Oligonucleotides targeting myostatin (MSTN)

Abstract

Provided herein are oligonucleotides (e.g., siRNA) targeting MSTN. Also provided are methods of treating a disease associated with MSTN expression.

Description

OLIGONUCLEOTIDES TARGETING MYOSTATIN (MSTN)RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 642,234, filed May 3, 2024, and U.S. Provisional Application Serial No. 63 / 696,246, filed September 18, 2024, the entire disclosures of which are hereby incorporated herein by reference.BACKGROUND

[0002] Cachexia and muscle wasting accompany about 80% of all cancer patients and ultimately lead to death in up to 30% of those affected (Argiles, Busquets, Stemmier, & Lopez- Soriano, 2014; Lim, Brown, Washington, & Greene, 2020). The discovery of myostatin, formerly also known as GDF8, in the context of tremendous muscle growth in knockout mice has generated great interest in the development of new therapeutics against all types of muscular disorders and atrophies.

[0003] Myostatin is predominantly present as an inactive form and can be processed into an active dimer by enzymatic cleavage. Binding of the active form to the activin receptor complex causes proteasomal degradation of proteins in the target cell and blocks the cell cycle, leading to atrophy. Current clinical treatment approaches aim to inhibit the mature myostatin dimer or its receptor by using antibodies against myostatin itself, decoy receptors, or propeptides to suspend the negative regulation of myostatin. However, these therapies fail due to high similarity among transforming growth factors, resulting in side effects or low efficacy of treatment (Suh & Lee, 2020). Accordingly, there exists a need for effective and safe therapies that inhibit myostatin.SUMMARY

[0004] In one aspect, the disclosure provides an RNA molecule comprising a sequence substantially complementary to MSTN nucleic acid sequence of any one of SEQ ID NOs: 1- 14 or 29-31.

[0005] In certain embodiments, the RNA molecule comprises a sequence substantially complementary to MSTN nucleic acid sequence of any one of SEQ ID NOs: 15-28 or 32-34.

[0006] In certain embodiments, the RNA molecule comprises complementarity to at least 10, 11, 12 or 13 contiguous nucleotides of the MSTN nucleic acid sequence of any one of SEQ ID NOs: 1-14 or 29-31.

[0007] In certain embodiments, the RNA molecule comprises no more than 3 mismatches with the MSTN nucleic acid sequence of any one of SEQ ID NOs: 1-14 or 29-31.

[0008] In certain embodiments, the RNA molecule comprises full complementarity to the MSTN nucleic acid sequence of any one of SEQ ID NOs: 1-14 or 29-31.

[0009] In certain embodiments, the RNA molecule comprises a single stranded RNA (ssRNA) or a double stranded RNA (dsRNA).

[0010] In certain embodiments, the RNA molecule comprises an antisense oligonucleotide.

[0011] In certain embodiments, the RNA molecule comprises a short hairpin RNA (shRNA).

[0012] In certain embodiments, the RNA molecule comprises an siRNA, the siRNA comprising an antisense strand comprising substantially complementary to the MSTN nucleic acid sequence and a sense strand.

[0013] In certain embodiments, the antisense strand comprises SEQ ID NO: 35, or a chemically modified version thereof, and the sense strand comprises SEQ ID NO: 36, or a chemically modified version thereof.

[0014] In certain embodiments, the antisense strand comprises SEQ ID NO: 37, or a chemically modified version thereof, and the sense strand comprises SEQ ID NO: 38, or a chemically modified version thereof.

[0015] In certain embodiments, the antisense strand comprises SEQ ID NO: 39, or a chemically modified version thereof, and the sense strand comprises SEQ ID NO: 40, or a chemically modified version thereof.

[0016] In certain embodiments, the antisense strand comprises SEQ ID NO: 41, or a chemically modified version thereof, and the sense strand comprises SEQ ID NO: 42, or a chemically modified version thereof.

[0017] In certain embodiments, the antisense strand comprises SEQ ID NO: 43, or a chemically modified version thereof, and the sense strand comprises SEQ ID NO: 44, or a chemically modified version thereof.

[0018] In certain embodiments, the antisense strand comprises SEQ ID NO: 45, or a chemically modified version thereof, and the sense strand comprises SEQ ID NO: 46, or a chemically modified version thereof.

[0019] In certain embodiments, the antisense strand comprises SEQ ID NO: 47, or a chemically modified version thereof, and the sense strand comprises SEQ ID NO: 48, or a chemically modified version thereof.

[0020] In certain embodiments, the antisense strand comprises SEQ ID NO: 49, or a chemically modified version thereof, and the sense strand comprises SEQ ID NO: 50, or a chemically modified version thereof.

[0021] In certain embodiments, the antisense strand comprises SEQ ID NO: 51, or a chemically modified version thereof, and the sense strand comprises SEQ ID NO: 52, or a chemically modified version thereof.

[0022] In certain embodiments, the antisense strand comprises SEQ ID NO: 53, or a chemically modified version thereof, and the sense strand comprises SEQ ID NO: 54, or a chemically modified version thereof.

[0023] In certain embodiments, the antisense strand comprises SEQ ID NO: 55, or a chemically modified version thereof, and the sense strand comprises SEQ ID NO: 56, or a chemically modified version thereof.

[0024] In certain embodiments, the antisense strand comprises SEQ ID NO: 57, or a chemically modified version thereof, and the sense strand comprises SEQ ID NO: 58, or a chemically modified version thereof.

[0025] In certain embodiments, the antisense strand comprises SEQ ID NO: 59, or a chemically modified version thereof, and the sense strand comprises SEQ ID NO: 60, or a chemically modified version thereof.

[0026] In certain embodiments, the antisense strand comprises about 15 nucleotides to 25 nucleotides in length. In certain embodiments, the sense strand comprises about 15 nucleotides to 25 nucleotides in length. In certain embodiments, the antisense strand is 20 nucleotides in length. In certain embodiments, the antisense strand is 21 nucleotides in length. In certain embodiments, the antisense strand is 22 nucleotides in length. In certain embodiments, the sense strand is 15 nucleotides in length. In certain embodiments, the sense strand is 16 nucleotides in length. In certain embodiments, the sense strand is 18 nucleotides in length. In certain embodiments, the sense strand is 20 nucleotides in length.

[0027] In certain embodiments, the siRNA comprises a double-stranded region of 15 base pairs to 20 base pairs. In certain embodiments, the siRNA comprises a double-stranded region of 15 base pairs. In certain embodiments, the siRNA comprises a double-stranded region of 16 basepairs. In certain embodiments, the siRNA comprises a double-stranded region of 18 base pairs. In certain embodiments, the siRNA comprises a double-stranded region of 20 base pairs.

[0028] In certain embodiments, the siRNA comprises at least one blunt-end.

[0029] In certain embodiments, the siRNA comprises at least one single stranded nucleotide overhang.

[0030] In certain embodiments, the siRNA comprises about a 2-nucleotide to 5-nucleotide single stranded nucleotide overhang. In certain embodiments, the siRNA comprises 2- nucleotide single stranded nucleotide overhang. In certain embodiments, the siRNA comprises 5-nucleotide single stranded nucleotide overhang.

[0031] In certain embodiments, the RNA molecule comprises naturally occurring nucleotides.

[0032] In certain embodiments, the RNA molecule comprises at least one modified nucleotide.

[0033] In certain embodiments, the modified nucleotide comprises a 2'-O-methyl modified nucleotide, a 2'-deoxy-2'-fluoro modified nucleotide, a 2'-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, a 2'-amino-modified nucleotide, a 2'-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base comprising nucleotide, or a mixture thereof.

[0034] In certain embodiments, the RNA molecule comprises at least one modified internucleotide linkage.

[0035] In certain embodiments, the modified internucleotide linkage comprises a phosphorothioate internucleotide linkage. In certain embodiments, the RNA molecule comprises 4-16 phosphorothioate intemucleotide linkages. In certain embodiments, the RNA molecule comprises 8-13 phosphorothioate internucleotide linkages. In certain embodiments, the antisense strand comprises 2-10 phosphorothioate internucleotide linkages.

[0036] In certain embodiments, the siRNA comprises at least 80% chemically modified nucleotides.

[0037] In certain embodiments, the siRNA is fully chemically modified.

[0038] In certain embodiments, the siRNA comprises at least 70% 2’-O-methyl nucleotide modifications.

[0039] In certain embodiments, the antisense strand comprises at least 70% 2’-O-methyl nucleotide modifications.

[0040] In certain embodiments, the antisense strand comprises about 70% to 90% 2’-O-methyl nucleotide modifications.

[0041] In certain embodiments, the sense strand comprises at least 65% 2’-O-methyl nucleotide modifications.

[0042] In certain embodiments, the sense strand comprises 100% 2’-O-methyl nucleotide modifications.

[0043] In certain embodiments, the sense strand comprises one or more nucleotide mismatches between the antisense strand and the sense strand.

[0044] In certain embodiments, the one or more nucleotide mismatches are present at positions 2, 6, and 12 from the 5’ end of sense strand.

[0045] In certain embodiments, the nucleotide mismatches are present at positions 2, 6, and 12 from the 5’ end of the sense strand.

[0046] In certain embodiments, the antisense strand comprises a 5’ phosphate, a 5 ’-alkyl phosphonate, a 5’ alkylene phosphonate, or a 5’ alkenyl phosphonate.

[0047] In certain embodiments, the antisense strand comprises a 5’ vinyl phosphonate.

[0048] In certain embodiments, the RNA molecule comprises at least one modified internucleotide linkage of Formula I:(i); wherein:B is a base pairing moiety;W is selected from the group consisting of O, OCH2, OCH, CH2, and CH;X is selected from the group consisting of halo, hydroxy, and C1-6 alkoxy;Y is selected from the group consisting of O , OH, OR, NH , NH2, S", and SH;Z is selected from the group consisting of O and CH2;R is a protecting group; and= is an optional double bond.

[0049] In certain embodiments, a functional moiety is linked to the 5’ end and / or 3’ end of the antisense strand.

[0050] In certain embodiments, a functional moiety is linked to the 5’ end and / or 3’ end of the sense strand.

[0051] In certain embodiments, a functional moiety is linked to the 3’ end of the sense strand.

[0052] In certain embodiments, the functional moiety comprises a hydrophobic moiety.

[0053] In certain embodiments, the hydrophobic moiety is selected from the group consisting of fatty acids, steroids, secosteroids, lipids, gangliosides, nucleoside analogs, endocannabinoids, vitamins, and a mixture thereof.

[0054] In certain embodiments, the fatty acid selected from the group consisting of Docosanoic acid (DCA), Eicosapentaenoic acid (EP A), and Docosahexaenoic acid (DHA).

[0055] In certain embodiments, the steroid selected from the group consisting of cholesterol and lithocholic acid (LA).

[0056] In certain embodiments, the vitamin is selected from the group consisting of choline, vitamin A, vitamin E, and derivatives or metabolites thereof.

[0057] In certain embodiments, the vitamin is selected from the group consisting of retinoic acid and alpha-tocopheryl succinate.

[0058] In certain embodiments, the functional moiety is linked to the antisense strand and / or sense strand by a linker.

[0059] In certain embodiments, the linker comprises an ethylene glycol chain, an alkyl chain, a peptide, an RNA, a DNA, a phosphodiester, a phosphorothioate, a phosphoramidate, an amide, a carbamate, or a combination thereof.

[0060] In certain embodiments, the linker is a cleavable linker.

[0061] In certain embodiments, the cleavable linker comprises a phosphodiester linkage, a disulfide linkage, an acid-labile linkage, or a photocleavable linkage.

[0062] In certain embodiments, the cleavable linker comprises a dTdT dinucleotide with phosphodiester internucleotide linkages.

[0063] In certain embodiments, the acid-labile linkage comprises a P-thiopropionate linkage or a carboxy dimethylmaleic anhydride (CDM) linkage.

[0064] In certain embodiments, the linker comprises a divalent or trivalent linker.

[0065] In certain embodiments, the divalent or trivalent linker is selected from the group consisting of:wherein n is 1, 2, 3, 4, or 5.

[0066] In certain embodiments, when the linker is a trivalent linker, the linker further links a phosphodiester or phosphodiester derivative.

[0067] In certain embodiments, the phosphodiester or phosphodiester derivative is selected from the group consisting of: ndwherein X is O, S or BH3.

[0068] In certain embodiments, the nucleotides at positions 1 and 2 from the 3’ end of sense strand, and the nucleotides at positions 1 and 2 from the 5’ end of antisense strand are connected to adjacent ribonucleotides via phosphorothioate linkages.

[0069] In certain embodiments, the sense strand comprises the following chemical modification pattern, from 5’ to 3’:(mN)#(mN)#(mN)(fN)(fN)(fN)(mN)(fN)(mN)(fN)(mN)(mN)(mN)(fN)#(mN)#(fN)(dT)(dT)- DCA; and the antisense strand comprises the following modification pattern, from 5’ to 3’:V(mN)#(fN)#(mN)(fN)(fN)(fN)(mN)(fN)(mN)(fN)(mN)(fN)(mN)(fN)#(mN)#(fN)#(mN)#(f N)#(mN)#(fxN)#(mxN) .In certain embodiments, the sense strand comprises the following chemical modification pattern, from 5’ to 3’:(mN)#(fN)#(mN)(fN)(mN)(fN)(mN)(fN)(mN)(fN)(mN)(fN)(mN)(fN)#(mN)#(fN)(dT)(dT)- DCA; and the antisense strand comprises the following modification pattern, from 5’ to 3 ’ : V(mN)#(fN)#(mN)(fN)(mN)(fN)(mN)(fN)(mN)(fN)(mN)(fN)(mN)(fN)#(mN)#(fN)#(mN) #(fN)#(mN)#(fxN)#(mxN) .

[0070] For the sense and antisense sequences above, “m” corresponds to a 2’-O-methyl modified nucleotide, “f” corresponds to a 2’ -fluoro modified nucleotide, “d” corresponds to a deoxynucleotide, “#” corresponds to a phosphorothioate internucleotide linkage, “x” corresponds to an exNA intemucleotide linkage of Formula IV, “V” corresponds to a 5’ vinyl phosphonate, and “DC A” corresponds to a docosanoic acid (DC A) moiety.

[0071] In one aspect, the disclosure provides a pharmaceutical composition for inhibiting the expression of MSTN gene in an organism, comprising the RNA molecule described herein and a pharmaceutically acceptable carrier.

[0072] In certain embodiments, the RNA molecule inhibits the expression of said MSTN gene by at least 20%.

[0073] In certain embodiments, the RNA molecule inhibits the expression of said MSTN gene by at least 50%.

[0074] In one aspect, the disclosure provides amethod for inhibiting expression of MSTN gene in a cell, the method comprising: (a) introducing into the cell the RNA molecule described herein; and (b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of the MSTN gene, thereby inhibiting expression of the MSTN gene in the cell.

[0075] In one aspect, the disclosure provides a method of treating or managing a muscle wasting disease comprising administering to a patient in need of such treatment a therapeutically effective amount of the RNA molecule described herein.

[0076] In certain embodiments, the RNA molecule is administered subcutaneously, intravenously, or intramuscularly.

[0077] In certain embodiments, the RNA molecule inhibits the expression of said MSTN gene by at least 20%.

[0078] In certain embodiments, the RNA molecule inhibits the expression of said MSTN gene by at least 50%.

[0079] In one aspect, the disclosure provides a vector comprising a regulatory sequence operably linked to a nucleotide sequence that encodes an RNA molecule substantially complementary to a. MSTN nucleic acid sequence of SEQ ID NOs: 1-14 or 29-31.

[0080] In certain embodiments, the RNA molecule inhibits the expression of said MSTN gene by at least 20%

[0081] In certain embodiments, the RNA molecule inhibits the expression of said MSTN gene by at least 50%.

[0082] In one aspect, the disclosure provides a cell comprising the vector described herein.

[0083] In one aspect, the disclosure provides a recombinant adeno-associated virus (rAAV) comprising the vector described herein and an AAV capsid.

[0084] In one aspect, the disclosure provides a branched RNA compound comprising two or more of the RNA molecules described herein covalently bound to one another.

[0085] In certain embodiments, the RNA molecules are covalently bound to one another by way of a linker, spacer, or branching point.BRIEF DESCRIPTION OF THE DRAWINGS

[0086] FIG. 1 shows relative MSTN expression from a primary screen of siRNAs targeting MSTN in human rhabdomyosarcoma cells (cell line SJCRh30). Each siRNA was used at a concentration of 1.5 pM with 7,500 cells per well. Cells were incubated with the siRNA for 72 hours before harvesting. Each siRNA was tested in triplicate.

[0087] FIG. 2 shows relative MSTN expression from dose response curves of select siRNA targeting MSTN. SJCRh30 cells (7,500 cells per well) were incubated with a concentrationrange of 23 nM to 1.5 pM of siRNA. Cells were incubated with the siRNA for 72 hours before harvesting. Each siRNA was tested in triplicate.

[0088] FIG. 3 shows relative MSTN expression from a primary screen of siRNAs targeting MSTN in mouse myoblast cells (cell line C2C12). Each siRNA was used at a concentration of 1.5 pM with 7,500 cells per well. Cells were incubated with the siRNA for 144 hours before harvesting. Each siRNA was tested in triplicate.

[0089] FIG. 4 shows a comparison of the human MSTN transcript and the mouse MSTN transcript. The transcripts were aligned and siRNA targets within the same region of the MSTN transcripts were compared, identifying areas of cross-homology and mismatches.

[0090] FIG. 5 shows relative MSTN mRNA expression in mice injected with select MSTN- targeting siRNA. Six- to eight-week-old FVBN mice were injected subcutaneously with siRNA targeting mouse MSTN mRNA targets 1192, 2507, 2093, 2057, and 1928. The siRNA were tested in either the P3 pattern or P2 pattern. MSTN mRNA levels were detected to quadriceps, gastrocnemius, and heart tissue.

[0091] FIG. 6 shows relative MSTN protein levels in plasma normalized to a PBS control in mice injected with an siRNA targeting a mouse MSTN. Mice were injected subcutaneously with siRNA in either pattern 1 or pattern 2.

[0092] FIG. 7 shows relative mouse MSTN mRNA levels normalized to non-target control (NTC) in mice. FVBNJ female mice (5 mice per group) were subcutaneously injected with 40 mg / kg of the 1928-targeting siRNA. Three-weeks post-injection, hearth, quadriceps, calf, biceps, diaphragm, and tongue tissues were collected and MSTN mRNA was quantified.

[0093] FIG. 8 shows mouse MSTN protein levels in plasma in mice. FVBNJ female mice (5 mice per group) were subcutaneously injected with 40 mg / kg of the 1928-targeting siRNA. MSTN protein levels in plasma was detected every two weeks for 29 weeks. The mice received either received a single 40 mg / kg dose or 40 mg / kg every 2 weeks.

[0094] FIG. 9 shows physical changes of the mice of FIG. 8 given the 1928-targeting siRNA. Body weight, lean mass, and grip strength were measured.

[0095] FIG. 10 shows Magnetic resonance imaging (MRI) of the mice of FIG. 8 given the 1928-targeting siRNA. Images are of calf and quadricep of mice given the single dose of the siRNA at 6-weeks post injection.

[0096] FIG. 11 shows the safety profile of the mice of FIG. 8 given the 1928-targeting siRNA. Blood levels of alanine transaminase (ALT), blood urea nitrogen (BUN), platelet count, and immune cell % (lymphocytes (“LYM”), monocytes (“MON”), and neutrophils (“NEU”)) was measured from mice at 29 weeks from either the single and repetitive (i.e., every two-week) dosing.

