to PKCζ and inactive to PKCμ.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include GSK2334470, or a derivative or an analog thereof. GSK2334470 is a novel PDK1 inhibitor with IC 50 of about 10 nM and with no activity at other close related AGC-kinases.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include GZD824 Dimesylate, or a derivative or an analog thereof. GZD824 is a novel orally bioavailable Bcr-Abl inhibitor for Bcr-Abl (wildtype) and Bcr-Abl (T315I) with IC 50 of 0.34 nM and 0.68 nM, respectively.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include INNO-206 (aldoxorubicin), or a derivative or an analog thereof. INNO-206, also known as Aldoxorubicin, is the 6-maleimidocaproyl hydrazone derivative prodrug of the anthracycline antibiotic doxorubicin (DOXO-EMCH) with antineoplastic activity.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include LDN193189, or a derivative or an analog thereof. LDN193189 is a selective BMP signaling inhibitor that inhibits the transcriptional activity of the BMP type I receptors ALK2 and ALK3 with IC 50 of 5 nM and 30 nM, respectively, exhibiting 200-fold selectivity for BMP versus TGF-B.

›DETAILED DESCRIPTION · 9 of 28

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include LDN-212854, or a derivative or an analog thereof. LDN-212854 is a potent and selective BMP receptor inhibitor with IC 50 of 1.3 nM for ALK2, exhibiting about 2-, 66-, 1641-, and 7135-fold selectivity over ALK1, ALK3, ALK4, and ALK5, respectively.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include Merestinib, or a derivative or an analog thereof. Merestinib, also known as LY2801653, is a type-II ATP competitive, slow-off inhibitor of MET tyrosine kinase with a Kd of 2 nM, a pharmacodynamic residence time (Koff) of 0.00132 min −1 and half life (t 1/2 ) of 525 min.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include MK-0752, or a derivative or an analog thereof. MK-0752 is a potent, reversible inhibitor of γ-secretase, reducing the cleavage of amyloid precursor protein (APP) to Δβ40 in human neuroblastoma SH-SYSY cells with an IC 50 value of 5 nM. It is orally bioavailable and crosses the blood-brain barrier, as orally administered MK-0752 dose-dependently reduces the generation of new amyloid β protein in the brain of rhesus monkeys. Through its effects on the NOTCH pathway, MK-0752 reduces the number of breast cancer stem cells in tumor grafts, enhancing the efficacy of the chemotherapy drug docetaxel in mice with breast cancer tumors.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include Momelotinib, or a derivative or an analog thereof. Momelotinib, also known as CYT387, is an ATP-competitive inhibitor of JAK1/JAK2 with IC 50 of 11 nM/18 nM and approximately 10-fold selectivity versus JAK3.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include Oligomycin A, or a derivative or an analog thereof. Oligomycin A is an inhibitor of ATP synthase, inhibits oxidative phosphorylation and all the ATP-dependent processes occurring on the coupling membrane of mitochondria.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include OSU-03012, or a derivative or an analog thereof. OSU-03012 is a potent inhibitor of recombinant PDK-1 with IC 50 of 5 μM and 2-fold increase in potency over OSU-02067.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include Pacritinib (SB1518), or a derivative or an analog thereof. Pacritinib, also known as SB1518, is a potent and selective inhibitor JAK2 and FLT3 with IC 50 s of 23 and 22 nM in cell-free assays, respectively.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include PHA-665752, or a derivative or an analog thereof. PHA-665752 is a potent, selective and ATP-competitive c-Met inhibitor with IC50 of 9 nM, >50-fold selectivity for c-Met than RTKs or STKs.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include Phenformin, or a derivative or an analog thereof. Phenformin hydrochloride is a hydrochloride salt of phenformin that is an anti-diabetic drug from the biguanide class.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include Phorbol 12,13-dibutyrate, or a derivative or an analog thereof. Phorbol 12,13-dibutyrate is a protein kinase C activator. It induces contraction of vascular smooth muscle and inhibits MLC phosphatase (MLCP) in vascular smooth muscle. The activity does not alter intracellular Ca 2+ concentration. It also inhibits the activity of Na+, K+ ATPase in opossum kidney cells.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include Pifithrin-μ, or a derivative or an analog thereof. Pifithrin-μ, specifically inhibits p53 activity by reducing its affinity to Bcl-xL and Bcl-2, and it also inhibits HSP70 function and autophagy.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include PND-1186, or a derivative or an analog thereof. PND-1186, VS-4718, is a reversible and selective focal adhesion kinase (FAK) inhibitor with IC 50 of 1.5 nM.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include prednisone, or a derivative or an analog thereof. Prednisone is a synthetic glucocorticoid with anti-inflammatory and immunosuppressive activity.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include R788 (fostamatinib disodium hexahydrate), or a derivative or an analog thereof. R788 sodium salt hydrate (fostamatinib), a prodrug of the active metabolite R406, is a potent Syk inhibitor with IC 50 of 41 nM.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include Rifampicin, or a derivative or an analog thereof. Rifampicin is a member of the rifamycin class of antibiotics, as it inhibits bacterial DNA-dependent RNA synthesis (Ki=˜1 nM). While this compound does not directly affect RNA synthesis in humans, its use as an antibiotic is limited by its potency toward activation of the pregnane X receptor (PXR, EC 50 =˜2 μM), which results in the up-regulation of enzymes that alter drug metabolism. Access of rifampicin to the nuclear receptor PXR requires its import into the cell via organic anion transporters (OATs) in the OAT polypeptide (OATP) family. By acting as a transporter substrate, rifampicin inhibits OATPs with Ki/IC 50 values ranging from 0.58-18 μM.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include Semaxanib, or a derivative or an analog thereof. Semaxanib is a quinolone derivative with potential antineoplastic activity. Semaxanib reversibly inhibits ATP binding to the tyrosine kinase domain of vascular endothelial growth factor receptor 2 (VEGFR2).

›DETAILED DESCRIPTION · 10 of 28

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include SIS3, or a derivative or an analog thereof. SIS3 is a specific inhibitor of Smad3. It inhibits TGF-B and activin signaling by suppressing Smad3 phosphorylation without affecting the MAPK/p38, ERK, or PI3-kinase signaling pathways.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include SKL2001, or a derivative or an analog thereof. SKL2001 is a novel agonist of the Wnt/β-catenin pathway that disrupts the Axin/β-catenin interaction.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include SMI-4a, or a derivative or an analog thereof. SMI-4a is a potent inhibitor of Pim1 with IC 50 of 17 nM, with modest potency to Pim-2. It does not significantly inhibit other serine/threonine- or tyrosine-kinases.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include T0901317, or a derivative or an analog thereof. T0901317 is a potent, non-selective LXR agonist with EC 50 of 50 nM. It increases ABCA1 expression associated with cholesterol efflux regulation and HDL metabolism. It also increases muscle expression of PPAR-6 and shows antiobesogenic effects in vivo.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include TFP, or a derivative or an analog thereof. Trifluoperazine (TFP) has central antiadrenergic, antidopaminergic, and minimal anticholinergic effects. It is thought to function by blockading dopamine D1 and D2 receptors in the mesocortical and mesolimbic pathways, relieving or minimizing such symptoms of schizophrenia as hallucinations, delusions, and disorganized thought and speech.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include Thalidomide, or a derivative or an analog thereof. Thalidomide was introduced as a sedative drug, immunomodulatory agent and also is investigated for treating symptoms of many cancers. Thalidomide inhibits an E3 ubiquitin ligase, which is a CRBN-DDB1-Cul4A complex.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include Tivozanib, or a derivative or an analog thereof. Tivozanib, also known as AV-951, is a potent and selective VEGFR inhibitor for VEGFR1/2/3 with IC 50 of 30 nM/6.5 nM/15 nM. It also inhibits PDGFR and c-Kit but exhibits low activity against FGFR-1, Flt3, c-Met, EGFR and IGF-1R.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include TP-434 (Eravacycline), or a derivative or an analog thereof. TP-434, also known as Eravacycline, is a novel, broad-spectrum fluorocycline antibiotic with activity against bacteria expressing major antibiotic resistance mechanisms including tetracycline-specific efflux and ribosomal-protection.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include WYE-125132 (WYE-132), or a derivative or an analog thereof. WYE-125132, also known as WYE-132, is a highly potent, ATP-competitive mTOR inhibitor with IC 50 of 0.19 nM. It is highly selective for mTOR versus PI3Ks or PI3K-related kinases hSMG1 and ATR.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include Zibotentan, or a derivative or an analog thereof. Zibotentan, also known as ZD4054, is an orally administered, potent and specific endothelin A receptor (ETA)-receptor antagonist with IC 50 of 21 nM.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include Mubritinib (TAK 165), or a derivative or an analog thereof. Mubritinib is a selective inhibitor of the human epidermal growth factor receptor 2 (HER2).

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include Dasatinib, or a derivative or an analog thereof. Dasatinib is a potent inhibitor of the non-receptor tyrosine kinases Abl, Src, and Lck.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include PF-00562271, or a derivative or an analog thereof. PF-00562271 is a potent, ATP-competitive, reversible inhibitor of focal adhesion kinase (FAK).

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include Lifirafenib (BGB-283), or a derivative or an analog thereof. Lifirafenib (BGB-283) is a potent inhibitor of the RAF family kinases and EGFR.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include BMS-214662, or a derivative or an analog thereof. BMS-214662 is a farnesyltransferase inhibitor, which results in inhibition of Ras function.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include Crenolanib, or a derivative or an analog thereof. Crenolanib is a potent inhibitor of class III receptor tyrosine kinases (RTK) FLT3 (FMS-like Tyrosine Kinase 3), PDGFR α (Platelet-Derived Growth Factor Receptor), and PDGFR β.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include XL228, or a derivative or an analog thereof. XL228 inhibits the insulin-like growth factor type-1 receptor (IGF1R), Src and Abl tyrosine kinases, as well as Aurora A.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include Echinomycin, or a derivative or an analog thereof. Echinomycin reversibly inhibits Hypoxia-inducible factor-1 (HIF-1) transcription activity.

Polypeptides

In some embodiments, compounds for altering expression of a urea cycle-related gene comprise a polypeptide. As used herein, the term “polypeptide” refers to a polymer of amino acid residues (natural or unnatural) linked together most often by peptide bonds. The term, as used herein, refers to proteins, polypeptides, and peptides of any size, structure, or function. In some instances, the polypeptide encoded is smaller than about 50 amino acids and the polypeptide is then termed a peptide. If the polypeptide is a peptide, it will be at least about 2, 3, 4, or at least 5 amino acid residues long. Thus, polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs of the foregoing. A polypeptide may be a single molecule or may be a multi-molecular complex such as a dimer, trimer or tetramer. They may also comprise single chain or multichain polypeptides and may be associated or linked. The term polypeptide may also apply to amino acid polymers in which one or more amino acid residues are an artificial chemical analog of a corresponding naturally occurring amino acid.

›DETAILED DESCRIPTION · 11 of 28

In some embodiments, polypeptide compounds capable of modulating expression of one or more urea cycle-related genes include, but are not limited to, Activin, Anti mullerian hormone, BMP2, EGF, FGF, GDF10 (BMP3b), GDF2 (BMP9), HGF/SF, IGF-1, Nodal, PDGF, TNF-α, and Wnt3a, or derivatives or analogs thereof. Any one of these compounds or a combination thereof may be administered to a subject to treat a urea cycle disorder, such as CPS1 deficiency, OTC deficiency, ASS1 Deficiency, ASL deficiency, NAGS deficiency, ARG1 deficiency, ORNT1 deficiency, and/or citrin deficiency.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include Activin, or a derivative or an analog thereof. Activins are homodimers or heterodimers of the different β subunit isoforms, part of the transforming growth factor-beta (TGF-B) family. Mature Activin A has two 116 amino acids residues PA subunits (PA-βA). Activin displays an extensive variety of biological activities, including mesoderm induction, neural cell differentiation, bone remodeling, hematopoiesis, and reproductive physiology. Activins takes part in the production and regulation of hormones such as FSH, LH, GnRH and ACTH. Cells that are identified to express Activin A include fibroblasts, endothelial cells, hepatocytes, vascular smooth muscle cells, macrophages, keratinocytes, osteoclasts, bone marrow monocytes, prostatic epithelium, neurons, chondrocytes, osteoblasts, Leydig cells, Sertoli cells, and ovarian granulosa cells.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include anti Mullerian hormone, or a derivative or an analog thereof. Anti Mullerian hormone is a member of the TGF-B gene family which mediates male sexual differentiation. Anti Mullerian hormone causes the regression of Mullerian ducts which would otherwise differentiate into the uterus and fallopian tubes. Some mutations in the anti-Mullerian hormone result in persistent Mullerian duct syndrome.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include BMP2, or a derivative or an analog thereof. Bone morphogenetic protein 2 (BMP2) belongs to the TGF-B superfamily. The BMP family members are regulators of cell growth and differentiation in both embryonic and adult tissues. BMP2 is a candidate gene for the autosomal dominant disease of fibrodysplasia (myositis) ossificans progressiva.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include EGF, or a derivative or an analog thereof. Epidermal Growth Factor (EGF) is a polypeptide growth factor which stimulates the proliferation of a wide range of epidermal and epithelial cells.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include FGF, or a derivative or an analog thereof. Fibroblast Growth Factor-acidic (FGF-acidic), also known as FGF-1 and endothelial cell growth factor, is a member of the FGF family which currently contain 23 members. FGF acidic and basic, unlike the other members of the family, lack signal peptides and are apparently secreted by mechanisms other than the classical protein secretion pathway. FGF acidic has been detected in large amounts in the brain. Other cells known to express FGF acidic include hepatocytes, vascular smooth muscle cells, CNS neurons, skeletal muscle cells, fibroblasts, keratinocytes, endothelial cells, intestinal columnar epithelium cells and pituitary basophils and acidophilus.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include GDF10 (BMP3b), or a derivative or an analog thereof. GDF10, also known as BMP3b, is a member of the BMP family and the TGF-B superfamily. GDF10 is expressed in femur, brain, lung, skeletal, muscle, pancreas and testis, and has a role in head formation and possibly multiple roles in skeletal morphogenesis. In humans, GDF10 mRNA is found in the cochlea and lung of fetuses, and in testis, retina, pineal gland, and other neural tissues of adults. These proteins are characterized by a polybasic proteolytic processing site which is cleaved to produce a mature protein containing 7 conserved cysteine residues.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include GDF2 (BMP9), or a derivative or an analog thereof. GDF2, also known as BMP9, is a member of BMP family and the TGF-B superfamily. BMP9 has a role in the maturation of basal forebrain cholinergic neurons (BFCN) as well as the induction and maintenance of the ability of these cells to respond to acetylcholine. BFCN are important for the processes of learning, memory and attention. BMP9 is a potent inducer of hepcidin (a cationic peptide that has an antimicrobial properties) in hepatocytes and can regulate iron metabolism. The physiological receptor of BMP9 is thought to be activin receptor-like kinase 1, ALK1 (also known as ACVRL1), an endothelial-specific type I receptor of the TGF-B receptor family. BMP9 is one of the most potent BMPs to induce orthotopic bone formation in vivo. BMP3, a blocker of most BMPs appears not to affect BMP9.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include HGF/SF, or a derivative or an analog thereof. Hepatocyte Growth Factor (HGF), also known as hepatopoietin-A and scatter factor (SF), is a pleiotropic mitogen belonging to the peptidase Si family (plasminogen subfamily). It is produced by mesenchymal cells and acts on epithelial cells, endothelial cells and hematopoietic progenitor cells. HGF binds to the proto-oncogenic c-Met receptor to activate a tyrosine kinase signaling cascade. It regulates cell growth, motility and morphogenesis, and it also plays a pivotal role in angiogenesis, tumorigenesis and tissue regeneration.

