Compositions and methods for treating sarcopenia

Abstract

This disclosure provides compositions and methods for treating sarcopenia, including age-related sarcopenia. Various embodiments include inhibitory nucleic acids, compositions comprising inhibitory nucleic acids, and methods for using inhibitory nucleic acids to treat such disorders. TIFF2026530600000002.tif128127

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 537,152, filed September 7, 2023, which is incorporated herein by reference in its entirety.

[0002] Inclusion by referencing a sequence listing provided as an XML file. The sequence listing is provided herein as a sequence listing XML file, "BERK-489WO_SEQLIST," created on September 6, 2024, with a size of 11,111 bytes. The entire contents of the sequence listing XML are incorporated herein by reference.

[0003] Introduction Sarcopenia is defined as the progressive loss of muscle mass and function with age. This loss of muscle mass and function is associated with an increased risk of adverse outcomes. Frailty and reduced mobility lead to increased falls, obesity, impaired recovery from illness, and ultimately, increased mortality. Currently, there are no approved medications for the treatment of sarcopenia. [Overview of the project]

[0004] overview This disclosure provides compositions and methods for treating sarcopenia, including age-related sarcopenia. Various embodiments include inhibitory nucleic acids, compositions comprising inhibitory nucleic acids, and methods for using inhibitory nucleic acids to treat such disorders. [Brief explanation of the drawing]

[0005] [Figure 1A] The nucleotide sequences of exemplary antisense oligonucleotides (ASOs) (SEQ ID NOs. 1-3, respectively) are provided. [Figure 1B] This provides the base pairing site (SEQ ID NO: 12) of the ASO within the target microRNA (miR-128-3p). [Figure 2A]Intermediate data showing the effect of LNA ASO targeting miR-128-3p on skeletal muscle function in aged mice is shown. [Figure 2B] See the description of Figure 2A. [Figure 2C] See the description of Figure 2A. [Figure 2D] See the description of Figure 2A. [Figure 2E] See the description of Figure 2A. [Figure 2F] See the description of Figure 2A. [Figure 2G] See the description of Figure 2A. [Figure 2H] See the description of Figure 2A. [Figure 2I] See the description of Figure 2A. [Figure 3-1] Complete experimental data showing the effect of LNA ASO targeting miR-128-3p on skeletal muscle function in aged mice is shown. [Figure 3-2] See the description of Figure 3-1. [Figure 3-3] See the description of Figure 3-1. [Figure 3-4] See the description of Figure 3-1. [Figure 3-5] See the description of Figure 3-1. [Figure 3-6] See the description of Figure 3-1. [Figure 4-1] This is experimental data showing the effect of LNA ASO targeting miR-128-3p on skeletal muscle mass in aged mice. [Figure 4-2] See the description of Figure 4-1. DETAILED DESCRIPTION OF THE INVENTION

[0006] Definitions As used herein, "antisense oligonucleotide" refers to a nucleic acid sequence complementary to a DNA or RNA sequence, such as that of a microRNA.

[0007] "RNA" refers to a molecule containing at least one ribonucleotide residue. A "ribonucleotide" is a nucleotide having a hydroxyl group at the 2' position of the beta-D-ribofuranose moiety. As used herein, the term RNA includes double-stranded RNA, single-stranded RNA, isolated RNA, e.g., partially purified RNA, essentially pure RNA, synthetic RNA, recombinant RNA, and modified RNA which differs from naturally occurring RNA by the addition, deletion, substitution, and / or alteration of one or more nucleotides. The nucleotides of an RNA molecule may also include non-standard nucleotides, such as non-natural nucleotides, chemically synthesized nucleotides, or deoxyribonucleotides.

[0008] MicroRNAs (miRNAs) are single-stranded RNA molecules approximately 21–23 nt in length. Generally, miRNAs regulate gene expression. While miRNAs are encoded by genes transcribed from DNA, miRNAs are not translated into proteins. 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 primary function is to downregulate gene expression.

[0009] As used herein, “interfering RNA” refers to any double-stranded or single-stranded RNA sequence that can inhibit or downregulate gene expression directly or indirectly (i.e., during conversion) by mediating RNA interference. Examples of interfering RNA include, but are not limited to, small interfering RNAs ("siRNAs") and small hairpin RNAs ("shRNAs"). “RNA interference” refers to the selective degradation of sequence-compatible messenger RNA transcripts.

[0010] As used herein, "shRNA" (low-molecular-weight hairpin RNA) refers to an RNA molecule comprising an antisense region, a loop region, and a sense region, the sense region having complementary nucleotides that base-pair with the antisense region to form a double-stranded stem. After post-transcriptional processing, low-molecular-weight hairpin RNA is converted to low-molecular-weight interfering RNA by a cleavage event mediated by the enzyme Dicer, a member of the RNase III family.

[0011] As used herein, “small interfering RNA” or “siRNA” refers to any small RNA molecule that can inhibit or downregulate gene expression by mediating RNA interference in a sequence-specific manner. Small RNAs may be, for example, about 18 to 21 nucleotides in length.

[0012] As used herein, "antagomil" refers to a small synthetic RNA complementary to a specific microRNA target, which involves either mispairing at the cleavage site or one or more base modifications to inhibit cleavage.

[0013] As used herein, the term "post-transcriptional processing" refers to mRNA processing that occurs after transcription and is mediated, for example, by the enzymes Dicer and / or Drosha.

[0014] "Effective dose" means the amount of an agent (e.g., the inhibitory nucleic acid of this disclosure) or a composition containing the agent (e.g., the composition containing the inhibitory nucleic acid of this disclosure) necessary to improve the symptoms of a disease in an untreated patient. The effective dose of an agent or composition used to carry out therapeutic treatment for a disease or disorder will vary depending on the mode of administration, the age, weight, and overall health of the subject. Ultimately, the attending physician, doctor, or veterinarian will determine the appropriate dose and administration regimen. Such a dose is called the "effective dose."

[0015] The term "sarcopenia" encompasses any disorder characterized by a progressive and systemic loss of skeletal muscle mass and strength, associated with a risk of adverse outcomes such as physical disability, reduced 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. Primary sarcopenia refers to sarcopenia without causes other than aging, and is also called age-related sarcopenia. Activity-related sarcopenia refers to sarcopenia caused by bed rest, a sedentary lifestyle, poor health, or weightlessness. Disease-related sarcopenia refers to sarcopenia caused by advanced organ failure (heart, lungs, liver, kidneys, brain), inflammatory diseases, malignancies, or endocrine disorders. Nutrition-related sarcopenia refers to sarcopenia caused by malabsorption, gastrointestinal disorders, the use of drugs that cause loss of appetite, or insufficient protein intake in the diet.

[0016] As used herein, “treatment,” “to treat,” and similar terms mean obtaining a desired pharmacological and / or physiological effect. This effect may be prophylactic in that it completely or partially prevents a disease or its symptoms, and / or therapeutic in that it partially or completely cures a disease and / or adverse effects resulting from the disease. As used herein, “treatment” encompasses any treatment of a disease in a mammal, e.g., a human, and includes (a) preventing the development of a disease in a subject that may be predisposed to the disease but has not yet been diagnosed with it, (b) inhibiting the disease, i.e., stopping its onset, and (c) reducing the disease, i.e., causing its regression.

[0017] As used herein, “AAV particle” is a virus comprising a viral genome having at least one payload region and at least one inverted end repeat (ITR) region.

[0018] As used herein, “viral genome” or “vector genome” refers to nucleic acids (sequenced) encapsulated in an AAV particle. A viral genome includes at least one payload region encoding nucleic acids (e.g., interfering RNA).

[0019] As used herein, “payload” or “payload region” is any nucleic acid molecule encoding one or more nucleic acids as described herein. The payload region comprises at least a nucleic acid sequence encoding an antibody, an antibody-based composition, or a fragment thereof, but may optionally include one or more functional or regulatory elements to promote transcription and / or nucleic acid expression.

[0020] As used interchangeably herein, the terms “individual,” “subject,” “host,” and “patient” refer to individual organisms, including but not limited to mammals such as mice, monkeys, humans, non-human primates, ungulates, cats, dogs, cattle, sheep, domesticated mammals, sporting mammals, and pet mammals. In some cases, “individual” refers to a human.

[0021] Before further describing the present invention, it should be understood that the present invention is not limited to the specific embodiments described and, therefore, can vary. The scope of the present invention is limited only by the appended "Claims," ​​and it should also be understood that the terms used herein are intended solely to describe specific embodiments and are not intended to limit them.

[0022] Where a range of values ​​is provided, unless the context explicitly indicates otherwise, each intermediary value up to one-tenth of the lower limit between the upper and lower limits of that range, and any other stated or intermediary values ​​within that stated range, are understood to be included in the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller range and are also included in the invention, subject to any specifically excluded limitations within the stated range. Where a stated range includes one or both limits, ranges excluding either or both of those included limits are also included in the invention.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the present invention pertains. Any methods and substances similar to or equivalent to those described herein may also be used in the practice or testing of the present invention, but preferred methods and substances are described herein. All publications referenced herein are incorporated herein by reference to disclose and describe methods and / or materials cited in connection therewith.

[0024] It should be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include multiple referents unless the context otherwise explicitly indicates otherwise. Thus, for example, a reference to “antisense oligonucleotides” includes multiple such oligonucleotides, and a reference to “nucleic acid modifications” includes references to one or more nucleic acid modifications, as well as references to their equivalents known to those skilled in the art. It should be further noted that the claims may be constructed to exclude any optional element. Therefore, this statement is intended to serve as an antecedent for the use of exclusive terms such as “alone” and “only,” or for the use of “negative” limitation, in relation to the enumeration of elements of the claims.

[0025] In the context describing this disclosure (particularly in the context of the appended claims), the terms “a,” “an,” and “the,” and similar reference subjects, should be interpreted as encompassing both singular and plural forms unless otherwise indicated herein or unless the context clearly contradicts this interpretation. The terms “comprising,” “having,” “including,” and “containing” should be interpreted as non-restrictive terms (i.e., “including, but not limited to”) unless otherwise indicated herein. Enumerations of value ranges herein are merely intended to serve as a concise way of referring individually to each distinct value that falls within that range, unless otherwise indicated herein, and each distinct value is incorporated herein as it would be if individually listed herein. For example, if the range 10–15 is disclosed, then 11, 12, 13, and 14 are also disclosed. All methods described herein may be carried out in any preferred order, unless otherwise indicated herein or unless the context clearly contradicts it. Any examples or exemplary language provided herein (e.g., "etc.") are intended solely to better illustrate the embodiments of the disclosure and, unless otherwise asserted, do not limit the scope of the disclosure. No language herein should be construed as indicating that any unclaimed element is essential for carrying out the embodiments of the disclosure.

[0026] Where used herein, the term “about” in relation to quantity indicates that the quantity may vary by 10% from the stated quantity. For example, “about 100” means a quantity between 90 and 110. Where “about” is used in the context of a range, “about” used in relation to a lower quantity within a range means that the lower quantity includes a quantity 10% lower than the lower quantity within the range, and “about” used in relation to a higher quantity within a range means that the higher quantity includes a quantity 10% higher than the higher quantity within the range. For example, “about 100 to about 1000” means that the range spans from 90 to 1100.

[0027] As used herein, the term "and / or" as used in phrases such as "A and / or B" is intended to include both A and B, A or B, A (alone), and B (alone). Similarly, as used herein, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to include 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).

[0028] The aspects and embodiments of the Disclosure described herein are understood to include the terms “including,” “consisting of,” and “essentially consisting of.”

[0029] For clarity, it is understood that certain features of the Disclosure described in the context of a separate embodiment may also be provided in combination with a single embodiment. Conversely, for brevity, various features of the Disclosure described in the context of a single embodiment may also be provided separately or in any preferred subcombination. All combinations of embodiments relating to the Disclosure are specifically encompassed by the Disclosure, and every conceivable combination is disclosed herein just as it is disclosed individually and expressly. Furthermore, all subcombinations of various embodiments and their elements are also specifically encompassed by the Disclosure, and are disclosed herein just as such subcombinations are disclosed individually and expressly herein.

[0030] The publications discussed herein are provided only with respect to their disclosures prior to the filing date of this application. Nothing herein should be construed as accepting that the present invention is not granted prior rights to such publications by prior inventions. Furthermore, the dates of the publications provided may differ from the actual publication dates which may need to be independently verified.

[0031] Detailed explanation This disclosure provides inhibitory nucleic acids that reduce the level of miR-128, including miR-128-3p, in cells, and compositions comprising these inhibitory nucleic acids. This disclosure also provides methods for treating various disorders using the inhibitory nucleic acids of this disclosure.

[0032] inhibitory nucleic acid This disclosure provides inhibitory nucleic acids that reduce the level of miR-128-3p in cells (e.g., in the cells of an organism). In some cases, the inhibitory nucleic acid is an antisense oligonucleotide (ASO). This disclosure provides ASOs that reduce the level of miR-128-3p in cells (e.g., in the cells of an organism).

[0033] In some cases, miR-128 contains the nucleotide sequence: tgagctgttg gattcggggc cgtagcactg tctgagaggt ttacatttct cacagtgaac cggtctcttt ttcagctgct tc (SEQ ID NO: 4). In some cases, miR-128 contains the nucleotide sequence: ugagcuguug gauucggggc cguagcacug ucugagaggu uuacauuucu cacagugaac cggucucuuu uucagcugcu uc (SEQ ID NO: 5). In some cases, miR-128 contains the nucleotide sequence: tcacagtgaa ccggtctctt t (SEQ ID NO: 6). In some cases, miR-128 contains the nucleotide sequence: ucacagugaa ccggucucuu u (SEQ ID NO: 7). In some cases, miR-128 contains the nucleotide sequence: tgtgcagtgg gaaggggggc cgatacactg tacgagagtg agtagcaggt ctcacagtga accggtctct ttccctactg tgtc (SEQ ID NO: 8). In some cases, miR-128 contains the nucleotide sequence: ugugcagugg gaaggggggc cgauacacug uacgagagug aguagcaggu cucacaguga accggugugu uuggguacug ugcu (SEQ ID NO: 9).