[0097] FIG. 12 shows the troponin levels and creatine kinase-myoglobin binding (CK-MB) levels of the mice of FIG. 8 given the 1928-targeting siRNA. Plasma levels of each heart biomarker were measured for up to 12 weeks.

[0098] FIG. 13 shows MSTN levels over time in mice injected with the 1928-targeting siRNA at a dose and frequency of 1) 10 mg / kg every 2 weeks; 2) 20 mg / kg every 4 weeks; or 3) 40 mg / kg every 8 weeks. The siRNA were injected via subcutaneous injection and plasma was collected at multiple points over 32 weeks.DETAILED DESCRIPTION

[0099] Unless otherwise specified, nomenclature used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. Unless otherwise specified, the methods and techniques provided herein are performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout thexampe present specification unless otherwise indicated. Enzymatic reactions and purification techniques are performed according to manufacturer’s specifications, as commonly accomplished in the art or as described herein. The nomenclature used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, delivery, and treatment of patients.

[0100] Unless otherwise defined herein, scientific and technical terms used herein have the meanings that are commonly understood by those of ordinary skill in the art. In the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. The use of “or” means “and / or” unless stated otherwise. The use of the term “including,” as well as other forms, such as “includes” and “included,” is not limiting.

[0101] So that the disclosure may be more readily understood, certain terms are first defined.

[0102] As used herein in the context of oligonucleotide sequences, “A” represents a nucleoside comprising the base adenine (e.g., adenosine or a chemically-modified derivative thereof), “G” represents a nucleoside comprising the base guanine (e.g., guanosine or a chemically-modified derivative thereof), “U” represents a nucleoside comprising the base uracil (e.g., uridine or a chemically-modified derivative thereof), and “C” represents a nucleoside comprising the base adenine (e.g., cytidine or a chemically-modified derivative thereof).

[0103] The term “nucleoside” refers to a molecule having a purine or pyrimidine base covalently linked to a ribose or deoxyribose sugar. Exemplary nucleosides include adenosine, guanosine, cytidine, uridine and thymidine. Additional exemplary nucleosides include inosine, 1 -methyl inosine, pseudouridine, 5,6-dihydrouridine, ribothymidine, 2N-methylguanosine and N2,N2-dimethylguanosine (also referred to as “rare” nucleosides). The term “nucleotide” refers to a nucleoside having one or more phosphate groups joined in ester linkages to the sugar moiety. Exemplary nucleotides include nucleoside monophosphates, diphosphates and triphosphates. The terms “polynucleotide” and “nucleic acid molecule” are used interchangeably herein and refer to a polymer of nucleotides joined together by a phosphodiester or phosphorothioate linkage between 5' and 3' carbon atoms.

[0104] The term “RNA” or “RNA molecule” or “ribonucleic acid molecule” refers to a polymer of ribonucleotides (e.g., 2, 3, 4, 5, 10, 15, 20, 25, 30, or more ribonucleotides). The term “DNA” or “DNA molecule” or “deoxyribonucleic acid molecule" refers to a polymer of deoxyribonucleotides. DNA and RNA can be synthesized naturally (e.g., by DNA replication or transcription of DNA, respectively). RNA can be post-transcriptionally modified. DNA and RNA can also be chemically synthesized. DNA and RNA can be single-stranded (i.e., ssRNA and ssDNA, respectively) or multi-stranded (e.g., double stranded, i.e., dsRNA and dsDNA, respectively). "mRNA" or "messenger RNA" is single-stranded RNA that specifies the amino acid sequence of one or more polypeptide chains. This information is translated during protein synthesis when ribosomes bind to the mRNA.

[0105] As used herein, the term "small interfering RNA" ("siRNA") (also referred to in the art as "short interfering RNAs") refers to an RNA (or RNA analog) comprising between about 10-50 nucleotides (or nucleotide analogs), which is capable of directing or mediating RNA interference. The siRNA is a duplex formed by a sense strand and antisense strand which have sufficient complementarity to each other to form said duplex. In certain embodiments, a siRNA comprises between about 15-30 nucleotides or nucleotide analogs, or between about 16-25 nucleotides (or nucleotide analogs), or between about 18-23 nucleotides (or nucleotide analogs), or between about 19-22 nucleotides (or nucleotide analogs) (e.g., 19, 20, 21 or 22 nucleotides or nucleotide analogs). The term "short" siRNA refers to a siRNA comprising about 21 nucleotides (or nucleotide analogs), for example, 19, 20, 21 or 22 nucleotides. The term "long" siRNA refers to a siRNA comprising about 24-25 nucleotides, for example, 23, 24, 25 or 26 nucleotides. Short siRNAs may, in some instances, include fewer than 19 nucleotides, e.g., 16, 17 or 18 nucleotides, provided that the shorter siRNA retains the ability to mediate RNAi. Likewise, long siRNAs may, in some instances, include more than 26 nucleotides, provided that the longer siRNA retains the ability to mediate RNAi absent further processing, e.g., enzymatic processing, to a short siRNA.

[0106] The term "nucleotide analog" or "altered nucleotide" or "modified nucleotide" or “chemically modified nucleotide” refers to a non-standard nucleotide, including non-naturally occurring ribonucleotides or deoxyribonucleotides. Exemplary nucleotide analogs are modified at any position so as to alter certain chemical properties of the nucleotide yet retain the ability of the nucleotide analog to perform its intended function. Examples of positions of the nucleotide, which may be derivatized include: the 5 position, e.g., 5-(2-amino)propyl uridine, 5-bromo uridine, 5-propyne uridine, 5-propenyl uridine, etc.; the 6 position, e.g., 6-(2- amino)propyl uridine; and the 8-position for adenosine and / or guanosines, e.g., 8-bromo guanosine, 8-chloro guanosine, 8-fluoroguanosine, etc. Nucleotide analogs also include deaza nucleotides, e.g., 7-deaza-adenosine; O- and N-modified (e.g., alkylated, e.g., N6-methyl adenosine, or as otherwise known in the art) nucleotides; and other heterocyclically modified nucleotide analogs, such as those described in Herdewijn, Antisense Nucleic Acid Drug Dev., 2000 Aug. 10(4):297-310.

[0107] Nucleotide analogs may also comprise modifications to the sugar portion of the nucleotides. For example, the 2' OH-group may be replaced by a group selected from H, OR, R, F, Cl, Br, I, SH, SR, NH2, NHR, NR2, or COOR, wherein R is substituted or unsubstituted Ci-Ce alkyl, alkenyl, alkynyl, aryl, etc. Other possible modifications include those described in U.S. Pat. Nos. 5,858,988, and 6,291,438. In certain embodiments, the nucleotide analogcomprises a 2’-O-methyl modification. In certain embodiments, the nucleotide analog comprises a 2’ -fluoro modification.

[0108] The phosphate group of the nucleotide may also be modified, e.g., by substituting one or more of the oxygens of the phosphate group with sulfur (e.g., phosphorothioate), or by making other substitutions, which allow the nucleotide to perform its intended function, such as described in, for example, Eckstein, Antisense Nucleic Acid Drug Dev. 2000 Apr. 10(2): 117-21, Rusckowski et al. Antisense Nucleic Acid Drug Dev. 2000 Oct. 10(5):333-45, Stein, Antisense Nucleic Acid Drug Dev. 2001 Oct. 11(5): 317-25, Vorobjev et al. Antisense Nucleic Acid Drug Dev. 2001 Apr. 11(2):77-85, and U.S. Pat. No. 5,684,143. Certain of the above-referenced modifications (e.g., phosphate group modifications) decrease the rate of hydrolysis of, for example, polynucleotides comprising said analogs in vivo or in vitro.

[0109] The term "oligonucleotide" refers to a short polymer of nucleotides and / or nucleotide analogs. The term “oligonucleotide” includes, but is not limited to, antisense oligonucleotide (ASO), siRNA, micro-RNA, and short hairpin RNA (shRNA).

[0110] The term "RNA analog" refers to a polynucleotide (e.g., a chemically synthesized polynucleotide) having at least one altered or modified nucleotide as compared to a corresponding unaltered or unmodified RNA, but retaining the same or similar nature or function as the corresponding unaltered or unmodified RNA. As discussed above, the oligonucleotides may be linked with linkages, which result in a lower rate of hydrolysis of the RNA analog as compared to an RNA molecule with phosphodiester linkages. For example, the nucleotides of the analog may comprise methylenediol, ethylene diol, oxymethylthio, oxyethylthio, oxycarbonyloxy, phosphorodiamidate, phosphoroamidate, and / or phosphorothioate linkages. Some RNA analogues include sugar- and / or backbone-modified ribonucleotides and / or deoxyribonucleotides. Such alterations or modifications can further include addition of non-nucleotide material, such as to the end(s) of the RNA or internally (at one or more nucleotides of the RNA). An RNA analog need only be sufficiently similar to natural RNA that it has the ability to mediate RNA interference.[OHl] As used herein, the term "RNA interference" ("RNAi") refers to a selective intracellular degradation of RNA. RNAi occurs in cells naturally to remove foreign RNAs (e.g., viral RNAs). Natural RNAi proceeds via fragments cleaved from free dsRNA, which direct the degradative mechanism to other similar RNA sequences. Alternatively, RNAi can be initiated by the hand of man, for example, to silence the expression of target genes.

[0112] An RNAi agent, e.g., an RNA silencing agent, having a strand, which is "sequence sufficiently complementary to a target mRNA sequence to direct target-specific RNA interference (RNAi)" means that the strand has a sequence sufficient to trigger the destruction of the target mRNA by the RNAi machinery or process.

[0113] As used herein, the term “isolated RNA” (e.g., "isolated siRNA" or "isolated siRNA precursor") refers to RNA molecules, which are substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.

[0114] As used herein, the term “RNA silencing” refers to a group of sequence-specific regulatory mechanisms (e.g., RNA interference (RNAi), transcriptional gene silencing (TGS), post-transcriptional gene silencing (PTGS), quelling, co-suppression, and translational repression) mediated by RNA molecules, which result in the inhibition or "silencing" of the expression of a corresponding protein-coding gene. RNA silencing has been observed in many types of organisms, including plants, animals, and fungi.

[0115] The term "in vitro" has its art recognized meaning, e.g., involving purified reagents or extracts, e.g., cell extracts. The term "in vivo" also has its art recognized meaning, e.g., involving living cells, e.g., immortalized cells, primary cells, cell lines, and / or cells in an organism.

[0116] As used herein, a “target” refers to a particular nucleic acid sequence (e.g., a gene, an mRNA, a miRNA or the like) that an oligonucleotide conjugate or branched oligonucleotide of the disclosure binds to and / or otherwise effects the expression of. In certain embodiments, the target is expressed in the eye. In certain embodiments, target is expressed in a specific eye cell. In other embodiments, a target is associated with a particular disease or disorder in a subject.

[0117] As used herein, the term "target gene" is a gene whose expression is to be substantially inhibited or "silenced." This silencing can be achieved by RNA silencing, e.g., by cleaving the mRNA of the target gene or translational repression of the target gene. The term "non-target gene" is a gene whose expression is not to be substantially silenced. In one embodiment, the polynucleotide sequences of the target and non-target gene (e.g., mRNA encoded by the target and non-target genes) can differ by one or more nucleotides. In another embodiment, the target and non-target genes can differ by one or more polymorphisms (e.g., Single Nucleotide Polymorphisms or SNPs). In another embodiment, the target and non-target genes can shareless than 100% sequence identity. In another embodiment, the non-target gene may be a homologue (e.g., an orthologue or paralogue) of the target gene.

[0118] As used herein, the term "RNA silencing agent" refers to an RNA, which is capable of inhibiting or "silencing" the expression of a target gene. In certain embodiments, the RNA silencing agent is capable of preventing complete processing (e.g., the full translation and / or expression) of a mRNA molecule through a post-transcriptional silencing mechanism. RNA silencing agents include small (<50 b.p.), noncoding RNA molecules, for example RNA duplexes comprising paired strands, as well as precursor RNAs from which such small noncoding RNAs can be generated. Exemplary RNA silencing agents include siRNAs, miRNAs, siRNA-like duplexes, antisense oligonucleotides, GAPMER molecules, short hairpin RNA (shRNA), and dual-function oligonucleotides, as well as precursors thereof. In one embodiment, the RNA silencing agent is capable of inducing RNA interference. In another embodiment, the RNA silencing agent is capable of mediating translational repression.

[0119] As used herein, the term "rare nucleotide" refers to a naturally occurring nucleotide that occurs infrequently, including naturally occurring deoxyribonucleotides or ribonucleotides that occur infrequently, e.g., a naturally occurring ribonucleotide that is not guanosine, adenosine, cytosine, or uridine. Examples of rare nucleotides include, but are not limited to, inosine, 1- methyl inosine, pseudouridine, 5,6-dihydrouridine, ribothymidine, 2N-methylguanosine and 2,2N,N-dimethylguanosine.

[0120] The term "engineered," as in an engineered RNA precursor, or an engineered nucleic acid molecule, indicates that the precursor or molecule is not found in nature, in that all or a portion of the nucleic acid sequence of the precursor or molecule is created or selected by a human. Once created or selected, the sequence can be replicated, translated, transcribed, or otherwise processed by mechanisms within a cell. Thus, an RNA precursor produced within a cell from a transgene that includes an engineered nucleic acid molecule is an engineered RNA precursor.

[0121] As used herein, the term "microRNA" ("miRNA"), also known in the art as "small temporal RNAs" ("stRNAs"), refers to a small (10-50 nucleotide) RNA, which are genetically encoded (e.g., by viral, mammalian, or plant genomes) and are capable of directing or mediating RNA silencing. An "miRNA disorder" shall refer to a disease or disorder characterized by an aberrant expression or activity of a miRNA.

[0122] As used herein, the term "antisense strand" of an RNA silencing agent, e.g., an siRNA, refers to a strand that is substantially complementary to a section of about 10-50 nucleotides, e.g., about 15-30, 16-25, 18-23 or 19-22 nucleotides of the mRNA of the gene targeted for silencing. The antisense strand or first strand has sequence sufficiently complementary to the desired target mRNA sequence to direct target-specific silencing, e.g., complementarity sufficient to trigger the destruction of the desired target mRNA by the RNAi machinery or process (RNAi interference) or complementarity sufficient to trigger translational repression of the desired target mRNA.

[0123] The term "sense strand" or "second strand" of an RNA silencing agent, e.g., an siRNA or RNA silencing agent, refers to a strand that is complementary to the antisense strand or first strand. Antisense and sense strands can also be referred to as first or second strands, the first or second strand having complementarity to the target sequence and the respective second or first strand having complementarity to said first or second strand. miRNA duplex intermediates or siRNA-like duplexes include a miRNA strand having sufficient complementarity to a section of about 10-50 nucleotides of the mRNA of the gene targeted for silencing and a miRNA* strand having sufficient complementarity to form a duplex with the miRNA strand.

[0124] As used herein, the term "guide strand" refers to a strand of an RNA silencing agent, e.g., an antisense strand of an siRNA duplex or siRNA sequence, that enters into the RISC complex and directs cleavage of the target mRNA.

[0125] As used herein, the term "asymmetry," as in the asymmetry of the duplex region of an RNA silencing agent (e.g., the stem of an shRNA), refers to an inequality of bond strength or base pairing strength between the termini of the RNA silencing agent (e.g., between terminal nucleotides on a first strand or stem portion and terminal nucleotides on an opposing second strand or stem portion), such that the 5' end of one strand of the duplex is more frequently in a transient unpaired, e.g., single- stranded, state than the 5' end of the complementary strand. This structural difference determines that one strand of the duplex is preferentially incorporated into a RISC complex. The strand whose 5' end is less tightly paired to the complementary strand will preferentially be incorporated into RISC and mediate RNAi.

[0126] As used herein, the term "bond strength" or "base pair strength" refers to the strength of the interaction between pairs of nucleotides (or nucleotide analogs) on opposing strands of an oligonucleotide duplex (e.g., an siRNA duplex), due primarily to H-bonding, van der Waals interactions, and the like, between said nucleotides (or nucleotide analogs).

[0127] As used herein, the "5' end," as in the 5' end of an antisense strand, refers to the 5' terminal nucleotides, e.g., between one and about 5 nucleotides at the 5' terminus of the antisense strand. As used herein, the "3' end," as in the 3' end of a sense strand, refers to the region, e.g., a region of between one and about 5 nucleotides, that is complementary to the nucleotides of the 5' end of the complementary antisense strand.

[0128] As used herein the term "destabilizing nucleotide" refers to a first nucleotide or nucleotide analog capable of forming a base pair with second nucleotide or nucleotide analog such that the base pair is of lower bond strength than a conventional base pair (i.e., Watson- Crick base pair). In certain embodiments, the destabilizing nucleotide is capable of forming a mismatch base pair with the second nucleotide. In other embodiments, the destabilizing nucleotide is capable of forming a wobble base pair with the second nucleotide. In yet other embodiments, the destabilizing nucleotide is capable of forming an ambiguous base pair with the second nucleotide.

[0129] As used herein, the term "base pair" refers to the interaction between pairs of nucleotides (or nucleotide analogs) on opposing strands of an oligonucleotide duplex (e.g., a duplex formed by a strand of a RNA silencing agent and a target mRNA sequence), due primarily to H-bonding, van der Waals interactions, and the like between said nucleotides (or nucleotide analogs). As used herein, the term "bond strength" or "base pair strength" refers to the strength of the base pair.

[0130] As used herein, the term "mismatched base pair" refers to a base pair consisting of non- complementary or non-Watson-Crick base pairs, for example, not normal complementary G:C, A:T or A:U base pairs. As used herein the term "ambiguous base pair" (also known as a non- discriminatory base pair) refers to a base pair formed by a universal nucleotide.

[0131] As used herein, term "universal nucleotide" (also known as a "neutral nucleotide") include those nucleotides (e.g., certain destabilizing nucleotides) having a base (a "universal base" or "neutral base") that does not significantly discriminate between bases on a complementary polynucleotide when forming a base pair. Universal nucleotides are predominantly hydrophobic molecules that can pack efficiently into antiparallel duplex nucleic acids (e.g., double-stranded DNA or RNA) due to stacking interactions. The base portion of universal nucleotides typically comprise a nitrogen-containing aromatic heterocyclic moiety.

[0132] As used herein, the terms "sufficient complementarity" or "sufficient degree of complementarity" mean that the RNA silencing agent has a sequence (e.g., in the antisensestrand, mRNA targeting moiety or miRNA recruiting moiety), which is sufficient to bind the desired target RNA, respectively, and to trigger the RNA silencing of the target mRNA.

[0133] As used herein, the term "translational repression" refers to a selective inhibition of mRNA translation. Natural translational repression proceeds via miRNAs cleaved from shRNA precursors. Both RNAi and translational repression are mediated by RISC. Both RNAi and translational repression occur naturally or can be initiated by the hand of man, for example, to silence the expression of target genes.