›DETAILED DESCRIPTION · 12 of 28

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include IGF-1, or a derivative or an analog thereof. Insulin-like growth factor I (IGF-I) also known as Somatamedin C is a hormone similar in molecular structure to insulin. Human IGF-I has two isoforms (IGF-IA and IGF-IB) which is differentially expressed by various tissues. Mature human IGF-I respectively shares 94% and 96% aa sequence identity with mouse and rat IGF-I. Both IGF-I and IGF-II (another ligand of IGF) can signal through the IGF-I receptor (IGFIR), but IGF-II can alone bind the IGF-II receptor (IGFIIR/Mannose-6-phosphate receptor). IGF-I plays an important role in childhood growth and continues to have anabolic effects in adults.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include Nodal, or a derivative or an analog thereof. Nodal is a 13 kDa member of the TGF-B superfamily of molecules. In human, it is synthesized as a 347 amino acid preproprecursor that contains a 26 amino acid signal sequence, a 211 amino acid prodomain, and a 110 amino acid mature region. Consistent with its TGF-B superfamily membership, it exists as a disulfide-linked homodimer and would be expected to demonstrate a cysteine-knot motif. Mature human Nodal is 99%, 98%, 96% and 98% amino acid identical to mature canine, rat, bovine and mouse Nodal, respectively. Nodal signals through two receptor complexes, both of which contain members of the TGF-beta family of Ser/Thr kinase receptors.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include PDGF, or a derivative or an analog thereof. the Platelet-derived growth factor (PDGF) is a disulfide-linked dimer consisting of two peptides-chain A and chain B. PDGF has three subforms: PDGF-AA, PDGF-BB, PDGF-AB. It is involved in a number of biological processes, including hyperplasia, embryonic neuron development, chemotaxis, and respiratory tubule epithelial cell development. The function of PDGF is mediated by two receptors (PDGFRα and PDGFRβ).

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include TNF-α, or a derivative or an analog thereof. TNF-α, the prototypical member of the TNF protein superfamily, is a homotrimeric type-II membrane protein. Membrane bound TNF-α is cleaved by the metalloprotease TACE/ADAM17 to generate a soluble homotrimer. Both membrane and soluble forms of TNF-α are biologically active. TNF-α is produced by a variety of immune cells including T cells, B cells, NK cells and macrophages. Cellular response to TNF-α is mediated through interaction with receptors TNF-R1 and TNF-R2 and results in activation of pathways that favor both cell survival and apoptosis depending on the cell type and biological context. Activation of kinase pathways (including JNK, ERK (p44/42), p38 MAPK and NF-kB) promotes the survival of cells, while TNF-α mediated activation of caspase-8 leads to programmed cell death. TNF-α plays a key regulatory role in inflammation and host defense against bacterial infection, notably Mycobacterium tuberculosis . The role of TNF-α in autoimmunity is underscored by blocking TNF-α action to treat rheumatoid arthritis and Crohn's disease.

In some embodiments, compounds capable of modulating the expression of one or more urea cycle-related genes may include Wnt3a, or a derivative or an analog thereof. The WNT gene family consists of structurally related genes which encode secreted signaling proteins. These proteins have been implicated in oncogenesis and in several developmental processes, including regulation of cell fate and patterning during embryogenesis. This gene is a member of the WNT gene family. It encodes a protein which shows 96% amino acid identity to mouse Wnt3a protein, and 84% to human WNT3 protein, another WNT gene product. This gene is clustered with WNT14 gene, another family member, in chromosome 1q42 region.

Antibodies

In some embodiments, compounds for altering expression of one or more urea cycle-related genes comprise an antibody. In one embodiment, antibodies provided herein comprising antibodies, antibody fragments, their variants or derivatives described herein are specifically immunoreactive with at least one component of the gene signaling networks associated with the urea cycle-related gene.

As used herein, the term “antibody” is used in the broadest sense and specifically covers various embodiments including, but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. bispecific antibodies formed from at least two intact antibodies), and antibody fragments such as diabodies so long as they exhibit a desired biological activity. Antibodies are primarily amino-acid based molecules but may also comprise one or more modifications such as with sugar moieties.

“Antibody fragments” comprise a portion of an intact antibody, preferably comprising an antigen binding region thereof. Examples of antibody fragments include Fab, Fab′, F(ab′)2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site. Also produced is a residual “Fc” fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab′)2 fragment that has two antigen-binding sites and is still capable of crosslinking antigen. Antibodies provided herein may comprise one or more of these fragments. For the purposes herein, an “antibody” may comprise a heavy and light variable domain as well as an Fc region.

“Native antibodies” are usually heterotetrameric glycoproteins of about 150,000 Daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (V H ) followed by a number of constant domains. Each light chain has a variable domain at one end (V L ) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain.

›DETAILED DESCRIPTION · 13 of 28

As used herein, the term “variable domain” refers to specific antibody domains that differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. As used herein, the term “Fv” refers to antibody fragments which contain a complete antigen-recognition and antigen-binding site. This region consists of a dimer of one heavy chain and one light chain variable domain in tight, non-covalent association.

Antibody “light chains” from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda based on amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains, antibodies can be assigned to different classes. There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.

“Single-chain Fv” or “scFv” as used herein, refers to a fusion protein of VH and VL antibody domains, wherein these domains are linked together into a single polypeptide chain. In some embodiments, the Fv polypeptide linker enables the scFv to form the desired structure for antigen binding.

The term “diabodies” refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain V H connected to a light chain variable domain V L in the same polypeptide chain. By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993), the contents of each of which are incorporated herein by reference in their entirety.

Antibodies provided herein may be polyclonal or monoclonal or recombinant, produced by methods known in the art or as described in this application. The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous cells (or clones), i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variants that may arise during production of the monoclonal antibody, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.

The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. The monoclonal antibodies herein include “chimeric” antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies.

“Humanized” forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from the hypervariable region from an antibody of the recipient are replaced by residues from the hypervariable region from an antibody of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity.

The term “hypervariable region” when used herein in reference to antibodies refers to regions within the antigen binding domain of an antibody comprising the amino acid residues that are responsible for antigen binding. The amino acids present within the hypervariable regions determine the structure of the complementarity determining region (CDR). As used herein, the “CDR” refers to the region of an antibody that comprises a structure that is complimentary to its target antigen or epitope.

In some embodiments, the compositions provided herein may be antibody mimetics. The term “antibody mimetic” refers to any molecule which mimics the function or effect of an antibody and which binds specifically and with high affinity to their molecular targets. As such, antibody mimics include nanobodies and the like.

In some embodiments, antibody mimetics may be those known in the art including, but are not limited to affibody molecules, affilins, affitins, anticalins, avimers, DARPins, Fynomers and Kunitz and domain peptides. In other embodiments, antibody mimetics may include one or more non-peptide region.

As used herein, the term “antibody variant” refers to a biomolecule resembling an antibody in structure and/or function comprising some differences in their amino acid sequence, composition or structure as compared to a native antibody.

The preparation of antibodies, whether monoclonal or polyclonal, is known in the art. Techniques for the production of antibodies are well known in the art and described, e.g. in Harlow and Lane “Antibodies, A Laboratory Manual,” Cold Spring Harbor Laboratory Press, 1988 and Harlow and Lane “Using Antibodies: A Laboratory Manual” Cold Spring Harbor Laboratory Press, 1999.

Antibodies provided herein may be characterized by their target molecule(s), by the antigens used to generate them, by their function (whether as agonists or antagonists) and/or by the cell niche in which they function.

Measures of antibody function may be made relative to a standard under normal physiologic conditions, in vitro or in vivo. Measurements may also be made relative to the presence or absence of the antibodies. Such methods of measuring include standard measurement in tissue or fluids such as serum or blood such as Western blot, enzyme-linked immunosorbent assay (ELISA), activity assays, reporter assays, luciferase assays, polymerase chain reaction (PCR) arrays, gene arrays, Real Time reverse transcriptase (RT) PCR and the like.

›DETAILED DESCRIPTION · 14 of 28

Antibodies provided herein exert their effects via binding (reversibly or irreversibly) to one or more target sites. While not wishing to be bound by theory, target sites which represent a binding site for an antibody, are most often formed by proteins or protein domains or regions. However, target sites may also include biomolecules such as sugars, lipids, nucleic acid molecules or any other form of binding epitope.

Alternatively, or additionally, antibodies provided herein may function as ligand mimetics or nontraditional payload carriers, acting to deliver or ferry bound or conjugated drug payloads to specific target sites.

Changes elicited by antibodies provided herein may result in a neomorphic change in the cell. As used herein, “a neomorphic change” is a change or alteration that is new or different. Such changes include extracellular, intracellular and cross cellular signaling.

In some embodiments, compounds or agents provided herein act to alter or control proteolytic events. Such events may be intracellular or extracellular.

Antibodies provided herein, as well as antigens used to generate them, are primarily amino acid-based molecules. These molecules may be “peptides,” “polypeptides,” or “proteins.”

As used herein, the term “peptide” refers to an amino-acid based molecule having from 2 to 50 or more amino acids. Special designators apply to the smaller peptides with “dipeptide” referring to a two amino acid molecule and “tripeptide” referring to a three amino acid molecule. Amino acid based molecules having more than 50 contiguous amino acids are considered polypeptides or proteins.

The terms “amino acid” and “amino acids” refer to all naturally occurring L-alpha-amino acids as well as non-naturally occurring amino acids. Amino acids are identified by either the one-letter or three-letter designations as follows: aspartic acid (Asp:D), isoleucine threonine (Thr:T), leucine (Leu:L), serine (Ser:S), tyrosine (Tyr:Y), glutamic acid (Glu:E), phenylalanine (Phe:F), proline (Pro:P), histidine (His:H), glycine (Gly:G), lysine (Lys:K), alanine (Ala:A), arginine (Arg:R), cysteine (Cys:C), tryptophan (Trp:W), valine (Val:V), glutamine (Gln:Q) methionine (Met:M), asparagines (Asn:N), where the amino acid is listed first followed parenthetically by the three and one letter codes, respectively.

Hybridizing Oligonucleotides

In some embodiments, oligonucleotides, including those which function via a hybridization mechanism, whether single of double stranded such as antisense molecules, RNAi constructs (including siRNA, saRNA, microRNA, etc.), aptamers and ribozymes may be used to alter or as perturbation stimuli of the gene signaling networks associated with a urea cycle-related gene.

As such oligonucleotides may also serve as therapeutics, their therapeutic liabilities and treatment outcomes may be ameliorated or predicted, respectively by interrogating the gene signaling networks.

In some embodiments, the compound comprises an siRNA. In some embodiments, compound is an siRNA compound that inhibits a target selected from the group consisting of JAK1, JAK2, JAK3, STAT5A, STAT5B, HSP90, and IRF9. In some embodiments, compound is an siRNA compound that inhibits JAK1. In some embodiments, compound is an siRNA compound that inhibits JAK2. In some embodiments, compound is an siRNA compound that inhibits JAK3. In some embodiments, compound is an siRNA compound that inhibits STAT5A. In some embodiments, compound is an siRNA compound that inhibits STAT5B. In some embodiments, compound is an siRNA compound that inhibits HSP90. In some embodiments, compound is an siRNA compound that inhibits IRF9. In some embodiments, the IRF9 siRNA is Dharmacon/Horizon Discovery #M-020858-02-0005, L-020858-00-0005, J-020858-06-0005, J-020858-07-0005, J-020858-08-0005, J-020858-09-0005, or LQ-020858-00-0005.

siRNA that target the indicated genes are commerically available from various vendors, including Horizon Disovery, which provides the Dharmacon line of siRNA. Multiple types of siRNA are available from Horizon Discovery under the Dharmacon brand, including the siRNA siGENOME and ON-TARGETplus reagents, which both can be purchased individually or as a set of SMARTpool siRNA reagents. Exemplary Dharmacon siRNA for targeting genes that modulate urea cycle genes include, but are not limited to, JAK1 siRNA Dharmacon #M-003145-02-0005; JAK2 siRNA Dharmacon #M-003146-02-0005; JAK3 siRNA Dharmacon #M-020858-02-0005; TYK2 siRNA Dharmacon #M-003182-02-0005; STAT1 siRNA Dharmacon #M-003543-01-0005; STAT2 siRNA Dharmacon #M-012064-00-0005; STAT3 siRNA Dharmacon #M-003544-02-0005; STAT4 siRNA Dharmacon #M-011784-01-0005; STAT5B siRNA Dharmacon #M-010539-02-0005; STAT5A siRNA Dharmacon #M-005169-02-0005; IRF1 siRNA Dharmacon #M-011704-01-0005; IRF2 siRNA Dharmacon #M-011705-01-0005; IRF7 siRNA Dharmacon #M-011810-02-0005; IRF8 siRNA Dharmacon #M-011699-01-0005; IRF9 siRNA Dharmacon #M-020858-02-0005.

Genome Editing Approaches

In certain embodiments, expression of a urea cycle-related gene may be modulated by altering the chromosomal regions defining the insulated neighborhood(s) and/or genome signaling center(s) associated with the urea cycle-related gene. For example, protein production may be increased by targeting a component of the gene signaling network that functions to repress the expression of the urea cycle-related gene.

Methods of altering the gene expression attendant to an insulated neighborhood include altering the signaling center (e.g., using CRISPR/Cas to change the signaling center binding site or repair/replace if mutated). These alterations may result in a variety of results including: activation of cell death pathways prematurely/inappropriately (key to many immune disorders), production of too little/much gene product (also known as the rheostat hypothesis), production of too little/much extracellular secretion of enzymes, prevention of lineage differentiation, switch of lineage pathways, promotion of stemness, initiation or interference with auto regulatory feedback loops, initiation of errors in cell metabolism, inappropriate imprinting/gene silencing, and formation of flawed chromatin states. Additionally, genome editing approaches including those well-known in the art may be used to create new signaling centers by altering the cohesin necklace or moving genes and enhancers.

›DETAILED DESCRIPTION · 15 of 28

In certain embodiments, genome editing approaches describe herein may include methods of using site-specific nucleases to introduce single-strand or double-strand DNA breaks at particular locations within the genome. Such breaks can be and regularly are repaired by endogenous cellular processes, such as homology-directed repair (HDR) and non-homologous end joining (NHEJ). HDR is essentially an error-free mechanism that repairs double-strand DNA breaks in the presence of a homologous DNA sequence. The most common form of HDR is homologous recombination. It utilizes a homologous sequence as a template for inserting or replacing a specific DNA sequence at the break point. The template for the homologous DNA sequence can be an endogenous sequence (e.g., a sister chromatid), or an exogenous or supplied sequence (e.g., plasmid or an oligonucleotide). As such, HDR may be utilized to introduce precise alterations such as replacement or insertion at desired regions. In contrast, NHEJ is an error-prone repair mechanism that directly joins the DNA ends resulting from a double-strand break with the possibility of losing, adding or mutating a few nucleotides at the cleavage site. The resulting small deletions or insertions (termed “Indels”) or mutations may disrupt or enhance gene expression. Additionally, if there are two breaks on the same DNA, NHEJ can lead to the deletion or inversion of the intervening segment. Therefore, NHEJ may be utilized to introduce insertions, deletions or mutations at the cleavage site.