[0034] In some cases, the inhibitory nucleic acids of this disclosure (e.g., ASO) reduce the level of miR-128-3p in cells more potently than inhibitory nucleic acids containing the nucleotide sequence: 5'-TTCACTGTG-3' (SEQ ID NO: 10) (e.g., ASO). In some cases, the inhibitory nucleic acids of this disclosure (e.g., ASO) are at least 2 times, at least 2.5 times, at least 5 times, at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, or at least 40 times more potent than inhibitory nucleic acids containing the nucleotide sequence: 5'-TTCACTGTG-3' (SEQ ID NO: 10) (e.g., ASO). For example, in some cases, the inhibitory nucleic acid of the present disclosure (e.g., ASO) reduces the level of miR-128-3p to a degree 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 degree to which the inhibitory nucleic acid containing the nucleotide sequence 5'-TTCACTGTG-3' (SEQ ID NO: 10) (e.g., ASO) reduces the level of miR-128-3p when administered in the same amount as the inhibitory nucleic acid of the present disclosure (e.g., ASO). In some cases, the inhibitory nucleic acid of the present disclosure (e.g., ASO) achieves a reduction of at least 10%, at least 15%, at least 20%, at least 25%, at least 50%, at least 75%, at least 90%, or at least 90% of the level of miR-128-3p in the cells of an individual when the inhibitory nucleic acid of the present disclosure (e.g., ASO) is administered to the individual in an amount at least 10%, at least 15%, at least 20%, at least 25%, at least 50%, at least 75%, at least 75%, or at least 90% less than the amount of the inhibitory nucleic acid (e.g., ASO) containing the nucleotide sequence: 5'-TTCACTGTG-3' (SEQ ID NO: 10) required to achieve the same reduction in the level of miR-128-3p.

[0035] In some cases, the inhibitory nucleic acids of this disclosure (e.g., ASO) reduce the level of miR-128-3p in cells more potently than inhibitory nucleic acids containing the nucleotide sequence: 5'-GGTTCACTGTG-3' (SEQ ID NO: 11) (e.g., ASO). In some cases, the inhibitory nucleic acids of this disclosure (e.g., ASO) are at least 2 times, at least 2.5 times, at least 5 times, at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, or at least 40 times more potent than inhibitory nucleic acids containing the nucleotide sequence: 5'-GGTTCACTGTG-3' (SEQ ID NO: 11) (e.g., ASO). For example, in some cases, the inhibitory nucleic acid of the present disclosure (e.g., ASO) reduces the level of miR-128-3p in cells by 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 reduction of miR-128-3p levels by the same amount of inhibitory nucleic acid of the present disclosure (e.g., ASO) that contains the nucleotide sequence 5'-GGTTCACTGTG-3' (SEQ ID NO: 11). In some cases, the inhibitory nucleic acid of the present disclosure (e.g., ASO) achieves a reduction of at least 10%, at least 15%, at least 20%, at least 25%, at least 50%, at least 75%, at least 90%, or at least 90% when administered to an individual, compared to the amount of the inhibitory nucleic acid (e.g., ASO) containing the nucleotide sequence: 5'-GGTTCACTGTG-3' (SEQ ID NO: 11) required to achieve the same reduction in miR-128-3p levels.

[0036] Useful inhibitory nucleic acids in this method and composition include antisense oligonucleotides, ribozymes, external guide sequence (EGS) oligonucleotides, siRNA compounds, single-stranded or double-stranded RNA interference (RNAi) compounds, such as siRNA compounds, modified base / locked nucleic acids (LNA), antagomyl, peptide nucleic acids (PNA), and other oligomeric compounds or oligonucleotide mimics that hybridize to at least a portion of the target nucleic acid (i.e., miR-128) and modulate its function. In some embodiments, inhibitory nucleic acids include antisense RNA, antisense DNA, chimeric antisense oligonucleotides, antisense oligonucleotides containing modified bindings, interfering RNA (RNAi), short interfering RNA (siRNA), microinterfering RNA (miRNA), small primary RNA (stRNA), short hairpin RNA (shRNA), small RNA-induced gene activation (RNAa), small activated RNA (saRNA), or combinations thereof. See, for example, International Publication No. 2010040112. In some cases, the inhibitory nucleic acids of this disclosure are ASOs.

[0037] In some cases, the inhibitory nucleic acid is 10–50, 13–50, or 13–30 nucleotides long. Those skilled in the art will understand that this embodies an oligonucleotide having an antisense portion that is 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 long, or any range of nucleotide lengths therein. In some cases, the inhibitory nucleic acid of this disclosure is 15 nucleotides long. In some cases, the inhibitory nucleic acid of this disclosure is 12–30, or 13–30 nucleotides long. Those skilled in the art will understand 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.

[0038] In some cases, the inhibitory nucleic acid is a chimeric oligonucleotide containing two or more chemically distinct regions each composed of at least one nucleotide. These oligonucleotides typically comprise at least one region of modified nucleotides that confers one or more beneficial properties (e.g., increased nuclease resistance, increased cellular uptake, increased binding affinity for a target, etc.), and a region that is a substrate for an enzyme capable of cleaving RNA:DNA hybrids or RNA:RNA hybrids. The chimeric inhibitory nucleic acids of the present disclosure can be formed as a composite structure of two or more oligonucleotides, modified oligonucleotides, oligonucleosides, and / or oligonucleotide mimetics as described above. Such compounds are also referred to in the art as hybrids or gapmers.

[0039] 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 of 14 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) locked nucleic acid (LNA), ii) a modified backbone, and iii) 5-methyldeoxycytosine. In some cases, a suitable inhibitory nucleic acid comprises the nucleotide sequence designated "NRC0090" shown in FIG. 1A and all modifications. The ASO designated NRC0090 in FIG. 1A is +A * +C * / iMe-dC / * G * G * +T * +T * +C * A * C * +T * G * +T * +G, wherein "+" precedes the LNA,* The / iMe-dC / prefix is ​​placed before the phosphorothioate base, indicating internal 5-methyldeoxycytosine.

[0040] As another non-limiting example, in some cases, the preferred inhibitory nucleic acid contains the following nucleotide sequence: 5'-GACCGGTTCACTGT-3' (SEQ ID NO: 2) and has a length of 14 to 20 nucleotides. In some cases, the inhibitory nucleic acid contains 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 contains one or more of i) locked nucleic acid (LNA), ii) a modified skeleton, and iii) 5-methyldeoxycytosine. In some cases, the preferred inhibitory nucleic acid contains the nucleotide sequence referred to as "NRC0091" shown in Figure 1A and all modifications. The ASO referred to as NRC0091 in Figure 1A is +G * +A * C * / iMe-dC / * +G * G * T * T * +C * +A * C * +T * +G * +T, and in the formula, "+" is placed before LNA. * The / iMe-dC / prefix is ​​placed before the phosphorothioate base, indicating internal 5-methyldeoxycytosine.

[0041] As another non-limiting example, in some cases, the preferred inhibitory nucleic acid contains the following nucleotide sequence: 5'-AGACCGGTTCACTGTG-3' (SEQ ID NO: 3) and has a length of 14 to 20 nucleotides. In some cases, the inhibitory nucleic acid contains the following nucleotide sequence: AGACCGGTTCACTGTG-3' (SEQ ID NO: 3) and has a length of 14 nucleotides. In some cases, the inhibitory nucleic acid contains one or more of i) locked nucleic acid (LNA), ii) a modified skeleton, and iii) 5-methyldeoxycytosine. In some cases, the preferred inhibitory nucleic acid contains the nucleotide sequence referred to as "NRC0119" shown in Figure 1A and all modifications. The ASO referred to as NRC0119 in Figure 1A is +A * +G * A * +C * / iMe-dC / * +G * G * +T * T * +C * A * C * +T * G * +T * It is +G, and in the formula, "+" is placed before LNA. * The / iMe-dC / prefix is ​​placed before the phosphorothioate base, indicating internal 5-methyldeoxycytosine.

[0042] 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, or 2'-methoxyethoxy modified nucleotide. In other cases, the RNA modification comprises 2'-fluoro, 2'-amino, and 2'O-methyl modifications to the ribose of a pyrimidine, or a debased residue or inverted base at the 3' end of the RNA. Such modifications have been conventionally incorporated into oligonucleotides, and these oligonucleotides have been shown to have a higher Tm (i.e., higher target binding affinity) for a given target than 2'-deoxyoligonucleotides.

[0043] Numerous nucleotide and nucleoside modifications have been shown to make the oligonucleotides they incorporate more resistant to nuclease digestion than native oligodeoxynucleotides. These modified oligonucleotides survive intact for longer periods than unmodified oligonucleotides. Specific examples of modified oligonucleotides include those containing modified skeletons such as phosphorothioates, phosphotriesters, methylphosphonates, short-chain alkyl or cycloalkyl intersaccharide bonds, or short-chain heteroatoms or heterocyclic intersaccharide bonds. In some cases, inhibitory nucleic acids include oligonucleotides with a phosphorothioate skeleton, as well as heteroatom skeletons, particularly CH2-NH-O-CH3, CH3-N(CH3)-O-CH2 (known as the methylene (methylimino) or MMI skeleton), CH2-ON(CH3)-CH2, CH2-N(CH3)-N(CH3)-CH2, and ON(CH3)-CH2-CH2 skeletons (where the native phosphodiester skeleton is represented as OPO-CH); amide skeletons (see De Mesmaeker et al. Ace.Chem.Res. 1995, 28:366-374); morpholino skeleton structures (see Summerton and Weller, USPat. No. 5, 034, 506); and peptide nucleic acid (PNA) skeletons (where the phosphodiester skeleton of an oligonucleotide is replaced by a polyamide skeleton, and the nucleotide is directly or indirectly bonded to the aza nitrogen atom of the polyamide skeleton, Nielsen et al. It is an oligonucleotide (see al., Science 1991, 254, 1497).Phosphorus-containing bonds include, but are not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-aminophospholamidates and aminoalkylphospholamidates, thiophospholamidates, thioalkyl phosphonates, thioalkyl phosphotriesters, and borane phosphates that usually have a 3'-5' bond, their 2'-5' bond analogues, and those having inverted polarity in which adjacent nucleoside unit pairs are bonded from 3'-5' to 5'-3' or from 2'-5' to 5'-2'.

[0044] Modified oligonucleotide skeletons that do not contain phosphorus atoms have skeletons formed by short-chain alkyl or cycloalkyl nucleoside bonds, mixed heteroatoms and alkyl or cycloalkyl nucleoside bonds, or one or more short-chain heteroatoms or heterocyclic nucleoside bonds. These include skeletons having morpholino bonds (partially formed from the sugar portion of a nucleoside), siloxane skeletons, sulfides, sulfoxides and sulfone skeletons, formacetyl and thioformacetyl skeletons, methyleneformacetyl and thioformacetyl skeletons, alkene-containing skeletons, sulfamate skeletons, methyleneimino and methylenehydrazino skeletons, sulfonate and sulfonamide skeletons, amide skeletons, and other skeletons having mixed N, O, S, and CH2 component portions.

[0045] One or more substituted sugar moieties may also be present, for example, one of the following at the 2' position: OH, SH, SCH3, F, OCN, OCH3OCH3, OCH3, O(CH2)nCH3, O(CH2)nNH 2、Or O(CH2)nCH3 (wherein n is 1 to about 10); C1-C10 lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkalyl, or aralkyl; Cl; Br; CN; CF3; OCF3; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; SOCH3; SO2CH3; ONO2; NO2; N3; NH2; heterocycloalkyl; heterocycloalkalyl; aminoalkylamino; polyalkylamino; substituted silyl; RNA cleavage group; reporter group; intercalator; group for improving the pharmacokinetic properties of oligonucleotides; or group for improving the pharmacodynamic properties of oligonucleotides and other substituents having similar properties. A preferred modification is 2'-methoxyethoxy [also known as 2'-O-CH2CH2OCH3, 2'-O-(2-methoxyethyl)] (Martin et al, Helv. Chim. Acta, 1995, 78, 486). Other modifications include 2'-methoxy (2'-O-CH3), 2'-propoxy (2'-OCH2CH2CH3), and 2'-fluoro (2'-F). Similar modifications can occur at other positions on oligonucleotides, particularly at the 3' position of the sugar on the 3'-terminal nucleotide and at the 5' position of the 5'-terminal nucleotide. Oligonucleotides may also have sugar mimetic groups such as cyclobutyl instead of the pentofuranosyl group.

[0046] Inhibitory nucleic acids may also, additionally or alternatively, include modifications or substitutions of nucleic acid bases (often simply referred to as “bases” in the art). As used herein, “unmodified” or “natural” nucleic acid bases include adenine (A), guanine (G), thymine (T), cytosine (C), and uracil (U). Modified nucleic acid bases include nucleic acid bases that are rarely or transiently found in natural nucleic acids, such as hypoxanthine, 6-methyladenine, 5-Me pyrimidine, especially 5-methylcytosine (also called 5-methyl-2'deoxycytosine, and often referred to as 5-Me-C in the art), 5-hydroxymethylcytosine (HMC), glycosyl HMC, and gentobiosyl HMC, as well as synthetic nucleic acid bases, such as 2-aminoadenine, 2-(methylamino)adenine, 2-(imidazolylalkyl)adenine, 2-(aminoalkylamino)adenine or other heterosubstituted alkyladenines, 2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6(6-aminohexyl)adenine, and 2,6-diaminopurine. Examples of "universal" bases known in the art include inosine. 5-Me-C substitution improves the stability of nucleic acid double helix by 0.6-12. <0> It has been shown that it increases C. In some cases, the inhibitory nucleic acids of this disclosure contain one or more 5-Me-C.

