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WO2025049599A1 - Methods for treating muscle-related disorders by modulating prolyl hydroxylase 3 - Google Patents

Methods for treating muscle-related disorders by modulating prolyl hydroxylase 3 Download PDF

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WO2025049599A1
WO2025049599A1 PCT/US2024/044218 US2024044218W WO2025049599A1 WO 2025049599 A1 WO2025049599 A1 WO 2025049599A1 US 2024044218 W US2024044218 W US 2024044218W WO 2025049599 A1 WO2025049599 A1 WO 2025049599A1
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phd3
subject
muscular dystrophy
agent
muscle
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French (fr)
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Marcia C. Haigis
Haejin YOON
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Harvard University
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Harvard University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • 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
    • 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
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes

Definitions

  • DMD Duchenne muscular dystrophy
  • PHD3 prolyl hydroxylase 3
  • a muscular dystrophy is DMD.
  • a muscular dystrophy is selected from Becker’s muscular dystrophy, congenital muscular dystrophy, distal muscular dystrophy, Emery-Dreifuss muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, myotonic muscular dystrophy, and oculopharyngeal muscular dystrophy.
  • FoleyHoagUS12471059.1 Attorney Docket No. HMV-32325
  • provided are methods of treating or preventing a muscle wasting disorder in a subject comprising administering to a subject an agent that reduces the levels of or inhibits the activity of PHD3.
  • a muscle wasting disorder is cachexia.
  • cachexia is an obesity-related cachexia.
  • a muscle wasting disorder is sarcopenia.
  • the sarcopenia is ageing-related sarcopenia.
  • ACC2 acetyl CoA carboxylase 2
  • the method increases fatty acid oxidation in muscle cells of the subject and/or decreases hydroxylation of ACC2 in muscle cells of a subject.
  • a subject is afflicted with a muscular dystrophy. In some embodiments, a subject is afflicted with DMD.
  • a subject is afflicted with a muscular dystrophy that is selected from Becker’s muscular dystrophy, congenital muscular dystrophy, distal muscular dystrophy, Emery-Dreifuss muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, myotonic muscular dystrophy, and oculopharyngeal muscular dystrophy.
  • a subject is afflicted with a muscle wasting disorder.
  • a muscle wasting disorder is cachexia.
  • cachexia is an obesity- related cachexia.
  • a muscle wasting disorder is sarcopenia.
  • a sarcopenia is ageing-related sarcopenia.
  • provided methods decrease creatine kinase activity in a subject.
  • provided methods restore mitochondrial size and/or morphology in a subject.
  • FoleyHoagUS12471059.1 Attorney Docket No. HMV-32325
  • an agent further increases Akt signaling.
  • Akt signaling is increase in muscle cells of a subject.
  • an agent is a small molecule.
  • an agent is a sgRNA specific for a nucleic acid sequence encoding PHD3.
  • an agent is an inhibitory polynucleotide.
  • an inhibitory polynucleotide is selected from the group consisting of siRNA, shRNA, and an antisense oligonucleotide, or a polynucleotide that encodes a molecule selected from the group consisting of siRNA, shRNA, and/or an antisense oligonucleotide.
  • an inhibitory polynucleotide is an shRNA.
  • shRNA targets a sequence listed in Table 2.
  • an shRNA is specific for PHD3.
  • an shRNA targets a sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2.
  • an shRNA comprises a sense strand comprising a sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2.
  • an shRNA comprises a sense strand comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or more identical to a 40-80 nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2.
  • an shRNA targets SEQ ID NO: 5 or SEQ ID NO: 6.
  • an shRNA comprises a sense strand set forth in SEQ ID NO: 5 or SEQ ID NO: 6. In some embodiments, an shRNA comprises a sense strand comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or more identical to SEQ ID NO: 5 or SEQ ID NO: 6.
  • FoleyHoagUS12471059.1 Attorney Docket No. HMV-32325 [0024]
  • an inhibitory polynucleotide is an siRNA. In some embodiments, an siRNA is specific for PHD3. In some embodiments, an siRNA is about 18 to 25 nucleotides in length.
  • an siRNA is a small double stranded RNA (dsRNA). In some embodiments, a dsRNA comprises overhangs. In some embodiment, a dsRNA comprises blunt ends.
  • an inhibitory polynucleotide is an antisense oligonucleotide (ASO). In some embodiments, an ASO is specific for PHD3. In some embodiments, an ASP is about 13 to 30 nucleotides in length.