CRISPR/Cas Systems

In certain embodiments, a CRISPR/Cas system may be used to delete CTCF anchor sites to modulate gene expression within the insulated neighborhood associated with that anchor site. See, Hnisz et al., Cell 167, Nov. 17, 2016, which is hereby incorporated by reference in its entirety. Disruption of the boundaries of insulated neighborhood prevents the interactions necessary for proper function of the associated signaling centers. Changes in the expression genes that are immediately adjacent to the deleted neighborhood boundary have also been observed due to such disruptions.

In certain embodiments, a CRISPR/Cas system may be used to modify existing CTCF anchor sites. For example, existing CTCF anchor sites may be mutated or inverted by inducing NHEJ with a CRISPR/Cas nuclease and one or more guide RNAs, or masked by targeted binding with a catalytically inactive CRISPR/Cas enzyme and one or more guide RNAs. Alteration of existing CTCF anchor sites may disrupt the formation of existing insulated neighborhoods and alter the expression of genes located within these insulated neighborhoods.

In certain embodiments, a CRISPR/Cas system may be used to introduce new CTCF anchor sites. CTCF anchor sites may be introduced by inducing HDR at a selected site with a CRISPR/Cas nuclease, one or more guide RNAs and a donor template containing the sequence of a CTCF anchor site. Introduction of new CTCF anchor sites may create new insulated neighborhoods and/or alter existing insulated neighborhoods, which may affect expression of genes that are located adjacent to these insulated neighborhoods.

In certain embodiments, a CRISPR/Cas system may be used to alter signaling centers by changing signaling center binding sites. For example, if a signaling center binding site contains a mutation that affects the assembly of the signaling center with associated transcription factors, the mutated site may be repaired by inducing a double strand DNA break at or near the mutation using a CRISPR/Cas nuclease and one or more guide RNAs in the presence of a supplied corrected donor template.

In certain embodiments, a CRISPR/Cas system may be used to modulate expression of neighborhood genes by binding to a region within an insulated neighborhood (e.g., enhancer) and block transcription. Such binding may prevent recruitment of transcription factors to signaling centers and initiation of transcription. The CRISPR/Cas system may be a catalytically inactive CRISPR/Cas system that do not cleave DNA.

In certain embodiments, a CRISPR/Cas system may be used to knockdown expression of neighborhood genes via introduction of short deletions in coding regions of these genes. When repaired, such deletions would result in frame shifts and/or introduce premature stop codons in mRNA produced by the genes followed by the mRNA degradation via nonsense-mediated decay. This may be useful for modulation of expression of activating and repressive components of signaling pathways that would result in decreased or increased expression of genes under control of these pathways including disease genes such as CPS1, OTC, ASS, ASL, NAGS, and ARG1.

In other embodiments, a CRISPR/Cas system may also be used to alter cohesion necklace or moving genes and enhancers.

CRISPR/Cas Enzymes

CRISPR/Cas systems are bacterial adaptive immune systems that utilize RNA-guided endonucleases to target specific sequences and degrade target nucleic acids. They have been adapted for use in various applications in the field of genome editing and/or transcription modulation. Any of the enzymes or orthologs known in the art or disclosed herein may be utilized in the methods herein for genome editing.

In certain embodiments, the CRISPR/Cas system may be a Type II CRISPR/Cas9 system. Cas9 is an endonuclease that functions together with a trans-activating CRISPR RNA (tracrRNA) and a CRISPR RNA (crRNA) to cleave double stranded DNAs. The two RNAs can be engineered to form a single-molecule guide RNA by connecting the 3′ end of the crRNA to the 5′ end of tracrRNA with a linker loop. Jinek et al., Science, 337(6096):816-821 (2012) showed that the CRISPR/Cas9 system is useful for RNA-programmable genome editing, and international patent application WO2013/176772 provides numerous examples and applications of the CRISPR/Cas endonuclease system for site-specific gene editing, which are incorporated herein by reference in their entirety. Exemplary CRISPR/Cas9 systems include those derived from Streptococcus pyogenes, Streptococcus thermophilus, Neisseria meningitidis, Treponema denticola, Streptococcus aureas , and Francisella tularensis.

›DETAILED DESCRIPTION · 16 of 28

In certain embodiments, the CRISPR/Cas system may be a Type V CRISPR/Cpf1 system. Cpf1 is a single RNA-guided endonuclease that, in contrast to Type II systems, lacks tracrRNA. Cpf1 produces staggered DNA double-stranded break with a 4 or 5 nucleotide 5′ overhang. Zetsche et al. Cell. 2015 Oct. 22; 163(3):759-71 provides examples of Cpf1 endonuclease that can be used in genome editing applications, which is incorporated herein by reference in its entirety. Exemplary CRISPR/Cpf1 systems include those derived from Francisella tularensis , Acidaminococcus sp., and Lachnospiraceae bacterium.

In certain embodiments, nickase variants of the CRISPR/Cas endonucleases that have one or the other nuclease domain inactivated may be used to increase the specificity of CRISPR-mediated genome editing. Nickases have been shown to promote HDR versus NHEJ. HDR can be directed from individual Cas nickases or using pairs of nickases that flank the target area.

In certain embodiments, catalytically inactive CRISPR/Cas systems may be used to bind to target regions (e.g., CTCF anchor sites or enhancers) and interfere with their function. Cas nucleases such as Cas9 and Cpf1 encompass two nuclease domains. Mutating critical residues at the catalytic sites creates variants that only bind to target sites but do not result in cleavage. Binding to chromosomal regions (e.g., CTCF anchor sites or enhancers) may disrupt proper formation of insulated neighborhoods or signaling centers and therefore lead to altered expression of genes located adjacent to the target region.

In certain embodiments, a CRISPR/Cas system may include additional functional domain(s) fused to the CRISPR/Cas enzyme. The functional domains may be involved in processes including but not limited to transcription activation, transcription repression, DNA methylation, histone modification, and/or chromatin remodeling. Such functional domains include but are not limited to a transcriptional activation domain (e.g., VP64 or KRAB, SID or SID4X), a transcriptional repressor, a recombinase, a transposase, a histone remodeler, a DNA methyltransferase, a cryptochrome, a light inducible/controllable domain or a chemically inducible/controllable domain.

In certain embodiments, a CRISPR/Cas enzyme may be administered to a cell or a patient as one or a combination of the following: one or more polypeptides, one or more mRNAs encoding the polypeptide, or one or more DNAs encoding the polypeptide.

Guide Nucleic Acid

In certain embodiments, guide nucleic acids may be used to direct the activities of an associated CRISPR/Cas enzymes to a specific target sequence within a target nucleic acid. Guide nucleic acids provide target specificity to the guide nucleic acid and CRISPR/Cas complexes by virtue of their association with the CRISPR/Cas enzymes, and the guide nucleic acids thus can direct the activity of the CRISPR/Cas enzymes.

In one aspect, guide nucleic acids may be RNA molecules. In one aspect, guide RNAs may be single-molecule guide RNAs. In one aspect, guide RNAs may be chemically modified.

In certain embodiments, more than one guide RNAs may be provided to mediate multiple CRISPR/Cas-mediated activities at different sites within the genome.

In certain embodiments, guide RNAs may be administered to a cell or a patient as one or more RNA molecules or one or more DNAs encoding the RNA sequences.

Ribonucleoprotein Complexes (RNPs)

In one embodiment, the CRISPR/Cas enzyme and guide nucleic acid may each be administered separately to a cell or a patient.

In another embodiment, the CRISPR/Cas enzyme may be pre-complexed with one or more guide nucleic acids. The pre-complexed material may then be administered to a cell or a patient. Such pre-complexed material is known as a ribonucleoprotein particle (RNP).

Zinc Finger Nucleases

In certain embodiments, genome editing approaches provided herein involve the use of Zinc finger nucleases (ZFNs). Zinc finger nucleases (ZFNs) are modular proteins comprised of an engineered zinc finger DNA binding domain linked to a DNA-cleavage domain. A typical DNA-cleavage domain is the catalytic domain of the type II endonuclease FokI. Because FokI functions only as a dimer, a pair of ZFNs must are required to be engineered to bind to cognate target “half-site” sequences on opposite DNA strands and with precise spacing between them to allow the two enable the catalytically active FokI domains to dimerize. Upon dimerization of the FokI domain, which itself has no sequence specificity per se, a DNA double-strand break is generated between the ZFN half-sites as the initiating step in genome editing.

Transcription Activator-Like Effector Nucleases (TALENs)

In certain embodiments, genome editing approaches provided herein involve the use of Transcription Activator-Like Effector Nucleases (TALENs). TALENs represent another format of modular nucleases which, similarly to ZFNs, are generated by fusing an engineered DNA binding domain to a nuclease domain, and operate in tandem to achieve targeted DNA cleavage. While the DNA binding domain in ZFN consists of Zinc finger motifs, the TALEN DNA binding domain is derived from transcription activator-like effector (TALE) proteins, which were originally described in the plant bacterial pathogen Xanthomonas sp. TALEs are comprised of tandem arrays of 33-35 amino acid repeats, with each repeat recognizing a single basepair in the target DNA sequence that is typically up to 20 bp in length, giving a total target sequence length of up to 40 bp. Nucleotide specificity of each repeat is determined by the repeat variable diresidue (RVD), which includes just two amino acids at positions 12 and 13. The bases guanine, adenine, cytosine and thymine are predominantly recognized by the four RVDs: Asn-Asn, Asn-Ile, His-Asp and Asn-Gly, respectively. This constitutes a much simpler recognition code than for zinc fingers, and thus represents an advantage over the latter for nuclease design. Nevertheless, as with ZFNs, the protein-DNA interactions of TALENs are not absolute in their specificity, and TALENs have also benefitted from the use of obligate heterodimer variants of the FokI domain to reduce off-target activity.

›DETAILED DESCRIPTION · 17 of 28

Modulation of Urea Cycle-Related Genes

Compositions and methods described herein may be effective in modulating the expression of one or more urea cycle-related genes. Such compositions and methods may be used to treat a urea cycle disorder.

In some embodiments, compositions and methods provided herein may be used to treat a urea cycle disorder by modulating the expression of CPS1. Compounds that may be used to modulate the CPS1 expression include, but are not limited to, Dasatinib, R788 (fostamatinib disodium hexahydrate), Bosutinib, Epinephrine, FRAX597, Merestinib, Deoxycorticosterone, 17-AAG (Tanespimycin), GDF2 (BMP9), GZD824 Dimesylate, PND-1186, Wnt3a, Nodal, Anti mullerian hormone, TNF-α, Activin, IGF-1, prednisone, PDGF, HGF/SF, EGF, BAY 87-2243, CP-673451, FGF, GDF10 (BMP3b), LDN193189, Amuvatinib, Momelotinib, Echinomycin, Pacritinib (SB1518), BMP2, Crizotinib, LDN-212854, Thalidomide, CO-1686 (Rociletinib), Zibotentan, and derivatives or analogs thereof. In some embodiments, Dasatinib perturbs the ABL signaling pathway to modulate CPS1 expression. In some embodiments, R788 (fostamatinib disodium hexahydrate) perturbs the Protein Tyrosine Kinase/RTK signaling pathway to modulate CPS1 expression. In some embodiments, Bosutinib perturbs the Src signaling pathway to modulate CPS1 expression. In some embodiments, Epinephrine perturbs the Adrenergic receptor signaling pathway to modulate CPS1 expression. In some embodiments, FRAX597 perturbs the PAK signaling pathway to modulate CPS1 expression. In some embodiments, Merestinib perturbs the c-MET signaling pathway to modulate CPS1 expression. In some embodiments, Corticosterone perturbs the Mineralcorticoid receptor signaling pathway to modulate CPS1 expression. In some embodiments, 17-AAG (Tanespimycin) perturbs the Cell Cycle/DNA Damage Metabolic Enzyme/Protease signaling pathway to modulate CPS1 expression. In some embodiments, GDF2 (BMP9) perturbs the TGF-B signaling pathway to modulate CPS1 expression. In some embodiments, GZD824 Dimesylate perturbs the ABL signaling pathway to modulate CPS1 expression. In some embodiments, PND-1186 perturbs the FAK signaling pathway to modulate CPS1 expression. In some embodiments, Wnt3a perturbs the WNT signaling pathway to modulate CPS1 expression. In some embodiments, Nodal perturbs the TGF-B signaling pathway to modulate CPS1 expression. In some embodiments, Anti mullerian hormone perturbs the TGF-B signaling pathway to modulate CPS1 expression. In some embodiments, TNF-α perturbs the NF-kB, MAPK, or Apoptosis pathway to modulate CPS1 expression. In some embodiments, Activin perturbs the TGF-B signaling pathway to modulate CPS1 expression. In some embodiments, IGF-1 perturbs the IGF-1R/InsR signaling pathway to modulate CPS1 expression. In some embodiments, prednisone perturbs the GR signaling pathway to modulate CPS1 expression. In some embodiments, PDGF perturbs the PDGFR signaling pathway to modulate CPS1 expression. In some embodiments, HGF/SF perturbs the c-MET signaling pathway to modulate CPS1 expression. In some embodiments, EGF perturbs the EGFR signaling pathway to modulate CPS1 expression. In some embodiments, BAY 87-2243 perturbs the Hypoxia activated signaling pathway to modulate CPS1 expression. In some embodiments, CP-673451 perturbs the PDGFR signaling pathway to modulate CPS1 expression. In some embodiments, FGF perturbs the FGFR signaling pathway to modulate CPS1 expression. In some embodiments, GDF10 (BMP3b) perturbs the TGF-B signaling pathway to modulate CPS1 expression. In some embodiments, LDN193189 perturbs the TGF-B signaling pathway to modulate CPS1 expression. In some embodiments, Amuvatinib perturbs the PDGFR signaling pathway to modulate CPS1 expression. In some embodiments, Momelotinib perturbs the JAK/STAT signaling pathway to modulate CPS1 expression. In some embodiments, Echinomycin perturbs the Hypoxia activated signaling pathway to modulate CPS1 expression. In some embodiments, Pacritinib (SB1518) perturbs the JAK/STAT signaling pathway to modulate CPS1 expression. In some embodiments, BMP2 perturbs the TGF-B signaling pathway to modulate CPS1 expression. In some embodiments, Crizotinib perturbs the c-MET signaling pathway to modulate CPS1 expression. In some embodiments, LDN-212854 perturbs the TGF-B signaling pathway to modulate CPS1 expression. In some embodiments, Thalidomide perturbs the NF-kB signaling pathway to modulate CPS1 expression. In some embodiments, CO-1686 (Rociletinib) perturbs the JAK/STAT and/or Tyrosine Kinase/RTK signaling pathway to modulate CPS1 expression. In some embodiments, Zibotentan perturbs the GPCR/G protein signaling pathway to modulate CPS1 expression.

In some embodiments, methods provided herein involve altering the composition and/or the structure of the insulated neighborhood containing the CPS1 gene. The CPS1 gene has a cytogenetic location of 2q34 and the genomic coordinate are on Chromosome 2 on the forward strand at position 210,477,682-210,679,107. Any chromatin mark, chromatin-associated protein, transcription factor and/or signaling protein that is associated with the insulated neighborhood, and/or any regions within or near the insulated neighborhood, may be targeted or altered to change the composition and/or structure of the insulated neighborhood, thereby modulating the expression of CPS1. The chromatin marks and/or chromatin-associated proteins may include but are not limited to H3K27ac, BRD4, p300, and SMC1. The transcription factors may include but are not limited to FOXA2, HNF4A, ONECUT1, ONECUT2, and YY1. The signaling proteins may include but are not limited to TCF7L2, ESRA, FOS, NR3C1, JUN, NR5A2, RBPJK, RXR, STAT3, NR1I1, NF-kB, SMAD2/3, SMAD4, STAT1, TEAD1, and TP53.

