# Patent

## Title
Inhibitors of MYH7B and uses thereof

## Abstract

The present invention provides nucleic acid inhibitors of MYH7B and compositions thereof. The present invention also provides methods of treating or preventing a cardiac disorder such as cardiac hypertrophy, myocardial infarction, or heart failure in a subject by administering to the subject an inhibitor of MYH7B. The present invention further provides methods of modulating the activity or expression of β-MHC in cardiac cells of a subject by administering to the subject an inhibitor of MYH7B.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
The present application claims the benefit of priority to U.S. Provisional Application No. 62/033,018, filed on Aug. 4, 2014, the contents of which are hereby incorporated by reference in their entirety.


FIELD OF THE INVENTION
The present invention relates to inhibitors of a myosin heavy chain gene, MYH7B, and compositions thereof. The invention also provides methods for treating or preventing cardiac disorders such as cardiac hypertrophy, myocardial infarction, and heart failure by administering inhibitors of MYH7B. In particular, the invention discloses nucleic acid inhibitors of MYH7B and compositions thereof, and methods for treating or preventing cardiac disorders by inhibiting the expression or activity of MYH7B in a subject in need thereof.
BACKGROUND
Heart disease is the leading cause of a death in the United States and presents a major health risk for millions of people across the world. The cost to diagnose, treat and support patients suffering from various types of heart disease, including hypertrophic cardiomyopathy, myocardial infarction, and heart failure, is very high and puts a serious burden on the healthcare system.
Hypertrophic cardiomyopathy (HCM) is of particular concern because it is a significant cause of sudden unexpected cardiac death and is frequently asymptomatic prior to onset of cardiac arrest. HCM is characterized by a thickening of myocardial cells, which can affect ventricular function and give rise to cardiac arrhythmias. HCM is a primary genetic disease of the heart, resulting from a dominant mutation in a number of sarcomeric genes, of which 8 have been extensively studied (Teekakirikul et al., “Hypertrophic cardiomyopathy: translating cellular cross talk into therapeutics,” J. Cell. Biol., 2012, 199(3), 417-421). Of these dominant mutations in HCM, roughly 40% are missense mutations in the β-myosin gene, MYH7. Genetic and pharmacologic studies have shown reductions in expression of this dominant allele, even to a small degree, can have robust phenotypic effects (Jiang et al., “Allele-specific silencing of mutant MYH6 transcripts in mice suppresses hypertrophic cardiomyopathy,” Science, 2013, 342(6154), 111-114).
Myosin is the major contractile protein of cardiac and skeletal muscle cells. Cardiac muscle contraction depends on the expression and relative ratios of two myosin heavy chain (MHC) proteins, α-MHC (MYH6) and β-MHC (MYH7). In rodents, α-MHC, a fast-twitching MHC, is the predominant myosin isoform in the adult heart, whereas β-MHC, a slow-twitching MHC, is predominantly expressed in the developing heart and is downregulated after birth (Morkin, E., “Control of cardiac myosin heavy chain gene expression,” Microsc. Res. Tech., 2000, 50, 522-531). In contrast, in human heart, the β-MHC isoform is heavily expressed and the α-MHC isoform accounts for less than 8% of total ventricular MHC (Miyata et al., “Myosin heavy chain isoform expression in the failing and nonfailing human heart,” Circ. Res., 2000, 86(4):386-90). Irrespective of the differences in the expression of alpha- and beta-myosins in various species, studies indicate that expression of beta-myosin is upregulated in cardiac disorders in these species including humans, rats, and rabbits. For example, in failing adult mouse hearts, a shift from the normally predominant alpha-MHC toward beta-MHC is often observed (Harada et al., Circulation, 1999, 100, 2093-2099). Similarly, in rats, congestive heart failure was associated with increased expression of beta-myosin and decreased expression of alpha-MHC. Consistent with rodent studies, beta-myosin expression was upregulated and alpha-myosin expression was significantly down-regulated in failing human hearts (Miyata et al., “Myosin heavy chain isoform expression in the failing and nonfailing human heart”, Circ Res. 2000 Mar. 3; 86(4):386-90). These studies show that although the expression of alpha- and beta-myosins is species-dependent, downregulating the expression of beta-myosin is likely to play a cardioprotective role in various species. For example, blunting the increase in β-MHC expression and increasing α-MHC expression has shown to be cardioprotective in rabbits (James et al., “Forced expression of alpha-myosin heavy chain in the rabbit ventricle results in cardioprotection under cardiomyopathic conditions,” Circulation, 2005, 111(18), 2339-2346).
The genes encoding α-MHC (MYH6), β-MHC (MYH7), and a related myosin, MYH7B, also encode a family of intronic miRNAs, miR-208a, miR-208b, and miR-499, respectively. These three miRs share sequence homology and are called the “MyomiRs” (van Rooji et al., “A family of microRNAs encoded by myosin genes governs myosin expression and muscle performance,” Dev. Cell, 2009, 17, 662-673). MyomiRs have been shown to control pathological cardiac remodeling, muscle myosin content, myofiber identity, and muscle performance (Liu and Olson, “MicroRNA regulatory networks in cardiovascular development,” Dev. Cell, 2010, 510-525).
Although the role of α-MHC, β-MHC, and myomiRs (miR-208a, miR-208b, and miR-499) in cardiac and muscle development have been extensively studied, the role of the third myosin, MYH7B, is largely unknown. The MYH7B gene is expressed in skeletal muscle, heart, and in a subset of cells in the brain where it regulates synapse structure and function in the brain (Yeung et al., “Myh7b/miR-499 gene expression is transcriptionally regulated by MRFs and Eos,” Nucleic Acids Res., 2012, 40(15):7303-18). A recent report suggests that MYH7b protein is detected in a minor fibre population in extraocular muscles, corresponding to slow-tonic fibres, and in bag fibres of muscle spindles (Rossi et al., “Two novel/ancient myosins in mammalian skeletal muscles: MYH14/7b and MYH15 are expressed in extraocular muscles and muscle spindles”, J Physiol., Jan. 15, 2010; 588(Pt 2):353-64). However, in human heart, MYH7B gene is considered to undergo non-productive splicing, and result in a RNA that may not encode a functional MYH7B protein (Bell et al., “Uncoupling of expression of an intronic microRNA and its myosin host gene by exon skipping,” Mol. Cell. Biol., 30, 1937-1945). Interestingly, the non-productive splicing of MYH7B mRNA in heart cells was not associated with altered expression of intronic miR-499. Thus, until the present invention, MYH7B was considered to be a non-functioning carrier of miR-499 in cardiac cells (Gerald Dorn, II, “MicroRNAs: redefining mechanisms in cardiac disease,” J. Cardiovasc. Pharmacol., 2010, 56(6), 589-595). Moreover, unlike miR-499, MYH7B is considered not to play an important role in cardiac and skeletal muscle biology.
SUMMARY OF THE INVENTION
The present invention is based, in part, on the surprising discovery that MYH7B regulates the expression of β-MHC in cardiac cells and thus regulates cardiac contractility and stress-induced cardiac remodeling. Specifically, the inhibition of MYH7B results in the down-regulation of β-MHC mRNA and protein in cardiac cells. Accordingly, in one embodiment, the present invention provides inhibitors of MYH7B, compositions thereof, and methods for modulating the expression and/or activity of β-MHC in a subject in need thereof comprising administering to the subject the inhibitor of MYH7B. In one embodiment, administration of an inhibitor of MYH7B to the subject downregulates the expression or activity of MYH7B in cardiac cells of the subject following administration. In an embodiment, administration of an inhibitor of MYH7B to the subject downregulates the expression or activity of β-MHC (MYH7) in cardiac cells of the subject following administration. In another embodiment, administration of an inhibitor of MYH7B to the subject upregulates the expression or activity of α-MHC (MYH6) in cardiac cells of the subject following administration. In yet another embodiment, administration of an inhibitor of MYH7B to the subject induces a “switch” in the myosins whereby the expression or activity of β-MHC is downregulated and the expression or activity of α-MHC is upregulated in the subject following administration. In one embodiment, administration of an inhibitor of MYH7B to the subject does not change the expression or activity of miR-208a, miR-208b, or miR-499 in cardiac cells of the subject.
In another embodiment, the present invention provides compositions and methods for treating or preventing cardiac disorders such as pathologic cardiac hypertrophy, myocardial infarction, heart failure, or hypertrophic cardiomyopathy in a subject in need thereof comprising administering an inhibitor of MYH7B to the subject. In one embodiment, the subject having a cardiac disorder has increased levels of β-MHC in cardiac cells compared to a healthy subject. In another embodiment, the subject having a cardiac disorder has an altered ratio of β-MHC/α-MHC in cardiac cells compared to a healthy subject. In one embodiment, the administration of an inhibitor of MYH7B to a subject having a cardiac disorder inhibits the expression or activity of MYH7B and/or the expression or activity of MYH7 in cardiac cells of the subject following administration. In another embodiment, the administration of a MYH7B inhibitor to a subject having a cardiac disorder restores the β-MHC/α-MHC ratio in cardiac cells of the subject fully or partially to normal levels following administration. In one embodiment, the cardiac disorder is cardiac hypertrophy, myocardial infarction, heart failure, or hypertrophic cardiomyopathy.
In one embodiment, an inhibitor of MYH7B is a nucleic acid inhibitor selected from an antisense oligonucleotide, an aptamer, a ribozyme, a small interfering RNA (siRNA), or a small hairpin RNA (shRNA). In another embodiment, an inhibitor of MYH7B is an antibody or a binding fragment thereof that specifically binds to MYH7B protein. In a particular embodiment, an inhibitor of MYH7B is an antisense oligonucleotide that comprises a sequence that is at least partially complementary to a MYH7B coding sequence. The antisense oligonucleotides used in the compositions and methods of the invention may have a length of from about 6 to about 22 nucleotides. In some embodiments, the antisense oligonucleotides comprise one or more chemical modifications, such as sugar, backbone, and/or base modifications.
In one embodiment, an antisense oligonucleotide inhibitor of MYH7B has a length of 8 to 18 nucleotides, and wherein the sequence of the antisense oligonucleotide is substantially complementary to the sequence of SEQ ID NO: 6.
In some embodiments, an antisense oligonucleotide inhibitor of MYH7B has a length of 8-18 nucleotides and the sequence of the antisense oligonucleotide is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 4300-4335.
In some other embodiments, the antisense oligonucleotide inhibitor of MYH7B has a length of 12-18 nucleotides and the sequence of the antisense oligonucleotide is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 4300-4317.
In still other embodiments, the antisense oligonucleotide inhibitor of MYH7B has a length of 12-18 nucleotides and the sequence of the antisense oligonucleotide is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 4316-4333.
In one embodiment, the antisense oligonucleotide inhibitor of MYH7B has a length of 14 nucleotides and the sequence of the antisense oligonucleotide is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 4302-4315.
In another embodiment, the antisense oligonucleotide inhibitor of MYH7B has a length of 14 nucleotides and the sequence of the antisense oligonucleotide is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 4318-4331.
In some embodiments, antisense oligonucleotide inhibitors of the invention contain at least one modified nucleotide. The modified nucleotide may include a sugar, base, and/or a backbone modification.
In one embodiment, the modified nucleotide is a locked nucleotide. In some embodiments, the antisense oligonucleotide inhibitor contains one to six locked nucleotides. In some embodiments, at least the first three nucleotides at the 5′ end of the antisense oligonucleotide inhibitor are locked nucleotides. In another embodiment, at least the first three nucleotides at the 3′ end of the antisense oligonucleotide inhibitor are locked nucleotides.
In some embodiments, at least the first three nucleotides at the 5′ end of the antisense oligonucleotide inhibitor are locked or non-locked ribonucleotides. In some embodiments, the at least first three nucleotides at the 3′ end of the antisense oligonucleotide inhibitor are locked or non-locked ribonucleotides.
In one embodiment, the antisense oligonucleotide inhibitor contains at least one deoxyribonucleotide. In other embodiments, the antisense oligonucleotide inhibitor contains two to eight deoxyribonucleotides.
In various embodiments, the antisense oligonucleotide inhibitor of MYH7B may include a sugar modification selected from the group consisting of 2′-O, 4′-C methylene bridge, 2′-O, 4′-C ethylene bridge, 2′-CH2—NH—CH2-4′ bridge, 2′-deoxy, 2′-O-alkyl, and 2′-halo modifications.
In various embodiments, the antisense oligonucleotide inhibitor of MYH7B may include a backbone modification. In one embodiment, the antisense oligonucleotide inhibitor contains at least one phosphorothioate linkage. In another embodiment, the antisense oligonucleotide inhibitor contains two or more phosphorothioate linkages. In still other embodiments, the antisense oligonucleotide is fully phosphorothioate linked.
In one embodiment, the antisense oligonucleotide inhibitor of MYH7B further comprises a peptide or a sugar moiety connected through a linker. The linker could be a 3′thiol-modified-C3-disulfide linker, (C6)2 disulfide linker, or 1-3 phosphodiester linkages.
In certain embodiments, the antisense oligonucleotide inhibitor of MYH7B comprises a sequence selected from Tables 1-5.
In one embodiment, the antisense oligonucleotide inhibitor of MYH7B comprises the sequence of 5′-lTslTslGsdAsdTsdCsdTsdTsdGsdGsdCslCslTslC-3′ (SEQ ID NO: 146) or 5′-lCslTslGsdCsdAsdGsdCsdTsdCsdCsdTslCslCslA-3′ (SEQ ID NO: 148).
The invention also provides pharmaceutical compositions comprising an inhibitor of MYH7B and a pharmaceutically acceptable excipient. In one embodiment, the inhibitor of MYH7B is an antisense oligonucleotide. In one aspect, the pharmaceutical composition further comprises a second therapeutic agent, wherein the second therapeutic agent is an antisense oligonucleotide inhibitor of miR-208a, miR-208b, miR-499, miR-15a, miR-15b, miR-16, miR-195, or a mixture thereof.
The invention further provides a method for treating or preventing pathologic cardiac hypertrophy, myocardial infarction, or heart failure in a subject in need thereof, comprising administering to the subject an antisense oligonucleotide inhibitor of MYH7B. In one embodiment, the pathologic cardiac hypertrophy is hypertrophic cardiomyopathy. In some embodiments, the methods according to the invention may comprise administering a second cardiac therapeutic agent such as an antisense oligonucleotide inhibitor of miR-208a, miR-208b, miR-499, miR-15a, miR-15b, miR-16, miR-195, or combinations thereof.
In some embodiments, the invention provides a method of treating a subject who is at risk for pathologic cardiac hypertrophy. In one embodiment, the subject at risk for pathologic cardiac hypertrophy has a mutation in a beta myosin heavy chain gene.
In one aspect, administration of the antisense oligonucleotide inhibitor of MYH7B reduces the expression or activity of MYH7B in cardiac cells of the subject following administration. In another aspect, administration of the antisense oligonucleotide inhibitor of MYH7B reduces the expression of beta myosin heavy chain in cardiac cells of the subject following administration. In yet another aspect, administration of the antisense oligonucleotide inhibitor of MYH7B does not significantly alter the expression of miR-499, miR-208a, and/or miR-208b in cardiac cells of the subject following administration.
In some embodiments, the invention provides a method for treating or preventing pathologic cardiac hypertrophy, myocardial infarction, or heart failure in a subject in need thereof, comprising administering to the subject an inhibitor of MYH7B. In various embodiments, the inhibitor of MYH7B is a nucleic acid inhibitor selected from an antisense oligonucleotide, an aptamer, a ribozyme, a small interfering RNA, or a small hairpin RNA.
In one embodiment, the nucleic acid inhibitor of MYH7B is a small interfering RNA or a small hairpin RNA comprising a double-stranded region of about 10 to about 30 nucleotides, said double-stranded region comprising (i) a first RNA strand having a sequence that is at least 70% identical to a sequence of the human Myh7b gene and (ii) a second RNA strand that is partially, substantially, or fully complementary to the first RNA strand. In one aspect, the first RNA strand of the siRNA or shRNA has a sequence that is at least 70% identical to a sequence of 5′-GAGGCCAAGATCAA-3′ (SEQ ID NO: 4). In another aspect, the first RNA strand of the siRNA or shRNA has a sequence that is at least 70% identical to a sequence of 5′-TGGAGGAGCTGCAG-3′ (SEQ ID NO: 5).



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A shows levels of MYH7B mRNA in human iPS cardiomyocytes 48 hours after transfection with antisense oligonucleotides to MYH7B. FIG. 1B shows levels of MYH7 (13-MHC) mRNA in human iPS cardiomyocytes 48 hours after transfection with antisense oligonucleotides to MYH7B.
FIG. 2A shows levels of miR-208a in human iPS cardiomyocytes 48 hours after transfection with antisense oligonucleotides to MYH7B. FIG. 2B shows levels of miR-499 in human iPS cardiomyocytes 48 hours after transfection with antisense oligonucleotides to MYH7B. FIG. 2C shows levels of miR-208b in human iPS cardiomyocytes 48 hours after transfection with antisense oligonucleotides to MYH7B.
FIG. 3A shows levels of MYH7 (β-MHC) mRNA in control and HCM-R663H cardiomyocytes 48 hours after transfection with antisense oligonucleotides to MYH7B. FIG. 3B shows levels of MYH7 (β-MHC) mRNA in control and HCM-R663H cardiomyocytes 168 hours (1 week) after transfection with antisense oligonucleotides to MYH7B.
FIG. 4 shows a Western blot analysis of MYH7 (β-MHC) or GAPDH from control and HCM iPS cardiomyocytes at 96 hours post-treatment.
FIG. 5 shows levels of MYH7B mRNA in human iPS cardiomyocytes 48 hrs after transfection with test ASO compounds to MYH7B.
FIG. 6A shows levels of MYH7B mRNA in human iPS cardiomyocytes 168 hrs after passive administration of 1, 5, or 10 μM test ASO compounds (SEQ ID NOs. 92, 146, 32, 46, and 30) targeted to MYH7B. FIG. 6B shows levels of MYH7 (β-MHC) mRNA in human iPS cardiomyocytes 168 hrs after passive administration of 1, 5, or 10 μM the test ASO compounds. FIG. 6C shows levels of MYH6 (α-MHC) mRNA in human iPS cardiomyocytes 168 hrs after passive administration of 1, 5, or 10 μM of the test ASO compounds.
FIG. 7 shows levels of MYH7B, MYH7 (β-MHC), and MYH6 (α-MHC) mRNAs in human iPS cardiomyocytes 168 hrs after passive administration of 5 μM test ASO having the sequence of SEQ ID NO: 148.
FIG. 8 shows levels of MYH7B mRNA in left ventricles of rats injected with 25 mg/kg of test ASOs per day for three consecutive days and sacrificed 48 hours after the last dose.
FIG. 9A shows levels of MYH7B mRNA in left ventricles of rabbits treated with ASO compounds comprising the sequence of SEQ ID NOs: 146 and 148. FIG. 9B shows levels of MYH7B mRNA in right ventricles of rabbits treated with ASO compounds comprising the sequence of SEQ ID NOs: 146 and 148. FIG. 9C shows levels of MYH7 (β-MHC) mRNA in left ventricles of rabbits treated with ASO compounds comprising the sequence of SEQ ID NOs: 146 and 148. FIG. 9D shows levels of MYH7 (β-MHC) mRNA in right ventricles of rabbits treated with ASO compounds comprising the sequence of SEQ ID NOs: 146 and 148.
FIG. 10A shows levels of MYH7B mRNA in human iPS cardiomyocytes 48 hrs after passive administration of ENA-modified ASOs based on the compound comprising the sequence of SEQ ID NO: 146. FIG. 10B shows levels of MYH7B mRNA in human iPS cardiomyocytes 48 hrs after passive administration of 5-methyl cytidine and 2′-O-methyl modified ASOs of the compound comprising the sequence of SEQ ID NO: 146. FIG. 10C shows levels of MYH7B mRNA in human iPS cardiomyocytes 48 hrs after passive administration of backbone-modified ASOs of the compound comprising the sequence of SEQ ID NO: 146.
FIGS. 11A-11D show levels of MYH7B mRNA in human iPS cardiomyocytes 48 hrs after passive administration of test ASO compounds directed to MYH7B.



DETAILED DESCRIPTION OF THE INVENTION
The inventors have found that MYH7B, which was considered until now to act merely as a non-functional carrier of miR-499, plays an important role in regulating the expression of β-MHC in cardiac cells. Particularly, the inventors have found that the inhibition of MYH7B down-regulates the expression of β-MHC mRNA and protein in cardiac cells. Thus, the present invention provides MYH7B as a new target for therapeutic intervention for cardiac disorders, particularly cardiac muscle disorders.
The present invention provides agents that inhibit the expression or activity of MYH7B. In various embodiments, the agent that inhibits MYH7B is a nucleic acid inhibitor targeted to MYH7B gene, mRNA, or pre-mRNA or an antibody or a binding fragment thereof targeted to MYH7B protein. In certain embodiments, a nucleic acid inhibitor of MYH7B is selected from an antisense oligonucleotide, an aptamer, a ribozyme, a small interfering RNA, or a small hairpin RNA.
In one embodiment, an inhibitor of MYH7B is an antisense oligonucleotide (“ASO”). In the context of the present invention, the term “antisense oligonucleotide” or “ASO” is used broadly and encompasses an oligomer comprising ribonucleotides, deoxyribonucleotides, modified ribonucleotides, modified deoxyribonucleotides or a combination thereof that inhibits the information transfer from gene to protein. The term “antisense oligonucleotide” or “ASO” as used herein also includes oligomers comprising ribonucleotides, deoxyribonucleotides, modified ribonucleotides, modified deoxyribonucleotides or a combination thereof that facilitate transcriptional arrest; disruption of mRNA synthesis from pre-mRNA at various stages including capping, splicing, transport from nucleus to cytoplasm; degradation of mRNA such as that mediated by gapmer ASOs or other RNAse H-inducing ASOs; translational arrest where the ASO binds to target mRNA and makes it unavailable for translation; or steric blocking of target mRNA by hybridizing to it. The term “antisense oligonucleotide” as used herein also includes antisense oligonucleotide conjugates that comprise oligomers containing natural and/or modified nucleotides conjugated to or connected to a peptide or a sugar through a linker. The peptide or the sugar moiety and the linkers that can be used to prepare antisense oligonucleotide conjugates are described below in more detail.
In various embodiments, antisense oligonucleotides useful for inhibiting the activity of MYH7B are about 5 to about 25 nucleotides in length, about 6 to about 22 nucleotides in length, about 10 to about 30 nucleotides in length, about 12 to about 20 nucleotides in length, or about 20 to about 25 nucleotides in length. In certain embodiments, antisense oligonucleotides targeting MYH7B are about 8 to about 18 nucleotides in length, in other embodiments about 12 to about 18 nucleotides in length, and in still other embodiments about 12 to about 16 nucleotides in length. In some embodiments, antisense oligonucleotides targeting MYH7B are about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length.
In some embodiments, antisense oligonucleotide inhibitors directed to MYH7B are about 50 to about 150 nucleotides in length, about 60 to about 140 nucleotides in length, about 70 to about 130 nucleotides in length, about 80 to about 120 nucleotides in length, about 90 to about 110 nucleotides in length, or about 100 to about 120 nucleotides in length. In one embodiment, antisense oligonucleotide inhibitors are about 100 to about 120 nucleotides in length. In certain embodiments, antisense oligonucleotide inhibitors targeting MYH7B are about 80, 90, 100, 110, or 120 nucleotides in length.
Antisense oligonucleotides targeting MYH7B comprise a sequence that is at least partially or substantially complementary to a MYH7B sequence (DNA, mRNA, or pre-mRNA) to hybridize to MYH7B under physiological conditions and inhibit the expression or activity of MYH7B in the cells of a subject. For instance, in some embodiments, antisense oligonucleotides comprise a sequence that is at least partially complementary to a MYH7B gene, mRNA or pre-mRNA sequence, e.g. at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary to a MYH7B gene, mRNA or pre-mRNA sequence. In some embodiments, the antisense oligonucleotide can be substantially complementary to a MYH7B gene, mRNA or pre-mRNA sequence, that is at least about 90%, 95%, 96%, 97%, 98%, or 99% complementary to a target polynucleotide sequence. In one embodiment, the antisense oligonucleotide comprises a sequence that is 100% or completely complementary to a MYH7B gene, mRNA or pre-mRNA sequence. In certain embodiments, the antisense oligonucleotide targeting MYH7B comprises a sequence that is at least partially, substantially, or completely complementary to the sequence of the human MYH7B gene. In some embodiments, the antisense oligonucleotide targeting MYH7B comprises a sequence that is at least partially, substantially, or completely complementary to the coding sequence of the human MYH7B gene (Genebank Accession No. NM_020884.4; SEQ ID NO: 6).








