Compositions and their uses directed to acetyl-CoA carboxylases

This application claims priority to U.S. Provisional Patent Application Ser. No. 60/669,530, filed Apr. 8, 2005, the entirety of which is herein incorporated by reference.

A paper copy of the sequence listing and a computer-readable form of the sequence listing, on diskette, containing the file named BIOL0058WOSEQ, which was created on Apr. 10, 2006, are herein incorporated by reference

Disclosed herein are compounds, compositions and methods useful for modulating the expression of ACC1 or ACC2 in a cell, tissue or animal.

Acetyl-CoA carboxylase (ACC) activity is responsible for the ATP-dependent carboxylation of acetyl-CoA to malonyl-CoA, the rate-limiting step in fatty acid synthesis. This reaction proceeds in two half-reactions: a biotin carboxylase reaction and a carboxyltransferase reaction. Malonyl-CoA is the carbon donor in the synthesis of long chain fatty acids and in their elongation into very long chain fatty acids, and is also a regulator of the palmitoyl-CoA-carnitine shuttle system that is involved in the mitochondrial oxidation of long chain fatty acids (Harwood, Curr. Opin. Investig. Drugs, 2004, 5, 283-289; McGarry et al., J. Biol. Chem., 1978, 253, 8294-8300).

Malonyl-CoA, the product of ACC activity, is the key metabolic signal for the control of fatty acid oxidation and synthesis in response to dietary changes. The carboxylases are highly regulated by diet, hormones, and other physiological factors. Food intake induces the synthesis of ACC and increases ACC activity. Starvation or diabetes mellitus represses the expression of the genes and decreases the activities of the enzymes. Treating diabetic animals with insulin increases the activity of the enzyme, and prolonged insulin treatment stimulates the synthesis of ACC protein.

ACC exists as two tissue-specific isozymes: ACC1 (also known as acetyl-CoA carboxylase alpha; ACAC; ACACA; and tgf) which is present in lipogenic tissues such as liver and adipose and ACC2 (also known as acetyl-Coenzyme A carboxylase beta, ACACB, ACCB, HACC275, and acetyl-CoA carboxylase 2) which is present in oxidative tissues such as liver, heart and skeletal muscle. ACC1 and ACC2 are encoded by separate genes (Harwood, Curr. Opin. Investig. Drugs, 2004, 5, 283-289).

Use of antisense oligonucleotides to decrease ACC1 and ACC2 for therapeutics is advantageous over small molecules in that antisense oligonucleotides will not decrease ACC levels in the central nervous system or pancreas, thus preventing side effects observed with small molecule inhibition of ACC1 and ACC2.

The present invention is directed to methods of using compounds, particularly antisense compounds, targeted to a nucleic acid molecule encoding ACC1 or ACC2 and which modulate the expression of ACC1, ACC2, or both ACC1 and ACC2 to achieve particular phenotypic endpoints. Further provided are methods of reducing ACC1 or ACC2 or both ACC1 and ACC2 concurrently in cells, tissues or animals comprising contacting said cells, tissues or animals with one or more of the compounds or compositions of the present invention.

The present invention provides methods of lowering blood glucose, triglycerides, or cholesterol, in a subject in need of such treatment, by administering to said subject an antisense compound which reduces ACC1 or ACC2. In a preferred embodiment, the antisense compound reduces both ACC1 and ACC2. In another embodiment, the present invention provides methods of lowering adiposity. In another embodiment, the present invention provides methods of lowering liver triglyceride levels. Another embodiment of the present invention is a method of inhibiting fatty acid synthesis. Other embodiments include methods of stimulating fatty acid oxidation, improving insulin sensitivity, and inhibiting hepatic glucose output in a subject in need of such treatment, by administering to said subject an antisense compound which reduces ACC1 or ACC2 or both ACC1 and ACC2.

In other embodiments, the present invention provides methods of ameliorating or lessening the severity of a condition in an animal comprising contacting said animal with an effective amount of an antisense compound which reduces ACC1, ACC2, or both ACC1 and ACC2 so that measurement of one or more physical indices of said condition indicates a lessening of the severity of said condition. In one embodiment, the condition is obesity. In another embodiment, the obesity is diet-induced. In other embodiments, conditions include, but are not limited to, diabetes, insulin resistance, insulin deficiency, hypercholesterolemia, hyperglycemia, hypertriglyceridemia, hyperfattyacidemia, metabolic syndrome, and cardiovascular disease. In another embodiment, the condition is liver steatosis. In another embodiment, the liver steatosis is steatohepatitis, non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH). Also contemplated herein is the use of a compound of the invention in the preparation of a medicament for amelioration or treatment of a condition associated with ACC1 or ACC2 or both.

In some embodiments, antisense compounds modulate ACC1, ACC2, or both ACC1 and ACC2. Contemplated and provided herein are antisense compounds comprising sequences of 13 to 30 nucleotides in length. Also provided herein are antisense compounds with at least two modifications selected from a modified internucleoside linkage, a modified nucleobase, or a modified sugar. Provided herein are chimeric oligonucleotides comprising a deoxy nucleotide region flanked on each of the 5′ and 3′ ends with at least one 2′-O-methoxylethyl nucleotide. Further provided are chimeric oligonucleotides comprising ten deoxynucleotides and flanked on both the 5′ and 3′ ends with five 2′-O-methoxyethyl nucleotides wherein each internucleoside linkage is a phosphorothioate. In a further embodiment, the antisense compounds of the present invention may have at least one 5-methylcytosine.

Overview

Disclosed herein are antisense compounds and methods of using said antisense compounds, including antisense oligonucleotides which reduce ACC1 or ACC2 or both ACC1 and ACC2. Such methods are accomplished by providing antisense compounds which are complementary to one or more target nucleic acid molecules encoding ACC1 or ACC2.