In some embodiments, compositions and methods provided herein may be used to treat a urea cycle disorder by modulating the expression of OTC. Compounds that may be used to modulate the OTC expression include, but are not limited to, CP-673451, Pacritinib (SB1518), Echinomycin, Crenolanib, Thalidomide, Amuvatinib, Dasatinib, Momelotinib, Activin, Wnt3a, INNO-206 (aldoxorubicin), TNF-α, Anti mullerian hormone, Pifithrin-μ, PDGF, IGF-1, FRAX597, Nodal, FGF, HGF/SF, BIRB 796, and derivatives or analogs thereof. In some embodiments, CP-673451 perturbs the PDGFR signaling pathway to modulate OTC expression. In some embodiments, Pacritinib (SB1518) perturbs the JAK/STAT signaling pathway to modulate OTC expression. In some embodiments. Echinomycin perturbs the Hypoxia activated signaling pathway to modulate OTC expression. In some embodiments, Crenolanib perturbs the PDGFR signaling pathway to modulate OTC expression. In some embodiments, Thalidomide perturbs the NF-kB signaling pathway to modulate OTC expression. In some embodiments, Amuvatinib perturbs the PDGFR signaling pathway to modulate OTC expression. In some embodiments, Dasatinib perturbs the ABL signaling pathway to modulate OTC expression. In some embodiments, Momelotinib perturbs the JAK/STAT signaling pathway to modulate OTC expression. In some embodiments, Activin perturbs the TGF-B signaling pathway to modulate OTC expression. In some embodiments, Wnt3a perturbs the WNT signaling pathway to modulate OTC expression. In some embodiments, INNO-206 (aldoxorubicin) perturbs the Cell Cycle/DNA Damage pathway to modulate OTC expression. In some embodiments, TNF-α perturbs the INF-kB, MAPK, or Apoptosis signaling pathway to modulate OTC expression. In some embodiments, Anti mullerian hormone perturbs the TGF-B signaling pathway to modulate OTC expression. In some embodiments, Pifithrin-μ perturbs the p53 signaling pathway to modulate OTC expression. In some embodiments, PDGF perturbs the PDGFR signaling pathway to modulate OTC expression. In some embodiments, IGF-1 perturbs the IGF-1R/InsR signaling pathway to modulate OTC expression. In some embodiments, FRAX597 perturbs the PAK signaling pathway to modulate OTC expression. In some embodiments. Nodal perturbs the TGF-B signaling pathway to modulate OTC expression. In some embodiments, EGF perturbs the EGFR signaling pathway to modulate OTC expression. In some embodiments, perturbs the FGFR signaling pathway to modulate OTC expression. In some embodiments, HGF/SF perturbs the c-MET signaling pathway to modulate OTC expression. In some embodiments, BIRB 796 perturbs the MAPK signaling pathway to modulate OTC expression.

›DETAILED DESCRIPTION · 18 of 28

In some embodiments, methods provided herein involve altering the composition and/or the structure of the insulated neighborhood containing the OTC gene. The OTC gene has a cytogenetic location of Xp11.4 and the genomic coordinate are on Chromosome X on the forward strand at position 38,352,545-38,421,450. Any chromatin mark, chromatin-associated protein, transcription factor and/or signaling protein that is associated with the insulated neighborhood, and/or any regions within or near the insulated neighborhood, may be targeted or altered to change the composition and/or structure of the insulated neighborhood, thereby modulating the expression of OTC. The chromatin marks and/or chromatin-associated proteins may include but are not limited to H3K27ac and BRD4. The transcription factors may include but are not limited to FOXA2, HNF4A, ONECUT1, ONECUT2, YY1, and HNF1A. The signaling proteins may include but are not limited to TCF7L2, HIF1a, ESRA, NR3C1, JUN, RXR, STAT3, NF-kB, SMAD2/3, SMAD4, and TEAD1.

In some embodiments, compositions and methods provided herein may be used to treat a urea cycle disorder by modulating the expression of ASS1. Compounds that may be used to modulate the ASS1 expression include, but are not limited to, Dasatinib, CP-673451, Echinomycin, GDF2 (BMP9), Pacritinib (SB1518), Epinephrine, FRAX597, Bosutinib, TP-434 (Eravacycline), BMP2, SMI-4a, Amuvatinib, Crenolanib, Deoxycorticosterone, INNO-206 (aldoxorubicin), TNF-α, T0901317, and derivatives or analogs thereof. In some embodiments, Dasatinib perturbs the ABL signaling pathway to modulate ASS1 expression. In some embodiments, CP-673451 perturbs the PDGFR signaling pathway to modulate ASS1 expression. In some embodiments, Echinomycin perturbs the Hypoxia activated signaling pathway to modulate ASS1 expression. In some embodiments, GDF2 (BMP9) perturbs the TGF-B signaling pathway to modulate ASS1 expression. In some embodiments, Pacritinib (SB1518) perturbs the JAK/STAT signaling pathway to modulate ASS1 expression. In some embodiments, Epinephrine perturbs the Adrenergic receptor signaling pathway to modulate ASS1 expression. In some embodiments, FRAX597 perturbs the PAK signaling pathway to modulate ASS1 expression. In some embodiments, Bosutinib perturbs the Src signaling pathway to modulate ASS1 expression. In some embodiments, TP-434 (Eravacycline) perturbs the Tetracycline-specific efflux signaling pathway to modulate ASS1 expression. In some embodiments, BMP2 perturbs the TGF-B signaling pathway to modulate ASS1 expression. In some embodiments, SMI-4a perturbs the PIM signaling pathway to modulate ASS1 expression. In some embodiments, Amuvatinib perturbs the PDGFR signaling pathway to modulate ASS1 expression. In some embodiments, Crenolanib perturbs the PDGFR signaling pathway to modulate ASS1 expression. In some embodiments, Corticosterone perturbs the Mineralcorticoid receptor signaling pathway to modulate ASS1 expression. In some embodiments, INNO-206 (aldoxorubicin) perturbs the Cell Cycle/DNA Damage pathway to modulate ASS1 expression. In some embodiments, TNF-α perturbs the NF-kB, MAPK, or apoptosis pathway to modulate ASS1 expression. In some embodiments, T0901317 perturbs the LXR signaling pathway to modulate ASS1 expression.

In some embodiments, methods provided herein involve altering the composition and/or the structure of the insulated neighborhood containing the ASS1 gene. The ASS1 gene has a cytogenetic location of 9q34.11 and the genomic coordinate are on Chromosome 9 on the forward strand at position 130,444,929-130,501,274. Any chromatin mark, chromatin-associated protein, transcription factor and/or signaling protein that is associated with the insulated neighborhood, and/or any regions within or near the insulated neighborhood, may be targeted or altered to change the composition and/or structure of the insulated neighborhood, thereby modulating the expression of ASS1. The chromatin marks and/or chromatin-associated proteins may include but are not limited to H3K27ac, BRD4, p300, and SMC1. The transcription factors may include but are not limited to FOXA2, HNF4A, ONECUT1, MYC, and YY1. The signaling proteins may include but are not limited to CREB1, NR1H4, HIF1a, ESRA, JUN, RXR, STAT3, NR1I1, NF-kB, NR3C1, SMAD2/3, SMAD4, and TEAD1.

In some embodiments, compositions and methods provided herein may be used to treat a urea cycle disorder by modulating the expression of ASL. Compounds that may be used to modulate the ASL expression include, but are not limited to, CP-673451, Echinomycin, Pacritinib (SB1518), Dasatinib, Oligomycin A, Merestinib, Amuvatinib, Crenolanib, Epinephrine, BAY 87-2243, Thalidomide, and derivatives or analogs thereof. In some embodiments, CP-673451 perturbs the PDGFR signaling pathway to modulate ASL expression. In some embodiments, Echinomycin perturbs the Hypoxia activated signaling pathway to modulate ASL expression. In some embodiments, Pacritinib (SB1518) perturbs the JAK/STAT signaling pathway to modulate ASL expression. In some embodiments, Dasatinib perturbs the ABL signaling pathway to modulate ASL expression. In some embodiments, Oligomycin A perturbs the ATP channel signaling pathway to modulate ASL expression. In some embodiments, Merestinib perturbs the c-MET signaling pathway to modulate ASL expression. In some embodiments, Amuvatinib perturbs the PDGFR signaling pathway to modulate ASL expression. In some embodiments, Crenolanib perturbs the PDGFR signaling pathway to modulate ASL expression. In some embodiments, Epinephrine perturbs the Adrenergic receptor signaling pathway to modulate ASL expression. In some embodiments, BAY 87-2243 perturbs the Hypoxia activated signaling pathway to modulate ASL expression. In some embodiments, Thalidomide perturbs the NF-kB signaling pathway to modulate ASL expression.

In some embodiments, methods provided herein involve altering the composition and/or the structure of the insulated neighborhood containing the ASL gene. The ASL gene has a cytogenetic location of 7q11.21 and the genomic coordinate are on Chromosome 7 on the forward strand at position 66,075,798-66,093,558. Any chromatin mark, chromatin-associated protein, transcription factor and/or signaling protein that is associated with the insulated neighborhood, and/or any regions within or near the insulated neighborhood, may be targeted or altered to change the composition and/or structure of the insulated neighborhood, thereby modulating the expression of ASL. The chromatin marks and/or chromatin-associated proteins may include but are not limited to H3K27ac, BRD4, and p300. The transcription factors may include but are not limited to HNF3, HNF4A, ONECUT1, HNF1A, and MYC. The signaling proteins may include but are not limited to TCF7L2, CREB1, NR1H4, HIF1a, ESRA, FOS, JUN, RBPJK, RXR, STAT3, NR1I1, NF-kB, NR3C1, SMAD2/3, SMAD4, STAT1, TEAD1, and TP53.

›DETAILED DESCRIPTION · 19 of 28

In some embodiments, compositions and methods provided herein may be used to treat a urea cycle disorder by modulating the expression of NAGS. Compounds that may be used to modulate the NAGS expression include, but are not limited to, AZD2858, Enzastaurin, Bosutinib, Semaxanib, INNO-206 (aldoxorubicin), TP-434 (Eravacycline), Phenformin, Crizotinib, SMI-4a, Dasatinib, Calcitriol, Pifithrin-μ, PHA-665752, Darapladib, Thalidomide, CO-1686 (Rociletinib), OSU-03012, prednisone, GSK2334470, Afatinib, Tivozanib, SKL2001, GDC-0879, EVP-6124 (hydrochloride) (encenicline), Amlodipine Besylate, T0901317, 006983, Activin, WYE-125132 (WYE-132), SIS3, GDF2 (BMP9), Phorbol 12,13-dibutyrate, CD 2665, Erlotinib, Ceritinib, BMP2, HGF/SF, C1-4 AS-1, and derivatives or analogs thereof. In some embodiments, AZD2858 perturbs the GSK-3 signaling pathway to modulate NAGS expression. In some embodiments, Enzastaurin perturbs the Epigenetics or TGF-beta/Smad signaling pathway to modulate NAGS expression. In some embodiments, Bosutinib perturbs the Src signaling pathway to modulate NAGS expression. In some embodiments, Semaxanib perturbs the VEGFR signaling pathway to modulate NAGS expression. In some embodiments, INNO-206 (aldoxorubicin) perturbs the Cell Cycle/DNA Damage pathway to modulate NAGS expression. In some embodiments, TP-434 (Eravacycline) perturbs the Tetracycline-specific efflux signaling pathway to modulate NAGS expression. In some embodiments, Phenformin perturbs the AMPK signaling pathway to modulate NAGS expression. In some embodiments, Crizotinib perturbs the c-MET signaling pathway to modulate NAGS expression. In some embodiments, SMI-4a perturbs the PIM signaling pathway to modulate NAGS expression. In some embodiments, Dasatinib perturbs the ABL signaling pathway to modulate NAGS expression. In some embodiments, Calcitriol perturbs the Vitamin I) Receptor signaling pathway to modulate NAGS expression. In some embodiments, Pifithrin-μ perturbs the p53 signaling pathway to modulate NAGS expression. In some embodiments, PHA-665752 perturbs the c-MET signaling pathway to modulate NAGS expression. In some embodiments, Thalidomide perturbs the NF-kB signaling pathway to modulate NAGS expression. In some embodiments, CO-1686 (Rociletinib) perturbs the JAK/STAT or Tyrosine Kinase/RTK signaling pathway to modulate NAGS expression. In some embodiments, OSU-03012 perturbs the PDK-1 signaling pathway to modulate NAGS expression. In some embodiments, prednisone perturbs the GR signaling pathway to modulate NAGS expression. In some embodiments, GSK2334470 perturbs the PDK-1 signaling pathway to modulate NAGS expression. In some embodiments, Afatinib perturbs the EGFR signaling pathway to modulate NAGS expression. In some embodiments, Tivozanib perturbs the Protein Tyrosine Kinase/RTK signaling pathway to modulate NAGS expression. In some embodiments, SKL2001 perturbs the WNT signaling pathway to modulate NAGS expression. In some embodiments, GDC-0879 perturbs the MAPK signaling pathway to modulate NAGS expression. In some embodiments, EVP-6124 (hydrochloride) (encenicline) perturbs the Membrane Transporter/Ion Channel signaling pathway to modulate NAGS expression. In some embodiments, Amlodipine Besylate perturbs the Calcium channel signaling pathway to modulate NAGS expression. In some embodiments, 10901317 perturbs the Da signaling pathway to modulate NAGS expression. In some embodiments, G06983 perturbs the PKC signaling; pathway to modulate NAGS expression. In some embodiments, Activin perturbs the TGF-B signaling pathway to modulate NAGS expression. In some embodiments, WYE-125132 (WYE-132) perturbs the mTOR signaling pathway to modulate NAGS expression. In some embodiments, SIS3 perturbs the TGF-B signaling pathway to modulate NAGS expression. In some embodiments, GDF2 (BMP9) perturbs the TGF-B signaling pathway to modulate NAGS expression. In some embodiments, Phorbol 12,13-dibutyrate perturbs the PKC signaling pathway to modulate NAGS expression. In some embodiments, CD 2665 perturbs the RAR signaling pathway to modulate NAGS expression. In some embodiments. Erlotinib perturbs the EGFR signaling pathway to modulate NAGS expression. In some embodiments, Ceritinib perturbs the ALK signaling pathway to modulate NAGS expression. In some embodiments, BMP2 perturbs the TGF-B signaling pathway to modulate NAGS expression. In some embodiments, TFP perturbs the Calmodulin signaling pathway to modulate NAGS expression. In some embodiments, HGF/SF perturbs the c-MET signaling pathway to modulate NAGS expression. In some embodiments, CI-4AS-1 perturbs the Androgen receptor signaling; pathway to modulate NAGS expression.

In some embodiments, methods provided herein involve altering the composition and/or the structure of the insulated neighborhood containing the NAGS gene. The NAGS gene has a cytogenetic location of 17q21.31 and the genomic coordinate are on Chromosome 17 on the forward strand at position 44,004,546-44,009,063. Any chromatin mark, chromatin-associated protein, transcription factor and/or signaling protein that is associated with the insulated neighborhood, and/or any regions within or near the insulated neighborhood, may be targeted or altered to change the composition and/or structure of the insulated neighborhood, thereby modulating the expression of NAGS. The chromatin marks and/or chromatin-associated proteins may include but are not limited to H3K27ac, BRD4, and p300. The transcription factors may include but are not limited to FOXA2, HNF4A, ONECUT1, ONECUT2, YY1, and HNF1A. The signaling proteins may include but are not limited to TCF7L2, HIF1a, AHR, ESRA, JUN, RXR, STAT3, NR1I1, NF-kB, NR3C1, SMAD2/3, SMAD4, TEAD1, and TP53.