Human MYH7B coding sequence (SEQ ID NO: 6):










   1
taggcttaaa gagcaggcgg ggcagagacc catacctttg gcgctgagtc gaggccagga



 


  61
aggggcggtc gatgagaggg cggccttagc attaggagtg cgtcggctgc accctggcgg


 


 121
atggaccttg ggaaggaggg gaggggacca tcggggtcgg cggggagcta cgggttcccc


 


 181
tcttccaaga cgggtgccaa gtgatgagta tcaggtgtct atggtgagat ttggggagga


 


 241
tcccagacct taacagccct ttggttgaaa cctctcacaa tgtggcagag atggggtttt


 


 301
gatgtgttgc ccaggctggt cttgaactcc tgagcttaag tgatgtgtcc gcctgggcct


 


 361
cccaaagtgc tggaattaca gacatgagtg gcaataaaag gggtagcaga gcttcctgcc


 


 421
ctcaccgtgg tgccgagtgc ctgctgcctt gggccgcctt gaacctccag ggtttccagc


 


 481
tcctcctcct tcaccccagt gccactgcca tgatggatgt gagtgaactt ggggagtctg


 


 541
cccgctacct ccgccagggc taccaggaga tgacgaaggt gcacactatc ccatgggacg


 


 601
ggaagaagcg agtctgggtg cctgatgaac aggacgccta cgtggaggcc gaggtcaagt


 


 661
cggaggctac cgggggcaga gtcaccgtgg agaccaaaga ccagaaggtg ctgatggtgc


 


 721
gtgaagccga gctgcagccc atgaacccgc ctcgcttcga cttactggag gacatggcca


 


 781
tgatgacgca cctgaacgag gcctctgtgc tgcacaacct gcgccagcgc tatgcccgct


 


 841
ggatgatcta tacctactca ggcctcttct gtgtcaccat caacccctac aaatggctcc


 


 901
cagtctatac ggcctccgta gtggctgctt acaagggaaa gcgccgctca gattccccgc


 


 961
cccatatata tgcggtggcg gacaacgcct acaacgacat gctgcgcaac cgagacaacc


 


1021
agtccatgct gatcaccgga gagtcggggg ccggtaagac ggttaacacc aagcgggtca


 


1081
ttcagtactt tgccatcgtc gctgccctgg gagacgggcc gggcaagaag gcccaatttc


 


1141
tggcaacaaa gacggggggc acccttgagg atcaaatcat cgaggccaac cctgccatgg


 


1201
aggcctttgg caacgccaag accctgagga atgataactc ctcccgcttt ggcaagttca


 


1261
tccgcattca ctttggtccc tctgggaagc tggcatccgc ggatattgac agctatctcc


 


1321
tggagaagtc gcgggtgatc ttccagttgc ctggtgagcg cagctaccat gtctactacc


 


1381
agatcctctc agggaggaag ccagagctgc aggacatgct gcttctgtct atgaacccct


 


1441
atgactacca cttctgcagc cagggcgtca tcaccgtgga caacatgaat gatggggagg


 


1501
agctcatcgc caccgaccat gccatggaca tcctaggctt cagcgtggat gagaaatgtg


 


1561
cctgctataa gatcgtgggc gccctcctgc actttggcaa catgaagttc aagcagaagc


 


1621
agcgggagga gcaggcggag gccgatggca ctgagagtgc tgacaaggct gcctacctga


 


1681
tgggggtcag cagtggggac ctcctcaaag gccttttgca cccccgggtg cgtgtaggga


 


1741
acgagtacgt gaccaagggc cagagtgtgg agcaggtggt gtttgctgtg ggggctctgg


 


1801
ccaaggccac ctatgaccgg ctgttcaggt ggctggtgtc tcggatcaac cagaccctgg


 


1861
acacaaagct gccccggcag ttcttcatcg gggttctgga catcgctggg tttgagatct


 


1921
ttgagttcaa cagcttcgaa cagctgtgca tcaacttcac caatgagaaa ttgcagcagt


 


1981
tcttcaacca gcacatgttt gtgctggagc aggaggagta caagcgggag ggcatcgact


 


2041
gggtcttcat cgacttcggc cttgacctgc agccttgcat cgacctcatc gagaagccac


 


2101
tgggcatcct gtccatcctg gaggaggaat gcatgttccc caaggcctca gacgccagct


 


2161
tccgggccaa gctctacgac aaccacgcgg ggaagtcacc caatttccag cagcctcggc


 


2221
ctgacaagaa gcgcaagtac caggcccact tcgaggtggt ccactacgca ggcgtggtgc


 


2281
cttacagcat tgtgggctgg ctggagaaaa acaaggatcc cctgaatgag accgtggtcc


 


2341
ccatcttcca gaagtcacag aataggctcc tggcgactct ctatgagaat tatgcgggct


 


2401
cctgctccac tgagcccccc aagtctgggg tgaaagagaa gcgtaagaag gcagcatcgt


 


2461
tccagacggt gtcccagctg cacaaggaga acctcaacaa gctgatgacc aacctgcggg


 


2521
ccacacagcc ccacttcgtc cgctgcattg tccccaacga gaacaaaacc ccaggggtca


 


2581
tggatgcctt cttggtgcta caccagctgc gctgcaatgg ggtcctggag gggatccgga


 


2641
tctgccgcca agggttcccc aacaggttgc tctacaccga cttccggcag cggtaccgta


 


2701
tcctgaaccc cagtgccatc ccggatgaca ccttcatgga cagcaggaag gccacagaga


 


2761
aactgctggg ctcgctggac ttggatcaca cccagtacca gtttggccac accaaggtgt


 


2821
tcttcaaggc tgggcttcta ggcgtcctgg aagagctccg tgaccagcgc ctggccaagg


 


2881
tgctgacgct gctgcaggcg cggagccgtg gccgcctcat gcgccttgag taccagcgcc


 


2941
tgctgggagg cagggatgcg ctgttcacca tccagtggaa catccgtgcc ttcaatgccg


 


3001
tcaagaactg gtcatggatg aagctctttt tcaagatgaa gccgctgctg cgctcggcgc


 


3061
aggctgagga ggagctggcg gccctgcggg cagagctgcg ggggttgcga ggggcgctgg


 


3121
ctgcggccga ggccaagcgc caggaactgg aggagacgca cgtcagcatc acccaggaga


 


3181
agaatgacct ggccctgcag ctgcaggctg agcaggacaa cctggcagat gccgaggagc


 


3241
gctgccactt gctgatcaag tccaaggtgc agctggaggg gaaggtgaag gagctgagtg


 


3301
agcggctgga ggatgaggag gaggtgaacg ctgacctggc cgcccgccgg cgcaagctgg


 


3361
aggacgagtg cacggagctc aagaaggaca ttgatgacct ggagctgaca ctggccaaag


 


3421
ctgagaagga gaagcaagcc actgagaaca aggtgaagaa cctgacggaa gagatggctg


 


3481
cgctggacga gtcagtggcc cggctgacca aggagaagaa ggcgttgcag gaggcccacc


 


3541
aacaggccct gggtgacctg caggccgagg aggaccgtgt gagcgcgctg accaaggcca


 


3601
agctccggct ggagcaacag gtggaggacc tggaatgctc cctggagcag gagaagaagc


 


3661
tgcgcatgga cacggagcgg gccaagcgca agctggaggg tgacctgaag ctgacgcagg


 


3721
agtcggtggc tgatgctgct caagacaagc agcagctgga ggagaagctc aagaagaagg


 


3781
actccgagct gagccagctg agcctgcggg tggaagacga gcagctcttg ggggcccaga


 


3841
tgcagaagaa gatcaaggag ctgcaggctc gggcggagga gctggaagag gagctggagg


 


3901
cagagcgggc agcccgggcc cgcgtggaga agcagcgtgc agaggcggcg cgggagctgg


 


3961
aggagctgag cgagcggctg gaggaggcag gcggcgcatc cgcggggcag cgcgagggct


 


4021
gccgcaagcg ggaggcggag ctggggaggc tgcggcggga gctggaggag gcggcgctgc


 


4081
ggcacgaggc cacagtggcg gcactgcggc gcaagcaggc ggagggcgcg gcggagctgg


 


4141
gggagcaggt ggacagcctg cagcgggtgc ggcagaagct ggagaaggag aagagtgagc


 


4201
tgcgcatgga ggtggacgac ctggctgcca acgtggagac tctgacccgc gccaaggcca


 


4261
gtgcagagaa gctgtgccgg acctatgagg atcagctaag cgaggccaag atcaaggtgg


 


4321
aggagctgca gcggcagctg gcggacgcaa gcacgcagcg tgggcgacta cagacggaaa


 


4381
gcggggagct gagtcgcctg ctagaggaga aggagtgtct gatcagtcag ctgagccgtg


 


4441
gaaaggccct ggccgcccaa agcctggaag agttgcggcg ccagctagag gaggaaagca


 


4501
aggccaagag tgccctggcc cacgccgtgc aggctctgcg gcacgactgt gacctcctgc


 


4561
gggagcaaca cgaggaggag gctgaggccc aggctgagct gcagcggctg ctgtccaagg


 


4621
ccaatgccga ggtggcccag tggaggagca agtacgaagc agatgccatc cagaggaccg


 


4681
aggagctgga ggaggccaaa aaaaagctgg cactgcggct gcaggaggca gaggagggcg


 


4741
tggaggctgc caacgccaag tgctcatcgt tggagaaggc caagctgcgg ctacagacag


 


4801
agtcagagga tgtaaccctg gagctggagc gggcgacctc agcagctgct gcgctggaca


 


4861
agaagcagcg gcacttggaa cgggcactgg aggaacggcg gcggcaggag gaggagatgc


 


4921
agcgggagct ggaggcggca cagagggagt cccgtggcct gggcaccgag ctcttccggc


 


4981
tgcggcacgg ccacgaggag gcacttgaag ccctggagac gctcaagegg gagaacaaga


 


5041
acctgcagga ggagatcagc gacctcacag accaggtgag tctcagtggg aagagcatcc


 


5101
aggaactgga gaaaaccaag aaggcgctgg aaggcgagaa gagtgagatc caggctgcac


 


5161
tggaggaggc agagggggcc ctggagctgg aggagaccaa gacgctgcgg atccagctgg


 


5221
agctctccca ggtcaaagca gaagtggacc ggaagctggc agagaaagac gaggagtgcg


 


5281
ctaacctgag gcgcaaccac cagcgagctg tggagtccct gcaggcctcc ctggatgcag


 


5341
agacacgggc ccgcaatgag gcgctgcggc tcaagaagaa gatggagggt gacctcaacg


 


5401
acctggagct gcagctgggc catgccaccc gtcaggccac agaggcccag gctgccacgc


 


5461
ggctgatgca ggcacagctc aaggaggagc aggcagggcg ggacgaggag cagcggctgg


 


5521
cagctgagct ccacgagcag gcgcaggctc tggagcgccg ggcctcgctg ctggctgcgg


 


5581
agctggagga gctgcgggct gccctggagc agggcgagcg cagccggcga ctggcagagc


 


5641
aggagctttt ggaggccacc gagcgcctca accttctgca ttcgcagaac acaggcctcc


 


5701
taaaccagaa gaagaagctg gaggcggact tggcccagct gagcggggag gtggaggagg


 


5761
ctgcacagga gaggcgggag gctgaggaga aggccaaaaa ggccatcact gatgcggcca


 


5821
tgatggccga ggagctgaag aaggagcagg acacaagtgc acacctggaa cggatgaaga


 


5881
agacgctgga gcagacggtg cgcgagctcc aggcccgcct tgaggaggca gaacaggccg


 


5941
ccctccgtgg cgggaagaag caggtgcaga agctggaggc caaggtacgg gagctggagg


 


6001
ctgagcttga tgcagagcag aagaagcacg ccgaggccct taagggcgtg cgcaagcatg


 


6061
agcgccgtgt caaggagctc gcataccagg ccgaggagga caggaagaac ctggctcgca


 


6121
tgcaggacct ggtggacaag ctgcagagca aggtcaagag ctacaagcgc cagtttgagg


 


6181
aggcggagca gcaggccaac accaacctgg ccaagtatcg caaggcccag cacgagctgg


 


6241
atgatgcgga ggagcgggca gacatggcgg aaacccaggc caacaagctg cgggcacgga


 


6301
cccgggacgc cctgggcccc aagcacaagg agtgacggcc tgaccccctg ggctctaaag


 


6361
aggaatgtct gctgttgcac atctggctga ggccacctgc cccgatcctg ccatctctgc


 


6421
atcgccccct gctgccttca gccttccctg ggccctgaat aaacaccaca gccagtttcc


 


6481
ttctcattct tttctttggg gttcaggagg aaaaacacag tcctagggac aaaagccagg


 


6541
tccacagcag tcatttttaa aataaagtta tttaatagtc tccaaaaaaa aaaaaaaaaa


 


6601
aa






It is understood that the sequence of the antisense oligonucleotide inhibitor is considered to be complementary to a MYH7B gene, mRNA or pre-mRNA sequence even if the antisense oligonucleotide sequence includes a modified nucleotide instead of a naturally-occurring nucleotide. For example, if a MYH7B gene, mRNA or pre-mRNA sequence comprises a guanosine nucleotide at a specific position, the antisense oligonucleotide inhibitor may comprise a modified cytidine nucleotide, such as a locked cytidine nucleotide or 2′-fluoro-cytidine, at the corresponding position.
In one embodiment, the invention provides an antisense oligonucleotide inhibitor of MYH7B, wherein said antisense oligonucleotide has a length of 8 to 18 nucleotides, and wherein the sequence of the antisense oligonucleotide is substantially complementary to the sequence of SEQ ID NO: 6.
In one embodiment, the antisense oligonucleotide inhibitor has a length of 8 to 18 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 4300-4335.
In certain embodiments, the antisense oligonucleotide inhibitor has a length of 12-18 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 4300-4317, 4300-4316, 4301-4316, 4301-4315, 4302-4317, 4302-4316, or 4302-4315. In one embodiment, the antisense oligonucleotide inhibitor has a length of 12-14 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 4302-4315. In some embodiments, the antisense oligonucleotide inhibitor has a length of 14 nucleotides and the sequence of the antisense oligonucleotide is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 4302-4315. In one embodiment, the antisense oligonucleotide inhibitor comprises a sequence of 5′-lTslTslGsdAsdTsdCsdTsdTsdGsdGsdCslCslTslC-3′ (SEQ ID NO: 146), where l=locked nucleic acid modification, d=deoxynucleotide, and s=phosphorothioate linkage.
In certain embodiments, the antisense oligonucleotide inhibitor has a length of 12-18 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 4316-4333, 4316-4332, 4317-4332, 4317-4331, 4318-4333, 4318-4332, or 4318-4331. In one embodiment, the antisense oligonucleotide inhibitor has a length of 12-14 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 4318-4331. In some embodiments, the antisense oligonucleotide has a length of 14 nucleotides and the sequence of the antisense oligonucleotide is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 4318-4331. In one embodiment, the antisense oligonucleotide inhibitor comprises a sequence of 5′-lCslTslGsdCsdAsdGsdCsdTsdCsdCsdTslCslCslA-3′ (SEQ ID NO: 148).
In some embodiments, the antisense oligonucleotide inhibitor has a length of 8 to 18 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 1762-1783. In one embodiment, the antisense oligonucleotide inhibitor has a length of 12-18 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 1762-1779, 1763-1779, 1764-1781, 1765-1780, or 1766-1779. In one embodiment, the antisense oligonucleotide inhibitor has a length of 12-14 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 1764-1777 or 1766-1779. In one embodiment, the antisense oligonucleotide inhibitor comprises a sequence of 5′-lAslCslCsdTsdGsdCsdTsdCsdCsdAsdCslAslCslT-3′ (SEQ ID NO: 62). In another embodiment, the antisense oligonucleotide inhibitor comprises a sequence of 5′-lCslCslAsdCsdCsdTsdGsdCsdTsdCsdCslAslCslA-3′ (SEQ ID NO: 64).
In some embodiments, the antisense oligonucleotide inhibitor has a length of 8 to 18 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 511-538. In one embodiment, the antisense oligonucleotide inhibitor has a length of 12-18 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 511-532, 512-530, 513-526, 514-528, 514-527, 515-530, 515-529, or 515-528. In one embodiment, the antisense oligonucleotide inhibitor has a length of 12-14 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 513-526, 514-527, or 515-528. In yet another embodiment, the antisense oligonucleotide inhibitor has a length of 12-18 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 521-538, 522-538, 522-537, 523-538, 523-537, or 523-536. In yet another embodiment, the antisense oligonucleotide inhibitor has a length of 12-14 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 522-537 or 523-536. In one embodiment, the antisense oligonucleotide inhibitor comprises a sequence of 5′-lTslCslAsmdCsdTsmdCsdAsmdCsdAsdTsmdCslCslAslT-3′, (SEQ ID NO: 184), where and =5-methylcytosine. In another embodiment, the antisense oligonucleotide inhibitor comprises a sequence of 5′-lTslTslCsdAsmdCsdTsmdCsdAsmdCsdAsdTslCslCslA-3′ (SEQ ID NO: 186). In yet another embodiment, the antisense oligonucleotide inhibitor comprises a sequence of 5′-lGslTslTsmdCsdAsmdCsdTsmdCsdAsmdCsdAslTslCslC-3′ (SEQ ID NO: 188). In still other embodiment, the antisense oligonucleotide inhibitor comprises a sequence of 5′-lCslTslCsmdCsmdCsmdCsdAsdAsdGsdTsdTslCslAslC-3′ (SEQ ID NO: 204).
In certain embodiments, the antisense oligonucleotide inhibitor has a length of 8 to 18 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 1640-1701. In one embodiment, the antisense oligonucleotide inhibitor has a length of 12 to 18 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 1643-1660, 1648-1665, 1653-1670, or 1694-1711. In some embodiments, the antisense oligonucleotide inhibitor has a length of 12 to 14 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 1645-1658, 1650-1663, 1655-1668, or 1696-1709. In one embodiment, the antisense oligonucleotide inhibitor comprises a sequence of 5′-lAslCslTsmdCsdTsmdCsdAsdGsdTsdGsmdCslCslAslT-3′ (SEQ ID NO: 230). In another embodiment, the antisense oligonucleotide inhibitor comprises a sequence of 5′-lTslCslAsdGsmdCsdAsmdCsdTsmdCsdTsmdCslAslGslT-3′ (SEQ ID NO: 238). In yet another embodiment, the antisense oligonucleotide inhibitor comprises a sequence of 5′-lCslCslTsdTsdGsdTsmdCsdAsdGsmdCsdAslCslTslC-3′ (SEQ ID NO: 244). In yet another embodiment, the antisense oligonucleotide inhibitor comprises a sequence of 5′-lTslTslTsdGsdAsdGsdGsdAsdGsdGsdTslCslCslC-3′ (SEQ ID NO: 272).
In some embodiments, the antisense oligonucleotide inhibitor has a length of 12 to 18 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 4280-4300. In one embodiment, the antisense oligonucleotide inhibitor has a length of 12 to 18 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 4280-4297, 4282-4295, 4283-4300 or 4283-4296. In one embodiment, the antisense oligonucleotide inhibitor comprises a sequence of 5′-lCslTslGsdAsdTsmdCsmdCsdTsmdCsdAsdTslAslGslG-3′ (SEQ ID NO: 304). In another embodiment, the antisense oligonucleotide inhibitor comprises a sequence of 5′-lGslCslTsdGsdAsdTsmdCsmdCsdTsmdCsdAslTslAslG-3′ (SEQ ID NO: 306).
In some embodiments, the antisense oligonucleotide inhibitor has a length of 12 to 18 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 688-705, 689-706, 690-707, or 690-703. In one embodiment, the antisense oligonucleotide inhibitor comprises a sequence of 5′-lTslGslGsdTsmdCsdTsdTsdTsdGsdGsdTslCslTslC-3′ (SEQ ID NO: 344). In other embodiments, the antisense oligonucleotide inhibitor has a length of 12 to 18 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 890-907, 892-909, or 892-905. In one embodiment, the antisense oligonucleotide inhibitor comprises a sequence of 5′-lGslAslCsdTsdGsdGsdGsdAsdGsmdCsmdCslAslTslT-3′ (SEQ ID NO: 350).
In some embodiments, the antisense oligonucleotide inhibitor has a length of 12 to 18 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 1217-1234, 1218-1235, 1219-1236, or 1219-1232. In one embodiment, the antisense oligonucleotide inhibitor comprises a sequence of 5′-lAslTslTsmdCsmdCsdTsmdCsdAsdGsdGsdGslTslCslT-3′ (SEQ ID NO: 362). In other embodiments, the antisense oligonucleotide inhibitor has a length of 12 to 18 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 1224-1241, 1225-1242, 1226-1243, or 1226-1239. In one embodiment, the antisense oligonucleotide inhibitor comprises a sequence of 5′-lAslGslTsdTsdAsdTsmdCsdAsdTsdTsmdCslCslTslC-3′ (SEQ ID NO: 370).
In some embodiments, the antisense oligonucleotide inhibitor has a length of 12 to 18 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 1415-1441. In some embodiments, the antisense oligonucleotide inhibitor has a length of 12 to 18 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 1415-1432, 1416-1433, 1417-1434, or 1417-1430. In one embodiment, the antisense oligonucleotide inhibitor comprises a sequence of 5′-lAslGslAsmdCsdAsdGsdAsdAsdGsmdCsdAslGslCslA-3′ (SEQ ID NO: 396). In some embodiments, the antisense oligonucleotide inhibitor has a length of 12 to 18 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 1422-1439, 1423-1440, 1424-1441, or 1424-1437. In one embodiment, the antisense oligonucleotide inhibitor comprises a sequence of 5′-lGslGslTsdTsmdCsdAsdTsdAsdGsdAsmdCslAslGslA-3′ (SEQ ID NO: 410).
In some embodiments, the antisense oligonucleotide inhibitor has a length of 8 to 18 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 2348-2371. In one embodiment, the antisense oligonucleotide inhibitor has a length of 12-18 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 2348-2365, 2349-2364, 2348-2361, 2349-2366, 2350-2365, 2351-2366, 2352-2367, 2352-2365, 2353-2370, 2354-2369, or 2354-2368. In one embodiment, the antisense oligonucleotide inhibitor has a length of 12-14 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 2349-2362 or 2350-2363. In yet another embodiment, the antisense oligonucleotide inhibitor has a length of 12-14 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 2350-2363 or 2351-2364. In yet another embodiment, the antisense oligonucleotide inhibitor has a length of 12-14 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 2352-2365. In yet another embodiment, the antisense oligonucleotide inhibitor has a length of 12-14 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 2353-2366 or 2354-2367. In one embodiment, the antisense oligonucleotide inhibitor comprises a sequence of 5′-lAslTslTsmdCsdTsdGsdTsdGsdAsmdCsdTslTslCslT-3′ (SEQ ID NO: 474). In another embodiment, the antisense oligonucleotide inhibitor comprises a sequence of 5′-lTslAslTsdTsmdCsdTsdGsdTsdGsdAsmdCslTslTslC-3′ (SEQ ID NO: 476). In yet another embodiment, the antisense oligonucleotide inhibitor comprises a sequence of 5′-lCslTslAsdTsdTsmdCsdTsdGsdTsdGsdAslCslTslT-3′ (SEQ ID NO: 478). In still other embodiment, the antisense oligonucleotide inhibitor comprises a sequence of 5′-lGslCslCsdTsdAsdTsdTsmdCsdTsdGsdTslGslAslC-3′ (SEQ ID NO: 482).
In some embodiments, the antisense oligonucleotide inhibitor has a length of 12 to 18 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 2442-2462. In certain embodiments, the antisense oligonucleotide inhibitor has a length of 12 to 14 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 2444-2457 or 2445-2458. In one embodiment, the antisense oligonucleotide inhibitor comprises a sequence of 5′-lAslTslGsmdCsdTsdGsmdCsmdCsdTsdTsmdCslTslTslA-3′ (SEQ ID NO: 508). In another embodiment, the antisense oligonucleotide inhibitor comprises a sequence of 5′-lGslAslTsdGsmdCsdTsdGsmdCsmdCsdTsdTslCslTslT-3′ (SEQ ID NO: 510).
In some other embodiments, the antisense oligonucleotide inhibitor has a length of 12 to 18 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 4408-4432. In various embodiments, the antisense oligonucleotide inhibitor has a length of 12 to 18 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 4408-4425, 4409-4426, 4410-4425, 4411-4428, 4411-4427, 4412-4429, 4412-4428, or 4413-4430. In one embodiment, the antisense oligonucleotide inhibitor has a length of 12 to 16 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 4409-4424 or 4410-4425. In another embodiment, the antisense oligonucleotide inhibitor has a length of 12 to 16 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 4411-4426 or 4412-4427. In yet another embodiment, the antisense oligonucleotide inhibitor has a length of 12 to 16 nucleotides and comprises a sequence that is substantially complementary to the sequence of SEQ ID NO: 6 from nucleotides 4413-4428. In one embodiment, the antisense oligonucleotide inhibitor comprises a sequence of 5′-lAslTslCsdAsdGsdAsmdCsdAsmdCsdTsmdCslCslTslT-3′ (SEQ ID NO: 570). In another embodiment, the antisense oligonucleotide inhibitor comprises a sequence of 5′-lTslGslAsdTsmdCsdAsdGsdAsmdCsdAsmdCslTslCslC-3′ (SEQ ID NO: 572). In yet another embodiment, the antisense oligonucleotide inhibitor comprises a sequence of 5′-lCslTslGsdAsdTsmdCsdAsdGsdAsmdCsdAslCslTslC-3′ (SEQ ID NO: 574).
In some embodiments, the antisense oligonucleotide targeting MYH7B comprises a sequence that is at least partially complementary or completely complementary to the sequence of human MYH7B gene from nucleotides 1182 to 1213 in SEQ ID NO: 6. In other embodiments, the antisense oligonucleotide targeting MYH7B comprises a sequence that is at least partially complementary or completely complementary to the sequence of human MYH7B gene from nucleotides 1365 to 1393 in SEQ ID NO: 6.
In one embodiment, the antisense oligonucleotide targeting MYH7B has a sequence of 5′-GTGAATGCGGATGAA-3′ (SEQ ID NO: 1). In another embodiment, the antisense oligonucleotide of MYH7B has a sequence of 5′-GAAGTGGTAGTCATA-3′ (SEQ ID NO: 2). In yet another embodiment, the antisense oligonucleotide of MYH7B has a sequence of 5′-lGslTslGsdAsdAsdTsdGsdCsdGsdGsdAsdTsdGslAslA-3′ (SEQ ID NO: 3).
The term “about” as used herein encompasses variations of +/−10% and more preferably +/−5%, as such variations are appropriate for practicing the present invention.
In one embodiment, the antisense oligonucleotide inhibitor of MYH7B contains at least one backbone modification, such as at least one phosphorothioate, morpholino, or phosphonocarboxylate internucleotide linkage (see, for example, U.S. Pat. Nos. 6,693,187 and 7,067,641, which are herein incorporated by reference in their entireties). In some embodiments, the antisense oligonucleotide inhibitor of MYH7B contains two or more phosphorothioate linkages. In certain embodiments, the antisense oligonucleotide inhibitor of MYH7B is fully phosphorothioate-linked. Alternatively, the antisense oligonucleotides can comprise peptide nucleic acids (PNAs), which contain a peptide-based backbone rather than a sugar-phosphate backbone.
In one embodiment, the antisense oligonucleotide inhibitor of MYH7B contains at least one modified nucleotide such as a locked nucleotide or a nucleotide containing other sugar, base, and/or backbone modifications. The terms “locked nucleotide,” “locked nucleic acid unit,” “locked nucleic acid residue,” “LNA unit”, “bridged nucleic acid,” “bridged nucleotide,” “BNA” may be used interchangeably throughout the disclosure and refer to a bicyclic nucleoside/nucleotide analogue. Specifically, a “bridged nucleic acid” or “bridged nucleotide” refers to a nucleotide containing a bridge at the 2′,4′-position or at the 3′,4′-position of the sugar residue. For instance, suitable antisense oligonucleotide inhibitors can be comprised of one or more “conformationally constrained” or 2′,4′-bridged nucleotide modifications that confer enhanced thermal stability to complexes formed between the oligonucleotide containing bridged nucleotides and their complementary target strand. In one embodiment, antisense oligonucleotide inhibitors contain a 2′-O, 4′-C-methylene bridge ribonucleoside (structure A), conventionally known as “locked nucleotide” or “LNA”. In another embodiment, antisense oligonucleotide inhibitors contain at least one 2′,4′-C-bridged 2′ deoxyribonucleoside (CDNA, structure B). See, e.g., U.S. Pat. No. 6,403,566 and Wang et al. (1999) Bioorganic and Medicinal Chemistry Letters, Vol. 9: 1147-1150, both of which are herein incorporated by reference in their entireties. In yet another embodiment, antisense oligonucleotide inhibitors contain at least one modified nucleoside having the structure shown in structure C. In yet another embodiment, antisense oligonucleotide inhibitors contain at least one 2′O, 4′-C-ethylene bridge ribonucleoside (structure D). A nucleic acid or an oligonucleotide or a nucleotide containing the 2′O, 4′-C-ethylene bridge is also known as ethylene-bridged nucleic acid (ENA). In still another embodiment, antisense oligonucleotide inhibitors contain at least one 2′-CH2—NH—CH2-4′-bridged nucleotide, also known as “amino-2′-C-Bridged Bicyclic Nucleotide” or “amino-CBBN”. The antisense oligonucleotide inhibitors targeting MYH7B can contain combinations of bridged nucleotides described above or other modified nucleotides, and unmodified ribonucleotides or deoxyribonucleotides.