[0134] Various methodologies of the instant disclosure include a step that involves comparing a value, level, feature, characteristic, property, etc. to a "suitable control," referred to interchangeably herein as an "appropriate control." A "suitable control" or "appropriate control" is any control or standard familiar to one of ordinary skill in the art useful for comparison purposes. In one embodiment, a "suitable control" or "appropriate control" is a value, level, feature, characteristic, property, etc. determined prior to performing an RNAi methodology, as described herein. For example, a transcription rate, mRNA level, translation rate, protein level, biological activity, cellular characteristic or property, genotype, phenotype, etc. can be determined prior to introducing an RNA silencing agent of the disclosure into a cell or organism. In another embodiment, a "suitable control" or "appropriate control" is a value, level, feature, characteristic, property, etc. determined in a cell or organism, e.g., a control or normal cell or organism, exhibiting, for example, normal traits. In yet another embodiment, a "suitable control" or "appropriate control" is a predefined value, level, feature, characteristic, property, etc.

[0135] In one aspect, instead of the RNA silencing agent being an interfering ribonucleic acid, e.g., an siRNA or shRNA as described above, the RNAi agent can encode an interfering ribonucleic acid, e.g., an shRNA, as described above. In other words, the RNAi agent can be a transcriptional template of the interfering ribonucleic acid. Thus, RNAi agents of the present disclosure can also include small hairpin RNAs (shRNAs), and expression constructs engineered to express shRNAs. Transcription of shRNAs is initiated at a polymerase III (pol III) promoter, and is thought to be terminated at position 2 of a 4-5-thymine transcription termination site. Upon expression, shRNAs are thought to fold into a stem-loop structure with 3' UU-overhangs; subsequently, the ends of these shRNAs are processed, converting the shRNAs into siRNA-like molecules of about 21-23 nucleotides (Brummelkamp et al., 2002; Lee et al., 2002, Supra, Miyagishi et al., 2002; Paddison et al., 2002, supra, Paul et al., 2002,supra, Sui et al., 2002 supra, Yu et al., 2002, supra. More information about shRNA design and use can be found on the internet at the following addresses: katandin.cshl.org:9331 / RNAi / docs / BseRI-BamHI Strategy.pdf and katandin.cshl.org:9331 / RNAi / docs / W eb_version_of_PCR_strategy 1.pdf).

[0136] Expression constructs of the present disclosure include any construct suitable for use in the appropriate expression system and include, but are not limited to, retroviral vectors, linear expression cassettes, plasmids and viral or virally-derived vectors, as known in the art. Such expression constructs can include one or more inducible promoters, RNA Pol III promoter systems, such as U6 snRNA promoters or Hl RNA polymerase III promoters, or other promoters known in the art. The constructs can include one or both strands of the siRNA. Expression constructs expressing both strands can also include loop structures linking both strands, or each strand can be separately transcribed from separate promoters within the same construct. Each strand can also be transcribed from a separate expression construct. (Tuschl, T., 2002, Supra).

[0137] Synthetic siRNAs can be delivered into cells by methods known in the art, including cationic liposome transfection and electroporation. To obtain longer term suppression of the target genes (e.g., MSTN gene) and to facilitate delivery under certain circumstances, one or more siRNA can be expressed within cells from recombinant DNA constructs. Such methods for expressing siRNA duplexes within cells from recombinant DNA constructs to allow longer- term target gene suppression in cells are known in the art, including mammalian Pol III promoter systems (e.g., Hl or U6 / snRNA promoter systems (Tuschl, T., 2002, supra) capable of expressing functional double-stranded siRNAs; (Bagella et al., 1998; Lee et al., 2002, supra, Miyagishi et al., 2002, supra, Paul et al., 2002, supra, Yu et al., 2002, supra, Sui et al., 2002, supra). Transcriptional termination by RNA Pol III occurs at runs of four consecutive T residues in the DNA template, providing a mechanism to end the siRNA transcript at a specific sequence. The siRNA is complementary to the sequence of the target gene in 5'-3' and 3'-5' orientations, and the two strands of the siRNA can be expressed in the same construct or in separate constructs. Hairpin siRNAs, driven by Hl or U6 snRNA promoter and expressed in cells, can inhibit target gene expression (Bagella et al., 1998; Lee et al., 2002, supra, Miyagishi et al., 2002, supra, Paul et al., 2002, supra, Yu et al., 2002), supra,' Sui et al., 2002, supra). Constructs containing siRNA sequence under the control of T7 promoter also make functional siRNAs when co-transfected into the cells with a vector expressing T7 RNA polymerase(Jacque et al., 2002, supra). A single construct may contain multiple sequences coding for siRNAs, such as multiple regions of the gene encoding MSTN, targeting the same gene or multiple genes, and can be driven, for example, by separate PolIII promoter sites.

[0138] Animal cells express a range of noncoding RNAs of approximately 22 nucleotides termed micro-RNA (miRNAs), which can regulate gene expression at the post transcriptional or translational level during animal development. One common feature of miRNAs is that they are all excised from an approximately 70 nucleotide precursor RNA stem-loop, probably by Dicer, an RNase Ill-type enzyme, or a homolog thereof. By substituting the stem sequences of the miRNA precursor with sequence complementary to the target mRNA, a vector construct that expresses the engineered precursor can be used to produce siRNAs to initiate RNAi against specific mRNA targets in mammalian cells (Zeng et al., 2002, supra). When expressed by DNA vectors containing polymerase III promoters, micro-RNA designed hairpins can silence gene expression (McManus et al., 2002, supra). MicroRNAs targeting polymorphisms may also be useful for blocking translation of mutant proteins, in the absence of siRNA-mediated genesilencing. Such applications may be useful in situations, for example, where a designed siRNA caused off-target silencing of wild type protein.

[0139] Viral-mediated delivery mechanisms can also be used to induce specific silencing of targeted genes through expression of siRNA, for example, by generating recombinant adenoviruses harboring siRNA under RNA Pol II promoter transcription control (Xia et al., 2002, supra). Infection of HeLa cells by these recombinant adenoviruses allows for diminished endogenous target gene expression. Injection of the recombinant adenovirus vectors into transgenic mice expressing the target genes of the siRNA results in in vivo reduction of target gene expression. Id. In an animal model, whole-embryo electroporation can efficiently deliver synthetic siRNA into post-implantation mouse embryos (Calegari et al., 2002). In adult mice, efficient delivery of siRNA can be accomplished by "high-pressure" delivery technique, a rapid injection (within 5 seconds) of a large volume of siRNA containing solution into animal via the tail vein (Liu et al., 1999, supra,' McCaffrey et al., 2002, supra, Lewis et al., 2002. Nanoparticles and liposomes can also be used to deliver siRNA into animals. In certain exemplary embodiments, recombinant adeno-associated viruses (rAAVs) and their associated vectors can be used to deliver one or more siRNAs into cells, e.g., neural cells (e.g., brain cells) (US Patent Applications 2014 / 0296486, 2010 / 0186103, 2008 / 0269149, 2006 / 0078542 and 2005 / 0220766).

[0140] The nucleic acid compositions of the disclosure include both unmodified siRNAs and modified siRNAs, such as crosslinked siRNA derivatives or derivatives having non-nucleotide moieties linked, for example to their 3' or 5' ends. Modifying siRNA derivatives in this way may improve cellular uptake or enhance cellular targeting activities of the resulting siRNA derivative, as compared to the corresponding siRNA, and are useful for tracing the siRNA derivative in the cell, or improving the stability of the siRNA derivative compared to the corresponding siRNA.

[0141] Engineered RNA precursors, introduced into cells or whole organisms as described herein, will lead to the production of a desired siRNA molecule. Such an siRNA molecule will then associate with endogenous protein components of the RNAi pathway to bind to and target a specific mRNA sequence for cleavage and destruction. In this fashion, the mRNA, which will be targeted by the siRNA generated from the engineered RNA precursor, and will be depleted from the cell or organism, leading to a decrease in the concentration of the protein encoded by that mRNA in the cell or organism. The RNA precursors are typically nucleic acid molecules that individually encode either one strand of a dsRNA or encode the entire nucleotide sequence of an RNA hairpin loop structure.

[0142] The nucleic acid compositions of the disclosure can be unconjugated or can be conjugated to another moiety, such as a nanoparticle, to enhance a property of the compositions, e.g., a pharmacokinetic parameter such as absorption, efficacy, bioavailability and / or half-life. The conjugation can be accomplished by methods known in the art, e.g., using the methods of Lambert et al., Drug Deliv. Rev.: 47(1), 99-112 (2001) (describes nucleic acids loaded to polyalkylcyanoacrylate (PACA) nanoparticles); Fattal et al., J. Control Release 53(1- 3): 137-43 (1998) (describes nucleic acids bound to nanoparticles); Schwab et al., Ann. Oncol. 5 Suppl. 4:55-8 (1994) (describes nucleic acids linked to intercalating agents, hydrophobic groups, polycations or PACA nanoparticles); and Godard et al., Eur. J. Biochem. 232(2):404- 10 (1995) (describes nucleic acids linked to nanoparticles).

[0143] The nucleic acid molecules of the present disclosure can also be labeled using any method known in the art. For instance, the nucleic acid compositions can be labeled with a fluorophore, e.g., Cy3, fluorescein, or rhodamine. The labeling can be carried out using a kit, e.g., the SILENCER™ siRNA labeling kit (Ambion). Additionally, the siRNA can be radiolabeled, e.g., using3H,32P or another appropriate isotope.

[0144] Moreover, because RNAi is believed to progress via at least one single-stranded RNA intermediate, the skilled artisan will appreciate that ss-siRNAs (e.g., the antisense strand of a ds-siRNA) can also be designed (e.g., for chemical synthesis), generated (e.g., enzymatically generated), or expressed (e.g., from a vector or plasmid) as described herein and utilized according to the claimed methodologies. Moreover, in invertebrates, RNAi can be triggered effectively by long dsRNAs (e.g., dsRNAs about 100-1000 nucleotides in length, such as about 200-500, for example, about 250, 300, 350, 400 or 450 nucleotides in length) acting as effectors of RNAi. (Brondani et al., Proc Natl Acad Sci USA. 2001 Dec. 4; 98(25): 14428-33. Epub 2001 Nov. 27.)MSTN-Targeting Oligonucleotides

[0145] Myostatin, also known as MSTN, GDF8, or growth differentiation factor 8, is a myokine that is produced and released by myocytes and acts on muscle cells to inhibit muscle growth (see, Saunders et al. American Journal of Human Genetics. 2006. 79(6): 1089-1097). Myostatin is a secreted growth differentiation factor that is a member of the TGF beta protein family (see, Carnac et al. Mini Reviews in Medicinal Chemistry. 2006 6(7): 765-770; Joulia- Ekaza et al. Current Opinion in Pharmacology. 2007. 7(3): 310-315). The human MSTN gene is provided in NCBI Reference Sequence NG_009800.1. The mouse MSTN gene is provided in NCBI Reference Sequence NC_000067.7. Described herein are oligonucleotides (e.g., siRNA, antisense oligonucleotides, short hairpin RNA (shRNA)) that target MSTN mRNA and effectively silence MSTN expression.

[0146] In one aspect, the disclosure provides an RNA molecule (e.g., an siRNA) comprising a sequence substantially complementary to a MSTN nucleic acid sequence of any one of SEQ ID NOs: 1-14 or 29-31.

[0147] In one aspect, the disclosure provides an RNA molecule (e.g., an siRNA) comprising a sequence substantially complementary to a MSTN nucleic acid sequence of SEQ ID NO: 1.

[0148] In one aspect, the disclosure provides an RNA molecule (e.g., an siRNA) comprising a sequence substantially complementary to a MSTN nucleic acid sequence of SEQ ID NO: 2.

[0149] In one aspect, the disclosure provides an RNA molecule (e.g., an siRNA) comprising a sequence substantially complementary to a MSTN nucleic acid sequence of SEQ ID NO: 3.

[0150] In one aspect, the disclosure provides an RNA molecule (e.g., an siRNA) comprising a sequence substantially complementary to a MSTN nucleic acid sequence of SEQ ID NO: 4.

[0151] In one aspect, the disclosure provides an RNA molecule (e.g., an siRNA) comprising a sequence substantially complementary to a MSTN nucleic acid sequence of SEQ ID NO: 5.

[0152] In one aspect, the disclosure provides an RNA molecule (e.g., an siRNA) comprising a sequence substantially complementary to a MSTN nucleic acid sequence of SEQ ID NO: 6.

[0153] In one aspect, the disclosure provides an RNA molecule (e.g., an siRNA) comprising a sequence substantially complementary to a MSTN nucleic acid sequence of SEQ ID NO: 7.

[0154] In one aspect, the disclosure provides an RNA molecule (e.g., an siRNA) comprising a sequence substantially complementary to a MSTN nucleic acid sequence of SEQ ID NO: 8.

[0155] In one aspect, the disclosure provides an RNA molecule (e.g., an siRNA) comprising a sequence substantially complementary to a MSTN nucleic acid sequence of SEQ ID NO: 9.

[0156] In one aspect, the disclosure provides an RNA molecule (e.g., an siRNA) comprising a sequence substantially complementary to a MSTN nucleic acid sequence of SEQ ID NO: 10.

[0157] In one aspect, the disclosure provides an RNA molecule (e.g., an siRNA) comprising a sequence substantially complementary to a MSTN nucleic acid sequence of SEQ ID NO: 11.

[0158] In one aspect, the disclosure provides an RNA molecule (e.g., an siRNA) comprising a sequence substantially complementary to a MSTN nucleic acid sequence of SEQ ID NO: 12.

[0159] In one aspect, the disclosure provides an RNA molecule (e.g., an siRNA) comprising a sequence substantially complementary to a MSTN nucleic acid sequence of SEQ ID NO: 13.

[0160] In one aspect, the disclosure provides an RNA molecule (e.g., an siRNA) comprising a sequence substantially complementary to a MSTN nucleic acid sequence of SEQ ID NO: 14.

[0161] In one aspect, the disclosure provides an RNA molecule (e.g., an siRNA) comprising a sequence substantially complementary to a MSTN nucleic acid sequence of SEQ ID NO: 29.

[0162] In one aspect, the disclosure provides an RNA molecule (e.g., an siRNA) comprising a sequence substantially complementary to a MSTN nucleic acid sequence of SEQ ID NO: 30.

[0163] In one aspect, the disclosure provides an RNA molecule (e.g., an siRNA) comprising a sequence substantially complementary to a MSTN nucleic acid sequence of SEQ ID NO: 31.

[0164] In certain embodiments, the RNA molecule comprises a sequence substantially complementary to a MSTN nucleic acid sequence of any one of SEQ ID NOs: 15-28 or 32-34. In certain embodiments, the RNA molecule comprises a sequence substantially complementary to a MSTN nucleic acid sequence of SEQ ID NO: 15. In certain embodiments, the RNA molecule comprises a sequence substantially complementary to a MSTN nucleic acid sequence of SEQ ID NO: 16. In certain embodiments, the RNA molecule comprises a sequence substantially complementary to a MSTN nucleic acid sequence of SEQ ID NO: 17. In certain embodiments, the RNA molecule comprises a sequence substantially complementary to a MSTN nucleic acid sequence of SEQ ID NO: 18. In certain embodiments, the RNA molecule comprises a sequence substantially complementary to a MSTN nucleic acid sequence of SEQ ID NO: 19. In certain embodiments, the RNA molecule comprises a sequence substantially complementary to a MSTN nucleic acid sequence of SEQ ID NO: 20. In certain embodiments, the RNA molecule comprises a sequence substantially complementary to a MSTN nucleic acid sequence of SEQ ID NO: 21. In certain embodiments, the RNA molecule comprises a sequence substantially complementary to a MSTN nucleic acid sequence of SEQ ID NO: 22. In certain embodiments, the RNA molecule comprises a sequence substantially complementary to a MSTN nucleic acid sequence of SEQ ID NO: 23. In certain embodiments, the RNA molecule comprises a sequence substantially complementary to a MSTN nucleic acid sequence of SEQ ID NO: 24. In certain embodiments, the RNA molecule comprises a sequence substantially complementary to a MSTN nucleic acid sequence of SEQ ID NO: 25. In certain embodiments, the RNA molecule comprises a sequence substantially complementary to a MSTN nucleic acid sequence of SEQ ID NO: 26. In certain embodiments, the RNA molecule comprises a sequence substantially complementary to a MSTN nucleic acid sequence of SEQ ID NO: 27. In certain embodiments, the RNA molecule comprises a sequence substantially complementary to a MSTN nucleic acid sequence of SEQ ID NO: 28. In certain embodiments, the RNA molecule comprises a sequence substantially complementary to a MSTN nucleic acid sequence of SEQ ID NO: 32. In certain embodiments, the RNA molecule comprises a sequence substantially complementary to a MSTN nucleic acid sequence of SEQ ID NO: 33. In certain embodiments, the RNA molecule comprises a sequence substantially complementary to a MSTN nucleic acid sequence of SEQ ID NO: 34.

[0165] In one aspect, the disclosure provides an RNA molecule (e.g., an siRNA) comprising a sequence substantially complementary to a MSTN nucleic acid sequence recited in Table 1, Table 2, Table 6, or Table 7.

[0166] In one aspect, the disclosure provides an RNA molecule (e.g., an siRNA) comprising a sequence substantially complementary to a MSTN nucleic acid sequence recited in Table 1, Table 2, Table 6, or Table 7 that achieves at least 50% reduction in MSTN mRNA levels relative to a control as recited in Table 5 or Table 10. By way of example, an RNA molecule with a sequence substantially complementary to a MSTN target position 296 achieves 18.39% expression of MSTN mRNA relative to a control, as shown in Table 5. Thus, the disclosure provides an RNA molecule with a sequence substantially complementary to target ID 296 of Table 1 or Table 2.

[0167] In certain embodiments, the RNA molecule comprises complementarity to at least 10, 11, 12 or 13 contiguous nucleotides of the MSTN nucleic acid sequence of any one of SEQ ID NOs: 1-14 or 29-31.

[0168] In certain embodiments, the RNA molecule comprises no more than 3 mismatches with the MSTN nucleic acid sequence of any one of SEQ ID NOs: 1-14 or 29-31.

[0169] In certain embodiments, the RNA molecule comprises full complementarity to the MSTN nucleic acid sequence of any one of SEQ ID NOs: 1-14 or 29-31.

[0170] In certain embodiments, the RNA molecule comprises a single stranded RNA (ssRNA) or a double stranded RNA (dsRNA).

[0171] In certain embodiments, the RNA molecule comprises an antisense oligonucleotide.

[0172] In certain embodiments, the RNA molecule comprises an siRNA, the siRNA comprising an antisense strand comprising substantially complementary to the MSTN nucleic acid sequence and a sense strand.

[0173] In certain embodiments, the antisense strand comprises about 15 nucleotides to 25 nucleotides in length (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length). In certain embodiments, the antisense strand is 20 nucleotides in length, 21 nucleotides in length, or 22 nucleotides in length. In certain embodiments, the sense strand comprises about 15 nucleotides to 25 nucleotides in length (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length). In certain embodiments, the sense strand is 15 nucleotides in length, 16 nucleotides in length, 18 nucleotides in length, or 20 nucleotides in length.

[0174] In certain embodiments, the siRNA comprises a double-stranded region of 15 base pairs to 20 base pairs (e.g., 15, 16, 17, 18, 19, or 20 base pairs). In certain embodiments, the siRNAcomprises a double-stranded region of 15 base pairs, 16 base pairs, 18 base pairs, or 20 base pairs.

[0175] In certain embodiments, the siRNA comprises at least one blunt-end. In certain embodiments, the siRNA comprises two blunt-ends.