In some embodiments, compositions and methods provided herein may be used to treat a urea cycle disorder by modulating the expression of ARG1. Compounds that may be used to modulate the ARG1 expression include, but are not limited to, R788 (fostamatinib disodium hexahydrate), Dasatinib, CP-673451, Merestinib, Echinomycin, Amuvatinib, Epinephrine, Bosutinib, Wnt3a, Anti mullerian Hormone, Nodal, Activin, 7-AAG (Tanespimycin), TNF-a, Pifithrin-μ, PDGF, Pacritinib (SB1518), GDF2 (BMP9), Crenolanib, prednisone, HGF/SF, Momelotinib, EGF, Deoxycorticosterone, FGF, Thalidomide, Phenformin, Tivozanib, BAY 87-2243, GZD824 Dimesylate, GDF10 (BMP3b), PND-1186, FRAX597, BMP2, Oligomycin A, Rifampicin, MK-0752, and derivatives or analogs thereof. In some embodiments, R788 (fostamatinib disodium hexahydrate) perturbs the Protein Tyrosine Kinase/RTK signaling pathway to modulate ARG1 expression. In some embodiments, Dasatinib perturbs the ABL signaling pathway to modulate ARG1 expression. In some embodiments, CP-673451 perturbs the PDGFR signaling pathway to modulate ARG1 expression. In some embodiments, Merestinib perturbs the c-MET signaling pathway to modulate ARG1 expression. In some embodiments, Echinomycin perturbs the Hypoxia activated signaling pathway to modulate ARG1 expression. In some embodiments, Amuvatinib perturbs the PDGFR signaling pathway to modulate ARG1 expression. In some embodiments, Epinephrine perturbs the Adrenergic receptor signaling pathway to modulate ARG1 expression. In some embodiments, Bosutinib perturbs the Src signaling pathway to modulate ARG1 expression. In some embodiments, Wnt3a perturbs the WNT signaling pathway to modulate ARG1 expression. In some embodiments, Anti mullerian Hormone perturbs the TGF-B signaling pathway to modulate ARG1 expression. In some embodiments, Nodal perturbs the TGF-B signaling pathway to modulate ARG1 expression. In some embodiments, Activin perturbs the TGF-B signaling pathway to modulate ARG1 expression. In some embodiments, IGF-1 perturbs the IGF-1R/InsR signaling pathway to modulate ARG1 expression. In some embodiments, 17-AAG (Tanespimycin) perturbs the Cell Cycle/DNA Damage Metabolic Enzyme/Protease signaling pathway to modulate ARG1 expression. In some embodiments, TNF-α perturbs the NE-kB, MAPK, or Apoptosis pathway to modulate ARG1 expression. In some embodiments, Pifithrin-μ perturbs the p53 signaling pathway to modulate ARG1 expression. In some embodiments, PDGF perturbs the PDGFR signaling pathway to modulate ARG1 expression. In some embodiments, Pacritinib (SB1518) perturbs the JAK/STAT signaling pathway to modulate ARG1 expression. In some embodiments, GDF2 (BMP9) perturbs the TGF-B signaling pathway to modulate ARG1 expression. In some embodiments, Crenolanib perturbs the PDGFR signaling pathway to modulate ARG1 expression. In some embodiments, prednisone perturbs the GR signaling pathway to modulate ARG1 expression. In some embodiments, HGF/SF perturbs the c-MET signaling pathway to modulate ARG1 expression. In some embodiments, Momelotinib perturbs the JAK/STAT signaling pathway to modulate ARG1 expression. In some embodiments, EGF perturbs the EGFR signaling pathway to modulate ARG1 expression. In some embodiments, Corticosterone perturbs the Mineralcorticoid receptor signaling pathway to modulate ARG1 expression. In some embodiments, FGF perturbs the FGFR signaling pathway to modulate ARG1 expression. In some embodiments, Thalidomide perturbs the NF-kB signaling pathway to modulate ARG1 expression. In some embodiments, Phenformin perturbs the AMPK signaling pathway to modulate ARG1 expression. In some embodiments, Tivozanib perturbs the Protein Tyrosine Kinase/RTK signaling pathway to modulate ARG1 expression. In some embodiments, BAY 87-2243 perturbs the Hypoxia activated signaling pathway to modulate ARG1 expression. In some embodiments, GZD824 Dimesylate perturbs the ABL signaling pathway to modulate ARG1 expression. In some embodiments, GDF10 (BMP3b) perturbs the TGF-B signaling pathway to modulate ARG1 expression. In some embodiments, PND-1186 perturbs the FAK signaling pathway to modulate ARG1 expression. In some embodiments, FRAX597 perturbs the PAK signaling pathway to modulate ARG1 expression. In some embodiments, BMP2 perturbs the TGF-B signaling pathway to modulate ARG1 expression. In some embodiments, Oligomycin A perturbs the ATP channel signaling pathway to modulate ARG1 expression. In some embodiments, Rifampicin perturbs the PXR signaling pathway to modulate ARG1 expression. In some embodiments, MK-0752 perturbs the NOTCH signaling pathway to modulate ARG1 expression.

›DETAILED DESCRIPTION · 20 of 28

In some embodiments, methods provided herein involve altering the composition and/or the structure of the insulated neighborhood containing the ARG1 gene. The ARG1 gene has a cytogenetic location of 6q23.2 and the genomic coordinate are on Chromosome 6 on the forward strand at position 131,573,144-131,584,332. Any chromatin mark, chromatin-associated protein, transcription factor and/or signaling protein that is associated with the insulated neighborhood, and/or any regions within or near the insulated neighborhood, may be targeted or altered to change the composition and/or structure of the insulated neighborhood, thereby modulating the expression of ARG1. The chromatin marks and/or chromatin-associated proteins may include but are not limited to H3K27ac, BRD4, and p300. The transcription factors may include but are not limited to FOXA2, HNF4A, ONECUT1, ONECUT2, YY1, HNF1A, and MYC. The signaling proteins may include but are not limited to HIF1a, ESRA, NR3C1, JUN, RXR, STAT3, NR1I1, SMAD2/3, STAT1, and TEAD1.

In some embodiments, compositions and methods provided herein may be used to treat a urea cycle disorder by modulating the expression of SLC25A15. Compounds that may be used to modulate the SLC25A15 expression include, but are not limited to, Dasatinib, FRAX597, Merestinib, R788 (fostamatinib disodium hexahydrate), Bosutinib, bms-986094 (inx-189), Epinephrine, GDF2 (BMP9), Echinomycin, Corticosterone, IGF-1, CP-673451, GZD824 Dimesylate, EW-7197, PDGF, Wnt3a, and derivatives or analogs thereof. In some embodiments, Dasatinib perturbs the ABL signaling pathway to modulate SLC25A15 expression. In some embodiments, FRAX597 perturbs the PAK signaling pathway to modulate SLC25A15 expression. In some embodiments, Merestinib perturbs the c-MET signaling pathway to modulate SLC25A15 expression. In some embodiments, R788 (fostamatinib disodium hexahydrate) perturbs the Protein Tyrosine Kinase/RTK signaling pathway to modulate SLC25M 5 expression. In some embodiments, Bosutinib perturbs the Src signaling pathway to modulate SLC25A15 expression. In some embodiments, Epinephrine perturbs the Adrenergic receptor signaling pathway to modulate SLC25A15 expression. In some embodiments, GDF2 (BMP9) perturbs the TGF-B signaling pathway to modulate SLC25A15 expression. In some embodiments, Echinomycin perturbs the Hypoxia activated signaling pathway to modulate SLC25A15 expression. In some embodiments, Corticosterone perturbs the Mineralcorticoid receptor signaling pathway to modulate SLC25A15 expression. In some embodiments, IGF-1 perturbs the IGF-1R/InsR signaling pathway to modulate SLC25A15 expression. In some embodiments, CP-673451 perturbs the PDGFR signaling pathway to modulate SLC25A15 expression. In some embodiments, GZD824 Dimesylate perturbs the ABL signaling pathway to modulate SLC25A15 expression. In some embodiments, EW-7197 perturbs the TGF-B signaling pathway to modulate SLC25A15 expression. In some embodiments, PDGF perturbs the PDGFR signaling pathway to modulate SLC25A15 expression. In some embodiments, Wnt3a perturbs the WNT signaling pathway to modulate SLC25A15 expression.

In some embodiments, methods provided herein involve altering the composition and/or the structure of the insulated neighborhood containing the SLC25A15 gene. SLC25A15 has a cytogenetic location of 13q14.11 and the genomic coordinate are on Chromosome 13 on the forward strand at position 40,789,412-40,810,111. Any chromatin mark, chromatin-associated protein, transcription factor and/or signaling protein that is associated with the insulated neighborhood, and/or any regions within or near the insulated neighborhood, may be targeted or altered to change the composition and/or structure of the insulated neighborhood, thereby modulating the expression of SLC25A15. The chromatin marks and/or chromatin-associated proteins may include but are not limited to H3K27ac, and BRD4. The transcription factors may include but are not limited to FOXA2, HNF4A, ONECUT1, ONECUT2, and YY1. The signaling proteins may include but are not limited to ESRA, Jun, RXR, NR1I1, NF-kB, NR3C1, SMAD2/3, and TP53.

In some embodiments, compositions and methods provided herein may be used to treat a urea cycle disorder by modulating the expression of SLC25A13. Compounds that may be used to modulate the SLC25A13 expression include, but are not limited to, TFP, 17-AAG (Tanespimycin), and derivatives or analogs thereof. In some embodiments, TFP perturbs the Calmodulin signaling pathway to modulate SLC25A13 expression. In some embodiments, 17-AAG (Tanespimycin) perturbs the Cell Cycle/DNA Damage Metabolic Enzyme/Protease signaling pathway to modulate SLC25A13 expression.

In some embodiments, methods provided herein involve altering the composition and/or the structure of the insulated neighborhood containing the SLC25A13 gene. SLC25A13 has a cytogenetic location of 7q21.3 and the genomic coordinate are on Chromosome 7 on the reverse strand at position 96,120,220-96,322,147. Any chromatin mark, chromatin-associated protein, transcription factor and/or signaling protein that is associated with the insulated neighborhood, and/or any regions within or near the insulated neighborhood, may be targeted or altered to change the composition and/or structure of the insulated neighborhood, thereby modulating the expression of SLC25A13. The chromatin marks and/or chromatin-associated proteins may include but are not limited to H3K27ac, BRD4, p300, and SMC1. The transcription factors may include but are not limited to FOXA2, HNF4A, ONECUT1, ATF5, ONECUT2, YY1, HNF1A, and MYC. The signaling proteins may include but are not limited to TCF7L2, HIF1a, ESRA, NR3C1, JUN, RXR, STAT3, NR1I1, NF-kB, SMAD2/3, STAT1, TEAD1, and TP53.

In some embodiments, compositions and methods provided herein may be used to treat a urea cycle disorder by modulating the expression of multiple urea cycle-related genes. As a non-limiting example, methods provided herein may be used to modulate the expression of any one of the following groups of genes: NAGS, CPS1, ASS1, ASL, OTC, ARG1, and SLC25A15; CPS1, ASS1, ASL, OTC, ARG1, and SLC25A15; ASS1, CPS1, NAGS, ARG1, and SLC25A15; CPS1, ASS1, ASL, ARG1, and SLC25A15; CPS1, ASS1, ASL, OTC, and ARG1; CPS1, ASS1, OTC, ARG1, and SLC25A15; NAGS, CPS1, ALS, OTC, and ARG1; ASS1, ASL, OTC, and ARG1; ASS1, CPS1, NAGS, and ARG1; CPS1, ASS1, ARG1, and SLC25A15; CPS1, ASS1, OTC, and ARG1; CPS1, OTC, ARG1, and SLC25A15; NAGS, CPS1, OTC, and ARG1; CPS1, ARG1, and SLC25A13; CPS1, ARG1, and SLC25A15; CPS1, ASL, and ARG1; CPS1, NAGS, and ARG1; CPS1, OTC, and ARG1; NAGS, ASS1, and OTC; OTC, NAGS, and ARG1. Compounds that may be used to modulate multiple urea cycle-related genes include, but are not limited to, Dasatinib, Echinomycin, CP-673451, GDF2 (BMP9), Bosutinib, Epinephrine, Pacritinib (SB1518), Amuvatinib, FRAX597, Thalidomide, Crenolanib, BMP2, Deoxycorticosterone, TNF-α, Wnt3a, PDGF, IGF-1, Activin, HGF/SF, 17-AAG (Tanespimycin), R788 (fostamatinib disodium hexahydrate), GZD824 Dimesylate, BAY 87-2243, prednisone, Nodal, Momelotinib, FGF, EGF, Anti mullerian hormone, INNO-206 (aldoxorubicin), and Pifithrin-μ.

›DETAILED DESCRIPTION · 21 of 28

In some embodiments, targeting multiple urea cycle-related genes may be accomplished by utilizing a combination of compounds that each specifically modulates a urea cycle-related gene. In some embodiments, targeting multiple urea cycle-related genes may be accomplished by utilizing a single compound that is capable of modulating multiple urea cycle-related genes.

In some embodiment, compounds provided herein may be used in combination with other drugs, such as Sodium phenylbutyrate (BUPHENYL®), glycerol phenylbutyrate (RAVICTI®), and sodium benzoate, to treat a urea cycle disorder.

In certain embodiments, the compound increases the expression of OTC, CPS1, NAGS, ASS1, ARG1, SLC25A15, or SLC25A13 in the cell or subject by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 125%, at least about 150%, at least about 175%, at least about 200%, at least about 250%, at least about 300%, at least about 400%, at least about 500%, from about 25% to about 50%, from about 40% to about 60%, from about 50% to about 70%, from about 60% to about 80%, from about 80% to about 100%, from about 100% to about 125%, from about 100 to about 150%, from about 150% to about 200%, from about 200% to about 300%, from about 300% to about 400%, from about 400% to about 500%, or more than 500% as compared to OTC, CPS1, NAGS, ASS1, ARG1, SLC25A15, or SLC25A13 expression in an untreated cell, untreated subject, or untreated population or a cell, subject, or population treated with a control compound. In some embodiments, the increased expression is in a cell in a subject. In some embodiments, the compound increases the expression of OTC, CPS1, NAGS, ASS1, ARG1, SLC25A15, or SLC25A13 in the cell by about 2 fold, about 3 fold, about 4 fold, about 5 fold, about 6 fold, about 7 fold, about 8 fold, about 9 fold, about 10 fold, about 12 fold, about 15 fold, about 18 fold, about 20 fold, about 25 fold, or more than 30 fold as compared to OTC, CPS1, NAGS, ASS1, ARG1, SLC25A15, or SLC25A13 expression in an untreated cell, untreated subject, or untreated population, or a cell, subject, or population treated with a control compound.

In some embodiments, modulation of OTC, CPS1, NAGS, ASS1, ARG1, SLC25A15, or SLC25A13 expression is an increase in OTC, CPS1, NAGS, ASS1, ARG1, SLC25A15, or SLC25A13 expression in a cell or a subject. In some embodiments, compositions and methods of the present invention cause an increase in the expression of a OTC, CPS1, NAGS, ASS1, ARG1, SLC25A15, or SLC25A13 gene as measured in a cell-based assay of cells exposed to the compound at a level corresponding to the plasma level achieved at steady state in a subject dosed with the effective amount as compared to cells exposed to a placebo.

