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WO2025054607A1 - Compositions et méthodes de traitement de la sarcopénie - Google Patents

Compositions et méthodes de traitement de la sarcopénie Download PDF

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Publication number
WO2025054607A1
WO2025054607A1 PCT/US2024/045849 US2024045849W WO2025054607A1 WO 2025054607 A1 WO2025054607 A1 WO 2025054607A1 US 2024045849 W US2024045849 W US 2024045849W WO 2025054607 A1 WO2025054607 A1 WO 2025054607A1
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nucleic acid
mir
capsid
oligonucleotide
therapeutically effective
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Anders Michael NÄÄR
Melissa Ann BOLDRIDGE
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University of California Berkeley
University of California San Diego UCSD
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University of California Berkeley
University of California San Diego UCSD
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system

Definitions

  • Sarcopenia is defined as the progressive loss of muscle mass and function with aging. This loss of muscle mass and function is associated with an increase in adverse outcomes. Frailty and decreased mobility lead to falls, obesity, impaired ability to recover from illness, and ultimately increased mortality. Hie re are currently no approved drugs for the treatment of sarcopenia.
  • FIG. 1A-1B provide nucleotide sequences of exemplary antisense oligonucleotides
  • FIG. 1A SEQ ID NOs: 1-3, respectively
  • FIG. IB SEQ ID NO: 12
  • FIG. 2A-2I depict interim data showing the effect of a miR-128-3p targeting LNA ASO on skeletal muscle function in aged mice.
  • FIG. 3A-3I depict full experimental data showing the effect of a miR-128-3p targeting LNA ASO on skeletal muscle function in aged mice.
  • FIG. 4A-4D experimental data showing the effect of a miR-128-3p targeting LNA ASO on skeletal muscle mass in aged mice.
  • an “antisense oligonucleotide” refers to a nucleic acid sequence that is complementary to a DNA or RNA sequence, such as that of a microRNA.
  • RNA refers to a molecule comprising at least one or more ribonucleotide residues.
  • a "ribonucleotide” is a nucleotide with a hydroxyl group at the 2' position of a beta-D-ribofuranose moiety.
  • Tire tenn RNA includes double-stranded RNA, single -stranded RNA, isolated RNA, such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as altered RNA that differs from naturally occurring RNA by tire addition, deletion, substitution and/or alteration of one or more nucleotides. Nucleotides of the RNA molecules can also comprise nonstandard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides.
  • a "microRNA” is a single -stranded RNA molecule of about 21-23 nts in length.
  • miRNAs regulate gene expression. miRNAs are encoded by genes from whose DNA they are transcribed, but miRNAs are not translated into protein. Each primary miRNA transcript is processed into a short stem-loop structure before undergoing further processing into a functional miRNA. Mature miRNA molecules are partially complementary to one or more messenger RNA (mRNA) molecules, and their main function is to down-regulate gene expression.
  • mRNA messenger RNA
  • an interfering RNA refers to any double stranded or single stranded RNA sequence, capable, either directly or indirectly (i.e.. upon conversion), of inhibiting or down regulating gene expression by mediating RNA interference.
  • Interfering RNA includes but is not limited to small interfering RNA ("siRNA”) and small hairpin RNA (“shRNA”).
  • siRNA small interfering RNA
  • shRNA small hairpin RNA
  • RNA interference refers to the selective degradation of a sequence-compatible messenger RNA transcript.
  • an shRNA small hairpin RNA refers to an RNA molecule comprising an antisense region, a loop portion and a sense region, wherein the sense, region has complementary nucleotides that base pair with the antisense region to form a duplex stem.
  • the small hairpin RNA is converted into a small interfering RNA by a cleavage event mediated by the enzyme Dicer, which is a member of the RNase III family.
  • Dicer which is a member of the RNase III family.
  • a "small interfering RNA” or “siRNA” as used herein refers to any small RNA molecule capable of inhibiting or down regulating gene expression by mediating RNA interference in a sequence specific manner.
  • the small RNA can be for example, about 18 to 21 nucleotides long.
  • an “antagomir” refers to a small synthetic RNA having complementarity to a specific microRNA target, with either mispairing at the cleavage site or one or more base modifications to inhibit cleavage.
  • post-transcriptional processing refers to mRNA processing that occurs after transcription and is mediated, for example, by the enzymes Dicer and/or Drosha.
  • an effective amount is meant the amount of a required agent (e.g., an inhibitory nucleic acid of the present disclosure) or composition (e.g., composition comprising an inhibitory nucleic acid of the present disclosure), comprising the agent to ameliorate the symptoms of a disease relative, to an untreated patient.
  • a required agent e.g., an inhibitory nucleic acid of the present disclosure
  • composition e.g., composition comprising an inhibitory nucleic acid of the present disclosure
  • the effective amount of an agent or a composition used to practice a therapeutic treatment of a disease or disorder varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending, physician or veterinarian will decide the appropriate amount and dosage regimen.
  • sarcopenia encompasses any disorder characterized by progressive and generalized loss of skeletal muscle mass and strength with a risk of adverse outcomes such as physical disability, poor quality of life and death. Sarcopenia can be classified according to its cause and includes primary sarcopenia. activity-related sarcopenia. disease-related sarcopenia and nutrition-related sarcopenia. The primary sarcopenia refers to sarcopenia without any cause other than aging and is also referred to as age-related sarcopenia.
  • the activity -related sarcopenia refers to sarcopenia caused by bed rest, sedentary' lifestyle, deconditioning or zero-gravity conditions.
  • the disease-related sarcopenia refers to sarcopenia caused by advanced organ failure (heart, lung, liver, kidney, brain), inflammatory disease, malignancy or endocrine disease.
  • Hie nutrition-related sarcopenia refers to sarcopenia caused by malabsorption, gastrointestinal disorders, use of medications that cause anorexia or insufficient dietary intake of protein.
  • tire effect may be prophylactic in tenns of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease.
  • Treatment covers any treatment of a disease in a mammal, e.g., in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease.
  • an “AAV particle” is a virus which comprises a. viral genome with at least one payload region and at least one inverted, terminal repeat (ITR) region.
  • viral genome refers to the nucleic acid sequenced) encapsulated in an AAV particle.
  • Viral genomes comprise at least one payload region encoding a nucleic acid (e g., interfering RNA, etc.).
  • a “payload” or “payload region” is any nucleic acid molecule which encodes one or more nucleic acid as described herein.
  • a payload region comprises nucleic acid sequences that encode an antibody, an antibody-based composition, or a fragment thereof, but may also optionally comprise one or more functional or regulatory elements to facilitate transcription and/or nucleic acid expression.
  • the terms “individual,” “subject,” “host,” and “patient,” used interchangeably herein, refer to an individual organism, e.g., a mammal, including, but not limited to, murines, simians, humans, non-human primates, ungulates, felines, canines, bovines. ovines, mammalian fann animals, mammalian sport animals, and mammalian pets. In some cases, an “individual” is a human.
  • the term “about” used in connection with an amount indicates that the amount can vary by 10% of the stated amount.
  • “about 100” means an amount of from 90- 110.
  • the “about” used in reference to the lower amount of the range means that the lower amount includes an amount that is 10% lower than the lower amount of the range
  • “about” used in reference to the higher amount of the range means that the higher amount includes an amount 10% higher than the higher amount of the range.
  • from about 100 to about 1000 means that the range extends from 90 to 1100.
  • a and/or B is intended to include both A and B; A or B; A (alone); and B (alone).
  • the term “and/or” as used herein a phrase such as “A, B, and/or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).
  • the present disclosure provides inhibitory nucleic acids that reduce the level of miR- 128, including miR-128-3p, in a cell, and compositions comprising the inhibitory nucleic acids.
  • the present disclosure provides methods of treating various disorders, using an inhibitory nucleic acid of the present disclosure.
  • the present disclosure provides inhibitory nucleic acids that reduce the level of miR- 128-3p in a cell (e.g., a cell in an individual).
  • the inhibitory nucleic acids are antisense oligonucleotides (ASOs).
  • ASOs antisense oligonucleotides
  • the present disclosure provides ASOs that reduce the level of miR-128-3p in a cell (e.g., in a cell in an individual).
  • an miR-128 comprises the nucleotide sequence: tgagctgttg gattcggggc cgtagcactg tetgagaggt ttacatttct cacagtgaac eggtetettt ttcagctgct tc (SEQ ID NO:4).
  • an miR-128 comprises the nucleotide sequence: ugagcuguug gauucggggc cguagcacug ucugagaggu uuacauuucu cacagugaac cggucucuuuuucagcugcu uc (SEQ ID NO:5).
  • an miR-128 comprises the nucleotide sequence: tcacagtgaa ccggtctctt t (SEQ ID NO:6). In some cases, an miR-128 comprises the nucleotide sequence: ucacagugaa ccggucucuu u (SEQ ID NO:7). In some cases, an miR- 128 comprises the nucleotide sequence: tgtgcagtgg gaaggggggc cgatacactg taegagagtg agtagcaggt ctcacagtga accggtctct ttccctactg tgte (SEQ ID NO:8).
  • an miR-128 comprises the nucleotide sequence: ugugcagugg gaaggggggc cgauacacug uacgagagug aguagcaggu cucacaguga accggugugugu uuggguacug ugcu (SEQ ID NO:9).
  • an inhibitory nucleic acid e.g., an ASO
  • an ASO reduces the level of miR-128-3p in a cell more potently than an inhibitory nucleic acid (e.g., an ASO) comprising the nucleotide sequence: 5’-TTCACTGTG-3' (SEQ ID NO: 10.
  • an inhibitory nucleic acid (e.g., an ASO) of the present disclosure is at least 2-fold, at least 2.5-fold, at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, or at least 40-fold, more potent than an inhibitory nucleic acid (e.g., an ASO) comprising the nucleotide sequence: 5’-TTCACTGTG-3’ (SEQ ID NO: 10).
  • an inhibitory nucleic acid (e.g., an ASO) of the present disclosure reduces the level of miR-128-3p in a cell to an extent that is at least 10%, at least 15%, at least 20%, at least 25%, at least 50%, at least 75%, at least 90%, or more than 90%, greater than the extent to which an inhibitory nucleic acid (e.g., an ASO) comprising the nucleotide sequence 5'-TTCACTGTG-3’ (SEQ ID NO: 10) reduces the level of miR-128-3p in the cell, when administered in the same amount as the mhibi tory nucleic acid (e.g., an ASO) of the present disclosure.
  • an inhibitory nucleic acid e.g., an ASO
  • an inhibitory nucleic acid (e.g., an ASO) of the present disclosure achieves a reduction of at least 10%, at least 15%, at least 20%, at least 25%, at least 50%, at least 75%, or at least 90%, of the level of miR-128-3p in a cell in an individual when the individual is administered with an inhibitory nucleic acid (e.g., an ASO) of the present disclosure in an amount that is at least 10%.