  • an agent is an antibody-siRNA conjugate. In some embodiments, an antibody-siRNA conjugate is specific for muscle tissue. [0028] In some embodiments, an agent is an antibody-ASO conjugate.
  • an antibody-ASO conjugate is specific for muscle tissue.
  • an agent is administered intravenously. In some embodiments, an agent is administered at least once a week or at least once a month. In some embodiments an agent is administered for at least 7 days, for at least 30 days, for at least 60 days, or for at least 90 days. [0030] In some embodiments, a subject is a human.
  • FIG. 1A shows PHD3 expression levels in human muscle biopsies in control or DMD patient group (GSE38417). Data represent means ⁇ SEM. FoleyHoagUS12471059.1 Attorney Docket No. HMV-32325 [0034]
  • FIG. 1B shows Gene targeting strategy for Phd3 in muscular dystrophy mouse (MDX) model. Phd3 +/+ were bred with CMV-Cre for phd3 -/- mice generation. phd3 -/- and mdx mice for phd3 -/- mdx generation. [0035] FIG.
  • FIG. 1C shows immunofluorescence of laminin (green), Evans blue (EBD; red), and DAPI (blue) in phd3 +/+ , mdx, phd3 -/- mdx mice quadriceps. Scale is 500 ⁇ m.
  • FIG. 1D-E show the relative intensity of laminin (d) and EBD (e) were normalized by the relative intensity of DAPI for each condition.
  • FIG. 1C shows immunofluorescence of laminin (green), Evans blue (EBD; red), and DAPI (blue) in phd3 +/+ , mdx, phd3 -/- mdx mice quadriceps. Scale
  • FIG. 2A shows qPCR analyses to confirm the lack of WT mRNA transcripts in pdh3 -/- and phd3 -/- mdx mouse quadriceps.
  • FIG. 3B shows principal components analysis (PCA) plot showing clusters of samples based on similarity. The first two components (PC1 and PC2) of gene expression variance are displayed.
  • FIG. 3C shows enrichment of KEGG metabolic signature scores in all RNA transcriptomes for phd3 -/- versus phd3 +/+ .
  • FIG. 3D shows a heatmap of the differentially expressed genes enriched in the quadricep tissues from phd3 +/+ , phd3 -/- , mdx, phd3 -/- mdx animals.
  • FoleyHoagUS12471059.1 Attorney Docket No. HMV-32325 [0047] FIG.
  • FIG. 4B shows a Venn diagram depicting overlap between significantly altered genes comparing phd3 +/+ vs. phd3 -/- and mdx vs. phd3 -/- mdx.
  • FIG. 4C shows the RNA transcriptomes from quadriceps in muscular dystrophy model was segregated into nine clusters based on gene expression level following KEGG pathway analysis for the specific functional pathways enriched within each cluster.
  • FIG. 4D-E shows enrichment of Gene Ontology term enrichment scores that are decreased (d) or increased (e) by PHD3 loss in mdx skeletal muscles.
  • FIG. 4F shows the significant gene alteration of positive (blue) and negative (red) directions by PHD3 loss in RNA transcriptomes from quadriceps in muscular dystrophy model.
  • FIG. 4D-E shows enrichment of Gene Ontology term enrichment scores that are decreased (d) or increased (e) by PHD3 loss in mdx skeletal muscles.
  • FIG. 4F shows the significant gene alteration of positive (blue) and negative (red) directions by PHD3 loss in RNA transcriptomes from quadriceps in muscular dystrophy model.
  • Relative Myoglobin expression is shown in FIG. 5A.
  • MyoD expression is shown in FIG. 5B.
  • MyoG expression is shown in FIG.5C.
  • Myf5 expression is shown in FIG. 5D.
  • MyHCl expression is shown in FIG. 5E.
  • MyHClla expression is shown in FIG.5F.
  • MyHCllx expression is shown in FIG. 5G.
  • FIG. 5H MyHCllb expression is shown in FIG. 5H.
  • FIG. 6A shows relative Phd3 expression using mouse ⁇ -actin as a reference gene in
  • FIG. 6B shows immunoblots of ACC2 hydroxylation from C2C12s expressing either control, Phd3, shRNA control, or Phd3 shRNA, and incubated with high glucose DMEM containing 2% horse serum and ITS Liquid Media Supplement for two days.
  • FoleyHoagUS12471059.1 Attorney Docket No. HMV-32325
  • FIG. 6C shows C2C12s expressing shRNA against Phd3 or shRNA control, after then cells were in myogenic differentiation for 2 days. Proteins in cell lysates were immunoblotted using PHD3, dystrophin, myosin heavy chain (MHC), or actin.
