Compositions and methods for treating sarcopenia

Abstract

The present disclosure provides compositions and methods for treating sarcopenia, including age-related sarcopenia. Various embodiments include inhibitory nucleic acids, compositions comprising the inhibitory nucleic acids, and methods of using the inhibitory nucleic acids to treat such disorders.

Description

COMPOSITIONS AND METHODS FOR TREATING SARCOPENIACROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No.63 / 537,152, filed September 7, 2023, the disclosure of which application is incorporated herein by reference in its entirety.INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED AS A SEQUENCE LISTING XNL FILE

[0002] A Sequence Listing is provided herewith as a Sequence Listing XML, ‘BERK- 489WO SEQLIST”, created on September 6. 2024 and having a size of 11,111 bytes. The contents of the Sequence Listing XML are incorporated herein by reference in their entirety.INTRODUCTION

[0003] Sarcopenia is defined as the progressive loss of muscle mass and function with aging. This loss of muscle mass and function is associated with an increase in adverse outcomes. Frailty and decreased mobility lead to falls, obesity, impaired ability to recover from illness, and ultimately increased mortality. Hie re are currently no approved drugs for the treatment of sarcopenia.SUMMARY

[0004] The present disclosure provides compositions and methods for treating sarcopenia, including age-related sarcopenia. Various embodiments include inhibitory nucleic acids, compositions comprising the inhibitory nucleic acids, and methods of using the inhibitory nucleic acids to treat such disorders.BRIEF DESCRIPTION OF THE DRAWINGS

[0005] FIG. 1A-1B provide nucleotide sequences of exemplary antisense oligonucleotides(ASOs) (FIG. 1A: SEQ ID NOs: 1-3, respectively) and the base-pairing location of the ASOs within a target microRNA (miR-128-3p) (FIG. IB: SEQ ID NO: 12).

[0006] FIG. 2A-2I depict interim data showing the effect of a miR-128-3p targeting LNA ASO on skeletal muscle function in aged mice.

[0007] FIG. 3A-3I depict full experimental data showing the effect of a miR-128-3p targeting LNA ASO on skeletal muscle function in aged mice.

[0008] FIG. 4A-4D experimental data showing the effect of a miR-128-3p targeting LNA ASO on skeletal muscle mass in aged mice.DEFINITIONS

[0009] As used herein, an "‘antisense oligonucleotide” refers to a nucleic acid sequence that is complementary to a DNA or RNA sequence, such as that of a microRNA.

[0010] “RNA” refers to a molecule comprising at least one or more ribonucleotide residues. A "ribonucleotide" is a nucleotide with a hydroxyl group at the 2' position of a beta-D-ribofuranose moiety. Tire tenn RNA, as used herein, includes double-stranded RNA, single -stranded RNA, isolated RNA, such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as altered RNA that differs from naturally occurring RNA by tire addition, deletion, substitution and / or alteration of one or more nucleotides. Nucleotides of the RNA molecules can also comprise nonstandard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides.

[0011] A "microRNA" (miRNA) is a single -stranded RNA molecule of about 21-23 nts in length. In general, miRNAs regulate gene expression. miRNAs are encoded by genes from whose DNA they are transcribed, but miRNAs are not translated into protein. Each primary miRNA transcript is processed into a short stem-loop structure before undergoing further processing into a functional miRNA. Mature miRNA molecules are partially complementary to one or more messenger RNA (mRNA) molecules, and their main function is to down-regulate gene expression.

[0012] As used herein "an interfering RNA" refers to any double stranded or single stranded RNA sequence, capable, either directly or indirectly (i.e.. upon conversion), of inhibiting or down regulating gene expression by mediating RNA interference. Interfering RNA includes but is not limited to small interfering RNA ("siRNA") and small hairpin RNA ("shRNA"). "RNA interference" refers to the selective degradation of a sequence-compatible messenger RNA transcript.

[0013] As used herein "an shRNA" (small hairpin RNA) refers to an RNA molecule comprising an antisense region, a loop portion and a sense region, wherein the sense, region has complementary nucleotides that base pair with the antisense region to form a duplex stem. Following post-transcriptional processing, the small hairpin RNA is converted into a small interfering RNA by a cleavage event mediated by the enzyme Dicer, which is a member of the RNase III family.

[0014] A "small interfering RNA" or "siRNA" as used herein refers to any small RNA molecule capable of inhibiting or down regulating gene expression by mediating RNA interference in a sequence specific manner. The small RNA can be for example, about 18 to 21 nucleotides long.

[0015] As used herein, an "antagomir" refers to a small synthetic RNA having complementarity to a specific microRNA target, with either mispairing at the cleavage site or one or more base modifications to inhibit cleavage.

[0016] As used herein, the phrase "post-transcriptional processing" refers to mRNA processing that occurs after transcription and is mediated, for example, by the enzymes Dicer and / or Drosha.

[0017] By "an effective amount" is meant the amount of a required agent (e.g., an inhibitory nucleic acid of the present disclosure) or composition (e.g., composition comprising an inhibitory nucleic acid of the present disclosure), comprising the agent to ameliorate the symptoms of a disease relative, to an untreated patient. The effective amount of an agent or a composition used to practice a therapeutic treatment of a disease or disorder varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending, physician or veterinarian will decide the appropriate amount and dosage regimen. Such an amount is referred to as an "effective" amount."

[0018] The term “sarcopenia” encompasses any disorder characterized by progressive and generalized loss of skeletal muscle mass and strength with a risk of adverse outcomes such as physical disability, poor quality of life and death. Sarcopenia can be classified according to its cause and includes primary sarcopenia. activity-related sarcopenia. disease-related sarcopenia and nutrition-related sarcopenia. The primary sarcopenia refers to sarcopenia without any cause other than aging and is also referred to as age-related sarcopenia. The activity -related sarcopenia refers to sarcopenia caused by bed rest, sedentary' lifestyle, deconditioning or zero-gravity conditions. The disease-related sarcopenia refers to sarcopenia caused by advanced organ failure (heart, lung, liver, kidney, brain), inflammatory disease, malignancy or endocrine disease. Hie nutrition-related sarcopenia refers to sarcopenia caused by malabsorption, gastrointestinal disorders, use of medications that cause anorexia or insufficient dietary intake of protein.

[0019] As used herein, the terms “treatment,” “treating,” and the like, refer to obtaining a desired pharmacologic and / or physiologic effect. Tire effect may be prophylactic in tenns of completely or partially preventing a disease or symptom thereof and / or may be therapeutic in terms of a partial or complete cure for a disease and / or adverse effect attributable to the disease. “Treatment,” as used herein, covers any treatment of a disease in a mammal, e.g., in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease.

[0020] As used herein, an “AAV particle” is a virus which comprises a. viral genome with at least one payload region and at least one inverted, terminal repeat (ITR) region.

[0021] As used herein, “viral genome” or “vector genome” refers to the nucleic acid sequenced) encapsulated in an AAV particle. Viral genomes comprise at least one payload region encoding a nucleic acid (e g., interfering RNA, etc.).

[0022] As used herein, a “payload” or “payload region” is any nucleic acid molecule which encodes one or more nucleic acid as described herein. At a minimum, a payload region comprises nucleic acid sequences that encode an antibody, an antibody-based composition, or a fragment thereof, but may also optionally comprise one or more functional or regulatory elements to facilitate transcription and / or nucleic acid expression.

[0023] The terms “individual,” “subject,” “host,” and “patient,” used interchangeably herein, refer to an individual organism, e.g., a mammal, including, but not limited to, murines, simians, humans, non-human primates, ungulates, felines, canines, bovines. ovines, mammalian fann animals, mammalian sport animals, and mammalian pets. In some cases, an “individual” is a human.

[0024] Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0025] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. Tire upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0026] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection w ith which the publications are cited.

[0027] It must be noted that as used herein and in the appended claims, the singular fomrs “a,” “an.” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example,reference to “an antisense oligonucleotide” includes a plurality of such oligonucleotides and reference to “the nucleic acid modification” includes reference to one or more nucleic acid modifications and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

[0028] The use of the terms “a,” “an,” and “the,” and similar referents in the context of describing the disclosure (especially in the context of die following claims) arc to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (z.e., meaning “including, but not limited to,”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if the range 10-15 is disclosed, then 11, 12, 13, and 14 are also disclosed. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherw ise clearly contradicted by context. Tire use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the embodiments of the disclosure and does not pose a limitation on the scope of the disclosure unless otherw ise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the embodiments of the disclosure.

[0029] As used herein, the term “about” used in connection with an amount indicates that the amount can vary by 10% of the stated amount. For example, “about 100” means an amount of from 90- 110. Where about is used in the context of a range, the “about” used in reference to the lower amount of the range means that the lower amount includes an amount that is 10% lower than the lower amount of the range, and “about” used in reference to the higher amount of the range means that the higher amount includes an amount 10% higher than the higher amount of the range. For example, from about 100 to about 1000 means that the range extends from 90 to 1100.

[0030] The term “and / or” as used herein a phrase such as “A and / or B” is intended to include both A and B; A or B; A (alone); and B (alone). Likewise, the term “and / or” as used herein a phrase such as “A, B, and / or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0031] It is understood that aspects and embodiments of the present disclosure described herein include “comprising,” “consisting,” and “consisting essentially of’ aspects and embodiments.

[0032] It is appreciated that certain features of the present disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a singleembodiment. Conversely, various features of this disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to this disclosure are specifically embraced by tire present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0033] The publications discussed herein arc provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.DETAILED DESCRIPTION

[0034] The present disclosure provides inhibitory nucleic acids that reduce the level of miR- 128, including miR-128-3p, in a cell, and compositions comprising the inhibitory nucleic acids. The present disclosure provides methods of treating various disorders, using an inhibitory nucleic acid of the present disclosure.INHIBITORY NUCLEIC ACIDS

[0035] The present disclosure provides inhibitory nucleic acids that reduce the level of miR- 128-3p in a cell (e.g., a cell in an individual). In some cases, the inhibitory nucleic acids are antisense oligonucleotides (ASOs). The present disclosure provides ASOs that reduce the level of miR-128-3p in a cell (e.g., in a cell in an individual).

[0036] In some cases, an miR-128 comprises the nucleotide sequence: tgagctgttg gattcggggc cgtagcactg tetgagaggt ttacatttct cacagtgaac eggtetettt ttcagctgct tc (SEQ ID NO:4). In some cases, an miR-128 comprises the nucleotide sequence: ugagcuguug gauucggggc cguagcacug ucugagaggu uuacauuucu cacagugaac cggucucuuu uucagcugcu uc (SEQ ID NO:5). In some cases, an miR-128 comprises the nucleotide sequence: tcacagtgaa ccggtctctt t (SEQ ID NO:6). In some cases, an miR-128 comprises the nucleotide sequence: ucacagugaa ccggucucuu u (SEQ ID NO:7). In some cases, an miR- 128 comprises the nucleotide sequence: tgtgcagtgg gaaggggggc cgatacactg taegagagtg agtagcaggt ctcacagtga accggtctct ttccctactg tgte (SEQ ID NO:8). In some cases, an miR-128 comprises the nucleotide sequence: ugugcagugg gaaggggggc cgauacacug uacgagagug aguagcaggu cucacaguga accggugugu uuggguacug ugcu (SEQ ID NO:9).

[0037] In some cases, an inhibitory nucleic acid (e.g., an ASO) of the present disclosure reduces the level of miR-128-3p in a cell more potently than an inhibitory nucleic acid (e.g., an ASO) comprising the nucleotide sequence: 5’-TTCACTGTG-3' (SEQ ID NO: 10. In some cases, an inhibitory nucleic acid (e.g., an ASO) of the present disclosure is at least 2-fold, at least 2.5-fold, at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, or at least 40-fold, more potent than an inhibitory nucleic acid (e.g., an ASO) comprising the nucleotide sequence: 5’-TTCACTGTG-3’ (SEQ ID NO: 10). For example, in some cases an inhibitory nucleic acid (e.g., an ASO) of the present disclosure reduces the level of miR-128-3p in a cell to an extent that is at least 10%, at least 15%, at least 20%, at least 25%, at least 50%, at least 75%, at least 90%, or more than 90%, greater than the extent to which an inhibitory nucleic acid (e.g., an ASO) comprising the nucleotide sequence 5'-TTCACTGTG-3’ (SEQ ID NO: 10) reduces the level of miR-128-3p in the cell, when administered in the same amount as the mhibi tory nucleic acid (e.g., an ASO) of the present disclosure. In some cases, an inhibitory nucleic acid (e.g., an ASO) of the present disclosure achieves a reduction of at least 10%, at least 15%, at least 20%, at least 25%, at least 50%, at least 75%, or at least 90%, of the level of miR-128-3p in a cell in an individual when the individual is administered with an inhibitory nucleic acid (e.g., an ASO) of the present disclosure in an amount that is at least 10%. at least 15%, at least 20%, at least 25%, at least 50%, at least 75%, at least 90%, or more than 90%, less than the amount of an inhibitory nucleic acid (e.g., an ASO) comprising the nucleotide sequence: 5’-TTCACTGTG-3’ (SEQ ID NO: 10) required to achieve the same reduction in tire level of miR-128-3p.