  • an inhibitory nucleic acid e.g., an ASO
  • an ASO comprising the nucleotide sequence: 5’-TTCACTGTG-3’ (SEQ ID NO: 10) required to achieve the same reduction in tire level of miR-128-3p.
  • an inhibitory nucleic acid (e.g ., an ASO) of the present disclosure reduces the level of miR-128-3p in a cell more potently than an inhibitory nucleic acid (e.g., an ASO) comprising the nucleotide sequence: 5’-GGTTCACTGTG-3’ (SEQ ID NO: 11).
  • an inhibitory nucleic acid (e.g., an ASO) of the present disclosure is at least 2-fold, at least 2.5-fold, at least 5-fold, at least 10- fold, at least 15 -fold, at least 20-fold, at least 25-fold, at least 30-fold, or at least 40-fold, more potent than an inhibitory nucleic acid (e.g...
  • an inhibitory nucleic acid e.g., an ASO
  • an ASO reduces the level of miR-128-3p in a cell to an extent that is at least 10%, at least 15%.
  • an inhibitory nucleic acid e.g., an ASO
  • an inhibitory nucleic acid comprising the nucleotide sequence 5’- GGTTCACTGTG-3’ (SEQ ID NO:11) reduces the level of miR-128-3p in the cell, when administered in the same amount as the inhibitory nucleic acid (e.g., an ASO) of the present disclosure.
  • an inhibitory nucleic acid e.g., an ASO
  • an inhibitory nucleic acid e.g., an ASO
  • an inhibitory nucleic acid e.g., an ASO
  • an ASO an inhibitory nucleic acid of the present disclosure in an amount that is at least 10%, at least 15%, at least 20%, at least 25%, at least 50%, at least 75%, at least 90%, or more than 90%, less than the amount of an inhibitory nucleic acid (e.g., an ASO) comprising the nucleotide sequence: 5’-GGTTCACTGTG-3’ (SEQ ID NO: 11) required to achieve the same reduction in the level of miR-128-3p.
  • Inhibitory nucleic acids useful in the present methods and compositions include antisense oligonucleotides, ribozymes, external guide sequence (EGS) oligonucleotides, siRNA compounds, single- or double -stranded RNA interference (RNAi) compounds such as siRNA compounds, modified bases/locked nucleic acids (LNAs), antagomirs, peptide nucleic acids (PNAs), and other oligomeric compounds or oligonucleotide mimetics which hybridize to at least a portion of the target nucleic acid (i.e., miR-128) and modulate its function.
  • the inhibitory nucleic acids include antisense RNA, antisense DNA.
  • RNAi interference RNA
  • siRNA short interfering RNA
  • miRNA micro, interfering RNA
  • stRNA small, temporal RNA
  • shRNA short, hairpin RNA
  • RNAa small RNA-induced gene activation
  • saRNAs small activating RNAs
  • the inhibitory nucleic acids are 10 to 50, 13 to 50, or 13 to 30 nucleotides in length.
  • One having ordinary skill in the art will appreciate that this embodies oligonucleotides having antisense portions of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length, or any range therewithin.
  • an inhibitory nucleic acid of the present disclosure is 15 nucleotides in length.
  • an inhibitory nucleic acid of the present disclosure is 12 to 30 or 13 to 30 nucleotides in length.
  • One having ordinary skill in the art will appreciate that this embodies inhibitory nucleic acids having a length of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides.
  • tire inhibitory nucleic acids are chimeric oligonucleotides that contain two or more chemically distinct regions, each made up of at least one nucleotide. These oligonucleotides typically contain at least one region of modified nucleotides that confers one or more beneficial properties (such as, for example, increased nuclease resistance, increased uptake into cells, increased binding affinity for the target) and a region that is a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids.
  • beneficial properties such as, for example, increased nuclease resistance, increased uptake into cells, increased binding affinity for the target
  • Chimeric inhibitory nucleic acids of the present disclosure may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide mimetics as described above. Such compounds have also been referred to in the art as hybrids or gapmers.
  • a suitable inhibitory nucleic acid comprises the following nucleotide sequence: 5’-ACCGGTTCACTGTG-3’ (SEQ ID NO: 1) and has a length of from 14 nucleotides to 20 nucleotides.
  • the inhibitory nucleic acid comprises the following nucleotide sequence: 5’-ACCGGTTCACTGTG-3’ (SEQ ID NO: 1) and has a length of 14 nucleotides.
  • the inhibitory nucleic acid comprises one or more of: i) a locked nucleic acid (LNA); ii) a modified backbone: and iii) a 5 -methyl deoxy cytosine.
  • a suitable inhibitory nucleic acid comprises the nucleotide sequence, and all of the modifications, depicted in FIG. 1A and designated •‘NRC0090.”
  • the ASO referred to in FIG. 1A as NRC0090 is: +A*+C*/iMe- dC/*G*G*+T*+T*+C*A*C*+T*G*+T*+G, where “+” precedes an LNA; * precedes a phosphorothioated base, and /iMe-dC/ denotes an internal 5-methyl deoxy cytosine.
  • a suitable inhibitory nucleic acid comprises the following nucleotide sequence: 5 -GACCGGTTCACTGT-3’ (SEQ ID NO:2) and has a length of from 14 nucleotides to 20 nucleotides.
  • the inhibitory nucleic acid comprises the following nucleotide sequence: 5’-GACCGGTTCACTGT-3’ (SEQ ID NO:2) and has a length of 14 nucleotides.
  • the inhibitory' nucleic acid comprises one or more of: i) a locked nucleic acid (LNA); ii) a modified backbone; and iii) a 5-methyl deoxy cytosine.
  • a suitable inhibitory nucleic acid comprises the nucleotide sequence, and all of the modifications, depicted in FIG. 1A and designated “NRC0091.”
  • the ASO referred to in FIG. 1A as NRC0091 is +G*+A*C*/iMe- dC/*+G*G*T*T*+C*+A*C*+T*+G*+T, where ‘"+” precedes an LNA; * precedes a phosphorothioated base, and /iMe-dC/ denotes an internal 5-methyl deoxy cytosine.
  • a suitable inhibitory nucleic acid comprises the following nucleotide sequence: 5’-AGACCGGTTCACTGTG-3‘ (SEQ ID NO:3) and has a length of from 14 nucleotides to 20 nucleotides.
  • the inhibitory nucleic acid comprises the following nucleotide sequence: AGACCGGTTCACTGTG-3 ’ (SEQ ID NO:3) and has a length of 14 nucleotides.
  • the inhibitory nucleic acid comprises one or more of: i) a locked nucleic acid (LNA); ii) a modified backbone; and iii) a 5-methyl deoxy cytosine.
  • LNA locked nucleic acid
  • a suitable inhibitory- nucleic acid comprises the nucleotide sequence, and all of the modifications, depicted in FIG. 1 A and designated “NRC0119.”
  • the ASO referred to in FIG. 1A as NRC0119 is +A*+G*A*+C*/iMe- dC/*+G*G*+T*T*+C*A*C*+T*G*+T*+G, where “+” precedes an LNA; * precedes a phosphorothioated base, and /iMe-dC/ denotes an internal 5-methyl deoxy cytosine.
  • the inhibitory nucleic acid comprises at least one nucleotide modified at the 2' position of the sugar, e.g., a 2'-O-alkyl, 2'-O-alkyl-O-alkyl, 2'-fluoro, 2'-methoxy, or2’- methoxyethoxy -modified nucleotide.
  • RNA modifications include 2'-fluoro, 2'-amino and 2'O-methyl modifications on the ribose of pyrimidines, abasic residues or an inverted base at the 3' end of the RNA.
  • oligonucleotides Such modifications are routinely incorporated into oligonucleotides and these oligonucleotides have been shown to have a higher Tm (i .e., higher target binding affinity) than: 2'- deoxyoligonucleotides against a given target.
  • Tm i .e., higher target binding affinity
  • nucleotide and nucleoside modifications have been shown to make the oligonucleotide into which they arc incorporated more resistant to nuclease digestion than the native oligodeoxy nucleotide; these modified oligos survive intact for a longer time than unmodified oligonucleotides.
  • modified oligonucleotides include those comprising modified backbones, for example, phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages.
  • inhibitory nucleic acids are oligonucleotides with phosphorothioate backbones and those with hctcroatom backbones, particularly CH 2 — NH— 0— CH 3 , CH 3 — N(CH 3 )— 0— CH 2 (known as a methylene(methylimino) or MMI backbone], CH 2 — O— N (CH 3 )— CH2, CH 2 — N (CH 3 )— N (CH 3 )— CH 2 and O-N (CH 3 )-CH 2 -CH 2 backbones, wherein the native-phosphodiester backbone is represented as O-P— O— CH); amide backbones (see De Mesmaeker et al. Ace.
  • PNA peptide nucleic acid
  • Phosphorus-containing linkages include, but are not limited to. phosphorothioates. chiral phosphorothioates.
  • phosphorodithioates phosphotriesters; aminoalkylphosphotriesters, methyl and other alkyl phosphonates comprising 3'alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates comprising 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having nonnal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'.
  • Modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl intemucleoside linkages, mixed heteroatom and alkyl or cycloalkyl intemucleoside linkages, or one or more short chain heteroatomic or heterocyclic intemucleoside linkages.
  • These comprise those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide; sulfoxide and sulfone backbones; fonnacetyl and thioformacetyl backbones; methylene fonnacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH 2 component parts.
  • siloxane backbones siloxane backbones
  • sulfide sulfoxide and sulfone backbones
  • fonnacetyl and thioformacetyl backbones methylene fonnacetyl and thioformacetyl backbones
  • One or more substituted sugar moieties can also be included, e.g., one of the following at the 2' position: OH, SH. SCH 3 , F, OCN, OCH 3 OCH 3 , 0CH 3 , 0(CH 2 )n CH 3 , O(CH 2 )nNH 2 or O(CH 2 )nCH 3 where n is from 1 to about 10; Cl to CIO lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN; CF 3 ; OCF 3 ; O-, S— , or N-alkyl; O-, S— , or N-alkenyl; SOCH 3 ; SO 2 CH 3 ; ONO 2 ; NO2; N3; NH2; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleaving group; a reporter
  • a suitable modification includes 2'-methoxyethoxy [2'— O — CH2CH2OCH3, also known as: 2'-O-(2- methoxyethyl)] (Martin et al, Helv. Chim. Acta, 1995, 78, 486).
  • Other modifications include 2'-methoxy (2'-O--CH3), 2'-propoxy ⁇ '-OCFLCFhCIt) and 2'-fluoro (2'-F) .
  • Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar on the 3' terminal nucleotide and the 5' position of 5' terminal nucleotide.
  • Oligonucleotides may also have sugar mimetics such as cyclobutyls in place of the pentofuranosyl group.
  • Inhibitory nucleic acids can also include, additionally or alternatively, nucleobase (often referred to in the art simply as “base”) modifications or substitutions.
  • nucleobase often referred to in the art simply as “base”
  • “unmodified” or “natural” nucleobases include adenine (A), guanine (G), thymine (T), cytosine, (C) and uracil (U).
  • Modified nucleobases include nucleobases found only infrequently or transiently in natural nucleic acids, e.g., hypoxanthine, 6-methyladenine, 5-Me pyrimidines, particularly 5-methylcytosine (also referred to as 5-methyl-2' deoxycytosine and often referred to in the art as 5-Me-C), 5 -hydroxymethylcytosine (HMC).
  • nucleobases found only infrequently or transiently in natural nucleic acids e.g., hypoxanthine, 6-methyladenine, 5-Me pyrimidines, particularly 5-methylcytosine (also referred to as 5-methyl-2' deoxycytosine and often referred to in the art as 5-Me-C), 5 -hydroxymethylcytosine (HMC).
  • hypoxanthine 6-methyladenine
  • 5-Me pyrimidines particularly 5-methylcytosine (also referred to as 5-methyl-2' deoxycytosine and often referred to in the art as 5-
  • glycosyl HMC and gentobiosyl HMC as well as synthetic nucleobases, e.g., 2-aminoadenine, 2- (methylamino)adenine, 2-(imidazolylalkyl)adenine, 2-(aminoalklyamino)adenine or other heterosubstituted alkyladenines, 2-thiouracil, 2-thiothymine, 5 -bromouracil, -hydroxymethyluracil, 8- azaguanine, 7-deazaguanine, N6 (6-aminohexyl)adenine and 2,6-diaminopurine.
  • 2-aminoadenine 2- (methylamino)adenine, 2-(imidazolylalkyl)adenine, 2-(aminoalklyamino)adenine or other heterosubstituted alkyladenines
  • 2-thiouracil 2-thiothymine
  • 5 -bromouracil -
  • an inhibitory nucleic acid of the present disclosure comprises one or more 5-Me-C.
  • both a sugar and an intemucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups.
  • the base units are maintained for hybridization with an appropriate nucleic acid target compound.
  • an oligomeric compound an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA).
  • PNA peptide nucleic acid
  • the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, for example, an aminoethylglycine backbone.
  • Hie nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.
  • Representative United States patents that teach the preparation of PNA compounds comprise, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331 ; and 5,719,262, each of which is herein incorporated by reference.
  • Inhibitory nucleic acids can also include one or more nucleobase (often referred to in the art simply as “base”) modifications or substitutions.
  • base nucleobase
  • “unmodified” or “natural” nucleobases comprise the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T). cytosine (C) and uracil (U).
  • Modified nucleobases comprise other synthetic and natural nucleobases such as 5 -methylcytosine (5-me-C), 5 -hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosinc. 5-halouracil and cytosine, 5- propynyl uracil and cytosine.
  • 5-halo particularly 5 -bromo, 5 -trifluoromethyl and other 5 -substituted uracils and cytosines, 7-methylquanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7 -deazaadenine and 3- deazaguanine and 3 -deazaadenine.
  • nucleobases comprise those disclosed in U.S. Pat. No. 3,687,808, those disclosed in 'The Concise Encyclopedia of Polymer Science And Engineering', pages 858-859, Kroschwitz. J. I., ed. John Wiley & Sons, 1990. those disclosed by English et al., Angewandle Chemie. International Edition', 1991, 30, page 613, and those disclosed by Sanghvi. Y. S.. Chapter 15, Antisense Research and Applications', pages 289-302, Crooke, S. T. and Lebleu, B. ea., CRC Press, 1993. Certain of these nucleobases are particularly usefid for increasing the binding affinity of an inhibitory nucleic acid.
  • 5-substitutcd pyrimidines 6-azapyrimidincs and N-2, N-6 and 0-6 substituted purines, comprising 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine, 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2 ⁇ 0>C (Sanghvi. Y. S., Crooke, S. T. and Lebleu, B., eds. Antisense Research and Applications', CRC Press, Boca Raton, 1993, pp. 276-278) and are suitable for inclusion in an inhibitory nucleic acid, e.g., alone or together with 2 -O- methoxyethyl sugar modifications.
  • tire inhibitory nucleic acids are chemically linked to one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide.
  • moieties comprise but are not limited to, lipid moieties such as a cholesterol moiety; cholic acid; a thioether, e.g., hexyl-S-tritylthiol; a thiocholesterol; an aliphatic chain, e g., dodecandiol or undecyl residues; a phospholipid; e.g., di-hexadecyl-rac -glycerol or triethylammonium 1,2-di-O-hexadecyl-rac- glycero-3-H-phosphonate; a polyamine or a polyethylene glycol chain; adamantane acetic acid; a palmityl moiety; an octa
  • conjugate groups can include conjugate groups covalently bound to functional groups such as primary' or secondary hydroxyl groups.
  • Conjugate groups suitable for use include intercalators, importer molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers.
  • Typical conjugate groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes.
  • Groups that enhance the pharmacodynamic properties include groups that improve uptake, enhance resistance to degradation and/or strengthen sequence-specific hybridization with the target nucleic acid.
  • Groups that enhance the pharmacokinetic properties include groups that improve uptake, distribution, metabolism or excretion of an inhibitory nucleic acid. Representative conjugate groups are disclosed in International Patent Application No. PCT/US92/09196, filed Oct. 23, 1992, and U.S. Pat. No. 6,287,860, which are incorporated herein by reference.
  • Conjugate moieties include, but are not limited to, lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-5 -tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1.2-di-O-hexadecyl-rac-glycero-3-H- phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxy cholesterol moiety.
  • lipid moieties such as a cholesterol moiety, cholic acid, a thio
  • the inhibitory nucleic acids useful in the present methods are sufficiently complementary to all or part of miR-128, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired effect.
  • “Complementary” refers to the capacity for pairing, through hydrogen bonding, between two sequences comprising naturally or non-naturally occurring bases or analogs thereof. For example, if a base at one position of an inhibitory nucleic acid is capable of hydrogen bonding with a base at the corresponding position of a miR-128 sequence, then the bases are considered to be complementary to each other at that position. 100% complementary to is not required.
  • hybridization means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases.
  • adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds.
  • “Complementary,” as used herein, refers to the capacity for precise pairing between two nucleotides. Tire inhibitory nucleic acids and the miR-128 are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied, by nucleotides that can hydrogen bond with each other.
  • “specifically hybridizable” and “complementary” are terms which are used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between the inhibitory nucleic acid and the miR-128 target sequence. For example, if a base at one position of an inhibitory nucleic acid is capable of hydrogen bonding with a base at the corresponding position of a miR-128 molecule; then the bases are considered complementary to each other at that position.
  • a complementary nucleic acid sequence need not be 100% complementary’ to that of its target nucleic acid to be specifically hybridizable.
  • a complementary' nucleic acid sequence for purposes of the present methods is specifically hybridizable when binding of the sequence to the target miR-128 molecule interferes with the normal function of the target miR-128 to cause a loss of activity, and there is a sufficient degree of complementarity to avoid non-specific binding of the sequence to non-target miR- 128 sequences under conditions in which specific binding is desired, e.g., under physiological conditions in the case of in vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed under suitable conditions, of stringency.
  • stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, less than about 500 mM NaCl and 50 mM trisodium citrate, or less than about 250 mM NaCl and 25 mM trisodium citrate.
  • Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, or at least about 50% formamide.
  • Stringent temperature conditions will ordinarily include temperatures of at least 30 degrees C, at least about 37 degrees C, or at least about 42 degrees C.
  • Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art.
  • Various levels of stringency are accomplished by combining these various conditions as needed.
  • hybridization will occur at 30 degrees C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS.
  • hybridization will occur at 37 degrees C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 pg/ml denatured salmon sperm DNA (ssDNA).
  • hybridization will occur at 42 degrees C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 pg/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.
  • the inhibitory nucleic acids useful in the methods described herein have at least 80% sequence complementarity to a target region within the target nucleic acid, e.g., 90%, 95%, or 100% sequence complementarity to the target region within miR-128 (e.g., a target region comprising the seed sequence).
  • a target region within the target nucleic acid
  • miR-128 e.g., a target region comprising the seed sequence
  • an antisense compound in which 18 of 20 nucleobases of the antisense oligonucleotide are complementary, and would therefore specifically hybridize, to a target region would represent 90 percent complementarity.
  • Percent complementarity of an inhibitory nucleic acid with a region of a target nucleic acid can be determined routinely using basic local alignment search tools (BLAST programs) (Altschul et aL, J. Mol. BioL, 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656).
  • An inhibitory nucleic acid e.g., an ASO
  • tire inhibitory nucleic acids must retain specificity for their target, i.e., must not directly bind to, or directly significantly affect expression levels of transcripts other than tire intended target.
  • Antisense e.g., an ASO
  • an inhibitory’ nucleic acid of the present disclosure arc ASOs.
  • ASOs are typically designed to block expression of a DNA or RNA target by binding to the target and halting expression at the level of transcription, translation, or splicing.
  • ASOs of the present disclosure are complementary nucleic acid sequences designed to hybridize under stringent conditions to a miR-128 target sequence.
  • oligonucleotides are chosen that are sufficiently complementary to the target, i.e., that hybridize sufficiently well and with sufficient specificity, to give the desired effect.
  • Modified Bases/Locked Nucleic Acids (LNAs) Modified Bases/Locked Nucleic Acids (LNAs)
  • an inhibitory nucleic acid of the present disclosure comprises one or more modified bonds or bases.
  • Modified bases include phosphorothioate. methylphosphonate, peptide nucleic acids, bridged nucleic acid (BNA) and/or locked nucleic acid (LNA) molecules.
  • BNA bridged nucleic acid
  • LNA locked nucleic acid
  • certain implementations use a bridged nucleic acid that includes a linkage across the ribose ring.
  • Bridges can include an ethylene bridged nucleic acid.
  • Some BNAs include LNA molecules, where the modified nucleotides are locked nucleic acid molecules, including [alpha] -L-LNAs.
  • LNAs comprise ribonucleic acid analogues wherein the ribose ring is "locked" by a methylene bridge between the 2'-oxygen and the 4'-carbon-i.e., oligonucleotides, containing at least one LNA monomer, that is. one 2'-O.4'-C-methylene- 0-D-ribofiiranosyl nucleotide.