In accordance with the present invention are compositions and methods for modulating the expression of ACC1 (also known as acetyl-CoA carboxylase alpha, ACAC, ACACA, and tgf) or ACC2 (also known as acetyl-Coenzyme A carboxylase beta, ACACB, ACCB, HACC275, and acetyl-CoA carboxylase 2). Listed in Table 1 are GENBANK® accession numbers of sequences useful for design of antisense compounds targeted to ACC1 or ACC2 or both ACC1 and ACC2. Each GENBANK® sequence is herein incorporated by reference. Shown in Table 1 is the SEQ ID NO of such sequences if assigned. Antisense compounds of the invention include those which reduce one or more target nucleic acid molecules shown in Table 1, as well as antisense compounds which reduce other nucleic acid molecules encoding ACC1 or ACC2. The antisense compounds may target any region, segment, or site of nucleic acid molecules which encode ACC1 or ACC2. Suitable target regions are described herein.

Embodiments of the present invention include antisense compounds comprising sequences of 12 to 50 nucleotides in length. It is understood that sequences of 12 to 50 nucleotides in length encompass sequences of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50 nucleotides in length. Preferred are antisense oligonucleotides 13 to 30 nucleotides in length. Also preferred are antisense oligonucleotides 12 to 30 nucleotides in length. One of ordinary skill will readily appreciate that the nucleotide length range 12 to 50 includes all nucleotide lengths within that range as well any range falling within the bounds of 12 to 50 nucleotides.

It is well known by those skilled in the art that it is possible to increase or decrease the length of an antisense compound and/or introduce mismatch bases without eliminating activity. For example, in Woolf et al. (Proc. Natl. Acad. Sci. USA 89:7305-7309, 1992, incorporated herein by reference), a series of ASOs 13-25 nucleobases in length were tested for their ability to induce cleavage of a target RNA in an oocyte injection model. ASOs 25 nucleobases in length with 8 or 11 mismatch bases near the ends of the ASOs were able to direct specific cleavage of the target mRNA, albeit to a lesser extent than the ASOs that contained no mismatches. Similarly, target specific cleavage was achieved using a 13 nucleobase ASOs, including those with 1 or 3 mismatches. Maher and Dolnick (Nuc. Acid. Res. 16:3341-3358, 1988, incorporated herein by reference) tested a series of tandem 14 nucleobase ASOs, and a 28 and 42 nucleobase ASOs comprised of the sequence of two or three of the tandem ASOs, respectively, for their ability to arrest translation of human DHFR in a rabbit reticulocyte assay. Each of the three 14 nucleobase ASOs alone were able to inhibit translation, albeit at a more modest level than the 28 or 42 nucleobase ASOs.

Therapeutics

Compounds of the invention can be used to modulate ACC1 or ACC2 in an animal, such as a human. In one non-limiting embodiment, the methods comprise the step of administering to said animal an effective amount of an antisense compound that decreases expression of ACC1 or ACC2 or, preferably, both. In one embodiment, the antisense compounds of the present invention effectively decrease the levels or function of ACC1 or ACC2 RNA. Because reduction in ACC1 or ACC2 mRNA levels can lead to reduction in ACC1 or ACC2 protein products of expression as well, such resultant alterations can also be measured. Antisense compounds of the present invention that effectively decrease levels or function of an ACC1 or ACC2 RNA or protein products of expression are considered active antisense compounds. In one embodiment, the antisense compounds of the invention target ACC1 and/or ACC2 causing a reduction of target RNA by at least 10%, by at least 20%, by at least 25%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 70%, by at least 75%, by at least 80%, by at least 85%, by at least 90%, by at least 95%, by at least 98%, by at least 99%, or by 100%. It is understood that the compounds of the invention may reduce the expression level of ACC1 RNA, ACC2 RNA, or both to such a degree.

For example, the reduction of ACC1 or ACC2 expression can be measured in a tissue or organ of the animal. Tissues or organs include, but are not limited to, skeletal muscle, liver, kidney, and adipose (white and brown). Samples of tissues or organs can be routinely obtained by biopsy.

The cells contained within said fluids, tissues or organs being analyzed can contain a nucleic acid molecule encoding ACC1 or ACC2 protein and/or the ACC1- or ACC2-encoded protein itself. For example, tissues or organs procured from an animal can be evaluated for levels of the target mRNA or protein. mRNA levels can be measured or evaluated by real-time PCR, Northern blot, in situ hybridization or DNA array analysis. Protein levels can be measured or evaluated by ELISA, immunoblotting, quantitative protein assays, protein activity assays (for example, caspase activity assays) immunohistochemistry or immunocytochemistry. Furthermore, the effects of treatment can be assessed by measuring biomarkers associated with the target gene expression in the aforementioned tissues or organs or bodily fluids such as blood, collected from an animal contacted with one or more compounds of the invention, by routine clinical methods known in the art. These biomarkers include but are not limited to: glucose, cholesterol, lipoproteins, triglycerides, free fatty acids, and other markers of glucose and lipid metabolism; liver transaminases, bilirubin, albumin, blood urea nitrogen, creatine, and other markers of kidney and liver function.

The compounds of the present invention can be utilized in pharmaceutical compositions by adding an effective amount of a compound to a suitable pharmaceutically acceptable diluent or carrier. In one aspect, the compounds of the present invention decrease expression of ACC1 or ACC2. In preferred aspect, the compounds of the invention decrease expression of both ACC1 and ACC2. The compounds of the invention can also be used in the manufacture of a medicament for the treatment of diseases and disorders treatable by reducing ACC1 or ACC2 expression.