[0047] Not all positions in a given oligonucleotide need to be uniformly modified; in fact, two or more of the aforementioned modifications can be incorporated into a single oligonucleotide, or even into a single nucleoside within an oligonucleotide.

[0048] In some embodiments, both the sugar and nucleoside bonds (i.e., the backbone) of a nucleotide unit are replaced with novel groups. The base unit is maintained for hybridization with a suitable nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimetic shown to have excellent hybridization properties, is called a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of the oligonucleotide is replaced with an amide-containing backbone, such as an aminoethylglycine backbone. The nucleic acid bases are retained and bonded directly or indirectly to the aza nitrogen atom of the amide portion of the backbone. Representative U.S. patents teaching the preparation of PNA compounds include, but are not limited to, U.S. Patents 5,539,082, 5,714,331, and 5,719,262 (each of which is incorporated herein by reference). Further teachings of PNA compounds can be found in Nielsen et al, Science, 1991, 254, 1497-1500.

[0049] Inhibitory nucleic acids may also include modifications or substitutions of one or more nucleic acid bases (often simply referred to in the art as “bases”). As used herein, “unmodified” or “natural” nucleic acid bases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleic acid bases include other synthetic and natural nucleic acid bases, such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine, and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and Examples include thymine, 5-uracil (pseudolacil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl, and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine.

[0050] Furthermore, nucleic acid bases include those disclosed in U.S. Patent No. 3,687,808, those disclosed in "The Concise Encyclopedia of Polymer Science and Engineering," pp. 858-859, Kroschwitz, J.I., ed. John Wiley & Sons, 1990, those disclosed in English et al., Angewandle Chemie, International Edition, 1991, pp. 30, 613, and those disclosed in Sanghvi, Y.S., Chapter 15, "Antisense Research and Applications," pp. 289-302, Crooke, ST. and Lebleu, B.ea., CRC Press, 1993. Some of these nucleic acid bases are particularly useful for increasing the binding affinity of inhibitory nucleic acids. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, containing 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. The 5-methylcytosine substitution improves nucleic acid double-strand stability from 0.6 to 1.2. <0> It has been shown to increase C (Sanghvi, YS, Crooke, STand Lebleu, B., eds, `Antisense Research and Applications`, CRC Press, Boca Raton, 1993, pp.276-278), and is suitable for inclusion in inhibitory nucleic acids, for example, alone or together with 2'-O-methoxyethyl sugar modification.

[0051] In some cases, inhibitory nucleic acids are chemically linked to one or more moieties or conjugates that enhance the activity, cell distribution, or cell uptake of oligonucleotides. Such moieties include, but are not limited to, lipid moieties such as cholesterol moieties, cholic acid, thioethers such as hexyl-S-tritylthiol, thiocholesterol, aliphatic chains such as dodecanediol or undecyl residues, phospholipids such as di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, polyamine or polyethylene glycol chains, adamantane acetate, palmityl moieties, octadecylamine moieties, or hexylamino-carbonyl-t-oxycholesterol moieties.

[0052] These parts or conjugates may include conjugate groups covalently bonded to functional groups such as primary or secondary hydroxyl groups. Suitable conjugates for use include intercalators, importer molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical conjugates include, but are not limited to, cholesterol, lipids, phospholipids, biotin, phenazine, folic acid, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin, and dyes. Groups that enhance pharmacodynamic properties include those that improve uptake, enhance resistance to degradation, and / or enhance sequence-specific hybridization with target nucleic acids. Groups that enhance pharmacokinetic properties include those that improve the uptake, distribution, metabolism, or efflux of inhibitory nucleic acids. Representative conjugate groups are disclosed in International Patent Application PCT / US92 / 09196, filed October 23, 1992, and U.S. Patent No. 6,287,860, which are incorporated herein by reference. Conjugate moieties include, but are not limited to, lipid moieties such as cholesterol moieties, cholic acid, thioethers such as hexyl-5-tritylthiol, thiocholesterol, aliphatic chains such as dodecanediol or undecyl residues, phospholipids such as di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, polyamine or polyethylene glycol chains, or adamantane acetate, palmityl moieties, or octadecylamine or hexylamino-carbonyl-oxycholesterol moieties.

[0053] In this method, the inhibitory nucleic acid is sufficiently complementary to all or part of miR-128 to produce the desired effect; that is, it hybridizes sufficiently specificly and well. "Complementary" refers to the ability of two sequences, including naturally occurring or non-naturally occurring bases, or analogs thereof, to pair via hydrogen bonding. For example, if a base at one position of the inhibitory nucleic acid can hydrogen bond with a base at a corresponding position in the miR-128 sequence, these bases are considered complementary at that position. 100% complementarity is not required.

[0054] In the context of this disclosure, hybridization means hydrogen bonding between complementary nucleosides or nucleotide bases, which may be Watson-Crick, Hoogsteen, or reverse Hoogsteen hydrogen bonds. For example, adenine and thymine are complementary nucleic acid bases that pair through the formation of hydrogen bonds. As used herein, “complementary” refers to the ability of two nucleotides to pair precisely. Inhibitory nucleic acids and miR-128 are complementary if the corresponding positions in each molecule are occupied by a sufficient number of nucleotides that can hydrogen bond with each other. Thus, “specifically hybridizable” and “complementary” are terms used to indicate a degree of complementarity or precise pairing sufficient to result in stable and specific binding between the inhibitory nucleic acid and the miR-128 target sequence. For example, if a base at one position of an inhibitory nucleic acid can hydrogen bond with a base at a corresponding position of the miR-128 molecule, then these bases are considered complementary at that position.

[0055] While 100% complementarity is desirable in some embodiments, it is understood in the art that complementary nucleic acid sequences do not need to be 100% complementary to the target nucleic acid sequence because they are specifically hybridizable. For the purposes of the present method, a complementary nucleic acid sequence is specifically hybridizable if its binding to the target miR-128 molecule interferes with the normal function of the target miR-128 and causes loss of activity, and has sufficient complementarity to avoid nonspecific binding of the sequence to non-target miR-128 sequences under conditions where specific binding is desired, such as physiological conditions in the case of in vivo assays or therapeutic treatments, and stringent conditions under which the assay is performed in the case of in vitro assays. For example, stringent salt concentrations are typically less than about 750 mM NaCl and less than 75 mM trisodium citrate, less than about 500 mM NaCl and less than 50 mM trisodium citrate, or less than about 250 mM NaCl and less than 25 mM trisodium citrate. Low-stringency hybridization can be obtained in the absence of organic solvents, such as 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 typically include temperatures of at least 30°C, at least about 37°C, or at least about 42°C. Various additional parameters, such as hybridization time, surfactant concentration, such as sodium dodecyl sulfate (SDS), and inclusion or exclusion of carrier DNA, are well known to those skilled in the art. By combining these various conditions as needed, various levels of stringency can be achieved. As a non-limiting example, in some cases hybridization occurs at 30°C with 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. As another non-limiting example, in some cases hybridization occurs at 37°C with 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg / ml denatured salmon sperm DNA (ssDNA).As another non-limiting example, in some cases, hybridization occurs at 42°C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg / ml ssDNA. Useful modifications to these conditions will be readily apparent to those skilled in the art.

[0056] In general, inhibitory nucleic acids useful in the methods described herein have at least 80% sequence complementarity to the target region within the target nucleic acid, for example, 90%, 95%, or 100% sequence complementarity to the target region within miR-128 (e.g., the target region containing the seed sequence). For example, an antisense compound in which 18 of the 20 nucleic acid bases of an antisense oligonucleotide are complementary to the target region and therefore specifically hybridize exhibits 90% complementarity. The percentage of complementarity of an inhibitory nucleic acid to the region of the target nucleic acid can conventionally be determined using a basic local alignment search tool (BLAST program) (Altschul et al., J.Mol.Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656). Inhibitory nucleic acids (e.g., ASOs) that hybridize to the miR-128 target sequence can be identified through conventional experiments. Generally, inhibitory nucleic acids retain specificity for their targets; that is, they do not directly bind to transcripts other than the intended target, or directly and significantly affect their expression levels.

[0057] Antisense As described above, in some cases the inhibitory nucleic acids of this disclosure are ASOs. ASOs are typically designed to block the expression of a DNA or RNA target by binding to the target and stopping its expression at the level of transcription, translation, or splicing. The ASOs of this disclosure are complementary nucleic acid sequences designed to hybridize to the miR-128 target sequence under stringent conditions. Therefore, an oligonucleotide is selected that is sufficiently complementary to the target, i.e., hybridizes sufficiently specificly and sufficiently well, in order to produce the desired effect.

[0058] Modified bases / Locked nucleic acids (LNAs) In some cases, the inhibitory nucleic acids of this disclosure include one or more modified bonds or bases. Examples of modified bases include phosphorothioates, methylphosphonates, peptide nucleic acids, crosslinked nucleic acids (BNAs), and / or locked nucleic acid (LNA) molecules. For example, certain implementations use crosslinked nucleic acids that include a bond across a ribose ring. Examples of crosslinks include ethylene-crosslinked nucleic acids. Some BNAs include an LNA molecule, and the modified nucleotide is a locked nucleic acid molecule containing [alpha]-L-LNA. The LNA includes an oligonucleotide containing a ribonucleic acid analog in which the ribose ring is "locked" by a methylene crosslink between 2'-oxygen and 4'-carbon, i.e., at least one LNA monomer, i.e., one 2'-O,4'-C-methylene-β-D-ribofuranosylnucleotide. LNA bases form standard Watson-Crick base pairs, but their locked configuration increases the rate and stability of base pairing reactions (Jepsen et al., Oligonucleotides, 14, 130-146 (2004)). LNA also exhibits increased affinity for base pairing with RNA compared to DNA. These properties make LNA particularly useful as a probe for fluorescence in situ hybridization (FISH) and comparative genomic hybridization, as a knockdown tool for miRNAs, and as an antisense oligonucleotide targeting mRNA or other RNAs.

[0059] LNA molecules may include molecules with 10 to 30, for example, 12 to 24, for example, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in each chain, where one of the chains is substantially identical to the miR-128 target sequence, for example, at least 80% (or more, for example, 85%, 90%, 95%, or 100%) identical and has, for example, 3, 2, 1, or 0 mismatched nucleotides. LNA molecules can be chemically synthesized using methods known in the art.

[0060] Antagomiru In some cases, the inhibitory nucleic acid is an antagomyl. An antagomyl is a chemically modified antisense oligonucleotide that targets the miR-128 target sequence. For example, an antagomyl for use in the method described herein may contain a nucleotide sequence of about 12–25 nucleotides, or about 15–23 nucleotides, that is sufficiently complementary to hybridize to the miR-128 target sequence.

[0061] Generally, antagomils contain a cholesterol moiety, for example, at the 3' end. In some embodiments, antagomils have various modifications for pharmacological properties such as RNase protection and enhanced tissue and cellular uptake. For example, in addition to the modifications considered above for antisense oligos, antagomils may have one or more of the following: complete or partial 2'-O-methylation of sugars and / or a phosphorothioate skeleton. The phosphorothioate modifications provide protection against RNase activity, and their lipophilicity contributes to enhanced tissue uptake. In some embodiments, antagomils may contain six phosphorothioate skeleton modifications, with two phosphorothioates located at the 5' end and four at the 3' end. Antagomils useful in this method may also be modified in terms of their length or, otherwise, the number of nucleotides constituting the antagomil. The antagomils shall retain specificity for their targets, i.e., they shall not directly bind to transcripts other than the intended target or directly and significantly affect their expression levels. In some embodiments, the inhibitory nucleic acid is locked and contains a cholesterol moiety (e.g., locked antagomil).

[0062] Gapmar In some cases, the inhibitory nucleic acid is a gapmer. The gapmer is a chemically modified antisense oligonucleotide that targets the miR-128 target sequence. In certain embodiments, the gapmer for use in the method described herein may include nucleotide sequences of about 7–50 nucleotides, about 7–30 nucleotides, and / or about 14–23 nucleotides, which are sufficiently complementary to hybridize to the miR-128 target sequence.

[0063] Generally, gapmers are short DNA antisense oligonucleotide structures having RNA-like segments on both sides of the sequence (i.e., 5'-wings and 3'-wings). The DNA antisense structure is called a gap. The RNA-like structure may contain modified nucleotides, such as LNA, 2'-methoxy, and / or 2'-fluoromodified nucleotides. In various embodiments, gapmers utilize nucleotides modified with phosphorothioate bonds (e.g., a backbone). The wing structures (e.g., 5'-wings and / or 3'-wings) each contain 1 to 5 nucleotides. The wing structures may be the same size (e.g., both the 5'-wing and 3'-wings contain 5 nucleotides), or they may be of different sizes (e.g., the 5'-wing contains 5 nucleotides while the 3'-wing contains 4 nucleotides).