  • LNA bases form standard Watson-Crick base pairs but the locked configuration increases the rate and stability of the basepairing reaction (Jepsen et al., Oligonucleotides, 14, 130-146 (2004)).
  • LNAs also have increased affinity to base pair with RNA as compared to DNA.
  • LNAs especially useful as probes for fluorescence in situ hybridization (FISH) and comparative genomic hybridization, as knockdown tools for miRNAs, and as antisense oligonucleotides to target mRNAs or other RNAs.
  • the LNA molecules can include molecules comprising 10-30, e.g., 12-24, e.g., 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in each strand, wherein one of the strands is substantially identical, e.g., at least 80% (or more, e.g., 85%, 90%, 95%, or 100%) identical, e.g.. having 3. 2, 1, or 0 mismatched nucleotide(s), to a miR-128 target sequence.
  • Tire LNA molecules can be chemically synthesized using methods known in the art.
  • the inhibitory’ nucleic acid is an antagomir.
  • Antagomirs are chemically modified antisense oligonucleotides that target a miR-128 target sequence.
  • an antagomir for use in the methods described herein can include a nucleotide sequence sufficiently complementary to hybridize to a miR-128 target sequence of about 12 to 25 nucleotides, or from about 15 to 23 nucleotides.
  • antagomirs include a cholesterol moiety, e g., at the 3'-end.
  • antagomirs have various modifications for RNase protection and pharmacologic properties such as enhanced tissue and cellular uptake.
  • an antagomir can have one or more of complete or partial 2'-O-methylation of sugar and/or a phosphorothioate backbone. Phosphorothioate modifications provide protection against RNase activity and their lipophilicity contributes to enhanced tissue uptake.
  • the antagomir can include six phosphorothioate backbone modifications; two phosphorothioates are located at the 5'-end and four at the 3'-end.
  • Antagomirs useful in the present methods can also be modified with respect to their length or otherwise the number of nucleotides making up the antagomir.
  • the antagomirs must retain specificity for their target, i.e., must not directly bind to, or directly significantly affect expression levels of, transcripts other than the intended target.
  • the inhibitory nucleic acid is locked and includes a cholesterol moiety (e.g., a locked antagomir).
  • the inhibitory nucleic acid is a gapmer.
  • Gapmers are chemically modified antisense oligonucleotides that target a miR-128 target sequence.
  • a gapmer for use in tire methods described herein can include a nucleotide sequence sufficiently complementary to hybridize to a miR-128 target sequence of about 7 to 50 nucleotides, of about 7 to 30 nucleotides, and/or of about 14-23 nucleotides.
  • Tire wing structures may be of the same size (e.g., both 5'- and 3’- wings are 5 nucleotides), or the wing structures may be of the different sizes (e.g., ’-wing is 5 nucleotides, while 3’-wing is 4 nucleotides).
  • siRNA/shRNA siRNA/shRNA
  • an inhibitory nucleic acid of the present disclosure is an interfering RNA, including but not limited to a small interfering RNA (“siRNA”) or a small hairpin RNA (“shRNA”).
  • siRNA small interfering RNA
  • shRNA small hairpin RNA
  • the interfering RNA can be assembled from two separate oligonucleotides, where one strand is the sense strand and the other is the antisense strand, wherein the antisense and sense strands are self-complementary (i.e., each strand comprises nucleotide sequence that is complementary to nucleotide sequence in the other strand; such as where the antisense strand and sense strand form a duplex or double stranded structure); the antisense strand comprises nucleotide sequence that is complementary to a nucleotide sequence in a target nucleic acid molecule or a portion thereof (i.e., an undesired gene) and the sense strand comprises nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof.
  • interfering RNA is assembled from a single oligonucleotide, where the self-complementary sense and antisense regions are linked by means of nucleic acid based or non-nucleic acid-based linker(s).
  • the interfering RNA can be a polynucleotide with a duplex, asymmetric duplex, hairpin or asymmetric hairpin secondary structure, having self-complementary sense and antisense regions, wherein the antisense region comprises a nucleotide sequence that is complementary to nucleotide sequence in a separate target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof.
  • the interfering can be a circular single-stranded polynucleotide having two or more loop structures and a stem comprising self- complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic-acid sequence or a portion thereof, and wherein the circular polynucleotide can be processed either in vivo or in vitro to generate an active siRNA molecule capable of mediating RNA interference.
  • the interfering RNA coding region encodes a self-complementary RNA molecule having a sense region, an antisense region and a loop region.
  • RNA molecule when expressed, desirably forms a "hairpin" structure, and is referred to herein as an "shRNA,"
  • the loop region is generally between about 2 and about 10 nucleotides in length. In some embodiments, the loop region is from about 6 to about 9 nucleotides in length. In some embodiments, the sense region and the antisense region are between about 15 and about 20 nucleotides in length.
  • the small hairpin RNA is converted into a siRNA by a cleavage event mediated by the enzyme Dicer, which is a member of the RNase III family.
  • Dicer which is a member of the RNase III family.
  • the siRNA is then capable of inhibiting the expression of a gene with which it shares homology.
  • siRNAs The target RNA cleavage reaction guided by siRNAs is highly sequence specific.
  • siRNAs containing a nucleotide sequence identical to a portion of the target nucleic acid are used for inhibition.
  • 100% sequence identity between the siRNA and the target gene is not required.
  • the present disclosure has the advantage of being able to tolerate sequence variations that might be expected due to genetic mutation, strain polymorphism, or evolutionary divergence.
  • siRNA sequences with insertions, deletions, and single point mutations relative to the target sequence have also been found to be effective for inhibition.
  • siRNA sequences with nucleotide analog substitutions or insertions can be effective for inhibition.
  • the siRNAs must retain specificity for their target, i.e., must not directly bind to, or directly significantly affect expression levels of, transcripts other than the intended target.
  • Inhibitory nucleic acids can be synthesized in vitro by well-known chemical synthesis techniques, as described in, e.g., Adams (1983) J. Am. Chem. Soc. 105:661; Belousov (1997) 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; U.S. Pat. No. 4,458,066.
  • compositions including pharmaceutical compositions, comprising an inhibitory nucleic acid of the present disclosure.
  • An inhibitory nucleic acid of the present disclosure may be referred to below as an “agent” or an “active agent.”
  • tire compositions are formulated with a pharmaceutical acceptable carrier.
  • the pharmaceutical compositions and fonnulations can be administered parenterally, topically, orally or by local administration, such as by aerosol or transdermally.
  • the pharmaceutical compositions can be formulated in any way and can be administered in a variety of unit dosage forms depending upon the condition or disease and the degree of illness, the general medical condition of each patient, the resulting preferred method of administration and the like. Details on techniques for formulation and administration of pharmaceuticals are well described in the scientific and patent literature, see, e.g., Remington: The Science and Practice of Pharmacy, 21st ed.. 2005.
  • the inhibitory nucleic acids can be administered alone or as a component of a pharmaceutical formulation (composition).
  • An inhibitory nucleic acid can be formulated for administration, in any convenient way for use in human or veterinary medicine.
  • Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
  • Formulations of an inhibitory 7 nucleic acid of the present disclosure include those suitable for intradermal, inhalation, oral/nasal, topical, parenteral, rectal, and/or intravaginal administration.
  • Tire fonnulations may conveniently be presented in unit dosage fonn and may be prepared by any methods well known in the art of pharmacy.
  • the amount, of active ingredient (e.g., an inhibitory nucleic acid of the present disclosure) which can be combined with a carrier material to produce a single dosage fonn will vary depending upon the host being treated, the particular mode of administration, e.g., intradermal or inhalation.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form w ill generally be that amount of the compound which produces a therapeutic effect, e.g., reduction or suspension of muscle loss.
  • compositions can be prepared according to any method known to the art for the manufacture of pharmaceuticals.
  • Such drugs can contain sweetening agents, flavoring agents, coloring agents and preserving agents.
  • a formulation can be admixed with nontoxic pharmaceutically acceptable excipients which are suitable for manufacture.
  • Formulations may comprise one or more diluents, emulsifiers, preservatives, buffers, excipients, flavorants, colorants, bulking agents, solvents, lubricants, binders, surfactants, disintegrants, etc. and may be provided in such forms as liquids, powders, emulsions, lyophilized powders, sprays, creams, lotions, controlled release formulations, tablets, pills, gels, on patches, in implants, etc.
  • compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in appropriate and suitable dosages. Such carriers enable the pharmaceuticals to be formulated in unit dosage forms as tablets, pills, powder, dragees, capsules, liquids, lozenges, gels, syrups, slurries, suspensions, etc., suitable for ingestion by the patient.
  • Pharmaceutical preparations for oral use can be formulated as a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable additional compounds, if desired, to obtain tablets or dragee cores.
  • Suitable solid excipients are carbohydrate or protein fillers include, e.g., sugars, including lactose, sucrose, mannitol, or sorbitol; starch from com, wheat, rice, potato, or other plants; cellulose such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxy-methylcellulose; and gums including arabic and tragacanth; and proteins, e.g., gelatin and collagen.
  • Disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.
  • Push-fit capsules can contain active agents mixed with a filler or binders such as lactose or starches, lubricants such as talc or magnesium stearate, and, optionally, stabilizers.
  • the active agents can be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycol with or without stabilizers.
  • polyoxyethylene stearate a condensation product of ethylene oxide with a long chain aliphatic alcohol (e.g., heptadecaethylene oxycetanol), a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol (e.g., polyoxyethylene sorbitol mono-oleate), or a condensation product of ethylene oxide with a partial ester derived from fatty acid and a hexitol anhydride (e.g., polyoxyethylene sorbitan mono-oleate).
  • a condensation product of ethylene oxide with a long chain aliphatic alcohol e.g., heptadecaethylene oxycetanol
  • a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol e.g., polyoxyethylene sorbitol mono-oleate
  • the aqueous suspension can also contain one or more preservatives such as ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents and one or more Sweetening agents, such as sucrose, aspartame or saccharin. Formulations can be adjusted for osmolarity.
  • oil-based pharmaceuticals are used for administration of an inhibitory nucleic acid. Oil-based suspensions can be formulated by suspending an active agent in a vegetable oil, such as arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin; or a mixture of these. See e.g.. U.S. Pat. No.
  • the oil suspensions can contain a thickening agent, such as beeswax, hard paraffin or cetyl alcohol.
  • Sweetening agents can be added to provide a palatable oral preparation, such as glycerol, sorbitol or sucrose.
  • an antioxidant such as ascorbic acid.
  • an injectable oil vehicle see Minto (1997) J. Pharmacol. Exp. Ther. 281:93-102.
  • compositions can also be in the form of oil-in-water emulsions.
  • the oily phase can be a vegetable oil or a mineral oil, described above, or a mixture of these.