Another embodiment of the present invention is a method of decreasing expression of ACC1 or ACC2 or both wherein expression of ACC1 and ACC2 are not decreased in the central nervous system. Another embodiment of the present invention is a method of decreasing expression of ACC1 or ACC2 or both wherein expression of ACC1 and ACC2 are not decreased in the pancreas. Another embodiment of the present invention is a method of decreasing expression of ACC1 or ACC2 or both wherein expression of ACC1 and ACC2 are not decreased in the islet cells of the pancreas. Other embodiments of the invention include ameliorating or lessening the severity of a condition in an animal by administering an antisense compound which decreases expression of ACC1 or ACC2 or both wherein food intake is not increased. Other embodiments of the invention include ameliorating or lessening the severity of a condition in an animal by administering an antisense compound which decreases expression of ACC1 or ACC2 or both wherein appetite is not increased. In a preferred embodiment, expression of both ACC1 and ACC2 are decreased. Contemplated herein is use of an antisense oligonucleotide of the invention in the preparation of a medicament for ameliorating or lessening the severity of a condition in an animal wherein said condition is treatable by reducing expression of ACC1, ACC2, or both.

Other embodiments of the present invention are methods of lowering plasma lipid levels in an animal by administering an antisense compound which decreases expression of ACC1 or ACC2 or both. Plasma lipids include, but are not limited to, fatty acids, triglycerides, cholesterol, LDL, and VLDL. Contemplated herein is use of an antisense oligonucleotide of the invention in the preparation of a medicament to lower plasma lipid levels in an animal.

Another embodiment of the present invention is a method of lowering liver triglyceride levels in an animal by administering an antisense compound which decreases expression of ACC1 or ACC2 or both ACC1 and ACC2. Contemplated herein is use of an antisense oligonucleotide of the invention in the preparation of a medicament to lower liver triglyceride levels in an animal. NAFLD encompasses a disease spectrum ranging from simple triglyceride accumulation in hepatocytes (hepatic steatosis) to hepatic steatosis with inflammation (steatohepatitis), fibrosis, and cirrhosis. NAFLD can progress to NASH. Therefore, other embodiments of the present invention include ameliorating hepatic steatosis in an animal by administering an antisense compound which decreases expression of ACC1 or ACC2 or, preferably, both. The hepatic steatosis may be NAFLD, steatohepatitis, or NASH. Contemplated herein is use of an antisense oligonucleotide of the invention in the preparation of a medicament to treat hepatic steatosis in an animal. In some embodiments, the steatosis is NAFLD, steatohepatitis, or NASH.

Another embodiment of the present invention is a method of reducing adiposity in an animal in need thereof by administering an antisense compound which decreases expression of ACC1 or ACC2 or both. Indicators of reduction of adiposity of an animal include, but are not limited to, decreases in body fat. Another embodiment of the present invention is a method of ameliorating or lessening the severity of obesity in an animal by administering an antisense compound which decreases expression of ACC1 or ACC2 or both. Another embodiment of the present invention is a method of ameliorating or lessening the severity of diet-induced obesity in an animal by administering an antisense compound which decreases expression of ACC1 or ACC2 or both. Contemplated herein is use of an antisense oligonucleotide of the invention in the preparation of a medicament to treat obesity in an animal. In one embodiment, the medicament is used to reduce body fat in an animal. In other embodiment, the obesity is diet-induced.

Other embodiments of the present invention include, but are not limited to, methods of lowering plasma glucose and methods of lowering plasma triglyceride levels in an animal by administering an antisense compound which decreases expression of ACC1 or ACC2 or both. Provided herein is use of an antisense oligonucleotide of the invention in the preparation of a medicament to lower blood glucose or plasma triglycerides in an animal. Other embodiments include, but are not limited to, methods of improving insulin or leptin sensitivity in an animal by administering an antisense compound which decreases ACC1 or ACC2 expression selectively, or ACC1 and ACC2 expression concurrently. Provided herein is use of an antisense oligonucleotide of the invention in the preparation of a medicament to improve insulin or leptin sensitivity in an animal. In one embodiment, improved insulin sensitivity results in consequent reduction in insulin levels. In one embodiment improved insulin sensitivity is measured by a reduction in insulin. Other embodiments include, but are not limited to, methods of increasing fatty acid oxidation, methods of decreasing fatty acid synthesis, and methods of inhibiting hepatic glucose output in an animal by administering an antisense compound which decreases expression of ACC1 or ACC2 or both. Provided herein is use of an antisense oligonucleotide of the invention in the preparation of a medicament which increases fatty acid oxidation, decreases fatty acid synthesis or inhibits hepatic glucose output. Another embodiment includes methods of reducing hepatic malonyl-CoA levels in an animal by administering an antisense compound which decreases expression of ACC1 or ACC2 or both. Another embodiment includes methods of reducing malonyl-CoA levels in lipogenic or oxidative tissues in an animal by administering an antisense compound which decreases expression of ACC1 or ACC2 or both. Another embodiment includes improvement of hepatic insulin sensitivity in an animal by administering an antisense compound which decreases expression of ACC1 or ACC2 or both. Provided herein is use of an antisense oligonucleotide of the invention in the preparation of a medicament for improving insulin sensitivity in an animal. Another embodiment is a method of improving plasma ketone levels in an animal by administering an antisense compound which decreases expression of ACC1 or ACC2 or both. Plasma ketones include 3-betahydroxybutarate, acetone, and acetoacetate. Another embodiment is a method of increasing hepatic fat oxidation in an animal by administering an antisense compound which decreases expression of ACC1 or ACC2 or both.

Other embodiments of the invention include ameliorating or lessening the severity of a condition in an animal by administering an antisense compound which decreases expression of ACC1 or ACC2 or both. Conditions include, but are not limited to, metabolic and cardiovascular disorders. Metabolic disorders include, but are not limited to, obesity, diet-induced obesity, diabetes, insulin resistance, insulin deficiency, dyslipidemia, hypercholesterolemia, hyperglycemia, hypertriglyceridemia, hyperfattyacidemia, liver steatosis and metabolic syndrome. Cardiovascular disorders include, but are not limited to, coronary heart disease. Provided herein is use of an antisense oligonucleotide of the invention in the preparation of a medicament for preventing or treating a metabolic or cardiovascular disorder. Contemplated herein is use of an antisense oligonucleotide of the invention for amelioration or treatment of a condition selected from obesity, diabetes, insulin resistance, insulin deficiency, hypercholesterolemia, hyperglycemia, hypertriglyceridemia, hyperfattyacidemia, liver steatosis, metabolic syndrome, or cardiovascular disease.