[0176] In certain embodiments, the siRNA comprises at least one single stranded nucleotide overhang (also referred to herein as a “single-stranded tail”). In certain embodiments, the siRNA comprises two single stranded nucleotide overhangs. In certain embodiments, the siRNA comprises about a 2-nucleotide to 5-nucleotide single stranded nucleotide overhang (e.g., a 2-, 3-, 4-, or 5-nucleotide overhang). In certain embodiments, the siRNA comprises a 2-nucleotide single stranded nucleotide overhang or a 5-nucleotide single stranded nucleotide overhang.

[0177] In certain embodiments, the siRNA comprises naturally occurring nucleotides (i.e., unmodified ribonucleotides).

[0178] In certain embodiments, the siRNA comprises at least one modified nucleotide. In certain embodiments, the modified nucleotide comprises a 2'-O-methyl modified nucleotide, a 2'-deoxy-2'-fluoro modified nucleotide, a 2'-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, a 2'-amino-modified nucleotide, a 2'-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base comprising nucleotide, or a mixture thereof.

[0179] In certain embodiments, the siRNA comprises at least one modified intemucleotide linkage. In certain embodiments, the modified intemucleotide linkage comprises a phosphorothioate intemucleotide linkage. In certain embodiments, the siRNA comprises 4-16 phosphorothioate intemucleotide linkages. In certain embodiments, the siRNA comprises 8-13 phosphorothioate intemucleotide linkages.

[0180] In certain embodiments, the siRNA comprises at least 80% chemically modified nucleotides (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% chemically modified nucleotides). In certain embodiments, the siRNA is fully chemically modified.

[0181] In certain embodiments, the siRNA comprises at least 70% 2’-O-methyl nucleotide modifications (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or 100% % 2’-O-methyl nucleotide modifications). In certain embodiments, the antisense strand comprises at least 70% 2’-O-methyl nucleotide modifications (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% % 2’-O-methyl nucleotide modifications). In certain embodiments, the antisense strand comprises about 70% to 90% 2’-O-methyl nucleotide modifications. In certain embodiments, the sense strand comprises at least 65% 2’-O-methyl nucleotide modifications (e.g., 65%, 66%, 67%, 68%, 69% 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% % 2’-O-methyl nucleotide modifications). In certain embodiments, the sense strand comprises 100% 2’-O-methyl nucleotide modifications.

[0182] In certain embodiments, the sense strand comprises one or more nucleotide mismatches between the antisense strand and the sense strand.

[0183] In certain embodiments, the antisense strand comprises a 5’ phosphate, a 5 ’-alkyl phosphonate, a 5’ alkylene phosphonate, or a 5’ alkenyl phosphonate. In certain embodiments, the antisense strand comprises a 5’ vinyl phosphonate.Anti-MSTN Short Hairpin RNA (shRNA) Molecules

[0184] In certain featured embodiments, the instant disclosure provides shRNAs capable of mediating RNA silencing of an MSTN target sequence with enhanced selectivity. In contrast to siRNAs, shRNAs mimic the natural precursors of micro RNAs (miRNAs) and enter at the top of the gene silencing pathway. For this reason, shRNAs are believed to mediate gene silencing more efficiently by being fed through the entire natural gene silencing pathway.

[0185] miRNAs are noncoding RNAs of approximately 22 nucleotides, which can regulate gene expression at the post transcriptional or translational level during plant and animal development. One common feature of miRNAs is that they are all excised from an approximately 70 nucleotide precursor RNA stem-loop termed pre-miRNA, probably by Dicer, an RNase Ill-type enzyme, or a homolog thereof. Naturally-occurring miRNA precursors (pre- miRNA) have a single strand that forms a duplex stem including two portions that are generally complementary, and a loop, which connects the two portions of the stem. In typical pre- miRNAs, the stem includes one or more bulges, e.g., extra nucleotides that create a singlenucleotide "loop" in one portion of the stem, and / or one or more unpaired nucleotides that create a gap in the hybridization of the two portions of the stem to each other. Short hairpin RNAs, or engineered RNA precursors, of the present application are artificial constructs based on these naturally occurring pre-miRNAs, but which are engineered to deliver desired RNA silencing agents (e.g., siRNAs of the disclosure). By substituting the stem sequences of the pre- miRNA with sequence complementary to the target mRNA, a shRNA is formed. The shRNA is processed by the entire gene silencing pathway of the cell, thereby efficiently mediating RNAi.

[0186] The requisite elements of a shRNA molecule include a first portion and a second portion, having sufficient complementarity to anneal or hybridize to form a duplex or doublestranded stem portion. The two portions need not be fully or perfectly complementary. The first and second "stem" portions are connected by a portion having a sequence that has insufficient sequence complementarity to anneal or hybridize to other portions of the shRNA. This latter portion is referred to as a "loop" portion in the shRNA molecule. The shRNA molecules are processed to generate siRNAs. shRNAs can also include one or more bulges, i.e., extra nucleotides that create a small nucleotide "loop" in a portion of the stem, for example a one-, two- or three-nucleotide loop. The stem portions can be the same length, or one portion can include an overhang of, for example, 1-5 nucleotides. The overhanging nucleotides can include, for example, uracils (Us), e.g., all Us. Such Us are notably encoded by thymidines (Ts) in the shRNA-encoding DNA which signal the termination of transcription.

[0187] In shRNAs (or engineered precursor RNAs) of the instant disclosure, one portion of the duplex stem is a nucleic acid sequence that is complementary (or anti-sense) to the AP target sequence. In certain embodiments, one strand of the stem portion of the shRNA is sufficiently complementary (e.g., antisense) to a target RNA (e.g., mRNA) sequence to mediate degradation or cleavage of said target RNA via RNA interference (RNAi). Thus, engineered RNA precursors include a duplex stem with two portions and a loop connecting the two stem portions. The antisense portion can be on the 5' or 3' end of the stem. The stem portions of a shRNA are about 15 to about 50 nucleotides in length. In certain embodiments, the two stem portions are about 18 or 19 to about 21, 22, 23, 24, 25, 30, 35, 37, 38, 39, or 40 or more nucleotides in length. In certain embodiments, the length of the stem portions should be 21 nucleotides or greater. When used in mammalian cells, the length of the stem portions should be less than about 30 nucleotides to avoid provoking non-specific responses like the interferonpathway. In non-mammalian cells, the stem can be longer than 30 nucleotides. In fact, the stem can include much larger sections complementary to the target mRNA (up to, and including the entire mRNA). In fact, a stem portion can include much larger sections complementary to the target mRNA (up to, and including the entire mRNA).

[0188] The two portions of the duplex stem must be sufficiently complementary to hybridize to form the duplex stem. Thus, the two portions can be, but need not be, fully or perfectly complementary. In addition, the two stem portions can be the same length, or one portion can include an overhang of 1, 2, 3, or 4 nucleotides. The overhanging nucleotides can include, for example, uracils (Us), e.g., all Us. The loop in the shRNAs or engineered RNA precursors may differ from natural pre-miRNA sequences by modifying the loop sequence to increase or decrease the number of paired nucleotides, or replacing all or part of the loop sequence with a tetraloop or other loop sequences. Thus, the loop in the shRNAs or engineered RNA precursors can be 2, 3, 4, 5, 6, 7, 8, 9, or more, e.g., 15 or 20, or more nucleotides in length.

[0189] The loop in the shRNAs or engineered RNA precursors may differ from natural pre- miRNA sequences by modifying the loop sequence to increase or decrease the number of paired nucleotides, or replacing all or part of the loop sequence with a tetraloop or other loop sequences. Thus, the loop portion in the shRNA can be about 2 to about 20 nucleotides in length, i.e., about 2, 3, 4, 5, 6, 7, 8, 9, or more, e.g., 15 or 20, or more nucleotides in length. In certain embodiments, a loop consists of or comprises a "tetraloop" sequence. Exemplary tetraloop sequences include, but are not limited to, the sequences GNRA, where N is any nucleotide and R is a purine nucleotide, GGGG, and UUUU.

[0190] In certain embodiments, shRNAs of the present application include the sequences of a desired siRNA molecule described supra. In other embodiments, the sequence of the antisense portion of a shRNA can be designed essentially as described above or generally by selecting an 18, 19, 20, 21 nucleotide, or longer, sequence from within the target RNA (e.g., APP mRNA), for example, from a region 100 to 200 or 300 nucleotides upstream or downstream of the start of translation. In general, the sequence can be selected from any portion of the target RNA (e.g., mRNA) including the 5' UTR (untranslated region), coding sequence, or 3' UTR. This sequence can optionally follow immediately after a region of the target gene containing two adjacent AA nucleotides. The last two nucleotides of the nucleotide sequence can be selected to be UU. This 21 or so nucleotide sequence is used to create one portion of a duplex stem in the shRNA. This sequence can replace a stem portion of a wild-type pre-miRNAsequence, e.g., enzymatically, or is included in a complete sequence that is synthesized. For example, one can synthesize DNA oligonucleotides that encode the entire stem-loop engineered RNA precursor, or that encode just the portion to be inserted into the duplex stem of the precursor, and using restriction enzymes to build the engineered RNA precursor construct, e.g., from a wild-type pre-miRNA.

[0191] Engineered RNA precursors include, in the duplex stem, the 21-22 or so nucleotide sequences of the siRNA or siRNA-like duplex desired to be produced in vivo. Thus, the stem portion of the engineered RNA precursor includes at least 18 or 19 nucleotide pairs corresponding to the sequence of an exonic portion of the gene whose expression is to be reduced or inhibited. The two 3' nucleotides flanking this region of the stem are chosen so as to maximize the production of the siRNA from the engineered RNA precursor and to maximize the efficacy of the resulting siRNA in targeting the corresponding mRNA for translational repression or destruction by RNAi in vivo and in vitro.

[0192] In certain embodiments, shRNAs of the disclosure include miRNA sequences, optionally end-modified miRNA sequences, to enhance entry into RISC. The miRNA sequence can be similar or identical to that of any naturally occurring miRNA (see e.g. The miRNA Registry; Griffiths-Jones S, Nuc. Acids Res., 2004). Over one thousand natural miRNAs have been identified to date and together they are thought to comprise about 1% of all predicted genes in the genome. Many natural miRNAs are clustered together in the introns of pre-mRNAs and can be identified in silico using homology-based searches (Pasquinelli et al., 2000; Lagos- Quintana et al., 2001; Lau et al., 2001; Lee and Ambros, 2001) or computer algorithms (e.g. MiRScan, MiRSeeker) that predict the capability of a candidate miRNA gene to form the stem loop structure of a pri-mRNA (Grad et al., Mol. Cell., 2003; Lim et al., Genes Dev., 2003; Lim et al., Science, 2003; Lai E C et al., Genome Bio., 2003). An online registry provides a searchable database of all published miRNA sequences (The miRNA Registry at the Sanger Institute website; Griffiths-Jones S, Nuc. Acids Res., 2004). Exemplary, natural miRNAs include lin-4, let-7, miR-10, mirR-15, miR-16, miR-168, miR-175, miR-196 and their homologs, as well as other natural miRNAs from humans and certain model organisms including Drosophila melanogaster, Caenorhabditis elegans. zebrafish, Arabidopsis thalania. Mus musculus, and Rattus norvegicus as described in International PCT Publication No. WO 03 / 029459.

[0193] Naturally-occurring miRNAs are expressed by endogenous genes in vivo and are processed from a hairpin or stem-loop precursor (pre-miRNA or pri-miRNAs) by Dicer or other RNAses (Lagos-Quintana et al., Science, 2001; Lau et al., Science, 2001; Lee and Ambros, Science, 2001; Lagos-Quintana et al., Curr. Biol., 2002; Mourelatos et al., Genes Dev., 2002; Reinhart et al., Science, 2002; Ambros et al., Curr. Biol., 2003; Brennecke et al., 2003; Lagos- Quintana et al., RNA, 2003; Lim et al., Genes Dev., 2003; Lim et al., Science, 2003). miRNAs can exist transiently in vivo as a double-stranded duplex, but only one strand is taken up by the RISC complex to direct gene silencing. Certain miRNAs, e.g., plant miRNAs, have perfect or near-perfect complementarity to their target mRNAs and, hence, direct cleavage of the target mRNAs. Other miRNAs have less than perfect complementarity to their target mRNAs and, hence, direct translational repression of the target mRNAs. The degree of complementarity between a miRNA and its target mRNA is believed to determine its mechanism of action. For example, perfect or near-perfect complementarity between a miRNA and its target mRNA is predictive of a cleavage mechanism (Yekta et al., Science, 2004), whereas less than perfect complementarity is predictive of a translational repression mechanism. In certain embodiments, the miRNA sequence is that of a naturally-occurring miRNA sequence, the aberrant expression or activity of which is correlated with a miRNA disorder.Modified Anti-MSTN RNA Silencing Agents

[0194] In certain aspects of the disclosure, an RNA silencing agent (or any portion thereof) of the present application, as described supra, may be modified, such that the activity of the agent is further improved. For example, the RNA silencing agents described in Section II supra, may be modified with any of the modifications described infra. The modifications can, in part, serve to further enhance target discrimination, to enhance stability of the agent (e.g., to prevent degradation), to promote cellular uptake, to enhance the target efficiency, to improve efficacy in binding (e.g., to the targets), to improve patient tolerance to the agent, and / or to reduce toxicity.1) Modifications to Enhance Target Discrimination

[0195] In certain embodiments, the RNA silencing agents of the present application may be substituted with a destabilizing nucleotide to enhance single nucleotide target discrimination(see U.S. application Ser. No. 11 / 698,689, filed Jan. 25, 2007, and U.S. Provisional Application No. 60 / 762,225 filed Jan. 25, 2006, both of which are incorporated herein by reference). Such a modification may be sufficient to abolish the specificity of the RNA silencing agent for a non-target mRNA (e.g., wild-type mRNA), without appreciably affecting the specificity of the RNA silencing agent for a target mRNA (e.g., gain-of-function mutant mRNA).

[0196] In certain embodiments, the RNA silencing agents of the present application are modified by the introduction of at least one universal nucleotide in the antisense strand thereof. Universal nucleotides comprise base portions that are capable of base pairing indiscriminately with any of the four conventional nucleotide bases (e.g., A, G, C, U). A universal nucleotide is contemplated because it has relatively minor effect on the stability of the RNA duplex or the duplex formed by the guide strand of the RNA silencing agent and the target mRNA. Exemplary universal nucleotides include those having an inosine base portion or an inosine analog base portion selected from the group consisting of deoxyinosine (e.g., 2'-deoxyinosine), 7-deaza-2'-deoxyinosine, 2'-aza-2'-deoxyinosine, PNA-inosine, morpholino-inosine, LNA- inosine, phosphoramidate-inosine, 2'-O-methoxyethyl-inosine, and 2'-OMe-inosine. In certain embodiments, the universal nucleotide is an inosine residue or a naturally occurring analog thereof.

[0197] In certain embodiments, the RNA silencing agents of the disclosure are modified by the introduction of at least one destabilizing nucleotide within 5 nucleotides from a specificitydetermining nucleotide (i.e., the nucleotide which recognizes the disease-related polymorphism). For example, the destabilizing nucleotide may be introduced at a position that is within 5, 4, 3, 2, or 1 nucleotide(s) from a specificity-determining nucleotide. In exemplary embodiments, the destabilizing nucleotide is introduced at a position which is 3 nucleotides from the specificity-determining nucleotide (i.e., such that there are 2 stabilizing nucleotides between the destabilizing nucleotide and the specificity-determining nucleotide). In RNA silencing agents having two strands or strand portions (e.g., siRNAs and shRNAs), the destabilizing nucleotide may be introduced in the strand or strand portion that does not contain the specificity-determining nucleotide. In certain embodiments, the destabilizing nucleotide is introduced in the same strand or strand portion that contains the specificity-determining nucleotide.2) Modifications to Enhance Efficacy and Specificity

[0198] In certain embodiments, the RNA silencing agents of the disclosure may be altered to facilitate enhanced efficacy and specificity in mediating RNAi according to asymmetry design rules (see U.S. Patent Nos. 8,309,704, 7,750,144, 8,304,530, 8,329,892 and 8,309,705). Such alterations facilitate entry of the antisense strand of the siRNA (e.g., a siRNA designed using the methods of the present application or an siRNA produced from a shRNA) into RISC in favor of the sense strand, such that the antisense strand preferentially guides cleavage or translational repression of a target mRNA, and thus increasing or improving the efficiency of target cleavage and silencing. In certain embodiments, the asymmetry of an RNA silencing agent is enhanced by lessening the base pair strength between the antisense strand 5' end (AS 5') and the sense strand 3' end (S 3') of the RNA silencing agent relative to the bond strength or base pair strength between the antisense strand 3' end (AS 3') and the sense strand 5' end (S '5) of said RNA silencing agent.

[0199] In one embodiment, the asymmetry of an RNA silencing agent of the present application may be enhanced such that there are fewer G:C base pairs between the 5' end of the first or antisense strand and the 3' end of the sense strand portion than between the 3' end of the first or antisense strand and the 5' end of the sense strand portion. In another embodiment, the asymmetry of an RNA silencing agent of the disclosure may be enhanced such that there is at least one mismatched base pair between the 5' end of the first or antisense strand and the 3' end of the sense strand portion. In certain embodiments, the mismatched base pair is selected from the group consisting of G:A, C:A, C:U, G:G, A:A, C:C and U:U. In another embodiment, the asymmetry of an RNA silencing agent of the disclosure may be enhanced such that there is at least one wobble base pair, e.g., G:U, between the 5' end of the first or antisense strand and the 3' end of the sense strand portion. In another embodiment, the asymmetry of an RNA silencing agent of the disclosure may be enhanced such that there is at least one base pair comprising a rare nucleotide, e.g., inosine (I). In certain embodiments, the base pair is selected from the group consisting of an I: A, I:U and I:C. In yet another embodiment, the asymmetry of an RNA silencing agent of the disclosure may be enhanced such that there is at least one base pair comprising a modified nucleotide. In certain embodiments, the modified nucleotide is selected from the group consisting of 2-amino-G, 2-amino-A, 2,6-diamino-G, and 2,6-diamino-A.3) RNA Silencing Agents with Enhanced Stability

[0200] The RNA silencing agents of the present application can be modified to improve stability in serum or in growth medium for cell cultures. In order to enhance the stability, the 3 '-residues may be stabilized against degradation, e.g., they may be selected such that they consist of purine nucleotides, such as adenosine or guanosine nucleotides. Alternatively, substitution of pyrimidine nucleotides by modified analogues, e.g., substitution of uridine by 2'-deoxythymidine is tolerated and does not affect the efficiency of RNA interference.

[0201] In a one aspect, the present application features RNA silencing agents that include first and second strands wherein the second strand and / or first strand is modified by the substitution of internal nucleotides with modified nucleotides, such that in vivo stability is enhanced as compared to a corresponding unmodified RNA silencing agent. As defined herein, an "internal" nucleotide is one occurring at any position other than the 5' end or 3' end of nucleic acid molecule, polynucleotide or oligonucleotide. An internal nucleotide can be within a singlestranded molecule or within a strand of a duplex or double-stranded molecule. In one embodiment, the sense strand and / or antisense strand is modified by the substitution of at least one internal nucleotide. In another embodiment, the sense strand and / or antisense strand is modified by the substitution of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more internal nucleotides. In another embodiment, the sense strand and / or antisense strand is modified by the substitution of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of the internal nucleotides. In yet another embodiment, the sense strand and / or antisense strand is modified by the substitution of all of the internal nucleotides.