[0038] In some cases, an inhibitory nucleic acid (e.g ., an ASO) of the present disclosure reduces the level of miR-128-3p in a cell more potently than an inhibitory nucleic acid (e.g., an ASO) comprising the nucleotide sequence: 5’-GGTTCACTGTG-3’ (SEQ ID NO: 11). In some cases, an inhibitory nucleic acid (e.g., an ASO) of the present disclosure is at least 2-fold, at least 2.5-fold, at least 5-fold, at least 10- fold, at least 15 -fold, at least 20-fold, at least 25-fold, at least 30-fold, or at least 40-fold, more potent than an inhibitory nucleic acid (e.g.. an ASO) comprising the nucleotide sequence: 5’-GGTTCACTGTG- 3' (SEQ ID NO: 11). For example, in some cases an inhibitory nucleic acid (e.g., an ASO) of the present disclosure reduces the level of miR-128-3p in a cell to an extent that is at least 10%, at least 15%. at least 20%, at least 25%, at least 50%, at least 75%, at least 90%, or more than 90%, greater than the extent to which an inhibitory nucleic acid (e.g., an ASO) comprising the nucleotide sequence 5’- GGTTCACTGTG-3’ (SEQ ID NO:11) reduces the level of miR-128-3p in the cell, when administered in the same amount as the inhibitory nucleic acid (e.g., an ASO) of the present disclosure. In some cases, an inhibitory nucleic acid (e.g., an ASO) of the present disclosure achieves a reduction of at least 10%, at least 15%, at least 20%. at least 25%, at least 50%, at least 75%, or at least 90%, of the level of miR-128- 3p in a cell in an individual when the individual is administered with an inhibitory nucleic acid (e.g., an ASO) of the present disclosure in an amount that is at least 10%, at least 15%, at least 20%, at least 25%,at least 50%, at least 75%, at least 90%, or more than 90%, less than the amount of an inhibitory nucleic acid (e.g., an ASO) comprising the nucleotide sequence: 5’-GGTTCACTGTG-3’ (SEQ ID NO: 11) required to achieve the same reduction in the level of miR-128-3p.

[0039] Inhibitory nucleic acids useful in the present methods and compositions include antisense oligonucleotides, ribozymes, external guide sequence (EGS) oligonucleotides, siRNA compounds, single- or double -stranded RNA interference (RNAi) compounds such as siRNA compounds, modified bases / locked nucleic acids (LNAs), antagomirs, peptide nucleic acids (PNAs), and other oligomeric compounds or oligonucleotide mimetics which hybridize to at least a portion of the target nucleic acid (i.e., miR-128) and modulate its function. In some embodiments, the inhibitory nucleic acids include antisense RNA, antisense DNA. chimeric antisense oligonucleotides, antisense oligonucleotides comprising modified linkages, interference RNA (RNAi), short interfering RNA (siRNA); a micro, interfering RNA (miRNA); a small, temporal RNA (stRNA); or a short, hairpin RNA (shRNA); small RNA-induced gene activation (RNAa); small activating RNAs (saRNAs), or combinations thereof. See, e.g., WO 2010040112. In some cases, an inhibitory nucleic acid of the present disclosure is an ASO.

[0040] In some cases, the inhibitory nucleic acids are 10 to 50, 13 to 50, or 13 to 30 nucleotides in length. One having ordinary skill in the art will appreciate that this embodies oligonucleotides having antisense portions of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length, or any range therewithin. In some cases, an inhibitory nucleic acid of the present disclosure is 15 nucleotides in length. In some cases, an inhibitory nucleic acid of the present disclosure is 12 to 30 or 13 to 30 nucleotides in length. One having ordinary skill in the art will appreciate that this embodies inhibitory nucleic acids having a length of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides.

[0041] In some cases, tire inhibitory nucleic acids are chimeric oligonucleotides that contain two or more chemically distinct regions, each made up of at least one nucleotide. These oligonucleotides typically contain at least one region of modified nucleotides that confers one or more beneficial properties (such as, for example, increased nuclease resistance, increased uptake into cells, increased binding affinity for the target) and a region that is a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. Chimeric inhibitory nucleic acids of the present disclosure may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and / or oligonucleotide mimetics as described above. Such compounds have also been referred to in the art as hybrids or gapmers.

[0042] As one non-limiting example, in some cases, a suitable inhibitory nucleic acid comprises the following nucleotide sequence: 5’-ACCGGTTCACTGTG-3’ (SEQ ID NO: 1) and has a length offrom 14 nucleotides to 20 nucleotides. In some cases, the inhibitory nucleic acid comprises the following nucleotide sequence: 5’-ACCGGTTCACTGTG-3’ (SEQ ID NO: 1) and has a length of 14 nucleotides. In some cases, the inhibitory nucleic acid comprises one or more of: i) a locked nucleic acid (LNA); ii) a modified backbone: and iii) a 5 -methyl deoxy cytosine. In some cases, a suitable inhibitory nucleic acid comprises the nucleotide sequence, and all of the modifications, depicted in FIG. 1A and designated •‘NRC0090.” The ASO referred to in FIG. 1A as NRC0090 is: +A*+C* / iMe- dC / *G*G*+T*+T*+C*A*C*+T*G*+T*+G, where “+” precedes an LNA; * precedes a phosphorothioated base, and / iMe-dC / denotes an internal 5-methyl deoxy cytosine.

[0043] As another non-limiting example, in some cases, a suitable inhibitory nucleic acid comprises the following nucleotide sequence: 5 -GACCGGTTCACTGT-3’ (SEQ ID NO:2) and has a length of from 14 nucleotides to 20 nucleotides. In some cases, the inhibitory nucleic acid comprises the following nucleotide sequence: 5’-GACCGGTTCACTGT-3’ (SEQ ID NO:2) and has a length of 14 nucleotides. In some cases, the inhibitory' nucleic acid comprises one or more of: i) a locked nucleic acid (LNA); ii) a modified backbone; and iii) a 5-methyl deoxy cytosine. In some cases, a suitable inhibitory nucleic acid comprises the nucleotide sequence, and all of the modifications, depicted in FIG. 1A and designated “NRC0091.” The ASO referred to in FIG. 1A as NRC0091 is +G*+A*C* / iMe- dC / *+G*G*T*T*+C*+A*C*+T*+G*+T, where ‘"+” precedes an LNA; * precedes a phosphorothioated base, and / iMe-dC / denotes an internal 5-methyl deoxy cytosine.

[0044] As another non-limiting example, in some cases, a suitable inhibitory nucleic acid comprises the following nucleotide sequence: 5’-AGACCGGTTCACTGTG-3‘ (SEQ ID NO:3) and has a length of from 14 nucleotides to 20 nucleotides. In some cases, the inhibitory nucleic acid comprises the following nucleotide sequence: AGACCGGTTCACTGTG-3 ’ (SEQ ID NO:3) and has a length of 14 nucleotides. In some cases, the inhibitory nucleic acid comprises one or more of: i) a locked nucleic acid (LNA); ii) a modified backbone; and iii) a 5-methyl deoxy cytosine. In some cases, a suitable inhibitory- nucleic acid comprises the nucleotide sequence, and all of the modifications, depicted in FIG. 1 A and designated “NRC0119." The ASO referred to in FIG. 1A as NRC0119 is +A*+G*A*+C* / iMe- dC / *+G*G*+T*T*+C*A*C*+T*G*+T*+G, where “+” precedes an LNA; * precedes a phosphorothioated base, and / iMe-dC / denotes an internal 5-methyl deoxy cytosine.

[0045] In some embodiments, the inhibitory nucleic acid comprises at least one nucleotide modified at the 2' position of the sugar, e.g., a 2'-O-alkyl, 2'-O-alkyl-O-alkyl, 2'-fluoro, 2'-methoxy, or2’- methoxyethoxy -modified nucleotide. In other cases, RNA modifications include 2'-fluoro, 2'-amino and 2'O-methyl modifications on the ribose of pyrimidines, abasic residues or an inverted base at the 3' end of the RNA. Such modifications are routinely incorporated into oligonucleotides and these oligonucleotides have been shown to have a higher Tm (i .e., higher target binding affinity) than: 2'- deoxyoligonucleotides against a given target.

[0046] A number of nucleotide and nucleoside modifications have been shown to make the oligonucleotide into which they arc incorporated more resistant to nuclease digestion than the native oligodeoxy nucleotide; these modified oligos survive intact for a longer time than unmodified oligonucleotides. Specific examples of modified oligonucleotides include those comprising modified backbones, for example, phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages. In some cases, inhibitory nucleic acids are oligonucleotides with phosphorothioate backbones and those with hctcroatom backbones, particularly CH2— NH— 0— CH3, CH3— N(CH3)— 0— CH2(known as a methylene(methylimino) or MMI backbone], CH2— O— N (CH3)— CH2, CH2— N (CH3)— N (CH3)— CH2and O-N (CH3)-CH2-CH2backbones, wherein the native-phosphodiester backbone is represented as O-P— O— CH); amide backbones (see De Mesmaeker et al. Ace. Chem. Res. 1995, 28:366-374); morpholino backbone structures (see Summerton and Weller, U.S. Pat. No. 5,034,506); peptide nucleic acid (PNA) backbone (wherein the phosphodiester backbone of the oligonucleotide is replaced with a polyamide backbone, the nucleotides being bound directly or indirectly to the aza nitrogen atoms of the polyamide backbone, see Nielsen et al., Science 1991, 254, 1497). Phosphorus-containing linkages include, but are not limited to. phosphorothioates. chiral phosphorothioates. phosphorodithioates, phosphotriesters; aminoalkylphosphotriesters, methyl and other alkyl phosphonates comprising 3'alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates comprising 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having nonnal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'.

[0047] Modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl intemucleoside linkages, mixed heteroatom and alkyl or cycloalkyl intemucleoside linkages, or one or more short chain heteroatomic or heterocyclic intemucleoside linkages. These comprise those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide; sulfoxide and sulfone backbones; fonnacetyl and thioformacetyl backbones; methylene fonnacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2component parts.

[0048] One or more substituted sugar moieties can also be included, e.g., one of the following at the 2' position: OH, SH. SCH3, F, OCN, OCH3OCH3, 0CH3, 0(CH2)n CH3, O(CH2)nNH2or O(CH2)nCH3where n is from 1 to about 10; Cl to CIO lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN; CF3; OCF3; O-, S— , or N-alkyl; O-, S— , or N-alkenyl; SOCH3; SO2CH3; ONO2; NO2; N3; NH2; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino;polyalkylamino; substituted silyl; an RNA cleaving group; a reporter group; an intercalator; a group for improving the pharmacokinetic properties of an oligonucleotide; or a group for improving the pharmacodynamic properties of an oligonucleotide and other substituents having similar properties. A suitable modification includes 2'-methoxyethoxy [2'— O — CH2CH2OCH3, also known as: 2'-O-(2- methoxyethyl)] (Martin et al, Helv. Chim. Acta, 1995, 78, 486). Other modifications include 2'-methoxy (2'-O--CH3), 2'-propoxy ^'-OCFLCFhCIt) and 2'-fluoro (2'-F) . Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar on the 3' terminal nucleotide and the 5' position of 5' terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyls in place of the pentofuranosyl group.

[0049] Inhibitory nucleic acids can also include, additionally or alternatively, nucleobase (often referred to in the art simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include adenine (A), guanine (G), thymine (T), cytosine, (C) and uracil (U). Modified nucleobases include nucleobases found only infrequently or transiently in natural nucleic acids, e.g., hypoxanthine, 6-methyladenine, 5-Me pyrimidines, particularly 5-methylcytosine (also referred to as 5-methyl-2' deoxycytosine and often referred to in the art as 5-Me-C), 5 -hydroxymethylcytosine (HMC). glycosyl HMC and gentobiosyl HMC, as well as synthetic nucleobases, e.g., 2-aminoadenine, 2- (methylamino)adenine, 2-(imidazolylalkyl)adenine, 2-(aminoalklyamino)adenine or other heterosubstituted alkyladenines, 2-thiouracil, 2-thiothymine, 5 -bromouracil, -hydroxymethyluracil, 8- azaguanine, 7-deazaguanine, N6 (6-aminohexyl)adenine and 2,6-diaminopurine. A "universal" base known in the art, e.g., inosine, can also be included. 5-Me-C substitutions have been shown to increase nucleic acid duplex stability by 0.6-12<0>C. In some cases, an inhibitory nucleic acid of the present disclosure comprises one or more 5-Me-C.

[0050] It is not necessary for all positions in a given oligonucleotide to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single oligonucleotide or even at within a single nucleoside within an oligonucleotide.

[0051] In some embodiments, both a sugar and an intemucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, for example, an aminoethylglycine backbone. Hie nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative United States patents that teach the preparation of PNA compounds comprise, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331 ; and 5,719,262, each of which is herein incorporated by reference.Further teaching of PNA compounds can be found in Nielsen et al, Science, 1991, 254, 1497-1500.

[0052] Inhibitory nucleic acids can also include one or more nucleobase (often referred to in the art simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases comprise the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T). cytosine (C) and uracil (U). Modified nucleobases comprise other synthetic and natural nucleobases such as 5 -methylcytosine (5-me-C), 5 -hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosinc. 5-halouracil and cytosine, 5- propynyl uracil and cytosine. 6-azo uracil, cytosine and thymine, 5-uracil (pseudo-uracil), 4-thiouracil, 8- halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines. 5-halo particularly 5 -bromo, 5 -trifluoromethyl and other 5 -substituted uracils and cytosines, 7-methylquanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7 -deazaadenine and 3- deazaguanine and 3 -deazaadenine.