[0064] siRNA / shRNA In some cases, the inhibitory nucleic acids of this disclosure are interfering RNAs, including but not limited to small interfering RNAs ("siRNAs") or small hairpin RNAs ("shRNAs"). Methods for constructing interfering RNAs are well known in the art. For example, an interfering RNA may be assembled from two distinct oligonucleotides, one strand being a sense strand and the other an antisense strand, wherein the antisense and sense strands are self-complementary (i.e., if the antisense and sense strands form a double-stranded or bistranded structure, each strand contains a nucleotide sequence complementary to the nucleotide sequence of the other strand), the antisense strand containing a nucleotide sequence complementary to the nucleotide sequence in the target nucleic acid molecule or a portion thereof (i.e., an unwanted gene), and the sense strand containing a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. Alternatively, an interfering RNA may be assembled from a single oligonucleotide in which self-complementary sense and antisense regions are linked by a nucleic acid-based or non-nucleic acid-based linker. Interfering RNA can be a polynucleotide having a double-stranded, asymmetric double-stranded, hairpin, or asymmetric hairpin secondary structure, having a self-complementary sense region and an antisense region, wherein the antisense region contains a nucleotide sequence complementary to the nucleotide sequence in a separate target nucleic acid molecule or a portion thereof, and the sense region contains a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. Interference can be a cyclic single-stranded polynucleotide having two or more loop structures and a stem containing a self-complementary sense region and an antisense region, wherein the antisense region contains a nucleotide sequence complementary to the nucleotide sequence in a target nucleic acid molecule or a portion thereof, and the sense region contains a nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof, and the cyclic polynucleotide can be processed either in vivo or in vitro to produce an active siRNA molecule capable of mediating RNA interference.

[0065] 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. When expressed, such an RNA molecule preferably forms a “hairpin” structure, which is referred to herein as “shRNA”. The loop region is generally about 2 to about 10 nucleotides long. In some embodiments, the loop region is about 6 to about 9 nucleotides long. In some embodiments, the sense and antisense regions are about 15 to about 20 nucleotides long. After post-transcriptional processing, the small hairpin RNA is converted to siRNA by a cleavage event mediated by the enzyme Dicer, a member of the RNase III family. The siRNA can then inhibit the expression of genes sharing homology.

[0066] The target RNA cleavage reaction induced by siRNA is highly sequence-specific. Generally, siRNAs containing nucleotide sequences 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. Therefore, this disclosure has the advantage of being able to tolerate sequence variations that may be expected due to gene mutations, strain polymorphisms, or evolutionary divergence. For example, siRNA sequences with insertions, deletions, and single-point mutations to the target sequence have also been found to be effective for inhibition. Alternatively, siRNA sequences with nucleotide analog substitutions or insertions may be effective for inhibition. Generally, siRNAs should retain specificity to their targets, i.e., they should not directly bind to transcripts other than the intended target or directly and significantly affect their expression levels.

[0067] Production of inhibitory nucleic acids Inhibitory nucleic acids can be synthesized in vitro by known chemical synthesis techniques, for example, as described in Adams (1983) J.Am. Chem. Soc. 105:661; Belousov (1997) Nucleic Acids 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; and U.S. Patent No. 4,458,066.

[0068] Pharmaceutical composition This disclosure provides compositions comprising a pharmaceutical composition containing the inhibitory nucleic acid of this disclosure. The inhibitory nucleic acid of this disclosure may hereafter be referred to as “agent” or “active agent.”

[0069] In some cases, the composition is formulated with a pharmaceutically acceptable carrier. Pharmaceutical compositions and formulations may be administered parenterally, topically, or orally, for example, by aerosol or transdermally. Pharmaceutical compositions may be formulated in any way and may be administered in various unit dosage forms depending on the condition or disease, the severity of the disease, the overall medical condition of each patient, and the preferred method of administration. Further details regarding the techniques for the formulation and administration of pharmaceuticals are well described in the scientific literature and patent literature, see, for example, Remington: The Science and Practice of Pharmacy, 21st ed., 2005.

[0070] Inhibitory nucleic acids may be administered alone or as components of pharmaceutical formulations (compositions). Inhibitory nucleic acids may be formulated for administration by any convenient method for use in humans or veterinary medicine. Wetting agents, emulsifiers, and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as colorants, release agents, coating agents, sweeteners, flavorings, fragrances, preservatives, and antioxidants may also be present in the composition.

[0071] Formulations of the inhibitory nucleic acids of this disclosure include those suitable for intradermal, inhalation, oral / nasal, topical, parenteral, rectal, and / or vaginal administration. The formulations may be conveniently provided in unit dosage forms and may be prepared by any method well known in the field of pharmacy. The amount of the active ingredient (e.g., the inhibitory nucleic acid of this disclosure) that can be combined with a carrier material to produce a single dosage form will vary depending on the host being treated, the specific mode of administration, e.g., intradermal or inhalation. The amount of the active ingredient that can be combined with a carrier material to produce a single dosage form will generally be the amount of the compound that produces a therapeutic effect, e.g., reduction or cessation of muscle loss.

[0072] Pharmaceutical preparations may be prepared according to any method known in the art for the manufacture of pharmaceuticals. Such drugs may contain sweeteners, flavorings, colorings, and preservatives. Preparations may be mixed with non-toxic, pharmaceutically acceptable excipients suitable for manufacture. Preparations may contain one or more diluents, emulsifiers, preservatives, buffers, excipients, flavorings, colorings, fillers, solvents, lubricants, binders, surfactants, disintegrants, etc., and may be provided in the form of liquids, powders, emulsions, lyophilized powders, sprays, creams, lotions, controlled-release formulations, tablets, pills, gels, patches, implants, etc.

[0073] Pharmaceutical preparations for oral administration can be formulated in appropriate and suitable doses using pharmaceutically acceptable carriers well known in the art. Such carriers enable the formulation of pharmaceuticals in unit dosage forms such as tablets, pills, powders, sugar-coated tablets, capsules, liquids, lozenges, gels, syrups, slurries, and suspensions suitable for patient ingestion. Pharmaceutical preparations for oral use can be formulated as solid excipients, and optionally, the resulting mixture may be ground, and if desired, further suitable compounds may be added, after which the granular mixture may be processed to obtain tablets or sugar-coated tablet cores. Suitable solid excipients are carbohydrate or protein fillers, such as sugars including lactose, sucrose, mannitol, or sorbitol; starches from corn, wheat, rice, potato, or other plants; cellulose, such as methylcellulose, hydroxypropyl methylcellulose, or carboxymethylcellulose sodium; and gums including gum arabic and tragacanth gum; and proteins, such as gelatin and collagen. Disintegrants or solubilizers, such as cross-linked polyvinylpyrrolidone, agar, alginic acid, or salts thereof such as sodium alginate, may be added. Push-fit capsules may contain an active agent mixed with a filler or binder such as lactose or starch, a lubricant such as talc or magnesium stearate, and optionally a stabilizer. In soft capsules, the active agent may be dissolved or suspended in a suitable liquid such as fatty oil, liquid paraffin, or liquid polyethylene glycol, with or without a stabilizer.

[0074] The aqueous suspension may contain an active agent (e.g., the inhibitory nucleic acid of this disclosure) mixed with an excipient suitable for the preparation of the aqueous suspension, for example, an excipient suitable for aqueous intradermal injection. Such excipients include suspending agents, for example, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, tragacanth gum, and acacia gum, and dispersing or wetting agents, for example, naturally occurring phosphatides (e.g., lecithin), condensation products of alkylene oxides and fatty acids (e.g., polyoxyethylene stearate), condensation products of ethylene oxides and long-chain aliphatic alcohols (e.g., heptadecaethyleneoxycetanol), condensation products of ethylene oxides and partial esters derived from fatty acids and hexitol (e.g., polyoxyethylene sorbitol monooleate), or condensation products of ethylene oxides and partial esters derived from fatty acids and hexitol anhydride (e.g., polyoxyethylene sorbitan monooleate). The aqueous suspension may also contain one or more preservatives, such as ethyl p-hydroxybenzoate or n-propyl p-hydroxybenzoate, one or more colorants, one or more flavorings, and one or more sweeteners, such as sucrose, aspartame, or saccharin. The formulation may be adjusted for osmotic pressure.

[0075] In some cases, oil-based pharmaceuticals are used for the administration of inhibitory nucleic acids. Oily suspensions can be formulated by suspending the active agent in vegetable oils such as peanut oil, olive oil, sesame oil, or coconut oil, or in mineral oils such as liquid paraffin, or in mixtures thereof. See, for example, U.S. Patent No. 5,716,928 (see also U.S. Patent No. 5,858,401), which describes the use of essential oils or essential oil components to increase bioavailability and reduce inter- and intra-individual variability of orally administered hydrophobic pharmaceutical compounds. Oily suspensions may contain thickeners such as beeswax, solid paraffin, or cetyl alcohol. Sweeteners, such as glycerol, sorbitol, or sucrose, may be added to provide a palatable oral formulation. These formulations can be preserved by the addition of antioxidants such as ascorbic acid. For an example of an injectable oil vehicle, see Minto (1997) J. Pharmacol. Pharmacol. Exp. Ther. 281:93-102.

[0076] Pharmaceutical formulations may also be in the form of oil-in-water emulsions. The oil phase may be the vegetable oils or mineral oils described above, or mixtures thereof. Suitable emulsifiers include naturally occurring gums (e.g., acacia gum and tragacanth gum), naturally occurring phosphatides (e.g., soy lecithin), esters or partial esters derived from fatty acids and hexitol anhydrides (e.g., sorbitan monooleate), and condensation products of these partial esters with ethylene oxide (e.g., polyoxyethylene sorbitan monooleate). The emulsion may also contain sweeteners and flavorings, as in the case of syrup and elixir formulations. Such formulations may also contain sedatives, preservatives, or colorants. In alternative embodiments, the injectable oil-in-water emulsion comprises paraffinic oil, sorbitan monooleate, ethoxylated sorbitan monooleate, and / or ethoxylated sorbitan trioleate.

[0077] Pharmaceutical compositions include suppositories, inhalants, powders, and aerosol formulations, which can be administered via intranasal, intraocular, and intravaginal routes (see, for example, inhaled steroids, e.g., Rohatagi (1995) J. Clin. Pharmacol. 35:1187-1193; Tjwa (1995) Ann. Allergy, Asthma & Immunol. 75:107-111). Suppository formulations can be prepared by mixing the drug with a suitable non-irritating excipient, the excipient being solid at room temperature but liquid at body temperature, and thus melting in the body to release the drug. Such materials include cocoa butter and polyethylene glycol.

[0078] In some cases, pharmaceutically acceptable excipients may be selected from lipids or polymers, including poly(amidoamine), poly(propyleneimine), and poly(L-lysine).

[0079] In some cases, pharmaceutical compositions may be formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols, and delivered transdermally via topical routes.

[0080] In some cases, pharmaceutical compositions may be delivered as microspheres for slow release within the body. For example, microspheres may be administered via intradermal injection of a drug that is slowly released subcutaneously (see Rao (1995) J. Biomator Sci. Polym. Ed. 7:623-645), as a biodegradable and injectable gel formulation (see, for example, Gao (1995) Pharm. Res. 12:857-863 (1995)), or as microspheres for oral administration. See, for example, Eyles (1997) J. Pharm. Pharmacol. 49:669-674.

[0081] In some cases, pharmaceutical compositions may be administered parenterally, such as by intravenous (IV) administration or by administration into body cavities or organ lumens. These formulations may contain a solution of the active agent (e.g., the inhibitory nucleic acid of this disclosure) in a pharmaceutically acceptable carrier. Acceptable vehicles and solvents that may be used are water and Ringer's solution (isotonic sodium chloride). Furthermore, sterile fixatives may be used as solvents or suspension media. For this purpose, any low-irritation fixative, including synthetic mono or diglycerides, may be used. Furthermore, fatty acids such as oleic acid may also be used in the preparation of injectable formulations. These solutions are sterile and generally free of undesirable substances. These formulations may be sterilized by conventional, well-known sterilization techniques. The formulations may contain pharmaceutically acceptable auxiliary substances necessary to approximate physiological conditions, such as pH adjusters and buffers, toxicity modifiers, and sodium acetate, sodium chloride, sodium chloride, sodium lactate, etc. The concentration of the active agent in these formulations can vary widely and is selected primarily based on fluid volume, viscosity, body weight, etc., according to the specific mode of administration chosen and the patient's needs. For intravenous administration, the formulation may be a sterile injection preparation, such as a sterile aqueous or oily suspension for injection. This suspension may be formulated using suitable dispersants or wetting agents and suspending agents. The sterile injection preparation may be a suspension in a non-toxic, parenterally acceptable diluent or solvent, such as a solution of 1,3-butanediol. Administration may be by bolus or continuous infusion (e.g., substantially uninterrupted introduction into a blood vessel for a specific period of time).

[0082] In some cases, pharmaceutical compositions may be lyophilized. Stable lyophilized compositions containing inhibitory nucleic acids may be prepared by lyophilizing a solution containing the pharmaceutical composition of this disclosure and an expander, such as mannitol, trehalose, raffinose, and sucrose, or a mixture thereof. A process for preparing a stable lyophilized formulation may involve lyophilizing a solution of about 2.5 mg / mL of nucleic acid, about 15 mg / mL of sucrose, about 19 mg / mL of NaCl, and a sodium citrate buffer having a pH higher than 5.5 but lower than 6.5. See, for example, U.S. Patent Application Publication No. 20040028670.

[0083] Compositions and formulations may be delivered by the use of liposomes. By using liposomes, the delivery of active agents (e.g., inhibitory nucleic acids of this disclosure) to target cells can be concentrated in vivo, particularly when the liposome surface carries a ligand specific to the target cell or otherwise preferentially directed to a particular organ. See, for example, U.S. Patent Nos. 6,063,400 and 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 multiple bilayers. Liposomes are monolayer or multilayer vesicles having a membrane formed from a lipophilic material and an aqueous interior containing the composition to be delivered. Cationic liposomes are positively charged liposomes that are thought to interact with negatively charged DNA molecules to form stable complexes. pH-sensitive or negatively charged liposomes are thought to capture DNA rather than form complexes with it. Both cationic and non-cationic liposomes are used to deliver DNA to cells.