[0202] In one aspect, the present application features RNA silencing agents that are at least 80% chemically modified. In certain embodiments, the RNA silencing agents may be fully chemically modified, i.e., 100% of the nucleotides are chemically modified. In another aspect, the present application features RNA silencing agents comprising 2’-OH ribose groups that are at least 80% chemically modified. In certain embodiments, the RNA silencing agents comprise 2’ -OH ribose groups that are about 80%, 85%, 90%, 95%, or 100% chemically modified.

[0203] In certain embodiments, the RNA silencing agents may contain at least one modified nucleotide analogue. The nucleotide analogues may be located at positions where the targetspecific silencing activity, e.g., the RNAi mediating activity or translational repression activityis not substantially affected, e.g., in a region at the 5'-end and / or the 3'-end of the siRNA molecule. Moreover, the ends may be stabilized by incorporating modified nucleotide analogues.

[0204] Exemplary nucleotide analogues include sugar- and / or backbone-modified ribonucleotides (i.e., include modifications to the phosphate-sugar backbone). For example, the phosphodiester linkages of natural RNA may be modified to include at least one of a nitrogen or sulfur heteroatom. In exemplary backbone-modified ribonucleotides, the phosphoester group connecting to adjacent ribonucleotides is replaced by a modified group, e.g., of phosphothioate group. In exemplary sugar-modified ribonucleotides, the 2' OH-group is replaced by a group selected from H, OR, R, halo, SH, SR, NH2, NHR, NR2 or ON, wherein R is Ci-Ce alkyl, alkenyl or alkynyl and halo is F, Cl, Br or I.

[0205] In certain embodiments, the modifications are 2'-fluoro, 2'-amino and / or 2'-thio modifications. Modifications include 2'-fluoro-cytidine, 2'-fluoro-uridine, 2'-fluoro-adenosine, 2'-fluoro-guanosine, 2'-amino-cytidine, 2'-amino-uridine, 2'-amino-adenosine, 2'-amino- guanosine, 2,6-diaminopurine, 4-thio-uridine, and / or 5-amino-allyl-uridine. In a certain embodiment, the 2'-fluoro ribonucleotides are every uridine and cytidine. Additional exemplary modifications include 5-bromo-uridine, 5-iodo-uridine, 5-methyl-cytidine, ribothymidine, 2-aminopurine, 2'-amino-butyryl-pyrene-uridine, 5-fluoro-cytidine, and 5-fluoro- uridine. 2'-deoxy-nucleotides and 2'-0me nucleotides can also be used within modified RNA- silencing agent moieties of the instant disclosure. Additional modified residues include, deoxy - abasic, inosine, N3-methyl-uridine, N6,N6-dimethyl-adenosine, pseudouridine, purine ribonucleoside and ribavirin. In a certain embodiment, the 2' moiety is a methyl group such that the linking moiety is a 2'-O-methyl oligonucleotide.

[0206] In a certain embodiment, the RNA silencing agent of the present application comprises Locked Nucleic Acids (LNAs). LNAs comprise sugar-modified nucleotides that resist nuclease activities (are highly stable) and possess single nucleotide discrimination for mRNA (Elmen et al., Nucleic Acids Res., (2005), 33(1): 439-447; Braasch et al. (2003) Biochemistry 42:7967- 7975, Petersen et al. (2003) Trends Biotechnol 21 :74-81). These molecules have 2'-O,4'-C- ethylene-bridged nucleic acids, with possible modifications such as 2'-deoxy-2"-fluorouridine. Moreover, LNAs increase the specificity of oligonucleotides by constraining the sugar moiety into the 3'-endo conformation, thereby pre-organizing the nucleotide for base pairing and increasing the melting temperature of the oligonucleotide by as much as 10 °C per base.

[0207] In another exemplary embodiment, the RNA silencing agent of the present application comprises Peptide Nucleic Acids (PNAs). PNAs comprise modified nucleotides in which the sugar-phosphate portion of the nucleotide is replaced with a neutral 2-amino ethylglycine moiety capable of forming a polyamide backbone , which is highly resistant to nuclease digestion and imparts improved binding specificity to the molecule (Nielsen, et al., Science, (2001), 254: 1497-1500).

[0208] Also contemplated are nucleobase-modified ribonucleotides, i.e., ribonucleotides, containing at least one non-naturally occurring nucleobase instead of a naturally occurring nucleobase. Bases may be modified to block the activity of adenosine deaminase. Exemplary modified nucleobases include, but are not limited to, uridine and / or cytidine modified at the 5- position, e.g., 5-(2-amino)propyl uridine, 5-bromo uridine; adenosine and / or guanosines modified at the 8 position, e.g., 8-bromo guanosine; deaza nucleotides, e.g., 7-deaza-adenosine; O- and N-alkylated nucleotides, e.g., N6-methyl adenosine are suitable. It should be noted that the above modifications may be combined.

[0209] In other embodiments, cross-linking can be employed to alter the pharmacokinetics of the RNA silencing agent, for example, to increase half-life in the body. Thus, the present application includes RNA silencing agents having two complementary strands of nucleic acid, wherein the two strands are crosslinked. The present application also includes RNA silencing agents which are conjugated or unconjugated (e.g., at its 3' terminus) to another moiety (e.g., a non-nucleic acid moiety such as a peptide), an organic compound (e.g., a dye), or the like). Modifying siRNA derivatives in this way may improve cellular uptake or enhance cellular targeting activities of the resulting siRNA derivative as compared to the corresponding siRNA, are useful for tracing the siRNA derivative in the cell, or improve the stability of the siRNA derivative compared to the corresponding siRNA.

[0210] Other exemplary modifications include: (a) 2' modification, e.g., provision of a 2' OMe moiety on a U in a sense or antisense strand, but especially on a sense strand, or provision of a 2' OMe moiety in a 3' overhang, e.g., at the 3' terminus (3' terminus means at the 3' atom of the molecule or at the most 3' moiety, e.g., the most 3' P or 2' position, as indicated by the context); (b) modification of the backbone, e.g., with the replacement of an 0 with an S, in the phosphate backbone, e.g., the provision of a phosphorothioate modification, on the U or the A or both, especially on an antisense strand; e.g., with the replacement of a O with an S; (c) replacement of the U with a C5 amino linker; (d) replacement of an A with a G (sequence changes can belocated on the sense strand and not the antisense strand in certain embodiments); and (d) modification at the 2', 6', 7', or 8' position. Exemplary embodiments are those in which one or more of these modifications are present on the sense but not the antisense strand, or embodiments where the antisense strand has fewer of such modifications. Yet other exemplary modifications include the use of a methylated P in a 3' overhang, e.g., at the 3' terminus; combination of a 2' modification, e.g., provision of a 2' O Me moiety and modification of the backbone, e.g., with the replacement of a O with an S, e.g., the provision of a phosphorothioate modification, or the use of a methylated P, in a 3' overhang, e.g., at the 3' terminus; modification with a 3' alkyl; modification with an abasic pyrrolidone in a 3' overhang, e.g., at the 3' terminus; modification with naproxen, ibuprofen, or other moi eties which inhibit degradation at the 3' terminus.Heavily modified RNA silencing agents

[0211] In certain embodiments, the RNA silencing agent comprises at least 80% chemically modified nucleotides. In certain embodiments, the RNA silencing agent is fully chemically modified, i.e., 100% of the nucleotides are chemically modified.

[0212] In certain embodiments, the RNA silencing agent is 2’-O-methyl rich, i.e., comprises greater than 50% 2’-O-methyl content. In certain embodiments, the RNA silencing agent comprises at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% 2’-O- methyl nucleotide content. In certain embodiments, the RNA silencing agent comprises at least about 70% 2’-O-methyl nucleotide modifications. In certain embodiments, the RNA silencing agent comprises between about 70% and about 90% 2’-O-methyl nucleotide modifications. In certain embodiments, the RNA silencing agent is a dsRNA comprising an antisense strand and sense strand. In certain embodiments, the antisense strand comprises at least about 70% 2’-O- methyl nucleotide modifications. In certain embodiments, the antisense strand comprises between about 70% and about 90% 2’-O-methyl nucleotide modifications. In certain embodiments, the sense strand comprises at least about 70% 2’-O-methyl nucleotide modifications. In certain embodiments, the sense strand comprises between about 70% and about 90% 2’-O-methyl nucleotide modifications. In certain embodiments, the sense strand comprises between 100% 2’-O-methyl nucleotide modifications.

[0213] 2’ -O-methyl rich RNA silencing agents and specific chemical modification patterns are further described in US20200087663 and US20210115442, each of which is incorporated herein by reference.Internucleotide linkage modifications

[0214] In certain embodiments, at least one internucleotide linkage, intersubunit linkage, or nucleotide backbone is modified in the RNA silencing agent. In certain embodiments, all of the intemucleotide linkages in the RNA silencing agent are modified. In certain embodiments, the modified internucleotide linkage comprises a phosphorothioate internucleotide linkage. In certain embodiments, the RNA silencing agent comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 phosphorothioate intemucleotide linkages. In certain embodiments, the RNA silencing agent comprises 4-16 phosphorothioate intemucleotide linkages. In certain embodiments, the RNA silencing agent comprises 8-13 phosphorothioate intemucleotide linkages. In certain embodiments, the RNA silencing agent is a dsRNA comprising an antisense strand and a sense strand, each comprising a 5’ end and a 3’ end. In certain embodiments, the nucleotides at positions 1 and 2 from the 5’ end of sense strand are connected to adjacent ribonucleotides via phosphorothioate intemucleotide linkages. In certain embodiments, the nucleotides at positions 1 and 2 from the 3’ end of sense strand are connected to adjacent ribonucleotides via phosphorothioate intemucleotide linkages. In certain embodiments, the nucleotides at positions 1 and 2 from the 5’ end of antisense strand are connected to adjacent ribonucleotides via phosphorothioate intemucleotide linkages. In certain embodiments, the nucleotides at positions 1-2 to 1-8 from the 3’ end of antisense strand are connected to adjacent ribonucleotides via phosphorothioate intemucleotide linkages. In certain embodiments, the nucleotides at positions 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, or 1-8 from the 3’ end of antisense strand are connected to adjacent ribonucleotides via phosphorothioate intemucleotide linkages. In certain embodiments, the nucleotides at positions 1-2 to 1-7 from the 3’ end of antisense strand are connected to adjacent ribonucleotides via phosphorothioate intemucleotide linkages.

[0215] In one aspect, the disclosure provides a modified oligonucleotide, said oligonucleotide having a 5’ end, a 3’ end, that is complementary to a target, wherein the oligonucleotide comprises a sense and antisense strand, and at least one modified intersubunit linkage of Formula (I):wherein:B is a base pairing moiety;W is selected from the group consisting of O, OCH2, OCH, CH2, and CH;X is selected from the group consisting of halo, hydroxy, and C1-6 alkoxy;Y is selected from the group consisting of O , OH, OR, NH , NH2, S", and SH;Z is selected from the group consisting of O and CH2;R is a protecting group; and= is an optional double bond.

[0216] In an embodiment of Formula (I), when W is CH, = is a double bond.

[0217] In an embodiment of Formula (I), when W selected from the group consisting of O, OCH2, OCH, CH2, = is a single bond.

[0218] In an embodiment of Formula (I), when Y is O , either Z or W is not O.

[0219] In an embodiment of Formula (I), Z is CH2 and W is CH2. In another embodiment, the modified intersubunit linkage of Formula (I) is a modified intersubunit linkage of Formula (II):

[0220] In an embodiment of Formula (I), Z is CH2 and W is O. In another embodiment, wherein the modified intersubunit linkage of Formula (I) is a modified intersubunit linkage of Formula (III):

[0221] In an embodiment of Formula (I), Z is O and W is CH2. In another embodiment, the modified intersubunit linkage of Formula (I) is a modified intersubunit linkage of Formula (IV):

[0222] In an embodiment of Formula (I), Z is O and W is CH. In another embodiment, the modified intersubunit linkage of Formula (I) is a modified intersubunit linkage of Formula V:

[0223] In an embodiment of Formula (I), Z is O and W is OCH2. In another embodiment, the modified intersubunit linkage of Formula (I) is a modified intersubunit linkage of Formula VI:

[0224] In an embodiment of Formula (I), Z is CH2 and W is CH. In another embodiment, the modified intersubunit linkage of Formula (I) is a modified intersubunit linkage of Formula VII:

[0225] In an embodiment of Formula (I), the base pairing moiety B is selected from the group consisting of adenine, guanine, cytosine, and uracil.

[0226] In an embodiment, the modified oligonucleotide is incorporated into siRNA, said modified siRNA having a 5 ’ end, a 3 ’ end, that is complementary to a target, wherein the siRNA comprises a sense and antisense strand, and at least one modified intersubunit linkage of any one or more of Formula (I), Formula (II), Formula (III), Formula (IV), Formula (V), Formula (VI), or Formula (VII).

[0227] In an embodiment, the modified oligonucleotide is incorporated into siRNA, said modified siRNA having a 5’ end, a 3’ end, that is complementary to a target and comprises a sense and antisense strand, wherein the siRNA comprises at least one modified intersubunit linkage is of Formula VIII:(VIII); wherein:D is selected from the group consisting of O, OCH2, OCH, CH2, and CH;C is selected from the group consisting of O , OH, OR1, NH , NH2, S", and SH;A is selected from the group consisting of O and CH2;R1is a protecting group;= is an optional double bond; and the intersubunit is bridging two optionally modified nucleosides.

[0228] In an embodiment, when C is O , either A or D is not O.

[0229] In an embodiment, D is CH2. In another embodiment, the modified intersubunit linkage of Formula VIII is a modified intersubunit linkage of Formula (IX):

[0230] In an embodiment, D is O. In another embodiment, the modified intersubunit linkage of Formula VIII is a modified intersubunit linkage of Formula (X):(X).

[0231] In an embodiment, D is CH2. In another embodiment, the modified intersubunit linkage of Formula (VIII) is a modified intersubunit linkage of Formula (XI):(XI).

[0232] In an embodiment, D is CH. In another embodiment, the modified intersubunit linkage of Formula VIII is a modified intersubunit linkage of Formula (XII):

[0233] In another embodiment, the modified intersubunit linkage of Formula (VII) is a modified intersubunit linkage of Formula (XIV):(XIV).

[0234] In an embodiment, D is OCH2. In another embodiment, the modified intersubunit linkage of Formula (VII) is a modified intersubunit linkage of Formula (XIII):

[0235] In another embodiment, the modified intersubunit linkage of Formula (VII) is a modified intersubunit linkage of Formula (XXa):(XXa).

[0236] In an embodiment of the modified siRNA linkage, each optionally modified nucleoside is independently, at each occurrence, selected from the group consisting of adenosine, guanosine, cytidine, and uridine.

[0237] In certain exemplary embodiments of Formula (I), W is O. In another embodiment, W is CH2. In yet another embodiment, W is CH.

[0238] In certain exemplary embodiments of Formula (I), X is OH. In another embodiment, X is OCH3. In yet another embodiment, X is halo.

[0239] In a certain embodiment of Formula (I), the modified siRNA does not comprise a 2’- fluoro substituent.

[0240] In an embodiment of Formula (I), Y is O . In another embodiment, Y is OH. In yet another embodiment, Y is OR. In still another embodiment, Y is NH". In an embodiment, Y is NH2. In another embodiment, Y is S . In yet another embodiment, Y is SH.

[0241] In an embodiment of Formula (I), Z is O. In another embodiment, Z is CH2.

[0242] In an embodiment, the modified intersubunit linkage is inserted on position 1-2 of the antisense strand. In another embodiment, the modified intersubunit linkage is inserted on position 6-7 of the antisense strand. In yet another embodiment, the modified intersubunit linkage is inserted on position 10-11 of the antisense strand. In still another embodiment, the modified intersubunit linkage is inserted on position 19-20 of the antisense strand. In an embodiment, the modified intersubunit linkage is inserted on positions 5-6 and 18-19 of the antisense strand.

[0243] In an exemplary embodiment of the modified siRNA linkage of Formula (VIII), C is O . In another embodiment, C is OH. In yet another embodiment, C is OR1. In still another embodiment, C is NH . In an embodiment, C is NH2. In another embodiment, C is S . In yet another embodiment, C is SH.

[0244] In an exemplary embodiment of the modified siRNA linkage of Formula (VIII), A is O. In another embodiment, A is CH2. In yet another embodiment, C is OR1. In still another embodiment, C is NH . In an embodiment, C is NH2. In another embodiment, C is S . In yet another embodiment, C is SH.

[0245] In a certain embodiment of the modified siRNA linkage of Formula (VIII), the optionally modified nucleoside is adenosine. In another embodiment of the modified siRNA linkage of Formula (VIII), the optionally modified nucleoside is guanosine. In another embodiment of the modified siRNA linkage of Formula (VIII), the optionally modified nucleoside is cytidine. In another embodiment of the modified siRNA linkage of Formula (VIII), the optionally modified nucleoside is uridine.

[0246] In an embodiment of the modified siRNA linkage, wherein the linkage is inserted on position 1-2 of the antisense strand. In another embodiment, the linkage is inserted on position 6-7 of the antisense strand. In yet another embodiment, the linkage is inserted on position 10- 11 of the antisense strand. In still another embodiment, the linkage is inserted on position 19- 20 of the antisense strand. In an embodiment, the linkage is inserted on positions 5-6 and 18- 19 of the antisense strand.

[0247] In certain embodiments of Formula (I), the base pairing moiety B is adenine. In certain embodiments of Formula (I), the base pairing moiety B is guanine. In certain embodiments of Formula (I), the base pairing moiety B is cytosine. In certain embodiments of Formula (I), the base pairing moiety B is uracil.

[0248] In an embodiment of Formula (I), W is O. In an embodiment of Formula (I), W is CH2. In an embodiment of Formula (I), W is CH.

[0249] In an embodiment of Formula (I), X is OH. In an embodiment of Formula (I), X is OCH3. In an embodiment of Formula (I), X is halo.

[0250] In an exemplary embodiment of Formula (I), the modified oligonucleotide does not comprise a 2’ -fluoro substituent.

[0251] In an embodiment of Formula (I), Y is O . In an embodiment of Formula (I), Y is OH. In an embodiment of Formula (I), Y is OR. In an embodiment of Formula (I), Y is NH". In an embodiment of Formula (I), Y is NH2. In an embodiment of Formula (I), Y is S". In an embodiment of Formula (I), Y is SH.

[0252] In an embodiment of Formula (I), Z is O. In an embodiment of Formula (I), Z is CH2.

[0253] In an embodiment of the Formula (I), the linkage is inserted on position 1-2 of the antisense strand. In another embodiment of Formula (I), the linkage is inserted on position 6-7 of the antisense strand. In yet another embodiment of Formula (I), the linkage is inserted on position 10-11 of the antisense strand. In still another embodiment of Formula (I), the linkage is inserted on position 19-20 of the antisense strand. In an embodiment of Formula (I), the linkage is inserted on positions 5-6 and 18-19 of the antisense strand.