[0053] Further, nucleobases comprise those disclosed in U.S. Pat. No. 3,687,808, those disclosed in 'The Concise Encyclopedia of Polymer Science And Engineering', pages 858-859, Kroschwitz. J. I., ed. John Wiley & Sons, 1990. those disclosed by English et al., Angewandle Chemie. International Edition', 1991, 30, page 613, and those disclosed by Sanghvi. Y. S.. Chapter 15, Antisense Research and Applications', pages 289-302, Crooke, S. T. and Lebleu, B. ea., CRC Press, 1993. Certain of these nucleobases are particularly usefid for increasing the binding affinity of an inhibitory nucleic acid. These include 5-substitutcd pyrimidines, 6-azapyrimidincs and N-2, N-6 and 0-6 substituted purines, comprising 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine, 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2<0>C (Sanghvi. Y. S., Crooke, S. T. and Lebleu, B., eds. Antisense Research and Applications', CRC Press, Boca Raton, 1993, pp. 276-278) and are suitable for inclusion in an inhibitory nucleic acid, e.g., alone or together with 2 -O- methoxyethyl sugar modifications.

[0054] In some cases, tire inhibitory nucleic acids are chemically linked to one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide. Such moieties comprise but are not limited to, lipid moieties such as a cholesterol moiety; cholic acid; a thioether, e.g., hexyl-S-tritylthiol; a thiocholesterol; an aliphatic chain, e g., dodecandiol or undecyl residues; a phospholipid; e.g., di-hexadecyl-rac -glycerol or triethylammonium 1,2-di-O-hexadecyl-rac- glycero-3-H-phosphonate; a polyamine or a polyethylene glycol chain; adamantane acetic acid; a palmityl moiety; an octadecylamine moiety; or a hexylamino-carbonyl-t oxycholesterol moiety.

[0055] These moieties or conjugates can include conjugate groups covalently bound to functional groups such as primary' or secondary hydroxyl groups. Conjugate groups suitable for use include intercalators, importer molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance thepharmacokinetic properties of oligomers. Typical conjugate groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties include groups that improve uptake, enhance resistance to degradation and / or strengthen sequence-specific hybridization with the target nucleic acid. Groups that enhance the pharmacokinetic properties include groups that improve uptake, distribution, metabolism or excretion of an inhibitory nucleic acid. Representative conjugate groups are disclosed in International Patent Application No. PCT / US92 / 09196, filed Oct. 23, 1992, and U.S. Pat. No. 6,287,860, which are incorporated herein by reference. Conjugate moieties include, but are not limited to, lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-5 -tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1.2-di-O-hexadecyl-rac-glycero-3-H- phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxy cholesterol moiety.

[0056] The inhibitory nucleic acids useful in the present methods are sufficiently complementary to all or part of miR-128, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired effect. "Complementary" refers to the capacity for pairing, through hydrogen bonding, between two sequences comprising naturally or non-naturally occurring bases or analogs thereof. For example, if a base at one position of an inhibitory nucleic acid is capable of hydrogen bonding with a base at the corresponding position of a miR-128 sequence, then the bases are considered to be complementary to each other at that position. 100% complementary to is not required.

[0057] In tlie context of this disclosure, hybridization means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases. For example, adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds. “Complementary,” as used herein, refers to the capacity for precise pairing between two nucleotides. Tire inhibitory nucleic acids and the miR-128 are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied, by nucleotides that can hydrogen bond with each other. Thus, "specifically hybridizable" and "complementary" are terms which are used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between the inhibitory nucleic acid and the miR-128 target sequence. For example, if a base at one position of an inhibitory nucleic acid is capable of hydrogen bonding with a base at the corresponding position of a miR-128 molecule; then the bases are considered complementary to each other at that position.

[0058] Although in some embodiments, 100% complementarity is desirable, it is understood in the art that a complementary nucleic acid sequence need not be 100% complementary’ to that of its target nucleic acid to be specifically hybridizable. A complementary' nucleic acid sequence for purposes of thepresent methods is specifically hybridizable when binding of the sequence to the target miR-128 molecule interferes with the normal function of the target miR-128 to cause a loss of activity, and there is a sufficient degree of complementarity to avoid non-specific binding of the sequence to non-target miR- 128 sequences under conditions in which specific binding is desired, e.g., under physiological conditions in the case of in vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed under suitable conditions, of stringency. For example, stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, less than about 500 mM NaCl and 50 mM trisodium citrate, or less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, or at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least 30 degrees C, at least about 37 degrees C, or at least about 42 degrees C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. As a non-limiting example, in some cases, hybridization will occur at 30 degrees C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. As another non-limiting example, in some cases, hybridization will occur at 37 degrees C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 pg / ml denatured salmon sperm DNA (ssDNA). As another non-limiting example, in some cases, hybridization will occur at 42 degrees C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 pg / ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.

[0059] In general, the inhibitory nucleic acids useful in the methods described herein have at least 80% sequence complementarity to a target region within the target nucleic acid, e.g., 90%, 95%, or 100% sequence complementarity to the target region within miR-128 (e.g., a target region comprising the seed sequence). For example, an antisense compound in which 18 of 20 nucleobases of the antisense oligonucleotide are complementary, and would therefore specifically hybridize, to a target region would represent 90 percent complementarity. Percent complementarity of an inhibitory nucleic acid with a region of a target nucleic acid can be determined routinely using basic local alignment search tools (BLAST programs) (Altschul et aL, J. Mol. BioL, 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656). An inhibitory nucleic acid (e.g., an ASO) that hybridizes to a miR-128 target sequence can be identified through routine experimentation. In general, tire inhibitory nucleic acids must retain specificity for their target, i.e., must not directly bind to, or directly significantly affect expression levels of transcripts other than tire intended target.Antisense

[0060] As noted above, in some cases, an inhibitory’ nucleic acid of the present disclosure arc ASOs. ASOs are typically designed to block expression of a DNA or RNA target by binding to the target and halting expression at the level of transcription, translation, or splicing. ASOs of the present disclosure are complementary nucleic acid sequences designed to hybridize under stringent conditions to a miR-128 target sequence. Thus, oligonucleotides are chosen that are sufficiently complementary to the target, i.e., that hybridize sufficiently well and with sufficient specificity, to give the desired effect. Modified Bases / Locked Nucleic Acids (LNAs)

[0061] In some cases, an inhibitory nucleic acid of the present disclosure comprises one or more modified bonds or bases. Modified bases include phosphorothioate. methylphosphonate, peptide nucleic acids, bridged nucleic acid (BNA) and / or locked nucleic acid (LNA) molecules. For example, certain implementations use a bridged nucleic acid that includes a linkage across the ribose ring. Bridges can include an ethylene bridged nucleic acid. Some BNAs include LNA molecules, where the modified nucleotides are locked nucleic acid molecules, including [alpha] -L-LNAs. LNAs comprise ribonucleic acid analogues wherein the ribose ring is "locked" by a methylene bridge between the 2'-oxygen and the 4'-carbon-i.e., oligonucleotides, containing at least one LNA monomer, that is. one 2'-O.4'-C-methylene- 0-D-ribofiiranosyl nucleotide. LNA bases form standard Watson-Crick base pairs but the locked configuration increases the rate and stability of the basepairing reaction (Jepsen et al., Oligonucleotides, 14, 130-146 (2004)). LNAs also have increased affinity to base pair with RNA as compared to DNA. These properties render LNAs especially useful as probes for fluorescence in situ hybridization (FISH) and comparative genomic hybridization, as knockdown tools for miRNAs, and as antisense oligonucleotides to target mRNAs or other RNAs.

[0062] The LNA molecules can include molecules comprising 10-30, e.g., 12-24, e.g., 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in each strand, wherein one of the strands is substantially identical, e.g., at least 80% (or more, e.g., 85%, 90%, 95%, or 100%) identical, e.g.. having 3. 2, 1, or 0 mismatched nucleotide(s), to a miR-128 target sequence. Tire LNA molecules can be chemically synthesized using methods known in the art.Antagomirs

[0063] In some cases, the inhibitory’ nucleic acid is an antagomir. Antagomirs are chemically modified antisense oligonucleotides that target a miR-128 target sequence. For example, an antagomir for use in the methods described herein can include a nucleotide sequence sufficiently complementary to hybridize to a miR-128 target sequence of about 12 to 25 nucleotides, or from about 15 to 23 nucleotides.

[0064] In general, antagomirs include a cholesterol moiety, e g., at the 3'-end. In some embodiments, antagomirs have various modifications for RNase protection and pharmacologic propertiessuch as enhanced tissue and cellular uptake. For example, in addition to the modifications discussed above for antisense oligos, an antagomir can have one or more of complete or partial 2'-O-methylation of sugar and / or a phosphorothioate backbone. Phosphorothioate modifications provide protection against RNase activity and their lipophilicity contributes to enhanced tissue uptake. In some embodiments, the antagomir can include six phosphorothioate backbone modifications; two phosphorothioates are located at the 5'-end and four at the 3'-end. Antagomirs useful in the present methods can also be modified with respect to their length or otherwise the number of nucleotides making up the antagomir. The antagomirs must retain specificity for their target, i.e., must not directly bind to, or directly significantly affect expression levels of, transcripts other than the intended target. In some embodiments, the inhibitory nucleic acid is locked and includes a cholesterol moiety (e.g., a locked antagomir).Gapmers

[0065] In some cases, the inhibitory nucleic acid is a gapmer. Gapmers are chemically modified antisense oligonucleotides that target a miR-128 target sequence. In certain embodiments, a gapmer for use in tire methods described herein can include a nucleotide sequence sufficiently complementary to hybridize to a miR-128 target sequence of about 7 to 50 nucleotides, of about 7 to 30 nucleotides, and / or of about 14-23 nucleotides.

[0066] In general, gapmers are short DNA antisense oligonucleotide structures with RNA-like segments on both sides of the sequence — i.e., a 5’-wing and a 3’-wing. Tire DNA antisense structure is referred to as the gap. The RNA-like structures can include modified nucleotides, such as LNAs, 2’- methoxy, and / or 2’-fluoro modified nucleotides. In various embodiments, gapmers utilize nucleotides modified with phosphorothioate linkages (e.g., backbone). The wing structures (e.g., 5’- and / or 3 ’-wings) comprise 1 to 5 nucleotides each. Tire wing structures may be of the same size (e.g., both 5'- and 3’- wings are 5 nucleotides), or the wing structures may be of the different sizes (e.g., ’-wing is 5 nucleotides, while 3’-wing is 4 nucleotides). siRNA / shRNA

[0067] In some cases, an inhibitory nucleic acid of the present disclosure is an interfering RNA, including but not limited to a small interfering RNA ("siRNA") or a small hairpin RNA ("shRNA"). Methods for constructing interfering RNAs are well known in the art. For example, the interfering RNA can be assembled from two separate oligonucleotides, where one strand is the sense strand and the other is the antisense strand, wherein the antisense and sense strands are self-complementary (i.e., each strand comprises nucleotide sequence that is complementary to nucleotide sequence in the other strand; such as where the antisense strand and sense strand form a duplex or double stranded structure); the antisense strand comprises nucleotide sequence that is complementary to a nucleotide sequence in a target nucleic acid molecule or a portion thereof (i.e., an undesired gene) and the sense strand comprises nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. Alternatively, interferingRNA is assembled from a single oligonucleotide, where the self-complementary sense and antisense regions are linked by means of nucleic acid based or non-nucleic acid-based linker(s). The interfering RNA can be a polynucleotide with a duplex, asymmetric duplex, hairpin or asymmetric hairpin secondary structure, having self-complementary sense and antisense regions, wherein the antisense region comprises a nucleotide sequence that is complementary to nucleotide sequence in a separate target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. The interfering can be a circular single-stranded polynucleotide having two or more loop structures and a stem comprising self- complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic-acid sequence or a portion thereof, and wherein the circular polynucleotide can be processed either in vivo or in vitro to generate an active siRNA molecule capable of mediating RNA interference.

[0068] In some cases, the interfering RNA coding region encodes a self-complementary RNA molecule having a sense region, an antisense region and a loop region. Such an RNA molecule, when expressed, desirably forms a "hairpin" structure, and is referred to herein as an "shRNA," The loop region is generally between about 2 and about 10 nucleotides in length. In some embodiments, the loop region is from about 6 to about 9 nucleotides in length. In some embodiments, the sense region and the antisense region are between about 15 and about 20 nucleotides in length. Following post-transcriptional processing, the small hairpin RNA is converted into a siRNA by a cleavage event mediated by the enzyme Dicer, which is a member of the RNase III family. The siRNA is then capable of inhibiting the expression of a gene with which it shares homology.

[0069] The target RNA cleavage reaction guided by siRNAs is highly sequence specific. In general, siRNAs containing a nucleotide sequence identical to a portion of the target nucleic acid are used for inhibition. However, 100% sequence identity between the siRNA and the target gene is not required. Thus, the present disclosure has the advantage of being able to tolerate sequence variations that might be expected due to genetic mutation, strain polymorphism, or evolutionary divergence. For example, siRNA sequences with insertions, deletions, and single point mutations relative to the target sequence have also been found to be effective for inhibition. Alternatively, siRNA sequences with nucleotide analog substitutions or insertions can be effective for inhibition. In general, the siRNAs must retain specificity for their target, i.e., must not directly bind to, or directly significantly affect expression levels of, transcripts other than the intended target.Making Inhibitory Nucleic Acids

[0070] Inhibitory nucleic acids can be synthesized in vitro by well-known chemical synthesis techniques, as described in, e.g., Adams (1983) J. Am. Chem. Soc. 105:661; Belousov (1997) NucleicAcids Res. 25:3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19:373-380; Blommers (1994) Biochemistry 33:7886-7896; Narang (1979) Meth. Enzymol. 68:90; Brown (1979) Meth. Enzymol. 68: 109; Beaucage (1981) Tetra. Lett. 22: 1859; U.S. Pat. No. 4,458,066.PHARMACEUTICAL COMPOSITIONS

[0071] The present disclosure provides compositions, including pharmaceutical compositions, comprising an inhibitory nucleic acid of the present disclosure. An inhibitory nucleic acid of the present disclosure may be referred to below as an “agent” or an “active agent.”