  • TEM transmission electron microscopy
  • FIG. 7B shows quantitation of mitochondrial size and cristae density in five independent TEM images as shown in FIG.7A.
  • FIG. 7C shows a PCA plot showing clusters of samples based on similarity. The first two components (PC1 and PC2) of metabolite levels variance are displayed. Each dot represents a sample color-coded by phd3 +/+ , phd3 -/- , mdx, phd3 -/- mdx group.
  • FIG. 7C shows quantitation of mitochondrial size and cristae density in five independent TEM images as shown in FIG.7A.
  • FIG. 7C shows a PCA plot showing clusters of samples based on similarity. The first two components (PC1 and PC2) of metabolite levels variance are displayed. Each dot represents a sample color-coded by phd3 +/+ , phd3 -/- , mdx, phd3 -/- m
  • OCR oxygen consumption rate
  • PC(30:0) (a), PE(32:0) (b), LPC(20:0) (c), LPC(20:4) (d), LPC(20:5) (e), ceramides (f), or amino acids (g) are identified by untargeted metabolomics method.
  • FIG. 8H shows volcano plots demonstrating that mdx have numerous statistically significant metabolic changes compared to control group.
  • FIG. 8H shows volcano plots demonstrating that mdx have numerous statistically significant metabolic changes compared to control group.
  • FIG. 8I shows volcano plots demonstrating that PHD3 depleted mdx quadricep muscles have numerous statistically significant metabolic changes compared to mdx group.
  • FIG. 9C-F show the individual exhaustion time (FIG. 9C), total running distance (FIG. 9D), time to maximum oxygen consumption rate (FIG. 9E), and maximum oxygen uptake (FIG. 9F) of both genotypes were calculated from the individual performances during treadmill running. Data represent mean ⁇ SEM. * P ⁇ 0.05.
  • FIG. 10A shows a schematic of a protocol for generating mice and treating them with a high fat diet condition).
  • FIG. 10B-C show a relative ratio of weight gain in WT and PHD3 KO mice on a high fat diet (FIG.10B) and low fat diet (FIG. 10C).
  • FIG. 10D-E show a glucose as a percent of basal glucose in WT and PHD3 KO mice over time after a high glucose injection. Mice on a high fat diet are shown in FIG. 10D and on low fat diet in FIG. 10E.
  • FIG. 10A shows a schematic of a protocol for generating mice and treating them with a high fat diet condition).
  • FIG. 10B-C show a relative ratio of weight gain in WT and PHD3 KO mice on a high fat diet (FIG.10B) and low fat diet (FIG. 10C).
  • FoleyHoagUS12471059.1 Attorney Docket No. HMV-32325
  • FIG. 10D-E show
  • FIG. 11A-D show that loss of PHD3 prevents accumulating fat in organ under HFD.
  • Epidiymal and inguinal white adipose tissue from WT and PHD3 KO mice on a high fat diet are shown in FIG. 11A, and quantified in FIG. 11B; livers tissue and H&E stained liver tissue from these mice are shown in FIG.11C and FIG.11D, respectively.
  • FIG. 12 shows blood glucose and serum insulin levels in WT and PHD3 KO mice on both low fat diet and high fat diet.
  • FIG. 13A-E show metabolic profiling of PHD3 KO mice under high fat diet.
  • FIG. 13A-E show metabolic profiling of PHD3 KO mice under high fat diet.
  • FIG. 14A-B show analysis of Akt signaling pathway in the liver of PHD3 KO mice on a high fat diet.
  • FIG. 15A-B show analysis of Akt signaling pathway in the muscle of PHD3 KO mice on a high fat diet.
  • administering means providing a pharmaceutical agent or composition to a subject, and includes, but is not limited to, administration by a medical professional and self- administration.
  • routes of administration of agents that modulate PHD3 described herein include topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically.
  • routes of administration for agents that modulate PHD3 described herein include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion.
  • Administering may be performed, for example, once, a plurality of times, and/or over one or more extended periods.
  • pharmaceutically-acceptable carrier means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body.
  • prevent refers to reducing the probability of developing a disease, disorder, or condition in a subject, who does not have, but is at risk of or susceptible to developing a disease, disorder, or condition.
  • the term “subject” means a human or non-human animal selected for treatment or therapy.
  • a subject encompasses, but is not limited to, a mammal.