[0254] Modified intersubunit linkages are further described in U.S. Patent Publication No. 2020 / 0385740A1, and U.S. Patent Publication No. 2022 / 0010309, each of which is incorporated herein by reference.4) Conjugated Functional Moieties

[0255] In other embodiments, RNA silencing agents may be modified with one or more functional moieties. A functional moiety is a molecule that confers one or more additional activities to the RNA silencing agent. In certain embodiments, the functional moieties enhance cellular uptake by target cells (e.g., neuronal cells). Thus, the disclosure includes RNA silencing agents which are conjugated or unconjugated (e.g., at its 5’ and / or 3' terminus) to another moiety (e.g., a non-nucleic acid moiety such as a peptide), an organic compound (e.g., a dye), or the like. The conjugation can be accomplished by methods known in the art, e.g., using the methods of Lambert et al., Drug Deliv. Rev.: 47(1), 99-112 (2001) (describes nucleic acids loaded to polyalkylcyanoacrylate (PACA) nanoparticles); Fattal et al., J. Control Release 53(1-3): 137-43 (1998) (describes nucleic acids bound to nanoparticles); Schwab et al., Ann. Oncol. 5 Suppl. 4:55-8 (1994) (describes nucleic acids linked to intercalating agents, hydrophobic groups, polycations or PACA nanoparticles); and Godard et al., Eur. J. Biochem. 232(2):404-10 (1995) (describes nucleic acids linked to nanoparticles).

[0256] In a certain embodiment, the functional moiety is a hydrophobic moiety. In a certain embodiment, the hydrophobic moiety is selected from the group consisting of fatty acids, steroids, secosteroids, lipids, gangliosides and nucleoside analogs, endocannabinoids, and vitamins. In a certain embodiment, the steroid selected from the group consisting of cholesterol and lithocholic acid (LA). In a certain embodiment, the fatty acid selected from the group consisting of Eicosapentaenoic acid (EP A), Docosahexaenoic acid (DHA) and Docosanoic acid (DC A). In a certain embodiment, the vitamin selected from the group consisting of choline,vitamin A, vitamin E, and derivatives or metabolites thereof. In a certain embodiment, the vitamin is selected from the group consisting of retinoic acid and alpha-tocopheryl succinate.

[0257] In a certain embodiment, an RNA silencing agent of disclosure is conjugated to a lipophilic moiety. In one embodiment, the lipophilic moiety is a ligand that includes a cationic group. In another embodiment, the lipophilic moiety is attached to one or both strands of an siRNA. In an exemplary embodiment, the lipophilic moiety is attached to one end of the sense strand of the siRNA. In another exemplary embodiment, the lipophilic moiety is attached to the 3' end of the sense strand. In certain embodiments, the lipophilic moiety is selected from the group consisting of cholesterol, vitamin E, vitamin K, vitamin A, folic acid, a cationic dye (e.g., Cy3). In an exemplary embodiment, the lipophilic moiety is cholesterol. Other lipophilic moi eties include cholic acid, adamantane acetic acid, 1 -pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyl oxy hexyl group, hexadecylglycerol, borneol, menthol, 1,3 -propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine.

[0258] In certain embodiments, the functional moieties may comprise one or more ligands tethered to an RNA silencing agent to improve stability, hybridization thermodynamics with a target nucleic acid, targeting to a particular tissue or cell-type, or cell permeability, e.g., by an endocytosis-dependent or -independent mechanism. Ligands and associated modifications can also increase sequence specificity and consequently decrease off-site targeting. A tethered ligand can include one or more modified bases or sugars that can function as intercal ators. These can be located in an internal region, such as in a bulge of RNA silencing agent / target duplex. The intercalator can be an aromatic, e.g., a polycyclic aromatic or heterocyclic aromatic compound. A polycyclic intercalator can have stacking capabilities, and can include systems with 2, 3, or 4 fused rings. The universal bases described herein can be included on a ligand. In one embodiment, the ligand can include a cleaving group that contributes to target gene inhibition by cleavage of the target nucleic acid. The cleaving group can be, for example, a bleomycin (e.g., bleomycin-A5, bleomycin-A2, or bleomycin-B2), pyrene, phenanthroline (e.g., O-phenanthroline), a polyamine, a tripeptide (e.g., lys-tyr-lys tripeptide), or a metal ion chelating group. The metal ion chelating group can include, e.g., an Lu(III) or EU(III) macrocyclic complex, a Zn(II) 2,9-dimethylphenanthroline derivative, a Cu(II) terpyridine, or acridine, which can promote the selective cleavage of target RNA at the site of the bulge byfree metal ions, such as Lu(III). In some embodiments, a peptide ligand can be tethered to an RNA silencing agent to promote cleavage of the target RNA, e.g., at the bulge region. For example, l,8-dimethyl-l,3,6,8,10,13-hexaazacyclotetradecane (cyclam) can be conjugated to a peptide (e.g., by an amino acid derivative) to promote target RNA cleavage. A tethered ligand can be an aminoglycoside ligand, which can cause an RNA silencing agent to have improved hybridization properties or improved sequence specificity. Exemplary aminoglycosides include glycosylated polylysine, galactosylated polylysine, neomycin B, tobramycin, kanamycin A, and acridine conjugates of aminoglycosides, such as Neo-N-acridine, Neo-S- acridine, Neo-C-acridine, Tobra-N-acridine, and KanaA-N-acridine. Use of an acridine analog can increase sequence specificity. For example, neomycin B has a high affinity for RNA as compared to DNA, but low sequence-specificity. An acridine analog, neo-5-acridine, has an increased affinity for the HIV Rev-response element (RRE). In some embodiments, the guanidine analog (the guanidinoglycoside) of an aminoglycoside ligand is tethered to an RNA silencing agent. In a guanidinoglycoside, the amine group on the amino acid is exchanged for a guanidine group. Attachment of a guanidine analog can enhance cell permeability of an RNA silencing agent. A tethered ligand can be a poly-arginine peptide, peptoid or peptidomimetic, which can enhance the cellular uptake of an oligonucleotide agent.

[0259] Exemplary ligands are coupled, either directly or indirectly, via an intervening tether, to a ligand-conjugated carrier. In certain embodiments, the coupling is through a covalent bond. In certain embodiments, the ligand is attached to the carrier via an intervening tether. In certain embodiments, a ligand alters the distribution, targeting or lifetime of an RNA silencing agent into which it is incorporated. In certain embodiments, a ligand provides an enhanced affinity for a selected target, e.g., molecule, cell or cell type, compartment, e.g., a cellular or organ compartment, tissue, organ or region of the body, as, e.g., compared to a species absent such a ligand.

[0260] Exemplary ligands can improve transport, hybridization, and specificity properties and may also improve nuclease resistance of the resultant natural or modified RNA silencing agent, or a polymeric molecule comprising any combination of monomers described herein and / or natural or modified ribonucleotides. Ligands in general can include therapeutic modifiers, e.g., for enhancing uptake; diagnostic compounds or reporter groups e.g., for monitoring distribution; cross-linking agents; nuclease-resistance conferring moieties; and natural or unusual nucleobases. General examples include lipophiles, lipids, steroids (e.g., uvaol,hecigenin, diosgenin), terpenes (e.g., triterpenes, e.g., sarsasapogenin, Friedelin, epifriedelanol derivatized lithocholic acid), vitamins (e.g., folic acid, vitamin A, biotin, pyridoxal), carbohydrates, proteins, protein binding agents, integrin targeting molecules, polycationics, peptides, polyamines, and peptide mimics. Ligands can include a naturally occurring substance, (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); amino acid, or a lipid. The ligand may also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid. Examples of polyamino acids include polyamino acid is a polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N- isopropyl acrylamide polymers, or polyphosphazine. Example of polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptidepolyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an alpha helical peptide.

[0261] Ligands can also include targeting groups, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a kidney cell. A targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl- galactosamine (GalNAc) or derivatives thereof, N-acetyl-glucosamine, multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, biotin, or an RGD peptide or RGD peptide mimetic. Other examples of ligands include dyes, intercalating agents (e.g. acridines and substituted acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine, phenanthroline, pyrenes), lys-tyr-lys tripeptide, aminoglycosides, guanidium aminoglycodies, artificial endonucleases (e.g. EDTA), lipophilic molecules, e.g, cholesterol (and thio analogs thereof), cholic acid, cholanic acid, lithocholic acid, adamantane acetic acid, 1 -pyrene butyric acid, dihydrotestosterone, glycerol (e.g., esters (e.g., mono, bis, or tris fatty acid esters, e.g., Cio, Cn, C12, C13, C14, C15, Ci6, C17, Cis, C19, or C20 fatty acids) and ethers thereof, e.g., Cio, Cn, C12, C13, C14, C15, Ci6, C17, Cis, C19, or C20 alkyl; e.g., l,3-bis-O(hexadecyl)glycerol, l,3-bis-O(octaadecyl)glycerol),geranyl oxy hexyl group, hexadecylglycerol, borneol, menthol, 1,3 -propanediol, heptadecyl group, palmitic acid, stearic acid (e.g., glyceryl distearate), oleic acid, myristic acid, 03- (oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport / absorption facilitators (e.g., aspirin, naproxen, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+complexes of tetraazamacrocycles), dinitrophenyl, HRP or AP. In certain embodiments, the ligand is GalNAc or a derivative thereof.

[0262] Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, or bone cell. Ligands may also include hormones and hormone receptors. They can also include non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl- galactosamine, N-acetyl-glucosamine multivalent mannose, or multivalent fucose. The ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-kB.

[0263] The ligand can be a substance, e.g., a drug, which can increase the uptake of the RNA silencing agent into the cell, for example, by disrupting the cell's cytoskeleton, e.g., by disrupting the cell's microtubules, microfilaments, and / or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin. The ligand can increase the uptake of the RNA silencing agent into the cell by activating an inflammatory response, for example. Exemplary ligands that would have such an effect include tumor necrosis factor alpha (TNF-alpha), interleukin-1 beta, or gamma interferon. In one aspect, the ligand is a lipid or lipid-based molecule. Such a lipid or lipid-based molecule can bind a serum protein, e.g., human serum albumin (HSA). An HSA binding ligand allows for distribution of the conjugate to a target tissue, e.g., a non-kidney target tissue of the body. For example, the target tissue can be the liver, including parenchymal cells of the liver. Other molecules that can bind HSA can also be used as ligands. For example, neproxin or aspirin can be used. A lipid or lipid-based ligand can (a) increase resistance to degradation of the conjugate, (b) increase targeting ortransport into a target cell or cell membrane, and / or (c) can be used to adjust binding to a serum protein, e.g., HSA. A lipid-based ligand can be used to modulate, e.g., control the binding of the conjugate to a target tissue. For example, a lipid or lipid-based ligand that binds to HSA more strongly will be less likely to be targeted to the kidney and therefore less likely to be cleared from the body. A lipid or lipid-based ligand that binds to HSA less strongly can be used to target the conjugate to the kidney. In a certain embodiment, the lipid-based ligand binds HSA. A lipid-based ligand can bind HSA with a sufficient affinity such that the conjugate will be distributed to a non-kidney tissue. However, it is contemplated that the affinity is not so strong that the HSA-ligand binding cannot be reversed. In another embodiment, the lipid-based ligand binds HSA weakly or not at all, such that the conjugate will be distributed to the kidney. Other moieties that target to kidney cells can also be used in place of or in addition to the lipid- based ligand.

[0264] In another aspect, the ligand is a moiety, e.g., a vitamin, which is taken up by a target cell, e.g., a proliferating cell. These can be useful for treating disorders characterized by unwanted cell proliferation, e.g., of the malignant or non-malignant type, e.g., cancer cells. Exemplary vitamins include vitamin A, E, and K. Other exemplary vitamins include are B vitamin, e.g., folic acid, B12, riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up by cancer cells. Also included are HSA and low-density lipoprotein (LDL).

[0265] In another aspect, the ligand is a cell-permeation agent, such as a helical cell-permeation agent. In certain embodiments, the agent is amphipathic. An exemplary agent is a peptide such as tat or antennopedia. If the agent is a peptide, it can be modified, including a peptidylmimetic, invertomers, non-peptide or pseudo-peptide linkages, and use of D-amino acids. The helical agent can be an alpha-helical agent, which may have a lipophilic and a lipophobic phase.

[0266] The ligand can be a peptide or peptidomimetic. A peptidomimetic (also referred to herein as an oligopeptidomimetic) is a molecule capable of folding into a defined three- dimensional structure similar to a natural peptide. The attachment of peptide and peptidomimetics to oligonucleotide agents can affect pharmacokinetic distribution of the RNA silencing agent, such as by enhancing cellular recognition and absorption. The peptide or peptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long. A peptide or peptidomimetic can be, for example, a cell permeation peptide, cationic peptide, amphipathic peptide, or hydrophobic peptide (e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety can be a dendrimer peptide,constrained peptide or crosslinked peptide. The peptide moiety can be an L-peptide or D- peptide. In another alternative, the peptide moiety can include a hydrophobic membrane translocation sequence (MTS). A peptide or peptidomimetic can be encoded by a random sequence of DNA, such as a peptide identified from a phage-display library, or one-bead-one- compound (OBOC) combinatorial library (Lam et al., Nature 354:82-84, 1991). In exemplary embodiments, the peptide or peptidomimetic tethered to an RNA silencing agent via an incorporated monomer unit is a cell targeting peptide such as an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. A peptide moiety can range in length from about 5 amino acids to about 40 amino acids. The peptide moieties can have a structural modification, such as to increase stability or direct conformational properties. Any of the structural modifications described below can be utilized.

[0267] In certain embodiments, the functional moiety is a muscle-targeting functional moiety. In certain embodiments, the muscle-targeting functional moiety is an antibody or antigen binding fragment. In certain embodiments, the antibody or antigen binding fragment binds to a target on the surface of muscle cells. In certain embodiments, the target on the surface of muscle cells is transferrin receptor 1 (TfRl). Exemplary TfRl antibodies and TfRl antibody- oligonucleotide conjugates are described in greater detail in Malecova et al. (Nucleic Acids Research. 51(12): 5901-5910. 2023). In certain embodiments, the target on the surface of muscle cells is Calcium Voltage-Gated Channel Auxiliary Subunit Gamma 1 (CACNG1).

[0268] In certain embodiments, the functional moiety is linked to the 5’ end and / or 3’ end of the RNA silencing agent of the disclosure. In certain embodiments, the functional moiety is linked to the 5’ end and / or 3’ end of an antisense strand of the RNA silencing agent of the disclosure. In certain embodiments, the functional moiety is linked to the 5’ end and / or 3’ end of a sense strand of the RNA silencing agent of the disclosure. In certain embodiments, the functional moiety is linked to the 3’ end of a sense strand of the RNA silencing agent of the disclosure.

[0269] In certain embodiments, the functional moiety is linked to the RNA silencing agent by a linker. In certain embodiments, the functional moiety is linked to the antisense strand and / or sense strand by a linker. In certain embodiments, the functional moiety is linked to the 3’ end of a sense strand by a linker. In certain embodiments, the linker comprises a divalent or trivalent linker. In certain embodiments, the linker comprises an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphodiester, a phosphorothioate, a phosphoramidate, an amide, acarbamate, or a combination thereof. In certain embodiments, the divalent or trivalent linker is selected from:

[0270] In certain embodiments, the linker further comprises a phosphodiester or phosphodiester derivative. In certain embodiments, the phosphodiester or phosphodiester derivative is selected from the group consisting of:; andwherein X is O, S or BH3.

[0271] The various functional moieties of the disclosure and means to conjugate them to RNA silencing agents are described in further detail in W02017 / 030973A1 and WO2018 / 031933A2, incorporated herein by reference.Branched RNA Molecules (i.e.. Branched Oligonucleotides)

[0272] The MSTN-targeting RNA molecules described herein may be contained in a branched RNA molecule structure. The branched oligonucleotides comprise two or more RNA molecule linked together.

[0273] In certain embodiments, the two or more RNA molecule in the branched RNA molecule are connected to one another by one or more moi eties independently selected from a linker, a spacer and a branching point.

[0274] In certain embodiments, the linker comprises an ethylene glycol chain, an alkyl chain, a peptide, an RNA, a DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, or combinations thereof.

[0275] In certain embodiments, the branching point comprises a polyvalent organic species or derivative thereof.

[0276] In another embodiment, the branching point is an amino acid derivative. In another embodiment of the branching point is selected from the formulas of:

[0277] Polyvalent organic species are moieties comprising carbon and three or more valencies (i.e., points of attachment with moieties such as S, L or N, as defined above). Non-limiting examples of polyvalent organic species include triols (e.g., glycerol, phloroglucinol, and the like), tetrols (e.g., ribose, pentaerythritol, 1,2,3,5-tetrahydroxybenzene, and the like), tricarboxylic acids (e.g., citric acid, 1,3,5-cyclohexanetricarboxylic acid, trimesic acid, and the like), tetra-carboxylic acids (e.g., ethylenediaminetetraacetic acid, pyromellitic acid, and the like), tertiary amines (e.g., tripropargylamine, triethanolamine, and the like), triamines (e.g., diethylenetriamine and the like), tetramines, and species comprising a combination of hydroxyl, thiol, amino, and / or carboxyl moieties (e.g., amino acids such as lysine, serine, cysteine, and the like).

[0278] In certain embodiments, the spacer comprises an ethylene glycol chain, an alkyl chain, a peptide, an RNA, a DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, or a combination thereof.

[0279] In certain embodiments, the linker comprises the structure LI :

[0281] In certain embodiments, the linker comprises the structure L2:

[0283] In certain embodiments, the branched RNA molecule consists of two RNA molecules. In certain embodiments, the branched RNA molecule consists of three RNA molecules. In certain embodiments, the branched RNA molecule consists of four RNA molecules. In certain embodiments, the RNA molecules are siRNA.

[0284] In certain embodiments, the branched RNA molecule comprises the structure:Oigonucleotide-(dimer);Oligonucleotide.ungonucieouae(trimer); or(tetramer).

[0285] For any of the above recited structures, the term “oligonucleotide” corresponds to any of the oligonucleotides recited herein, e.g., an ASO or siRNA. In certain embodiments, the term “oligonucleotide” in the structures recited above corresponds to the sense strand of an siRNA. In certain embodiments, the oxygen immediately adjacent to the term “oligonucleotide” in the structures is linked to the 3’ end of a sense strand of an siRNA.

[0286] Branched oligonucleotides, including synthesis and methods of use, are described in greater detail in WO2017 / 132669, incorporated herein by reference. Further details regarding synthesis are provided in the Materials and Methods section of the Examples.Methods of Introducing Nucleic Acids, Vectors and Host Cells

[0287] RNA silencing agents of the disclosure may be directly introduced into the cell (e.g., an eye cell) (i.e., intracellularly); or introduced extracellularly into a cavity, interstitial space, into the circulation of an organism, introduced orally, or may be introduced by bathing a cell or organism in a solution containing the nucleic acid. Vascular or extravascular circulation, the blood or lymph system, and the cerebrospinal fluid are sites where the nucleic acid may be introduced.

[0288] The RNA silencing agents of the disclosure can be introduced using nucleic acid delivery methods known in art including injection of a solution containing the nucleic acid, bombardment by particles covered by the nucleic acid, soaking the cell or organism in a solution of the nucleic acid, or electroporation of cell membranes in the presence of the nucleic acid. Other methods known in the art for introducing nucleic acids to cells may be used, such as lipid-mediated carrier transport, chemical-mediated transport, and cationic liposome transfection such as calcium phosphate, and the like. The nucleic acid may be introduced along with other components that perform one or more of the following activities: enhance nucleic acid uptake by the cell or other-wise increase inhibition of the target gene.

[0289] Physical methods of introducing nucleic acids include injection of a solution containing the RNA, bombardment by particles covered by the RNA, soaking the cell or organism in a solution of the RNA, or electroporation of cell membranes in the presence of the RNA. A viral construct packaged into a viral particle would accomplish both efficient introduction of an expression construct into the cell and transcription of RNA encoded by the expression construct. Other methods known in the art for introducing nucleic acids to cells may be used, such as lipid-mediated carrier transport, chemical-mediated transport, such as calcium phosphate, and the like. Thus, the RNA may be introduced along with components that perform one or more of the following activities: enhance RNA uptake by the cell, inhibit annealing of single strands, stabilize the single strands, or other-wise increase inhibition of the target gene.