[0072] In some cases, tire compositions are formulated with a pharmaceutical acceptable carrier. The pharmaceutical compositions and fonnulations can be administered parenterally, topically, orally or by local administration, such as by aerosol or transdermally. The pharmaceutical compositions can be formulated in any way and can be administered in a variety of unit dosage forms depending upon the condition or disease and the degree of illness, the general medical condition of each patient, the resulting preferred method of administration and the like. Details on techniques for formulation and administration of pharmaceuticals are well described in the scientific and patent literature, see, e.g., Remington: The Science and Practice of Pharmacy, 21st ed.. 2005.

[0073] The inhibitory nucleic acids can be administered alone or as a component of a pharmaceutical formulation (composition). An inhibitory nucleic acid can be formulated for administration, in any convenient way for use in human or veterinary medicine. Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

[0074] Formulations of an inhibitory7nucleic acid of the present disclosure include those suitable for intradermal, inhalation, oral / nasal, topical, parenteral, rectal, and / or intravaginal administration. Tire fonnulations may conveniently be presented in unit dosage fonn and may be prepared by any methods well known in the art of pharmacy. The amount, of active ingredient (e.g., an inhibitory nucleic acid of the present disclosure) which can be combined with a carrier material to produce a single dosage fonn will vary depending upon the host being treated, the particular mode of administration, e.g., intradermal or inhalation. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form w ill generally be that amount of the compound which produces a therapeutic effect, e.g., reduction or suspension of muscle loss.

[0075] Pharmaceutical formulations can be prepared according to any method known to the art for the manufacture of pharmaceuticals. Such drugs can contain sweetening agents, flavoring agents, coloring agents and preserving agents. A formulation can be admixed with nontoxic pharmaceutically acceptable excipients which are suitable for manufacture. Formulations may comprise one or morediluents, emulsifiers, preservatives, buffers, excipients, flavorants, colorants, bulking agents, solvents, lubricants, binders, surfactants, disintegrants, etc. and may be provided in such forms as liquids, powders, emulsions, lyophilized powders, sprays, creams, lotions, controlled release formulations, tablets, pills, gels, on patches, in implants, etc.

[0076] Pharmaceutical formulations for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in appropriate and suitable dosages. Such carriers enable the pharmaceuticals to be formulated in unit dosage forms as tablets, pills, powder, dragees, capsules, liquids, lozenges, gels, syrups, slurries, suspensions, etc., suitable for ingestion by the patient. Pharmaceutical preparations for oral use can be formulated as a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable additional compounds, if desired, to obtain tablets or dragee cores. Suitable solid excipients are carbohydrate or protein fillers include, e.g., sugars, including lactose, sucrose, mannitol, or sorbitol; starch from com, wheat, rice, potato, or other plants; cellulose such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxy-methylcellulose; and gums including arabic and tragacanth; and proteins, e.g., gelatin and collagen. Disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate. Push-fit capsules can contain active agents mixed with a filler or binders such as lactose or starches, lubricants such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active agents can be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycol with or without stabilizers.

[0077] Aqueous suspensions can contain an active agent (e.g., an inhibitory nucleic acid of tire present disclosure) in admixture with excipients suitable for the manufacture of aqueous suspensions, e.g., for aqueous intradermal injections. Such excipients include a suspending agent, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing or wetting agents such as a naturally occurring phosphatide (e.g., lecithin), a condensation product of an alkylene oxide with a fatly acid (e.g.. polyoxyethylene stearate), a condensation product of ethylene oxide with a long chain aliphatic alcohol (e.g., heptadecaethylene oxycetanol), a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol (e.g., polyoxyethylene sorbitol mono-oleate), or a condensation product of ethylene oxide with a partial ester derived from fatty acid and a hexitol anhydride (e.g., polyoxyethylene sorbitan mono-oleate). The aqueous suspension can also contain one or more preservatives such as ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents and one or more Sweetening agents, such as sucrose, aspartame or saccharin. Formulations can be adjusted for osmolarity.

[0078] In some cases, oil-based pharmaceuticals are used for administration of an inhibitory nucleic acid. Oil-based suspensions can be formulated by suspending an active agent in a vegetable oil, such as arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin; or a mixture of these. See e.g.. U.S. Pat. No. 5,716,928 describing using essential oils or essential oil components for increasing bioavailability and reducing inter- and intra-individual variability of orally administered hydrophobic pharmaceutical compounds (see also U.S. Pat. No. 5,858,401). The oil suspensions can contain a thickening agent, such as beeswax, hard paraffin or cetyl alcohol. Sweetening agents can be added to provide a palatable oral preparation, such as glycerol, sorbitol or sucrose. These formulations can be preserved by the addition of an antioxidant such as ascorbic acid. As an example of an injectable oil vehicle, see Minto (1997) J. Pharmacol. Exp. Ther. 281:93-102.

[0079] Pharmaceutical formulations can also be in the form of oil-in-water emulsions. The oily phase can be a vegetable oil or a mineral oil, described above, or a mixture of these. Suitable emulsifying agents include naturally occurring gums, such as gum acacia and gum tragacanth, naturally occurring phosphatides, such as soybean lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan mono-oleate, and condensation products of these partial esters with ethylene oxide, such as polyoxyethylene sorbitan mono-oleate. The emulsion can also contain sweetening agents and flavoring agents, as in the formulation of syrups and elixirs. Such formulations can also contain a demulcent, a preservative, or a coloring agent. In alternative embodiments, an injectable oil-in-water emulsion comprises a paraffin oil, a sorbitan monoolcatc, an ethoxylated sorbitan monoolcatc and / or an ethoxylated sorbitan trioleate.

[0080] The pharmaceutical compositions can be administered by intranasal, intraocular and intravaginal routes including suppositories, insufflation, powders and aerosol formulations (for examples of steroid inhalants, see e.g., Rohatagi (1995) J. Clin. Pharmacol. 35: 1187-1193; Tjwa (1995) Ann. Allergy Asthma Immunol. 75: 107-111). Suppositories formulations can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary’ temperatures but liquid at body temperatures and will therefore melt in the body to release the drug. Such materials are cocoa butter and polyethylene glycols.

[0081] In some instances, a pharmaceutically acceptable excipient can be selected from lipids or polymers, including poly(amidoamine), poly(propyleneimine), poly (E-ly sine).

[0082] In some cases, the pharmacal compositions can be delivered transdermally, by a topical route, formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes jellies, paints, powders, and aerosols.

[0083] In some cases, the pharmaceutical compositions can be delivered as microspheres for slow release in the body. For example, microspheres can be administered via intradermal injection ofdrug which slowly release subcutaneously; see Rao (1995) J. Biomater Sci. Polyrn. Ed. 7:623-645; as biodegradable and injectable gel formulations, see, e.g., Gao (1995) Phann. Res. 12:857-863 (1995); or, as microspheres for oral administration, see, e.g., Eyles (1997) J. Pham. Pharmacol. 49:669-674.

[0084] In some cases, the pharmacal compositions can be parenterally administered, such as by intravenous (IV) administration or administration into a body cavity or lumen of an organ. These formulations can comprise a solution of active agent (e.g., an inhibitory nucleic acid of the present disclosure) in a pharmaceutically acceptable carrier. Acceptable vehicles and solvents that can be employed arc water and Ringer's solution, an isotonic sodium chloride. In addition, sterile fixed, oils can be employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid can likewise be used in the preparation of injectables. These solutions are sterile and generally free of undesirable matter. These formulations may be sterilized by conventional, well known sterilization techniques. The formulations may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. Tire concentration of active agent in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight, and the like, in accordance with the particular mode of administration selected and the patient's needs. For intravenous administration, the formulation can be a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension. This suspension can be formulated using those suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can also be a suspension in a nontoxic parenterally acceptable diluent or solvent, such as a solution of 1,3 -butanediol. Tire administration can be by bolus or continuous infusion (e.g., substantially uninterrupted introduction into a blood vessel for a specified period of time).

[0085] In some cases, the pharmaceutical composition can be lyophilized. Stable lyophilized compositions comprising an inhibitory nucleic acid can be made by lyophilizing a solution comprising a pharmaceutical composition of the present disclosure and a bulking agent, e.g., mannitol, trehalose, raffinose, and sucrose or mixtures thereof. A process for preparing a stable lyophilized formulation can include lyophilizing a solution of about 2.5 mg / mL nucleic acid, about 15 mg / mL sucrose, about 19 mg / mL NaCl, and a sodium citrate buffer having a pH greater than 5.5 but less than 6.5. See, e.g., U.S. 20040028670.

[0086] Tire compositions and fonnulations can be delivered by the use of liposomes. By using liposomes, particularly where the liposome surface carries ligands specific for target cells, or are otherwise preferentially directed to a specific organ, one can focus the delivery of the active agent (e.g., an inhibitory nucleic acid of the present disclosure) into target cells w vivo. See, e g., U.S. Pat. Nos. 6,063,400; 6,007,839; Al-Muhammed (1996) J. Microencapsul. 13:293-306; Chonn (1995) Curr. Opin.Biotechnol. 6:698-708; Ostro (1989) Am. J. Hosp. Pharm. 46: 1576-1587. As used herein, the term "liposome" means a vesicle composed of amphiphilic lipids arranged in a bilayer or bilayers. Liposomes are unilamellar or multilamellar vesicles that have a membrane fonned from a lipophilic material and an aqueous interior that contains the composition to be delivered. Cationic liposomes are positively charged liposomes that are believed to interact with negatively charged DNA molecules to form a stable complex. Liposomes that are pH-sensitive or negatively charged are believed to entrap DNA rather than complex with it. Both cationic and noncationic liposomes have been used to deliver DNA to cells.

[0087] Liposomes can also include "stcrically stabilized" liposomes, i.c., liposomes comprising one or more specialized lipids. When incorporated into liposomes, these specialized lipids result in liposomes with enhanced circulation lifetimes relative to liposomes lacking such specialized lipids.Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome comprises one or more glycolipids or is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. Liposomes and their uses are further described in U.S. Pat. No. 6,287,860.

[0088] Tire fonnulations of the present disclosure can be administered for prophylactic and / or therapeutic treatments. In some cases, for therapeutic applications, compositions are administered to a subject in need thereof (e.g., an individual who is at risk of (e.g., at greater risk than the general population) or has a disorder described herein) in an amount sufficient to cure, alleviate or partially arrest the clinical manifestations of the disorder or its complications; this can be called a therapeutically effective amount.

[0089] The amount of pharmaceutical composition adequate to accomplish this is a therapeutically effective dose. The dosage schedule and amounts effective for this use. i.e., the dosing regimen will depend upon a variety of factors, including the stage of the disease or condition, the severity of the disease or condition, the general state of the patient's health, the patient's physical status, age and the like. In calculating the dosage regimen for a patient, the mode of administration also is taken into consideration.

[0090] The dosage regimen also takes into consideration pharmacokinetics parameters well known in the art, i.e., the active agents' rate of absorption, bioavailability, metabolism, clearance, and the like (see, e.g., Remington: The Science and Practice of Pharmacy, 21st ed., 2005). The state of the art allows the clinician to determine the dosage regimen for each individual patient, active agent (e.g., inhibitory nucleic acid) and disease or condition treated. Guidelines provided for similar compositions used as pharmaceuticals can be used as guidance to determine the dosage regiment, i.e.. dose schedule and dosage levels.

[0091] Single or multiple administrations of formulations can be given depending on for example: the dosage and frequency as required and tolerated by the patient, the degree and amount of therapeutic effect generated after each administration (e.g., effect on blood glucose levels), and the like. The formulations should provide a sufficient quantity of active agent (e.g., inhibitory nucleic acid) to effectively treat, prevent or ameliorate conditions, diseases or symptoms.

[0092] In alternative embodiments, pharmaceutical formulations for oral administration are in a daily amount of between about 1 pg to about 100 mg nucleic acid per kilogram of body weight per day. Lower dosages can be used, in contrast to administration orally, into the blood stream, into a body cavity or into a lumen of an organ. Substantially higher dosages can be used in topical or oral administration or administering by powders, spray or inhalation. Actual methods for preparing parenterally or non- parenterally administrable formulations will be known or apparent to those skilled in the art and are described in more detail in such publications as Remington: The Science and Practice of Pharmacy, 21st ed., 2005.

[0093] In some embodiments, the methods described herein can include co-administration with other drugs or pharmaceuticals, e.g., compositions for reducing blood glucose levels. For example, the inhibitory nucleic acids can be co-administered with drugs for treating or reducing risk of a disorder described herein.METHODS OF TREATING SARCOPENIA

[0094] Tire present disclosure provides methods using an inhibitory nucleic acid of the present disclosure, e.g., in subjects suffering from sarcopenia. The methods comprise administering to an individual in need thereof an effective amount of an inhibitory nucleic acid of the present disclosure, or a composition (e.g., a pharmaceutical composition) comprising same. In some cases, an inhibitory nucleic acid of the present disclosure is administered in a lipid nanoparticle. In some cases, an inhibitory nucleic acid of the present disclosure is administered in a liposome and / or a recombinant adeno-associated virus (AAV) particle. Sarcopenia disorders that can be treated with a method of the present disclosure include, e.g.. age-related sarcopenia.

[0095] In some cases, a therapeutically effective amount of an inhibitory nucleic acid of the present disclosure is an amount that, when administered in one or more doses to a subject, results in one or more of: i) reduced oxidative stress; ii) improved muscular function; iii) decreased rate of atrophy; iv) reduced inflammation; and v) reduced fatty infiltration.