When referring to substituting a DNA or RNA nucleotide by its corresponding bridged or locked nucleotide in the context of the present invention, the terms “corresponding bridged nucleotide” or “corresponding locked nucleotide” are intended to mean that the DNA/RNA nucleotide has been replaced by a bridged or locked nucleotide containing the same naturally-occurring nitrogenous base as the DNA/RNA nucleotide that it has replaced or the same nitrogenous base that is chemically modified. For example, the corresponding bridged or locked nucleotide of a DNA nucleotide containing the nitrogenous base C may contain the same nitrogenous base C or the same nitrogenous base C that is chemically modified, such as 5-methylcytosine.
The terms “non-bridged nucleotide” or “non-locked nucleotide” refer to a nucleotide different from a bridged or locked-nucleotide, i.e. the terms “non-bridged nucleotide” or “non-locked nucleotide” include a DNA nucleotide, an RNA nucleotide as well as a modified nucleotide where a base and/or sugar is modified except that the modification is not a bridge or locked modification.
In one embodiment, the antisense oligonucleotide inhibitor of MYH7B contains at least one locked nucleotide. In some embodiments, the antisense oligonucleotide inhibitor of MYH7B contains one to six locked nucleotides. In one embodiment, at least the first three nucleotides from the 3′ end of the antisense oligonucleotide inhibitor are locked nucleotides. In another embodiment, at least the first three nucleotides from the 5′ end of the antisense oligonucleotide inhibitor are locked nucleotides.
In certain embodiments, the antisense oligonucleotide inhibitor contains one to six natural or modified ribonucleotides. In one embodiment, at least the first three nucleotides from the 3′ end of the antisense oligonucleotide inhibitor are natural or modified ribonucleotides. In another embodiment, at least the first three nucleotides from the 5′ end of the antisense oligonucleotide inhibitor are natural or modified ribonucleotides. In some embodiments, the first three nucleotides from the 3′ end of the antisense oligonucleotide inhibitor are locked ribonucleotides. In other embodiments, the first three nucleotides from the 5′ end of the antisense oligonucleotide inhibitor are locked ribonucleotides.
Oligonucleotide inhibitors of the present invention may include modified nucleotides that have a base modification or substitution. The natural or unmodified bases are the purine bases adenine (A) and guanine (G), and the pyrimidine bases cytosine (C), thymine (T) and uracil (U). Modified bases, also referred to as heterocyclic base moieties, include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo (including 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines), 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. In one embodiment, antisense oligonucleotide inhibitors of the present invention comprise one to five 5-methylcytidine or 5-methyldeoxycytidine nucleotides. In certain embodiments, antisense oligonucleotide inhibitors comprise one or more bridged nucleic acid modifications (e.g. LNA, ENA, etc.) in combination with a base modification (e.g. 5-methyl cytidine).
Oligonucleotide inhibitors of the present invention may include nucleotides with modified sugar moieties. Representative modified sugars include carbocyclic or acyclic sugars, sugars having substituent groups at one or more of their 2′, 3′ or 4′ positions and sugars having substituents in place of one or more hydrogen atoms of the sugar. In certain embodiments, the sugar is modified by having a substituent group at the 2′ position. In additional embodiments, the sugar is modified by having a substituent group at the 3′ position. In other embodiments, the sugar is modified by having a substituent group at the 4′ position. It is also contemplated that a sugar may have a modification at more than one of those positions, or that an oligonucleotide inhibitor may have one or more nucleotides with a sugar modification at one position and also one or more nucleotides with a sugar modification at a different position.
Sugar modifications contemplated in the oligonucleotide inhibitors of the present invention include, but are not limited to, a substituent group selected from: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted with C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl. In one embodiment, the modification includes 2′-methoxyethoxy (2′-O—CH2CH2OCH3, which is also known as 2′-O-(2-methoxyethyl) or 2′-MOE), that is, an alkoxyalkoxy group. Another modification includes 2′-dimethylaminooxyethoxy, that is, a O(CH2)2ON(CH3)2 group, also known as 2′-DMAOE and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), that is, 2′-O—CH2—O—CH2—N(CH3)2.
Additional sugar substituent groups include allyl (—CH2—CH═CH2), —O-allyl, methoxy (—O—CH3), aminopropoxy (—OCH2CH2CH2NH2), and fluoro (F). Sugar substituent groups on the 2′ position (2′-) may be in the arabino (up) position or ribo (down) position. One 2′-arabino modification is 2′-F. Other similar modifications may also be made at other positions on the sugar moiety, particularly the 3′ position of the sugar on the 3′ terminal nucleoside or in 2′-5′ linked oligonucleotides and the 5′ position of 5′ terminal nucleotide. In certain embodiments, the sugar modification is a 2′-O-alkyl (e.g. 2′-O-methyl, 2′-O-methoxyethyl), 2′-halo (e.g., 2′-fluoro, 2′-chloro, 2′-bromo), and 4′ thio modifications.
Other modifications of oligonucleotide inhibitors to enhance stability and improve efficacy, such as those described in U.S. Pat. No. 6,838,283, which is herein incorporated by reference in its entirety, are known in the art and are suitable for use in the methods of the invention. For instance, to facilitate in vivo delivery and stability, the oligonucleotide inhibitor can be linked to a steroid, such as cholesterol moiety, a vitamin, a fatty acid, a carbohydrate or glycoside, a peptide, or other small molecule ligand at its 3′ end. In some embodiments, the antisense oligonucleotide inhibitor is attached to a peptide to enhance the uptake of the inhibitor by target cells. For instance, in some embodiments, the 3′ end of an antisense oligonucleotide inhibitor is attached to a peptide having the sequence “WLSEAGPVVTVRALRGTGSW (SEQ ID NO: 788)”. This peptide is described in McGuire et al. (“In vitro selection of a peptide with high selectivity for cardiomyocytes in vivo”, J. Mol. Bio, 2004, vol. 342, 171-182), which is hereby incorporated by reference herein. In one embodiment, the peptide is attached to the antisense oligo via a linker such as “thiolC3,” whereby a thioether bond is formed between the SH group of the linker and the oligo and the maleimide on the peptide. In some embodiments, the 5′end of an antisense oligonucleotide inhibitor could be attached to a sugar moiety directly or via a linker. The linker could be a C6-disulfide linker or a 1-3 phosphodiester linker such as a phosphodiester dTdT linker.
In various embodiments, the antisense oligonucleotide of MYH7B has a sequence selected from Tables 1-5.








TABLE 1







Starting 










nucleotide




of target




sequence




in SEQ




ID NO: 6
Sequence(5′-3′)
Sequence(5′-3′) with modifications





 733
GTTCATGGGCTGCA
lGslTslTsdCsdAsdTsdGsdGsdGsdCsdTslGslCslA



(SEQ ID NO: 7)
(SEQ ID NO: 8)


 


 734
GGTTCATGGGCTGC
lGslGslTsdTsdCsdAsdTsdGsdGsdGsdCslTslGslC



(SEQ ID NO: 9)
(SEQ ID NO: 10)


 


 735
GGGTTCATGGGCTG
lGslGslGsdTsdTsdCsdAsdTsdGsdGsdGslCslTslG



(SEQ ID NO: 11)
(SEQ ID NO: 12)


 


 867
ATGGTGACACAGAA
lAslTslGsdGsdTsdGsdAsdCsdAsdCsdAslGslAslA



(SEQ ID NO: 13)
(SEQ ID NO: 14)


 


1020
ATCAGCATGGACTG
lAslTslCsdAsdGsdCsdAsdTsdGsdGsdAslCslTslG



(SEQ ID NO: 15)
(SEQ ID NO: 16)


 


1061
GCTTGGTGTTAACC
lGslCslTsdTsdGsdGsdTsdGsdTsdTsdAslAslCslC



(SEQ ID NO: 17)
(SEQ ID NO: 18)


 


1188
TCCATGGCAGGGTT
lTslCslCsdAsdTsdGsdGsdCsdAsdGsdGslGslTslT



(SEQ ID NO: 19)
(SEQ ID NO: 20)


 


1189
CTCCATGGCAGGGT
lCslTslCsdCsdAsdTsdGsdGsdCsdAsdGslGslGslT



(SEQ ID NO: 21)
(SEQ ID NO: 22)


 


1190
CCTCCATGGCAGGG
lCslCslTsdCsdCsdAsdTsdGsdGsdCsdAslGslGslG



(SEQ ID NO: 23)
(SEQ ID NO: 24)


 


1191
GCCTCCATGGCAGG
lGslCslCsdTsdCsdCsdAsdTsdGsdGsdCslAslGslG



(SEQ ID NO: 25)
(SEQ ID NO: 26)


 


1305
AGATAGCTGTCAAT
lAslGslAsdTsdAsdGsdCsdTsdGsdTsdCslAslAslT



(SEQ ID NO: 27)
(SEQ ID NO: 28)


 


1306
GAGATAGCTGTCAA
lGslAslGsdAsdTsdAsdGsdCsdTsdGsdTslCslAslA



(SEQ ID NO: 29)
(SEQ ID NO: 30)


 


1307
GGAGATAGCTGTCA
lGslGslAsdGsdAsdTsdAsdGsdCsdTsdGslTslCslA



(SEQ ID NO: 31)
(SEQ ID NO: 32)


 


1308
AGGAGATAGCTGTC
lAslGslGsdAsdGsdAsdTsdAsdGsdCsdTslGslTslC



(SEQ ID NO: 33)
(SEQ ID NO: 34)


 


1309
CAGGAGATAGCTGT
lCslAslGsdGsdAsdGsdAsdTsdAsdGsdCslTslGslT



(SEQ ID NO: 35)
(SEQ ID NO: 36)


 


1310
CCAGGAGATAGCTG
lCslCslAsdGsdGsdAsdGsdAsdTsdAsdGslCslTslG



(SEQ ID NO: 37)
(SEQ ID NO: 38)


 


1311
TCCAGGAGATAGCT
lTslCslCsdAsdGsdGsdAsdGsdAsdTsdAslGslCslT



(SEQ ID NO: 39)
(SEQ ID NO: 40)


 


1443
CAGAAGTGGTAGTC
lCslAslGsdAsdAsdGsdTsdGsdGsdTsdAslGslTslC



(SEQ ID NO: 41)
(SEQ ID NO: 42)


 


1445
TGCAGAAGTGGTAG
lTslGslCsdAsdGsdAsdAsdGsdTsdGsdGslTslAslG



(SEQ ID NO: 43)
(SEQ ID NO: 44)


 


1446
CTGCAGAAGTGGTA
lCslTslGsdCsdAsdGsdAsdAsdGsdTsdGslGslTslA



(SEQ ID NO: 45)
(SEQ ID NO: 46)


 


1647
GCACTCTCAGTGCC
lGslCslAsdCsdTsdCsdTsdCsdAsdGsdTslGslCslC



(SEQ ID NO: 47)
(SEQ ID NO: 48)


 


1662
TAGGCAGCCTTGTC
lTslAslGsdGsdCsdAsdGsdCsdCsdTsdTslGslTslC



(SEQ ID NO: 49)
(SEQ ID NO: 50)


 


1755
ACACTCTGGCCCTT
lAslCslAsdCsdTsdCsdTsdGsdGsdCsdCslCslTslT



(SEQ ID NO: 51)
(SEQ ID NO: 52)


 


1758
TCCACACTCTGGCC
lTslCslCsdAsdCsdAsdCsdTsdCsdTsdGslGslCslC



(SEQ ID NO: 53)
(SEQ ID NO: 54)


 


1759
CTCCACACTCTGGC
lCslTslCsdCsdAsdCsdAsdCsdTsdCsdTslGslGslC



(SEQ ID NO: 55)
(SEQ ID NO: 56)


 


1760
GCTCCACACTCTGG
lGslCslTsdCsdCsdAsdCsdAsdCsdTsdCslTslGslG



(SEQ ID NO: 57)
(SEQ ID NO: 58)


 


1761
TGCTCCACACTCTG
lTslGslCsdTsdCsdCsdAsdCsdAsdCsdTslCslTslG



(SEQ ID NO: 59)
(SEQ ID NO: 60)


 


1764
ACCTGCTCCACACT
lAslCslCsdTsdGsdCsdTsdCsdCsdAsdCslAslCslT



(SEQ ID NO: 61)
(SEQ ID NO: 62)


 


1766
CCACCTGCTCCACA
lCslCslAsdCsdCsdTsdGsdCsdTsdCsdCslAslCslA



(SEQ ID NO: 63)
(SEQ ID NO: 64)


 


1767
ACCACCTGCTCCAC
lAslCslCsdAsdCsdCsdTsdGsdCsdTsdCslCslAslC



(SEQ ID NO: 65)
(SEQ ID NO: 66)


 


1912
CTCAAAGATCTCAA
lCslTslCsdAsdAsdAsdGsdAsdTsdCsdTslCslAslA



(SEQ ID NO: 67)
(SEQ ID NO: 68)


 


1913
ACTCAAAGATCTCA
lAslCslTsdCsdAsdAsdAsdGsdAsdTsdCslTslCslA



(SEQ ID NO: 69)
(SEQ ID NO: 70)


 


1914
AACTCAAAGATCTC
lAslAslCsdTsdCsdAsdAsdAsdGsdAsdTslCslTslC



(SEQ ID NO: 71)
(SEQ ID NO: 72)


 


2037
ATGAAGACCCAGTC
lAslTslGsdAsdAsdGsdAsdCsdCsdCsdAslGslTslC



(SEQ ID NO: 73)
(SEQ ID NO: 74)


 


2038
GATGAAGACCCAGT
lGslAslTsdGsdAsdAsdGsdAsdCsdCsdCslAslGslT



(SEQ ID NO: 75)
(SEQ ID NO: 76)


 


2253
TAGTGGACCACCTC
lTslAslGsdTsdGsdGsdAsdCsdCsdAsdCslCslTslC



(SEQ ID NO: 77)
(SEQ ID NO: 78)


 


2481
AGGTTCTCCTTGTG
lAslGslGsdTsdTsdCsdTsdCsdCsdTsdTslGslTslG



(SEQ ID NO: 79)
(SEQ ID NO: 80)


 


2482
GAGGTTCTCCTTGT
lGslAslGsdGsdTsdTsdCsdTsdCsdCsdTslTslGslT



(SEQ ID NO: 81)
(SEQ ID NO: 82)


 


2483
TGAGGTTCTCCTTG
lTslGslAsdGsdGsdTsdTsdCsdTsdCsdCslTslTslG



(SEQ ID NO: 83)
(SEQ ID NO: 84)


 


2484
TTGAGGTTCTCCTT
lTslTslGsdAsdGsdGsdTsdTsdCsdTsdCslCslTslT



(SEQ ID NO: 85)
(SEQ ID NO: 86)


 


2485
GTTGAGGTTCTCCT
lGslTslTsdGsdAsdGsdGsdTsdTsdCsdTslCslCslT



(SEQ ID NO: 87)
(SEQ ID NO: 88)


 


2488
CTTGTTGAGGTTCT
lCslTslTsdGsdTsdTsdGsdAsdGsdGsdTslTslCslT



(SEQ ID NO: 89)
(SEQ ID NO: 90)


 


2518
GGGCTGTGTGGCCC
lGslGslGsdCsdTsdGsdTsdGsdTsdGsdGslCslCslC



(SEQ ID NO: 91)
(SEQ ID NO: 92)


 


2738
CCTTCCTGCTGTCC
lCslCslTsdTsdCsdCsdTsdGsdCsdTsdGslTslCslC



(SEQ ID NO: 93)
(SEQ ID NO: 94)


 


2740
GGCCTTCCTGCTGT
lGslGslCsdCsdTsdTsdCsdCsdTsdGsdCslTslGslT



(SEQ ID NO: 95)
(SEQ ID NO: 96)


 


2961
TGGATGGTGAACAG
lTslGslGsdAsdTsdGsdGsdTsdGsdAsdAslCslAslG



(SEQ ID NO: 97)
(SEQ ID NO: 98)


 


2962
CTGGATGGTGAACA
lCslTslGsdGsdAsdTsdGsdGsdTsdGsdAslAslCslA



(SEQ ID NO: 99)
(SEQ ID NO: 100)


 


2964
CACTGGATGGTGAA
lCslAslCsdTsdGsdGsdAsdTsdGsdGsdTslGslAslA



(SEQ ID NO: 101)
(SEQ ID NO: 102)


 


2966
TCCACTGGATGGTG
lTslCslCsdAsdCsdTsdGsdGsdAsdTsdGslGslTslG



(SEQ ID NO: 103)
(SEQ ID NO: 104)


 


2967
TTCCACTGGATGGT
lTslTslCsdCsdAsdCsdTsdGsdGsdAsdTslGslGslT



(SEQ ID NO: 105)
(SEQ ID NO: 106)


 


3186
TGCAGGGCCAGGTC
lTslGslCsdAsdGsdGsdGsdCsdCsdAsdGslGslTslC



(SEQ ID NO: 107)
(SEQ ID NO: 108)


 


3187
CTGCAGGGCCAGGT
lCslTslGsdCsdAsdGsdGsdGsdCsdCsdAslGslGslT



(SEQ ID NO: 109)
(SEQ ID NO: 110)


 


3255
ACCTTGGACTTGAT
lAslCslCsdTsdTsdGsdGsdAsdCsdTsdTslGslAslT



(SEQ ID NO: 111)
(SEQ ID NO: 112)


 


3257
GCACCTTGGACTTG
lGslCslAsdCsdCsdTsdTsdGsdGsdAsdCslTslTslG



(SEQ ID NO: 113)
(SEQ ID NO: 114)


 


3258
TGCACCTTGGACTT
lTslGslCsdAsdCsdCsdTsdTsdGsdGsdAslCslTslT



(SEQ ID NO: 115)
(SEQ ID NO: 116)


 


3259
CTGCACCTTGGACT
lCslTslGsdCsdAsdCsdCsdTsdTsdGsdGslAslCslT



(SEQ ID NO: 117)
(SEQ ID NO: 118)


 


3260
GCTGCACCTTGGAC
lGslCslTsdGsdCsdAsdCsdCsdTsdTsdGslGslAslC



(SEQ ID NO: 119)
(SEQ ID NO: 120)


 


3261
AGCTGCACCTTGGA
lAslGslCsdTsdGsdCsdAsdCsdCsdTsdTslGslGslA



(SEQ ID NO: 121)
(SEQ ID NO: 122)


 


4253
CTGCACTGGCCTTG
lCslTslGsdCsdAsdCsdTsdGsdGsdCsdCslTslTslG



(SEQ ID NO: 123)
(SEQ ID NO: 124)


 


4254
TCTGCACTGGCCTT
lTslCslTsdGsdCsdAsdCsdTsdGsdGsdCslCslTslT



(SEQ ID NO: 125)
(SEQ ID NO: 126)


 


4255
CTCTGCACTGGCCT
lCslTslCsdTsdGsdCsdAsdCsdTsdGsdGslCslCslT



(SEQ ID NO: 127)
(SEQ ID NO: 128)


 


4256
TCTCTGCACTGGCC
lTslCslTsdCsdTsdGsdCsdAsdCsdTsdGslGslCslC



(SEQ ID NO: 129)
(SEQ ID NO: 130)


 


4257
TTCTCTGCACTGGC
lTslTslCsdTsdCsdTsdGsdCsdAsdCsdTslGslGslC



(SEQ ID NO: 131)
(SEQ ID NO: 132)


 


4258
CTTCTCTGCACTGG
lCslTslTsdCsdTsdCsdTsdGsdCsdAsdCslTslGslG



(SEQ ID NO: 133)
(SEQ ID NO: 134)


 


4259
GCTTCTCTGCACTG
lGslCslTsdTsdCsdTsdCsdTsdGsdCsdAslCslTslG



(SEQ ID NO: 135)
(SEQ ID NO: 136)


 


4260
AGCTTCTCTGCACT
lAslGslCsdTsdTsdCsdTsdCsdTsdGsdCslAslCslT



(SEQ ID NO: 137)
(SEQ ID NO: 138)


 


4261
CAGCTTCTCTGCAC
lCslAslGsdCsdTsdTsdCsdTsdCsdTsdGslCslAslC



(SEQ ID NO: 139)
(SEQ ID NO: 140)


 


4262
ACAGCTTCTCTGCA
lAslCslAsdGsdCsdTsdTsdCsdTsdCsdTslGslCslA



(SEQ ID NO: 141)
(SEQ ID NO: 142)


 


4263
CACAGCTTCTCTGC
lCslAslCsdAsdGsdCsdTsdTsdCsdTsdCslTslGslC



(SEQ ID NO: 143)
(SEQ ID NO: l44)


 


4302
TTGATCTTGGCCTC
lTslTslGsdAsdTsdCsdTsdTsdGsdGsdCslCslTslC



(SEQ ID NO: 145)
(SEQ ID NO: l46)


 


4318
CTGCAGCTCCTCCA
lCslTslGsdCsdAsdGsdCsdTsdCsdCsdTslCslCslA



(SEQ ID NO: 147)
(SEQ ID NO: 148)


 