  • Suitable emulsifying agents include naturally occurring gums, such as gum acacia and gum tragacanth, naturally occurring phosphatides, such as soybean lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan mono-oleate, and condensation products of these partial esters with ethylene oxide, such as polyoxyethylene sorbitan mono-oleate.
  • the emulsion can also contain sweetening agents and flavoring agents, as in the formulation of syrups and elixirs.
  • an injectable oil-in-water emulsion comprises a paraffin oil, a sorbitan monoolcatc, an ethoxylated sorbitan monoolcatc and/or an ethoxylated sorbitan trioleate.
  • compositions can be administered by intranasal, intraocular and intravaginal routes including suppositories, insufflation, powders and aerosol formulations (for examples of steroid inhalants, see e.g., Rohatagi (1995) J. Clin. Pharmacol. 35: 1187-1193; Tjwa (1995) Ann. Allergy Asthma Immunol. 75: 107-111).
  • Suppositories formulations can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary’ temperatures but liquid at body temperatures and will therefore melt in the body to release the drug.
  • suitable non-irritating excipient which is solid at ordinary’ temperatures but liquid at body temperatures and will therefore melt in the body to release the drug.
  • Such materials are cocoa butter and polyethylene glycols.
  • a pharmaceutically acceptable excipient can be selected from lipids or polymers, including poly(amidoamine), poly(propyleneimine), poly (E-ly sine).
  • the phannaceutical compositions can be delivered transdermally, by a topical route, formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes jellies, paints, powders, and aerosols.
  • the pharmaceutical compositions can be delivered as microspheres for slow release in the body.
  • microspheres can be administered via intradermal injection of drug which slowly release subcutaneously; see Rao (1995) J. Biomater Sci. Polyrn. Ed. 7:623-645; as biodegradable and injectable gel formulations, see, e.g., Gao (1995) Phann. Res. 12:857-863 (1995); or, as microspheres for oral administration, see, e.g., Eyles (1997) J. Pham. Pharmacol. 49:669-674.
  • the phannaceutical compositions can be parenterally administered, such as by intravenous (IV) administration or administration into a body cavity or lumen of an organ.
  • IV intravenous
  • These formulations can comprise a solution of active agent (e.g., an inhibitory nucleic acid of the present disclosure) in a pharmaceutically acceptable carrier.
  • active agent e.g., an inhibitory nucleic acid of the present disclosure
  • a pharmaceutically acceptable carrier e.g., an inhibitory nucleic acid of the present disclosure
  • Acceptable vehicles and solvents that can be employed arc water and Ringer's solution, an isotonic sodium chloride.
  • sterile fixed, oils can be employed as a solvent or suspending medium.
  • any bland fixed oil can be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid can likewise be used in the preparation of injectables.
  • formulations may be sterilized by conventional, well known sterilization techniques.
  • the formulations may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like.
  • Tire concentration of active agent in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight, and the like, in accordance with the particular mode of administration selected and the patient's needs.
  • the formulation can be a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension.
  • This suspension can be formulated using those suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation can also be a suspension in a nontoxic parenterally acceptable diluent or solvent, such as a solution of 1,3 -butanediol.
  • Tire administration can be by bolus or continuous infusion (e.g., substantially uninterrupted introduction into a blood vessel for a specified period of time).
  • the pharmaceutical composition can be lyophilized.
  • Stable lyophilized compositions comprising an inhibitory nucleic acid can be made by lyophilizing a solution comprising a pharmaceutical composition of the present disclosure and a bulking agent, e.g., mannitol, trehalose, raffinose, and sucrose or mixtures thereof.
  • a process for preparing a stable lyophilized formulation can include lyophilizing a solution of about 2.5 mg/mL nucleic acid, about 15 mg/mL sucrose, about 19 mg/mL NaCl, and a sodium citrate buffer having a pH greater than 5.5 but less than 6.5. See, e.g., U.S. 20040028670.
  • Tire compositions and fonnulations can be delivered by the use of liposomes.
  • liposomes particularly where the liposome surface carries ligands specific for target cells, or are otherwise preferentially directed to a specific organ, one can focus the delivery of the active agent (e.g., an inhibitory nucleic acid of the present disclosure) into target cells w vivo.
  • the active agent e.g., an inhibitory nucleic acid of the present disclosure
  • liposome means a vesicle composed of amphiphilic lipids arranged in a bilayer or bilayers. Liposomes are unilamellar or multilamellar vesicles that have a membrane fonned from a lipophilic material and an aqueous interior that contains the composition to be delivered. Cationic liposomes are positively charged liposomes that are believed to interact with negatively charged DNA molecules to form a stable complex. Liposomes that are pH-sensitive or negatively charged are believed to entrap DNA rather than complex with it. Both cationic and noncationic liposomes have been used to deliver DNA to cells.
  • Liposomes can also include "stcrically stabilized" liposomes, i.c., liposomes comprising one or more specialized lipids. When incorporated into liposomes, these specialized lipids result in liposomes with enhanced circulation lifetimes relative to liposomes lacking such specialized lipids.
  • sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome comprises one or more glycolipids or is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety.
  • PEG polyethylene glycol
  • Tire fonnulations of the present disclosure can be administered for prophylactic and/or therapeutic treatments.
  • compositions are administered to a subject in need thereof (e.g., an individual who is at risk of (e.g., at greater risk than the general population) or has a disorder described herein) in an amount sufficient to cure, alleviate or partially arrest the clinical manifestations of the disorder or its complications; this can be called a therapeutically effective amount.
  • the amount of pharmaceutical composition adequate to accomplish this is a therapeutically effective dose.
  • the dosage schedule and amounts effective for this use. i.e., the dosing regimen will depend upon a variety of factors, including the stage of the disease or condition, the severity of the disease or condition, the general state of the patient's health, the patient's physical status, age and the like. In calculating the dosage regimen for a patient, the mode of administration also is taken into consideration.
  • the dosage regimen also takes into consideration pharmacokinetics parameters well known in the art, i.e., the active agents' rate of absorption, bioavailability, metabolism, clearance, and the like (see, e.g., Remington: The Science and Practice of Pharmacy, 21st ed., 2005).
  • active agent e.g., inhibitory nucleic acid
  • guidelines provided for similar compositions used as pharmaceuticals can be used as guidance to determine the dosage regiment, i.e.. dose schedule and dosage levels.
  • formulations can be given depending on for example: the dosage and frequency as required and tolerated by the patient, the degree and amount of therapeutic effect generated after each administration (e.g., effect on blood glucose levels), and the like.
  • the formulations should provide a sufficient quantity of active agent (e.g., inhibitory nucleic acid) to effectively treat, prevent or ameliorate conditions, diseases or symptoms.
  • active agent e.g., inhibitory nucleic acid
  • pharmaceutical formulations for oral administration are in a daily amount of between about 1 pg to about 100 mg nucleic acid per kilogram of body weight per day. Lower dosages can be used, in contrast to administration orally, into the blood stream, into a body cavity or into a lumen of an organ. Substantially higher dosages can be used in topical or oral administration or administering by powders, spray or inhalation. Actual methods for preparing parenterally or non- parenterally administrable formulations will be known or apparent to those skilled in the art and are described in more detail in such publications as Remington: The Science and Practice of Pharmacy, 21st ed., 2005.
  • the methods described herein can include co-administration with other drugs or pharmaceuticals, e.g., compositions for reducing blood glucose levels.
  • the inhibitory nucleic acids can be co-administered with drugs for treating or reducing risk of a disorder described herein.
  • Tire present disclosure provides methods using an inhibitory nucleic acid of the present disclosure, e.g., in subjects suffering from sarcopenia.
  • the methods comprise administering to an individual in need thereof an effective amount of an inhibitory nucleic acid of the present disclosure, or a composition (e.g., a pharmaceutical composition) comprising same.
  • an inhibitory nucleic acid of the present disclosure is administered in a lipid nanoparticle.
  • an inhibitory nucleic acid of the present disclosure is administered in a liposome and/or a recombinant adeno-associated virus (AAV) particle.
  • Sarcopenia disorders that can be treated with a method of the present disclosure include, e.g.. age-related sarcopenia.
  • a therapeutically effective amount of an inhibitory nucleic acid of the present disclosure is an amount that, when administered in one or more doses to a subject, results in one or more of: i) reduced oxidative stress; ii) improved muscular function; iii) decreased rate of atrophy; iv) reduced inflammation; and v) reduced fatty infiltration.
  • an effective amount of an inhibitory nucleic acid of the present disclosure is an amount that, when administered in one or more doses to a subject, provides for an increase in muscle strength in the subject.
  • an effective amount of an inhibitory' nucleic acid of the present disclosure is an amount that, when administered in one or more doses to a subject, provides for an increase of at least 10%, at least 25%, at least 50%, at least 2-fold, or more than 2-fold, in muscle strength in the subject, compared to the level of muscle strength in the subject before being treated with the inhibitory nucleic acid.
  • an effective amount of an inhibitory nucleic acid of the present disclosure is an amount that, when administered in one or more doses to a subject, provides for a reduction in skeletal muscle fibrosis and necrosis.
  • an effective amount of an inhibitory nucleic acid of the present disclosure is an amount that, when administered in one or more doses to a subject, provides for a reduction of at least 10%, at least 25%, at least 50%, at least 60%, at least 70%, or more than 70%, in skeletal muscle fibrosis and necrosis, compared to the level of skeletal muscle fibrosis and necrosis in the subject before being treated with the inhibitory nucleic acid.
  • Suitable routes of administration include oral, rectal, nasal, pulmonary, topical, subcutaneous, intramuscular, intraperitoneal, intravenous, intradermal, intrathecal, and epidural. In some cases, the route of administration is intramuscular. In some cases, the route of administration is intravenous.
  • the present disclosure contemplates the use of an inhibitory nucleic acid of the present disclosure in combination with one or more additional agents (e.g., one or more additional active therapeutic agents) or other prophylactic or therapeutic modalities.
  • additional agents e.g., one or more additional active therapeutic agents
  • tire various active agents frequently have different mechanisms of action.
  • Such combination therapy may be especially advantageous by allowing a dose reduction of one or more of the agents, thereby reducing or eliminating the adverse effects associated with one or more of the agents; furthermore, such combination therapy may have a synergistic therapeutic or prophylactic effect on the underlying disease, disorder, or condition.
  • “combination” is meant to include therapies that can be administered separately, for example, fonnulated separately for separate administration (e.g.. as may be provided in a kit), and therapies that can be administered together in a single formulation (i.e., a “co-formulation”).
  • an inhibitory nucleic acid of the present disclosure and the at least one additional agent are administered or applied sequentially, e.g., where one agent is administered prior to one or more other agents.