Contemplated herein is use of an antisense oligonucleotide of the invention for amelioration or treatment of a condition selected from obesity, diabetes, insulin resistance, insulin deficiency, hypercholesterolemia, hyperglycemia, hypertriglyceridemia, hyperfattyacidemia, liver steatosis, metabolic syndrome, or cardiovascular disease wherein said condition is treatable by reducing ACC1 and ACC2 expression.

Targets

As used herein, the terms “target nucleic acid” and “nucleic acid molecule encoding ACC1 or ACC2” have been used for convenience to encompass RNA (including pre-mRNA and mRNA or portions thereof) transcribed from DNA encoding ACC1 or ACC2, and also cDNA derived from such RNA.

The locations on the target nucleic acid to which active antisense compounds are complementary are hereinbelow referred to as “validated target segments.” As used herein the term “validated target segment” is defined as at least a portion of a target region to which an active antisense compound is targeted. While not wishing to be bound by theory, it is presently believed that these target segments represent portions of the target nucleic acid which are accessible for hybridization.

Regions, Segments, and Sites

The targeting process usually also includes determination of at least one target region, segment, or site within the target nucleic acid for the antisense interaction to occur such that the desired effect, e.g., reduction of expression of ACC1 or ACC2 or both, will result. “Region” is defined as a portion of the target nucleic acid having at least one identifiable structure, function, or characteristic. Within regions of target nucleic acids are segments. “Segments” are defined as smaller or sub-portions of regions within a target nucleic acid. “Sites,” as used in the present invention, are defined as unique nucleobase positions within a target nucleic acid.

Once one or more target regions, segments or sites have been identified, antisense compounds are designed which are sufficiently complementary to the target, i.e., hybridize with sufficient affinity and specificity to give the desired effect.

Target segments can include DNA or RNA sequences that comprise at least a portion of consecutive nucleobases from the 5′-terminus of a validated target segment (the remaining nucleobases being a consecutive stretch of the same DNA or RNA beginning immediately upstream of the 5′-terminus of the target segment and continuing until the DNA or RNA contains the desired number of nucleobases). Similarly validated target segments are represented by DNA or RNA sequences that comprise at least a portion of consecutive nucleobases from the 3′-terminus of a validated target segment (the remaining nucleobases being a consecutive stretch of the same DNA or RNA beginning immediately downstream of the 3′-terminus of the target segment and continuing until the DNA or RNA contains the desired number of nucleobases). It is also understood that a validated oligomeric target segment can be represented by DNA or RNA sequences that comprise at least a portion of consecutive nucleobases from an internal portion of the sequence of a validated target segment, and can extend in either or both directions until the oligonucleotide contains the desired number of nucleobases. Alternatively, target segments can include at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 consecutive nucleobases of a validated target segment, for example, as counted from the 5′ or 3′ terminus or from an internal site of a validated target segment. Therefore, oligonucleotides encompassed by the invention may comprise at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 consecutive nucleobases of an oligonucleotide exemplified herein. Preferred are oligonucleotides which comprise at least a 13 nucleobase portion of an exemplified antisense oligonucleotide. Also preferred are oligonucleotides which comprise at least an 8 nucleobase portion of an exemplified antisense oligonucleotide.

The validated target segments identified herein can be employed in a screen for additional compounds that modulate ACC1 or ACC2. The screening method comprises the steps of contacting a validated target segment of a nucleic acid molecule encoding ACC1 or ACC2 with one or more candidate modulators, and selecting for one or more candidate modulators which increase or decrease levels of a nucleic acid molecule encoding ACC1 or ACC2. Once it is shown that the candidate modulator or modulators are capable of such alteration of levels of a nucleic acid molecule encoding ACC1 or ACC2, the modulator can then be employed in further investigative studies of the function of ACC1 or ACC2, or for use as a research, diagnostic, or therapeutic agent.

The target regions to which the exemplified compounds are inhibitory are described herein by indicating the 5′-most position on the target nucleic acid. Coupled with the length of the exemplified compound, the target region for the exemplified compound is described. For example, the antisense compound having SEQ ID NO: 210 targets nucleotides 1932 to 1951 of the ACC2 sequence NM — 133904.1. As shown in the examples herein, the oligonucleotide reduces ACC2, thus nucleotides 1932 to 1951 is a validated target segment of NM 133904.1. Because compounds of the invention may be, for example, 13 to 30 nucleobases in length, and may comprise, for example, a 13 nucleobase portion of an exemplified sequence, it can be appreciated that a compound within the scope of the invention may target any portion of nucleotides 1915 to 1968 of NM — 133904.1. As a further example, a 30-mer targeting nucleotides 1915 to 1944 of NM — 133904.1 and comprising a 13-nucleobase portion of SEQ ID NO: 210 would still fall within the scope of the invention described herein.

The start codon and stop codon in mRNA molecules and their corresponding DNA molecules are readily identifiable to those of skill in the art. “Start codon region” or “stop codon region” as used herein refers to the portion of an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from a start or stop codon. Consequently, the “start codon region” and the “stop codon region” are all regions which may be targeted effectively with antisense compounds of the invention.

The open reading frame (ORF) or “coding region,” which is known in the art to refer to the region between the translation initiation codon and the translation termination codon, is also a region which may be targeted effectively. Within the context of the present invention, one region is the intragenic region encompassing the translation initiation or termination codon of the open reading frame (ORF) of a gene.

Other target regions include the “5′ untranslated region” (5′UTR) and the “3′ untranslated region” (3′UTR). The 5′ cap region of an mRNA is considered to include the 5′ cap structure itself as well as the first 50 nucleotides adjacent to the cap site. The 5′ cap region is also a target.