In some embodiments, the increase is determined in a population of test subjects and the amount of increase is determined by reference to a matched control population. In some embodiments, the increase is determined in a population of test subjects and the amount of reduction is determined by reference to a pre-treatment baseline measurement. In some embodiments, the increase is determined in a population of cells and the amount of increase is determined by reference to a matched control cell population.

In some embodiments, the urea cycle gene mutation status of the subject is or has been determined by a direct to consumer genetic test. Such tests are commercially available from multiple vendors, including, but not limited to, 23andMe, Ancestry.com, Futura Genetics, MyDNA, Pathway Genomics, Progenity, and Dante Labs.

In some embodiments, the urea cycle gene mutation status of the subject is or has been determined by a physician or healthcare provider ordered genetic test. Such tests can be provided by multiple vendors such as university internal labs, or a variety of established CLIA certified vendors, including but not limited to Invivoscribe, Cancer Genetics, Foundation Medicine, Centogene, and Quest Diagnostics.

IV. Formulations and Delivery

Pharmaceutical Compositions

Compositions of active ingredients may be prepared as pharmaceutical compositions. It will be understood that such compositions necessarily comprise one or more active ingredients and, most often, a pharmaceutically acceptable excipient.

Relative amounts of the active ingredient, a pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition in accordance with the present disclosure may vary, depending upon the identity, size, and/or condition of the subject being treated and further depending upon the route by which the composition is to be administered. For example, the composition may comprise between 0.1% and 99% (w/w) of the active ingredient. By way of example, the composition may comprise between 0.1% and 100%, e.g., between 0.5 and 50%, between 1-30%, between 5-80%, at least 80% (w/w) active ingredient.

In some embodiments, the pharmaceutical compositions described herein may comprise at least one payload. As a non-limiting example, the pharmaceutical compositions may contain 1, 2, 3, 4 or 5 payloads.

Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to any other animal, e.g., to non-human animals, e.g. non-human mammals. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions is contemplated include, but are not limited to, humans and/or other primates; mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice, rats, birds, including commercially relevant birds such as poultry, chickens, ducks, geese, and/or turkeys.

›DETAILED DESCRIPTION · 22 of 28

In some embodiments, compositions are administered to humans, human patients or subjects.

Formulations

Formulations provided herein can include, without limitation, saline, liposomes, lipid nanoparticles, polymers, peptides, proteins, cells transfected with viral vectors (e.g., for transfer or transplantation into a subject) and combinations thereof.

Formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. As used herein the term “pharmaceutical composition” refers to compositions comprising at least one active ingredient and optionally one or more pharmaceutically acceptable excipients.

In general, such preparatory methods include the step of associating the active ingredient with an excipient and/or one or more other accessory ingredients.

Formulations of the compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with an excipient and/or one or more other accessory ingredients, and then, if necessary and/or desirable, dividing, shaping and/or packaging the product into a desired single- or multi-dose unit.

A pharmaceutical composition in accordance with the present disclosure may be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses. As used herein, a “unit dose” refers to a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.

Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition in accordance with the present disclosure may vary, depending upon the identity, size, and/or condition of the subject being treated and further depending upon the route by which the composition is to be administered. For example, the composition may comprise between 0.1% and 99% (w/w) of the active ingredient. By way of example, the composition may comprise between 0.1% and 100%, e.g., between 0.5 and 50%, between 1-30%, between 5-80%, at least 80% (w/w) active ingredient.

Excipients and Diluents

In some embodiments, a pharmaceutically acceptable excipient may be at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure. In some embodiments, an excipient is approved for use for humans and for veterinary use. In some embodiments, an excipient may be approved by United States Food and Drug Administration. In some embodiments, an excipient may be of pharmaceutical grade. In some embodiments, an excipient may meet the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and/or the International Pharmacopoeia.

Excipients, as used herein, include, but are not limited to, any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, and the like, as suited to the particular dosage form desired. Various excipients for formulating pharmaceutical compositions and techniques for preparing the composition are known in the art (see Remington: The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro, Lippincott, Williams & Wilkins, Baltimore, Md., 2006; incorporated herein by reference in its entirety). The use of a conventional excipient medium may be contemplated within the scope of the present disclosure, except insofar as any conventional excipient medium may be incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition.

Exemplary diluents include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, etc., and/or combinations thereof.

Inactive Ingredients

In some embodiments, the pharmaceutical compositions formulations may comprise at least one inactive ingredient. As used herein, the term “inactive ingredient” refers to one or more agents that do not contribute to the activity of the active ingredient of the pharmaceutical composition included in formulations. In some embodiments, all, none or some of the inactive ingredients which may be used in the formulations provided herein may be approved by the US Food and Drug Administration (FDA).