[0084] Liposomes may also include “stereostabilized” liposomes, i.e., liposomes containing one or more specialized lipids. When incorporated into liposomes, these specialized lipids result in liposomes with improved circulating lifespan compared to liposomes lacking such specialized lipids. An example of a stereostabilized liposome is one in which a portion of the vesicle-forming lipid moiety of the liposome contains one or more glycolipids or is derivatized with one or more hydrophilic polymers (e.g., polyethylene glycol (PEG) moiety). Liposomes and their uses are further described in U.S. Patent No. 6,287,860.

[0085] The formulations of this disclosure may be administered for prophylactic and / or therapeutic purposes. In some cases, for therapeutic use, the composition is administered to a subject in need (e.g., an individual at or having a risk of the disorder described herein (e.g., a higher risk than the population)) in an amount sufficient to cure, alleviate, or partially prevent the clinical symptoms of the disorder or its complications, which may be referred to as a therapeutically effective dose.

[0086] The amount of pharmaceutical composition appropriate to achieve this is the therapeutically effective dose. The effective dosage schedule and amount for this use, i.e., the administration regimen, depends on various factors, including the stage of the disease or condition, the severity of the disease or condition, the patient's overall health status, the patient's physical condition, and age. The mode of administration is also considered when calculating the administration regimen for a patient.

[0087] Dosage regimens also take into account pharmacokinetic parameters well known in the art, namely the absorption rate, bioavailability, metabolism, and clearance of the active agent (see, e.g., Remington: The Science and Practice of Pharmacy, 21st ed., 2005). State-of-the-art technology enables clinicians to determine dosage regimens for each individual patient, active agent (e.g., inhibitory nucleic acid), and disease or condition being treated. Guidelines provided for similar compositions used as pharmaceuticals can be used as guidance for determining dosage regimens, i.e., dosing schedules and dosing levels.

[0088] The formulation may be administered as a single or multiple dose depending on, for example, the required and tolerable dosage and frequency for the patient, and the degree and amount of therapeutic effect (e.g., effect on blood glucose levels) that occurs after each dose. The formulation should provide an amount of the active agent (e.g., inhibitory nucleic acid) sufficient to effectively treat, prevent, or improve a condition, disease, or symptom.

[0089] In alternative embodiments, pharmaceutical formulations for oral administration are administered at a daily dose of approximately 1 μg to 100 mg of nucleic acid per kilogram of body weight. Lower doses may be used for administration into the bloodstream, body cavities, or organ lumens, in contrast to oral administration. Substantially higher doses may be used for topical or oral administration, or for administration by powder, spray, or inhalation. Practical methods for preparing formulations that can be administered parenterally or orally are known or obvious to those skilled in the art and are described in more detail in publications such as Remington: The Science and Practice of Pharmacy, 21st ed., 2005.

[0090] In some embodiments, the methods described herein may involve co-administration with other drugs or pharmaceuticals, such as compositions for lowering blood glucose levels. For example, inhibitory nucleic acids may be co-administered with drugs for treating or reducing the risk of the disorders described herein.

[0091] How to treat sarcopenia This disclosure provides a method for using the inhibitory nucleic acid of this disclosure in subjects suffering from, for example, sarcopenia. The method comprises administering an effective amount of the inhibitory nucleic acid of this disclosure or a composition containing it (e.g., a pharmaceutical composition) to an individual in need. In some cases, the inhibitory nucleic acid of this disclosure is administered in lipid nanoparticles. In some cases, the inhibitory nucleic acid of this disclosure is administered in liposomes and / or recombinant adeno-associated virus (AAV) particles. Examples of sarcopenic disorders that can be treated by the method of this disclosure include age-related sarcopenia.

[0092] In some cases, the therapeutically effective dose of the inhibitory nucleic acid of this disclosure is the amount administered to a subject in one or more doses that results in one or more of the following: i) reduction of oxidative stress, ii) improvement of muscle function, iii) slower rate of atrophy, iv) reduced inflammation, and v) reduced fat infiltration.

[0093] In some cases, an effective dose of the inhibitory nucleic acid of this disclosure is an amount that, when administered to a subject in one or more doses, results in an increase in muscle strength in the subject. For example, in some cases, an effective dose of the inhibitory nucleic acid of this disclosure is an amount that, when administered to a subject in one or more doses, provides an increase of at least 10%, at least 25%, at least 50%, at least 2 times, or more than 2 times in muscle strength in the subject compared to the level of muscle strength in the subject before treatment with the inhibitory nucleic acid.

[0094] In some cases, the effective dose of the inhibitory nucleic acid of this disclosure is the amount that, when administered to a subject in one or more doses, results in a reduction of skeletal muscle fibrosis and necrosis. For example, in some cases, the effective dose of the inhibitory nucleic acid of this disclosure is the amount that, when administered to a subject in one or more doses, results in a reduction of at least 10%, at least 25%, at least 50%, at least 60%, at least 70%, or more than 70% of skeletal muscle fibrosis and necrosis compared to the level of skeletal muscle fibrosis and necrosis in the subject before treatment with the inhibitory nucleic acid.

[0095] Route of administration Preferred routes of administration include oral, rectal, nasal, pulmonary, topical, subcutaneous, intramuscular, intraperitoneal, intravenous, intradermal, intrathecal, and epidural administration. In some cases, the route of administration is oral. In some cases, the route of administration is intravenous.

[0096] Combination therapy This disclosure envisions the use of the inhibitory nucleic acids of this 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 therapies, various active agents often have different mechanisms of action. Such combination therapies may be particularly advantageous by reducing the dose of one or more agents, thereby reducing or eliminating the adverse effects associated with one or more agents, and furthermore, such combination therapies may have synergistic therapeutic or prophylactic effects on the underlying disease, disorder, or condition.

[0097] As used herein, “combination” means therapies that can be administered separately, for example, therapies that can be formulated separately for separate administrations (as may be provided in a kit), and therapies that can be administered together in a single formulation (i.e., “co-formulations”).

[0098] In certain cases, the inhibitory nucleic acids of this disclosure and at least one additional agent are administered sequentially or applied, for example, one agent is administered before one or more other agents. In other cases, the inhibitory nucleic acids of this disclosure and at least one additional agent are administered simultaneously, for example, two or more agents are administered simultaneously or nearly simultaneously. Two or more agents may be present in two or more separate formulations or may be combined into a single formulation (i.e., a co-formulation). Regardless of whether the two or more agents are administered sequentially or simultaneously, they are considered to be administered in combination for the purposes of this disclosure.

[0099] The inhibitory nucleic acids of this disclosure may be used in combination with other agents useful for treating the disorders or conditions described herein, including those typically administered to subjects suffering from muscle atrophy, malnutrition, metabolic syndrome, and certain hormonal disorders.

[0100] This disclosure envisions combination therapy with a number of drugs (and classes thereof), including: 1) testosterone, testosterone mimes, and drugs that stimulate testosterone secretion. In certain embodiments, testosterone may be used directly or as a prodrug. Testosterone prodrugs may include testosterone esters, such as testosterone caproate, testosterone cypionicate, testosterone decanoate, testosterone enanthate, testosterone isobutyrate, testosterone isocaproate, testosterone phenylpropionate, testosterone propionate, testosterone undecanoate, testosterone acetate, testosterone cyclohexylpropionate, hydrazone testosterone benzyl enanthate, testosterone furoate, testosterone hexahydrobenzoate, testosterone hexahydrobenzylcarbonate, testosterone hexyloxyphenylpropionate, testosterone ketlaurate, testosterone nicotinate, testosterone phenylacetate, testosterone phosphate, testosterone undecylenate, and testosterone valerate. Trademarks for testosterone replacement therapy include Androgel®, Testim®, Depo-Testosterone®, Androderm®, Methyltestosterone, and Android®. Certain vitamins or nutritional supplements, including creatine, omega-3 fatty acids, vitamin D, and dehydroepiandrosterone (DHEA), may increase boost testosterone levels in individuals. 2) Hormone therapy including growth hormones and growth factors, such as growth hormone (e.g., somatotropin, human growth hormone, Somatropin®, Humatrope®), insulin-like growth factor (IGF-1), mechanical growth factor (MGF), and urocortin II.3) Anabolic steroids, such as danazol, drostanolone propionate, ethylestrenol, fluoxymesterone, mesterone, methandienone, metenolone acetate, metenolone enanthate, methyltestosterone, nandrolone decanoate, nandrolone phenylpropionate, norethandrolone, oxandrolone, oxymetholone, stanozolol, testosterone cypionate, testosterone enanthate, testosterone propionate, testosterone undecanoate, and trenbolone HBC.

[0101] For certain individuals with sarcopenia, lifestyle adjustments, including improvements in diet and exercise, may also be beneficial. Diet may include calorie-reducing (e.g., calorie-deficient) diets and / or any other diets that promote the reduction of body fat and / or excess weight. Exercise may include weight training, resistance training, and / or any other type of exercise for increasing muscle mass and / or strength. Weight training and / or resistance training may include the use of free weights (e.g., dumbbells, barbells, and kettlebells), punching bags, weight balls (e.g., medicine balls), weight machines, resistance bands, pull-ups, and bodyweight exercises.

[0102] Specific embodiments include the treatment of metabolic syndrome and related disorders, such as insulin resistance, obesity, and hypertension. Such therapeutic agents include 1) insulin, insulin mimes, and agents that stimulate insulin secretion (including sulfonylureas (e.g., chlorpropamide, trazamide, acetohexamide, tolbutamide, glibride, glimepiride, glipizide), and meglitinides (e.g., mitiglinide, repaglinide, and nateglinide)), and 2) biguanides (e.g., metformin and its pharmaceutically acceptable salts, in particular metformin hydrochloride, and its sustained-release formulations). 1) Other agents, e.g., Glumetza (trademark), Fortamet (trademark), and GlucophageXR (trademark), and other agents that act by promoting glucose utilization, reducing hepatic glucose production, and / or reducing intestinal glucose excretion, 3) α-glucosidase inhibitors (e.g., acarbose, voglibose, and miglitol), and other agents that slow carbohydrate digestion, thereby slowing absorption from the intestines and reducing postprandial hyperglycemia, 4) thiazoli Zingione (e.g., rosiglitazone, troglitazone, pioglitazone, glipizide, paraglitazone, riboglitazone, netoglitazone, AMG131, MBX2044, mitoglitazone, lobeglitazone, IDR-105, troglitazone, englitazone, siglitazone, adaglitazone, dalglitazone which enhances insulin action (e.g., by insulin sensitization), and insulin mimetic (e.g., insulin degludec, insulin 5) DPP-IV inhibitors (e.g., alogliptin, omaligliptin, linagliptin, vildagliptin, and sitagliptin), and glucagon-like peptide-1 (GLP-1), and GLP-1 agonists and analogs (e.g., exenatide (BYETTA), and ITCA 650 (a subcutaneously implanted osmotic pump that delivers exenatide analogs over a 12-month period), glucagon-like peptides, Intarcia, Boston,Mass)), and GLP-1 receptor agonists (e.g., dulaglutide, semaglutide, tilzepatide, albiglutide, exenatide, liraglutide, lixisenatide, taspogultide, CJC-1131, and BIM-51077, including their intranasal, transdermal, and once-weekly formulations), as well as 6) DPP-IV resistance analogues (incretin mimes), PPAR gamma agonists, fenofibre PPAR alpha agonists such as bric acid derivatives (e.g., gemfibrozil, clofibrate, ciprofibrate, fenofibrate, bezafibrate), dual-acting PPAR agonists (e.g., ZYH2, ZYH1, GFT505, tiglitazar, mulaglitazar, alleglitazar, sodelglitazar, and nabeglitazar), general-acting PPAR agonists, and PTP1B inhibitors. Drugs (e.g., ISIS-113715 and TTP814), SGLT inhibitors (e.g., ASP1941, SGLT-3, empagliflozin, dapagliflozin, canagliflozin, BI-10773, PF-04971729, remogliflozin, TS-071, tofogliflozin, ipragliflozin, and LX-4211), insulin secretagogues, angiotensin-converting enzyme inhibitors (e.g., alacepril, benazepril) Examples include captopril, seronapril, cilazapril, delapril, enalapril, enalaprilat, fosinopril, imidapril, lisinopril, movertipril, perindopril, quinapril, ramipril, spirapril, temocapril, or trandolapril), and angiotensin II receptor antagonists (e.g., losartan, valsartan, candesartan, olmesartan, termesartan).

[0103] Suitable subjects for treatment Suitable subjects for treatment using the methods described herein include individuals with sarcopenia, including age-related sarcopenia. In some cases, subjects have muscle weakness, muscle atrophy, muscle wasting, and / or decreased muscle mass. Certain subjects may have a high body mass index and / or sarcopenic obesity. Certain subjects may be elderly (e.g., over 60, 65, 70, 75, 80, 85, or older).

[0104] Some individuals may have one or more risk factors associated with sarcopenia, including lack of exercise, obesity, one or more chronic diseases (e.g., chronic obstructive pulmonary disease, kidney disease, diabetes, cancer, HIV infection), rheumatoid arthritis, insulin resistance, low hormone levels, malnutrition, insufficient protein intake, reduced ability to convert protein into energy, and a decrease in nerve cells between the brain and muscles.

[0105] Sarcopenia can be assessed using one of several methods, including strength tests, assistance with walking, standing up from a chair, climbing stairs, and falls. Such assessments may include grip strength tests, chair stand tests, walking speed tests, physical function assessment batteries, and time-up-and-go tests. In some situations, muscle mass can be measured using imaging techniques, including dual-energy X-ray absorptiometry and / or bioelectrical impedance analysis.