Suitable target regions thus include, but are not limited to, the 5′UTR, the start codon, the stop codon, the coding region, the 3′UTR, the 5′cap region, introns, exons, intron-exon junctions, exon-intron junctions, and exon-exon junctions. Suitable target regions encompass from about 25 to about 50 nucleotides in either direction (i.e. 5′ or 3′) of a junction site.

Variants

It is also known in the art that alternative RNA transcripts can be produced from the same genomic region of DNA. These alternative transcripts are generally known as “variants.” More specifically, “pre-mRNA variants” are transcripts produced from the same genomic DNA that differ from other transcripts produced from the same genomic DNA in either their start or stop position and contain both intronic and exonic sequence.

Upon excision of one or more exon or intron regions, or portions thereof during splicing, pre-mRNA variants produce smaller “mRNA variants.” Consequently, mRNA variants are processed pre-mRNA variants and each unique pre-mRNA variant must always produce a unique mRNA variant as a result of splicing. These mRNA variants are also known as “alternative splice variants.” If no splicing of the pre-mRNA variant occurs then the pre-mRNA variant is identical to the mRNA variant.

It is also known in the art that variants can be produced through the use of alternative signals to start or stop transcription and that pre-mRNAs and mRNAs can possess more that one start codon or stop codon. Variants that originate from a pre-mRNA or mRNA that use alternative start codons are known as “alternative start variants” of that pre-mRNA or mRNA. Those transcripts that use an alternative stop codon are known as “alternative stop variants” of that pre-mRNA or mRNA. One specific type of alternative stop variant is the “polyA variant” in which the multiple transcripts produced result from the alternative selection of one of the “polyA stop signals” by the transcription machinery, thereby producing transcripts that terminate at unique polyA sites. Consequently, the types of variants described herein are also suitable target nucleic acids.

Modulation of Target Expression

“Modulation” means a perturbation of function, for example, either an increase (stimulation or induction) or a decrease (inhibition or reduction) in expression or in level of target RNA. As another example, modulation of expression can include perturbing splice site selection of pre-mRNA processing. “Expression” includes all the functions by which a gene's coded information is converted into structures present and operating in a cell. These structures include the products of transcription and translation. “Modulation of expression” means the perturbation of such functions. “Modulators” are those compounds that modulate the expression of ACC1 or ACC2 and which comprise at least a portion which is complementary to a validated target segment.

Modulation of expression of a target nucleic acid can be achieved through alteration of any number of nucleic acid functions. The functions of RNA to be modulated can include translocation functions, which include, but are not limited to, translocation of the RNA to a site of protein translation, translocation of the RNA to sites within the cell which are distant from the site of RNA synthesis, and translation of protein from the RNA. RNA processing functions that can be modulated include, but are not limited to, splicing of the RNA to yield one or more RNA species, capping of the RNA, 3′ maturation of the RNA and catalytic activity or complex formation involving the RNA which may be engaged in or facilitated by the RNA. Modulation of expression can result in the increased level of one or more nucleic acid species or the decreased level of one or more nucleic acid species, either temporally or by net steady state level. One result of such interference with target nucleic acid function is modulation of the expression of ACC1 or ACC2 or both ACC1 and ACC2. Thus, in one embodiment modulation of expression can mean increase or decrease in target RNA or protein levels. In another embodiment modulation of expression can mean an increase or decrease of one or more RNA splice products, or a change in the ratio of two or more splice products.

Hybridization and Complementarity

“Hybridization” means the pairing of complementary strands of antisense compounds. While not limited to a particular mechanism, the most common mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases (nucleobases) of the strands of antisense compounds. For example, adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds. Hybridization can occur under varying circumstances. An antisense compound is “specifically hybridizable” when there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target nucleic acid sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and under conditions in which assays are performed in the case of in vitro assays.

“Complementarity,” as used herein, refers to the capacity for precise pairing between two nucleobases on one or two antisense compound strands. For example, if a nucleobase at a certain position of an antisense compound is capable of hydrogen bonding with a nucleobase at a certain position of a target nucleic acid, then the position of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be a complementary position. The antisense compound and the further DNA or RNA are complementary to each other when a sufficient number of complementary positions in each molecule are occupied by nucleobases which can hydrogen bond with each other. Thus, “specifically hybridizable” and “complementary” are terms which are used to indicate a sufficient degree of precise pairing or complementarity over a sufficient number of nucleobases such that stable and specific binding occurs between the antisense compound and a target nucleic acid to allow the compound to function.

It is understood in the art that the sequence of an antisense compound need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable or to be encompassed by the present invention. One of ordinary skill in the art will appreciate that complementarity less than 100% to a target nucleic acid or to a validated target segment within such a nucleic acid renders the compound with mismatches, thus, compounds having mismatches fall within the scope of the invention. Preferred oligonucleotides have fewer than about 3 mismatches.

Moreover, an oligonucleotide may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure, mismatch or hairpin structure). The antisense compounds of the present invention comprise at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 92%, or at least 95%, or at least 97%, or at least 98%, or at least 99% sequence complementarity to a target region within the target nucleic acid sequence to which they are targeted. For example, an antisense compound in which 18 of 20 nucleobases of the antisense compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. In this example, the remaining noncomplementary nucleobases may be clustered or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases. As such, an antisense compound which is 18 nucleobases in length having 4 (four) noncomplementary nucleobases which are flanked by two regions of complete complementarity with the target nucleic acid would have 77.8% overall complementarity with the target nucleic acid and would thus fall within the scope of the present invention. Percent complementarity of an antisense compound with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656). Percent homology, sequence identity or complementarity, can be determined by, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489).