In one embodiment, the pharmaceutical compositions comprise at least one inactive ingredient such as, but not limited to, 1,2,6-Hexanetriol; 1,2-Dimyristoyl-Sn-Glycero-3-(Phospho-S-(1-Glycerol)); 1,2-Dimyristoyl-Sn-Glycero-3-Phosphocholine; 1,2-Dioleoyl-Sn-Glycero-3-Phosphocholine; 1,2-Dipalmitoyl-Sn-Glycero-3-(Phospho-Rac-(1-Glycerol)); 1,2-Distearoyl-Sn-Glycero-3-(Phospho-Rac-(1-Glycerol)); 1,2-Distearoyl-Sn-Glycero-3-Phosphocholine; 1-O-Tolylbiguanide; 2-Ethyl-1,6-Hexanediol; Acetic Acid; Acetic Acid, Glacial; Acetic Anhydride; Acetone; Acetone Sodium Bisulfite; Acetylated Lanolin Alcohols; Acetylated Monoglycerides; Acetylcysteine; Acetyltryptophan, DL-; Acrylates Copolymer; Acrylic Acid-Isooctyl Acrylate Copolymer; Acrylic Adhesive 788; Activated Charcoal; Adcote 72A103; Adhesive Tape; Adipic Acid; Aerotex Resin 3730; Alanine; Albumin Aggregated; Albumin Colloidal; Albumin Human; Alcohol; Alcohol, Dehydrated; Alcohol, Denatured; Alcohol, Diluted; Alfadex; Alginic Acid; Alkyl Ammonium Sulfonic Acid Betaine; Alkyl Aryl Sodium Sulfonate; Allantoin; Allyl .Alpha.-Ionone; Almond Oil; Alpha-Terpineol; Alpha-Tocopherol; Alpha-Tocopherol Acetate, Dl-; Alpha-Tocopherol, Dl-; Aluminum Acetate; Aluminum Chlorohydroxy Allantoinate; Aluminum Hydroxide; Aluminum Hydroxide-Sucrose, Hydrated; Aluminum Hydroxide Gel; Aluminum Hydroxide Gel F 500; Aluminum Hydroxide Gel F 5000; Aluminum Monostearate; Aluminum Oxide; Aluminum Polyester; Aluminum Silicate; Aluminum Starch Octenylsuccinate; Aluminum Stearate; Aluminum Subacetate; Aluminum Sulfate Anhydrous; Amerchol C; Amerchol-Cab; Aminomethylpropanol; Ammonia; Ammonia Solution; Ammonia Solution, Strong; Ammonium Acetate; Ammonium Hydroxide; Ammonium Lauryl Sulfate; Ammonium Nonoxynol-4 Sulfate; Ammonium Salt Of C-12-C-15 Linear Primary Alcohol Ethoxylate; Ammonium Sulfate; Ammonyx; Amphoteric-2; Amphoteric-9; Anethole; Anhydrous Citric Acid; Anhydrous Dextrose; Anhydrous Lactose; Anhydrous Trisodium Citrate; Aniseed Oil; Anoxid Sbn; Antifoam; Antipyrine; Apaflurane; Apricot Kernel Oil Peg-6 Esters; Aquaphor; Arginine; Arlacel; Ascorbic Acid; Ascorbyl Palmitate; Aspartic Acid; Balsam Peru; Barium Sulfate; Beeswax; Beeswax, Synthetic; Beheneth-10; Bentonite; Benzalkonium Chloride; Benzenesulfonic Acid; Benzethonium Chloride; Benzododecinium Bromide; Benzoic Acid; Benzyl Alcohol; Benzyl Benzoate; Benzyl Chloride; Betadex; Bibapcitide; Bismuth Subgallate; Boric Acid; Brocrinat; Butane; Butyl Alcohol; Butyl Ester Of Vinyl Methyl Ether/Maleic Anhydride Copolymer (125000 Mw); Butyl Stearate; Butylated Hydroxyanisole; Butylated Hydroxytoluene; Butylene Glycol; Butylparaben; Butyric Acid; C20-40 Pareth-24; Caffeine; Calcium; Calcium Carbonate; Calcium Chloride; Calcium Gluceptate; Calcium Hydroxide; Calcium Lactate; Calcobutrol; Caldiamide Sodium; Caloxetate Trisodium; Calteridol Calcium; Canada Balsam; Caprylic/Capric Triglyceride; Caprylic/Capric/Stearic Triglyceride; Captan; Captisol; Caramel; Carbomer 1342; Carbomer 1382; Carbomer 934; Carbomer 934p; Carbomer 940; Carbomer 941; Carbomer 980; Carbomer 981; Carbomer Homopolymer Type B (Allyl Pentaerythritol Crosslinked); Carbomer Homopolymer Type C (Allyl Pentaerythritol Crosslinked); Carbon Dioxide; Carboxy Vinyl Copolymer; Carboxymethylcellulose; Carboxymethylcellulose Sodium; Carboxypolymethylene; Carrageenan; Carrageenan Salt; Castor Oil; Cedar Leaf Oil; Cellulose; Cellulose, Microcrystalline; Cerasynt-Se; Ceresin; Ceteareth-12; Ceteareth-15; Ceteareth-30; Cetearyl Alcohol/Ceteareth-20; Cetearyl Ethylhexanoate; Ceteth-10; Ceteth-2; Ceteth-20; Ceteth-23; Cetostearyl Alcohol; Cetrimonium Chloride; Cetyl Alcohol; Cetyl Esters Wax; Cetyl Palmitate; Cetylpyridinium Chloride; Chlorobutanol; Chlorobutanol Hemihydrate; Chlorobutanol, Anhydrous; Chlorocresol; Chloroxylenol; Cholesterol; Choleth; Choleth-24; Citrate; Citric Acid; Citric Acid Monohydrate; Citric Acid, Hydrous; Cocamide Ether Sulfate; Cocamine Oxide; Coco Betaine; Coco Diethanolamide; Coco Monoethanolamide; Cocoa Butter; Coco-Glycerides; Coconut Oil; Coconut Oil, Hydrogenated; Coconut Oil/Palm Kernel Oil Glycerides, Hydrogenated; Cocoyl Caprylocaprate; Cola Nitida Seed Extract; Collagen; Coloring Suspension; Corn Oil; Cottonseed Oil; Cream Base; Creatine; Creatinine; Cresol; Croscarmellose Sodium; Crospovidone; Cupric Sulfate; Cupric Sulfate Anhydrous; Cyclomethicone; Cyclomethicone/Dimethicone Copolyol; Cysteine; Cysteine Hydrochloride; Cysteine Hydrochloride Anhydrous; Cysteine, Dl-; D&C Red No. 28; D&C Red No. 33; D&C Red No. 36; D&C Red No. 39; D&C Yellow No. 10; Dalfampridine; Daubert 1-5 Pestr (Matte) 164z; Decyl Methyl Sulfoxide; Dehydag Wax Sx; Dehydroacetic Acid; Dehymuls E; Denatonium Benzoate; Deoxycholic Acid; Dextran; Dextran 40; Dextrin; Dextrose; Dextrose Monohydrate; Dextrose Solution; Diatrizoic Acid; Diazolidinyl Urea; Dichlorobenzyl Alcohol; Dichlorodifluoromethane; Dichlorotetrafluoroethane; Diethanolamine; Diethyl Pyrocarbonate; Diethyl Sebacate; Diethylene Glycol Monoethyl Ether; Diethylhexyl Phthalate; Dihydroxyaluminum Aminoacetate; Diisopropanolamine; Diisopropyl Adipate; Diisopropyl Dilinoleate; Dimethicone 350; Dimethicone Copolyol; Dimethicone Mdx4-4210; Dimethicone Medical Fluid 360; Dimethyl Isosorbide; Dimethyl Sulfoxide; Dimethylaminoethyl Methacrylate-Butyl Methacrylate-Methyl Methacrylate Copolymer; Dimethyldioctadecylammonium Bentonite; Dimethylsiloxane/Methylvinylsiloxane Copolymer; Dinoseb Ammonium Salt; Dipalmitoylphosphatidylglycerol, Dl-; Dipropylene Glycol; Disodium Cocoamphodiacetate; Disodium Laureth Sulfosuccinate; Disodium Lauryl Sulfosuccinate; Disodium Sulfosalicylate; Disofenin; Divinylbenzene Styrene Copolymer; Dmdm Hydantoin; Docosanol; Docusate Sodium; Duro-Tak 280-2516; Duro-Tak 387-2516; Duro-Tak 80-1196; Duro-Tak 87-2070; Duro-Tak 87-2194; Duro-Tak 87-2287; Duro-Tak 87-2296; Duro-Tak 87-2888; Duro-Tak 87-2979; Edetate Calcium Disodium; Edetate Disodium; Edetate Disodium Anhydrous; Edetate Sodium; Edetic Acid; Egg Phospholipids; Entsufon; Entsufon Sodium; Epilactose; Epitetracycline Hydrochloride; Essence Bouquet 9200; Ethanolamine Hydrochloride; Ethyl Acetate; Ethyl Oleate; Ethylcelluloses; Ethylene Glycol; Ethylene Vinyl Acetate Copolymer; Ethylenediamine; Ethylenediamine Dihydrochloride; Ethylene-Propylene Copolymer; Ethylene-Vinyl Acetate Copolymer (28% Vinyl Acetate); Ethylene-Vinyl Acetate Copolymer (9% Vinylacetate); Ethylhexyl Hydroxystearate; Ethylparaben; Eucalyptol; Exametazime; Fat, Edible; Fat, Hard; Fatty Acid Esters; Fatty Acid Pentaerythriol Ester; Fatty Acids; Fatty Alcohol Citrate; Fatty Alcohols; Fd&C Blue No. 1; Fd&C Green No. 3; Fd&C Red No. 4; Fd&C Red No. 40; Fd&C Yellow No. 10 (Delisted); Fd&C Yellow No. 5; Fd&C Yellow No. 6; Ferric Chloride; Ferric Oxide; Flavor 89-186; Flavor 89-259; Flavor Df-119; Flavor Df-1530; Flavor Enhancer; Flavor Fig 827118; Flavor Raspberry Pfc-8407; Flavor Rhodia Pharmaceutical No. Rf 451; Fluorochlorohydrocarbons; Formaldehyde; Formaldehyde Solution; Fractionated Coconut Oil; Fragrance 3949-5; Fragrance 520a; Fragrance 6.007; Fragrance 91-122; Fragrance 9128-Y; Fragrance 93498g; Fragrance Balsam Pine No. 5124; Fragrance Bouquet 10328; Fragrance Chemoderm 6401-B; Fragrance Chemoderm 6411; Fragrance Cream No. 73457; Fragrance Cs-28197; Fragrance Felton 066m; Fragrance Firmenich 47373; Fragrance Givaudan Ess 9090/1c; Fragrance H-6540; Fragrance Herbal 10396; Fragrance Nj-1085; Fragrance P O F1-147; Fragrance Pa 52805; Fragrance Pera Derm D; Fragrance Rbd-9819; Fragrance Shaw Mudge U-7776; Fragrance Tf 044078; Fragrance Ungerer Honeysuckle K 2771; Fragrance Ungerer N5195; Fructose; Gadolinium Oxide; Galactose; Gamma Cyclodextrin; Gelatin; Gelatin, Crosslinked; Gelfoam Sponge; Gellan Gum (Low Acyl); Gelva 737; Gentisic Acid; Gentisic Acid Ethanolamide; Gluceptate Sodium; Gluceptate Sodium Dihydrate; Gluconolactone; Glucuronic Acid; Glutamic Acid, Dl-; Glutathione; Glycerin; Glycerol Ester Of Hydrogenated Rosin; Glyceryl Citrate; Glyceryl Isostearate; Glyceryl Laurate; Glyceryl Monostearate; Glyceryl Oleate; Glyceryl Oleate/Propylene Glycol; Glyceryl Palmitate; Glyceryl Ricinoleate; Glyceryl Stearate; Glyceryl Stearate-Laureth-23; Glyceryl Stearate/Peg Stearate; Glyceryl Stearate/Peg-100 Stearate; Glyceryl Stearate/Peg-40 Stearate; Glyceryl Stearate-Stearamidoethyl Diethylamine; Glyceryl Trioleate; Glycine; Glycine Hydrochloride; Glycol Distearate; Glycol Stearate; Guanidine Hydrochloride; Guar Gum; Hair Conditioner (18n195-1m); Heptane; Hetastarch; Hexylene Glycol; High Density Polyethylene; Histidine; Human Albumin Microspheres; Hyaluronate Sodium; Hydrocarbon; Hydrocarbon Gel, Plasticized; Hydrochloric Acid; Hydrochloric Acid, Diluted; Hydrocortisone; Hydrogel Polymer; Hydrogen Peroxide; Hydrogenated Castor Oil; Hydrogenated Palm Oil; Hydrogenated Palm/Palm Kernel Oil Peg-6 Esters; Hydrogenated Polybutene 635-690; Hydroxide Ion; Hydroxyethyl Cellulose; Hydroxyethylpiperazine Ethane Sulfonic Acid; Hydroxymethyl Cellulose; Hydroxyoctacosanyl Hydroxystearate; Hydroxypropyl Cellulose; Hydroxypropyl Methylcellulose 2906; Hydroxypropyl-Beta-cyclodextrin; Hypromellose 2208 (15000 Mpa·S); Hypromellose 2910 (15000 Mpa·S); Hypromelloses; Imidurea; Iodine; Iodoxamic Acid; Iofetamine Hydrochloride; Irish Moss Extract; Isobutane; Isoceteth-20; Isoleucine; Isooctyl Acrylate; Isopropyl Alcohol; Isopropyl Isostearate; Isopropyl Myristate; Isopropyl Myristate—Myristyl Alcohol; Isopropyl Palmitate; Isopropyl Stearate; Isostearic Acid; Isostearyl Alcohol; Isotonic Sodium Chloride Solution; Jelene; Kaolin; Kathon Cg; Kathon Cg II; Lactate; Lactic Acid; Lactic Acid, Dl-; Lactic Acid, L-; Lactobionic Acid; Lactose; Lactose Monohydrate; Lactose, Hydrous; Laneth; Lanolin; Lanolin Alcohol—Mineral Oil; Lanolin Alcohols; Lanolin Anhydrous; Lanolin Cholesterols; Lanolin Nonionic Derivatives; Lanolin, Ethoxylated; Lanolin, Hydrogenated; Lauralkonium Chloride; Lauramine Oxide; Laurdimonium Hydrolyzed Animal Collagen; Laureth Sulfate; Laureth-2; Laureth-23; Laureth-4; Lauric Diethanolamide; Lauric Myristic Diethanolamide; Lauroyl Sarcosine; Lauryl Lactate; Lauryl Sulfate; Lavandula angustifolia Flowering Top; Lecithin; Lecithin Unbleached; Lecithin, Egg; Lecithin, Hydrogenated; Lecithin, Hydrogenated Soy; Lecithin, Soybean; Lemon Oil; Leucine; Levulinic Acid; Lidofenin; Light Mineral Oil; Light Mineral Oil (85 Ssu); Limonene, (+/−)-; Lipocol Sc-15; Lysine; Lysine Acetate; Lysine Monohydrate; Magnesium Aluminum Silicate; Magnesium Aluminum Silicate Hydrate; Magnesium Chloride; Magnesium Nitrate; Magnesium Stearate; Maleic Acid; Mannitol; Maprofix; Mebrofenin; Medical Adhesive Modified S-15; Medical Antiform A-F Emulsion; Medronate Disodium; Medronic Acid; Meglumine; Menthol; Metacresol; Metaphosphoric Acid; Methanesulfonic Acid; Methionine; Methyl Alcohol; Methyl Gluceth-10; Methyl Gluceth-20; Methyl Gluceth-20 Sesquistearate; Methyl Glucose Sesquistearate; Methyl Laurate; Methyl Pyrrolidone; Methyl Salicylate; Methyl Stearate; Methylboronic Acid; Methylcellulose (4000 Mpa·S); Methylcelluloses; Methylchloroisothiazolinone; Methylene Blue; Methylisothiazolinone; Methylparaben; Microcrystalline Wax; Mineral Oil; Mono And Diglyceride; Monostearyl Citrate; Monothioglycerol; Multisterol Extract; Myristyl Alcohol; Myristyl Lactate; Myristyl-.Gamma.-Picolinium Chloride; N-(Carbamoyl-Methoxy Peg-40)-1,2-Distearoyl-Cephalin Sodium; N,N-Dimethylacetamide; Niacinamide; Nioxime; Nitric Acid; Nitrogen; Nonoxynol Iodine; Nonoxynol-15; Nonoxynol-9; Norflurane; Oatmeal; Octadecene-1/Maleic Acid Copolymer; Octanoic Acid; Octisalate; Octoxynol-1; Octoxynol-40; Octoxynol-9; Octyldodecanol; Octylphenol Polymethylene; Oleic Acid; Oleth-10/Oleth-5; Oleth-2; Oleth-20; Oleyl Alcohol; Oleyl Oleate; Olive Oil; Oxidronate Disodium; Oxyquinoline; Palm Kernel Oil; Palmitamine Oxide; Parabens; Paraffin; Paraffin, White Soft; Parfum Creme 45/3; Peanut Oil; Peanut Oil, Refined; Pectin; Peg 6-32 Stearate/Glycol Stearate; Peg Vegetable Oil; Peg-100 Stearate; Peg-12 Glyceryl Laurate; Peg-120 Glyceryl Stearate; Peg-120 Methyl Glucose Dioleate; Peg-15 Cocamine; Peg-150 Distearate; Peg-2 Stearate; Peg-20 Sorbitan Isostearate; Peg-22 Methyl Ether/Dodecyl Glycol Copolymer; Peg-25 Propylene Glycol Stearate; Peg-4 Dilaurate; Peg-4 Laurate; Peg-40 Castor Oil; Peg-40 Sorbitan Diisostearate; Peg-45/Dodecyl Glycol Copolymer; Peg-5 Oleate; Peg-50 Stearate; Peg-54 Hydrogenated Castor Oil; Peg-6 Isostearate; Peg-60 Castor Oil; Peg-60 Hydrogenated Castor Oil; Peg-7 Methyl Ether; Peg-75 Lanolin; Peg-8 Laurate; Peg-8 Stearate; Pegoxol 7 Stearate; Pentadecalactone; Pentaerythritol Cocoate; Pentasodium Pentetate; Pentetate Calcium Trisodium; Pentetic Acid; Peppermint Oil; Perflutren; Perfume 25677; Perfume Bouquet; Perfume E-1991; Perfume Gd 5604; Perfume Tana 90/42 Scba; Perfume W-1952-1; Petrolatum; Petrolatum, White; Petroleum Distillates; Phenol; Phenol, Liquefied; Phenonip; Phenoxyethanol; Phenylalanine; Phenylethyl Alcohol; Phenylmercuric Acetate; Phenylmercuric Nitrate; Phosphatidyl Glycerol, Egg; Phospholipid; Phospholipid, Egg; Phospholipon 90g; Phosphoric Acid; Pine Needle Oil ( Pinus sylvestris ); Piperazine Hexahydrate; Plastibase-50w; Polacrilin; Polidronium Chloride; Poloxamer 124; Poloxamer 181; Poloxamer 182; Poloxamer 188; Poloxamer 237; Poloxamer 407; Poly(Bis(P-Carboxyphenoxy)Propane Anhydride): Sebacic Acid; Poly(Dimethylsiloxane/Methylvinylsiloxane/Methylhydrogensiloxane) Dimethylvinyl Or Dimethylhydroxy Or Trimethyl Endblocked; Poly(Dl-Lactic-Co-Glycolic Acid), (50:50; Poly(Dl-Lactic-Co-Glycolic Acid), Ethyl Ester Terminated, (50:50; Polyacrylic Acid (250000 Mw); Polybutene (1400 Mw); Polycarbophil; Polyester; Polyester Polyamine Copolymer; Polyester Rayon; Polyethylene Glycol 1000; Polyethylene Glycol 1450; Polyethylene Glycol 1500; Polyethylene Glycol 1540; Polyethylene Glycol 200; Polyethylene Glycol 300; Polyethylene Glycol 300-1600; Polyethylene Glycol 3350; Polyethylene Glycol 400; Polyethylene Glycol 4000; Polyethylene Glycol 540; Polyethylene Glycol 600; Polyethylene Glycol 6000; Polyethylene Glycol 8000; Polyethylene Glycol 900; Polyethylene High Density Containing Ferric Oxide Black (<1%); Polyethylene Low Density Containing Barium Sulfate (20-24%); Polyethylene T; Polyethylene Terephthalates; Polyglactin; Polyglyceryl-3 Oleate; Polyglyceryl-4 Oleate; Polyhydroxyethyl Methacrylate; Polyisobutylene; Polyisobutylene (1100000 Mw); Polyisobutylene (35000 Mw); Polyisobutylene 178-236; Polyisobutylene 241-294; Polyisobutylene 35-39; Polyisobutylene Low Molecular Weight; Polyisobutylene Medium Molecular Weight; Polyisobutylene/Polybutene Adhesive; Polylactide; Polyols; Polyoxyethylene-Polyoxypropylene 1800; Polyoxyethylene Alcohols; Polyoxyethylene Fatty Acid Esters; Polyoxyethylene Propylene; Polyoxyl 20 Cetostearyl Ether; Polyoxyl 35 Castor Oil; Polyoxyl 40 Hydrogenated Castor Oil; Polyoxyl 40 Stearate; Polyoxyl 400 Stearate; Polyoxyl 6 And Polyoxyl 32 Palmitostearate; Polyoxyl Distearate; Polyoxyl Glyceryl Stearate; Polyoxyl Lanolin; Polyoxyl Palmitate; Polyoxyl Stearate; Polypropylene; Polypropylene Glycol; Polyquaternium-10; Polyquaternium-7 (70/30 Acrylamide/Dadmac; Polysiloxane; Polysorbate 20; Polysorbate 40; Polysorbate 60; Polysorbate 65; Polysorbate 80; Polyurethane; Polyvinyl Acetate; Polyvinyl Alcohol; Polyvinyl Chloride; Polyvinyl Chloride-Polyvinyl Acetate Copolymer; Polyvinylpyridine; Poppy Seed Oil; Potash; Potassium Acetate; Potassium Alum; Potassium Bicarbonate; Potassium Bisulfite; Potassium Chloride; Potassium Citrate; Potassium Hydroxide; Potassium Metabisulfite; Potassium Phosphate, Dibasic; Potassium Phosphate, Monobasic; Potassium Soap; Potassium Sorbate; Povidone Acrylate Copolymer; Povidone Hydrogel; Povidone K17; Povidone K25; Povidone K29/32; Povidone K30; Povidone K90; Povidone K90f; Povidone/Eicosene Copolymer; Povidones; Ppg-12/Smdi Copolymer; Ppg-15 Stearyl Ether; Ppg-20 Methyl Glucose Ether Distearate; Ppg-26 Oleate; Product Wat; Proline; Promulgen D; Promulgen G; Propane; Propellant A-46; Propyl Gallate; Propylene Carbonate; Propylene Glycol; Propylene Glycol Diacetate; Propylene Glycol Dicaprylate; Propylene Glycol Monolaurate; Propylene Glycol Monopalmitostearate; Propylene Glycol Palmitostearate; Propylene Glycol Ricinoleate; Propylene Glycol/Diazolidinyl Urea/Methylparaben/Propylparben; Propylparaben; Protamine Sulfate; Protein Hydrolysate; Pvm/Ma Copolymer; Quaternium-15; Quaternium-15 Cis-Form; Quaternium-52; Ra-2397; Ra-3011; Saccharin; Saccharin Sodium; Saccharin Sodium Anhydrous; Safflower Oil; Sd Alcohol 3a; Sd Alcohol 40; Sd Alcohol 40-2; Sd Alcohol 40b; Sepineo P 600; Serine; Sesame Oil; Shea Butter; Silastic Brand Medical Grade Tubing; Silastic Medical Adhesive, Silicone Type A; Silica, Dental; Silicon; Silicon Dioxide; Silicon Dioxide, Colloidal; Silicone; Silicone Adhesive 4102; Silicone Adhesive 4502; Silicone Adhesive Bio-Psa Q7-4201; Silicone Adhesive Bio-Psa Q7-4301; Silicone Emulsion; Silicone/Polyester Film Strip; Simethicone; Simethicone Emulsion; Sipon Ls 20np; Soda Ash; Sodium Acetate; Sodium Acetate Anhydrous; Sodium Alkyl Sulfate; Sodium Ascorbate; Sodium Benzoate; Sodium Bicarbonate; Sodium Bisulfate; Sodium Bisulfite; Sodium Borate; Sodium Borate Decahydrate; Sodium Carbonate; Sodium Carbonate Decahydrate; Sodium Carbonate Monohydrate; Sodium Cetostearyl Sulfate; Sodium Chlorate; Sodium Chloride; Sodium Chloride Injection; Sodium Chloride Injection, Bacteriostatic; Sodium Cholesteryl Sulfate; Sodium Citrate; Sodium Cocoyl Sarcosinate; Sodium Desoxycholate; Sodium Dithionite; Sodium Dodecylbenzenesulfonate; Sodium Formaldehyde Sulfoxylate; Sodium Gluconate; Sodium Hydroxide; Sodium Hypochlorite; Sodium Iodide; Sodium Lactate; Sodium Lactate, L-; Sodium Laureth-2 Sulfate; Sodium Laureth-3 Sulfate; Sodium Laureth-5 Sulfate; Sodium Lauroyl Sarcosinate; Sodium Lauryl Sulfate; Sodium Lauryl Sulfoacetate; Sodium Metabisulfite; Sodium Nitrate; Sodium Phosphate; Sodium Phosphate Dihydrate; Sodium Phosphate, Dibasic; Sodium Phosphate, Dibasic, Anhydrous; Sodium Phosphate, Dibasic, Dihydrate; Sodium Phosphate, Dibasic, Dodecahydrate; Sodium Phosphate, Dibasic, Heptahydrate; Sodium Phosphate, Monobasic; Sodium Phosphate, Monobasic, Anhydrous; Sodium Phosphate, Monobasic, Dihydrate; Sodium Phosphate, Monobasic, Monohydrate; Sodium Polyacrylate (2500000 Mw); Sodium Pyrophosphate; Sodium Pyrrolidone Carboxylate; Sodium Starch Glycolate; Sodium Succinate Hexahydrate; Sodium Sulfate; Sodium Sulfate Anhydrous; Sodium Sulfate Decahydrate; Sodium Sulfite; Sodium Sulfosuccinated Undecyclenic Monoalkylolamide; Sodium Tartrate; Sodium Thioglycolate; Sodium Thiomalate; Sodium Thiosulfate; Sodium Thiosulfate Anhydrous; Sodium Trimetaphosphate; Sodium Xylenesulfonate; Somay 44; Sorbic Acid; Sorbitan; Sorbitan Isostearate; Sorbitan Monolaurate; Sorbitan Monooleate; Sorbitan Monopalmitate; Sorbitan Monostearate; Sorbitan Sesquioleate; Sorbitan Trioleate; Sorbitan Tristearate; Sorbitol; Sorbitol Solution; Soybean Flour; Soybean Oil; Spearmint Oil; Spermaceti; Squalane; Stabilized Oxychloro Complex; Stannous 2-Ethylhexanoate; Stannous Chloride; Stannous Chloride Anhydrous; Stannous Fluoride; Stannous Tartrate; Starch; Starch 1500, Pregelatinized; Starch, Corn; Stearalkonium Chloride; Stearalkonium Hectorite/Propylene Carbonate; Stearamidoethyl Diethylamine; Steareth-10; Steareth-100; Steareth-2; Steareth-20; Steareth-21; Steareth-40; Stearic Acid; Stearic Diethanolamide; Stearoxytrimethylsilane; Steartrimonium Hydrolyzed Animal Collagen; Stearyl Alcohol; Sterile Water For Inhalation; Styrene/Isoprene/Styrene Block Copolymer; Succimer; Succinic Acid; Sucralose; Sucrose; Sucrose Distearate; Sucrose Polyesters; Sulfacetamide Sodium; Sulfobutylether .Beta.-Cyclodextrin; Sulfur Dioxide; Sulfuric Acid; Sulfurous Acid; Surfactol Qs; Tagatose, D-; Talc; Tall Oil; Tallow Glycerides; Tartaric Acid; Tartaric Acid, Dl-; Tenox; Tenox-2; Tert-Butyl Alcohol; Tert-Butyl Hydroperoxide; Tert-Butylhydroquinone; Tetrakis(2-Methoxyisobutylisocyanide)Copper(I) Tetrafluoroborate; Tetrapropyl Orthosilicate; Tetrofosmin; Theophylline; Thimerosal; Threonine; Thymol; Tin; Titanium Dioxide; Tocopherol; Tocophersolan; Total parenteral nutrition, lipid emulsion; Triacetin; Tricaprylin; Trichloromonofluoromethane; Trideceth-10; Triethanolamine Lauryl Sulfate; Trifluoroacetic Acid; Triglycerides, Medium Chain; Trihydroxystearin; Trilaneth-4 Phosphate; Trilaureth-4 Phosphate; Trisodium Citrate Dihydrate; Trisodium Hedta; Triton 720; Triton X-200; Trolamine; Tromantadine; Tromethamine (TRIS); Tryptophan; Tyloxapol; Tyrosine; Undecylenic Acid; Union 76 Amsco-Res 6038; Urea; Valine; Vegetable Oil; Vegetable Oil Glyceride, Hydrogenated; Vegetable Oil, Hydrogenated; Versetamide; Viscarin; Viscose/Cotton; Vitamin E; Wax, Emulsifying; Wecobee Fs; White Ceresin Wax; White Wax; Xanthan Gum; Zinc; Zinc Acetate; Zinc Carbonate; Zinc Chloride; and Zinc Oxide.