[0106] In certain embodiments, the subjects have not been diagnosed with a metabolic disorder (e.g., metabolic syndrome). In additional embodiments, the subjects have been determined not to have a metabolic disorder.

[0107] Adeno-associated virus particles This disclosure provides recombinant adeno-associated virus (rAAV) particles comprising a capsid and a nucleic acid vector comprising a heterogeneous nucleic acid region containing a sequence encoding the interfering RNA described above, wherein the interfering RNA comprises a region complementary to the miR-128 target nucleic acid.

[0108] Viruses of the parvovirus family are small, non-enveloped icosahedral capsid viruses characterized by a single-stranded DNA genome. Examples of viruses in the parvovirus family include adeno-associated viruses (AAVs) that can replicate in vertebrate hosts, including but not limited to humans, primates, cattle, dogs, horses, and sheep.

[0109] In one embodiment, the AAV particles of the Disclosure are recombinant AAV viral vectors that are replication-deficient and lack sequences encoding functional Rep and Cap proteins within their viral genome. These deficient AAV vectors lack most or all parent coding sequences and may essentially contain only one or two AAV ITR sequences and the nucleic acid of interest (e.g., a nucleic acid containing a nucleotide sequence encoding the inhibitory nucleic acid of the Disclosure) for delivery to cells, tissues, organs, or organisms.

[0110] According to some embodiments, AAV particles for use in treatment and / or diagnosis contain a virus that has been purified or reduced to the minimum components necessary for the transduction of the nucleic acid payload or cargo of interest. In this way, the AAV particles are operated as a vehicle for specific delivery, while lacking the harmful replication and / or integration features found in wild-type viruses.

[0111] The AAV vectors of this disclosure may be produced by recombination and may be based on an adeno-associated virus (AAV) parent sequence or a reference sequence. As used herein, “vector” is any molecule or portion that transports, transduces, or otherwise acts as a carrier for a heterologous molecule, such as a nucleic acid described herein.

[0112] In addition to single-stranded AAV viral genomes (e.g., ssAAV), this disclosure also provides a self-complementary AAV (scAAV) viral genome, where the scAAV vector genome contains DNA strands that anneal together to form double-stranded DNA. By omitting the synthesis of the second strand, scAAV enables rapid expression within cells.

[0113] In one embodiment, the AAV particles of this disclosure are scAAVs.

[0114] In one embodiment, the AAV particles of this disclosure are ssAAV.

[0115] Methods for producing and / or modifying AAV particles are disclosed in the art, such as pseudotype AAV vectors (International Publication Nos. 200028004, 0200123001, 2004112727, 2005005610, and 2005072364, each of which is incorporated herein by reference in its entirety).

[0116] AAV particles can be modified to enhance delivery efficiency. Such modified AAV particles can be efficiently packaged and used to successfully infect target cells at high frequency and with minimal toxicity. In some embodiments, the capsid of the AAV particle is manipulated according to the method described in U.S. Patent Application Publication No. 20195801 (the contents of which are incorporated herein by reference in their entirety).

[0117] Viral genome components: Inverted terminal repeats (ITRs) The AAV particles of this disclosure comprise a viral genome having at least one ITR region and a payload region. In one embodiment, the viral genome has two ITRs. These two ITRs are adjacent to the payload region at their 5' and 3' ends. The ITRs function as origins of replication, including recognition sites for replication. The ITRs comprise sequence regions that may be complementary, and the symmetrically arranged ITRs incorporated into the viral genome of the present invention may consist of naturally occurring polynucleotide sequences or recombinant-derived polynucleotide sequences.

[0118] The ITR may originate from the same serotype as the capsid and may be selected from any other serotype or its derivative. The ITR may be of a different serotype than the capsid. In one embodiment, an AAV particle has two or more ITRs. In a non-limiting example, an AAV particle has a viral genome containing two ITRs. In one embodiment, the ITRs are of the same serotype as each other. In another embodiment, the ITRs are of different serotypes. A non-limiting example includes zero, one, or both ITRs having the same serotype as the capsid. In one embodiment, both ITRs of the viral genome in the AAV particle are AAV2 ITRs.

[0119] Each ITR can independently be about 100 to about 150 nucleotides long. An ITR can be about 100 to 105 nucleotides long, 106 to 110 nucleotides long, 111 to 115 nucleotides long, 116 to 120 nucleotides long, 121 to 125 nucleotides long, 126 to 130 nucleotides long, 131 to 135 nucleotides long, 136 to 140 nucleotides long, 141 to 145 nucleotides long, or 146 to 150 nucleotides long. In one embodiment, the ITR is 14 to 142 nucleotides long. Non-limiting examples of ITR lengths include 102, 140, 141, 142, 145 nucleotides long, and those having at least 95% identity to them.

[0120] Viral genome components: promoters In one embodiment, the payload region of the viral genome includes at least one element for enhancing transgene target specificity and expression (see, for example, Powell et al., Viral Expression Cassette Elements to Enhance Transgene Target Specificity and Expression in Gene Therapy, 2015; these disclosures are incorporated herein by reference in their entirety). Non-limiting examples of elements for enhancing transgene target specificity and expression include promoters, endogenous miRNAs, post-transcriptional regulatory elements (PREs), polyadenylation (PolyA) signal sequences, and upstream enhancers (USEs), CMV enhancers, and introns.

[0121] Those skilled in the art will recognize that the expression of nucleic acids described herein may require specific promoters, including but not limited to species-specific, inducible, tissue-specific, or cell cycle-specific promoters (Parr et al., Nat. Med. 3:1145-9 (1997), these disclosures are incorporated herein by reference in their entirety).

[0122] In one embodiment, the promoter is considered efficient in driving the transcription of the nucleic acid sequence encoded in the payload region of the viral genome of the AAV particle.

[0123] In one embodiment, the promoter is a promoter that is considered efficient in driving expression in targeted cells, tissues, and / or organs. In a particular embodiment, the promoter can be expressed in the muscle tissue of an organism, including skeletal muscle, smooth muscle, and / or cardiac muscle.

[0124] In various embodiments, tissue-specific expression is driven by one or more of the following promoters: the cytomegalovirus pre-early promoter (CMV), the hybrid chicken-beta-actin (CBA) promoter, the ubiquitin C (UBC) promoter, and the RNA polymerase III promoter.

[0125] AAV serotype The AAV particles of this disclosure may include or be derived from any natural or recombinant AAV serotype. According to this disclosure, the AAV particles may utilize or be based on serotypes selected from any of the following: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAVAAV2 / 2-7m8, AAV2 N587A, AAV2 E548A, AAV2 N708A, AAV V708K, goat AAV, AAV1 / AAV2 chimera, bovine AAV, mouse AAV, or rAAV2 / HBoV1 and their variants.