4717
TGCCTCCTGCAGCC
lTslGslCsdCsdTsdCsdCsdTsdGsdCsdAslGslCslC



(SEQ ID NO: 149)
(SEQ ID NO: 150)


 


4773
AGCTTGGCCTTCTC
lAslGslCsdTsdTsdGsdGsdCsdCsdTsdTslCslTslC



(SEQ ID NO: 151)
(SEQ ID NO: 152)


 


4904
TCTCCTCCTCCTGC
lTslCslTsdCsdCsdTsdCsdCsdTsdCsdCslTslGslC



(SEQ ID NO: 153)
(SEQ ID NO: 154)


 


6099
AGGTTCTTCCTGTC
lAslGslGsdTsdTsdCsdTsdTsdCsdCsdTslGslTslC



(SEQ ID NO: 155)
(SEQ ID NO: 156)


 


6101
CCAGGTTCTTCCTG
lCslCslAsdGsdGsdTsdTsdCsdTsdTsdCslCslTslG



(SEQ ID NO: 157)
(SEQ ID NO: 158)


 


6102
GCCAGGTTCTTCCT
lGslCslCsdAsdGsdGsdTsdTsdCsdTsdTslCslCslT



(SEQ ID NO: 159)
(SEQ ID NO: 160)


 


6103
AGCCAGGTTCTTCC
lAslGslCsdCsdAsdGsdGsdTsdTsdCsdTslTslCslC



(SEQ ID NO: 161)
(SEQ ID NO: 162)


 


6104
GAGCCAGGTTCTTC
lGslAslGsdCsdCsdAsdGsdGsdTsdTsdCslTslTslC



(SEQ ID NO: 163)
(SEQ ID NO: 164)


 


6142
GACCTTGCTCTGCA
lGslAslCsdCsdTsdTsdGsdCsdTsdCsdTslGslCslA



(SEQ ID NO: 165)
(SEQ ID NO: 166)


 


6143
TGACCTTGCTCTGC
lTslGslAsdCsdCsdTsdTsdGsdCsdTsdCslTslGslC



(SEQ ID NO: 167)
(SEQ ID NO: 168)


 


6144
TTGACCTTGCTCTG
lTslTslGsdAsdCsdCsdTsdTsdGsdCsdTslCslTslG



(SEQ ID NO: 169)
(SEQ ID NO: 170)


 


6146
TCTTGACCTTGCTC
lTslCslTsdTsdGsdAsdCsdCsdTsdTsdGslCslTslC



(SEQ ID NO: 171)
(SEQ ID NO: 172)


 


6149
AGCTCTTGACCTTG
lAslGslCsdTsdCsdTsdTsdGsdAsdCsdCslTslTslG



(SEQ ID NO: 173)
(SEQ ID NO: 174)


 


6151
GTAGCTCTTGACCT
lGslTslAsdGsdCsdTsdCsdTsdTsdGsdAslCslCslT



(SEQ ID NO: 175)
(SEQ ID NO: 176)


 


6220
GTGCTGGGCCTTGC
lGslTslGsdCsdTsdGsdGsdGsdCsdCsdTslTslGslC



(SEQ ID NO: 177)
(SEQ ID NO: 178)


 


6273
TTGTTGGCCTGGGT
lTslTslGsdTsdTsdGsdGsdCsdCsdTsdGslGslGslT



(SEQ ID NO: 179)
(SEQ ID NO: 180)


 


6277
CAGCTTGTTGGCCT
lCslAslGsdCsdTsdTsdGsdTsdTsdGsdGslCslCslT



(SEQ ID NO: 181)
(SEQ ID NO: 182)


 



GTGAATGCGGATGAA
lGslTslGsdAsdAsdTsdGsdCsdGsdGsdAsdTsdGslA



(SEQ ID NO: 1)
slA (SEQ ID NO: 3)





l = 1ocked nuc1eotide;


d = deoxynuc1eotide;


s = phosphorotioate 1inkage.















TABLE 2





Starting




nucleotide




of target




sequence




in SEQ




ID NO: 6
Sequence (5′-3′)
Sequence (5′-3′) with modifications







 513
TCACTCACATCCAT
lTslCslAsmdCsdTsmdCsdAsmdCsdAsdTsmdCslCslAslT



(SEQ ID NO: 183)
(SEQ ID NO: 184)


 


 514
TTCACTCACATCCA
lTslTslCsdAsmdCsdTsmdCsdAsmdCsdAsdTslCslCslA



(SEQ ID NO: 185)
(SEQ ID NO: 186)


 


 515
GTTCACTCACATCC
lGslTslTsmdCsdAsmdCsdTsmdCsdAsmdCsdAslTslCslC



(SEQ ID NO: 187)
(SEQ ID NO: 188)


 


 516
AGTTCACTCACATC
lAslGslTsdTsmdCsdAsmdCsdTsmdCsdAsmdCslAslTslC



(SEQ ID NO: 189)
(SEQ ID NO: 190)


 


 517
AAGTTCACTCACAT
lAslAslGsdTsdTsmdCsdAsmdCsdTsmdCsdAslCslAslT



(SEQ ID NO: 191)
(SEQ ID NO: 192)


 


 518
CAAGTTCACTCACA
lCslAslAsdGsdTsdTsmdCsdAsmdCsdTsmdCslAslCslA



(SEQ ID NO: 193)
(SEQ ID NO: 194)


 


 519
CCAAGTTCACTCAC
lCslCslAsdAsdGsdTsdTsmdCsdAsmdCsdTslCslAslC



(SEQ ID NO: 195)
(SEQ ID NO: 196)


 


 520
CCCAAGTTCACTCA
lCslCslCsdAsdAsdGsdTsdTsmdCsdAsmdCslTslCslA



(SEQ ID NO: 197)
(SEQ ID NO: 198)


 


 521
CCCCAAGTTCACTC
lCslCslCsmdCsdAsdAsdGsdTsdTsmdCsdAslCslTslC



(SEQ ID NO: 199)
(SEQ ID NO: 200)


 


 522
TCCCCAAGTTCACT
lTslCslCsmdCsmdCsdAsdAsdGsdTsdTsmdCslAslCslT



(SEQ ID NO: 201)
(SEQ ID NO: 202)


 


 523
CTCCCCAAGTTCAC
lCslTslCsmdCsmdCsmdCsdAsdAsdGsdTsdTslCslAslC



(SEQ ID NO: 203)
(SEQ ID NO: 204)


 


 524
ACTCCCCAAGTTCA
lAslCslTsmdCsmdCsmdCsmdCsdAsdAsdGsdTslTslCslA



(SEQ ID NO: 205)
(SEQ ID NO: 206)


 


 525
GACTCCCCAAGTTC
lGslAslCsdTsmdCsmdCsmdCsmdCsdAsdAsdGslTslTslC



(SEQ ID NO: 207)
(SEQ ID NO: 208)


 


 526
AGACTCCCCAAGTT
lAslGslAsmdCsdTsmdCsmdCsmdCsmdCsdAsdAslGslTslT



(SEQ ID NO: 209)
(SEQ ID NO: 210)


 


 527
CAGACTCCCCAAGT
lCslAslGsdAsmdCsdTsmdCsmdCsmdCsmdCsdAslAslGslT



(SEQ ID NO: 211)
(SEQ ID NO: 212)


 


 528
GCAGACTCCCCAAG
lGslCslAsdGsdAsmdCsdTsmdCsmdCsmdCsmdCslAslAslG



(SEQ ID NO: 213)
(SEQ ID NO: 214)


 


 610
AGGCACCCAGACTC
lAslGslGsmdCsdAsmdCsmdCsmdCsdAsdGsdAslCslTslC



(SEQ ID NO: 215)
(SEQ ID NO: 216)


 


 611
CAGGCACCCAGACT
lCslAslGsdGsmdCsdAsmdCsmdCsmdCsdAsdGslAslCslT



(SEQ ID NO: 217)
(SEQ ID NO: 218)


 


 612
TCAGGCACCCAGAC
lTslCslAsdGsdGsmdCsdAsmdCsmdCsmdCsdAslGslAslC



(SEQ ID NO: 219)
(SEQ ID NO: 220)


 


 613
ATCAGGCACCCAGA
lAslTslCsdAsdGsdGsmdCsdAsmdCsmdCsmdCslAslGslA



(SEQ ID NO: 221)
(SEQ ID NO: 222)


 


1447
GCTGCAGAAGTGGT
lGslCslTsdGsmdCsdAsdGsdAsdAsdGsdTslGslGslT



(SEQ ID NO: 223)
(SEQ ID NO: 224)


 


1518
ATGTCCATGGCATG
lAslTslGsdTsmdCsmdCsdAsdTsdGsdGsmdCslAslTslG



(SEQ ID NO: 225)
(SEQ ID NO: 226)


 


1560
ATCTTATAGCAGGC
lAslTslCsdTsdTsdAsdTsdAsdGsmdCsdAslGslGslC



(SEQ ID NO: 227)
(SEQ ID NO: 228)


 


1645
ACTCTCAGTGCCAT
lAslCslTsmdCsdTsmdCsdAsdGsdTsdGsmdCslCslAslT



(SEQ ID NO: 229)
(SEQ ID NO: 230)


 


1646
CACTCTCAGTGCCA
lCslAslCsdTsmdCsdTsmdCsdAsdGsdTsdGslCslCslA



(SEQ ID NO: 231)
(SEQ ID NO: 232)


 


1648
AGCACTCTCAGTGC
lAslGslCsdAsmdCsdTsmdCsdTsmdCsdAsdGslTslGslC



(SEQ ID NO: 233)
(SEQ ID NO: 234)


 


1649
CAGCACTCTCAGTG
lCslAslGsmdCsdAsmdCsdTsmdCsdTsmdCsdAslGslTslG



(SEQ ID NO: 235)
(SEQ ID NO: 236)


 


1650
TCAGCACTCTCAGT
lTslCslAsdGsmdCsdAsmdCsdTsmdCsdTsmdCslAslGslT



(SEQ ID NO: 237)
(SEQ ID NO: 238)


 


1651
GTCAGCACTCTCAG
lGslTslCsdAsdGsmdCsdAsmdCsdTsmdCsdTslCslAslG



(SEQ ID NO: 239)
(SEQ ID NO: 240)


 


1652
TGTCAGCACTCTCA
lTslGslTsmdCsdAsdGsmdCsdAsmdCsdTsmdCslTslCslA



(SEQ ID NO:241 )
(SEQ ID NO: 242)


 


1655
CCTTGTCAGCACTC
lCslCslTsdTsdGsdTsmdCsdAsdGsmdCsdAslCslTslC



(SEQ ID NO: 243)
(SEQ ID NO: 244)


 


1682
CACTGCTGACCCCC
lCslAslCsdTsdGsmdCsdTsdGsdAsmdCsmdCslCslCslC



(SEQ ID NO: 245)
(SEQ ID NO: 246)


 


1683
CCACTGCTGACCCC
lCslCslAsmdCsdTsdGsmdCsdTsdGsdAsmdCslCslCslC



(SEQ ID NO: 247)
(SEQ ID NO: 248)


 


1684
CCCACTGCTGACCC
lCslCslCsdAsmdCsdTsdGsmdCsdTsdGsdAslCslCslC



(SEQ ID NO: 249)
(SEQ ID NO: 250)


 


1685
CCCCACTGCTGACC
lCslCslCsmdCsdAsmdCsdTsdGsmdCsdTsdGslAslCslC



(SEQ ID NO: 251)
(SEQ ID NO: 252)


 


1686
TCCCCACTGCTGAC
lTslCslCsmdCsmdCsdAsmdCsdTsdGsmdCsdTslGslAslC



(SEQ ID NO: 253)
(SEQ ID NO: 254)


 


1687
GTCCCCACTGCTGA
lGslTslCsmdCsmdCsmdCsdAsmdCsdTsdGsmdCslTslGslA



(SEQ ID NO: 255)
(SEQ ID NO: 256)


 


1688
GGTCCCCACTGCTG
lGslGslTsmdCsmdCsmdCsmdCsdAsmdCsdTsdGslCslTslG



(SEQ ID NO: 257)
(SEQ ID NO: 258)


 


1689
AGGTCCCCACTGCT
lAslGslGsdTsmdCsmdCsmdCsmdCsdAsmdCsdTslGslCslT



(SEQ ID NO: 259)
(SEQ ID NO: 260)


 


1690
GAGGTCCCCACTGC
lGslAslGsdGsdTsmdCsmdCsmdCsmdCsdAsmdCslTslGslC



(SEQ ID NO: 261)
(SEQ ID NO: 262)


 


1691
GGAGGTCCCCACTG
lGslGslAsdGsdGsdTsmdCsmdCsmdCsmdCsdAslCslTslG



(SEQ ID NO: 263)
(SEQ ID NO: 264)


 


1692
AGGAGGTCCCCACT
lAslGslGsdAsdGsdGsdTsmdCsmdCsmdCsmdCslAslCslT



(SEQ ID NO: 265)
(SEQ ID NO: 266)


 


1693
GAGGAGGTCCCCAC
lGslAslGsdGsdAsdGsdGsdTsmdCsmdCsmdCslCslAslC



(SEQ ID NO: 267)
(SEQ ID NO: 268)


 


1694
TGAGGAGGTCCCCA
lTslGslAsdGsdGsdAsdGsdGsdTsmdCsmdCslCslCslA



(SEQ ID NO: 269)
(SEQ ID NO: 270)


 


1696
TTTGAGGAGGTCCC
lTslTslTsdGsdAsdGsdGsdAsdGsdGsdTslCslCslC



(SEQ ID NO: 271)
(SEQ ID NO: 272)


 


1702
AAGGCCTTTGAGGA
lAslAslGsdGsmdCsmdCsdTsdTsdTsdGsdAslGslGslA



(SEQ ID NO: 273)
(SEQ ID NO: 274)


 


1773
GCAAACACCACCTG
lGslCslAsdAsdAsmdCsdAsmdCsmdCsdAsmdCslCslTslG



(SEQ ID NO: 275)
(SEQ ID NO: 276)


 


1774
AGCAAACACCACCT
lAslGslCsdAsdAsdAsmdCsdAsmdCsmdCsdAslCslCslT



(SEQ ID NO: 277)
(SEQ ID NO: 278)


 


1775
CAGCAAACACCACC
lCslAslGsmdCsdAsdAsdAsmdCsdAsmdCsmdCslAslCslC



(SEQ ID NO:279 )
(SEQ ID NO: 280)


 


1776
ACAGCAAACACCAC
lAslCslAsdGsmdCsdAsdAsdAsmdCsdAsmdCslCslAslC



(SEQ ID NO: 281)
(SEQ ID NO: 282)


 


1828
AGACACCAGCCACC
lAslGslAsmdCsdAsmdCsmdCsdAsdGsmdCsmdCslAslCslC



(SEQ ID NO: 283)
(SEQ ID NO: 284)


 


1829
GAGACACCAGCCAC
lGslAslGsdAsmdCsdAsmdCsmdCsdAsdGsmdCslCslAslC



(SEQ ID NO: 285)
(SEQ ID NO: 286)


 


2218
CTTCTTGTCAGGCC
lCslTslTsmdCsdTsdTsdGsdTsmdCsdAsdGslGslCslC



(SEQ ID NO: 287)
(SEQ ID NO: 288)


 


2238
AAGTGGGCCTGGTA
lAslAslGsdTsdGsdGsdGsmdCsmdCsdTsdGslGslTslA



(SEQ ID NO: 289)
(SEQ ID NO: 290)


 


2254
GTAGTGGACCACCT
lGslTslAsdGsdTsdGsdGsdAsmdCsmdCsdAslCslCslT



(SEQ ID NO: 291)
(SEQ ID NO: 292)


 


2574
GCATCCATGACCCC
lGslCslAsdTsmdCsmdCsdAsdTsdGsdAsmdCslCslCslC



(SEQ ID NO: 293)
(SEQ ID NO: 294)


 


2575
GGCATCCATGACCC
lGslGslCsdAsdTsmdCsmdCsdAsdTsdGsdAslCslCslC



(SEQ ID NO: 295)
(SEQ ID NO: 296)


 


2576
AGGCATCCATGACC
lAslGslGsmdCsdAsdTsmdCsmdCsdAsdTsdGslAslCslC



(SEQ ID NO: 297)
(SEQ ID NO: 298)


 


2577
AAGGCATCCATGAC
lAslAslGsdGsmdCsdAsdTsmdCsmdCsdAsdTslGslAslC



(SEQ ID NO: 299)
(SEQ ID NO: 300)


 


4281
TGATCCTCATAGGT
lTslGslAsdTsmdCsmdCsdTsmdCsdAsdTsdAslGslGslT



(SEQ ID NO: 301)
(SEQ ID NO: 302)


 


4282
CTGATCCTCATAGG
lCslTslGsdAsdTsmdCsmdCsdTsmdCsdAsdTslAslGslG



(SEQ ID NO: 303)
(SEQ ID NO: 304)


 


4283
GCTGATCCTCATAG
lGslCslTsdGsdAsdTsmdCsmdCsdTsmdCsdAslTslAslG



(SEQ ID NO: 305)
(SEQ ID NO: 306)


 


4284
AGCTGATCCTCATA
lAslGslCsdTsdGsdAsdTsmdCsmdCsdTsmdCslAslTslA



(SEQ ID NO: 307)
(SEQ ID NO: 308)


 


4305
ACCTTGATCTTGGC
lAslCslCsdTsdTsdGsdAsdTsmdCsdTsdTslGslGslC



(SEQ ID NO: 309)
(SEQ ID NO: 310)


 


4656
ATGGCATCTGCTTC
lAslTslGsdGsmdCsdAsdTsmdCsdTsdGsmdCslTslTslC



(SEQ ID NO: 311)
(SEQ ID NO: 312)


 


4657
GATGGCATCTGCTT
lGslAslTsdGsdGsmdCsdAsdTsmdCsdTsdGslCslTslT



(SEQ ID NO: 313)
(SEQ ID NO: 314)


 


4661
TCTGGATGGCATCT
lTslCslTsdGsdGsdAsdTsdGsdGsmdCsdAslTslCslT



(SEQ ID NO: 315)
(SEQ ID NO: 316)


 


6141
ACCTTGCTCTGCAG
lAslCslCsdTsdTsdGsmdCsdTsmdCsdTsdGslCslAslG



(SEQ ID NO: 317)
(SEQ ID NO: 318)


 


436
AGGCAGCAGGCACT
lAslGslGsmdCsdAsdGsmdCsdAsdGsdGsmdCslAslCslT



(SEQ ID NO: 319)
(SEQ ID NO: 320)


 


437
AAGGCAGCAGGCAC
lAslAslGsdGsmdCsdAsdGsmdCsdAsdGsdGslCslAslC



(SEQ ID NO: 321)
(SEQ ID NO: 322)


 


438
CAAGGCAGCAGGCA
lCslAslAsdGsdGsmdCsdAsdGsmdCsdAsdGslGslCslA



(SEQ ID NO: 323)
(SEQ ID NO: 324)


 


439
CCAAGGCAGCAGGC
lCslCslAsdAsdGsdGsmdCsdAsdGsmdCsdAslGslGslC



(SEQ ID NO: 325)
(SEQ ID NO: 326)


 


482
GGTGAAGGAGGAGG
lGslGslTsdGsdAsdAsdGsdGsdAsdGsdGslAslGslG



(SEQ ID NO: 327)
(SEQ ID NO: 328)


 


483
GGGTGAAGGAGGAG
lGslGslGsdTsdGsdAsdAsdGsdGsdAsdGslGslAslG



(SEQ ID NO: 329)
(SEQ ID NO: 330)


 


509
TCACATCCATCATG
lTslCslAsmdCsdAsdTsmdCsmdCsdAsdTsmdCslAslTslG



(SEQ ID NO: 331)
(SEQ ID NO: 332)


 


511
ACTCACATCCATCA
lAslCslTsmdCsdAsmdCsdAsdTsmdCsmdCsdAslTslCslA



(SEQ ID NO: 333)
(SEQ ID NO: 334)


 


512
CACTCACATCCATC
lCslAslCsdTsmdCsdAsmdCsdAsdTsmdCsmdCslAslTslC



(SEQ ID NO: 335)
(SEQ ID NO: 336)


 


616
TTCATCAGGCACCC
lTslTslCsdAsdTsmdCsdAsdGsdGsmdCsdAslCslCslC



(SEQ ID NO: 337)
(SEQ ID NO: 338)


 


617
GTTCATCAGGCACC
lGslTslTsmdCsdAsdTsmdCsdAsdGsdGsmdCslAslCslC



(SEQ ID NO: 339)
(SEQ ID NO: 340)


 


622
GTCCTGTTCATCAG
lGslTslCsmdCsdTsdGsdTsdTsmdCsdAsdTslCslAslG



(SEQ ID NO: 341)
(SEQ ID NO: 342)


 


690
TGGTCTTTGGTCTC
lTslGslGsdTsmdCsdTsdTsdTsdGsdGsdTslCslTslC



(SEQ ID NO: 343)
(SEQ ID NO: 344)


 


804
TTGTGCAGCACAGA
lTslTslGsdTsdGsmdCsdAsdGsmdCsdAsmdCslAslGslA



(SEQ ID NO: 345)
(SEQ ID NO: 346)


 


805
GTTGTGCAGCACAG
lGslTslTsdGsdTsdGsmdCsdAsdGsmdCsdAslCslAslG



(SEQ ID NO: 347)
(SEQ ID NO: 348)


 


892
GACTGGGAGCCATT
lGslAslCsdTsdGsdGsdGsdAsdGsmdCsmdCslAslTslT



(SEQ ID NO: 349)
(SEQ ID NO: 350)


 


893
AGACTGGGAGCCAT
lAslGslAsmdCsdTsdGsdGsdGsdAsdGsmdCslCslAslT



(SEQ ID NO: 351)
(SEQ ID NO: 352)


 


1102
GTCTCCCAGGGCAG
lGslTslCsdTsmdCsmdCsmdCsdAsdGsdGsdGslCslAslG



(SEQ ID NO: 353)
(SEQ ID NO: 354)


 


1215
CTCAGGGTCTTGGC
lCslTslCsdAsdGsdGsdGsdTsmdCsdTsdTslGslGslC



(SEQ ID NO: 355)
(SEQ ID NO: 356)


 


1216
CCTCAGGGTCTTGG
lCslCslTsmdCsdAsdGsdGsdGsdTsmdCsdTslTslGslG



(SEQ ID NO: 357)
(SEQ ID NO: 358)


 


1217
TCCTCAGGGTCTTG
lTslCslCsdTsmdCsdAsdGsdGsdGsdTsmdCslTslTslG



(SEQ ID NO: 359)
(SEQ ID NO: 360)


 


1219
ATTCCTCAGGGTCT
lAslTslTsmdCsmdCsdTsmdCsdAsdGsdGsdGslTslCslT



(SEQ ID NO: 361)
(SEQ ID NO: 362)


 


1220
CATTCCTCAGGGTC
lCslAslTsdTsmdCsmdCsdTsmdCsdAsdGsdGslGslTslC



(SEQ ID NO: 363)
(SEQ ID NO: 364)


 


1222
ATCATTCCTCAGGG
lAslTslCsdAsdTsdTsmdCsmdCsdTsmdCsdAslGslGslG



(SEQ ID NO: 365)
(SEQ ID NO: 366)


 


1225
GTTATCATTCCTCA
lGslTslTsdAsdTsmdCsdAsdTsdTsmdCsmdCslTslCslA



(SEQ ID NO: 366)
(SEQ ID NO: 368)


 


1226
AGTTATCATTCCTC
lAslGslTsdTsdAsdTsmdCsdAsdTsdTsmdCslCslTslC



(SEQ ID NO: 369)
(SEQ ID NO: 370)


 


1268
AGGGACCAAAGTGA
lAslGslGsdGsdAsmdCsmdCsdAsdAsdAsdGslTslGslA



(SEQ ID NO: 371)
(SEQ ID NO: 372)


 


1273
CCCAGAGGGACCAA
lCslCslCsdAsdGsdAsdGsdGsdGsdAsmdCslCslAslA



(SEQ ID NO: 373)
(SEQ ID NO: 374)


 


1274
TCCCAGAGGGACCA
lTslCslCsmdCsdAsdGsdAsdGsdGsdGsdAslCslCslA



(SEQ ID NO: 375)
(SEQ ID NO: 376)


 


1275
TTCCCAGAGGGACC
lTslTslCsmdCsmdCsdAsdGsdAsdGsdGsdGslAslCslC



(SEQ ID NO: 377)
(SEQ ID NO: 378)


 


1276
CTTCCCAGAGGGAC
lCslTslTsmdCsmdCsmdCsdAsdGsdAsdGsdGslGslAslC



(SEQ ID NO: 379)
(SEQ ID NO: 380)


 


1277
GCTTCCCAGAGGGA
lGslCslTsdTsmdCsmdCsmdCsdAsdGsdAsdGslGslGslA



(SEQ ID NO: 381)
(SEQ ID NO: 382)


 


1301
AGCTGTCAATATCC
lAslGslCsdTsdGsdTsmdCsdAsdAsdTsdAslTslCslC



(SEQ ID NO: 383)
(SEQ ID NO: 384)