[0096] In some cases, an effective amount of an inhibitory nucleic acid of the present disclosure is an amount that, when administered in one or more doses to a subject, provides for an increase in muscle strength in the subject. For example, in some cases, an effective amount of an inhibitory' nucleic acid of the present disclosure is an amount that, when administered in one or more doses to a subject,provides for an increase of at least 10%, at least 25%, at least 50%, at least 2-fold, or more than 2-fold, in muscle strength in the subject, compared to the level of muscle strength in the subject before being treated with the inhibitory nucleic acid.

[0097] In some cases, an effective amount of an inhibitory nucleic acid of the present disclosure is an amount that, when administered in one or more doses to a subject, provides for a reduction in skeletal muscle fibrosis and necrosis. For example, in some cases, an effective amount of an inhibitory nucleic acid of the present disclosure is an amount that, when administered in one or more doses to a subject, provides for a reduction of at least 10%, at least 25%, at least 50%, at least 60%, at least 70%, or more than 70%, in skeletal muscle fibrosis and necrosis, compared to the level of skeletal muscle fibrosis and necrosis in the subject before being treated with the inhibitory nucleic acid.Routes of administration

[0098] Suitable routes of administration include oral, rectal, nasal, pulmonary, topical, subcutaneous, intramuscular, intraperitoneal, intravenous, intradermal, intrathecal, and epidural. In some cases, the route of administration is intramuscular. In some cases, the route of administration is intravenous.Combination therapy

[0099] The present disclosure contemplates the use of an inhibitory nucleic acid of the present disclosure in combination with one or more additional agents (e.g., one or more additional active therapeutic agents) or other prophylactic or therapeutic modalities. In such combination therapy, tire various active agents frequently have different mechanisms of action. Such combination therapy may be especially advantageous by allowing a dose reduction of one or more of the agents, thereby reducing or eliminating the adverse effects associated with one or more of the agents; furthermore, such combination therapy may have a synergistic therapeutic or prophylactic effect on the underlying disease, disorder, or condition.

[0100] As used herein, “combination” is meant to include therapies that can be administered separately, for example, fonnulated separately for separate administration (e.g.. as may be provided in a kit), and therapies that can be administered together in a single formulation (i.e., a “co-formulation”).

[0101] In certain cases, an inhibitory nucleic acid of the present disclosure and the at least one additional agent are administered or applied sequentially, e.g., where one agent is administered prior to one or more other agents. In other cases, an inhibitory nucleic acid of the present disclosure and the at least one additional agent are administered simultaneously, e.g., where two or more agents are administered at or about the same time; the two or more agents may be present in two or more separate formulations or combined into a single formulation (i.e., a co-formulation). Regardless of whether thetwo or more agents are administered sequentially or simultaneously, they are considered to be administered in combination for purposes of the present disclosure.

[0102] An inhibitory nucleic acid of the present disclosure can be used in combination with other agents useful in the treatment of the disorders or conditions set forth herein, including those that are normally administered to subjects suffering from muscular atrophy, malnutrition, metabolic syndrome, and certain hormonal disorders.

[0103] The present disclosure contemplates combination therapy with numerous agents (and classes thereof), including 1) testosterone, testosterone mimetics, and agents that stimulate testosterone secretion. In certain embodiments, testosterone can be used directly or as prodrug. Testosterone prodrugs can include testosterone esters, such as Testosterone caproate, Testosterone cypionate, Testosterone decanoate. Testosterone enanthate, Testosterone isobutyrate. Testosterone isocaproate, Testosterone phenylpropionate, Testosterone propionate, Testosterone undecanoate, Testosterone acetate, Testosterone cyclohexylpropionate, Testosterone enantate benzilic acid hydrazone, Testosterone furoate, Testosterone hexahydrobenzoate, Testosterone hexahydrobenzylcarbonate, Testosterone hexyloxyphenylpropionate, Testosterone ketolaurate, Testosterone nicotinate. Testosterone phenylacetate, Testosterone phosphate. Testosterone undecylenate, and Testosterone valerate. Trade names of testosterone replacement therapies include Androgel™, Testim™, Depo-Testosterone™, Androderm™, methyltestosterone, and Android™. Certain vitamins or nutritional supplements may increase boost testosterone levels in an individual, including creatine, omcga-3 fatty acids, vitamin D, and dehydroepiandrosterone (DHEA); 2) hormonal therapies, including growth honnones and gro th factors, including growth hormone (e.g., somatotropin, human growth hormone, Somatropin™.Humatrope™), insulin-like growth factor (IGF-1), mechanical growth factor (MGF), and urocortin II. And 3) anabolic steroids, such as Danazol, Drostanolone propionate, Ethylestrenol, Fluoxymesterone, Mesterolone, Metandienone, Metenolone acetate, Metenolone enanthate, Methyltestosterone, Nandrolone decanoate, Nandrolone phenylpropionate, Norethandrolone, Oxandrolone, Oxymetholone, Stanozolol, Testosterone cypionate, Testosterone enanthate, Testosterone propionate, Testosterone undecanoate, and Trenbolone HBC.

[0104] Lifestyle adjustments may also be beneficial for certain individuals with sarcopenia, including improved diet and exercise. Diet can include reduced calorie (e.g., caloric deficit) diets and / or any other diet that encourage a reduction in bodily fat and / or excess w eight. Exercise can include weight training, resistance training, and / or any other exercise type for the increase in muscle mass and / or muscle strength. Weight training and / or resistance training can include the use of free weights (e.g., dumbbells, barbells and kettlebells), sandbags, weighted balls (e.g.. medicine balls), weight machines, resistance bands, suspension equipment, the subject’s body weight.

[0105] Certain embodiments include treatments for metabolic syndrome and related disorders, such as insulin-rcsistancc, obesity, and hypertension. Such therapeutics can include: 1) insulin, insulin mimetics and agents that entail stimulation of insulin secretion, including sulfonylureas (e.g., chlorpropamide, tolazamide, acetohexamide, tolbutamide, glyburide, glimepiride. glipizide) and meglitinides (e.g., mitiglinide, repaglinide and nateglinide); 2) biguanides (e.g., metformin, and its pharmaceutically acceptable salts, in particular, metformin hydrochloride, and extended-release formulations thereof, such as Glumetza™, Fortamet™, and GlucophageXR™) and other agents that act by promoting glucose utilization, reducing hepatic glucose production and / or diminishing intestinal glucose output; 3) alpha-glucosidase inhibitors (e g., acarbose, voglibose and miglitol) and other agents that slow down carbohydrate digestion and consequently absorption from the gut and reduce postprandial hyperglycemia; 4) thiazolidinediones (e.g., rosiglitazone, troglitazone, pioglitazone, glipizide, balaglitazone, rivoglitazone, netoglitazone, AMG 131, MBX2044, mitoglitazone, lobeglitazone, IDR- 105, troglitazone, englitazone, ciglitazone, adaglitazone, darglitazone that enhance insulin action (e.g., by insulin sensitization) including insulin, and insulin mimetics (e.g., insulin degludec, insulin glargine, insulin lispro, insulin detemir, insulin glulisine and inhalable formulations of each), thus promoting glucose utilization in peripheral tissues; 5) glucagon-like-peptides including DPP -IV inhibitors (e.g., alogliptin, omarigliptin, linagliptin, vildagliptin and sitagliptin) and Glucagon-Like Peptide- 1 (GLP-1) and GLP-1 agonists and analogs (e.g., exenatide (BYETTA and ITCA 650 (an osmotic pump inserted subcutaneously that delivers an exenatide analog over a 12-month period; Intarcia, Boston, Mass.)) and GLP-1 receptor agonists (e.g., dulaglutide, semaglutide, tirzepatide, albiglutide, exenatide, liraglutide, lixisenatide. taspoglutide, CJC-1131, and BIM-51077, including intranasal, transdermal, and once- weekly formulations thereof); and 6) and DPP-IV-resistant analogues (incretin mimetics), PPAR gamma agonists, PPAR alpha agonists such as fenofibric acid derivatives (e.g., gemfibrozil, clofibrate, ciprofibrate, fenofibrate, bezafibrate), dual-acting PPAR agonists (e.g., ZYH2, ZYH1, GFT505, chiglitazar, muraglitazar, aleglitazar, sodelglitazar, and naveglitazar), pan-acting PPAR agonists, PTP1B inhibitors (e.g., ISIS-113715 and TTP814), SGLT inhibitors (e.g., ASP1941, SGLT-3, empagliflozin, dapagliflozin, canagliflozin, BI-10773, PF-04971729. remogloflozin, TS-071, tofogliflozin, ipragliflozin. and LX-4211), insulin secretagogues. angiotensin converting enzyme inhibitors (e.g.. alacepril. benazepril, captopril, ceronapril, cilazapril, delapril, enalapril, enalaprilat, fosinopril, imidapril, lisinopril, moveltipril, perindopril, quinapril, ramipril, spirapril, temocapril, ortrandolapril), angiotensin II receptor antagonists (e.g., losartan, valsartan, candesartan, olmesartan, telmesartan); and the like.Subjects suitable for treatment

[0106] Subjects suitable for treatment using a method of the present disclosure include individuals having sarcopenia, including age-related sarcopenia. In some instances, the subject has muscle weakness, muscle atrophy, muscle wasting, and / or decreased muscle mass. Certain subjects mayhave a high body mass index and / or possess sarcopenic obesity. Certain subjects may be of an advanced age (e.g., greater than 60 years of age, 65 years of age, 70 years of age, 75 years of age, 80 years of age, 85 years of age, or greater.

[0107] Some subjects may possess one or more risk factors associated with sarcopenia, including physical inactivity, obesity, one or more chronic diseases (e.g., chronic obstructive pulmonary disease, kidney disease, diabetes, cancer, human immunodeficiency virus infection, etc.), rheumatoid arthritis, insulin resistance, reduced hormone levels, malnutrition, inadequate protein intake, decreased ability to convert protein to energy, decline in nerve cells between brain and muscle.

[0108] Sarcopenia can be assessed using any of a number of methods, such as strength, assistance with walking, rising from a chair, climbing stairs, and falls. Such assessments can include a handgrip test, a chair stand test, a walking speed test, a physical performance battery, a timed-up and go test. In some situations, imaging can be used to measure muscle mass, including dual-energy X-ray absorptiometry and / or bioelectrical impedance analysis.

[0109] In certain embodiments, a subject has not been diagnosed with a metabolic disorder (e.g., metabolic syndrome). In additional embodiments, the subject has been determined to not have a metabolic disorder.ADENO-ASSOCIATED VIRUS PARTICLES

[0110] The present disclosure provides a recombinant adeno-associated viral (rAAV) particle comprising a capsid and a nucleic acid vector comprising a heterologous nucleic acid region comprising sequence encoding an interfering RNA. as described above, the interfering RNA comprising a region complementary to a miR-128 target nucleic acid.

[0111] Viruses of the Parvoviridae family are small non-enveloped icosahedral capsid viruses characterized by a single stranded DNA genome. Parvoviridae family viruses include adeno-associated viruses (AAV) capable of replication in vertebrate hosts including, but not limited to, human, primate, bovine, canine, equine, and ovine species.

[0112] In one embodiment, AAV particles of the present disclosure are recombinant AAV viral vectors which are replication defective and lacking sequences encoding functional Rep and Cap proteins within their viral genome. These defective AAV vectors may lack most or all parental coding sequences and essentially carry only one or two AAV ITR sequences and the nucleic acid of interest (e.g., a nucleic acid comprising a nucleotide sequence encoding an inhibitory nucleic acid of the present disclosure) for delivery to a cell, a tissue, an organ, or an organism.

[0113] According to some embodiments, AAV particles for use in therapeutics and / or diagnostics comprise a virus that has been distilled or reduced to the minimum components necessary for transduction of a nucleic acid payload or cargo of interest. In this manner, AAV particles are engineeredas vehicles for specific delivery while lacking the deleterious replication and / or integration features found in wild-type viruses.

[0114] AAV vectors of the present disclosure may be produced recombinantly and may be based on adeno-associated virus (AAV) parent or reference sequences. As used herein, a " vector” is any molecule or moiety which transports, transduces, or otherwise acts as a carrier of a heterologous molecule such as the nucleic acids described herein.

[0115] In addition to single stranded AAV viral genomes (e.g., ssAAVs), the present disclosure also provides for self-complementary AAV (scAAVs) viral genomes, scAAV vector genomes contain DNA strands which anneal together to form double stranded DNA. By skipping second strand synthesis, scAAVs allow for rapid expression in the cell.

[0116] In one embodiment, the AAV particle of the present disclosure is an scAAV.

[0117] In one embodiment, the AAV particle of the present disclosure is an ssAAV.

[0118] Methods for producing and / or modifying AAV particles are disclosed in the art such as pseudotyped AAV vectors (PCT Patent Publication Nos. W0200028004; W00200123001;W020041 12727; W02005005610; and W02005072364, the content of each of which is incorporated herein by reference in its entirety).

[0119] AAV particles may be modified to enhance the efficiency of delivery. Such modified AAV particles can be packaged efficiently and be used to successfully infect the target cells at high frequency and with minimal toxicity. In some embodiments, the capsids of the AAV particles are engineered according to the methods described in US Publication Number US20I95801, the contents of which are incorporated herein by reference in their entirety.Viral Genome Component: Inverted Terminal Repeats (ITRs)

[0120] Tire AAV particles of the present disclosure comprise a viral genome with at least one ITR region and a payload region. In one embodiment, the viral genome has two ITRs. These two ITRs flank the payload region at the 5' and 3' ends. The ITRs function as origins of replication comprising recognition sites for replication. ITRs comprise sequence regions which can be complementary find symmetrically arranged ITRs incorporated into viral genomes of the invention may be comprised of naturally occurring polynucleotide sequences or recombinantly derived polynucleotide sequences.