An antisense compound with complementarity to both ACC1 and ACC2 is considered to fall within the scope of the present invention. For example, an oligonucleotide which is 100% complementarity to ACC1 but has 85% complementarity or 3 mismatches to ACC2 (thus having 17 of 20 nucleotides which are complementary to the target site) is contemplated herein. Likewise, an oligonucleotide having 85% complementarity to both ACC1 and ACC2 is contemplated. Also contemplated are oligonucleotides having 100% complementarity to both ACC1 and ACC2. Preferred embodiments of the present invention include oligonucleotides having sufficient complementarity to ACC1 and ACC2 to cause a reduction in expression in both ACC1 and ACC2.

One of ordinary skill in the art will appreciate that a routine alignment of ACC1 and ACC2 sequences (such as an alignment performed by BLAST or other readily available programs) will show regions suitable for design of oligonucleotides targeting ACC1 and ACC2 independently or simultaneously.

Antisense Mechanisms and Compounds

“Antisense mechanisms” are all those involving hybridization of a compound with target nucleic acid, wherein the outcome or effect of the hybridization is either target degradation or target occupancy with concomitant stalling of the cellular machinery involving, for example, transcription or splicing. Such mechanisms are appreciated in the art and include, for example, RNase-H and RNAi-based mechanisms.

The term “antisense compound” refers to a polymeric structure capable of hybridizing to a region of a nucleic acid molecule. This term includes oligonucleotides, oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics and chimeric combinations of these. Antisense compounds are routinely prepared linearly but can be joined or otherwise prepared to be circular. Moreover, branched structures are known in the art. An “antisense compound” or “oligomeric antisense compound” refers to an antisense compound that is at least partially complementary to the region of a nucleic acid molecule to which it hybridizes and which modulates (increases or decreases) its expression. Consequently, while all antisense compounds can be said to be oligomeric compounds, not all oligomeric compounds are antisense compounds. An “antisense oligonucleotide” is an antisense compound that is a nucleic acid-based oligomer. An antisense oligonucleotide can be chemically modified. Nonlimiting examples of antisense compounds include primers, probes, antisense compounds, antisense oligonucleotides, external guide sequence (EGS) oligonucleotides, alternate splicers, and siRNAs. As such, these compounds can be introduced in the form of single-stranded, double-stranded, circular, branched or hairpins and can contain structural elements such as internal or terminal bulges or loops. Double-stranded compounds can be comprised of two strands hybridized to form double-stranded compounds or a single strand with sufficient self complementarity to allow for hybridization and formation of a fully or partially double-stranded compound. In a preferred embodiment, the compounds of the instant invention are non-autocatalytic.

“Chimeric” antisense compounds or “chimeras,” in the context of this invention, are single-or double-stranded antisense compounds, such as oligonucleotides, which contain two or more chemically distinct regions, each comprising at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide compound.

A “gapmer” is defined as an antisense compound, generally an oligonucleotide, having a 2′-deoxyoligonucleotide region flanked by non-deoxyoligonucleotide segments. The central region is referred to as the “gap.” The flanking segments are referred to as “wings.” If one of the wings has zero non-deoxyoligonucleotide monomers, a “hemimer” is described.

In one embodiment of the invention, double-stranded antisense compounds encompass short interfering RNAs (siRNAs). As used herein, the term “siRNA” is defined as a double-stranded compound having a first and second strand, each strand having a central portion and two independent terminal portions. The central portion of the first strand is complementary to the central portion of the second strand, allowing hybridization of the strands. The terminal portions are independently, optionally complementary to the corresponding terminal portion of the complementary strand. The ends of the strands may be modified by the addition of one or more natural or modified nucleobases to form an overhang. In one nonlimiting example, the first strand of the siRNA is antisense to the target nucleic acid, while the second strand is complementary to the first strand. Once the antisense strand is designed to target a particular nucleic acid target, the sense strand of the siRNA can then be designed and synthesized as the complement of the antisense strand and either strand may contain modifications or additions to either terminus. For example, in one embodiment, both strands of the siRNA duplex would be complementary over the central nucleobases, each having overhangs at one or both termini. It is possible for one end of a duplex to be blunt and the other to have overhanging nucleobases. In one embodiment, the number of overhanging nucleobases is from 1 to 6 on the 3′ end of each strand of the duplex. In another embodiment, the number of overhanging nucleobases is from 1 to 6 on the 3′ end of only one strand of the duplex. In a further embodiment, the number of overhanging nucleobases is from 1 to 6 on one or both 5′ ends of the duplexed strands. In another embodiment, the number of overhanging nucleobases is zero. In a preferred embodiment, each of the strands is 19 nucleobases in length, fully hybridizable with the complementary strand, and includes no overhangs.

Each strand of the siRNA duplex may be from about 12 to about 35 nucleobases. In a preferred embodiment, each strand of the siRNA duplex is about 17 to about 25 nucleobases. The central complementary portion may be from about 12 to about 35 nucleobases in length. In a preferred embodiment, the central complimentary portion is about 17 to about 25 nucleobases in length. It is understood that each the strand of the siRNA duplex and the central complementary portion may be about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleobases in length. The terminal portions can be from 1 to 6 nucleobases. It is understood that the terminal portions can be about 1, 2, 3, 4, 5, or 6 nucleobases in length. The siRNAs may also have no terminal portions. The two strands of an siRNA can be linked internally leaving free 3′ or 5′ termini, or can be linked to form a continuous hairpin structure or loop. The hairpin structure may contain an overhang on either the 5′ or 3′ terminus producing an extension of single-stranded character.

Double-stranded compounds can be made to include chemical modifications as discussed herein.

Chemical Modifications

Embodiments of the present invention include compounds comprising at least two modifications selected from a modified internucleoside linkage, a modified nucleobase, or a modified sugar. In one embodiment, the antisense compounds of the present invention are chimeric oligonucleotides. In one embodiment, the antisense compounds of the present invention are chimeric oligonucleotides comprising a deoxy nucleotide region flanked on each of the 5′ and 3′ ends with at least one 2′-O-(2-methoxyethyl) nucleotide. In another embodiment, the antisense compounds of the present invention are chimeric oligonucleotides comprising ten deoxynucleotides and flanked on both the 5′ and 3′ ends with five 2′-O-(2-methoxyethyl) nucleotides. In a further embodiment, the antisense compounds of the present invention may have at least one 5-methylcytosine.