[0290] The cell having the target gene may be from the germ line or somatic, totipotent or pluripotent, dividing or non-dividing, parenchyma or epithelium, immortalized or transformed, or the like. The cell may be a stem cell or a differentiated cell. Cell types that are differentiated include adipocytes, fibroblasts, myocytes, cardiomyocytes, endothelium, neurons, glia, blood cells, megakaryocytes, lymphocytes, macrophages, neutrophils, eosinophils, basophils, mastcells, leukocytes, granulocytes, keratinocytes, chondrocytes, osteoblasts, osteoclasts, hepatocytes, and cells of the endocrine or exocrine glands.

[0291] Depending on the particular target gene and the dose of double stranded RNA material delivered, this process may provide partial or complete loss of function for the target gene. A reduction or loss of gene expression in at least 50%, 60%, 70%, 80%, 90%, 95% or 99% or more of targeted cells is exemplary. Inhibition of gene expression refers to the absence (or observable decrease) in the level of protein and / or mRNA product from a target gene. Specificity refers to the ability to inhibit the target gene without manifest effects on other genes of the cell. The consequences of inhibition can be confirmed by examination of the outward properties of the cell or organism (as presented below in the examples) or by biochemical techniques such as RNA solution hybridization, nuclease protection, Northern hybridization, reverse transcription, gene expression monitoring with a microarray, antibody binding, Enzyme Linked ImmunoSorbent Assay (ELISA), Western blotting, RadioImmunoAssay (RIA), other immunoassays, and Fluorescence Activated Cell Sorting (FACS).

[0292] For RNA-mediated inhibition in a cell line or whole organism, gene expression is conveniently assayed by use of a reporter or drug resistance gene whose protein product is easily assayed. Such reporter genes include acetohydroxyacid synthase (AHAS), alkaline phosphatase (AP), beta galactosidase (LacZ), beta glucoronidase (GUS), chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP), horseradish peroxidase (HRP), luciferase (Luc), nopaline synthase (NOS), octopine synthase (OCS), and derivatives thereof. Multiple selectable markers are available that confer resistance to ampicillin, bleomycin, chloramphenicol, gentarnycin, hygromycin, kanamycin, lincomycin, methotrexate, phosphinothricin, puromycin, and tetracyclin. Depending on the assay, quantitation of the amount of gene expression allows one to determine a degree of inhibition which is greater than 10%, 33%, 50%, 90%, 95% or 99% as compared to a cell not treated according to the present disclosure. Lower doses of injected material and longer times after administration of RNAi agent may result in inhibition in a smaller fraction of cells (e.g., at least 10%, 20%, 50%, 75%, 90%, or 95% of targeted cells). Quantization of gene expression in a cell may show similar amounts of inhibition at the level of accumulation of target mRNA or translation of target protein. As an example, the efficiency of inhibition may be determined by assessing the amount of gene product in the cell; mRNA may be detected with a hybridization probe having a nucleotide sequence outside the region used for the inhibitory double-stranded RNA, ortranslated polypeptide may be detected with an antibody raised against the polypeptide sequence of that region.

[0293] The RNA may be introduced in an amount which allows delivery of at least one copy per cell. Higher doses (e.g., at least 5, 10, 100, 500 or 1000 copies per cell) of material may yield more effective inhibition; lower doses may also be useful for specific applications.

[0294] In an exemplary aspect, the efficacy of an RNAi agent of the disclosure (e.g., an siRNA targeting an MSTN target sequence) is tested for its ability to specifically degrade mRNA (e.g., MSTN mRNA and / or the production of MSTN protein) in cells, such as muscle cells. In certain embodiments, muscle cells include, but are not limited to, myocytes, myoblasts, skeletal muscle cells, cardiac muscle cells, smooth muscle cells, and myosatellite cells. Also suitable for cell-based validation assays are other readily transfectable cells, for example, HeLa cells or COS cells. Cells are transfected with human wild type or mutant cDNAs (e.g., human wild type or mutant MSTN). Standard siRNA, modified siRNA or vectors able to produce siRNA from U-looped mRNA are co-transfected. Selective reduction in target mRNA (e.g., MSTN and / or target protein (e.g., MSTN protein) is measured. Reduction of target mRNA or protein can be compared to levels of target mRNA or protein in the absence of an RNAi agent or in the presence of an RNAi agent that does not target MSTN mRNA. Exogenously-introduced mRNA or protein (or endogenous mRNA or protein) can be assayed for comparison purposes. When utilizing neuronal cells, which are known to be fairly resistant to standard transfection techniques, it may be desirable to introduce RNAi agents (e.g., siRNAs) by passive uptake.Recombinant Adeno- Associated Viruses and Vectors

[0295] In certain exemplary embodiments, recombinant adeno-associated viruses (rAAVs) and their associated vectors can be used to deliver one or more siRNAs into cells, e.g., neural cells (e.g., brain cells). AAV is able to infect many different cell types, although the infection efficiency varies based upon serotype, which is determined by the sequence of the capsid protein. Several native AAV serotypes have been identified, with serotypes 1-9 being the most commonly used for recombinant AAV. AAV-2 is the most well-studied and published serotype. The AAV-DJ system includes serotypes AAV-DJ and AAV-DJ / 8. These serotypes were created through DNA shuffling of multiple AAV serotypes to produce AAV with hybrid capsids that have improved transduction efficiencies in vitro (AAV-DJ) and in vivo (AAV- DJ / 8) in a variety of cells and tissues.

[0296] In certain embodiments, widespread central nervous system (CNS) delivery can be achieved by intravascular delivery of recombinant adeno-associated virus 7 (rAAV7), RAAV9 and rAAVIO, or other suitable rAAVs (Zhang et al. (2011) Mol. Ther. 19(8): 1440-8. doi: 10.1038 / mt.2011.98. Epub 2011 May 24). rAAVs and their associated vectors are well-known in the art and are described in US Patent Applications 2014 / 0296486, 2010 / 0186103, 2008 / 0269149, 2006 / 0078542 and 2005 / 0220766, each of which is incorporated herein by reference in its entirety for all purposes.

[0297] rAAVs may be delivered to a subject in compositions according to any appropriate methods known in the art. An rAAV can be suspended in a physiologically compatible carrier (i.e., in a composition), and may be administered to a subject, i.e., a host animal, such as a human, mouse, rat, cat, dog, sheep, rabbit, horse, cow, goat, pig, guinea pig, hamster, chicken, turkey, a non-human primate (e.g., Macaque) or the like. In certain embodiments, a host animal is a non-human host animal.

[0298] Delivery of one or more rAAVs to a mammalian subject may be performed, for example, by intramuscular injection or by administration into the bloodstream of the mammalian subject. Administration into the bloodstream may be by injection into a vein, an artery, or any other vascular conduit. In certain embodiments, one or more rAAVs are administered into the bloodstream by way of isolated limb perfusion, a technique well known in the surgical arts, the method essentially enabling the artisan to isolate a limb from the systemic circulation prior to administration of the rAAV virions. A variant of the isolated limb perfusion technique, described in U.S. Pat. No. 6,177,403, can also be employed by the skilled artisan to administer virions into the vasculature of an isolated limb to potentially enhance transduction into muscle cells or tissue. Moreover, in certain instances, it may be desirable to deliver virions to the central nervous system (CNS) of a subject. By “CNS” is meant all cells and tissue of the brain and spinal cord of a vertebrate. Thus, the term includes, but is not limited to, neuronal cells, glial cells, astrocytes, cerebrospinal fluid (CSF), interstitial spaces, bone, cartilage and the like. Recombinant AAVs may be delivered directly to the CNS or brain by injection into, e.g., the ventricular region, as well as to the striatum (e.g., the caudate nucleus or putamen of the striatum), spinal cord and neuromuscular junction, or cerebellar lobule, with a needle, catheter or related device, using neurosurgical techniques known in the art, such as by stereotactic injection (see, e.g., Stein et al., J Virol 73:3424-3429, 1999; Davidson et al.,PNAS 97:3428-3432, 2000; Davidson et al., Nat. Genet. 3:219-223, 1993; and Alisky and Davidson, Hum. Gene Ther. 11 :2315-2329, 2000).

[0299] The compositions of the disclosure may comprise an rAAV alone, or in combination with one or more other viruses (e.g., a second rAAV encoding having one or more different transgenes). In certain embodiments, a composition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different rAAVs each having one or more different transgenes.

[0300] An effective amount of an rAAV is an amount sufficient to target infect an animal, target a desired tissue. In some embodiments, an effective amount of an rAAV is an amount sufficient to produce a stable somatic transgenic animal model. The effective amount will depend primarily on factors such as the species, age, weight, health of the subject, and the tissue to be targeted, and may thus vary among animal and tissue. For example, an effective amount of one or more rAAVs is generally in the range of from about 1 ml to about 100 ml of solution containing from about 109to 1016genome copies. In some cases, a dosage between about 1011to 1012rAAV genome copies is appropriate. In certain embodiments, 1012rAAV genome copies is effective to target heart, liver, and pancreas tissues. In some cases, stable transgenic animals are produced by multiple doses of an rAAV.

[0301] In some embodiments, rAAV compositions are formulated to reduce aggregation of AAV particles in the composition, particularly where high rAAV concentrations are present (e.g., about 1013genome copies / mL or more). Methods for reducing aggregation of rAAVs are well known in the art and, include, for example, addition of surfactants, pH adjustment, salt concentration adjustment, etc. (See, e.g., Wright et al. (2005) Molecular Therapy 12: 171-178, the contents of which are incorporated herein by reference.)

[0302] “Recombinant AAV (rAAV) vectors” comprise, at a minimum, a transgene and its regulatory sequences, and 5' and 3' AAV inverted terminal repeats (ITRs). It is this recombinant AAV vector which is packaged into a capsid protein and delivered to a selected target cell. In some embodiments, the transgene is a nucleic acid sequence, heterologous to the vector sequences, which encodes a polypeptide, protein, functional RNA molecule (e.g., siRNA) or other gene product, of interest. The nucleic acid coding sequence is operatively linked to regulatory components in a manner which permits transgene transcription, translation, and / or expression in a cell of a target tissue.

[0303] The AAV sequences of the vector typically comprise the cis-acting 5' and 3' inverted terminal repeat (ITR) sequences (See, e.g., B. J. Carter, in "Handbook of Parvoviruses", ed., P.Tijsser, CRC Press, pp. 155 168 (1990)). The ITR sequences are usually about 145 basepairs in length. In certain embodiments, substantially the entire sequences encoding the ITRs are used in the molecule, although some degree of minor modification of these sequences is permissible. The ability to modify these ITR sequences is within the skill of the art. (See, e.g., texts such as Sambrook et al, "Molecular Cloning. A Laboratory Manual", 2d ed., Cold Spring Harbor Laboratory, New York (1989); and K. Fisher et al., J Virol., 70:520 532 (1996)). An example of such a molecule employed in the present disclosure is a “cis-acting” plasmid containing the transgene, in which the selected transgene sequence and associated regulatory elements are flanked by the 5' and 3' AAV ITR sequences. The AAV ITR sequences may be obtained from any known AAV, including mammalian AAV types described further herein.Methods of Treatment

[0304] In one aspect, the present disclosure provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) developing a disease associated with MSTN expression. In one embodiment, the disease is a muscle wasting disease. In one embodiment, the muscle wasting disease is cachexia. In one embodiment, the muscle wasting disease is a muscular dystrophy. In one embodiment, the muscle wasting disease is sarcopenia. In one embodiment, the muscle wasting disease is multiple sclerosis.

[0305] " Treatment," or "treating," as used herein, is defined as the application or administration of a therapeutic agent (e.g., a RNA agent or vector or transgene encoding same) to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient, who has the disease or disorder, a symptom of disease or disorder or a predisposition toward a disease or disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease or disorder, the symptoms of the disease or disorder, or the predisposition toward disease.

[0306] In one aspect, the disclosure provides a method for preventing in a subject, a disease or disorder as described above, by administering to the subject a therapeutic agent (e.g., an RNAi agent or vector or transgene encoding same). Subjects at risk for the disease can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the disease or disorder, such that the disease or disorder is prevented or, alternatively, delayed in its progression.

[0307] Another aspect of the disclosure pertains to methods treating subjects therapeutically, i.e., alter onset of symptoms of the disease or disorder. In an exemplary embodiment, the modulatory method of the disclosure involves contacting a muscle cell expressing MSTN with a therapeutic agent (e.g., a RNAi agent or vector or transgene encoding same) that is specific for a target sequence within the gene (e.g., MSTN target sequences of Table 1, Table 2, Table 6, or Table 7), such that sequence specific interference with the gene is achieved. These methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject).Pharmaceutical Compositions and Methods of Administration

[0308] The disclosure pertains to uses of the above-described agents for prophylactic and / or therapeutic treatments as described infra. Accordingly, the modulators (e.g., RNAi agents) of the present disclosure can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the nucleic acid molecule, protein, antibody, or modulatory compound and a pharmaceutically acceptable carrier. As used herein the language "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. 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 compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[0309] A pharmaceutical composition of the disclosure is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intravitreal, intradermal, subcutaneous, intraperitoneal, intramuscular, oral (e.g., inhalation), transdermal (topical), and transmucosal administration.

[0310] The nucleic acid molecules of the disclosure can be inserted into expression constructs, e.g., viral vectors, retroviral vectors, expression cassettes, or plasmid viral vectors, e.g., using methods known in the art, including but not limited to those described in Xia et al., (2002), Supra. Expression constructs can be delivered to a subject by, for example, inhalation, orally, intravenous injection, local administration (see U.S. Pat. No. 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994), Proc. Natl. Acad. Sci. USA, 91, 3054-3057). Thepharmaceutical preparation of the delivery vector can include the vector in an acceptable diluent, or can comprise a slow-release matrix in which the delivery vehicle is imbedded. Alternatively, where the complete delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.

[0311] The nucleic acid molecules of the disclosure can also include small hairpin RNAs (shRNAs), and expression constructs engineered to express shRNAs. Transcription of shRNAs is initiated at a polymerase III (pol III) promoter, and is thought to be terminated at position 2 of a 4-5-thymine transcription termination site. Upon expression, shRNAs are thought to fold into a stem-loop structure with 3' UU-overhangs; subsequently, the ends of these shRNAs are processed, converting the shRNAs into siRNA-like molecules of about 21 nucleotides. Brummelkamp et al. (2002), Science, 296, 550-553; Lee et al, (2002). supra, Miyagishi and Taira (2002), Nature Biotechnol., 20, 497-500; Paddison et al. (2002), supra, Paul (2002), supra, Sui (2002) supra, Yu et al. (2002), supra.

[0312] The expression constructs may be any construct suitable for use in the appropriate expression system and include, but are not limited to retroviral vectors, linear expression cassettes, plasmids and viral or virally-derived vectors, as known in the art. Such expression constructs may include one or more inducible promoters, RNA Pol III promoter systems such as U6 snRNA promoters or Hl RNA polymerase III promoters, or other promoters known in the art. The constructs can include one or both strands of the siRNA. Expression constructs expressing both strands can also include loop structures linking both strands, or each strand can be separately transcribed from separate promoters within the same construct. Each strand can also be transcribed from a separate expression construct, Tuschl (2002), Supra.EXAMPLESExample 1: Development of an siRNA against myostatin (MSTN)

[0313] An initial screen was performed to identify potential siRNA sequences for MSTN. Target human MSTN mRNA sequences that were tested are shown below in Table 1 and Table 2.Table 1 - Human MSTN 45-nucleotide gene region target sites

[0314] For the above recited 45-nucleotide gene regions, the sequences correspond to the DNA gene sequence, however the mRNA encoded by the MSTN gene will have the same sequences with T nucleotides replaced with U nucleotides. Accordingly, and by way of example, an siRNA with an antisense strand that targets SEQ ID NO: 1 will target the mRNA sequence that corresponds to the gene region of SEQ ID NO: 1.Table 2 - Human MSTN 20-nucleotide target sitesTable 3 - Human MSTN sense strandsTable 4 - Human MSTN antisense strands

[0315] For the sense and antisense sequences of Table 3 and 4, “m” corresponds to a 2’-O- methyl modified nucleotide, “f” corresponds to a 2’-fluoro modified nucleotide, “#” corresponds to a phosphorothioate intemucleotide linkage, “P” corresponds to a 5’ phosphate, and “TegChol” corresponds to a tri- or tetra-ethylene glycol linked cholesterol moiety.

[0316] Select unmodified antisense (“AS”) and sense (“S”) strands are provided below in Table 4B. The recited strands may be chemically modified with any modifications described herein.

[0317] Table 4B - select modified antisense and sense strands

[0318] A primary screen was performed in human rhabdomyosarcoma cells (cell line SJCRh30), which was previously shown to express myostatin (data not shown). Numerous siRNA targeting MSTN at different locations of the MSTN mRNA were screened. Each siRNA was used at a concentration of 1.5 pM with 7,500 cells per well. Cells were incubated with the siRNA for 72 hours before harvesting. Each siRNA was tested in triplicate.

[0319] As shown in FIG. 1, numerous siRNA were identified that robustly knocked down expression of MSTN, with several reducing MSTN expression to 20% or less relative to a nontarget control.

[0320] 12 different siRNA identified in the screen were further tested at various doses to generate dose response curves. Experiments were performed in the SJCRh30 cell line at a concentration range of 23 nM to 1.5 pM with 7,500 cells per well. Cells were incubated with the siRNA for 72 hours before harvesting. Each siRNA was tested in triplicate. As shown in FIG. 2, the tested siRNA each knocked down MSTN at various concentrations.

[0321] The relative expression levels of human MSTN from the primary screen of FIG. 1 are shown below in Table 5.Table 5 - Primary screen human MSTN expression values

[0322] Another screen was performed to identify potential siRNA sequences for mouse MSTN. Target mouse MSTN mRNA sequences that were tested are shown below in Table 6 and Table 7.Table 6 - Mouse MSTN 45-nucleotide gene region target sites

[0323] For the above recited 45-nucleotide gene regions, the sequences correspond to the DNA gene sequence, however the mRNA encoded by the MSTN gene will have the same sequences with T nucleotides replaced with U nucleotides. Accordingly, and by way of example, an siRNA with an antisense strand that targets SEQ ID NO: 1 will target the mRNA sequence that corresponds to the gene region of SEQ ID NO: 1.Table 7 - Mouse MSTN 20-nucleotide target sitesTable 8 - Mouse MSTN sense strandsTable 9 - Mouse MSTN antisense strands

[0324] For the sense and antisense sequences of Table 8 and 9, “m” corresponds to a 2’-O- methyl modified nucleotide, “f” corresponds to a 2’-fluoro modified nucleotide, “#” corresponds to a phosphorothioate intemucleotide linkage, “P” corresponds to a 5’ phosphate, and “TegChol” corresponds to a tri- or tetra-ethylene glycol linked cholesterol moiety.

[0325] A primary screen was performed in mouse myoblast cells (cell line C2C12). Numerous siRNA targeting MSTN at different locations of the MSTN mRNA were screened. Each siRNA was used at a concentration of 1.5 pM with 7,500 cells per well. Cells were incubated with the siRNA for 144 hours before harvesting. Each siRNA was tested in triplicate.