  • an inhibitory nucleic acid of the present disclosure and the at least one additional agent are administered simultaneously, e.g., where two or more agents are administered at or about the same time; the two or more agents may be present in two or more separate formulations or combined into a single formulation (i.e., a co-formulation). Regardless of whether the two or more agents are administered sequentially or simultaneously, they are considered to be administered in combination for purposes of the present disclosure.
  • An inhibitory nucleic acid of the present disclosure can be used in combination with other agents useful in the treatment of the disorders or conditions set forth herein, including those that are normally administered to subjects suffering from muscular atrophy, malnutrition, metabolic syndrome, and certain hormonal disorders.
  • the present disclosure contemplates combination therapy with numerous agents (and classes thereof), including 1) testosterone, testosterone mimetics, and agents that stimulate testosterone secretion.
  • testosterone can be used directly or as prodrug.
  • Testosterone prodrugs can include testosterone esters, such as Testosterone caproate, Testosterone cypionate, Testosterone decanoate. Testosterone enanthate, Testosterone isobutyrate.
  • testosterone replacement therapies include AndrogelTM, TestimTM, Depo-TestosteroneTM, AndrodermTM, methyltestosterone, and AndroidTM.
  • Certain vitamins or nutritional supplements may increase boost testosterone levels in an individual, including creatine, omcga-3 fatty acids, vitamin D, and dehydroepiandrosterone (DHEA); 2) hormonal therapies, including growth honnones and gro th factors, including growth hormone (e.g., somatotropin, human growth hormone, SomatropinTM.
  • HumatropeTM insulin-like growth factor (IGF-1), mechanical growth factor (MGF), and urocortin II.
  • anabolic steroids such as Danazol, Drostanolone propionate, Ethylestrenol, Fluoxymesterone, Mesterolone, Metandienone, Metenolone acetate, Metenolone enanthate, Methyltestosterone, Nandrolone decanoate, Nandrolone phenylpropionate, Norethandrolone, Oxandrolone, Oxymetholone, Stanozolol, Testosterone cypionate, Testosterone enanthate, Testosterone propionate, Testosterone undecanoate, and Trenbolone HBC.
  • Lifestyle adjustments may also be beneficial for certain individuals with sarcopenia, including improved diet and exercise.
  • Diet can include reduced calorie (e.g., caloric deficit) diets and/or any other diet that encourage a reduction in bodily fat and/or excess w eight.
  • Exercise can include weight training, resistance training, and/or any other exercise type for the increase in muscle mass and/or muscle strength.
  • Weight training and/or resistance training can include the use of free weights (e.g., dumbbells, barbells and kettlebells), sandbags, weighted balls (e.g.. medicine balls), weight machines, resistance bands, suspension equipment, the subject’s body weight.
  • Certain embodiments include treatments for metabolic syndrome and related disorders, such as insulin-rcsistancc, obesity, and hypertension.
  • Such therapeutics can include: 1) insulin, insulin mimetics and agents that entail stimulation of insulin secretion, including sulfonylureas (e.g., chlorpropamide, tolazamide, acetohexamide, tolbutamide, glyburide, glimepiride.
  • sulfonylureas e.g., chlorpropamide, tolazamide, acetohexamide, tolbutamide, glyburide, glimepiride.
  • glipizide and meglitinides (e.g., mitiglinide, repaglinide and nateglinide); 2) biguanides (e.g., metformin, and its pharmaceutically acceptable salts, in particular, metformin hydrochloride, and extended-release formulations thereof, such as GlumetzaTM, FortametTM, and GlucophageXRTM) and other agents that act by promoting glucose utilization, reducing hepatic glucose production and/or diminishing intestinal glucose output; 3) alpha-glucosidase inhibitors (e g., acarbose, voglibose and miglitol) and other agents that slow down carbohydrate digestion and consequently absorption from the gut and reduce postprandial hyperglycemia; 4) thiazolidinediones (e.g., rosiglitazone, troglitazone, pioglitazone, glipizide, balaglitazone, rivoglita
  • taspoglutide CJC-1131, and BIM-51077, including intranasal, transdermal, and once- weekly formulations thereof); and 6) and DPP-IV-resistant analogues (incretin mimetics), PPAR gamma agonists, PPAR alpha agonists such as fenofibric acid derivatives (e.g., gemfibrozil, clofibrate, ciprofibrate, fenofibrate, bezafibrate), dual-acting PPAR agonists (e.g., ZYH2, ZYH1, GFT505, chiglitazar, muraglitazar, aleglitazar, sodelglitazar, and naveglitazar), pan-acting PPAR agonists, PTP1B inhibitors (e.g., ISIS-113715 and TTP814), SGLT inhibitors (e.g., ASP1941, SGLT-3, empaglifl
  • angiotensin converting enzyme inhibitors e.g.. alacepril.
  • angiotensin II receptor antagonists e.g., losartan, valsartan, candesartan, olmesartan, telmesartan
  • angiotensin II receptor antagonists e.g., losartan, valsartan, candesartan, olmesartan, telmesartan
  • Subjects suitable for treatment using a method of the present disclosure include individuals having sarcopenia, including age-related sarcopenia. In some instances, the subject has muscle weakness, muscle atrophy, muscle wasting, and/or decreased muscle mass. Certain subjects may have a high body mass index and/or possess sarcopenic obesity. Certain subjects may be of an advanced age (e.g., greater than 60 years of age, 65 years of age, 70 years of age, 75 years of age, 80 years of age, 85 years of age, or greater.
  • Some subjects may possess one or more risk factors associated with sarcopenia, including physical inactivity, obesity, one or more chronic diseases (e.g., chronic obstructive pulmonary disease, kidney disease, diabetes, cancer, human immunodeficiency virus infection, etc.), rheumatoid arthritis, insulin resistance, reduced hormone levels, malnutrition, inadequate protein intake, decreased ability to convert protein to energy, decline in nerve cells between brain and muscle.
  • chronic diseases e.g., chronic obstructive pulmonary disease, kidney disease, diabetes, cancer, human immunodeficiency virus infection, etc.
  • rheumatoid arthritis e.g., insulin resistance, reduced hormone levels, malnutrition, inadequate protein intake, decreased ability to convert protein to energy, decline in nerve cells between brain and muscle.
  • Sarcopenia can be assessed using any of a number of methods, such as strength, assistance with walking, rising from a chair, climbing stairs, and falls. Such assessments can include a handgrip test, a chair stand test, a walking speed test, a physical performance battery, a timed-up and go test. In some situations, imaging can be used to measure muscle mass, including dual-energy X-ray absorptiometry and/or bioelectrical impedance analysis.
  • a subject has not been diagnosed with a metabolic disorder (e.g., metabolic syndrome). In additional embodiments, the subject has been determined to not have a metabolic disorder.
  • a metabolic disorder e.g., metabolic syndrome.
  • the present disclosure provides a recombinant adeno-associated viral (rAAV) particle comprising a capsid and a nucleic acid vector comprising a heterologous nucleic acid region comprising sequence encoding an interfering RNA. as described above, the interfering RNA comprising a region complementary to a miR-128 target nucleic acid.
  • rAAV adeno-associated viral
  • Viruses of the Parvoviridae family are small non-enveloped icosahedral capsid viruses characterized by a single stranded DNA genome.
  • Parvoviridae family viruses include adeno-associated viruses (AAV) capable of replication in vertebrate hosts including, but not limited to, human, primate, bovine, canine, equine, and ovine species.
  • AAV adeno-associated viruses
  • AAV particles of the present disclosure are recombinant AAV viral vectors which are replication defective and lacking sequences encoding functional Rep and Cap proteins within their viral genome. These defective AAV vectors may lack most or all parental coding sequences and essentially carry only one or two AAV ITR sequences and the nucleic acid of interest (e.g., a nucleic acid comprising a nucleotide sequence encoding an inhibitory nucleic acid of the present disclosure) for delivery to a cell, a tissue, an organ, or an organism.
  • the nucleic acid of interest e.g., a nucleic acid comprising a nucleotide sequence encoding an inhibitory nucleic acid of the present disclosure
  • AAV particles for use in therapeutics and/or diagnostics comprise a virus that has been distilled or reduced to the minimum components necessary for transduction of a nucleic acid payload or cargo of interest.
  • AAV particles are engineered as vehicles for specific delivery while lacking the deleterious replication and/or integration features found in wild-type viruses.
  • AAV vectors of the present disclosure may be produced recombinantly and may be based on adeno-associated virus (AAV) parent or reference sequences.
  • AAV adeno-associated virus
  • a " vector” is any molecule or moiety which transports, transduces, or otherwise acts as a carrier of a heterologous molecule such as the nucleic acids described herein.
  • scAAV vector genomes contain DNA strands which anneal together to form double stranded DNA. By skipping second strand synthesis, scAAVs allow for rapid expression in the cell.
  • the AAV particle of the present disclosure is an ssAAV.
  • AAV particles may be modified to enhance the efficiency of delivery. Such modified AAV particles can be packaged efficiently and be used to successfully infect the target cells at high frequency and with minimal toxicity.
  • the capsids of the AAV particles are engineered according to the methods described in US Publication Number US20I95801, the contents of which are incorporated herein by reference in their entirety.
  • ITRs Inverted Terminal Repeats
  • Tire AAV particles of the present disclosure comprise a viral genome with at least one ITR region and a payload region.
  • the viral genome has two ITRs. These two ITRs flank the payload region at the 5' and 3' ends.
  • the ITRs function as origins of replication comprising recognition sites for replication.
  • ITRs comprise sequence regions which can be complementary find symmetrically arranged ITRs incorporated into viral genomes of the invention may be comprised of naturally occurring polynucleotide sequences or recombinantly derived polynucleotide sequences.
  • the ITRs may be derived from the same serotype as the capsid, selected from any other serotype, or a derivative thereof.
  • the ITR may be of a different serotype than the capsid.
  • the AAV particle has more than one ITR.
  • the AAV particle has a viral genome comprising two ITRs.
  • the ITRs are of the same serotype as one another.
  • the ITRs are of different serotypes. Non-limiting examples include zero, one or both of the ITRs having the same serotype as the capsid.
  • both ITRs of the viral genome of the AAV particle are AAV2 ITRs.
  • each ITR may be about 100 to about 150 nucleotides in length.
  • An ITR may be about 100-105 nucleotides in length, 106-110 nucleotides in length. 111-115 nucleotides in length, 116-120 nucleotides in length, 121-125 nucleotides in length, 126-130 nucleotides in length, 131- 135 nucleotides in length, 136-140 nucleotides in length, 141-145 nucleotides in length or 146-150 nucleotides in length.
  • the ITRs are 14-142 nucleotides in length.
  • Non-limiting examples of ITR length arc 102, 140, 141, 142, 145 nucleotides in length, and those having at least 95% identity thereto.