[0121] The ITRs may be derived from the same serotype as the capsid, selected from any other serotype, or a derivative thereof. The ITR may be of a different serotype than the capsid. In one embodiment, the AAV particle has more than one ITR. In a non-limiting example, the AAV particle has a viral genome comprising two ITRs. In one embodiment, the ITRs are of the same serotype as one another. In another embodiment, the ITRs are of different serotypes. Non-limiting examples includezero, one or both of the ITRs having the same serotype as the capsid. In one embodiment both ITRs of the viral genome of the AAV particle are AAV2 ITRs.

[0122] Independently, each ITR may be about 100 to about 150 nucleotides in length. An ITR may be about 100-105 nucleotides in length, 106-110 nucleotides in length. 111-115 nucleotides in length, 116-120 nucleotides in length, 121-125 nucleotides in length, 126-130 nucleotides in length, 131- 135 nucleotides in length, 136-140 nucleotides in length, 141-145 nucleotides in length or 146-150 nucleotides in length. In one embodiment, the ITRs are 14-142 nucleotides in length. Non-limiting examples of ITR length arc 102, 140, 141, 142, 145 nucleotides in length, and those having at least 95% identity thereto.Viral Genome Component: Promoters

[0123] In one embodiment, the payload region of the viral genome comprises at least one element to enhance the transgene target specificity and expression (See e.g., Powell et al. Viral Expression Cassette Elements to Enhance Transgene Target Specificity and Expression in Gene Therapy, 2015; the contents of which are herein incorporated by reference in its entirety). Non-limiting examples of elements to enhance the transgene target specificity and expression include promoters, endogenous miRNAs. post-transcriptional regulatory elements (PREs), polyadenylation (Poly A) signal sequences and upstream enhancers (USEs), CMV enhancers and introns.

[0124] A person skilled in the art may recognize that expression of the nucleic acids described herein may require a specific promoter, including but not limited to, a promoter that is species specific, inducible, tissue-specific, or cell cycle-specific (Parr et al., Nat. Med.3: 1145-9 (1997); the contents of which are herein incorporated by reference in their entirety).

[0125] In one embodiment, the promoter is deemed to be efficient when it drives transcription of the nucleic acid sequence encoded in the payload region of the viral genome of the AAV particle.

[0126] In one embodiment, the promoter is a promoter deemed to be efficient when it drives expression in the cell, tissue, and / or organ being targeted. In certain embodiments, the promoter is capable of being expressed in muscle tissue of an individual, including skeletal muscle, smooth muscle, and / or cardiac muscle.

[0127] In various embodiments, tissue specific expression is driven by one or more of the following promotors: cytomegalovirus immediate early promoter (CMV). hybrid chicken-beta-actin (CBA) promoter, and ubiquitin C (UBC) promoter, and an RNA polymerase III promoter.AAV Serotypes

[0128] AAV particles of the present disclosure may comprise or be derived from any natural or recombinant AAV seroty pe. According to the present disclosure, the AAV particles may utilize or be based on a serotype selected from any of the following AAV1, AAV2, AAV3, AAV4, AAV5, AAV6,AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrhlO, AAV11, AAV12, AAV2R471A, AAVAAV2 / 2-7m8, AAV2 N587A, AAV2 E548A, AAV2 N708A, AAV V708K, goat AAV, AAV1 / AAV2 chimer, bovine AAV, mouse AAV, or rAAV2 / HBoVland variants thereof.Examples of Non-Limiting Aspects of the Disclosure

[0129] Aspects, including embodiments, of the present subject matter described above may be beneficial alone or in combination, with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of the disclosure are provided below. As will be apparent to those of skill in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any of the preceding or following individually numbered aspects. This is intended to provide support for all such combinations of aspects and is not limited to combinations of aspects explicitly provided below:Aspect 1. A method for treating age-related sarcopenia, comprising administering an effective amount of a miR-128 inhibitor to an individual diagnosed with age-related sarcopenia.Aspect 2. The method of Aspect 1, wherein the individual has not been diagnosed with a metabolic disorder.Aspect 3. The method of Aspect 1 or 2. wherein the miR-128 inhibitor comprises an inhibitory nucleic acid.Aspect 4. The method of Aspect 3, wherein the inhibitory nucleic acid comprises one or more of the following sequences:5 -ACCGGTTCACTGTG-3’ (SEQ ID NO: 1);5 -GACCGGTTCACTGT-3' (SEQ ID NO:2); and 5 -AGACCGGTTCACTGTG-3’ (SEQ ID NO:3).Aspect 5. The method of Aspect 3 or 4, wherein the inhibitory nucleic acid comprises one or more locked nucleic acids (LNA).Aspect 6. The method of any of Aspects 2-5, wherein the inhibitory nucleic acid comprises one or more of a modified backbone and one or more 5 -methyl deoxycytosine residues.Aspect 7. Tire method of any of Aspects 2-6, wherein the inhibitory nucleic acid has a length of from 14 nucleotides to 20 nucleotides.Aspect 8. The method of any of Aspects 2-7, wherein the inhibitory nucleic acid comprises the nucleotide sequence designated “NRC-0090.”Aspect 9. The method of any of Aspects 2-7, wherein the inhibitory nucleic acid comprises the nucleotide sequence designated “NRC-0091.”Aspect 10. Tire method of any of Aspects 2-7, wherein the inhibitory nucleic acid comprises the nucleotide sequence designated “NRC-0119.”Aspect 11. The method of any of Aspects 1-10, wherein the miR-128 inhibitor is formulated for subcutaneous delivery'.Aspect 12. The method of any of Aspects 1-11, wherein the miR-128 inhibitor is fonnulated as a pharmaceutical composition comprising one or more of the following: a pharmacally acceptable excipient, a buffering agent, a solvent, a bulking agent, a flavorant. a lubricant, a colorant, a binder, a surfactant, a binder, and a disintegrant.Aspect 13. The method of any of Aspects 1-11, wherein the miR-128 inhibitor is formulated as a pharmaceutical composition comprising a pharmacally acceptable excipient.Aspect 14. The method of Aspect 13, wherein the pharmacally acceptable excipient is selected from: poly(amidoamine), poly(propyleneimine), and poly(L-lysine).Aspect 15. The method of Aspect 13, wherein the pharmaceutically acceptable excipient comprises one or more lipids.Aspect 16. The method of any of Aspects 1-15, further comprising administering at least one additional therapeutic agent.Aspect 17. Hie method of Aspect 16, wherein the at least one additional therapeutic agent is selected from testosterone, growth hormone, insulin-like growtli factor, mechanical growth factor, urocortin II. anabolic steroids, omega-3 fatty acids, creatine, dehydroepiandrosterone.Aspect 18. The method of any of Aspects 1-17, further comprising directing the individual to perform weight training exercises or resistance training exercises.Aspect 19. A combination therapy for the treatment of age-related sarcopenia, comprising: a miR-128 inhibitor; and one or more of: testosterone, growth hormone, insulin-like growth factor, mechanical growth factor, urocortin II. anabolic steroids, omega-3 fatty acids, creatine, dehydroepiandrosterone.Aspect 20. The combination therapy of Aspect 19, wherein the miR-128 inhibitor comprises an inhibitory' nucleic acid.Aspect 21. The combination therapy of Aspect 20, wherein the inhibitory' nucleic acid comprises one or more of tire following sequences:5’-ACCGGTTCACTGTG-3’ (SEQ ID NO: 1);5 -GACCGGTTCACTGT-3’ (SEQ ID NO:2); and5 -AGACCGGTTCACTGTG-3’ (SEQ ID NO:3).Aspect 22. The combination therapy of Aspect 20 or 21, wherein the inhibitory' nucleic acid comprises one or more locked nucleic acids (LNA).Aspect 23. Hie method of any of Aspects 20-22, wherein the inhibitory nucleic acid comprises one or more of a modified backbone and one or more 5 -methyl deoxy cytosine residues.Aspect 24. The combination therapy of any of Aspects 20-23, wherein the inhibitory nucleic acid has a length of from 14 nucleotides to 20 nucleotides.Aspect 25. The combination therapy of any of Aspects 20-24, wherein the inhibitor}’ nucleic acid comprises the nucleotide designated "NRC-0090.’'Aspect 26. The combination therapy of any of Aspects 20-24, wherein the inhibitory nucleic acid comprises the nucleotide sequence designated “NRC-0091.”Aspect 27. The combination therapy of any of Aspects 20-24, wherein the inhibitory nucleic acid comprises the nucleotide sequence designated “NRC-0119.”Aspect 28. The combination therapy of any of Aspects 19-27, wherein tire miR-128 inhibitor is formulated for subcutaneous delivery.Aspect 29. The combination therapy of any of Aspects 19-28, wherein the miR-128 inhibitor is formulated as a pharmaceutical composition comprising one or more of the following: a pharmaceutically acceptable excipient, a buffering agent, a solvent, a bulking agent, a flavorant, a lubricant, a colorant, a binder, a surfactant, a binder, and a disintegrat'd.Aspect 30. Hie combination therapy of any of Aspects 19-29, wherein the miR-128 inhibitor is formulated as a pharmaceutical composition comprising a pharmaceutically acceptable excipient.Aspect 31. The combination therapy of Aspect 30, wherein the pharmaceutically acceptable excipient is selected from: poly(amidoamine), poly(propyleneimine), and poly(L-lysine).Aspect 32. The combination therapy of Aspect 30, wherein the pharmaceutically acceptable excipient comprises one or more lipids.Aspect 33. An oligonucleotide, comprising: a plurality of interlinked nucleotides, wherein a contiguous subset of the plurality of nucleotides is complementary to a portion of a miR-128 nucleic acid; wherein at least one nucleotide in the plurality of interlinked nucleotides is a bridged nucleic acid.Aspect 34. The oligonucleotide of Aspect 33, wherein the oligonucleotide is comprised of 7 to 50 interlinked nucleotides.Aspect 35. Tire oligonucleotide of Aspect 33 or 34, wherein the oligonucleotide is comprised of 7 to 30 interlinked nucleotides.Aspect 36. The oligonucleotide of any of Aspects 33-35. wherein the oligonucleotide is comprised of 14 to 23 interlinked nucleotides.Aspect 37. The oligonucleotide of any of Aspects 33-36, wherein the contiguous subset comprises at least 6 nuclcobascs that arc complementary to the miR-128 nucleic acid.Aspect 38. Tire oligonucleotide of any of Aspects 33-37. wherein the oligonucleotide is a unimer.Aspect 39. The oligonucleotide of Aspect 38, wherein each nucleotide is independently a bridged nucleic acid.Aspect 40. The oligonucleotide of any of Aspects 33-37, wherein the nucleobase sequence comprises one or more of the following sequences: 5 -ACCGGTTCACTGTG-3’ (SEQ ID NO: 1);5 -GACCGGTTCACTGT-3' (SEQ ID NO:2); and 5’-AGACCGGTTCACTGTG-3’ (SEQ ID NO:3).Aspect 41. A recombinant adeno-associated viral (rAAV) particle comprising a capsid and a nucleic acid vector comprising a heterologous nucleic acid region comprising sequence encoding an interfering RNA comprising a region complementary to a miR-128 target nucleic acid.Aspect 42. The rAAV of Aspect 41, wherein the miR-128 target nucleic acid is miR-128-3p, pri- miR-128-1, pre-miR-128-1. pri-miR- 128-2, or pre-miR- 128-2.Aspect 43. The rAAV of Aspect 41, wherein the interfering RNA is a short hairpin RNA or a small interfering RNA.Aspect 44. The rAAV of Aspect 41, wherein the nucleic acid vector comprises a promoter, wherein the sequence encoding an interfering RNA is operably linked to the promoter.Aspect 45. Hie rAAV of Aspect 44, wherein the promoter is capable of inducing expression of the interfering RNA in a skeletal muscle cell.Aspect 46. The rAAV of Aspect 44, wherein the promoter is a hybrid chicken P-actin (CBA) promoter or an RNA polymerase III promoter.Aspect 47. The rAAV of Aspect 41, wherein the nucleic acid vector comprises inverted terminal repeats from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrhlO, AAV11, AAV12, AAV2R471A, AAV DJ, goat AAV, bovine AAV, or mouse AAV serotypes.Aspect 48. The rAAV of Aspect 41, wherein the capsid is selected from: an AAV1 capsid, and AAV2 capsid, an AAV3 capsid, an AAV4 capsid, an AAV5 capsid, anAAV6 capsid, an AAV7 capsid, anAAV8 capsid, an AAVrh8 capsid, an AAVrh8R capsid, an AAV9 capsid, an AAV10 capsid, an AAVrhlO capsid, an AAV11 capsid, an AAV12 capsid, atyrosine capsid mutant, a heparin binding capsid mutant, an AAV2R471A capsid, an AAVAAV2 / 2-7m8 capsid, an AAV DJ capsid, an AAV2 N587A capsid, an AAV2 E548A capsid, an AAV2 N708A capsid, an AAV V708K capsid, a goat AAV capsid, an AAV 1 / AAV2 chimeric capsid, a bovine AAV capsid, a mouse AAV capsid, and a rAAV2 / HBoVl capsid.Aspect 49. A pharmaceutical composition comprising: a pharmaceutically acceptable excipient; and the oligonucleotide of any of Aspects 33-40 or the rAAV of any of Aspects 41-48. Aspect 50. A therapeutically effective composition for use in a method of treating age-related macular degeneration in a subject in need thereof, the method comprising administering to the subject thetherapeutically effective amount of the oligonucleotide of any of Aspects 1-8 or tire rAAV of any of Aspects 9-16.Aspect 51. A miR-128 inhibitor for use in a method of treating aging-related sarcopenia in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the miR-128 inhibitor.Aspect 52. The miR-128 inhibitor of Aspect 51, wherein the miR-128 inhibitor is selected from: an antisense oligonucleotide, a short hairpin RNA, a small interfering RNA, or a recombinant adeno-associated viral (rAAV) particle comprising a capsid and a nucleic acid vector comprising a heterologous nucleic acid region comprising sequence encoding an interfering RNA, wherein the interfering RNA is selected from an antisense oligonucleotide, a short hairpin RNA, or a small interfering RNA.Aspect 53. A therapeutically effective composition for use in a method of treating age-related sarcopenia in a subject in need thereof, the method comprising administering to the subject the therapeutically effective amount of the oligonucleotide of any of Aspects 1-8 or the rAAV of any of Aspects 9-16.Aspect 54. The therapeutically effective composition of Aspect 53, wherein the oligonucleotide is a single-stranded oligonucleotide.Aspect 55. The therapeutically effective composition of Aspect 53, wherein the oligonucleotide is an antisense oligonucleotide.Aspect 56. Tire therapeutically effective composition of Aspect 53, wherein the oligonucleotide comprises 7 to 30 interlinked nucleotides.Aspect 57. The therapeutically effective composition of Aspect 56, wherein the oligonucleotide comprises 14 to 23 interlinked nucleotides.Aspect 58. The therapeutically effective composition of any of Aspects 53-57, wherein the oligonucleotide comprises at least one modified sugar nucleoside.Aspect 59. Tire therapeutically effective composition of Aspect 58, wherein at least 50% of the nucleosides comprise a modified sugar nucleoside.Aspect 60. The therapeutically effective composition of Aspect 58 or 59, wherein 100% of the nucleosides comprise a modified sugar nucleoside.Aspect 61. The therapeutically effective composition of any of Aspects 58-60, wherein the modified sugar nucleoside is a 2 ’-modified sugar nucleoside.Aspect 62. The therapeutically effective composition of Aspect 61, wherein the 2’-modified sugar nucleoside comprises a 2 ’-modification independently selected from the group consisting of 2’-fluoro, 2'-methoxy, and 2 ’-methoxy ethoxy.Aspect 63. Tire therapeutically effective composition of any of Aspects 58-60, wherein tire modified sugar nucleoside is a bridged nucleic acid.Aspect 64. The therapeutically effective composition of Aspect 63, wherein the bridged nucleic acid is a locked nucleic acid.Aspect 65. The therapeutically effective composition of Aspect 63, wherein the bridged nucleic acid is an ethylene bridged nucleic acid.Aspect 66. Tire therapeutically effective composition of any of Aspects 58-60, wherein tire nucleobase sequence comprises one or more of the following sequences: 5 -ACCGGTTCACTGTG-3’ (SEQ ID NO: 1): 5’-GACCGGTTCACTGT-3’ (SEQ ID NO:2); and 5’-AGACCGGTTCACTGTG-3’ (SEQ ID NO:3).Aspect 67. Hie therapeutically effective composition of any of Aspects 53-66, wherein the oligonucleotide is a gapmer comprising a '-wing, a 3 ’-wing, and a gap, wherein the 5 ’-wing and the 3’- wing each comprise 1 to 5 nucleotides, wherein each nucleotide in the 5 ’-wing and the 3 ’-wing are independently a bridged nucleic acid, and wherein each nucleotide in the gap is a deoxyribonucleotide. Aspect 68. The therapeutically effective composition of any of Aspects 53-67, wherein at least one intemucleoside linkage in the oligonucleotide is a phosphorothioate diester.Aspect 69. Hie therapeutically effective composition of Aspect 68, wherein at least 50% of intemucleoside linkages in the oligonucleotide are phosphorothioate diesters.Aspect 70. The therapeutically effective composition of Aspect 68 or 69, wherein 100% of intemucleoside linkages in the oligonucleotide are phosphorothioate diesters.Aspect 71. The therapeutically effective composition of any of Aspects 53-70, wherein the method further comprises administering the oligonucleotide as a guide strand in a short interfering RNA.Aspect 72. Hie therapeutically effective composition of any of Aspects 53-71, wherein the miR-128 target nucleic acid is selected from: pri-miR-128-1, pre-miR-128-1. pri-miR- 128-2. pre-miR- 128-2, and miR-128-3p.Aspect 73. The therapeutically effective composition of any of Aspects 53-72, wherein the route of administration is an intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection.EXAMPLES