 


1302
TAGCTGTCAATATC
lTslAslGsmdCsdTsdGsdTsmdCsdAsdAsdTslAslTslC



(SEQ ID NO: 385)
(SEQ ID NO: 386)


 


1303
ATAGCTGTCAATAT
lAslTslAsdGsmdCsdTsdGsdTsmdCsdAsdAslTslAslT



(SEQ ID NO: 387)
(SEQ ID NO: 388)


 


1304
GATAGCTGTCAATA
lGslAslTsdAsdGsmdCsdTsdGsdTsmdCsdAslAslTslA



(SEQ ID NO: 389)
(SEQ ID NO: 390)


 


1379
CTGAGAGGATCTGG
lCslTslGsdAsdGsdAsdGsdGsdAsdTsmdCslTslGslG



(SEQ ID NO: 391)
(SEQ ID NO: 392)


 


1380
CCTGAGAGGATCTG
lCslCslTsdGsdAsdGsdAsdGsdGsdAsdTslCslTslG



(SEQ ID NO: 393)
(SEQ ID NO: 394)


 


1417
AGACAGAAGCAGCA
lAslGslAsmdCsdAsdGsdAsdAsdGsmdCsdAslGslCslA



(SEQ ID NO: 395)
(SEQ ID NO: 396)


 


1418
TAGACAGAAGCAGC
lTslAslGsdAsmdCsdAsdGsdAsdAsdGsmdCslAslGslC



(SEQ ID NO: 397)
(SEQ ID NO: 398)


 


1419
ATAGACAGAAGCAG
lAslTslAsdGsdAsmdCsdAsdGsdAsdAsdGslCslAslG



(SEQ ID NO: 399)
(SEQ ID NO: 400)


 


1420
CATAGACAGAAGCA
lCslAslTsdAsdGsdAsmdCsdAsdGsdAsdAslGslCslA



(SEQ ID NO: 401)
(SEQ ID NO: 402)


 


1421
TCATAGACAGAAGC
lTslCslAsdTsdAsdGsdAsmdCsdAsdGsdAslAslGslC



(SEQ ID NO: 403)
(SEQ ID NO: 404)


 


1422
TTCATAGACAGAAG
lTslTslCsdAsdTsdAsdGsdAsmdCsdAsdGslAslAslG



(SEQ ID NO: 405)
(SEQ ID NO: 406)


 


1423
GTTCATAGACAGAA
lGslTslTsmdCsdAsdTsdAsdGsdAsmdCsdAslGslAslA



(SEQ ID NO: 407)
(SEQ ID NO: 408)


 


1424
GGTTCATAGACAGA
lGslGslTsdTsmdCsdAsdTsdAsdGsdAsmdCslAslGslA



(SEQ ID NO: 409)
(SEQ ID NO: 410)


 


1425
GGGTTCATAGACAG
lGslGslGsdTsdTsmdCsdAsdTsdAsdGsdAslCslAslG



(SEQ ID NO: 411)
(SEQ ID NO: 412)


 


1484
CCCCATCATTCATG
lCslCslCsmdCsdAsdTsmdCsdAsdTsdTsmdCslAslTslG



(SEQ ID NO: 413)
(SEQ ID NO: 414)


 


1557
TTATAGCAGGCACA
lTslTslAsdTsdAsdGsmdCsdAsdGsdGsmdCslAslCslA



(SEQ ID NO: 415)
(SEQ ID NO: 416)


 


1558
CTTATAGCAGGCAC
lCslTslTsdAsdTsdAsdGsmdCsdAsdGsdGslCslAslC



(SEQ ID NO: 417)
(SEQ ID NO: 418)


 


1559
TCTTATAGCAGGCA
lTslCslTsdTsdAsdTsdAsdGsmdCsdAsdGslGslCslA



(SEQ ID NO: 419)
(SEQ ID NO: 420)


 


1561
GATCTTATAGCAGG
lGslAslTsmdCsdTsdTsdAsdTsdAsdGsmdCslAslGslG



(SEQ ID NO: 421)
(SEQ ID NO: 422)


 


1582
AAAGTGCAGGAGGG
lAslAslAsdGsdTsdGsmdCsdAsdGsdGsdAslGslGslG



(SEQ ID NO: 423)
(SEQ ID NO: 424)


 


1587
TTGCCAAAGTGCAG
lTslTslGsmdCsmdCsdAsdAsdAsdGsdTsdGslCslAslG



(SEQ ID NO: 425)
(SEQ ID NO: 426)


 


1703
AAAGGCCTTTGAGG
lAslAslAsdGsdGsmdCsmdCsdTsdTsdTsdGslAslGslG



(SEQ ID NO: 427)
(SEQ ID NO: 428)


 


1704
AAAAGGCCTTTGAG
lAslAslAsdAsdGsdGsmdCsmdCsdTsdTsdTslGslAslG



(SEQ ID NO: 429)
(SEQ ID NO: 430)


 


1705
CAAAAGGCCTTTGA
lCslAslAsdAsdAsdGsdGsmdCsmdCsdTsdTslTslGslA



(SEQ ID NO: 431)
(SEQ ID NO: 432)


 


1706
GCAAAAGGCCTTTG
lGslCslAsdAsdAsdAsdGsdGsmdCsmdCsdTslTslTslG



(SEQ ID NO: 433)
(SEQ ID NO: 434)


 


1707
TGCAAAAGGCCTTT
lTslGslCsdAsdAsdAsdAsdGsdGsmdCsmdCslTslTslT



(SEQ ID NO: 435)
(SEQ ID NO: 436)


 


1708
GTGCAAAAGGCCTT
lGslTslGsmdCsdAsdAsdAsdAsdGsdGsmdCslCslTslT



(SEQ ID NO: 437)
(SEQ ID NO: 438)


 


1709
GGTGCAAAAGGCCT
lGslGslTsdGsmdCsdAsdAsdAsdAsdGsdGslCslCslT



(SEQ ID NO: 439)
(SEQ ID NO: 440)


 


1710
GGGTGCAAAAGGCC
lGslGslGsdTsdGsmdCsdAsdAsdAsdAsdGslGslCslC



(SEQ ID NO: 441)
(SEQ ID NO: 442)


 


1804
GTCATAGGTGGCCT
lGslTslCsdAsdTsdAsdGsdGsdTsdGsdGslCslCslT



(SEQ ID NO: 443)
(SEQ ID NO: 444)


 


1805
GGTCATAGGTGGCC
lGslGslTsmdCsdAsdTsdAsdGsdGsdTsdGslGslCslC



(SEQ ID NO: 445)
(SEQ ID NO: 446)


 


1819
CCACCTGAACAGCC
lCslCslAsmdCsmdCsdTsdGsdAsdAsmdCsdAslGslCslC



(SEQ ID NO: 447)
(SEQ ID NO: 448)


 


1820
GCCACCTGAACAGC
lGslCslCsdAsmdCsmdCsdTsdGsdAsdAsmdCslAslGslC



(SEQ ID NO: 449)
(SEQ ID NO: 450)


 


1827
GACACCAGCCACCT
lGslAslCsdAsmdCsmdCsdAsdGsmdCsmdCsdAslCslCslT



(SEQ ID NO: 451)
(SEQ ID NO: 452)


 


1850
TGTCCAGGGTCTGG
lTslGslTsmdCsmdCsdAsdGsdGsdGsdTsmdCslTslGslG



(SEQ ID NO: 453)
(SEQ ID NO: 454)


 


1851
GTGTCCAGGGTCTG
lGslTslGsdTsmdCsmdCsdAsdGsdGsdGsdTslCslTslG



(SEQ ID NO: 455)
(SEQ ID NO: 456)


 


1852
TGTGTCCAGGGTCT
lTslGslTsdGsdTsmdCsmdCsdAsdGsdGsdGslTslCslT



(SEQ ID NO: 457)
(SEQ ID NO: 458)


 


2189
TGGGTGACTTCCCC
lTslGslGsdGsdTsdGsdAsmdCsdTsdTsmdCslCslCslC



(SEQ ID NO: 459)
(SEQ ID NO: 460)


 


2190
TTGGGTGACTTCCC
lTslTslGsdGsdGsdTsdGsdAsmdCsdTsdTslCslCslC



(SEQ ID NO: 461)
(SEQ ID NO: 462)


 


2191
ATTGGGTGACTTCC
lAslTslTsdGsdGsdGsdTsdGsdAsmdCsdTslTslCslC



(SEQ ID NO: 463)
(SEQ ID NO: 464)


 


2198
GCTGGAAATTGGGT
lGslCslTsdGsdGsdAsdAsdAsdTsdTsdGslGslGslT



(SEQ ID NO: 465)
(SEQ ID NO: 466)


 


2274
CTGTAAGGCACCAC
lCslTslGsdTsdAsdAsdGsdGsmdCsdAsmdCslCslAslC



(SEQ ID NO: 467)
(SEQ ID NO: 468)


 


2275
GCTGTAAGGCACCA
lGslCslTsdGsdTsdAsdAsdGsdGsmdCsdAslCslCslA



(SEQ ID NO: 469)
(SEQ ID NO: 470)


 


2276
TGCTGTAAGGCACC
lTslGslCsdTsdGsdTsdAsdAsdGsdGsmdCslAslCslC



(SEQ ID NO: 471)
(SEQ ID NO: 472)


 


2350
ATTCTGTGACTTCT
lAslTslTsmdCsdTsdGsdTsdGsdAsmdCsdTslTslCslT



(SEQ ID NO: 473)
(SEQ ID NO: 474)


 


2351
TATTCTGTGACTTC
lTslAslTsdTsmdCsdTsdGsdTsdGsdAsmdCslTslTslC



(SEQ ID NO: 475)
(SEQ ID NO: 476)


 


2352
CTATTCTGTGACTT
lCslTslAsdTsdTsmdCsdTsdGsdTsdGsdAslCslTslT



(SEQ ID NO: 477)
(SEQ ID NO: 478)


 


2353
CCTATTCTGTGACT
lCslCslTsdAsdTsdTsmdCsdTsdGsdTsdGslAslCslT



(SEQ ID NO: 479)
(SEQ ID NO: 480)


 


2354
GCCTATTCTGTGAC
lGslCslCsdTsdAsdTsdTsmdCsdTsdGsdTslGslAslC



(SEQ ID NO: 481)
(SEQ ID NO: 482)


 


2355
AGCCTATTCTGTGA
lAslGslCsmdCsdTsdAsdTsdTsmdCsdTsdGslTslGslA



(SEQ ID NO: 483)
(SEQ ID NO: 484)


 


2356
GAGCCTATTCTGTG
lGslAslGsmdCsmdCsdTsdAsdTsdTsmdCsdTslGslTslG



(SEQ ID NO: 485)
(SEQ ID NO: 486)


 


2357
GGAGCCTATTCTGT
lGslGslAsdGsmdCsmdCsdTsdAsdTsdTsmdCslTslGslT



(SEQ ID NO: 487)
(SEQ ID NO: 488)


 


2358
AGGAGCCTATTCTG
lAslGslGsdAsdGsmdCsmdCsdTsdAsdTsdTslCslTslG



(SEQ ID NO: 489)
(SEQ ID NO: 490)


 


2359
CAGGAGCCTATTCT
lCslAslGsdGsdAsdGsmdCsmdCsdTsdAsdTslTslCslT



(SEQ ID NO: 491)
(SEQ ID NO: 492)


 


2360
CCAGGAGCCTATTC
lCslCslAsdGsdGsdAsdGsmdCsmdCsdTsdAslTslTslC



(SEQ ID NO: 493)
(SEQ ID NO: 494)


 


2361
GCCAGGAGCCTATT
lGslCslCsdAsdGsdGsdAsdGsmdCsmdCsdTslAslTslT



(SEQ ID NO: 495)
(SEQ ID NO: 496)


 


2375
TCTCATAGAGAGTC
lTslCslTsmdCsdAsdTsdAsdGsdAsdGsdAslGslTslC



(SEQ ID NO: 497)
(SEQ ID NO: 498)


 


2376
TTCTCATAGAGAGT
lTslTslCsdTsmdCsdAsdTsdAsdGsdAsdGslAslGslT



(SEQ ID NO: 499)
(SEQ ID NO: 500)


 


2396
TGGAGCAGGAGCCC
lTslGslGsdAsdGsmdCsdAsdGsdGsdAsdGslCslCslC



(SEQ ID NO: 501)
(SEQ ID NO: 502)


 


2397
GTGGAGCAGGAGCC
lGslTslGsdGsdAsdGsmdCsdAsdGsdGsdAslGslCslC



(SEQ ID NO: 503)
(SEQ ID NO: 504)


 


2443
TGCTGCCTTCTTAC
lTslGslCsdTsdGsmdCsmdCsdTsdTsmdCsdTslTslAslC



(SEQ ID NO: 505)
(SEQ ID NO: 506)


 


2444
ATGCTGCCTTCTTA
lAslTslGsmdCsdTsdGsmdCsmdCsdTsdTsmdCslTslTslA



(SEQ ID NO: 507)
(SEQ ID NO: 508)


 


2445
GATGCTGCCTTCTT
lGslAslTsdGsmdCsdTsdGsmdCsmdCsdTsdTslCslTslT



(SEQ ID NO: 509)
(SEQ ID NO: 510)


 


2579
AGAAGGCATCCATG
lAslGslAsdAsdGsdGsmdCsdAsdTsmdCsmdCslAslTslG



(SEQ ID NO: 511)
(SEQ ID NO: 512)


 


2580
AAGAAGGCATCCAT
lAslAslGsdAsdAsdGsdGsmdCsdAsdTsmdCslCslAslT



(SEQ ID NO: 513)
(SEQ ID NO: 514)


 


2581
CAAGAAGGCATCCA
lCslAslAsdGsdAsdAsdGsdGsmdCsdAsdTslCslCslA



(SEQ ID NO: 515)
(SEQ ID NO: 516)


 


2584
CACCAAGAAGGCAT
lCslAslCsmdCsdAsdAsdGsdAsdAsdGsdGslCslAslT



(SEQ ID NO: 517)
(SEQ ID NO: 518)


 


2585
GCACCAAGAAGGCA
lGslCslAsmdCsmdCsdAsdAsdGsdAsdAsdGslGslCslA



(SEQ ID NO: 519)
(SEQ ID NO: 520)


 


2587
TAGCACCAAGAAGG
lTslAslGsmdCsdAsmdCsmdCsdAsdAsdGsdAslAslGslG



(SEQ ID NO: 521)
(SEQ ID NO: 522)


 


2588
GTAGCACCAAGAAG
lGslTslAsdGsmdCsdAsmdCsmdCsdAsdAsdGslAslAslG



(SEQ ID NO: 523)
(SEQ ID NO: 524)


 


2589
TGTAGCACCAAGAA
lTslGslTsdAsdGsmdCsdAsmdCsmdCsdAsdAslGslAslA



(SEQ ID NO: 525)
(SEQ ID NO: 526)


 


2590
GTGTAGCACCAAGA
lGslTslGsdTsdAsdGsmdCsdAsmdCsmdCsdAslAslGslA



(SEQ ID NO: 527)
(SEQ ID NO: 528)


 


2591
GGTGTAGCACCAAG
lGslGslTsdGsdTsdAsdGsmdCsdAsmdCsmdCslAslAslG



(SEQ ID NO: 529)
(SEQ ID NO: 530)


 


2592
TGGTGTAGCACCAA
lTslGslGsdTsdGsdTsdAsdGsmdCsdAsmdCslCslAslA



(SEQ ID NO: 531)
(SEQ ID NO: 532)


 


2709
GGGATGGCACTGGG
lGslGslGsdAsdTsdGsdGsmdCsdAsmdCsdTslGslGslG



(SEQ ID NO: 533)
(SEQ ID NO: 534)


 


2731
GCTGTCCATGAAGG
lGslCslTsdGsdTsmdCsmdCsdAsdTsdGsdAslAslGslG



(SEQ ID NO: 535)
(SEQ ID NO: 536)


 


2830
GCCTAGAAGCCCAG
lGslCslCsdTsdAsdGsdAsdAsdGsmdCsmdCslCslAslG



(SEQ ID NO: 537)
(SEQ ID NO: 538)


 


2986
GGCATTGAAGGCAC
lGslGslCsdAsdTsdTsdGsdAsdAsdGsdGslCslAslC



(SEQ ID NO: 539)
(SEQ ID NO: 540)


 


3248
ACTTGATCAGCAAG
lAslCslTsdTsdGsdAsdTsmdCsdAsdGsmdCslAslAslG



(SEQ ID NO: 541)
(SEQ ID NO: 542)


 


3249
GACTTGATCAGCAA
lGslAslCsdTsdTsdGsdAsdTsmdCsdAsdGslCslAslA



(SEQ ID NO: 543)
(SEQ ID NO: 544)


 


3625
GCATTCCAGGTCCT
lGslCslAsdTsdTsmdCsmdCsdAsdGsdGsdTslCslCslT



(SEQ ID NO: 545)
(SEQ ID NO: 546)


 


3626
AGCATTCCAGGTCC
lAslGslCsdAsdTsdTsmdCsmdCsdAsdGsdGslTslCslC



(SEQ ID NO: 547)
(SEQ ID NO: 548)


 


3628
GGAGCATTCCAGGT
lGslGslAsdGsmdCsdAsdTsdTsmdCsmdCsdAslGslGslT



(SEQ ID NO: 549)
(SEQ ID NO: 550)


 


3629
GGGAGCATTCCAGG
lGslGslGsdAsdGsmdCsdAsdTsdTsmdCsmdCslAslGslG



(SEQ ID NO: 551)
(SEQ ID NO: 552)


 


3631
CAGGGAGCATTCCA
lCslAslGsdGsdGsdAsdGsmdCsdAsdTsdTslCslCslA



(SEQ ID NO: 553)
(SEQ ID NO: 554)


 


3793
CAGGCTCAGCTGGC
lCslAslGsdGsmdCsdTsmdCsdAsdGsmdCsdTslGslGslC



(SEQ ID NO: 555)
(SEQ ID NO: 556)


 


3794
GCAGGCTCAGCTGG
lGslCslAsdGsdGsmdCsdTsmdCsdAsdGsmdCslTslGslG



(SEQ ID NO: 557)
(SEQ ID NO: 558)


 


4233
GTCAGAGTCTCCAC
lGslTslCsdAsdGsdAsdGsdTsmdCsdTsmdCslCslAslC



(SEQ ID NO: 559)
(SEQ ID NO: 560)


 


4234
GGTCAGAGTCTCCA
lGslGslTsmdCsdAsdGsdAsdGsdTsmdCsdTslCslCslA



(SEQ ID NO: 561)
(SEQ ID NO: 562)


 


4235
GGGTCAGAGTCTCC
lGslGslGsdTsmdCsdAsdGsdAsdGsdTsmdCslTslCslC



(SEQ ID NO: 563)
(SEQ ID NO: 564)


 


4286
TTAGCTGATCCTCA
lTslTslAsdGsmdCsdTsdGsdAsdTsmdCsmdCslTslCslA



(SEQ ID NO: 565)
(SEQ ID NO: 566)


 


4287
CTTAGCTGATCCTC
lCslTslTsdAsdGsmdCsdTsdGsdAsdTsmdCslCslTslC



(SEQ ID NO: 567)
(SEQ ID NO: 568)


 


4410
ATCAGACACTCCTT
lAslTslCsdAsdGsdAsmdCsdAsmdCsdTsmdCslCslTslT



(SEQ ID NO: 569)
(SEQ ID NO: 570)


 


4412
TGATCAGACACTCC
lTslGslAsdTsmdCsdAsdGsdAsmdCsdAsmdCslTslCslC



(SEQ ID NO: 571)
(SEQ ID NO: 572)


 


4413
CTGATCAGACACTC
lCslTslGsdAsdTsmdCsdAsdGsdAsmdCsdAslCslTslC



(SEQ ID NO: 573)
(SEQ ID NO: 574)


 


4414
ACTGATCAGACACT
lAslCslTsdGsdAsdTsmdCsdAsdGsdAsmdCslAslCslT



(SEQ ID NO: 575)
(SEQ ID NO: 576)


 


4415
GACTGATCAGACAC
lGslAslCsdTsdGsdAsdTsmdCsdAsdGsdAslCslAslC



(SEQ ID NO: 577)
(SEQ ID NO: 578)


 


4416
TGACTGATCAGACA
lTslGslAsmdCsdTsdGsdAsdTsmdCsdAsdGslAslCslA



(SEQ ID NO: 579)
(SEQ ID NO: 580)


 


4417
CTGACTGATCAGAC
lCslTslGsdAsmdCsdTsdGsdAsdTsmdCsdAslGslAslC



(SEQ ID NO: 581)
(SEQ ID NO: 582)


 


4418
GCTGACTGATCAGA
lGslCslTsdGsdAsmdCsdTsdGsdAsdTsmdCslAslGslA



(SEQ ID NO: 583)
(SEQ ID NO: 584)


 


4419
AGCTGACTGATCAG
lAslGslCsdTsdGsdAsmdCsdTsdGsdAsdTslCslAslG



(SEQ ID NO: 585)
(SEQ ID NO: 586)


 


4422
CTCAGCTGACTGAT
lCslTslCsdAsdGsmdCsdTsdGsdAsmdCsdTslGslAslT



(SEQ ID NO: 587)
(SEQ ID NO: 588)


 


4423
GCTCAGCTGACTGA
lGslCslTsmdCsdAsdGsmdCsdTsdGsdAsmdCslTslGslA



(SEQ ID NO: 589)
(SEQ ID NO: 590)


 


4424
GGCTCAGCTGACTG
lGslGslCsdTsmdCsdAsdGsmdCsdTsdGsdAslCslTslG



(SEQ ID NO: 591)
(SEQ ID NO: 592)


 


4797
TCCTCTGACTCTGT
lTslCslCsdTsmdCsdTsdGsdAsmdCsdTsmdCslTslGslT



(SEQ ID NO: 593)
(SEQ ID NO: 594)


 


4798
ATCCTCTGACTCTG
lAslTslCsmdCsdTsmdCsdTsdGsdAsmdCsdTslCslTslG



(SEQ ID NO: 595)
(SEQ ID NO: 596)


 


4799
CATCCTCTGACTCT
lCslAslTsmdCsmdCsdTsmdCsdTsdGsdAsmdCslTslCslT



(SEQ ID NO: 597)
(SEQ ID NO: 598)


 


4800
ACATCCTCTGACTC
lAslCslAsdTsmdCsmdCsdTsmdCsdTsdGsdAslCslTslC



(SEQ ID NO: 599)
(SEQ ID NO: 600)


 


4801
TACATCCTCTGACT
lTslAslCsdAsdTsmdCsmdCsdTsmdCsdTsdGslAslCslT



(SEQ ID NO: 601)
(SEQ ID NO: 602)


 


4802
TTACATCCTCTGAC
lTslTslAsmdCsdAsdTsmdCsmdCsdTsmdCsdTslGslAslC



(SEQ ID NO: 603)
(SEQ ID NO: 604)


 


4803
GTTACATCCTCTGA
lGslTslTsdAsmdCsdAsdTsmdCsmdCsdTsmdCslTslGslA



(SEQ ID NO: 605)
(SEQ ID NO: 606)


 


4804
GGTTACATCCTCTG
lGslGslTsdTsdAsmdCsdAsdTsmdCsmdCsdTslCslTslG



(SEQ ID NO: 607)
(SEQ ID NO: 608)


 


4805
GGGTTACATCCTCT
lGslGslGsdTsdTsdAsmdCsdAsdTsmdCsmdCslTslCslT



(SEQ ID NO: 609)
(SEQ ID NO: 610)


 


4806
AGGGTTACATCCTC
lAslGslGsdGsdTsdTsdAsmdCsdAsdTsmdCslCslTslC



(SEQ ID NO: 611)
(SEQ ID NO: 612)


 


4807
CAGGGTTACATCCT
lCslAslGsdGsdGsdTsdTsdAsmdCsdAsdTslCslCslT



(SEQ ID NO: 613)
(SEQ ID NO: 614)


 


4808
CCAGGGTTACATCC
lCslCslAsdGsdGsdGsdTsdTsdAsmdCsdAslTslCslC



(SEQ ID NO: 615)
(SEQ ID NO: 616)


 


4811
GCTCCAGGGTTACA
lGslCslTsmdCsmdCsdAsdGsdGsdGsdTsdTslAslCslA



(SEQ ID NO: 617)
(SEQ ID NO: 618)


 


4812
AGCTCCAGGGTTAC
lAslGslCsdTsmdCsmdCsdAsdGsdGsdGsdTslTslAslC



(SEQ ID NO: 619)
(SEQ ID NO: 620)


 


4836
GCAGCTGCTGAGGT
lGslCslAsdGsmdCsdTsdGsmdCsdTsdGsdAslGslGslT



(SEQ ID NO: 621)
(SEQ ID NO: 622)


 


4837
AGCAGCTGCTGAGG
lAslGslCsdAsdGsmdCsdTsdGsmdCsdTsdGslAslGslG



(SEQ ID NO: 623)
(SEQ ID NO: 624)


 