[0326] As shown in FIG. 3, numerous siRNA were identified that robustly knocked down expression of MSTN, with several reducing MSTN expression to 20% or less relative to a nontarget control.

[0327] The relative expression levels of mouse MSTN from the primary screen of FIG. 3 are shown below in Table 10.Table 10 - Primary screen mouse MSTN expression values

[0328] A comparison of the human MSTN transcript and the mouse MSTN transcript was performed. The transcripts were aligned and siRNA targets within the same region of the MSTN transcripts were compared, identifying areas of cross-homology and mismatches. The results are shown in FIG. 4.Example 2: Development of alternatively chemically modified siRNA against MSTN

[0329] Select siRNA from the screen in mouse cells were tested in an alternative chemical modification pattern. Two different patterns were tested in vivo in mice, and are recited below:P3 pattern sense strand:(mN)#(mN)#(mN)(fN)(mN)(fN)(mN)(fN)(mN)(fN)(mN)(mN)(mN)(fN)#(mN)#(mN)(dT)(d T)-DCAP3 pattern antisense strand:V(mN)#(fN)#(mN)(fN)(fN)(fN)(mN)(fN)(mN)(fN)(mN)(fN)(mN)(fN)#(mN)#(fN)#(mN)#( mN)#(mN)#(mxN)#(fxN)P2 pattern sense strand:(fN)#(mN)#(fN)(mN)(fN)(mN)(fN)(mN)(fN)(mN)(fN)(mN)(fN)#(mN)#(fN)(dT)(dT)-DCAP2 pattern antisense strand:V(mN)#(fN)#(mN)(fN)(mN)(fN)(mN)(fN)(mN)(fN)(mN)(fN)(mN)#(fN)#(mN)#(fN)#(mN)# (fN)#(mN)#(fN)

[0330] For the sense and antisense sequences above, “m” corresponds to a 2’-O-methyl modified nucleotide, “f” corresponds to a 2’ -fluoro modified nucleotide, “d” corresponds to a deoxynucleotide, “#” corresponds to a phosphorothioate internucleotide linkage, “x” corresponds to an exNA intemucleotide linkage of Formula IV, “V” corresponds to a 5’ vinyl phosphonate, and “DC A” corresponds to a docosanoic acid (DC A) moiety.

[0331] Six- to eight- week-old FVBN mice were injected subcutaneously with siRNA targeting mouse MSTN mRNA targets 1192, 2507, 2093, 2057, and 1928. The siRNA were tested ineither the P3 pattern or P2 pattern recited above. As shown in FIG. 5, siRNA with the exNA internucleotide modification achieved higher in vivo silencing of MSTN.

[0332] Two additional chemical modification patterns were tested in mice. The two different patterns are recited below:Pattern 1 sense strand:(mN)#(mN)#(mN)(fN)(fN)(fN)(mN)(fN)(mN)(fN)(mN)(mN)(mN)(fN)#(mN)#(fN)(dT)(dT)- DCAPattern 1 antisense strand:V(mN)#(fN)#(mN)(fN)(fN)(fN)(mN)(fN)(mN)(fN)(mN)(fN)(mN)(fN)#(mN)#(fN)#(mN)#(f N)#(mN)#(fxN)#(mxN)Pattern 2 sense strand:(mN)#(fN)#(mN)(fN)(mN)(fN)(mN)(fN)(mN)(fN)(mN)(fN)(mN)(fN)#(mN)#(fN)(dT)(dT)- DCAPattern 2 antisense strand:V(mN)#(fN)#(mN)(fN)(mN)(fN)(mN)(fN)(mN)(fN)(mN)(fN)(mN)(fN)#(mN)#(fN)#(mN)#( fN)#(mN)#(fxN)#(mxN)

[0333] For the sense and antisense sequences above, “m” corresponds to a 2’-O-methyl modified nucleotide, “f’ corresponds to a 2’ -fluoro modified nucleotide, “d” corresponds to a deoxynucleotide, “#” corresponds to a phosphorothioate internucleotide linkage, “x” corresponds to an exNA intemucleotide linkage of Formula IV, “V” corresponds to a 5’ vinyl phosphonate, and “DC A” corresponds to a docosanoic acid (DC A) moiety.

[0334] Mice were injected subcutaneously with siRNA targeting a mouse MSTN mRNA target. The siRNA were tested in either pattern 1 or pattern 2 recited above. As shown in FIG. 6, siRNA with the pattern 2 modifications achieved higher in vivo silencing of MSTN. Accordingly, any of the siRNAs described herein, for example the siRNAs of Table 4B, may be modified accordingly to Pattern 2 described above, for enhanced muscle silencing of MSTN in vivo.Example 3: In vivo targeting of mouse MSTN

[0335] Mouse MSTN target 1928 was selected for further in vivo studies. The following chemically modified siRNA were used.MSTN_1928 sense(mG)#(fG)#(mA)(fA)(mA)(fG)(mA)(fA)(mG)(fA)(mA)(fU)(mC)(fU)#(mU)#(fA)(dT)(dT)- DCAMSTN_1928 antisenseV(mU)#(fA)#(mA)(fG)(mA)(fU)(mU)(fC)(mU)(fU)(mC)(fU)(mU)(fU)(mC)(fC)(mU)#(fU)# (mA)#(mxU)#(fxU)

[0336] For the sense and antisense sequences above, “m” corresponds to a 2’-O-methyl modified nucleotide, “f” corresponds to a 2’ -fluoro modified nucleotide, “d” corresponds to a deoxynucleotide, “#” corresponds to a phosphorothioate internucleotide linkage, “x” corresponds to an exNA intemucleotide linkage of Formula IV, “V” corresponds to a 5’ vinyl phosphonate, and “DC A” corresponds to a docosanoic acid (DC A) moiety.

[0337] FVBNJ female mice (5 mice per group) were subcutaneously injected with 40 mg / kg of the 1928-targeting siRNA. Three-weeks post-injection, various mouse tissues were collected and MSTN mRNA was quantified. As shown in FIG. 7, MSTN mRNA levels were robustly reduced in several muscle tissues, including hearth, quadriceps, calf, biceps, diaphragm, and tongue.

[0338] To assess the durability of silencing from the siRNA, MSTN protein levels in plasma was detected every two weeks for 29 weeks. The mice received either received a single 40 mg / kg dose or 40 mg / kg every 2 weeks. As shown FIG. 8, a single injection was capable of repressing MSTN protein levels compared to non-target control for up to 14 weeks. Moreover, dosing the siRNA every two weeks greatly repressed MSTN protein levels for the full term of the experiment (29 weeks).

[0339] The physical changes of the mice of FIG. 8 given the 1928-targeting siRNA was also measured. Body weight, lean mass, and grip strength were measured. As shown in FIG. 9,mice receiving the 1928-targeting siRNA (either the single dose or bi-weekly dose) had increased body weight, increased lean mass, and increased grip strength. Magnetic resonance imaging (MRI) was also used to detect muscle growth. As shown in FIG. 10, mice given the single had calf and quadricep volume increase after 6-weeks relative to the non-target control.

[0340] The safety profile of the mice of FIG. 8 given the 1928-targeting siRNA was also measured. Blood levels of alanine transaminase (ALT), blood urea nitrogen (BUN), platelet count, and immune cell % (lymphocytes, monocytes, and neutrophils) was measured from mice at 29 weeks from either the single and repetitive (i.e., every two-week) dosing. As shown in FIG. 11, no significant alterations in blood chemistry or complete blood count was observed, indicating that the 1928-targeting siRNA is safe. Known markers of heart damage were also measured. As shown in FIG. 12, troponin levels and creatine kinase-myoglobin binding (CK- MB) levels were not significantly altered relative to a non-target control, indicating heart safety of the 1928-targeting siRNA.

[0341] Next, multiple different doses and dosing frequency were tested with the 1928-targeting siRNA. Specifically, mice were subcutaneously injected with 1) 10 mg / kg every 2 weeks; 2) 20 mg / kg every 4 weeks; or 3) 40 mg / kg every 8 weeks. Plasma was collected at multiple points over 32 weeks and the level of MSTN was measured. As shown in FIG. 13, MSTN levels were effectively reduced over a prolonged period of time in all three doses.

Claims

CLAIMS1. An RNA molecule comprising a sequence substantially complementary to MSTN nucleic acid sequence of any one of SEQ ID NOs: 1-14 or 29-31.

2. The RNA molecule of claim 1, comprising a sequence substantially complementary to a MSTN nucleic acid sequence of any one of SEQ ID NOs: 15-28 or 32-34.

3. The RNA molecule of claim 1 or 2, comprising complementarity to at least 10, 11, 12 or 13 contiguous nucleotides of the MSTN nucleic acid sequence of any one of SEQ ID NOs: 1-14 or 29-31.

4. The RNA molecule of any one of claims 1-3, comprising no more than 3 mismatches with the MSTN nucleic acid sequence of any one of SEQ ID NOs: 1-14 or 29-31.

5. The RNA molecule of claim 1 or 2, comprising full complementarity to the MSTN nucleic acid sequence of any one of SEQ ID NOs: 1-14 or 29-31.

6. The RNA molecule of any one of claims 1-5, wherein the RNA molecule comprises a single stranded RNA (ssRNA) or a double stranded RNA (dsRNA).

7. The RNA molecule of any one of claims 1-6, wherein the RNA molecule comprises an antisense oligonucleotide.

8. The RNA molecule of any one of claims 1-6, wherein the RNA molecule comprises an siRNA, the siRNA comprising an antisense strand comprising substantially complementary to the MSTN nucleic acid sequence and a sense strand.

9. The RNA molecule of claim 8, wherein the antisense strand comprises about 15 nucleotides to 25 nucleotides in length.

10. The RNA molecule of claim 8 or 9, wherein the sense strand comprises about 15 nucleotides to 25 nucleotides in length.

11. The RNA molecule of any one of claims 8-10, wherein the antisense strand is 20 nucleotides in length.

12. The RNA molecule of any one of claims 8-10, wherein the antisense strand is 21 nucleotides in length.

13. The RNA molecule of any one of claims 8-10, wherein the antisense strand is 22 nucleotides in length.

14. The RNA molecule of any one of claims 8-13, wherein the sense strand is 15 nucleotides in length.

15. The RNA molecule of any one of claims 8-13, wherein the sense strand is 16 nucleotides in length.

16. The RNA molecule of any one of claims 8-13, wherein the sense strand is 18 nucleotides in length.

17. The RNA molecule of any one of claims 8-13, wherein the sense strand is 20 nucleotides in length.

18. The RNA molecule of any one of claims 8-13, wherein the siRNA comprises a double-stranded region of 15 base pairs to 20 base pairs.

19. The RNA molecule of any one of claims 8-13, wherein the siRNA comprises a double-stranded region of 15 base pairs.

20. The RNA molecule of any one of claims 8-13, wherein the siRNA comprises a double-stranded region of 16 base pairs.

21. The RNA molecule of any one of claims 8-13, wherein the siRNA comprises a double-stranded region of 18 base pairs.

22. The RNA molecule of any one of claims 8-13, wherein the siRNA comprises a double-stranded region of 20 base pairs.

23. The RNA molecule of any one of claims 8-22, wherein the siRNA comprises at least one blunt-end.

24. The RNA molecule of any one of claims 8-23, wherein the siRNA comprises at least one single stranded nucleotide overhang.

25. The RNA molecule of claim 24, wherein the siRNA comprises about a 2-nucleotide to 5-nucleotide single stranded nucleotide overhang.

26. The RNA molecule of claim 24, wherein the siRNA comprises 2-nucleotide single stranded nucleotide overhang.

27. The RNA molecule of claim 24, wherein the siRNA comprises 5-nucleotide single stranded nucleotide overhang.

28. The RNA molecule of any one of claims 1-27, wherein the RNA molecule comprises naturally occurring nucleotides.

29. The RNA molecule of any one of claims 1-28, wherein the RNA molecule comprises at least one modified nucleotide.

30. The RNA molecule of claim 29, wherein said modified nucleotide comprises a 2'-O- methyl modified nucleotide, a 2'-deoxy-2'-fluoro modified nucleotide, a 2'-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, a 2'-amino-modified nucleotide, a 2'- alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base comprising nucleotide, or a mixture thereof.

31. The RNA molecule of any one of claims 1-27, wherein the RNA molecule comprises at least one modified intemucleotide linkage.

32. The RNA molecule of claim 31, wherein said modified intemucleotide linkage comprises a phosphorothioate intemucleotide linkage.

33. The RNA molecule of any one of claims 1-32, comprising 4-16 phosphorothioate internucleotide linkages.

34. The RNA molecule of any one of claims 1-32, comprising 8-13 phosphorothioate internucleotide linkages.

35. The RNA molecule of any one of claims 8-34, wherein the antisense strand comprises 2-10 phosphorothioate intemucleotide linkages.

36. The RNA molecule of any one of claims 8-35, wherein the siRNA comprises at least 80% chemically modified nucleotides.

37. The RNA molecule of any one of claims 8-36, wherein the siRNA is fully chemically modified.

38. The RNA molecule of any one of claims 8-37, wherein the siRNA comprises at least 70% 2’-O-methyl nucleotide modifications.

39. The RNA molecule of any one of claims 8-38, wherein the antisense strand comprises at least 70% 2’-O-methyl nucleotide modifications.

40. The RNA molecule of any one of claims 8-39, wherein the antisense strand comprises about 70% to 90% 2’-O-methyl nucleotide modifications.

41. The RNA molecule of any one of claims 8-40, wherein the sense strand comprises at least 65% 2’-O-methyl nucleotide modifications.

42. The RNA molecule of any one of claims 8-41, wherein the sense strand comprises 100% 2’-O-methyl nucleotide modifications.

43. The RNA molecule of any one of claims 8-42, wherein the sense strand comprises one or more nucleotide mismatches between the antisense strand and the sense strand.

44. The RNA molecule of claim 43, wherein the one or more nucleotide mismatches are present at positions 2, 6, and 12 from the 5’ end of sense strand.

45. The RNA molecule of claim 43, wherein the nucleotide mismatches are present at positions 2, 6, and 12 from the 5’ end of the sense strand.

46. The RNA molecule of any one of claims 8-45, wherein the antisense strand comprises a 5’ phosphate, a 5 ’-alkyl phosphonate, a 5’ alkylene phosphonate, or a 5’ alkenyl phosphonate.

47. The RNA molecule of claim 46, wherein the antisense strand comprises a 5’ vinyl phosphonate.

48. The RNA molecule of any one of claims 1-47, wherein said RNA molecule of comprises at least one modified intemucleotide linkage of Formula I:(i); wherein:B is a base pairing moiety;W is selected from the group consisting of O, OCH2, OCH, CH2, and CH;X is selected from the group consisting of halo, hydroxy, and C1-6 alkoxy;Y is selected from the group consisting of O , OH, OR, NH , NH2, S", and SH;Z is selected from the group consisting of O and CH2;R is a protecting group; and = is an optional double bond.

49. The RNA molecule of any one of claims 8-48, wherein a functional moiety is linked to the 5’ end and / or 3’ end of the antisense strand.

50. The RNA molecule of any one of claims 8-49, wherein a functional moiety is linked to the 5’ end and / or 3’ end of the sense strand.

51. The RNA molecule of any one of claims 8-50, wherein a functional moiety is linked to the 3’ end of the sense strand.

52. The RNA molecule of any one of claims 49-51, wherein the functional moiety comprises a hydrophobic moiety.

53. The RNA molecule of claim 52, wherein the hydrophobic moiety is selected from the group consisting of fatty acids, steroids, secosteroids, lipids, gangliosides, nucleoside analogs, endocannabinoids, vitamins, and a mixture thereof.

54. The RNA molecule of claim 53, wherein the fatty acid selected from the group consisting of Docosanoic acid (DCA), Eicosapentaenoic acid (EP A), and Docosahexaenoic acid (DHA).

55. The RNA molecule of claim 53, wherein the steroid selected from the group consisting of cholesterol and lithocholic acid (LA).

56. The RNA molecule of claim 53, wherein the vitamin is selected from the group consisting of choline, vitamin A, vitamin E, and derivatives or metabolites thereof.

57. The RNA molecule of claim 53, wherein the vitamin is selected from the group consisting of retinoic acid and alpha-tocopheryl succinate.

58. The RNA molecule of any one of claims 49-57, wherein the functional moiety is linked to the antisense strand and / or sense strand by a linker.

59. The RNA molecule of claim 58, wherein the linker comprises an ethylene glycol chain, an alkyl chain, a peptide, an RNA, a DNA, a phosphodiester, a phosphorothioate, a phosphoramidate, an amide, a carbamate, or a combination thereof.

60. The RNA molecule of claim 58 or 59, wherein the linker comprises a divalent or trivalent linker.

61. The RNA molecule of claim 60, wherein the divalent or trivalent linker is selected from the group consisting of:wherein n is 1, 2, 3, 4, or 5.

62. The RNA molecule of claim 60 or 61, wherein when the linker is a trivalent linker, the linker further links a phosphodiester or phosphodi ester derivative.

63. The RNA molecule of claim 62, wherein the phosphodiester or phosphodiester derivative is selected from the group consisting of:(Zc2);; and(Zc3)HCApXO©x'(Zc4) wherein X is O, S or BH3.

64. The RNA molecule of any one of claims 8-63, wherein the nucleotides at positions 1 and 2 from the 3’ end of sense strand, and the nucleotides at positions 1 and 2 from the 5’ end of antisense strand are connected to adjacent ribonucleotides via phosphorothioate linkages.

65. A pharmaceutical composition for inhibiting the expression of MSTN gene in an organism, comprising the RNA molecule of any one of claims 1-64 and a pharmaceutically acceptable carrier.

66. The pharmaceutical composition of claim 65, wherein the RNA molecule inhibits the expression of said MSTN gene by at least 20%.

67. The pharmaceutical composition of claim 65, wherein the siRNA inhibits the expression of said MSTN gene by at least 50%.

68. A method for inhibiting expression of MSTN gene in a cell, the method comprising:(a) introducing into the cell the RNA molecule of any one of claims 1-64; and(b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of the MSTN gene, thereby inhibiting expression of the MSTN gene in the cell.

69. A method of treating or managing a muscle wasting disease comprising administering to a patient in need of such treatment a therapeutically effective amount of the RNA molecule of any one of claims 1-64.

70. The method of claim 69, wherein the RNA molecule is administered subcutaneously, intravenously, or intramuscularly.

71. The method of claim 69 or 70, wherein the RNA molecule inhibits the expression of said MSTN gene by at least 20%.

72. The method of any one of claims 69-71, wherein the RNA molecule inhibits the expression of said MSTN gene by at least 50%.

73. A vector comprising a regulatory sequence operably linked to a nucleotide sequence that encodes an RNA molecule substantially complementary to a. MSTN nucleic acid sequence of SEQ ID NOs: 1-14 or 29-31.

74. The vector of claim 73, wherein said RNA molecule inhibits the expression of said MSTN gene by at least 20%75. The vector of claim 73, wherein said RNA molecule inhibits the expression of said MSTN gene by at least 50%.

76. A cell comprising the vector of any one of claims 73-75.77 A recombinant adeno-associated virus (rAAV) comprising the vector of any one of claims 73-75 and an AAV capsid.

78. A branched RNA compound comprising two or more of the RNA molecules of any one of claims 1-64 covalently bound to one another.

79. The branched RNA compound of claim 78, wherein the RNA molecules are covalently bound to one another by way of a linker, spacer, or branching point.