As is known in the art, a nucleoside is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base (sometimes referred to as a “nucleobase” or simply a “base”). The two most common classes of such heterocyclic bases are the purines and the pyrimidines. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to the 2′,3′ or 5′ hydroxyl moiety of the sugar. In forming oligonucleotides, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. In turn, the respective ends of this linear polymeric compound can be further joined to form a circular compound. Within oligonucleotides, the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiester linkage. It is often preferable to include chemical modifications in oligonucleotides to alter their activity. Chemical modifications can alter oligonucleotide activity by, for example: increasing affinity of an antisense oligonucleotide for its target RNA, increasing nuclease resistance, and/or altering the pharmacokinetics of the oligonucleotide. The use of chemistries that increase the affinity of an oligonucleotide for its target can allow for the use of shorter oligonucleotide compounds.

The term “nucleobase” or “heterocyclic base moiety” as used herein, refers to the heterocyclic base portion of a nucleoside. In general, a nucleobase is any group that contains one or more atom or groups of atoms capable of hydrogen bonding to a base of another nucleic acid. In addition to “unmodified” or “natural” nucleobases such as the purine nucleobases adenine (A) and guanine (G), and the pyrimidine nucleobases thymine (T), cytosine (C) and uracil (U), many modified nucleobases or nucleobase mimetics known to the art skilled are amenable to the present invention. The terms modified nucleobase and nucleobase mimetic can overlap but generally a modified nucleobase refers to a nucleobase that is fairly similar in structure to the parent nucleobase such as for example a 7-deaza purine or a 5-methylcytosine whereas a nucleobase mimetic would include more complicated structures such as for example a tricyclic phenoxazine nucleobase mimetic. Methods for preparation of the above noted modified nucleobases are well known to those skilled in the art.

Antisense compounds of the present invention may also contain one or more nucleosides having modified sugar moieties. The furanosyl sugar ring of a nucleoside can be modified in a number of ways including, but not limited to, addition of a substituent group, bridging of two non-germinal ring atoms to form a bicyclic nucleic acid (BNA) and substitution of an atom or group such as —S—, —N(R)— or —C(R1)(R2) for the ring oxygen at the 4′-position. Modified sugar moieties are well known and can be used to alter, typically increase, the affinity of the antisense compound for its target and/or increase nuclease resistance. A representative list of preferred modified sugars includes but is not limited to bicyclic modified sugars (BNA's), including LNA and ENA (4′-(CH2)2-O-2′ bridge); and substituted sugars, especially 2′-substituted sugars having a 2′-F, 2′-OCH2 or a 2′-O(CH2)2-OCH3 substituent group. Sugars can also be replaced with sugar mimetic groups among others. Methods for the preparations of modified sugars are well known to those skilled in the art.

The present invention includes internucleoside linking groups that link the nucleosides or otherwise modified monomer units together thereby forming an antisense compound. The two main classes of internucleoside linking groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus containing internucleoside linkages include, but are not limited to, phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidate, and phosphorothioates. Representative non-phosphorus containing internucleoside linking groups include, but are not limited to, methylenemethylimino (—CH2-N(CH3)-O—CH2-), thiodiester (—O—C(O)—S—), thionocarbamate (—O—C(O)(NH)—S—); siloxane (—O—Si(H)2-O—); and N,N′-dimethylhydrazine (—CH2-N(CH3)-N(CH3)-). Antisense compounds having non-phosphorus internucleoside linking groups are referred to as oligonucleosides. Modified internucleoside linkages, compared to natural phosphodiester linkages, can be used to alter, typically increase, nuclease resistance of the antisense compound. Internucleoside linkages having a chiral atom can be prepared racemic, chiral, or as a mixture. Representative chiral internucleoside linkages include, but are not limited to, alkylphosphonates and phosphorothioates. Methods of preparation of phosphorous-containing and non-phosphorous-containing linkages are well known to those skilled in the art.

As used herein the term “mimetic” refers to groups that are substituted for a sugar, a nucleobase, and/or internucleoside linkage. Mimetics are groups that are structurally quite different (not simply a modification) but functionally similar to the linked nucleosides of oligonucleotides. Generally, a mimetic is used in place of the sugar or sugar-internucleoside linkage combination, and the nucleobase is maintained for hybridization to a selected target. Representative examples of a sugar mimetic include, but are not limited to, cyclohexenyl or morpholino. Representative examples of a mimetic for a sugar-internucleoside linkage combination include, but are not limited to, peptide nucleic acids (PNA) and morpholino groups linked by uncharged achiral linkages. In some instances a mimetic is used in place of the nucleobase. Representative nucleobase mimetics are well known in the art and include, but are not limited to, tricyclic phenoxazine analogs and universal bases (Berger et al., Nuc Acid Res. 2000, 28:2911-14, incorporated herein by reference). Methods of synthesis of sugar, nucleoside and nucleobase mimetics are well known to those skilled in the art.

As used herein the term “nucleoside” includes, nucleosides, abasic nucleosides, modified nucleosides, and nucleosides having mimetic bases and/or sugar groups.

In the context of this invention, the term “oligonucleotide” refers to an antisense compound which is an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). This term includes oligonucleotides composed of naturally- and non-naturally-occurring nucleobases, sugars and covalent internucleoside linkages, possibly further including non-nucleic acid conjugates.

The present invention provides compounds having reactive phosphorus groups useful for forming internucleoside linkages including for example phosphodiester and phosphorothioate internucleoside linkages. Methods of preparation and/or purification of precursors or olgomeric compounds of the instant invention are not a limitation of the compositions or methods of the invention. Methods for synthesis and purification of DNA, RNA, and the antisense compounds of the instant invention are well known to those skilled in the art.