[0126] Examples of non-limiting aspects of this disclosure The embodiments described above, including those covered by this disclosure, may be useful on their own or in combination with one or more other embodiments or forms. Without limiting the foregoing description, certain non-limiting embodiments of this disclosure are provided below. As will be apparent to those skilled in the art by reading this disclosure, each of the individually numbered embodiments may be used together with or in combination with any of the individually numbered embodiments described above or thereafter. This is intended to provide a basis for all combinations of such embodiments, and is not limited to the combinations of embodiments expressly provided below. Embodiment 1. A method for treating age-related sarcopenia, comprising administering an effective dose of a miR-128 inhibitor to an individual diagnosed with age-related sarcopenia. Embodiment 2. The method according to Embodiment 1, wherein the individual has not been diagnosed with a metabolic disorder. Embodiment 3. The method according to Embodiment 1 or 2, wherein the miR-128 inhibitor comprises an inhibitory nucleic acid. Embodiment 4. The inhibitory nucleic acid has the following sequence: 5'-ACCGGTTCACTGTG-3'(Sequence ID 1), 5'-GACCGGTTCACTGT-3' (SEQ ID NO: 2), and 5'-AGACCGGTTCACTGTG-3'(Sequence ID 3) The method according to embodiment 3, comprising one or more of the following. Embodiment 5. The method according to Embodiment 3 or 4, wherein the inhibitory nucleic acid comprises one or more locked nucleic acids (LNAs). Embodiment 6. The method according to any one of Embodiments 2 to 5, wherein the inhibitory nucleic acid comprises one or more modified skeletons and one or more 5-methyldeoxycytosine residues. Embodiment 7. The method according to any one of Embodiments 2 to 6, wherein the inhibitory nucleic acid has a length of 14 to 20 nucleotides. Embodiment 8. The method according to any one of Embodiments 2 to 7, wherein the inhibitory nucleic acid comprises a nucleotide sequence referred to as "NRC-0090". Embodiment 9. The method according to any one of Embodiments 2 to 7, wherein the inhibitory nucleic acid comprises a nucleotide sequence referred to as "NRC-0091". Embodiment 10. The method according to any one of Embodiments 2 to 7, wherein the inhibitory nucleic acid comprises a nucleotide sequence referred to as "NRC-0119". Embodiment 11. The method according to any one of Embodiments 1 to 10, wherein the miR-128 inhibitor is formulated for subcutaneous delivery. Embodiment 12. The method according to any one of Embodiments 1 to 11, wherein the miR-128 inhibitor is formulated as a pharmaceutical composition comprising one or more pharmaceutically acceptable excipients, buffers, solvents, bulking agents, fragrances, lubricants, colorants, binders, surfactants, binders, and disintegrants. Embodiment 13. The method according to any one of Embodiments 1 to 11, wherein the miR-128 inhibitor is formulated as a pharmaceutical composition containing pharmaceutically acceptable excipients. Embodiment 14. The method according to Embodiment 13, wherein the pharmaceutically acceptable excipient is selected from poly(amidoamine), poly(propyleneimine), and poly(L-lysine). Embodiment 15. The method according to Embodiment 13, wherein the pharmaceutically acceptable excipient comprises one or more lipids. Embodiment 16. The method according to any one of Embodiments 1 to 15, further comprising administering at least one additional therapeutic agent. Embodiment 17. The method according to Embodiment 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 steroid, omega-3 fatty acid, creatine, and dehydroepiandrosterone. Embodiment 18. The method according to any one of Embodiments 1 to 17, further comprising instructing the individual to perform weight training exercises or resistance training exercises. Apparatus 19. Combination therapy for the treatment of age-related sarcopenia, miR-128 inhibitors, One or more of the following: testosterone, growth hormone, insulin-like growth factor, mechanical growth factor, urocortin II, anabolic steroids, omega-3 fatty acids, creatine, dehydroepiandrosterone Combination therapy, including Embodiment 20. The combination therapy according to Embodiment 19, wherein the miR-128 inhibitor comprises an inhibitory nucleic acid. Embodiment 21. The inhibitory nucleic acid has the following sequence: 5'-ACCGGTTCACTGTG-3'(Sequence ID 1), 5'-GACCGGTTCACTGT-3' (SEQ ID NO: 2), and 5'-AGACCGGTTCACTGTG-3'(Sequence ID 3) The combination therapy according to embodiment 20, comprising one or more of the following. Embodiment 22. The combination therapy according to Embodiment 20 or 21, wherein the inhibitory nucleic acid comprises one or more locked nucleic acids (LNAs). Embodiment 23. The method according to any one of Embodiments 20 to 22, wherein the inhibitory nucleic acid comprises one or more modified skeletons and one or more 5-methyldeoxycytosine residues. Embodiment 24. The combination therapy according to any one of Embodiments 20 to 23, wherein the inhibitory nucleic acid has a length of 14 to 20 nucleotides. Embodiment 25. The combination therapy according to any one of Embodiments 20 to 24, wherein the inhibitory nucleic acid comprises a nucleotide called "NRC-0090". Embodiment 26. The combination therapy according to any one of Embodiments 20 to 24, wherein the inhibitory nucleic acid comprises a nucleotide sequence referred to as "NRC-0091". Embodiment 27. The combination therapy according to any one of Embodiments 20 to 24, wherein the inhibitory nucleic acid contains a nucleotide sequence referred to as "NRC-0119". Embodiment 28. The combination therapy according to any one of Embodiments 19 to 27, wherein the miR-128 inhibitor is formulated for subcutaneous delivery. Embodiment 29. The combination therapy according to any one of Embodiments 19 to 28, wherein the miR-128 inhibitor is formulated as a pharmaceutical composition comprising one or more pharmaceutically acceptable excipients, buffers, solvents, bulking agents, fragrances, lubricants, colorants, binders, surfactants, and disintegrants. Embodiment 30. The combination therapy according to any one of Embodiments 19 to 29, wherein the miR-128 inhibitor is formulated as a pharmaceutical composition containing pharmaceutically acceptable excipients. Embodiment 31. The combination therapy according to Embodiment 30, wherein the pharmaceutically acceptable excipient is selected from poly(amidoamine), poly(propyleneimine), and poly(L-lysine). Embodiment 32. The combination therapy according to Embodiment 30, wherein the pharmaceutically acceptable excipient comprises one or more lipids. Embodiment 33. An oligonucleotide comprising a plurality of interconnected nucleotides, The aforementioned sequential subset of multiple nucleotides is complementary to a portion of the miR-128 nucleic acid. At least one nucleotide in the plurality of interconnected nucleotides is a crosslinked nucleic acid. Oligonucleotides. Embodiment 34.7 An oligonucleotide according to Embodiment 33, comprising 50 interconnected nucleotides. Embodiment 35.7 - An oligonucleotide according to Embodiment 33 or 34, comprising 30 interconnected nucleotides. Embodiment 36.14-23 oligonucleotides according to any one of Embodiments 33-35. Embodiment 37. The oligonucleotide according to any one of embodiments 33 to 36, wherein the consecutive subset comprises at least six nucleic acid bases complementary to the miR-128 nucleic acid. Embodiment 38. An oligonucleotide according to any one of Embodiments 33 to 37, which is a unimer. Embodiment 39. The oligonucleotide according to Embodiment 38, wherein each nucleotide is independently a crosslinked nucleic acid. Apparatus 40. The nucleic acid base sequence is as follows: 5'-ACCGGTTCACTGTG-3'(Sequence ID 1), 5'-GACCGGTTCACTGT-3' (SEQ ID NO: 2), and 5'-AGACCGGTTCACTGTG-3'(Sequence ID 3) An oligonucleotide according to any one of embodiments 33 to 37, comprising one or more of the above. Appearance 41. Capsid and, A nucleic acid vector containing a heterogeneous nucleic acid region containing a sequence encoding interfering RNA that includes a region complementary to the miR-128 target nucleic acid, and Recombinant adeno-associated virus (rAAV) particles, including those containing the virus. Embodiment 42. The rAAV according to Embodiment 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. Embodiment 43. The rAAV according to Embodiment 41, wherein the interfering RNA is a short hairpin RNA or a small molecular weight interfering RNA. Embodiment 44. The rAAV according to Embodiment 41, wherein the nucleic acid vector includes a promoter, and the sequence encoding the interfering RNA is functionally linked to the promoter. Embodiment 45. The rAAV according to Embodiment 44, wherein the promoter can induce the expression of the interfering RNA in skeletal muscle cells. Embodiment 46. The rAAV according to Embodiment 44, wherein the promoter is a hybrid chicken β-actin (CBA) promoter or an RNA polymerase III promoter. Embodiment 47. The rAAV according to Embodiment 41, wherein the nucleic acid vector comprises an inverted terminal repeat derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, goat AAV, bovine AAV, or mouse AAV serotype. Embodiment 48. The capsid is AAV1 capsid, and AAV2 capsid, AAV3 capsid, AAV4 capsid, AAV5 capsid, AAV6 capsid, AAV7 capsid, AAV8 capsid, AAVrh8 capsid, AAVrh8R capsid, AAV9 capsid, AAV10 capsid, AAVrh10 capsid, AAV11 capsid, AAV12 capsid, tyrosine capsid variant, heparin-binding capsid variant, AAV2R471A capsid, AAVAAV2 / 2-7m8 capsid, AAV DJ capsid, AAV2 N587A capsid, AAV2 E548A capsid, AAV2 N708A capsid, AAV rAAV according to embodiment 41, selected from V708K capsid, goat AAV capsid, AAV1 / AAV2 chimeric capsid, bovine AAV capsid, mouse AAV capsid, and rAAV2 / HBoV1 capsid. Appearance 49. Pharmacologically acceptable excipients, An oligonucleotide according to any one of embodiments 33 to 40 or an rAAV according to any one of embodiments 41 to 48 A pharmaceutical composition containing the above. Embodiment 50. A therapeutically effective composition for use in a method of treating age-related macular degeneration in a subject requiring such treatment, wherein the method comprises administering to the subject a therapeutically effective amount of an oligonucleotide according to any one of Embodiments 1 to 8 or rAAV according to any one of Embodiments 9 to 16. Embodiment 51. A miR-128 inhibitor for use in a method for treating age-related sarcopenia in a subject requiring such treatment, wherein the method comprises administering a therapeutically effective dose of the miR-128 inhibitor to the subject. Appearance 52. Antisense oligonucleotide, Short hairpin RNA, Small interfering RNA, or Recombinant adeno-associated virus (rAAV) particles comprising a capsid and a nucleic acid vector containing a heterogeneous nucleic acid region containing a sequence encoding interfering RNA, wherein the interfering RNA is selected from antisense oligonucleotides, short hairpin RNA, or small interfering RNA. A miR-128 inhibitor according to embodiment 51, selected from the above. Embodiment 53. A therapeutically effective composition for use in a method of treating age-related sarcopenia in a subject requiring such treatment, wherein the method comprises administering to the subject a therapeutically effective amount of an oligonucleotide according to any one of Embodiments 1 to 8 or rAAV according to any one of Embodiments 9 to 16. Embodiment 54. The therapeutic composition according to Embodiment 53, wherein the oligonucleotide is a single-stranded oligonucleotide. Embodiment 55. The therapeutic composition according to Embodiment 53, wherein the oligonucleotide is an antisense oligonucleotide. Embodiment 56. The therapeutically effective composition according to Embodiment 53, wherein the oligonucleotide comprises 7 to 30 interconnected nucleotides. Embodiment 57. The therapeutically effective composition according to Embodiment 56, wherein the oligonucleotide comprises 14 to 23 interconnected nucleotides. Embodiment 58. The therapeutically effective composition according to any one of Embodiments 53 to 57, wherein the oligonucleotide comprises at least one modified sugar nucleoside. Embodiment 59. The therapeutically effective composition according to Embodiment 58, wherein at least 50% of the nucleoside comprises a modified sugar nucleoside. Embodiment 60. The therapeutically effective composition according to Embodiment 58 or 59, wherein 100% of the nucleoside comprises a modified sugar nucleoside. Embodiment 61. The therapeutically effective composition according to any one of Embodiments 58 to 60, wherein the modified sugar nucleoside is a 2'-modified sugar nucleoside. Embodiment 62. The therapeutically effective composition according to Embodiment 61, wherein the 2'-modified sugar nucleoside comprises a 2'-modification independently selected from the group consisting of 2'-fluoro, 2'-methoxy, and 2'-methoxyethoxy. Embodiment 63. The therapeutically effective composition according to any one of Embodiments 58 to 60, wherein the modified sugar nucleoside is a crosslinked nucleic acid. Embodiment 64. The therapeutically effective composition according to Embodiment 63, wherein the crosslinked nucleic acid is locked nucleic acid. Embodiment 65. The therapeutically effective composition according to Embodiment 63, wherein the crosslinked nucleic acid is ethylene crosslinked nucleic acid. Apparatus 66. The nucleic acid base sequence is as follows: 5'-ACCGGTTCACTGTG-3'(Sequence ID 1), 5'-GACCGGTTCACTGT-3' (SEQ ID NO: 2), and 5'-AGACCGGTTCACTGTG-3'(Sequence ID 3) A therapeutically effective composition according to any one of embodiments 58 to 60, comprising one or more of the following. Embodiment 67. The oligonucleotide is a gapmer comprising a 5'-wing, a 3'-wing, and a gap, The 5'-wing and the 3'-wing each contain 1 to 5 nucleotides, Each nucleotide in the 5'-wing and the 3'-wing is independently a crosslinked nucleic acid, and Each nucleotide in the gap is a deoxyribonucleotide. A therapeutically effective composition according to any one of embodiments 53 to 66. Embodiment 68. The therapeutically effective composition according to any one of embodiments 53 to 67, wherein at least one nucleoside linkage in the oligonucleotide is a phosphorothioate diester. Embodiment 69. The therapeutically effective composition according to Embodiment 68, wherein at least 50% of the nucleoside bonds in the oligonucleotide are phosphorothioate diesters. Embodiment 70. The therapeutically effective composition according to Embodiment 68 or 69, wherein 100% of the nucleoside bonds in the oligonucleotide are phosphorothioate diesters. Embodiment 71. The therapeutically effective composition according to any one of Embodiments 53 to 70, wherein the method further comprises administering the oligonucleotide as a guide chain in a short interfering RNA. Embodiment 72. The therapeutically effective composition according to any one of Embodiments 53 to 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. Embodiment 73. The therapeutically effective composition according to any one of Embodiments 53 to 72, wherein the route of administration is intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection. [Examples]

[0127] The following examples are proposed to provide those skilled in the art with a complete disclosure and description of how the present invention may be prepared and used, and are not intended to limit the scope of what the inventors consider to be their invention, nor are they intended to indicate that the following experiments are all or the only experiments performed. While efforts have been made to ensure accuracy with respect to the figures used (e.g., quantity, temperature, etc.), some degree of experimental error and variation should be taken into account. Unless otherwise indicated, parts are parts by weight, molecular weight is weight-average molecular weight, temperature is Celsius, and pressure is atmospheric pressure or near-atmospheric pressure. Standard abbreviations, e.g., bp, base pair; kb, kilobase; pl, picoliter; s or sec, second; min, minute; h or hr, hour; aa, amino acid; kb, kilobase; bp, base pair; nt, nucleotide; im, intramuscular; ip, intraperitoneal; sc, subcutaneous.

[0128] Example 1: Figure 1A shows sequence information for three ASOs. ID indicates the drug name, oligo indicates the nucleotide sequence (uppercase letters indicate locked nucleic acid (LNA), lowercase letters indicate standard bases), and string indicates modifications (+ symbols precede LNA). * / iMe-dC / is prefixed to the phosphorothioate base (where / iMe-dC / indicates internal 5-methyldeoxycytosine), length indicates the number of nucleotides, and #LNA indicates the number of standard bases replaced by LNA. Figure 1B provides schematic diagrams of the LNA / DNA composition of each ASO and their base pairing positions within the target miRNA (miR-128-3p). The seed sequence of miR-128-3p is shown in red.

[0129] Example 2: Aged mice (21.5 months old) and young control mice (8 weeks old) were subcutaneously injected with either anti-miR-128-3p LNA ASO or scrambled control LNA ASO at a dose of 10 mg / kg body weight weekly. Muscle function was then measured at 1, 2, and 3 months by measuring the time spent hanging from a wire and the time taken to run until fatigue (using a high-intensity test protocol). Muscle function was also measured at 2 and 3 months by the four-limb hanging test. Muscle function was finally measured at 3 months by the time taken to run until fatigue using a low-intensity protocol (Figure 2A). After euthanasia at 5 months post-injection, miR-128-3p levels in the gastrocnemius muscle (GA) were measured by quantitative reverse transcription polymerase chain reaction (RT-qPCR).

[0130] Wire hanging time in aged mice decreases compared to juvenile mice up to 22.5 months of age. Treatment with anti-miR-128-3p LNA ASO improves hanging time, as measured by the mean of three trials, the maximum value of a trial, or a calculated score (Figures 2B-2D). Treatment with anti-miR-128-3p LNA ASO significantly improved hanging time (both maximum and mean) after 3 months of treatment. This observation is also replicated by the four-paw hang time test performed at 2 and 3 months of treatment (measured by mean and maximum hanging time; Figures 2E, 2F). One month after treatment, running time to fatigue decreases in aged mice compared to juvenile mice, and this running time increases with anti-miR-128-3p treatment (Figure 2G). At 23.5 months of age, this decrease in running time to fatigue increases. Anti-miR-128-3p LNA ASO treatment significantly improved running time after 2 months of treatment in these aged mice. When mice were tested for muscle endurance using a low-intensity running protocol, miR-128-3p LNA ASO treatment resulted in a more robust improvement over the functional decline observed in aged mice after 3 months of treatment (Figure 2H). As measured after harvesting at 26.5 months of age, miR-128-3p LNA ASO firmly knocked down miR-128-3p expression in the gastrocnemius muscle (Figure 2I).

[0131] Figures 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 value of three trials. (D) Two-limb wire hanging time, measured by the total score from three trials. Hanging time is scored as follows: 1-5 seconds = 1, 6-10 seconds = 2, 11-20 seconds = 3, 21-30 seconds = 4, >30 seconds or reaching support = 5, Total score = sum of all three trial scores. (E) Quadrilateral hanging time, measured by the average of all trials. (F) Quadrilateral hanging time, measured by the maximum value of all trials. (G) Running time to fatigue on a high-intensity treadmill test. (H) Running time to fatigue on a low-intensity treadmill test performed 3 months after injection. (I) miR-128-3p levels measured by RT-qPCR in the gastrocnemius muscle of 26.5-month-old mice. Data are presented as mean and standard deviation (Student's t-test).

[0132] Example 3: Aged mice (86 weeks old) were subcutaneously injected weekly with either anti-miR-128-3p LNA ASO or scrambled control LNA ASO at a dose of 10 mg / kg body weight. Muscle function was then measured at 5, 9, and 15 weeks by measuring the time spent suspended from a wire, and running time to fatigue (using a low-intensity test protocol) was measured at 13 weeks (Figure 3A). Quantitative data are presented as mean and standard deviation (Student's t-test). After euthanasia at 20 weeks post-injection, miR-128-3p levels in the gastrocnemius muscle (GA) were measured by quantitative reverse transcription polymerase chain reaction (RT-qPCR).

[0133] As measured after sampling at 106 weeks of age, miR-128-3p LNA ASO firmly knocks down miR-128-3p expression in the gastrocnemius muscle (Figure 3B). As an additional control under a parallel protocol, aged mice (86 weeks of age) were subcutaneously injected weekly at 10 mg / kg body weight with either miR-27 LNA ASO, an off-target miRNA with a sequence similar to miR-128-3p, or a scrambled control LNA ASO. At 106 weeks of age, RT-qPCR of miR-128-p3 showed no difference between mice injected with miR-27 LNA ASO and mice injected with the scrambled control LNA ASO (Figure 3C).

[0134] When measured by the average of three trials, treatment with anti-miR-128-3p LNA ASO improved bilimb hanging time (Figure 3D). Running time also improved after 13 weeks of treatment with anti-miR-128-3p LNA ASO (Figure 3E).

[0135] Figure 3F shows laminin staining of cross-sections of the tibialis anterior muscle in control mice and anti-miR-128-3p LNA ASO-treated mice. Quantification of the cross-sectional area of ​​laminin-stained muscle fibers shows that fiber size was increased in anti-miR-128-3p LNA ASO-treated mice compared to controls (Figure 3G).