6033
CCCTTAAGGGCCTC
lCslCslCsdTsdTsdAsdAsdGsdGsdGsmdCslCslTslC



(SEQ ID NO: 625)
(SEQ ID NO: 626)





l = locked nucleotide;


d = deoxynucleotide;


mdC = 5-Methyl cytosine;


m = 2′-O-methyl nucleotide;


s = phosphorothioate linkage















TABLE 3





Starting




nucleotide




of target




sequence




in SEQ




ID NO: 6
Sequence (5′-3′)
Sequence (5′-3′) with modifications







 436
AGGCAGCAGGCACT
lAsdGslGsdCslAslGsdCsdAslGsdGslCsdAslCslT



(SEQ ID NO: 627)
(SEQ ID NO: 628)


 


 513
TCACTCACATCCAT
lTsdCslAsdCslTslCsdAsdCslAsdTslCsdCslAslT



(SEQ ID NO: 629)
(SEQ ID NO: 630)


 


 524
ACTCCCCAAGTTCA
lAsdCslTsdCslCslCsdCsdAslAsdGslTsdTslCslA



(SEQ ID NO: 631)
(SEQ ID NO: 632)


 


 610
AGGCACCCAGACTC
lAsdGslGsdCslAslCsdCsdCslAsdGslAsdCslTslC



(SEQ ID NO: 633)
(SEQ ID NO: 634)


 


 735
GGGTTCATGGGCTG
lGsdGslGsdTslTslCsdAsdTslGsdGslGsdCslTslG



(SEQ ID NO: 635)
(SEQ ID NO: 636)


 


 867
ATGGTGACACAGAA
lAsdTslGsdGslTslGsdAsdCslAsdCslAsdGslAslA



(SEQ ID NO: 637)
(SEQ ID NO: 638)


 


1020
ATCAGCATGGACTG
lAsdTslCsdAslGslCsdAsdTslGsdGslAsdCslTslG



(SEQ ID NO: 639)
(SEQ ID NO: 640)


 


1061
GCTTGGTGTTAACC
lGsdCslTsdTslGslGsdTsdGslTsdTslAsdAslCslC



(SEQ ID NO: 641)
(SEQ ID NO: 642)


 


1188
TCCATGGCAGGGTT
lTsdCslCsdAslTslGsdGsdCslAsdGslGsdGslTslT



(SEQ ID NO: 643)
(SEQ ID NO: 644)


 


1305
AGATAGCTGTCAAT
lAsdGslAsdTslAslGsdCsdTslGsdTslCsdAslAslT



(SEQ ID NO: 645)
(SEQ ID NO: 646)


 


1443
CAGAAGTGGTAGTC
lCsdAslGsdAslAslGsdTsdGslGsdTslAsdGslTslC



(SEQ ID NO: 647)
(SEQ ID NO: 648)


 


1447
GCTGCAGAAGTGGT
lGsdCslTsdGslCslAsdGsdAslAsdGslTsdGslGslT



(SEQ ID NO: 649)
(SEQ ID NO: 650)


 


1518
ATGTCCATGGCATG
lAsdTslGsdTslCslCsdAsdTslGsdGslCsdAslTslG



(SEQ ID NO: 651)
(SEQ ID NO: 652)


 


1560
ATCTTATAGCAGGC
lAsdTslCsdTslTslAsdTsdAslGsdCslAsdGslGslC



(SEQ ID NO: 653)
(SEQ ID NO: 654)


 


1647
GCACTCTCAGTGCC
lGsdCslAsdCslTslCsdTsdCslAsdGslTsdGslCslC



(SEQ ID NO: 655)
(SEQ ID NO: 656)


 


1662
TAGGCAGCCTTGTC
lTsdAslGsdGslCslAsdGsdCslCsdTslTsdGslTslC



(SEQ ID NO: 657)
(SEQ ID NO: 658)


 


1694
TGAGGAGGTCCCCA
lTsdGslAsdGslGslAsdGsdGslTsdCslCsdCslCslA



(SEQ ID NO: 659)
(SEQ ID NO: 660)


 


1755
ACACTCTGGCCCTT
lAsdCslAsdCslTslCsdTsdGslGsdCslCsdCslTslT



(SEQ ID NO: 661)
(SEQ ID NO: 662)


 


1764
ACCTGCTCCACACT
lAsdCslCsdTslGslCsdTsdCslCsdAslCsdAslCslT



(SEQ ID NO: 663)
(SEQ ID NO: 664)


 


1828
AGACACCAGCCACC
lAsdGslAsdCslAslCsdCsdAslGsdCslCsdAslCslC



(SEQ ID NO: 665)
(SEQ ID NO: 666)


 


1912
CTCAAAGATCTCAA
lCsdTslCsdAslAslAsdGsdAslTsdCslTsdCslAslA



(SEQ ID NO: 667)
(SEQ ID NO: 668)


 


2037
ATGAAGACCCAGTC
lAsdTslGsdAslAslGsdAsdCslCsdCslAsdGslTslC



(SEQ ID NO: 669)
(SEQ ID NO: 670)


 


2218
CTTCTTGTCAGGCC
lCsdTslTsdCslTslTsdGsdTslCsdAslGsdGslCslC



(SEQ ID NO: 671)
(SEQ ID NO: 672)


 


2253
TAGTGGACCACCTC
lTsdAslGsdTslGslGsdAsdCslCsdAslCsdCslTslC



(SEQ ID NO: 673)
(SEQ ID NO: 674)


 


2481
AGGTTCTCCTTGTG
lAsdGslGsdTslTslCsdTsdCslCsdTslTsdGslTslG



(SEQ ID NO: 675)
(SEQ ID NO: 676)


 


2518
GGGCTGTGTGGCCC
lGsdGslGsdCslTslGsdTsdGslTsdGslGsdCslCslC



(SEQ ID NO: 677)
(SEQ ID NO: 678)


 


2738
CCTTCCTGCTGTCC
lCsdCslTsdTslCslCsdTsdGslCsdTslGsdTslCslC



(SEQ ID NO: 679)
(SEQ ID NO: 680)


 


2961
TGGATGGTGAACAG
lTsdGslGsdAslTslGsdGsdTslGsdAslAsdCslAslG



(SEQ ID NO: 681)
(SEQ ID NO: 682)


 


3186
TGCAGGGCCAGGTC
lTsdGslCsdAslGslGsdGsdCslCsdAslGsdGslTslC



(SEQ ID NO: 683)
(SEQ ID NO: 684)


 


3255
ACCTTGGACTTGAT
lAsdCslCsdTslTslGsdGsdAslCsdTslTsdGslAslT



(SEQ ID NO: 685)
(SEQ ID NO: 686)


 


3625
GCATTCCAGGTCCT
lGsdCslAsdTslTslCsdCsdAslGsdGslTsdCslCslT



(SEQ ID NO: 687)
(SEQ ID NO: 688)


 


3794
GCAGGCTCAGCTGG
lGsdCslAsdGslGslCsdTsdCslAsdGslCsdTslGslG



(SEQ ID NO: 689)
(SEQ ID NO: 690)


 


4253
CTGCACTGGCCTTG
lCsdTslGsdCslAslCsdTsdGslGsdCslCsdTslTslG



(SEQ ID NO: 691)
(SEQ ID NO: 692)


 


4259
GCTTCTCTGCACTG
lGsdCslTsdTslCslTsdCsdTslGsdCslAsdCslTslG



(SEQ ID NO: 693)
(SEQ ID NO: 694)


 


4302
TTGATCTTGGCCTC
lTsdTslGsdAslTslCsdTsdTslGsdGslCsdCslTslC



(SEQ ID NO: 695)
(SEQ ID NO: 696)


 


4318
CTGCAGCTCCTCCA
lCsdTslGsdCslAslGsdCsdTslCsdCslTsdCslCslA



(SEQ ID NO: 697)
(SEQ ID NO: 698)


 


4416
TGACTGATCAGACA
lTsdGslAsdCslTslGsdAsdTslCsdAslGsdAslCslA



(SEQ ID NO: 699)
(SEQ ID NO: 700)


 


4656
ATGGCATCTGCTTC
lAsdTslGsdGslCslAsdTsdCslTsdGslCsdTslTslC



(SEQ ID NO: 701)
(SEQ ID NO: 702)


 


4717
TGCCTCCTGCAGCC
lTsdGslCsdCslTslCsdCsdTslGsdCslAsdGslCslC



(SEQ ID NO: 703)
(SEQ ID NO: 704)


 


4773
AGCTTGGCCTTCTC
lAsdGslCsdTslTslGsdGsdCslCsdTslTsdCslTslC



(SEQ ID NO: 705)
(SEQ ID NO: 706)


 


4802
TTACATCCTCTGAC
lTsdTslAsdCslAslTsdCsdCslTsdCslTsdGslAslC



(SEQ ID NO: 707)
(SEQ ID NO: 708)


 


4837
AGCAGCTGCTGAGG
lAsdGslCsdAslGslCsdTsdGslCsdTslGsdAslGslG



(SEQ ID NO: 709)
(SEQ ID NO: 710)


 


4904
TCTCCTCCTCCTGC
lTsdCslTsdCslCslTsdCsdCslTsdCslCsdTslGslC



(SEQ ID NO: 711)
(SEQ ID NO: 712)


 


6033
CCCTTAAGGGCCTC
lCsdCslCsdTslTslAsdAsdGslGsdGslCsdCslTslC



(SEQ ID NO: 713)
(SEQ ID NO: 714)


 


6099
AGGTTCTTCCTGTC
lAsdGslGsdTslTslCsdTsdTslCsdCslTsdGslTslC



(SEQ ID NO: 715)
(SEQ ID NO: 716)


 


6142
GACCTTGCTCTGCA
lGsdAslCsdCslTslTsdGsdCslTsdCslTsdGslCslA



(SEQ ID NO: 717)
(SEQ ID NO: 718)


 


6149
AGCTCTTGACCTTG
lAsdGslCsdTslCslTsdTsdGslAsdCslCsdTslTslG



(SEQ ID NO: 719)
(SEQ ID NO: 720)


 


6220
GTGCTGGGCCTTGC
lGsdTslGsdCslTslGsdGsdGslCsdCslTsdTslGslC



(SEQ ID NO: 721)
(SEQ ID NO: 722)


 


6273
TTGTTGGCCTGGGT
lTsdTslGsdTslTslGsdGsdCslCsdTslGsdGslGslT



(SEQ ID NO: 723)
(SEQ ID NO: 724)


 


6277
CAGCTTGTTGGCCT
lCsdAslGsdCslTslTsdGsdTslTsdGslGsdCslCslT



(SEQ ID NO: 725)
(SEQ ID NO: 726)


















TABLE 4





Starting 




nuc1eotide




of target




sequence




in SEQ ID




NO: 6
Sequence (5′-3′)
Sequence (5′-3′) with modifications







4302
+TT+GA+T+CTT+GG+
lTsdTslGsdAsLTslCsdTsdTslGsdGslCsdCslTslC



CC+T+C
(SEQ ID NO: 728)



(SEQ ID NO: 727)



 


4302
+TT+GA+TC+T+T+GG
lTsdTsLGsdAslTsdCslTslTslGsdGsdCslCsdTslC



C+CT+C
(SEQ ID NO: 730)



(SEQ ID NO: 729)



 


4302

lTslTslGsdAsdTsmdCsdTsdTsdGsdGsmdCslCslTslC




(SEQ ID NO: 731)


 


4302
TTGATCTTGGCCTC
eTseTseGsdAsdTsmdCsdTsdTsdGsdGsmdCseCseTseC



(SEQ ID NO: 732)
(SEQ ID NO: 733)


 


4302
TTGATCTTGGCCTC
lTslTsmGsdAsdTsmdCsdTsdTsdGsdGsmdCsmCslTslC



(SEQ ID NO: 734)
(SEQ ID NO: 735)


 


4302
TTGATCTTGGCCTC
eTseTslGsdAsdTsmdCsdTsdTsdGsdGsmdCseCseTseC



(SEQ ID NO: 736)
(SEQ ID NO: 737)


 


4302
TTGATCTTGGCCTC
eTslTslGsdAsdTsmdCsdTsdTsdGsdGsmdCslCslTslC



(SEQ ID NO: 738)
(SEQ ID NO: 739)


 


4302
TTGATCTTGGCCTC
lTseTslGsdAsdTsmdCsdTsdTsdGsdGsmdCslCslTslC



(SEQ ID NO: 740)
(SEQ ID NO: 741)


 


4302
TTGATCTTGGCCTC
lTslTseGsdAsdTsmdCsdTsdTsdGsdGsmdCslCslTslC



(SEQ ID NO: 742)
(SEQ ID NO: 743)


 


4302
TTGATCTTGGCCTC
lTslTslGsdAsdTsmdCsdTsdTsdGsdGsmdCseCslTslC



(SEQ ID NO: 744)
(SEQ ID NO: 745)


 


4302
TTGATCTTGGCCTC
lTslTslGsdAsdTsmdCsdTsdTsdGsdGsmdCslCseTslC



(SEQ ID NO: 746)
(SEQ ID NO: 747)


 


4302
TTGATCTTGGCCTC
lTslTslGsdAsdTsmdCsdTsdTsdGsdGsmdCslCslTseC



(SEQ ID NO: 748)
(SEQ ID NO: 749)


 


4302
TTGATCTTGGCCTC
lTslTslGdAdTmdCdTdTdGdGmdClCslTslC



(SEQ ID NO: 750)
(SEQ ID NO: 751)


 


4302
TTGATCTTGGCCTC
lTslTslGsdAdTmdCdTdTdGdGmdCslCslTslC



(SEQ ID NO: 752)
(SEQ ID NO: 753)


 


4302
TTGATCTTGGCCTC
lTslTslGdAsdTmdCsdTdTsdGdGsmdClCslTslC



(SEQ ID NO: 754)
(SEQ ID NO: 755)


 


4302
TTGATCTTGGCCTC
lTslTlGsdAdTsmdCdTsdTdGsdGmdCslClTslC



(SEQ ID NO: 756)
(SEQ ID NO: 757)


 


4302
TTGATCTTGGCCTC
lTslTslGdAsdTsmdCsdTdTsdGsdGsmdClCslTsLC



(SEQ ID NO: 758)
(SEQ ID NO: 759)


 


4318
CTGCAGCTCCTCCA
lCslTsmGsmdCsdAsdGsmdCsdTsmdCsmdCsdTsmCslCslA



(SEQ ID NO: 760)
(SEQ ID NO: 761)


 


4318
CTGCAGCTCCTCCA
lCslTslGmdCsdAdGsmdCdTsmdCmdCsdTlCslCslA



(SEQ ID NO: 762)
(SEQ ID NO: 763)


 


4318
CTGCAGCTCCTCCA
lCslTslGmdCsdAsdGsmdCdTsmdCsmdCsdTlCslCslA



(SEQ ID NO: 764)
(SEQ ID NO: 765)


 


4302
TTGATCTTGGCCTC
lTsmUslGsdAsdTsmdCsdTsdTsdGsdGsmdCslCsmuslC



(SEQ ID NO: 766)
(SEQ ID NO: 767)


 


4302
TTGATCTTGGCCTC
lTslTsfGsdAsdTsmdCsdTsdTsdGsdGsmdCsfCslTslC



(SEQ ID NO: 768)
(SEQ ID NO: 769)


 


4661

lTslCsmUsdGsdGsdAsdTsdGsdGsmdCsdAsmUslCsLT




(SEQ ID NO: 770)


 


 892

lGslAsmCsdTsdGsdGsdGsdAsdGsmdCsmdCsmAslTslT




(SEQ ID NO: 771)


 


2360

lCslCsmAsdGsdGsdAsdGsmdCsmdCsdTsdAsmUslTslC




(SEQ ID NO: 772)


 


1417

lAslGsmAsmdCsdAsdGsdAsdAsdGsmdCsdAsmGslCslA




(SEQ ID NO: 773)


 


 516

lAslGsmUsdTsmdCsdAsmdCsdTsmdCsdAsmdCsmAslTslC




(SEQ ID NO: 774)


 


1582

lAslAsmAsdGsdTsdGsmdCsdAsdGsdGsdAsmGslGslG




(SEQ ID NO: 775)


 


1217

lTslCsmCsdTsmdCsdAsdGsdGsdGsdTsmdCsmUslTslG




(SEQ ID NO: 776)


 


 515

lGslTsmUsmdCsdAsmdCsdTsmdCsdAsmdCsdAsmUslCslC




(SEQ ID NO: 777)


 


 514

lTslTsmCsdAsmdCsdTsmdCsdAsmdCsdAsdTsmCslCslA




(SEQ ID NO: 778)


 


1424

lGslGsmUsdTsmdCsdAsdTsdAsdGsdAsmdCsmAslGslA




(SEQ ID NO: 779)


 


1219

lAslTsmUsmdCsmdCsdTsmdCsdAsdGsdGsdGsmUslCslT




(SEQ ID NO: 780)


 


513

lTslCsmAsmdCsdTsmdCsdAsmdCsdAsdTsmdCsmCslAslT




(SEQ ID NO: 781)


 


1226

lAslGsmUsdTsdAsdTsmdCsdAsdTsdTsmdCsmCslTslC




(SEQ ID NO: 782)


 


1225

lGslTsmUsdAsdTsmdCsdAsdTsdTsmdCsmdCsmUslCslA




(SEQ ID NO: 783)


 


1645

lAslCsmUsmdCsdTsmdCsdAsdGsdTsdGsmdCsmCslAslT




(SEQ ID NO: 784)


 


4302

avTsavTsavGsdAsdTsmdCsdTsdTsdGsdGsmdCsavCsavT




savC (SEQ ID NO: 785)


 


3257

avGsavCsavAsdCsdCsdTsdTsdGsdGsdAsdCsavTsavTsa




vG (SEQ ID NO: 786)


 


2483

avTsavGsavAsdGsdGsdTsdTsdCsdTsdCsdCsavTsavTsa




vG (SEQ ID NO: 787)





l = locked nucleotide;


d = deoxynucleotide;


mdC = 5-Methyl cytosine;


f = 2′-fluoro nucleotide;


av = amino-CBBN nucleotide;


s = phosphorothioate linkage;


e = ethylene-bridged nucleotide













TABLE 5





Sequence (5′-3′) with modifications

















lGslTsdGsdAsdAsdTsdGsdCsdGsdGsdAsdTslGslAslA



(SEQ ID NO: 789)



 



lGslAslAsdGsdTsdGsdGsdTsdAsdGsdTsdCsdAslTslA



(SEQ ID NO: 790)



 



lGsdTslGsdAsdAslTslGsdCsdGslGsdAslTsdGslAslA



(SEQ ID NO: 791)



 



lGsdAslAsdGsdTslGslGsdTsdAslGsdTslCsdAslTslA



(SEQ ID NO: 792)