[0130] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., bp, base pair(s); kb. kilobase(s); pl, picoliter(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly); and the like.Example 1:

[0131] FIG. 1A depicts sequence information for three ASOs. ID indicates drug name, OLIGO indicates nucleotide sequence (uppercase denotes locked nucleic acids (LNAs), lowercase denotes standard bases), STRING indicates modifications (+ sign precedes an LNA, * precedes a phosphorothioated base, / iMe-dC / denotes an internal 5-methyl dcoxycytosme. Length indicates number of nucleotides, #LNAs indicates number of standard bases that have been replaced with LNAs. FIG. IB provides a schematic LNA / DNA composition of each ASO and their base-pairing location within the target miRNA (miR-128-3p). The seed sequence of miR-128-3p is denoted in red.Example 2:

[0132] Aged mice (21.5 months old) and young control mice (8 weeks old) were subcutaneously injected with anti-miR-128-3p LNA ASO or a scramble control LNA ASO weekly at 10 mg / kg bodyweight. Muscle function was then measured 1, 2, and 3 months later by measuring the time hanging suspended from a wire and the run time until exhaustion (using a high intensity testing protocol). At 2 months and 3 months, muscle function was also measured by the four-limb hanging test. At 3 months, muscle function was finally measured by run time until exhaustion using a low intensity protocol (FIG. 2A). After sacrifice at 5 months of injections, miR-128-3p levels in gastrocnemius (GA) muscle were measured by reverse transcription -quantitative polymerase chain reaction (RT-qPCR).

[0133] The wire hanging time of aged mice is impaired compared with young mice by 22.5 months of age. Treatment with anti-miR-128-3p LNA ASO improves hang time when measured by the average of three trials, the maximum of the trials, or the calculated score (FIG. 2B-2D). Treatment with anti-miR-128-3p LNA ASO significantly improved hang time (both maximum and average) by 3 monthsof treatment. This observation is also repeated by the four-paw hang time test performed at 2 and 3 months of treatment (as measured by average and maximum hang time; FIG. 2E, FIG. 2F). The run time until exhaustion is decreased in aged mice compared with young mice one month after treatment and this run time is increased with anti-miR-128-3p treatment (FIG. 2G). At 23.5 months of age, this impainnent in run time until exhaustion is increased. Anti-miR-128-3p LNA ASO treatment significantly improves ran time after two months of treatment in these aged mice. When mice are tested with a low-intensity running protocol to examine muscle endurance, miR-128-3p LNA ASO treatment results in even more robust improvement to the functional impairment observed in aged mice after 3 months of treatment (FIG. 2H). miR-128-3p LNA ASO robustly knocks down miR-128-3p expression in gastrocnemius muscle as measured post-harvest at 26.5 months of age (FIG. 21).

[0134] FIG. 2A-2I. Anti-miR-128-3p treatment improves skeletal muscle function in aged mice. (A) Experimental design. (B) Two limb wire hanging time measured by the average of three trials. (C) Two limb wire hanging time measured by the maximum of three trials. (D) Two limb wire hanging time measured by total score from three trials. Hanging is scored by the following: 1-5 s= 1; 6-10s= 2; 11- 20s= 3; 21-30s=4; >30s or reaching support= 5; total score= sum of all three trial scores. (E) Four limb hanging time measured by the average of all trials. (F) Four limb hanging time measured by the maximum of all trials. (G) High intensity treadmill test running time until exhaustion. (H) Low intensity treadmill test running time until exhaustion performed at 3 months of injections. (I) miR-128-3p levels measured in gastrocnemius muscle of 26.5 -month-old mice by RT-qPCR. Data are presented as mean and standard deviation; student's t-test.Example 3:

[0135] Aged mice (86 weeks old) were subcutaneously injected with anti-miR-128-3p LNA ASO or a scramble control LNA ASO weekly at 10 mg / kg bodyweight. Muscle function was then measured 5. 9 and 15 weeks later by measuring the time hanging suspended from a wire and the ran time until exhaustion (using a low intensity testing protocol) was measured at 13 weeks (Fig. 3A).Quantitative data are presented as mean and standard deviation; student’s t-test. After sacrifice at 20 weeks of injections, miR-128-3p levels in gastrocnemius (GA) muscle were measured by reverse transcription-quantitative polymerase chain reaction (RT-qPCR).

[0136] miR-128-3p LNA ASO robustly knocks down miR-128-3p expression in gastrocnemius muscle as measured post-harvest at 106 weeks of age (FIG. 3B). As an additional control under a parallel protocol, aged mice (86 weeks old) were subcutaneously injected with miR-27 LNA ASO, an off-target miRNA with similar sequence to miR-128-3p, or a scramble control LNA ASO weekly at 10 mg / kgbodyweight. RT-qPCR of miR-128-p3 was not different between mice injected with miR-27 LNA ASO and scramble control LNA ASO at 106 weeks of age (Fig. 3C).

[0137] Treatment with anti-miR-128-3p LNA ASO improves two limb hang time when measured by the average of three trials (Fig. 3D). Run time was also improved after 13 weeks of treatment with anti-miR-128-3p LNA ASO (Fig. 3E).

[0138] Fig. 3F shows laminin staining of cross sections of tibialis anterior muscle in control and anti-miR-128-3p LNA ASO treated mice. Quantification of tire cross-sectional area of the laminin- stained muscle fibers shows fiber size was increased in anti-miR-128-3p LNA ASO treated mice as compared with control (Fig. 3G).

[0139] Fig. 3H shows gene set enrichment analysis of RNA sequencing results from GA muscle. Expression of several mitochondrial genes are increased in GA muscle, as measured by RT- qPCR (Fig. 31).Example 4:

[0140] Aged mice (86 weeks old) were subcutaneously injected with anti-miR-128-3p LNA ASO or a scramble control LNA ASO weekly at 10 mg / kg bodyweight. Skeletal muscle mass was measured by EchoMRI and tissue weight of GA and quadraceps muscles was measured after 15 weeks of treatment (Fig. 4A). Data are presented as mean and standard deviation; student’s t-test.

[0141] Aged mice treated with anti-miR-128-3p LNA ASO showed increase percentage of lean muscle mass after 15 weeks (Fig. 4B), and decreased percent fat mass (Fig. 4C).REFERENCES

[0142] 1. Cruz-Jentoft, A. J., Bahat, G., Bauer, J., Boirie, Y., Bruyere, O., Cederholm, T.,Cooper, C., Landi, F , Rolland, Y., Sayer, A. A., Schneider, S. M., Sieber, C. C., Topinkova, E., Vandewoude, M., Visser, M., Zamboni, M., Bautmans, L, Baeyens, J.-P., Cesari, M., Cherubini, A., Kanis, J., Maggio, M., Martin, F., Michel, J.-P., Pitkala, K., Rcginstcr, J.-Y., Rizzoli, R., Sanchcz- Rodriguez, D. & Schols, J., "Sarcopenia: revised European consensus on definition and diagnosis," Age Ageing 48, 16-31 (2019). DOI: 10.1093 / ageing / afyl69

[0143] 2. Metter, E. J., Conwit, R., Tobin, J. & Fozard, J. L., "Age-Associated Loss of Power and Strength in the Upper Extremities in Women and Men," J Gerontol A Biol Sci Med Sci 52A, B267- B276 (1997). DOI: 10.1093 / gerona / 52A.5.B267

[0144] 3. Walston. J. D., "Sarcopenia in older adults," Curr Opin Rheumatol 24, 623-627(2012). DOI: 10.1097 / BOR.0b013e328358d59b

[0145] 4. Papadopoulou, S., "Sarcopenia: A Contemporary' Health Problem among Older AdultPopulations," Nutrients 12, 1293 (2020). DOI: 10.3390 / nul2051293

[0146] 5. Cho, M.-R., Lee. S. & Song, S.-K., "A Review of Sarcopenia Pathophysiology,Diagnosis, Treatment and Future Direction." J Korean Med Sci 37, (2022). DOI: 10.3346 / jkms.2022.37.el46

[0147] 6. Ambros, V., "The functions of animal microRNAs," Nature 431, 350-355 (2004).DOI: 10.1038 / nature02871

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[0149] 8. Zaharieva, I. T., Calissano, M., Scoto, M., Preston, M., Cirak, S., Feng, L., Collins, J.,Kole, R., Guglieri, M., Straub, V., Bushby, K., Ferlini, A., Morgan, J. E. & Muntoni, F., "Dystromirs as Serum Biomarkers for Monitoring the Disease Severity in Duchenne Muscular Dystrophy," PLoS One 8, e80263 (2013). DOI: 10.1371 / joumal.pone.0080263

[0150] 9. Sannicandro, A. J., Soriano-Arroquia, A. & Goljanek-Whysall, K., "Micro(RNA)- managing muscle wasting," J Appl Physiol 127, 619-632 (2019). DOI: 10.1152 / japplphysiol.00961.2018

[0151] While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to tire objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

Claims

CLAIMSWhat is claimed is:

1. A method for treating age-related sarcopenia, comprising administering an effective amount of a miR-128 inhibitor to an individual diagnosed with age-related sarcopenia.