As used herein the term “chimeric antisense compound” refers to an antisense compound having at least one sugar, nucleobase and/or internucleoside linkage that is differentially modified as compared to the other sugars, nucleobases and internucleoside linkages within the same antisense compound. The remainder of the sugars, nucleobases and internucleoside linkages can be independently modified or unmodified provided that they are distinguishable from the differentially modified moiety or moieties. In general a chimeric antisense compound will have modified nucleosides that can be in isolated positions or grouped together in regions that will define a particular motif. Any combination of modifications and or mimetic groups can comprise a chimeric antisense compound of the present invention.

Chimeric antisense compounds typically contain at least one region modified so as to confer increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the antisense compound may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of inhibition of gene expression. Consequently, comparable results can often be obtained with shorter antisense compounds when chimeras are used, compared to for example phosphorothioate deoxyoligonucleotides hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.

Certain chimeric as well as non-chimeric antisense compounds can be further described as having a particular motif. As used in the present invention the term “motif” refers to the orientation of modified sugar moieties and/or sugar mimetic groups in an antisense compound relative to like or differentially modified or unmodified nucleosides. As used in the present invention, the terms “sugars”, “sugar moieties” and “sugar mimetic groups” are used interchangeably. Such motifs include, but are not limited to, gapped motifs, alternating motifs, fully modified motifs, hemimer motifs, blockmer motifs, and positionally modified motifs. The sequence and the structure of the nucleobases and type of internucleoside linkage is not a factor in determining the motif of an antisense compound.

In one aspect of the present invention antisense compounds are modified by covalent attachment of one or more conjugate groups. Conjugate groups may be attached by reversible or irreversible attachments. Conjugate groups may be attached directly to antisense compounds or by use of a linker. Linkers may be mono- or bifunctional linkers. Such attachment methods and linkers are well known to those skilled in the art. In general, conjugate groups are attached to antisense compounds to modify one or more properties. Such considerations are well known to those skilled in the art.

NAFLD and Metabolic Syndrome

The term “nonalcoholic fatty liver disease” (NAFLD) encompasses a disease spectrum ranging from simple triglyceride accumulation in hepatocytes (hepatic steatosis) to hepatic steatosis with inflammation (steatohepatitis), fibrosis, and cirrhosis. Nonalcoholic steatohepatitis (NASH) occurs from progression of NAFLD beyond deposition of triglycerides. A second-hit capable of inducing necrosis, inflammation, and fibrosis is required for development of NASH. Candidates for the second-hit can be grouped into broad categories: factors causing an increase in oxidative stress and factors promoting expression of proinflammatory cytokines. It has been suggested that increased liver triglycerides lead to increased oxidative stress in hepatocytes of animals and humans, indicating a potential cause-and-effect relationship between hepatic triglyceride accumulation, oxidative stress, and the progression of hepatic steatosis to NASH (Browning and Horton, J. Clin. Invest., 2004, 114, 147-152). Hypertriglyceridemia and hyperfattyacidemia can cause triglyceride accumulation in peripheral tissues (Shimamura et al., Biochem. Biophys. Res. Commun., 2004, 322, 1080-1085).

“Metabolic syndrome” is defined as a clustering of lipid and non-lipid cardiovascular risk factors of metabolic origin. It is closely linked to the generalized metabolic disorder known as insulin resistance. The National Cholesterol Education Program (NCEP) Adult Treatment Panel III (ATPIII) established citeria for diagnosis of metabolic syndrome when three or more of five risk determinants are present. The five risk determinants are abdominal obesity defined as waist circumference of greater than 102 cm for men or greater than 88 cm for women, triglyceride levels greater than or equal to 150 mg/dL, HDL cholesterol levels of less than 40 mg/dL for men and less than 50 mg/dL for women, blood pressure greater than or equal to 130/85 mm Hg and fasting glucose levels greater than or equal to 110 mg/dL. These determinants can be readily measured in clinical practice (JAMA, 2001, 285, 2486-2497).

Combinations

Compositions of the invention can contain two or more antisense compounds. In another related embodiment, compositions of the present invention can contain one or more antisense compounds, particularly oligonucleotides, targeted to a first nucleic acid and one or more additional antisense compounds targeted to a second nucleic acid target. Alternatively, compositions of the present invention can contain two or more antisense compounds targeted to different regions of the same nucleic acid target. Two or more combined compounds may be used together or sequentially.

Combination Therapy

Compounds of the invention may be used in combination therapies wherein an additive effect is achieved by administering one or more compounds of the invention and one or more other suitable therapeutic/prophylactic compounds to treat a condition. One or more of the therapeutic/prophylactic compounds may be combined with one or more of the antisense inhibitors of ACC1 or ACC2 to achieve an additive therapeutic effect.

Oligomer Synthesis

Oligomerization of modified and unmodified nucleosides can be routinely performed according to literature procedures for DNA (Protocols for Oligonucleotides and Analogs, Ed. Agrawal (1993), Humana Press) and/or RNA (Scaringe, Methods (2001), 23, 206-217. Gait et al., Applications of Chemically synthesized RNA in RNA: Protein Interactions, Ed. Smith (1998), 1-36. Gallo et al., Tetrahedron (2001), 57, 5707-5713).

Antisense compounds of the present invention can be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and alkylated derivatives. The invention is not limited by the method of oligomer synthesis.

Oligomer Purification and Analysis

Methods of oligonucleotide purification and analysis are known to those skilled in the art. Analysis methods include capillary electrophoresis (CE) and electrospray-mass spectroscopy. Such synthesis and analysis methods can be performed in multi-well plates. The method of the invention is not limited by the method of oligomer purification.

Nonlimiting Disclosure and Incorporation by Reference

While certain compounds, compositions and methods of the present invention have been described with specificity in accordance with certain embodiments, the examples herein serve only to illustrate the compounds of the invention and are not intended to limit the same. Each of the references, GENBANK® accession numbers, and the like recited in the present application is incorporated herein by reference in its entirety.