›DETAILED DESCRIPTION · 23 of 28

Pharmaceutical composition formulations disclosed herein may include cations or anions. In one embodiment, the formulations include metal cations such as, but not limited to, Zn2+, Ca2+, Cu2+, Mn2+, Mg+ and combinations thereof. As a non-limiting example, formulations may include polymers and complexes with a metal cation (See e.g., U.S. Pat. Nos. 6,265,389 and 6,555,525, each of which is herein incorporated by reference in its entirety).

Formulations may also include one or more pharmaceutically acceptable salts. As used herein, “pharmaceutically acceptable salts” refers to derivatives of the disclosed compounds wherein the parent compound is modified by converting an existing acid or base moiety to its salt form (e.g., by reacting the free base group with a suitable organic acid). Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. Representative acid addition salts include acetate, acetic acid, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzene sulfonic acid, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. The pharmaceutically acceptable salts of the present disclosure include the conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids.

Solvates may be prepared by crystallization, recrystallization, or precipitation from a solution that includes organic solvents, water, or a mixture thereof. Examples of suitable solvents are ethanol, water (for example, mono-, di-, and tri-hydrates), N-methylpyrrolidinone (NMP), dimethyl sulfoxide (DMSO), N,N′-dimethylformamide (DMF), N,N′-dimethylacetamide (DMAC), 1,3-dimethyl-2-imidazolidinone (DMEU), 1,3-dimethyl-3,4,5,6-tetrahydro-2-(1H)-pyrimidinone (DMPU), acetonitrile (ACN), propylene glycol, ethyl acetate, benzyl alcohol, 2-pyrrolidone, benzyl benzoate, and the like. When water is the solvent, the solvate is referred to as a “hydrate.”

V. Administration and Dosing

Administration

The terms “administering” and “introducing” are used interchangeably herein and refer to the delivery of the pharmaceutical composition into a cell or a subject. In the case of delivery to a subject, the pharmaceutical composition is delivered by a method or route that results in at least partial localization of the introduced cells at a desired site, such as hepatocytes, such that a desired effect(s) is produced.

In one aspect of the method, the pharmaceutical composition may be administered via a route such as, but not limited to, enteral (into the intestine), gastroenteral, epidural (into the dura matter), oral (by way of the mouth), transdermal, peridural, intracerebral (into the cerebrum), intracerebroventricular (into the cerebral ventricles), epicutaneous (application onto the skin), intradermal, (into the skin itself), subcutaneous (under the skin), nasal administration (through the nose), intravenous (into a vein), intravenous bolus, intravenous drip, intraarterial (into an artery), intramuscular (into a muscle), intracardiac (into the heart), intraosseous infusion (into the bone marrow), intrathecal (into the spinal canal), intraperitoneal, (infusion or injection into the peritoneum), intravesical infusion, intravitreal, (through the eye), intracavernous injection (into a pathologic cavity) intracavitary (into the base of the penis), intravaginal administration, intrauterine, extra-amniotic administration, transdermal (diffusion through the intact skin for systemic distribution), transmucosal (diffusion through a mucous membrane), transvaginal, insufflation (snorting), sublingual, sublabial, enema, eye drops (onto the conjunctiva), in ear drops, auricular (in or by way of the ear), buccal (directed toward the cheek), conjunctival, cutaneous, dental (to a tooth or teeth), electro-osmosis, endocervical, endosinusial, endotracheal, extracorporeal, hemodialysis, infiltration, interstitial, intra-abdominal, intra-amniotic, intra-articular, intrabiliary, intrabronchial, intrabursal, intracartilaginous (within a cartilage), intracaudal (within the cauda equine), intracisternal (within the cisterna magna cerebellomedularis), intracorneal (within the cornea), dental intracornal, intracoronary (within the coronary arteries), intracorporus cavernosum (within the dilatable spaces of the corporus cavernosa of the penis), intradiscal (within a disc), intraductal (within a duct of a gland), intraduodenal (within the duodenum), intradural (within or beneath the dura), intraepidermal (to the epidermis), intraesophageal (to the esophagus), intragastric (within the stomach), intragingival (within the gingivae), intraileal (within the distal portion of the small intestine), intralesional (within or introduced directly to a localized lesion), intraluminal (within a lumen of a tube), intralymphatic (within the lymph), intramedullary (within the marrow cavity of a bone), intrameningeal (within the meninges), intramyocardial (within the myocardium), intraocular (within the eye), intraovarian (within the ovary), intrapericardial (within the pericardium), intrapleural (within the pleura), intraprostatic (within the prostate gland), intrapulmonary (within the lungs or its bronchi), intrasinal (within the nasal or periorbital sinuses), intraspinal (within the vertebral column), intrasynovial (within the synovial cavity of a joint), intratendinous (within a tendon), intratesticular (within the testicle), intrathecal (within the cerebrospinal fluid at any level of the cerebrospinal axis), intrathoracic (within the thorax), intratubular (within the tubules of an organ), intratumor (within a tumor), intratympanic (within the aurus media), intravascular (within a vessel or vessels), intraventricular (within a ventricle), iontophoresis (by means of electric current where ions of soluble salts migrate into the tissues of the body), irrigation (to bathe or flush open wounds or body cavities), laryngeal (directly upon the larynx), nasogastric (through the nose and into the stomach), occlusive dressing technique (topical route administration which is then covered by a dressing which occludes the area), ophthalmic (to the external eye), oropharyngeal (directly to the mouth and pharynx), parenteral, percutaneous, periarticular, peridural, perineural, periodontal, rectal, respiratory (within the respiratory tract by inhaling orally or nasally for local or systemic effect), retrobulbar (behind the pons or behind the eyeball), intramyocardial (entering the myocardium), soft tissue, subarachnoid, subconjunctival, submucosal, topical, transplacental (through or across the placenta), transtracheal (through the wall of the trachea), transtympanic (across or through the tympanic cavity), ureteral (to the ureter), urethral (to the urethra), vaginal, caudal block, diagnostic, nerve block, biliary perfusion, cardiac perfusion, photopheresis and spinal.

›DETAILED DESCRIPTION · 24 of 28

Modes of administration include injection, infusion, instillation, and/or ingestion. “Injection” includes, without limitation, intravenous, intramuscular, intra-arterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal, intracerebro spinal, and intrasternal injection and infusion. In some examples, the route is intravenous. For the delivery of cells, administration by injection or infusion can be made.

In some embodiments, compounds provided herein can be administered to cells systemically. The phrases “systemic administration,” “administered systemically,” “peripheral administration,” and “administered peripherally” refer to the administration other than directly into a target site, tissue, or organ, such that it enters, instead, the subject's circulatory system and, thus, is subject to metabolism and other like processes. In other embodiments, compounds provided herein can be administered to cells ex vivo, i.e., the compounds can be administered to cells that have been removed from an organ or tissue and held outside the subject's body e.g., in primary culture.

Dosing

The term “effective amount” refers to the amount of the active ingredient needed to prevent or alleviate at least one or more signs or symptoms of a specific disease and/or condition, and relates to a sufficient amount of a composition to provide the desired effect. The term “therapeutically effective amount” therefore refers to an amount of active ingredient or a composition comprising the active ingredient that is sufficient to promote a particular effect when administered to a typical subject. An effective amount would also include an amount sufficient to prevent or delay the development of a symptom of the disease, alter the course of a symptom of the disease (for example but not limited to, slow the progression of a symptom of the disease), or reverse a symptom of the disease. It is understood that for any given case, an appropriate “effective amount” can be determined by one of ordinary skill in the art using routine experimentation.

The pharmaceutical, diagnostic, or prophylactic compositions provided herein may be administered to a subject using any amount and any route of administration effective for preventing, treating, managing, or diagnosing diseases, disorders and/or conditions. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the particular composition, its mode of administration, its mode of activity, and the like. The subject may be a human, a mammal, or an animal. Compositions are typically formulated in unit dosage form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the compositions provided herein may be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective, prophylactically effective, or appropriate diagnostic dose level for any particular individual will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific payload employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, and route of administration; the duration of the treatment; drugs used in combination or coincidental with the active ingredient; and like factors well known in the medical arts.

In certain embodiments, pharmaceutical compositions may be administered at dosage levels sufficient to deliver from about 0.01 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 0.05 mg/kg, from about 0.05 mg/kg to about 0.5 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, from about 0.1 mg/kg to about 40 mg/kg, from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, or from about 1 mg/kg to about 25 mg/kg, of subject body weight per day, one or more times a day, to obtain the desired therapeutic, diagnostic, or prophylactic, effect.

The desired dosage of the composition may be delivered only once, three times a day, two times a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks. In certain embodiments, the desired dosage may be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations). When multiple administrations are employed, split dosing regimens such as those described herein may be used. As used herein, a “split dose” is the division of “single unit dose” or total daily dose into two or more doses, e.g., two or more administrations of the “single unit dose”. As used herein, a “single unit dose” is a dose of any therapeutic administered in one dose/at one time/single route/single point of contact, i.e., single administration event.

VI. Additional Embodiments

A method for increasing OTC gene expression in a cell harboring an OTC mutation associated with a partial reduction of OTC function, comprising: contacting the cell with an effective amount of a compound that inhibits a target selected from the group consisting of JAK1, JAK2, JAK3, HSP90, MAPK, EGFR, FGFR, BRAF, RAF1, KDR, FLT1, TBK1, IKBKE, PRKAA1, PRKAA2, PRKAB1, BMPR1A, BMPR1B, FAK, and HIF1a.

The method of the above embodiment, wherein the cell is a hepatocyte.

The method of the above embodiment, wherein the target is JAK1, JAK2 and JAK3 and the compound selected from the group consisting of Momelotinib and Baricitinib.

The method of the above embodiment, wherein the compound is Momelotinib.

The method of the above embodiment, wherein the compound is Baricitinib.

The method of the above embodiments, wherein the target is HSP90 and the compound is selected from the group consisting of 17-AAG, BIIB021, HSP-990, and Retaspimycin HCl.

›DETAILED DESCRIPTION · 25 of 28

The method of the above embodiments, wherein the target is MAPK and the compound is selected from the group consisting of BIRB796, Pamapimod and PH-797804.

The method of the above embodiments, wherein the target is EGFR and the compound is Mubritinib (TAK 165).

The method of the above embodiments, wherein the target is FGFR and the compound is XL228.

The method of the above embodiments, wherein the target is BRAF or RAF1 and the compound is selected from the group consisting of Lifirafenib (BGB-283) and BMS-214662.

The method of the above embodiments, wherein the target is KDR or FLT1 and the compound is Foretinib/XL880 (GSK1363089).

The method of the above embodiments, wherein the target is TBK1 or IKBKE and the compound is BX795.

The method of the above embodiments, wherein the target is PRKAA1, PRKAA2, or PRKAB1 and the compound is Dorsomorphin.

The method of the above embodiments, wherein the target is FAK and the compound is PF-00562271.

The method of the above embodiments, wherein the target is HIF1a and the compound is BAY 87-2243.

A method for increasing OTC gene expression in a cell harboring an OTC mutation associated with a partial reduction of OTC function, comprising: contacting the cell with an siRNA compound that inhibits a target selected from the group consisting of JAK1, WSTR1, YAP1, CSF1R, LYN, SMAD3, NTRK1, EPHB3, EPHB4, FGFR4, INSR, KDR, FLT1, FGFR2, EPHB2, PDGFRB, IRF5, FGFR1, EPHB1, FYN, FLT4, YY1, IRF1, IGF-1, SMAD1, DDR1, HSP90AA1, and SMAD2.

A method for increasing OTC expression in a human subject harboring an OTC mutation associated with a partial reduction of OTC function, comprising: administering to the subject an effective amount of a compound that inhibits a target selected from the group consisting of JAK1, JAK2, JAK3, HSP90, MAPK, EGFR, FGFR, BRAF, RAF1, KDR, FLT1, TBK1, IKBKE, PRKAA1, PRKAA2, PRKAB1, BMPR1A, BMPR1B, FAK, and HIF1a.

The method of the above embodiments, wherein the target is JAK1, JAK2 or JAK3 and the compound selected from the group consisting of Momelotinib and Baricitinib.

The method of the above embodiments, wherein the compound is Momelotinib.

The method of the above embodiments, wherein the compound is Baricitinib.