2. Tire method of claim 1, wherein tire individual has not been diagnosed with a metabolic disorder.

3. The method of claim 1 or 2, wherein the miR-128 inhibitor comprises an inhibitory nucleic acid.

4. Tire method of claim 3. wherein the inhibitory nucleic acid comprises one or more of the following sequences:5’-ACCGGTTCACTGTG-3’ (SEQ ID NOT);5’-GACCGGTTCACTGT-3’ (SEQ ID NO:2); and5 -AGACCGGTTCACTGTG-3’ (SEQ ID NOT).

5. The method of claim 3 or 4, wherein the inhibitory nucleic acid comprises one or more locked nucleic acids (LNA).

6. Tire method of any of claims 2-5. wherein the inhibitory nucleic acid comprises one or more of a modified backbone and one or more 5-methyl deoxycytosine residues.

7. The method of any of claims 2-6, wherein the inhibitory nucleic acid has a length of from 14 nucleotides to 20 nucleotides.

8. The method of any of claims 2-7, wherein the inhibitory nucleic acid comprises the nucleotide sequence designated “NRC-0090.”9. The method of any of claims 2-7, wherein the inhibitory nucleic acid comprises the nucleotide sequence designated “NRC-009I ”10. Hie method of any of claims 2-7, wherein the inhibitory nucleic acid comprises the nucleotide sequence designated “NRC-0119.”11. The method of any of claims 1-10, wherein the miR-128 inhibitor is formulated for subcutaneous delivery.

12. The method of any of claims 1-11, wherein the miR-128 inhibitor is formulated as a pharmaceutical composition comprising one or more of the following: a pharmaceutically acceptable excipient, a buffering agent, a solvent, a bulking agent, a flavorant, a lubricant, a colorant, a binder, a surfactant, a binder, and a disintegrant.

13. The method of any of claims 1-11, wherein the miR-128 inhibitor is formulated as a pharmaceutical composition comprising a pharmaceutically acceptable excipient.

14. Tire method of claim 13, wherein the pharmaceutically acceptable excipient is selected from: poly(amidoamine), poly(propyleneimine), and poly(L-lysine).

15. Hie method of claim 13, wherein the pharmaceutically acceptable excipient comprises one or more lipids.

16. The method of any of claims 1-15, further comprising administering at least one additional therapeutic agent.

17. The method of claim 16, wherein the at least one additional therapeutic agent is selected from testosterone, growth hormone, insulin-like growth factor, mechanical growth factor, urocortin II, anabolic steroids, omega-3 fatty acids, creatine, dehydroepiandrosterone.

18. The method of any of claims 1-17, further comprising directing the individual to perform weight training exercises or resistance training exercises.

19. A combination therapy for the treatment of age-related sarcopenia, comprising: a miR-128 inhibitor; and one or more of: testosterone, growth hormone, insulin-like growth factor, mechanical growth factor, urocortin II, anabolic steroids, omega-3 fatty acids, creatine, dehydroepiandrosterone.

20. Hie combination therapy of claim 19. wherein the miR-128 inhibitor comprises an inhibitory nucleic acid.

21. The combination therapy of claim 20, wherein the inhibitory nucleic acid comprises one or more of the following sequences:5’-ACCGGTTCACTGTG-3’ (SEQ ID NOT);5 -GACCGGTTCACTGT-3’ (SEQ ID NOT); and5 -AGACCGGTTCACTGTG-3’ (SEQ ID NOT).

22. The combination therapy of claim 20 or 21, wherein the inhibitory nucleic acid comprises one or more locked nucleic acids (LNA).

23. Tire method of any of claims 20-22, wherein the inhibitory nucleic acid comprises one or more of a modified backbone and one or more 5-methyl deoxycytosine residues.

24. Tire combination therapy of any of claims 20-23, wherein the inhibitory nucleic acid has a length of from 14 nucleotides to 20 nucleotides.

25. The combination therapy of any of claims 20-24, wherein the inhibitory nucleic acid comprises the nucleotide designated “NRC-0090.”26. The combination therapy of any of claims 20-24, wherein the inhibitory' nucleic acid comprises the nucleotide sequence designated "‘NRC-0091.”27. Hie combination therapy of any of claims 20-24, wherein the inhibitory nucleic acid comprises the nucleotide sequence designated “NRC-0119.”28. The combination therapy of any of claims 19-27, wherein the miR-128 inhibitor is formulated for subcutaneous delivery.

29. The combination therapy of any of claims 19-28, wherein the miR-128 inhibitor is formulated as a pharmaceutical composition comprising one or more of the following: a pharmaceutically acceptable excipient, a buffering agent, a solvent, a bulking agent, a flavorant, a lubricant, a colorant, a binder, a surfactant, a binder, and a disintegrant.

30. The combination therapy of any of claims 19-29, wherein the miR-128 inhibitor is formulated as a pharmaceutical composition comprising a pharmaceutically acceptable excipient.

31. Tire combination therapy of claim 30, wherein the pharmaceutically acceptable excipient is selected from: poly(amidoamine), poly(propyleneimine), and poly(L-lysine).

32. Hie combination therapy of claim 30, wherein the pharmaceutically acceptable excipient comprises one or more lipids.

33. An oligonucleotide, comprising: a plurality of interlinked nucleotides, wherein a contiguous subset of the plurality of nucleotides is complementary to a portion of a miR-128 nucleic acid; wherein at least one nucleotide in the plurality of interlinked nucleotides is a bridged nucleic acid.

34. The oligonucleotide of claim 33, wherein the oligonucleotide is comprises 7 to 50 interlinked nucleotides.

35. The oligonucleotide of claim 33 or 34, wherein the oligonucleotide is comprises 7 to 30 interlinked nucleotides.

36. Tire oligonucleotide of any of claims 33-35, wherein the oligonucleotide is comprises 14 to 23 interlinked nucleotides.

37. The oligonucleotide of any of claims 33-36, wherein the contiguous subset comprises at least 6 nucleobases that are complementary to the miR-128 nucleic acid.

38. The oligonucleotide of any of claims 33-37, wherein the oligonucleotide is a unimer.

39. Tire oligonucleotide of claim 38, wherein each nucleotide is independently a bridged nucleic acid.

40. The oligonucleotide of any of claims 33-37, wherein the nucleobase sequence comprises one or more of the following sequences:5 -ACCGGTTCACTGTG-3’ (SEQ ID NOT);5’-GACCGGTTCACTGT-3’ (SEQ ID NOT); and5’-AGACCGGTTCACTGTG-3’ (SEQ ID NOT).

41. A recombinant adeno-associated viral (rAAV) particle comprising a capsid and a nucleic acid vector comprising a heterologous nucleic acid region comprising sequence encoding an interfering RNA comprising a region complementary to a miR-128 target nucleic acid.

42. The rAAV of claim 41, wherein the miR-128 target nucleic acid is miR-128-3p, pri-miR-128-1, pre-miR-128-1, pri-miR- 128-2, or pre-miR-128-2.

43. The rAAV of claim 41 , wherein the interfering RNA is a short hairpin RNA or a small interfering RNA.

44. Tire rAAV of claim 41. wherein the nucleic acid vector comprises a promoter, wherein the sequence encoding an interfering RNA is operably linked to the promoter.

45. The rAAV of claim 44, wherein the promoter is capable of inducing expression of the interfering RNA in a skeletal muscle cell.

46. The rAAV of claim 44, wherein the promoter is a hybrid chicken P-actin (CBA) promoter or an RNA polymerase III promoter.

47. Tire rAAV of claim 41. wherein the nucleic acid vector comprises inverted terminal repeats from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrhlO, AAV11, AAV12, AAV2R471A, AAV DJ, goat AAV, bovine AAV, or mouse AAV serotypes.

48. Tire rAAV of claim 41. wherein the capsid is selected from: an AAV1 capsid, and AAV2 capsid, an AAV3 capsid, an AAV4 capsid, an AAV5 capsid, anAAV6 capsid, an AAV7 capsid, anAAV8 capsid, an AAVrh8 capsid, an AAVrh8R capsid, an AAV9 capsid, an AAV 10 capsid, an AAVrhlO capsid, an AAV11 capsid, an AAV 12 capsid, a tyrosine capsid mutant, a heparin binding capsid mutant, an AAV2R471A capsid, an AAVAAV2 / 2-7m8 capsid, an AAV DJ capsid, an AAV2 N587A capsid, an AAV2 E548A capsid, an AAV2 N708A capsid, an AAV V708K capsid, a goat AAV capsid, an AAV1 / AAV2 chimeric capsid, a bovine AAV capsid, a mouse AAV capsid, and a rAAV2 / HBoV 1 capsid.

49. A pharmaceutical composition comprising: a pharmaceutically acceptable excipient; and the oligonucleotide of any of claims 33-40 or the rAAV of any of claims 41-48.

50. A therapeutically effective composition for use in a method of treating age-related macular degeneration in a subject in need thereof, the method comprising administering to the subject the therapeutically effective amount of the oligonucleotide of any of claims 1-8 or the rAAV of any of claims 9-16.

51. A miR-128 inhibitor for use in a method of treating aging -related sarcopenia in a subject in need thereof, the method comprising administering to tire subject a therapeutically effective amount of the miR-128 inhibitor.

52. The miR-128 inhibitor of claim 51, wherein the miR-128 inhibitor is selected from: an antisense oligonucleotide, a short hairpin RNA, a small interfering RNA, or a recombinant adeno-associated viral (rAAV) particle comprising a capsid and a nucleic acid vector comprising a heterologous nucleic acid region comprising sequence encoding an interfering RNA,wherein the interfering RNA is selected from an antisense oligonucleotide, a short hairpin RNA, or a small interfering RNA.

53. A therapeutically effective composition for use in a method of treating age-related sarcopenia in a subject in need thereof, the method comprising administering to the subject the therapeutically effective amount of the oligonucleotide of any of claims 1-8 or the rAAV of any of claims 9-16.

54. The therapeutically effective composition of claim 53, wherein the oligonucleotide is a singlestranded oligonucleotide.

55. The therapeutically effective composition of claim 53, wherein the oligonucleotide is an antisense oligonucleotide.

56. The therapeutically effective composition of claim 53, wherein the oligonucleotide comprises 7 to 30 interlinked nucleotides.

57. Tire therapeutically effective composition of claim 56, wherein the oligonucleotide comprises 14 to 23 interlinked nucleotides.

58. Hie therapeutically effective composition of any of claims 53-57. wherein the oligonucleotide comprises at least one modified sugar nucleoside.

59. The therapeutically effective composition of claim 58, wherein at least 50% of the nucleosides comprise a modified sugar nucleoside.

60. The therapeutically effective composition of claim 58 or 59, wherein 100% of the nucleosides comprise a modified sugar nucleoside.

61. The therapeutically effective composition of any of claims 58-60. wherein the modified sugar nucleoside is a 2 ’-modified sugar nucleoside.

62. The therapeutically effective composition of claim 61, wherein the 2'-modified sugar nucleoside comprises a 2 ’-modification independently selected from the group consisting of 2'-fluoro, 2'-methoxy, and 2 ’-methoxy ethoxy.

63. The therapeutically effective composition of any of claims 58-60, wherein the modified sugar nucleoside is a bridged nucleic acid.

64. The therapeutically effective composition of claim 63, wherein the bridged nucleic acid is a locked nucleic acid.

65. Tire therapeutically effective composition of claim 63, wherein the bridged nucleic acid is an ethylene bridged nucleic acid.

66. Tire therapeutically effective composition of any of claims 58-60, wherein the nucleobase sequence comprises one or more of the following sequences:5 -ACCGGTTCACTGTG-3’ (SEQ ID NO: 1);5 -GACCGGTTCACTGT-3’ (SEQ ID NO:2); and5’-AGACCGGTTCACTGTG-3’ (SEQ ID NO:3).

67. Tire therapeutically effective composition of any of claims 53-66. wherein the oligonucleotide is a gapmer comprising a 5 ’-wing, a 3 ’-wing, and a gap, wherein the 5 ’-wing and the 3 ’-wing each comprise 1 to 5 nucleotides, wherein each nucleotide in the 5’-wing and the 3’-wing are independently a bridged nucleic acid, and wherein each nucleotide in the gap is a deoxyribonucleotide.

68. Tire therapeutically effective composition of any of claims 53-67. wherein at least one intemucleoside linkage in the oligonucleotide is a phosphorothioate diester.

69. Tire therapeutically effective composition of claim 68, wherein at least 50% of intemucleoside linkages in the oligonucleotide are phosphorothioate diesters.

70. The therapeutically effective composition of claim 68 or 69, wherein 100% of intemucleoside linkages in the oligonucleotide are phosphorothioate diesters.

71. The therapeutically effective composition of any of claims 53-70, wherein the method further comprises administering the oligonucleotide as a guide strand in a short interfering RNA.

72. Hie therapeutically effective composition of any of claims 53-71. wherein the miR-128 target nucleic acid is selected from: pri-miR- 128-1, pre-miR- 128-1. pri-miR- 128-2, pre-miR-128-2. or miR- 128-3p.

73. The therapeutically effective composition of any of claims 53-72. wherein the route of administration is an intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection.

74. A method of treating age-related sarcopenia, comprising administering to an individual diagnosed with age-related sarcopenia a combination of the therapeutically effective composition of claim 53 and an incretin mimetic.

75. The method of claim 74. wherein said incretin mimetic is a Glucagon-Like Peptide -1 (GLP-1) receptor agonist.

76. The method of claim 75, wherein the GLP-1 receptor agonist is selected from dulaglutide, semaglutide, tirzepatide, albiglutide, exenatide, liraglutide, lixisenatide. taspoglutide. CJC-1131, and BIM-51077.

77. The method of claim 76, wherein the GLP-1 receptor agonist is semaglutide or tirzepatide.

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