  • the payload region of the viral genome comprises at least one element to enhance the transgene target specificity and expression (See e.g., Powell et al. Viral Expression Cassette Elements to Enhance Transgene Target Specificity and Expression in Gene Therapy, 2015; the contents of which are herein incorporated by reference in its entirety).
  • elements to enhance the transgene target specificity and expression include promoters, endogenous miRNAs. post-transcriptional regulatory elements (PREs), polyadenylation (Poly A) signal sequences and upstream enhancers (USEs), CMV enhancers and introns.
  • nucleic acids described herein may require a specific promoter, including but not limited to, a promoter that is species specific, inducible, tissue-specific, or cell cycle-specific (Parr et al., Nat. Med.3: 1145-9 (1997); the contents of which are herein incorporated by reference in their entirety).
  • the promoter is deemed to be efficient when it drives transcription of the nucleic acid sequence encoded in the payload region of the viral genome of the AAV particle.
  • the promoter is a promoter deemed to be efficient when it drives expression in the cell, tissue, and/or organ being targeted.
  • the promoter is capable of being expressed in muscle tissue of an individual, including skeletal muscle, smooth muscle, and/or cardiac muscle.
  • tissue specific expression is driven by one or more of the following promotors: cytomegalovirus immediate early promoter (CMV). hybrid chicken-beta-actin (CBA) promoter, and ubiquitin C (UBC) promoter, and an RNA polymerase III promoter.
  • CMV cytomegalovirus immediate early promoter
  • CBA hybrid chicken-beta-actin
  • UBC ubiquitin C
  • AAV particles of the present disclosure may comprise or be derived from any natural or recombinant AAV seroty pe.
  • the AAV particles may utilize or be based on a serotype selected from any of the following AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrhlO, AAV11, AAV12, AAV2R471A, AAVAAV2/2-7m8, AAV2 N587A, AAV2 E548A, AAV2 N708A, AAV V708K, goat AAV, AAV1/AAV2 chimer, bovine AAV, mouse AAV, or rAAV2/HBoVland variants thereof.
  • a method for treating age-related sarcopenia comprising administering an effective amount of a miR-128 inhibitor to an individual diagnosed with age-related sarcopenia.
  • Aspect 2 The method of Aspect 1, wherein the individual has not been diagnosed with a metabolic disorder.
  • Aspect 3 The method of Aspect 1 or 2. wherein the miR-128 inhibitor comprises an inhibitory nucleic acid.
  • Aspect 4 The method of Aspect 3, wherein the inhibitory nucleic acid comprises one or more of the following sequences:
  • Aspect 5 The method of Aspect 3 or 4, wherein the inhibitory nucleic acid comprises one or more locked nucleic acids (LNA).
  • LNA locked nucleic acids
  • Aspect 6 The method of any of Aspects 2-5, wherein the inhibitory nucleic acid comprises one or more of a modified backbone and one or more 5 -methyl deoxycytosine residues.
  • Aspect 7 Tire method of any of Aspects 2-6, wherein the inhibitory nucleic acid has a length of from 14 nucleotides to 20 nucleotides.
  • Aspect 8 The method of any of Aspects 2-7, wherein the inhibitory nucleic acid comprises the nucleotide sequence designated “NRC-0090.”
  • Aspect 9 The method of any of Aspects 2-7, wherein the inhibitory nucleic acid comprises the nucleotide sequence designated “NRC-0091.”
  • Aspect 10 Tire method of any of Aspects 2-7, wherein the inhibitory nucleic acid comprises the nucleotide sequence designated “NRC-0119.”
  • Aspect 11 The method of any of Aspects 1-10, wherein the miR-128 inhibitor is formulated for subcutaneous delivery'.
  • Aspect 12 The method of any of Aspects 1-11, wherein the miR-128 inhibitor is fonnulated as a pharmaceutical composition comprising one or more of the following: a phannaceutically acceptable excipient, a buffering agent, a solvent, a bulking agent, a flavorant. a lubricant, a colorant, a binder, a surfactant, a binder, and a disintegrant.
  • Aspect 13 The method of any of Aspects 1-11, wherein the miR-128 inhibitor is formulated as a pharmaceutical composition comprising a phannaceutically acceptable excipient.
  • Aspect 14 The method of Aspect 13, wherein the phannaceutically acceptable excipient is selected from: poly(amidoamine), poly(propyleneimine), and poly(L-lysine).
  • Aspect 15 The method of Aspect 13, wherein the pharmaceutically acceptable excipient comprises one or more lipids.
  • Aspect 16 The method of any of Aspects 1-15, further comprising administering at least one additional therapeutic agent.
  • Aspect 17 Hie method of Aspect 16, wherein the at least one additional therapeutic agent is selected from testosterone, growth hormone, insulin-like growtli factor, mechanical growth factor, urocortin II. anabolic steroids, omega-3 fatty acids, creatine, dehydroepiandrosterone.
  • Aspect 18 The method of any of Aspects 1-17, further comprising directing the individual to perform weight training exercises or resistance training exercises.
  • a combination therapy for the treatment of age-related sarcopenia comprising: a miR-128 inhibitor; and one or more of: testosterone, growth hormone, insulin-like growth factor, mechanical growth factor, urocortin II. anabolic steroids, omega-3 fatty acids, creatine, dehydroepiandrosterone.
  • Aspect 20 The combination therapy of Aspect 19, wherein the miR-128 inhibitor comprises an inhibitory' nucleic acid.
  • Aspect 21 The combination therapy of Aspect 20, wherein the inhibitory' nucleic acid comprises one or more of tire following sequences:
  • Aspect 22 The combination therapy of Aspect 20 or 21, wherein the inhibitory' nucleic acid comprises one or more locked nucleic acids (LNA).
  • LNA locked nucleic acids
  • Aspect 23 Hie method of any of Aspects 20-22, wherein the inhibitory nucleic acid comprises one or more of a modified backbone and one or more 5 -methyl deoxy cytosine residues.
  • Aspect 24 The combination therapy of any of Aspects 20-23, wherein the inhibitory nucleic acid has a length of from 14 nucleotides to 20 nucleotides.
  • Aspect 25 The combination therapy of any of Aspects 20-24, wherein the inhibitor ⁇ ’ nucleic acid comprises the nucleotide designated "NRC-0090.’'
  • Aspect 26 The combination therapy of any of Aspects 20-24, wherein the inhibitory nucleic acid comprises the nucleotide sequence designated “NRC-0091.”
  • Aspect 27 The combination therapy of any of Aspects 20-24, wherein the inhibitory nucleic acid comprises the nucleotide sequence designated “NRC-0119.”
  • Aspect 28 The combination therapy of any of Aspects 19-27, wherein tire miR-128 inhibitor is formulated for subcutaneous delivery.
  • Aspect 29 The combination therapy of any of Aspects 19-28, wherein the miR-128 inhibitor is formulated as a pharmaceutical composition comprising one or more of the following: a pharmaceutically acceptable excipient, a buffering agent, a solvent, a bulking agent, a flavorant, a lubricant, a colorant, a binder, a surfactant, a binder, and a disintegrat'd.
  • Aspect 30 Hie combination therapy of any of Aspects 19-29, wherein the miR-128 inhibitor is formulated as a pharmaceutical composition comprising a pharmaceutically acceptable excipient.
  • Aspect 31 The combination therapy of Aspect 30, wherein the pharmaceutically acceptable excipient is selected from: poly(amidoamine), poly(propyleneimine), and poly(L-lysine).
  • Aspect 32 The combination therapy of Aspect 30, wherein the pharmaceutically acceptable excipient comprises one or more lipids.
  • An oligonucleotide comprising: a plurality of interlinked nucleotides, wherein a contiguous subset of the plurality of nucleotides is complementary to a portion of a miR-128 nucleic acid; wherein at least one nucleotide in the plurality of interlinked nucleotides is a bridged nucleic acid.
  • Aspect 34 The oligonucleotide of Aspect 33, wherein the oligonucleotide is comprised of 7 to 50 interlinked nucleotides.
  • Aspect 35 Tire oligonucleotide of Aspect 33 or 34, wherein the oligonucleotide is comprised of 7 to 30 interlinked nucleotides.
  • Aspect 36 The oligonucleotide of any of Aspects 33-35. wherein the oligonucleotide is comprised of 14 to 23 interlinked nucleotides.
  • Aspect 37 The oligonucleotide of any of Aspects 33-36, wherein the contiguous subset comprises at least 6 nuclcobascs that arc complementary to the miR-128 nucleic acid.
  • Aspect 38 Tire oligonucleotide of any of Aspects 33-37. wherein the oligonucleotide is a unimer.
  • Aspect 39 The oligonucleotide of Aspect 38, wherein each nucleotide is independently a bridged nucleic acid.
  • Aspect 40 The oligonucleotide of any of Aspects 33-37, wherein the nucleobase sequence comprises one or more of the following sequences: 5 -ACCGGTTCACTGTG-3’ (SEQ ID NO: 1);
  • a recombinant adeno-associated viral (rAAV) particle comprising a capsid and a nucleic acid vector comprising a heterologous nucleic acid region comprising sequence encoding an interfering RNA comprising a region complementary to a miR-128 target nucleic acid.
  • rAAV adeno-associated viral
  • Aspect 42 The rAAV of Aspect 41, wherein the miR-128 target nucleic acid is miR-128-3p, pri- miR-128-1, pre-miR-128-1. pri-miR- 128-2, or pre-miR- 128-2.
  • Aspect 43 The rAAV of Aspect 41, wherein the interfering RNA is a short hairpin RNA or a small interfering RNA.
  • Aspect 44 The rAAV of Aspect 41, wherein the nucleic acid vector comprises a promoter, wherein the sequence encoding an interfering RNA is operably linked to the promoter.
  • Aspect 45 Hie rAAV of Aspect 44, wherein the promoter is capable of inducing expression of the interfering RNA in a skeletal muscle cell.
  • Aspect 46 The rAAV of Aspect 44, wherein the promoter is a hybrid chicken P-actin (CBA) promoter or an RNA polymerase III promoter.
  • CBA hybrid chicken P-actin
  • Aspect 47 The rAAV of Aspect 41, wherein the nucleic acid vector comprises inverted terminal repeats from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrhlO, AAV11, AAV12, AAV2R471A, AAV DJ, goat AAV, bovine AAV, or mouse AAV serotypes.