[0136] Figure 3H shows the gene set enrichment analysis of RNA sequencing results from GA muscle. As measured by RT-qPCR, the expression of several mitochondrial genes is increased in GA muscle (Figure 3I).

[0137] Example 4: Aged mice (86 weeks old) were subcutaneously injected weekly with either anti-miR-128-3p LNA ASO or scrambled control LNA ASO at a dose of 10 mg / kg body weight. After 15 weeks of treatment, skeletal muscle mass was measured by EchoMRI, and tissue weights of the GA and quadriceps femoris muscles were determined (Figure 4A). Data are presented as mean and standard deviation (Student's t-test).

[0138] Aged mice treated with anti-miR-128-3p LNA ASO showed an increase in the percentage of lean muscle mass (Figure 4B) and a decrease in the percentage of fat mass (Figure 4C) after 15 weeks.

[0139] References 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, I., Baeyens, J.-P., Cesari, M., Cherubini, A., Kanis, J., Maggio, M., Martin, F., Michel, J.-P., Pitkala, K., Reginster, J.-Y., Rizzoli, R., Sanchez-Rodriguez, D. & Schols, J., "Sarcopenia: revised European consensus on definition and diagnosis," Age Ageing 48, 16-31(2019). DOI:10.1093 / ageing / afy169 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 3. Walston, J.D., "Sarcopenia in older adults," Curr Opin Rheumatol 24, 623-627(2012). DOI:10.1097 / BOR.0b013e328358d59b 4.Papadopoulou,S.,「Sarcopenia:A Contemporary Health Problem among Older Adult Populations,」Nutrients 12,1293(2020).DOI:10.3390 / nu12051293 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.e146 6.Ambros,V.,「The functions of animal microRNAs,」Nature 431,350-355(2004).DOI:10.1038 / nature02871 7.Bartel,D.P.,「MicroRNAs:Target Recognition and Regulatory Functions,」Cell 136,215-233(2009).DOI:10.1016 / j.cell.2009.01.002 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 / journal.pone.0080263 9. Sannicandro, AJ, Soriano-Arroquia, A. & Goljanek-Whysall, K., “Micro(RNA)-managing muscle wasting,” J Appl Physiol 127,619-632(2019).DOI:10.1152 / japplphysiol.00961.2018

[0140] While the present invention has been described in relation to its particular embodiments, it should be understood by those skilled in the art that various modifications may be made and equivalents may be substituted without departing from the true spirit and scope of this disclosure. Furthermore, many modifications may be made to adapt specific circumstances, materials, compositions of substances, processes, process steps, or steps to the spirit and scope of this disclosure. All such modifications are intended to be included within the scope of the appended claims.

Claims

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

2. The method according to claim 1, wherein the individual has not been diagnosed with a metabolic disorder.

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

4. The inhibitory nucleic acid has the following sequence: 5'-ACCGGTTCACTGTG-3' (Sequence ID 1), 5'-GACCGGTTCACTGT-3' (SEQ ID NO: 2), and 5'-AGACCGGTTCACTGTG-3' (Sequence ID 3) The method according to claim 3, comprising one or more of the above.

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

6. The method according to any one of claims 2 to 5, wherein the inhibitory nucleic acid comprises one or more modified skeletons and one or more 5-methyldeoxycytosine residues.

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

8. The method according to any one of claims 2 to 7, wherein the inhibitory nucleic acid comprises a nucleotide sequence referred to as "NRC-0090".

9. The method according to any one of claims 2 to 7, wherein the inhibitory nucleic acid comprises a nucleotide sequence referred to as "NRC-0091".

10. The method according to any one of claims 2 to 7, wherein the inhibitory nucleic acid comprises a nucleotide sequence referred to as "NRC-0119".

11. The method according to any one of claims 1 to 10, wherein the miR-128 inhibitor is formulated for subcutaneous delivery.

12. The method according to any one of claims 1 to 11, wherein the miR-128 inhibitor is formulated as a pharmaceutical composition comprising one or more pharmaceutically acceptable excipients, buffers, solvents, bulking agents, fragrances, lubricants, colorants, binders, surfactants, binders, and disintegrants.

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

14. The method according to claim 13, wherein the pharmaceutically acceptable excipient is selected from poly(amidoamine), poly(propyleneimine), and poly(L-lysine).

15. The method according to claim 13, wherein the pharmaceutically acceptable excipient comprises one or more lipids.

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

17. The method according to 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, and dehydroepiandrosterone.

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

19. A combination therapy for the treatment of age-related sarcopenia, miR-128 inhibitors, One or more of the following: testosterone, growth hormone, insulin-like growth factor, mechanical growth factor, urocortin II, anabolic steroids, omega-3 fatty acids, creatine, dehydroepiandrosterone Combination therapy, including

20. The combination therapy according to claim 19, wherein the miR-128 inhibitor comprises an inhibitory nucleic acid.

21. The inhibitory nucleic acid has the following sequence: 5'-ACCGGTTCACTGTG-3' (Sequence ID 1), 5'-GACCGGTTCACTGT-3' (SEQ ID NO: 2), and 5'-AGACCGGTTCACTGTG-3' (Sequence ID 3) The combination therapy according to claim 20, comprising one or more of the above.

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

23. The method according to any one of claims 20 to 22, wherein the inhibitory nucleic acid comprises one or more modified skeletons and one or more 5-methyldeoxycytosine residues.

24. The combination therapy according to any one of claims 20 to 23, wherein the inhibitory nucleic acid has a length of 14 to 20 nucleotides.

25. The combination therapy according to any one of claims 20 to 24, wherein the inhibitory nucleic acid comprises a nucleotide referred to as "NRC-0090".

26. The combination therapy according to any one of claims 20 to 24, wherein the inhibitory nucleic acid comprises a nucleotide sequence referred to as "NRC-0091".

27. The combination therapy according to any one of claims 20 to 24, wherein the inhibitory nucleic acid comprises a nucleotide sequence referred to as "NRC-0119".

28. The combination therapy according to any one of claims 19 to 27, wherein the miR-128 inhibitor is formulated for subcutaneous delivery.

29. The combination therapy according to any one of claims 19 to 28, wherein the miR-128 inhibitor is formulated as a pharmaceutical composition comprising one or more pharmaceutically acceptable excipients, buffers, solvents, bulking agents, fragrances, lubricants, colorants, binders, surfactants, binders, and disintegrants.

30. The combination therapy according to any one of claims 19 to 29, wherein the miR-128 inhibitor is formulated as a pharmaceutical composition containing pharmaceutically acceptable excipients.

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

32. The combination therapy according to claim 30, wherein the pharmaceutically acceptable excipient comprises one or more lipids.

33. An oligonucleotide comprising multiple interconnected nucleotides, The aforementioned sequential subset of multiple nucleotides is complementary to a portion of the miR-128 nucleic acid. At least one nucleotide in the plurality of interconnected nucleotides is a crosslinked nucleic acid. Oligonucleotides.

34. The oligonucleotide according to claim 33, comprising 7 to 50 interconnected nucleotides.

35. The oligonucleotide according to claim 33 or 34, comprising 7 to 30 interconnected nucleotides.

36. An oligonucleotide according to any one of claims 33 to 35, comprising 14 to 23 interconnected nucleotides.

37. The oligonucleotide according to any one of claims 33 to 36, wherein the consecutive subset comprises at least six nucleic acid bases complementary to the miR-128 nucleic acid.

38. An oligonucleotide according to any one of claims 33 to 37, which is a unimer.

39. The oligonucleotide according to claim 38, wherein each nucleotide is independently a crosslinked nucleic acid.

40. The nucleic acid base sequence is as follows: 5'-ACCGGTTCACTGTG-3' (Sequence ID 1), 5'-GACCGGTTCACTGT-3' (SEQ ID NO: 2), and 5'-AGACCGGTTCACTGTG-3' (Sequence ID 3) An oligonucleotide according to any one of claims 33 to 37, comprising one or more of the above.

41. Capsid and, A nucleic acid vector containing a heterogeneous nucleic acid region containing a sequence encoding interfering RNA that includes a region complementary to the miR-128 target nucleic acid, and Recombinant adeno-associated virus (rAAV) particles, including those containing the virus.

42. The rAAV according to 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 according to claim 41, wherein the interfering RNA is a short hairpin RNA or a low molecular weight interfering RNA.

44. The rAAV according to claim 41, wherein the nucleic acid vector includes a promoter, and the sequence encoding the interfering RNA is functionally linked to the promoter.

45. The rAAV according to claim 44, wherein the promoter can induce the expression of the interfering RNA in skeletal muscle cells.

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

47. The rAAV according to claim 41, wherein the nucleic acid vector comprises AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV2R471A, AAV DJ, goat AAV, bovine AAV, or reversed terminal repeat derived from a mouse AAV serotype.

48. The capsids include AAV1 capsid, AAV2 capsid, AAV3 capsid, AAV4 capsid, AAV5 capsid, AAV6 capsid, AAV7 capsid, AAV8 capsid, AAVrh8 capsid, AAVrh8R capsid, AAV9 capsid, AAV10 capsid, AAVrh10 capsid, AAV11 capsid, AAV12 capsid, tyrosine capsid variant, heparin-binding capsid variant, AAV2R471A capsid, AAV2 / 2-7m8 capsid, AAV DJ capsid, AAV2 N587A capsid, and AAV2 rAAV according to claim 41, selected from E548A capsid, AAV2 N708A capsid, AAV V708K capsid, goat AAV capsid, AAV1 / AAV2 chimeric capsid, bovine AAV capsid, mouse AAV capsid, and rAAV2 / HBoV1 capsid.

49. Pharmacologically acceptable excipients, Oligonucleotide according to any one of claims 33 to 40 or rAAV according to any one of claims 41 to 48 A pharmaceutical composition containing the above.

50. A therapeutically effective composition for use in a method of treating age-related macular degeneration in a subject requiring such treatment, wherein the method comprises administering to the subject a therapeutically effective amount of an oligonucleotide according to any one of claims 1 to 8 or an rAAV according to any one of claims 9 to 16.

51. An miR-128 inhibitor for use in a method of treating age-related sarcopenia in a subject requiring such treatment, wherein the method comprises administering a therapeutically effective amount of the miR-128 inhibitor to the subject.

52. Antisense oligonucleotide, Short hairpin RNA, Small interfering RNA, or Recombinant adeno-associated virus (rAAV) particles comprising a capsid and a nucleic acid vector containing a heterogeneous nucleic acid region including a sequence encoding interfering RNA, wherein the interfering RNA is selected from antisense oligonucleotides, short hairpin RNA, or small interfering RNA. A miR-128 inhibitor according to claim 51, selected from the above.

53. A therapeutically effective composition for use in a method of treating age-related sarcopenia in a subject requiring such treatment, wherein the method comprises administering to the subject a therapeutically effective amount of an oligonucleotide according to any one of claims 1 to 8 or an rAAV according to any one of claims 9 to 16.

54. The therapeutically effective composition according to claim 53, wherein the oligonucleotide is a single-stranded oligonucleotide.

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

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

57. The therapeutically effective composition according to claim 56, wherein the oligonucleotide comprises 14 to 23 interconnected nucleotides.

58. The therapeutically effective composition according to any one of claims 53 to 57, wherein the oligonucleotide comprises at least one modified sugar nucleoside.

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

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

61. The therapeutically effective composition according to any one of claims 58 to 60, wherein the modified sugar nucleoside is a 2'-modified sugar nucleoside.

62. The therapeutically effective composition according to 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'-methoxyethoxy.

63. The therapeutically effective composition according to any one of claims 58 to 60, wherein the modified sugar nucleoside is a crosslinked nucleic acid.

64. The therapeutically effective composition according to claim 63, wherein the crosslinked nucleic acid is locked nucleic acid.

65. The therapeutically effective composition according to claim 63, wherein the crosslinked nucleic acid is ethylene crosslinked nucleic acid.

66. The nucleic acid base sequence is as follows: 5'-ACCGGTTCACTGTG-3' (Sequence ID 1), 5'-GACCGGTTCACTGT-3' (SEQ ID NO: 2), and 5'-AGACCGGTTCACTGTG-3' (Sequence ID 3) A therapeutically effective composition according to any one of claims 58 to 60, comprising one or more of the above.

67. The aforementioned oligonucleotide is a gapmer comprising a 5'-wing, a 3'-wing, and a gap. The 5'-wing and the 3'-wing each contain 1 to 5 nucleotides, Each nucleotide in the 5'-wing and the 3'-wing is independently a crosslinked nucleic acid, and Each nucleotide in the gap is a deoxyribonucleotide. A therapeutically effective composition according to any one of claims 53 to 66.

68. The therapeutically effective composition according to any one of claims 53 to 67, wherein at least one nucleoside bond in the oligonucleotide is a phosphorothioate diester.

69. The therapeutically effective composition according to claim 68, wherein at least 50% of the nucleoside bonds in the oligonucleotide are phosphorothioate diesters.

70. The therapeutically effective composition according to claim 68 or 69, wherein 100% of the nucleoside bonds in the oligonucleotide are phosphorothioate diesters.

71. The therapeutically effective composition according to any one of claims 53 to 70, further comprising administering the oligonucleotide as a guide strand in short interfering RNA.

72. The therapeutically effective composition according to any one of claims 53 to 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 according to any one of claims 53 to 72, wherein the route of administration is intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection.

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

75. The method according to claim 74, wherein the incretin mimetic is a glucagon-like peptide-1 (GLP-1) receptor agonist.

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

77. The method according to claim 76, wherein the GLP-1 receptor agonist is semaglutide or tilzepatide.

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