In one embodiment, the present invention provides methods for treating or preventing a cardiac disorder in which the expression of β-MHC is up-regulated in cardiac cells of a subject comprising administering a composition comprising an inhibitor of MYH7B to the subject. In another embodiment, the present invention provides methods for treating or preventing a cardiac disorder in which the ratio of β-MHC/α-MHC is altered in cardiac cells of a subject comprising administering a composition comprising an inhibitor of MYH7B to the subject. For instance, the present invention provides compositions and methods for treating or preventing a cardiac disorder selected from the group consisting of pathologic cardiac hypertrophy, myocardial infarction, heart failure, or hypertrophic cardiomyopathy by inhibiting the expression or activity of β-MHC and/or altering the ratio of β-MHC/α-MHC in cardiac cells of a subject using an inhibitor of MYH7B. The administration of an inhibitor of MYH7B to the subject down-regulates the expression or activity of MYH7B in cardiac cells of the subject following administration. In one embodiment, the administration of an inhibitor of MYH7B does not significantly alter the expression or activity of myomiRs, particularly, miR-499, in cardiac cells of the subject. Thus, in some embodiments, the expression of miR-499, miR-208a, and/or miR-208b in cardiac cells is not statistically different after the administration of the MYH7B inhibitor.
In various embodiments, methods provided by the invention comprise administering one or more nucleic acid inhibitors disclosed herein. For instance, in one embodiment, methods provided by the invention comprise administering an antisense oligonucleotide inhibitor selected from Tables 1-5. In another embodiment, methods according to the invention comprise administering an antisense oligonucleotide inhibitor comprising the sequence of SEQ ID NO: 146. In yet another embodiment, methods according to the invention comprise administering an antisense oligonucleotide inhibitor comprising the sequence of SEQ ID NO: 148.
In a particular embodiment, the present invention provides methods for treating or preventing hypertrophic cardiomyopathy in a subject in need thereof comprising administering a composition comprising an inhibitor of MYH7B to the subject, wherein the expression or activity of MYH7B is reduced in cardiac cells of the subject following administration of the inhibitor. In one embodiment, the administration of an inhibitor of MYH7B to the subject reduces the expression or activity of β-MHC in cardiac cells of the subject following administration.
In one embodiment, the present invention provides methods for treating or preventing pathologic cardiac hypertrophy or hypertrophic cardiomyopathy in a subject at risk of developing pathologic cardiac hypertrophy or hypertrophic cardiomyopathy. The patient at risk of developing pathologic cardiac hypertrophy or hypertrophic cardiomyopathy may exhibit one or more risk factors including, for example, long standing uncontrolled hypertension, uncorrected valvular disease, chronic angina, recent myocardial infarction, congenital predisposition to heart disease or pathological hypertrophy. In some embodiments, the subject at risk may be diagnosed as having a genetic predisposition to pathologic cardiac hypertrophy or hypertrophic cardiomyopathy. For instance, in certain embodiments, the subject at risk may carry one or more mutations in sarcomeric genes, e.g., mutations in the β-MHC (MYH7) gene. In one embodiment, the subject at risk has one or more mutations in the β-MHC (MYH7) gene selected from Arg663His, Lys207Asn, Gly256Glu, Arg403Gln, Arg453Cys, Gly584Arg, Arg719Trp, Arg723Gly, Ile736Thr, Gly741Arg, Asp778Gly, Asp778Val, Asp906Gly, and Leu908Val. In certain embodiments, the subject at risk has an Arg403Gln, Arg453Cys, or Arg719Trp mutation in the β-MHC (MYH7) gene. Other mutations in sarcomeric genes associated with hypertrophic cardiomyopathy are known to those of skill in the art (see, e.g., Moore et al., Circulation Research, Vol. 111: 375-385, 2012, which hereby incorporated by reference in its entirety), and can be used to identify a subject at risk of developing pathologic cardiac hypertrophy or hypertrophic cardiomyopathy. In other particular embodiments, the subject at risk may have a familial history of pathologic cardiac hypertrophy or hypertrophic cardiomyopathy.
In another embodiment, the present invention provides compositions and methods for inhibiting the expression or activity of β-MHC in cardiac cells of a subject comprising administering an inhibitor of MYH7B to the subject. In yet another embodiment, the present invention provides compositions and methods for modulating the ratio of β-MHC/α-MHC in cardiac cells of a subject comprising administering an inhibitor of MYH7B to the subject. The administration of an inhibitor of MYH7B to the subject reduces the expression or activity of MYH7B and/or the expression or activity of β-MHC in cardiac cells of the subject compared to the expression or activity of MYH7B and β-MHC prior to the treatment with an inhibitor of MYH7B. In one embodiment, the administration of an inhibitor of MYH7B to the subject fully or partially restores the β-MHC/α-MHC ratio in cardiac cells of the subject to normal levels found in a healthy subject.
According to one aspect of the invention, administration of an inhibitor of MYH7B to the subject results in the improvement of one or more symptoms of the cardiac disorder. For instance, in one embodiment, administration of a MYH7B inhibitor results in the improvement of one or more symptoms of pathologic cardiac hypertrophy, myocardial infarction, or heart failure in the subject, or in the delay in the transition from pathologic cardiac hypertrophy to heart failure. In another embodiment, administration of a MYH7B inhibitor results in the delay in the onset of pathologic cardiac hypertrophy, myocardial infarction, or heart failure in the subject. The one or more improved symptoms may be, for example, increased exercise capacity, increased cardiac ejection volume, decreased left ventricular end diastolic pressure, decreased pulmonary capillary wedge pressure, increased cardiac output, increased cardiac index, lowered pulmonary artery pressures, decreased left ventricular end systolic and diastolic dimensions, decreased cardiac fibrosis, decreased collagen deposition in cardiac muscle, decreased left and right ventricular wall stress, decreased wall tension, increased quality of life, and decreased disease-related morbidity or mortality.
In some embodiments, an inhibitor of MYH7B is an aptamer. The term “aptamer” as used herein refers to a nucleic acid molecule having a specific binding affinity for a particular target molecule through interactions other than classic Watson-Crick base pairing. The aptamer can inhibit the activity or function of a target molecule by binding to the target molecule. In some embodiments, the aptamer of the present invention binds to one or more epitopes of the MYH7B protein. In certain embodiments, the aptamer binds to the human MYH7B protein. The anti-MYH7B aptamers may be comprised of ribonucleotides, deoxyribonucleotides, modified nucleotides, or mixtures thereof. For example, the aptamers may contain one or more 2′-O-alkyl (e.g. 2′-O-methyl) or 2′-halo (e.g. 2′-fluoro) modifications. In some embodiments the aptamer may be conjugated to a polymer, e.g. PEG polymer, to enhance the circulating half-life of the aptamer.
An aptamer for MYH7B can be identified using systematic evolution of ligands by exponential enrichment (SELEX) (Tuerk & Gold, Science 249:505-510, 1990; Ellington & Szostak, Nature 346:818-822, 1990), such as described in U.S. Pat. No. 5,270,163. A library of nucleic acids can be contacted with a target MYH7B, and those nucleic acids specifically bound to the target are partitioned from the remainder of nucleic acids in the library which do not specifically bind the target. The partitioned nucleic acids are amplified to yield a ligand-enriched pool. Multiple cycles of binding, partitioning, and amplifying (i.e., selection) result in identification of one or more aptamers with the desired activity. Modified methods, such as Laser SELEX or deSELEX as described in U.S. Patent Publication No. 20090264508 can also be used.
In certain embodiments, the length of an aptamer useful for inhibiting the expression or activity of MYH7B is about 10 to about 30 nucleotides, about 15 to about 35 nucleotides, about 20 to about 35 nucleotides, about 20 to about 40 nucleotides, or about 25 to about 50 nucleotides.
In some embodiments, an inhibitor of MYH7B is a ribozyme. The term “ribozyme” refers to an RNA molecule having catalytic activity. Ribozymes with a variety of activities are known. Alternatively, ribozymes having a target specific RNA-cleaving activity can be designed. In certain embodiments, ribozymes that can be used as an inhibitor of MYH7B include known or artificially-created RNase P ribozymes such as group I and group II intron-type ribozymes, hammerhead ribozymes and hairpin ribozymes. In some embodiments, a ribozyme that can be used as an inhibitor of MYH7B has a length of about 200 to about 500 nucleotides or about 300 to about 450 nucleotides. In other embodiments, a ribozyme inhibitor of MYH7B may have a length of about 30 to about 100 nucleotides, about 40 to about 80 nucleotides, or about 40 to about 60 nucleotides.
In one embodiment, an inhibitor of MYH7B is a small interfering RNA. As used herein, the terms “small interfering” or “short interfering RNA” or “siRNA” refer to an RNA duplex of nucleotides that is targeted to MYH7B gene and is capable of inhibiting the expression of MYH7B gene. In certain embodiments, the siRNA that may be used as an inhibitor of MYH7B comprises two complementary single-stranded RNAs of about 10 to about 30 nucleotides, particularly, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 30 nucleotides that form 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 30 base pairs and possess 5′ and/or 3′ overhangs of one or two nucleotides. The siRNA duplex comprises a first stand that has a sequence that is substantially identical to a sequence of MYH7B gene and a second strand that has a sequence that is partially, substantially, or fully complementary to the first strand. In certain embodiments, the first strand of a MYH7B siRNA comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to a sequence of MYH7B gene and the second strand comprises a sequence that is substantially or completely complementary to the first strand. In some embodiments, the first RNA strand of an siRNA targeted to MYH7B gene is at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to a coding region of the human MYH7B gene (SEQ ID NO: 6). In certain embodiments, the first RNA strand of an siRNA targeted to MYH7B gene is at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to a sequence of 5′-GAGGCCAAGATCAA-3′ (SEQ ID NO: 4). In other embodiments, the first RNA strand of an siRNA targeted to MYH7B gene is at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to a sequence of 5′-TGGAGGAGCTGCAG-3′ (SEQ ID NO: 5). An siRNA inhibitor of MYH7B can be administered to cardiac cells or expressed in cardiac cells from one or more vectors.
In another embodiment, an inhibitor of MYH7B is a small hairpin RNA (shRNA). As used herein, the term “shRNA” refers to an RNA duplex wherein a portion of the duplex is part of a hairpin structure (shRNA). In addition to the duplex portion, the hairpin structure may contain a loop portion positioned between the two first and the second strand that form the duplex. The loop can vary in length. In some embodiments the loop is 5, 6, 7, 8, 9, 10, 11, 12 or 13 nucleotides in length. The hairpin structure can also contain 3′ or 5′ overhang portions. In some aspects, the overhang is a 3′ or a 5′ overhang 0, 1, 2, 3, 4 or 5 nucleotides in length. The duplex portion or double-stranded region of an shRNA targeting MYH7B is from about 10 to about 30 nucleotides in length and comprises a first RNA strand that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to a sequence of the MYH7B gene and a second RNA strand that is substantially or fully complementary to the first strand. In certain embodiments, the first RNA strand of the duplex portion of an shRNA targeted to MYH7B gene is at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to a coding region of the human MYH7B gene (SEQ ID NO: 6). In some embodiments, the first RNA strand of the duplex portion of an shRNA targeted to MYH7B gene is at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to a sequence of 5′-GAGGCCAAGATCAA-3′ (SEQ ID NO: 4). In other embodiments, the first RNA strand of an shRNA targeted to MYH7B gene is at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to a sequence of 5′-TGGAGGAGCTGCAG-3′ (SEQ ID NO: 5). An shRNA inhibitor of MYH7B can be administered to cardiac cells or expressed in cardiac cells from a vector.
Similar to antisense oligonucleotides, other inhibitory nucleotide molecules described herein (aptamers, ribozymes, siRNA, shRNA) may contain one or more chemically-modified nucleotides described above. For example, an inhibitory nucleotide molecule targeted to MYH7B can contain bicyclic sugar nucleoside (BSN) modifications, locked nucleic acid (LNA) modifications, sugar modifications, such as 2′-O-alkyl (e.g. 2′-O-methyl, 2′-O-methoxyethyl), 2′-halo (e.g., 2′-fluoro, 2′-chloro, 2′-bromo), and 4′ thio modifications, backbone modifications, such as one or more phosphorothioate, morpholino, or phosphonocarboxylate linkages, base modifications such as 5-methyl cytidine or 2′-4′-bridged bicyclic nucleoside modifications.
In yet another embodiment, an inhibitor of MYH7B is an antibody or a binding fragment thereof that specifically binds to one or more epitopes of MYH7B protein. For instance, in certain embodiments, an inhibitor of MYH7B is a monoclonal or polyclonal antibody that specifically binds to one or more epitopes of MYH7B protein. The monoclonal antibody may be chimeric or humanized. In another embodiment, an inhibitor of MYH7B is a fragment derived from an MYH7B-specific antibody, wherein the fragment specifically binds to one or more epitopes of MYH7B protein. Such binding fragments may include a Fab fragment, scFv fragment, or engineered antibody fragments such as a diabody, triabody, minibody or a single-domain antibody. Anti-MYH7B antibodies encompass all immunoglobulin classes, such as IgM, IgG, IgD, IgE, and IgA or their subclasses, including IgG subclasses (e.g., IgG1, IgG2, IgG2a, IgG2b, IgG3, or IgGM). Preferably, the anti-MYH7B antibodies or binding fragments thereof have a binding affinity for MYH7B in a range from about 1×10−7 M to about 1×10−12 M, from about 1×10−8 M to about 1×10−11 M, or from about 1×10−9 M to about 5×10−10 M.
As used herein, the term “subject” or “patient” refers to any vertebrate including, without limitation, humans and other primates (e.g., chimpanzees and other apes and monkey species), farm animals (e.g., cattle, sheep, pigs, goats and horses), domestic mammals (e.g., dogs and cats), laboratory animals (e.g., rodents such as mice, rats, and guinea pigs), and birds (e.g., domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like). In some embodiments, the subject is a mammal. In other embodiments, the subject is a human and an inhibitory molecule is targeted to human MYH7B gene, mRNA, pre-mRNA, or protein.
Any of the nucleic acid inhibitors of MYH7B described herein can be delivered to the target cell (e.g. cardiac cells and skeletal muscle cells) by delivering to the cell an expression vector encoding the MYH7B inhibitors. A “vector” is a composition of matter which can be used to deliver a nucleic acid of interest to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “vector” includes an autonomously replicating plasmid or a virus. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, and the like. In one particular embodiment, the viral vector is a lentiviral vector or an adenoviral vector. An expression construct can be replicated in a living cell, or it can be made synthetically. For purposes of this application, the terms “expression construct,” “expression vector,” and “vector,” are used interchangeably to demonstrate the application of the invention in a general, illustrative sense, and are not intended to limit the invention.
In one embodiment, an expression vector for expressing an inhibitor of MYH7B comprises a promoter operably linked to a polynucleotide encoding an antisense oligonucleotide comprising a sequence of 5′-TTGATCTTGGCCTC-3′ (SEQ ID NO: 145) . In another embodiment, an expression vector for expressing an inhibitor of MYH7B comprises a promoter operably linked to a polynucleotide encoding an antisense oligonucleotide comprising a sequence of 5′-CTGCAGCTCCTCCA-3′ (SEQ ID NO: 147). The phrase “operably linked” or “under transcriptional control” as used herein means that the promoter is in the correct location and orientation in relation to a polynucleotide to control the initiation of transcription by RNA polymerase and expression of the polynucleotide.
As used herein, a “promoter” refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene. Suitable promoters include, but are not limited to RNA pol I, pol II, pol III, and viral promoters (e.g. human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter, and the Rous sarcoma virus long terminal repeat). In one embodiment, the promoter is a tissue-specific promoter. Of particular interest are muscle specific promoters, and more particularly, endothelial and cardiac specific promoters. These include the myosin light chain-2 promoter (Franz et al. (1994) Cardioscience, Vol. 5(4):235-43; Kelly et al. (1995) J. Cell Biol., Vol. 129(2):383-396), the alpha actin promoter (Moss et al. (1996) Biol. Chem., Vol. 271(49):31688-31694), the troponin 1 promoter (Bhaysar et al. (1996) Genomics, Vol. 35(1):11-23); the Na+/Ca2+ exchanger promoter (Barnes et al. (1997) J. Biol. Chem., Vol. 272(17):11510-11517), the dystrophin promoter (Kimura et al. (1997) Dev. Growth Differ., Vol. 39(3):257-265), the alpha7 integrin promoter (Ziober and Kramer (1996) J. Bio. Chem., Vol. 271(37):22915-22), the brain natriuretic peptide promoter (LaPointe et al. (1996) Hypertension, Vol. 27(3 Pt 2):715-22) and the alpha B-crystallin/small heat shock protein promoter (Gopal-Srivastava (1995) J. Mol. Cell. Biol., Vol. 15(12):7081-7090), alpha myosin heavy chain promoter (Yamauchi-Takihara et al. (1989) Proc. Natl. Acad. Sci. USA, Vol. 86(10):3504-3508) and the ANF promoter (LaPointe et al. (1988) J. Biol. Chem., Vol. 263(19):9075-9078).
In certain embodiments, the promoter operably linked to a polynucleotide encoding a MYH7B inhibitor can be an inducible promoter. Inducible promoters are known in the art and include, but are not limited to, tetracycline promoter, metallothionein HA promoter, heat shock promoter, steroid/thyroid hormone/retinoic acid response elements, the adenovirus late promoter, and the inducible mouse mammary tumor virus LTR.
Methods of delivering expression constructs and nucleic acids to cells are known in the art and can include, for example, calcium phosphate co-precipitation, electroporation, microinjection, DEAE-dextran, lipofection, transfection employing polyamine transfection reagents, cell sonication, gene bombardment using high velocity microprojectiles, and receptor-mediated transfection.
The present invention also includes pharmaceutical compositions comprising an inhibitor of MYH7B (e.g. antisense oligonucleotide, aptamer, ribozyme, siRNA, shRNA, or antibody) and a pharmaceutically acceptable carrier or excipient. For instance, the present invention provides compositions comprising an antisense oligonucleotide of MYH7B and a pharmaceutically acceptable carrier or excipient.
In one embodiment, the pharmaceutical composition comprises an effective dose of a MYH7B inhibitor and a pharmaceutically acceptable carrier. For instance, the pharmaceutical composition comprises an effective dose of an antisense oligonucleotide targeting MYH7B or an effective dose of a modified antisense oligonucleotide targeting MYH7B as described herein. In one embodiment, the pharmaceutical composition comprises an antisense oligonucleotide having a sequence of 5′-lTslTslGsdAsdTsdCsdTsdTsdGsdGsdCslCslTslC-3′ (SEQ ID NO: 146). In other embodiments, the pharmaceutical composition comprises an antisense oligonucleotide having a sequence of 5′-lCslTslGsdCsdAsdGsdCsdTsdCsdCsdTslCslCslA-3′ (SEQ ID NO: 148). In some other embodiments, the pharmaceutical composition comprises an antisense oligonucleotide a modified antisense oligonucleotide having a sequence selected from the sequences listed in Tables 1-5.
An “effective dose” is an amount sufficient to effect a beneficial or desired clinical result. An effective dose of an MYH7B inhibitor of the invention may be from about 1 mg/kg to about 100 mg/kg, about 2.5 mg/kg to about 50 mg/kg, or about 5 mg/kg to about 25 mg/kg. The precise determination of what would be considered an effective dose may be based on factors individual to each patient, including their size, age, type of disorder (e.g. hypertrophic cardiomyopathy, myocardial infarction, heart failure, or cardiac hypertrophy), and nature of inhibitor (e.g. antisense oligonucleotide, siRNA, shRNA, expression construct, antibody, etc). Therefore, dosages can be readily ascertained by those of ordinary skill in the art from this disclosure and the knowledge in the art.
In certain embodiments, the invention provides methods for treating or preventing pathologic cardiac hypertrophy, myocardial infarction, heart failure, or hypertrophic cardiomyopathy comprising administering an inhibitor of MYH7B and a second therapeutic agent. In some embodiments, the second therapeutic agent is a cardiac therapeutic agent such as an antisense oligonucleotide inhibitor of miR-208a, miR-208b, miR-499, miR-15a, miR-15b, miR-16, miR-195, or combinations thereof. Exemplary inhibitors of such miRNAs are described in WO 2012/083005 and WO 2013/192486, which are hereby incorporated by reference in their entireties. In embodiments where a second therapeutic agent is included, the second agent may be administered concurrently but in separate formulations or sequentially. In other embodiments, the second therapeutic agent may be administered at different times prior to or after administration of a MYH7B inhibitor. Prior administration includes, for instance, administration of the first agent within the range of about one week to up to 30 minutes prior to administration of the second agent. Prior administration may also include, for instance, administration of the first agent within the range of about 2 weeks to up to 30 minutes prior to administration of the second agent. After or later administration includes, for instance, administration of the second agent within the range of about one week to up to 30 minutes after administration of the first agent. After or later administration may also include, for instance, administration of the second agent within the range of about 2 weeks to up to 30 minutes after administration of the first agent.
The present invention includes pharmaceutical compositions comprising an inhibitor of MYH7B, a second therapeutic agent and a pharmaceutically acceptable carrier or excipient. In certain embodiments, the pharmaceutical composition may comprise two or more inhibitors of MYH7B. In some embodiments, the second therapeutic agent is a cardiac therapeutic agent such as an antisense oligonucleotide inhibitor of miR-208a, miR-208b, miR-499, miR-15a, miR-15b, miR-16, miR-195, or combinations thereof. In these embodiments, the second therapeutic agent may be included in the same formulation as an inhibitor of MYH7B or in a separate formulation. Where clinical applications are contemplated, pharmaceutical compositions will be prepared in a form appropriate for the intended application. Generally, this will entail preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals.
Colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes, may be used as delivery vehicles for the oligonucleotide inhibitors of MYH7B, or constructs expressing MYH7B nucleotide inhibitors. Commercially available fat emulsions that are suitable for delivering the nucleic acids of the invention to cardiac and skeletal muscle tissues include Intralipid®, Liposyn®, Liposyn® II, Liposyn® III, Nutrilipid, and other similar lipid emulsions. A preferred colloidal system for use as a delivery vehicle in vivo is a liposome (i.e., an artificial membrane vesicle). The preparation and use of such systems is well known in the art. Exemplary formulations are also disclosed in U.S. Pat. Nos. 5,981,505; 6,217,900; 6,383,512; 5,783,565; 7,202,227; 6,379,965; 6,127,170; 5,837,533; 6,747,014; and WO03/093449, which are herein incorporated by reference in their entireties.
In certain embodiments, liposomes used for delivery are amphoteric liposomes such SMARTICLES® (Marina Biotech, Inc.) which are described in detail in U.S. Pre-grant Publication No. 20110076322. The surface charge on the SMARTICLES® is fully reversible which make them particularly suitable for the delivery of nucleic acids. SMARTICLES® can be delivered via injection, remain stable, and aggregate free and cross cell membranes to deliver the nucleic acids
In some embodiments, appropriate salts and buffers are used to render delivery vehicles stable and allow for uptake by target cells. Aqueous compositions of the present invention comprise an effective amount of the delivery vehicle comprising the inhibitor polynucleotides (e.g. liposomes or other complexes or expression vectors) dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. The phrases “pharmaceutically acceptable” or “pharmacologically acceptable” refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human. As used herein, “pharmaceutically acceptable carrier” includes solvents, buffers, solutions, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like acceptable for use in formulating pharmaceuticals, such as pharmaceuticals suitable for administration to humans. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredients of the present invention, its use in therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions, provided they do not inactivate the vectors or polynucleotides of the compositions.
The active compositions of the present invention may include classic pharmaceutical preparations. Administration of these compositions according to the present invention may be via any common route so long as the target tissue is available via that route. This includes oral, nasal (inhalational), or buccal. Alternatively, administration may be by intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection, or by direct injection into cardiac tissue. Pharmaceutical compositions comprising MYH7B inhibitors or expression constructs comprising MYH7B inhibitors may also be administered by catheter systems or systems that isolate coronary circulation for delivering therapeutic agents to the heart. Various catheter systems for delivering therapeutic agents to the heart and coronary vasculature are known in the art. Some non-limiting examples of catheter-based delivery methods or coronary isolation methods suitable for use in the present invention are disclosed in U.S. Pat. Nos. 6,416,510; 6,716,196; 6,953,466, WO 2005/082440, WO 2006/089340, U.S. Patent Publication No. 2007/0203445, U.S. Patent Publication No. 2006/0148742, and U.S. Patent Publication No. 2007/0060907, which are all herein incorporated by reference in their entireties. Such compositions would normally be administered as pharmaceutically acceptable compositions as described herein.
In another embodiment of the invention, compositions comprising MYH7B inhibitors as described herein may be formulated as a coating for a medical device, such as a stent, balloon, or catheter. Particularly useful in methods of treating cardiac disorders in a subject, the inhibitors of MYH7B can be used to coat a metal stent to produce a drug-eluting stent. A drug-eluting stent is a scaffold that holds open narrowed or diseased arteries and releases a compound to prevent cellular proliferation and/or inflammation. The inhibitors of MYH7B may be applied to a metal stent imbedded in a thin polymer for release of the inhibitors over time. Methods for device-based delivery and methods of coating devices are well known in the art, as are drug-eluting stents and other implantable devices. See, e.g., U.S. Pat. Nos. 7,294,329, 7,273,493, 7,247,313, 7,236,821, 7,232,573, 7,156,869, 7,144,422, 7,105,018, 7,087,263, 7,083,642, 7,055,237, 7,041,127, 6,716,242, and 6,589,286, and WO 2004/004602, which are herein incorporated by reference in their entireties. Thus, the present invention includes a medical device, such as a balloon, catheter, or stent, coated with a MYH7B inhibitor. In some embodiments, the MYH7B inhibitor can be used in combination with other therapeutic agents (e.g. anti-restenosis compounds) to produce a formulation for incorporation into drug-eluting stents.
The active compounds may also be administered parenterally or intraperitoneally. By way of illustration, solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations generally contain a preservative to prevent the growth of microorganisms.
The pharmaceutical forms suitable for injectable use or catheter delivery include, for example, sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Generally, these preparations are sterile and fluid to the extent that easy injectability exists. Preparations should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Appropriate solvents or dispersion media may contain, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions may be prepared by incorporating the active compounds in an appropriate amount into a solvent along with any other ingredients (for example as enumerated above) as desired, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the desired other ingredients, e.g., as enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation include vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient(s) plus any additional desired ingredient from a previously sterile-filtered solution thereof.
The compositions of the present invention generally may be formulated in a neutral or salt form. Pharmaceutically-acceptable salts include, for example, acid addition salts (formed with the free amino groups of the protein) derived from inorganic acids (e.g., hydrochloric or phosphoric acids), or from organic acids (e.g., acetic, oxalic, tartaric, mandelic, and the like). Salts formed with the free carboxyl groups of the protein can also be derived from inorganic bases (e.g., sodium, potassium, ammonium, calcium, or ferric hydroxides) or from organic bases (e.g., isopropylamine, trimethylamine, histidine, procaine and the like).
Upon formulation, solutions are preferably administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations may easily be administered in a variety of dosage forms such as injectable solutions, drug release capsules, eye drops, intravitreal injections, drug-eluting stents or other coated vascular devices, and the like. For parenteral administration in an aqueous solution, for example, the solution generally is suitably buffered and the liquid diluent first rendered isotonic for example with sufficient saline or glucose. Such aqueous solutions may be used, for example, for intravenous, intramuscular, subcutaneous, intravitreal and intraperitoneal administration. Preferably, sterile aqueous media are employed as is known to those of skill in the art, particularly in light of the present disclosure. By way of illustration, a single dose may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, “Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by regulatory agencies.
In certain embodiments of the invention, the pharmaceutical compositions of the invention are packaged with or stored within a device for administration. Devices for injectable formulations include, but are not limited to, injection ports, autoinjectors, injection pumps, and injection pens. Devices for aerosolized or powder formulations include, but are not limited to, inhalers, insufflators, aspirators, and the like. Thus, the present invention includes administration devices comprising a pharmaceutical composition of the invention for treating or preventing one or more of the disorders described herein.
This invention is further illustrated by the following additional examples that should not be construed as limiting. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made to the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
All patent and non-patent documents referenced throughout this disclosure are incorporated by reference herein in their entirety for all purposes.
EXAMPLES
Example 1
Inhibition of MYH7B Down-Regulates MYH7
Human iPS cardiomyocytes were transfected with a control oligonucleotide or antisense oligonucleotides (Anti-7b #1 (5′-GTGAATGCGGATGAA-3′; SEQ ID NO: 1) and Anti-7b #2 (5′-GAAGTGGTAGTCATA-3′; SEQ ID NO: 2) targeted to MYH7B. The levels of MYH7B and MYH7 (β-MHC) mRNA were measured at 48 hours using real-time PCR. Human iPS cardiomyocytes treated with a thyroid hormone, T3, were used as a positive control. It is known that T3 down-regulates the expression of β-MHC. The real-time PCR analysis of RNA isolated from the transfected cells showed that the inhibition of MYH7B resulted in the down-regulation of MYH7B as well as MYH7 (β-MHC) (FIG. 1). As expected, T3 down-regulated MYH7 (β-MHC) (FIG. 1B) but did not affect the levels MYH7B or miR-208a, miR-208b, and miR-499 (FIGS. 1A and 2).
The observation that MYH7B knockdown results in the myosin switch could, in part, be through the regulation of the myosin's intronic miRNAs (MYH7B, miR-499; MYH7, miR-208b; MYH6, miR-208a). Therefore, we tested whether the inhibition of MYH7B in human iPS cardiomyocytes affects the expression of the myomiRs, miR-208a, miR-208b, or miR-499.
Specifically, to determine if the down-regulation of MYH7B had any effect on the expression of myomiRs (miR-208a, miR-499, and miR-208b), the levels of miR-208a, miR-499, and miR-208b were measured 48 hours after transfection. No significant effect was observed on the expression of miR-208a, miR-499, or miR-208b (FIG. 2). Over time, there could be a down-regulation of miR-208b because of the downregulation of MYH7; however, the data in FIG. 2 shows that antisense oligonucleotides directed to MYH7B do not directly or immediately affect the expression of myomiRs. Therefore, the regulation of the myosin switch by inhibition of MYH7B represents a novel, miRNA-independent mechanism in cardiac cells.
It has been shown that miR-208a acts upstream of miR-499 and is required for post-natal miR-499, MYH7B, and β-MHC expression in rodents (van Rooji et al., “Control of stress-dependent cardiac growth and gene expression by a microRNA,” Science, 2007, 316(5824), 575-579; Montgomery et al., “Therapeutic inhibition of miR-208a improves cardiac function and survival during heart failure,” Circulation, 2011, 124(14), 1537-1547). Loss of miR-499 and MYH7B are required for β-MHC down-regulation in the heart in rodents (van Rooji et al., “A family of microRNAs encoded by myosin genes governs myosin expression and muscle performance,” Dev. Cell, 2009, 17, 662-673). Although these studies show that myomiRs play an important role in the regulation of β-MHC expression in α-MHC predominant species such as rodents, studies in β-MHC predominant species such as pigs, rabbits, and humans indicate that alternative mechanisms are likely involved in regulating the expression of β-MHC in β-MHC predominant species.
Example 2
Inhibition of MYH7B in iPS Cardiac Cells Derived from a Patient with Hypertrophic Cardiomyopathy Down-Regulates MYH7
Control iPS cardiomyocytes and iPS cardiomyocytes derived from patients with hypertrophic cardiomyopathy (HCM) having a mutation R663H were transfected with a control oligonucleotide or MYH7B antisense oligonucleotides (Anti-7b #1 and Anti-7b #2; SEQ ID NOs: 1 and 2). The levels of MYH7 (β-MHC) mRNA were measured at 48 hours and 1 week (168 hours) post-transfection using real-time PCR. The real-time PCR analysis showed that the inhibition of MYH7B resulted in the down-regulation of MYH7 (β-MHC) expression in HCM cardiomyocytes (FIG. 3A). Moreover, the down-regulation in the expression of MYH7 (β-MHC) was sustained up to 1 week post-treatment (FIG. 3B).
Example 3
Inhibition of MYH7B Down-Regulates β-MHC Protein
To determine if inhibition of MYH7B results in the down-regulation of MYH7 at the protein level, control and HCM R663H human iPS cardiomyocytes were transfected with a control oligonucleotide, MYH7B ASO #1 (SEQ ID NO: 1), MYH7B ASO and TINY. TINY is a 8mer oligo complementary to the seed regions of miR-208a, miR-208b, and miR-499. The levels of MYH7 (β-MHC) protein were assessed by western blot analysis. The results showed that the treatment of control or HCM iPS cardiomyocytes using MYH7B antisense oligonucleotides significantly down-regulated the MYH7 (β-MHC) protein at 96 hours post-treatment (FIG. 4). A control oligo showed no effect. T3 was used as a positive control.
Example 4
Screening of Antisense Oligonucleotides (ASOs) Directed to MYH7B
Human iPS cardiomyocytes were transfected with a control oligonucleotide (Mock) or 1.5 nM of test ASOs targeted to MYH7B. The levels of MYH7B mRNA were measured at 48 hours using real-time PCR (FIG. 5). A number of ASOs showed greater than 50% inhibition of MYH7B levels. Specifically, the following ASO compounds were tested:










TABLE 6







CPD #
SEQ ID NO:









Comp-1
SEQ ID NO: 52



Comp-2
SEQ ID NO: 8



Comp-3
SEQ ID NO: 22



Comp-4
SEQ ID NO: 82



Comp-5
SEQ ID NO: 96



Comp-6
SEQ ID NO: 110



Comp-7
SEQ ID NO: 3



Comp-8
SEQ ID NO: 140



Comp-9
SEQ ID NO: 154



Comp-10
SEQ ID NO: 170



Comp-11
SEQ ID NO: 38



Comp-12
SEQ ID NO: 54



Comp-13
SEQ ID NO: 24



Comp-14
SEQ ID NO: 68



Comp-15
SEQ ID NO: 84



Comp-16
SEQ ID NO: 98



Comp-17
SEQ ID NO: 112



Comp-18
SEQ ID NO: 126



Comp-19
SEQ ID NO: 142



Comp-20
SEQ ID NO: 156



Comp-21
SEQ ID NO: 172



Comp-22
SEQ ID NO: 56



Comp-23
SEQ ID NO: 10



Comp-24
SEQ ID NO: 26



Comp-25
SEQ ID NO: 70



Comp-26
SEQ ID NO: 86



Comp-27
SEQ ID NO: 100



Comp-28
SEQ ID NO: 114



Comp-29
SEQ ID NO: 128



Comp-30
SEQ ID NO: 144



Comp-31
SEQ ID NO: 158



Comp-32
SEQ ID NO: 174



Comp-33
SEQ ID NO: 42



Comp-34
SEQ ID NO: 58



Comp-35
SEQ ID NO: 12



Comp-36
SEQ ID NO: 28



Comp-37
SEQ ID NO: 72



Comp-38
SEQ ID NO: 88



Comp-39
SEQ ID NO: 102



Comp-40
SEQ ID NO: 116



Comp-41
SEQ ID NO: 130



Comp-42
SEQ ID NO: 146



Comp-43
SEQ ID NO: 160



Comp-44
SEQ ID NO: 176



Comp-45
SEQ ID NO: 44



Comp-46
SEQ ID NO: 60



Comp-47
SEQ ID NO: 14



Comp-48
SEQ ID NO: 30



Comp-49
SEQ ID NO: 74



Comp-50
SEQ ID NO: 90



Comp-51
SEQ ID NO: 104



Comp-52
SEQ ID NO: 118



Comp-53
SEQ ID NO: 132



Comp-54
SEQ ID NO: 148



Comp-55
SEQ ID NO: 162



Comp-56
SEQ ID NO: 178



Comp-57
SEQ ID NO: 46



Comp-58
SEQ ID NO: 62



Comp-59
SEQ ID NO: 16



Comp-60
SEQ ID NO: 32



Comp-61
SEQ ID NO: 76



Comp-62
SEQ ID NO: 92



Comp-63
SEQ ID NO: 3



Comp-64
SEQ ID NO: 120



Comp-65
SEQ ID NO: 134



Comp-66
SEQ ID NO: 150



Comp-67
SEQ ID NO: 164



Comp-68
SEQ ID NO: 180



Comp-69
SEQ ID NO: 48



Comp-70
SEQ ID NO: 64



Comp-71
SEQ ID NO: 18



Comp-72
SEQ ID NO: 34



Comp-73
SEQ ID NO: 78



Comp-74
SEQ ID NO: 106



Comp-75
SEQ ID NO: 122



Comp-76
SEQ ID NO: 136



Comp-77
SEQ ID NO: 152



Comp-78
SEQ ID NO: 166



Comp-79
SEQ ID NO: 182



Comp-80
SEQ ID NO: 50



Comp-81
SEQ ID NO: 66



Comp-82
SEQ ID NO: 36



Comp-83
SEQ ID NO: 80



Comp-84
SEQ ID NO: 94



Comp-85
SEQ ID NO: 108



Comp-86
SEQ ID NO: 124



Comp-87
SEQ ID NO: 138



Comp-88
SEQ ID NO: 168










Example 5
Inhibition of MYH7B Upon Passive Administration of ASOs
ASOs that showed more than 50% inhibition of MYH7B levels in Example 4 were further tested for their inhibitory potential upon passive uptake. Specifically, selected ASOs were incubated with human iPS cardiomyocytes for 168 hours at 1, 5, or 10 μM ASO concentrations. The levels of MYH7B, MYH7 (β-MHC), and MYH6 (α-MHC) mRNAs were measured at 168 hours using real-time PCR. The real-time PCR analysis showed that many ASOs downregulated MYH7B mRNA levels (FIG. 6A). Moreover, antisense oligonucleotide having the sequence of SEQ ID NO: 146 showed a “switch” in the levels of myosins, where β-MHC is down-regulated and α-MHC is up-regulated following inhibition of MYH7B (FIGS. 6B and 6C). Some other ASOs also showed a similar trend in the levels of β- and α-MHC (FIGS. 6B and 6C).
Another ASO compound having the sequence of SEQ ID NO: 146, was also tested for its inhibitory activity upon passive uptake. Human iPS cardiomyocytes were incubated with 5 μM concentration of the compound with SEQ ID NO: 146 for 168 hours and the levels of MYH7B, MYH7 (β-MHC), and MYH6 (α-MHC) mRNAs were measured using real-time PCR. As shown in FIG. 7, the compound with SEQ ID NO: 146 also showed a “switch” in the levels of myosins, where β-MHC is down-regulated and α-MHC is up-regulated following inhibition of MYH7B.
Example 6
In Vivo Activity of Test ASOs
ASOs that showed in vitro inhibition of MYH7B were further tested for their inhibitory potential in vivo. Specifically, selected ASOs were injected subcutaneously into rats at a dose of 25 mg/kg per day for three consecutive days and rats were sacrificed 48 hours after the last dose. The levels of MYH7B mRNA in left ventricles of rats were measured using real-time PCR (FIG. 8). As shows in FIG. 8, antisense oligonucleotide compounds that showed robust activity via passive administration (e.g. SEQ ID NO: 146) also showed downregulation of MYH7B in vivo, whereas compounds that showed no activity via passive administration (e.g. SEQ ID NO: 32) also showed no activity in vivo.
Compounds having the sequence of SEQ ID NOs: 146 and 148 that showed robust activity in rats were selected for rabbit studies for assessment in β-MHC-predominant species. Rabbits were injected with 10, 15, 20, or 25 mg/kg of compounds with SEQ ID NOs: 146 and 148 at days 1, 3, 5, 10, and 17 subcutaneously and sacrificed at day 18. Real-time PCR data for MYH7B shows a dose-dependent reduction in MYH7B in both ventricles (FIGS. 9A-9B). Further, the rabbit with the most robust inhibition of MYH7B (5003) also showed the most robust reduction in β-MHC (MYH7). See FIGS. 9C and 9D.
Example 7
Effect of Nucleotide Modifications on the Activity of MYH7B ASOs
The ASO compound with SEQ ID NO: 146 was modified at various positions to assess the effect of modifications on its activity. Modifications included substitution of LNA nucleotides with corresponding ENA nucleotides and other base and sugar modifications. FIG. 10A shows that substitution of ENA for LNA retained passive activity targeting MYH7B. Likewise, substitution of cytidine with 5′-methyl cytidine and substitution of an LNA base with 2′O-methyl modified nucleotide retain activity (FIG. 10B). FIG. 10C shows backbone modifications greatly affect the passive activity of compounds.
Example 8
Screening of Additional ASOs
Additional antisense oligonucleotide compounds were screened for their inhibitory potential upon passive administration. Human iPS cardiomyocytes were passively treated with test compounds at 5 μM for 48 hours. The levels of MYH7B mRNA were measured at 48 hours using real-time PCR. A number of ASOs showed robust inhibition of MYH7B levels whereas other compounds showed no effect on MYH7B levels. FIGS. 11A-11D show results from 4 separate studies. Specifically, the following ASO compounds were tested.










TABLE 7







CPD #
SEQ ID NO:









Comp-1
SEQ ID NO: 184



Comp-2
SEQ ID NO: 186



Comp-3
SEQ ID NO: 188



Comp-4
SEQ ID NO: 190



Comp-5
SEQ ID NO: 192



Comp-6
SEQ ID NO: 194



Comp-7
SEQ ID NO: 196



Comp-8
SEQ ID NO: 198



Comp-9
SEQ ID NO: 200



Comp-10
SEQ ID NO: 202



Comp-11
SEQ ID NO: 204



Comp-12
SEQ ID NO: 206



Comp-13
SEQ ID NO: 208



Comp-14
SEQ ID NO: 210



Comp-15
SEQ ID NO: 212



Comp-16
SEQ ID NO: 214



Comp-17
SEQ ID NO: 216



Comp-18
SEQ ID NO: 218



Comp-19
SEQ ID NO: 220



Comp-20
SEQ ID NO: 222



Comp-21
SEQ ID NO: 224



Comp-22
SEQ ID NO: 226



Comp-23
SEQ ID NO: 228



Comp-24
SEQ ID NO: 230



Comp-25
SEQ ID NO: 232



Comp-26
SEQ ID NO: 234



Comp-27
SEQ ID NO: 236



Comp-28
SEQ ID NO: 238



Comp-29
SEQ ID NO: 240



Comp-30
SEQ ID NO: 242



Comp-31
SEQ ID NO: 244



Comp-32
SEQ ID NO: 246



Comp-33
SEQ ID NO: 248



Comp-34
SEQ ID NO: 250



Comp-35
SEQ ID NO: 252



Comp-36
SEQ ID NO: 254



Comp-37
SEQ ID NO: 256



Comp-38
SEQ ID NO: 258



Comp-39
SEQ ID NO: 260



Comp-40
SEQ ID NO: 262



Comp-41
SEQ ID NO: 264



Comp-42
SEQ ID NO: 266



Comp-43
SEQ ID NO: 268



Comp-44
SEQ ID NO: 270



Comp-45
SEQ ID NO: 272



Comp-46
SEQ ID NO: 274



Comp-47
SEQ ID NO: 276



Comp-48
SEQ ID NO: 278



Comp-49
SEQ ID NO: 280



Comp-50
SEQ ID NO: 282



Comp-51
SEQ ID NO: 284



Comp-52
SEQ ID NO: 286



Comp-53
SEQ ID NO: 288



Comp-54
SEQ ID NO: 290



Comp-55
SEQ ID NO: 292



Comp-56
SEQ ID NO: 294



Comp-57
SEQ ID NO: 296



Comp-58
SEQ ID NO: 298



Comp-59
SEQ ID NO: 300



Comp-60
SEQ ID NO: 302



Comp-61
SEQ ID NO: 304



Comp-62
SEQ ID NO: 306



Comp-63
SEQ ID NO: 308



Comp-64
SEQ ID NO: 310



Comp-65
SEQ ID NO: 312



Comp-66
SEQ ID NO: 314



Comp-67
SEQ ID NO: 330



Comp-68
SEQ ID NO: 342



Comp-69
SEQ ID NO: 320



Comp-70
SEQ ID NO: 332



Comp-71
SEQ ID NO: 344



Comp-72
SEQ ID NO: 322



Comp-73
SEQ ID NO: 334



Comp-74
SEQ ID NO: 346



Comp-75
SEQ ID NO: 324



Comp-76
SEQ ID NO: 336



Comp-77
SEQ ID NO: 348



Comp-78
SEQ ID NO: 326



Comp-79
SEQ ID NO: 338



Comp-80
SEQ ID NO: 350



Comp-81
SEQ ID NO: 328



Comp-82
SEQ ID NO: 340



Comp-83
SEQ ID NO: 362



Comp-84
SEQ ID NO: 374



Comp-85
SEQ ID NO: 352



Comp-86
SEQ ID NO: 364



Comp-87
SEQ ID NO: 376



Comp-88
SEQ ID NO: 354



Comp-89
SEQ ID NO: 366



Comp-90
SEQ ID NO: 378



Comp-91
SEQ ID NO: 356



Comp-92
SEQ ID NO: 368



Comp-93
SEQ ID NO: 380



Comp-94
SEQ ID NO: 358



Comp-95
SEQ ID NO: 370



Comp-96
SEQ ID NO: 382



Comp-97
SEQ ID NO: 360



Comp-98
SEQ ID NO: 372



Comp-99
SEQ ID NO: 394



Comp-100
SEQ ID NO: 406



Comp-101
SEQ ID NO: 384



Comp-102
SEQ ID NO: 396



Comp-103
SEQ ID NO: 408



Comp-104
SEQ ID NO: 386



Comp-105
SEQ ID NO: 398



Comp-106
SEQ ID NO: 410



Comp-107
SEQ ID NO: 388



Comp-108
SEQ ID NO: 400



Comp-109
SEQ ID NO: 412



Comp-110
SEQ ID NO: 390



Comp-111
SEQ ID NO: 402



Comp-112
SEQ ID NO: 414



Comp-113
SEQ ID NO: 392



Comp-114
SEQ ID NO: 404



Comp-115
SEQ ID NO: 426



Comp-116
SEQ ID NO: 416



Comp-117
SEQ ID NO: 316



Comp-118
SEQ ID NO: 418



Comp-119
SEQ ID NO: 318



Comp-120
SEQ ID NO: 420



Comp-121
SEQ ID NO: 422



Comp-122
SEQ ID NO: 424



Comp-123
SEQ ID NO: 428



Comp-124
SEQ ID NO: 430



Comp-125
SEQ ID NO: 432



Comp-126
SEQ ID NO: 434



Comp-127
SEQ ID NO: 436



Comp-128
SEQ ID NO: 438



Comp-129
SEQ ID NO: 440



Comp-130
SEQ ID NO: 442



Comp-131
SEQ ID NO: 444



Comp-132
SEQ ID NO: 446



Comp-133
SEQ ID NO: 448



Comp-134
SEQ ID NO: 450



Comp-135
SEQ ID NO: 452



Comp-136
SEQ ID NO: 454



Comp-137
SEQ ID NO: 456



Comp-138
SEQ ID NO: 458



Comp-139
SEQ ID NO: 460



Comp-140
SEQ ID NO: 462



Comp-141
SEQ ID NO: 464



Comp-142
SEQ ID NO: 466



Comp-143
SEQ ID NO: 468



Comp-144
SEQ ID NO: 470



Comp-145
SEQ ID NO: 472



Comp-146
SEQ ID NO: 474



Comp-147
SEQ ID NO: 476



Comp-148
SEQ ID NO: 478



Comp-149
SEQ ID NO: 480



Comp-150
SEQ ID NO: 482



Comp-151
SEQ ID NO: 484



Comp-152
SEQ ID NO: 486



Comp-153
SEQ ID NO: 488



Comp-154
SEQ ID NO: 490



Comp-155
SEQ ID NO: 492



Comp-156
SEQ ID NO: 494



Comp-157
SEQ ID NO: 496



Comp-158
SEQ ID NO: 498



Comp-159
SEQ ID NO: 500



Comp-160
SEQ ID NO: 502



Comp-161
SEQ ID NO: 504



Comp-162
SEQ ID NO: 506



Comp-163
SEQ ID NO: 508



Comp-164
SEQ ID NO: 510



Comp-165
SEQ ID NO: 512



Comp-166
SEQ ID NO: 514



Comp-167
SEQ ID NO: 516



Comp-168
SEQ ID NO: 518



Comp-169
SEQ ID NO: 522



Comp-170
SEQ ID NO: 544



Comp-171
SEQ ID NO: 524



Comp-172
SEQ ID NO: 534



Comp-173
SEQ ID NO: 546



Comp-174
SEQ ID NO: 526



Comp-175
SEQ ID NO: 536



Comp-176
SEQ ID NO: 548



Comp-177
SEQ ID NO: 528



Comp-178
SEQ ID NO: 538



Comp-179
SEQ ID NO: 550



Comp-180
SEQ ID NO: 530



Comp-181
SEQ ID NO: 540



Comp-182
SEQ ID NO: 552



Comp-183
SEQ ID NO: 532



Comp-184
SEQ ID NO: 542



Comp-185
SEQ ID NO: 554



Comp-186
SEQ ID NO: 576



Comp-187
SEQ ID NO: 556



Comp-188
SEQ ID NO: 566



Comp-189
SEQ ID NO: 578



Comp-190
SEQ ID NO: 558



Comp-191
SEQ ID NO: 568



Comp-192
SEQ ID NO: 580



Comp-193
SEQ ID NO: 560



Comp-194
SEQ ID NO: 570



Comp-195
SEQ ID NO: 582



Comp-196
SEQ ID NO: 562



Comp-197
SEQ ID NO: 572



Comp-198
SEQ ID NO: 584



Comp-199
SEQ ID NO: 564



Comp-200
SEQ ID NO: 574



Comp-201
SEQ ID NO: 586



Comp-202
SEQ ID NO: 598



Comp-203
SEQ ID NO: 588



Comp-204
SEQ ID NO: 600



Comp-205
SEQ ID NO: 610



Comp-206
SEQ ID NO: 590



Comp-207
SEQ ID NO: 602



Comp-208
SEQ ID NO: 612



Comp-209
SEQ ID NO: 592



Comp-210
SEQ ID NO: 604



Comp-211
SEQ ID NO: 614



Comp-212
SEQ ID NO: 594



Comp-213
SEQ ID NO: 606



Comp-214
SEQ ID NO: 616



Comp-215
SEQ ID NO: 596



Comp-216
SEQ ID NO: 608



Comp-217
SEQ ID NO: 618



Comp-218
SEQ ID NO: 620



Comp-219
SEQ ID NO: 622



Comp-220
SEQ ID NO: 624



Comp-221
SEQ ID NO: 626



Comp-222
SEQ ID NO: 136



Comp-223
SEQ ID NO: 146



Comp-224
SEQ ID NO: 148



Comp-225
SEQ ID NO: 520

## Claims

1. An antisense oligonucleotide inhibitor of MYH7B containing at least one modified nucleotide, wherein the antisense oligonucleotide is 8 to 18 nucleotides and wherein the sequence of the antisense oligonucleotide is completely complementary to a sequence of SEQ ID NO: 6 from nucleotides 4300-4317, nucleotides 4302-4315, or nucleotides 4318-4331.

2. The antisense oligonucleotide inhibitor of claim 1, wherein the antisense oligonucleotide is 14 nucleotides and the sequence of the antisense oligonucleotide is completely complementary to a sequence of SEQ ID NO: 6 from nucleotides 4300-4317.

3. The antisense oligonucleotide inhibitor of claim 1, wherein the antisense oligonucleotide is 14 nucleotides and the sequence of the antisense oligonucleotide is completely complementary to the sequence of SEQ ID NO: 6 from nucleotides 4302-4315.

4. The antisense oligonucleotide inhibitor of claim 1, wherein the antisense oligonucleotide is 14 nucleotides and the sequence of the antisense oligonucleotide is completely complementary to the sequence of SEQ ID NO: 6 from nucleotides 4318-4331.

5. The antisense oligonucleotide inhibitor of claim 1, wherein said modified nucleotide includes a sugar, base, or a backbone modification.

6. The antisense oligonucleotide inhibitor of claim 5, wherein the sugar modification is selected from the group consisting of 2′-O, 4′-C methylene bridge, 2′-O, 4′-C ethylene bridge, 2′-CH2—NH—CH2-4′ bridge, 2′-deoxy, 2′-O-alkyl, and 2′-halo modifications.

7. The antisense oligonucleotide inhibitor of claim 5, wherein the backbone modification is a phosphorothioate linkage.

8. The antisense oligonucleotide inhibitor of claim 7, wherein said antisense oligonucleotide contains two or more phosphorothioate linkages.

9. The antisense oligonucleotide inhibitor of claim 8, wherein said antisense oligonucleotide is fully phosphorothioate linked.

10. The antisense oligonucleotide inhibitor of claim 1, wherein said modified nucleotide is a locked nucleotide.

11. The antisense oligonucleotide inhibitor of claim 1, wherein said antisense oligonucleotide contains one to six locked nucleotides.

12. The antisense oligonucleotide inhibitor of claim 1, wherein said antisense oligonucleotide contains at least three locked nucleotides at the 5′ end.

13. The antisense oligonucleotide inhibitor of claim 12, wherein the at least three locked nucleotides at the 5′ end are ribonucleotides.

14. The antisense oligonucleotide inhibitor of claim 1, wherein said antisense oligonucleotide contains at least three locked nucleotides at the 3′ end.

15. The antisense oligonucleotide inhibitor of claim 14, wherein the at least three locked nucleotides at the 3′ end are ribonucleotides.

16. The antisense oligonucleotide inhibitor of claim 1, wherein said antisense oligonucleotide contains at least one deoxyribonucleotide.

17. The antisense oligonucleotide inhibitor of claim 16, wherein said antisense oligonucleotide contains two to eight deoxyribonucleotides.

18. The antisense oligonucleotide inhibitor of claim 1, wherein said modified nucleotide is 5′-methyl cytidine.

19. A pharmaceutical composition comprising the antisense oligonucleotide inhibitor of MYH7B of claim 1 and a pharmaceutically acceptable excipient.

20. The pharmaceutical composition of claim 19, further comprising a second therapeutic agent, wherein the second therapeutic agent is an antisense oligonucleotide inhibitor of miR-208a, miR-208b, miR-499, miR-15a, miR-15b, miR-16, miR-195, or a mixture thereof.

21. An antisense oligonucleotide inhibitor of MYH7B containing at least one modified nucleotide, and wherein said antisense oligonucleotide comprises a sequence selected from Tables 1-5.

22. The antisense oligonucleotide inhibitor of claim 21, wherein said antisense oligonucleotide comprises the sequence of 5′-lTslTslGsdAsdTsdCsdTsdTsdGsdGsdCslCslTslC-3′ (SEQ ID NO: 146) or 5′ -lCslTslGsdCsdAsdGsdCsdTsdCsdCsdTslCslCslA-3′ (SEQ ID NO: 148).

23. The antisense oligonucleotide inhibitor of claim 21, wherein said antisense oligonucleotide comprises the sequence of 5′-lCslCslAsdGsdGsdAsdGsmdCsmdCsdTsdAslTslTslC-3′ (SEQ ID NO: 494).

24. The antisense oligonucleotide inhibitor of claim 21, wherein said antisense oligonucleotide comprises the sequence of 5′-lGslTslTsmdCsdAsmdCsdTsmdCsdAsmdCsdAslTslCslC-3′ (SEQ ID NO: 188).

25. The antisense oligonucleotide inhibitor of claim 21, wherein said antisense oligonucleotide comprises the sequence of 5′-lAslGslTsdTsmdCsdAsmdCsdTsmdCsdAsmdCslAslTslC-3′ (SEQ ID NO: 190).

26. The antisense oligonucleotide inhibitor of claim 21, wherein said antisense oligonucleotide comprises the sequence of 5′-lAslGslTsdTsdAsdTsmdCsdAsdTsdTsmdCslCslTslC-3′ (SEQ ID NO: 370).

27. The antisense oligonucleotide inhibitor of claim 21, wherein said antisense oligonucleotide comprises the sequence of 5′-lCslTslTsdAsdGsmdCsdTsdGsdAsdTsmdCslCslTslC-3′ (SEQ ID NO: 568).

28. A pharmaceutical composition comprising the antisense oligonucleotide inhibitor of MYH7B of claim 21 and a pharmaceutically acceptable excipient.

29. The pharmaceutical composition of claim 28, further comprising a second therapeutic agent, wherein the second therapeutic agent is an antisense oligonucleotide inhibitor of miR-208a, miR-208b, miR-499, miR-15a, miR-15b, miR-16, miR-195, or a mixture thereof.