  • Aspect 48 The rAAV of Aspect 41, wherein the capsid is selected from: an AAV1 capsid, and AAV2 capsid, an AAV3 capsid, an AAV4 capsid, an AAV5 capsid, anAAV6 capsid, an AAV7 capsid, anAAV8 capsid, an AAVrh8 capsid, an AAVrh8R capsid, an AAV9 capsid, an AAV10 capsid, an AAVrhlO capsid, an AAV11 capsid, an AAV12 capsid, atyrosine capsid mutant, a heparin binding capsid mutant, an AAV2R471A capsid, an AAVAAV2/2-7m8 capsid, an AAV DJ capsid, an AAV2 N587A capsid, an AAV2 E548A capsid, an AAV2 N708A capsid, an AAV V708K capsid,
  • a pharmaceutical composition comprising: a pharmaceutically acceptable excipient; and the oligonucleotide of any of Aspects 33-40 or the rAAV of any of Aspects 41-48.
  • Aspect 50. A therapeutically effective composition for use in a method of treating age-related macular degeneration in a subject in need thereof, the method comprising administering to the subject the therapeutically effective amount of the oligonucleotide of any of Aspects 1-8 or tire rAAV of any of Aspects 9-16.
  • a miR-128 inhibitor for use in a method of treating aging-related sarcopenia in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the miR-128 inhibitor.
  • Aspect 52 The miR-128 inhibitor of Aspect 51, wherein the miR-128 inhibitor is selected from: an antisense oligonucleotide, a short hairpin RNA, a small interfering RNA, or a recombinant adeno-associated viral (rAAV) particle comprising a capsid and a nucleic acid vector comprising a heterologous nucleic acid region comprising sequence encoding an interfering RNA, wherein the interfering RNA is selected from an antisense oligonucleotide, a short hairpin RNA, or a small interfering RNA.
  • rAAV recombinant adeno-associated viral
  • Aspect 54 The therapeutically effective composition of Aspect 53, wherein the oligonucleotide is a single-stranded oligonucleotide.
  • Aspect 55 The therapeutically effective composition of Aspect 53, wherein the oligonucleotide is an antisense oligonucleotide.
  • Aspect 56 Tire therapeutically effective composition of Aspect 53, wherein the oligonucleotide comprises 7 to 30 interlinked nucleotides.
  • Aspect 57 The therapeutically effective composition of Aspect 56, wherein the oligonucleotide comprises 14 to 23 interlinked nucleotides.
  • Aspect 58 The therapeutically effective composition of any of Aspects 53-57, wherein the oligonucleotide comprises at least one modified sugar nucleoside.
  • Aspect 59 Tire therapeutically effective composition of Aspect 58, wherein at least 50% of the nucleosides comprise a modified sugar nucleoside.
  • Aspect 60 The therapeutically effective composition of Aspect 58 or 59, wherein 100% of the nucleosides comprise a modified sugar nucleoside.
  • Aspect 61 The therapeutically effective composition of any of Aspects 58-60, wherein the modified sugar nucleoside is a 2 ’-modified sugar nucleoside.
  • Aspect 62. The therapeutically effective composition of Aspect 61, wherein the 2’-modified sugar nucleoside comprises a 2 ’-modification independently selected from the group consisting of 2’-fluoro, 2'-methoxy, and 2 ’-methoxy ethoxy.
  • Aspect 63 Tire therapeutically effective composition of any of Aspects 58-60, wherein tire modified sugar nucleoside is a bridged nucleic acid.
  • Aspect 64 The therapeutically effective composition of Aspect 63, wherein the bridged nucleic acid is a locked nucleic acid.
  • Aspect 65 The therapeutically effective composition of Aspect 63, wherein the bridged nucleic acid is an ethylene bridged nucleic acid.
  • Aspect 66 Tire therapeutically effective composition of any of Aspects 58-60, wherein tire nucleobase sequence comprises one or more of the following sequences: 5 -ACCGGTTCACTGTG-3’ (SEQ ID NO: 1): 5’-GACCGGTTCACTGT-3’ (SEQ ID NO:2); and 5’-AGACCGGTTCACTGTG-3’ (SEQ ID NO:3).
  • Aspect 67 Hie therapeutically effective composition of any of Aspects 53-66, wherein the oligonucleotide is a gapmer comprising a '-wing, a 3 ’-wing, and a gap, wherein the 5 ’-wing and the 3’- wing each comprise 1 to 5 nucleotides, wherein each nucleotide in the 5 ’-wing and the 3 ’-wing are independently a bridged nucleic acid, and wherein each nucleotide in the gap is a deoxyribonucleotide.
  • Aspect 68 The therapeutically effective composition of any of Aspects 53-67, wherein at least one intemucleoside linkage in the oligonucleotide is a phosphorothioate diester.
  • Aspect 69 Hie therapeutically effective composition of Aspect 68, wherein at least 50% of intemucleoside linkages in the oligonucleotide are phosphorothioate diesters.
  • Aspect 70 The therapeutically effective composition of Aspect 68 or 69, wherein 100% of intemucleoside linkages in the oligonucleotide are phosphorothioate diesters.
  • Aspect 71 The therapeutically effective composition of any of Aspects 53-70, wherein the method further comprises administering the oligonucleotide as a guide strand in a short interfering RNA.
  • Aspect 72 Hie therapeutically effective composition of any of Aspects 53-71, wherein the miR-128 target nucleic acid is selected from: pri-miR-128-1, pre-miR-128-1. pri-miR- 128-2. pre-miR- 128-2, and miR-128-3p.
  • Aspect 73 The therapeutically effective composition of any of Aspects 53-72, wherein the route of administration is an intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection.
  • FIG. 1A depicts sequence information for three ASOs.
  • ID indicates drug name
  • OLIGO indicates nucleotide sequence
  • uppercase denotes locked nucleic acids (LNAs)
  • lowercase denotes standard bases
  • STRING indicates modifications (+ sign precedes an LNA
  • /iMe-dC/ denotes an internal 5-methyl dcoxycytosme.
  • Length indicates number of nucleotides
  • #LNAs indicates number of standard bases that have been replaced with LNAs.
  • FIG. IB provides a schematic LNA/DNA composition of each ASO and their base-pairing location within the target miRNA (miR-128-3p). The seed sequence of miR-128-3p is denoted in red.
  • mice Aged mice (21.5 months old) and young control mice (8 weeks old) were subcutaneously injected with anti-miR-128-3p LNA ASO or a scramble control LNA ASO weekly at 10 mg/kg bodyweight. Muscle function was then measured 1, 2, and 3 months later by measuring the time hanging suspended from a wire and the run time until exhaustion (using a high intensity testing protocol). At 2 months and 3 months, muscle function was also measured by the four-limb hanging test. At 3 months, muscle function was finally measured by run time until exhaustion using a low intensity protocol (FIG. 2A).
  • FIG. 2A-2I Anti-miR-128-3p treatment improves skeletal muscle function in aged mice.
  • A Experimental design.
  • B Two limb wire hanging time measured by the average of three trials.
  • C Two limb wire hanging time measured by the maximum of three trials.
  • E Four limb hanging time measured by the average of all trials.
  • F Four limb hanging time measured by the maximum of all trials.
  • G High intensity treadmill test running time until exhaustion.
  • mice Aged mice (86 weeks old) were subcutaneously injected with anti-miR-128-3p LNA ASO or a scramble control LNA ASO weekly at 10 mg/kg bodyweight. Muscle function was then measured 5. 9 and 15 weeks later by measuring the time hanging suspended from a wire and the ran time until exhaustion (using a low intensity testing protocol) was measured at 13 weeks (Fig. 3A).
  • miR-128-3p LNA ASO robustly knocks down miR-128-3p expression in gastrocnemius muscle as measured post-harvest at 106 weeks of age (FIG. 3B).
  • aged mice 86 weeks old
  • miR-27 LNA ASO an off-target miRNA with similar sequence to miR-128-3p
  • scramble control LNA ASO weekly at 10 mg/kg bodyweight.
  • RT-qPCR of miR-128-p3 was not different between mice injected with miR-27 LNA ASO and scramble control LNA ASO at 106 weeks of age (Fig. 3C).
  • Fig. 3F shows laminin staining of cross sections of tibialis anterior muscle in control and anti-miR-128-3p LNA ASO treated mice. Quantification of tire cross-sectional area of the laminin- stained muscle fibers shows fiber size was increased in anti-miR-128-3p LNA ASO treated mice as compared with control (Fig. 3G).
  • Fig. 3H shows gene set enrichment analysis of RNA sequencing results from GA muscle. Expression of several mitochondrial genes are increased in GA muscle, as measured by RT- qPCR (Fig. 31).
  • mice Aged mice (86 weeks old) were subcutaneously injected with anti-miR-128-3p LNA ASO or a scramble control LNA ASO weekly at 10 mg/kg bodyweight. Skeletal muscle mass was measured by EchoMRI and tissue weight of GA and quadraceps muscles was measured after 15 weeks of treatment (Fig. 4A). Data are presented as mean and standard deviation; student’s t-test.

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Abstract

La présente divulgation concerne des compositions et des méthodes de traitement de la sarcopénie, y compris de la sarcopénie liée à l'âge. Divers modes de réalisation comprennent des acides nucléiques inhibiteurs, des compositions comprenant les acides nucléiques inhibiteurs, ainsi que des méthodes d'utilisation des acides nucléiques inhibiteurs pour traiter de tels troubles.
PCT/US2024/045849 2023-09-07 2024-09-09 Compositions et méthodes de traitement de la sarcopénie Pending WO2025054607A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120172416A1 (en) * 2009-09-10 2012-07-05 Flemming Velin Method for the preparation of micro-rna and its therapeutic application
US20140303236A1 (en) * 2011-10-06 2014-10-09 The Board Of Regents, The University Of Texas System Control of whole body energy homeostasis by microrna regulation
US20200368162A1 (en) * 2017-02-24 2020-11-26 Modernatx, Inc. Nucleic Acid-Based Therapy of Muscular Dystrophies
US20210170052A1 (en) * 2010-11-23 2021-06-10 Presage Biosciences, Inc. Therapeutic methods and compositions for solid delivery
US20220213477A1 (en) * 2016-10-27 2022-07-07 The General Hospital Corporation Therapeutic Targeting of a microRNA to Treat Duchenne Muscular Dystrophy

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120172416A1 (en) * 2009-09-10 2012-07-05 Flemming Velin Method for the preparation of micro-rna and its therapeutic application
US20210170052A1 (en) * 2010-11-23 2021-06-10 Presage Biosciences, Inc. Therapeutic methods and compositions for solid delivery
US20140303236A1 (en) * 2011-10-06 2014-10-09 The Board Of Regents, The University Of Texas System Control of whole body energy homeostasis by microrna regulation
US20220213477A1 (en) * 2016-10-27 2022-07-07 The General Hospital Corporation Therapeutic Targeting of a microRNA to Treat Duchenne Muscular Dystrophy
US20200368162A1 (en) * 2017-02-24 2020-11-26 Modernatx, Inc. Nucleic Acid-Based Therapy of Muscular Dystrophies

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