  • a subject can be a human, a non-human primate (e.g., monkey, baboon, or chimpanzee), a domestic animal or a livestock (e.g., a horse, a cow, a pig, a sheep, a goat, a dog, a cat, a rabbit, a guinea pig, a gerbil, a hamster, a rat, and a mouse).
  • the subject is an infant (e.g., a human infant).
  • therapeutically-effective amount refers to an amount necessary (for example, at dosages and for periods of time and for the means of enteral or oral administration) to achieve the desired therapeutic result.
  • An effective amount of an agent that modulates PHD3 may vary according to factors such as the disease state, age, sex, and weight of the individual.
  • An effective amount is also one in which medical provider, e.g., the attending physician, determines that any toxic or detrimental effects of the agonist are outweighed by the therapeutically beneficial effects.
  • treating a disease, disorder or condition in a subject or “treating” a subject having a disease, disorder or condition refers to subjecting the subject to a pharmaceutical treatment, e.g., administration of a composition, such that at least one symptom of the disease, disorder, or condition is decreased or prevented from worsening.
  • a pharmaceutical treatment e.g., administration of a composition
  • FoleyHoagUS12471059.1 Attorney Docket No. HMV-32325 DETAILED DESCRIPTION
  • the present disclosure provides, among other things agents and methods useful for treating a number of conditions including, but not limited to, muscular dystrophies, muscle wasting disorders, obesity, and metabolic disorders. While in no way intended to be limiting, exemplary compositions, kits, and applications are elaborated on below.
  • PHD3 [0092] The present disclosure provides methods that include administration of an agent that modulates prolyl hydroxylase domain 3 (PHD3), as well as compositions and uses encompassing the same.
  • PHD3 also known as EGLN3, egl-9 family hypoxia inducible factor 3, HIFPH3, and HIFP4H3, is an alpha-ketoglutarate-dependent dioxygenase (Gorres and Raines, 2010; Chan et al., 2002).
  • Mitochondrial fatty acid oxidation (FAO) is negatively regulated by PHD3 (German et al., 2016).
  • PHD3 plays a significant role in muscle metabolism as it has been found to hydroxylate the acetyl CoA carboxylase 2 (ACC2) enzyme, leading to the inhibition of fatty acid oxidation (Yoon et al., 2020). Intriguingly, loss of PHD3 improves metabolic health, promotes fat oxidation, and results in a remarkable 30% increase in exercise capacity by de-repressing the hydroxylation of ACC2 (Yoon et al., 2020). Despite these promising findings, the physiological implications of PHD3- dependent FAO in muscle disease, particularly in the context of DMD, remain unexplored. Consequently, the precise role of PHD3 in the development of DMD, as well as its potential as a therapeutic target for DMD remains elusive.
  • ACC2 acetyl CoA carboxylase 2
  • the present disclosure demonstrates that the deletion of PHD3 leads to notable improvements in both mitochondrial and muscle function in a mouse model of DMD. As described in detail in the examples below, a significant upregulation of genes associated with mitochondrial biogenesis, fatty acid oxidation (FAO), and skeletal muscle structure was observed upon PHD3 deletion. These findings indicate that agents that modulate PHD3 may be useful for enhancing mitochondrial function in individuals affected by DMD. [0095] The disclosure reveals that the loss of PHD3 in an MDX model increases exercise capacity and protects against characteristic muscle damage observed in DMD.
  • Exemplary polynucleotide sequences encoding PHD3 and PHD3 polypeptide sequences are set forth in Table 1 below. 12 FoleyHoagUS12471059.1 Attorney Docket No. HMV-32325 13 FoleyHoagUS12471059.1 Attorney Docket No. HMV-32325 Methods of Treating and/or Preventing [0098]
  • the present disclosure provides the insight inhibition of PHD3 may be useful for treating and/or preventing a number of conditions including, but not limited to, muscular dystrophies, muscle wasting disorders, obesity, and metabolic disorders.
  • Provided herein are methods of treating and/or preventing a disease, condition, or disorder, that includes administration of an agent that modulates PHD3.
  • provided methods include administering one or more agents that reduce the level of or inhibits the activity of PHD3.
  • Muscle Wasting and DMD [0099]
  • the present disclosure provides methods of treating and/or preventing muscle wasting in a subject.
  • Muscle wasting is characterized by weakness and/or wasting away of muscle tissue, which may occur with or without the breakdown of nerve tissue.
  • muscle wasting disorders including muscular dystrophies. Muscular dystrophies are all associated an eventual loss of strength, increasing disability, and possible deformity. Muscle wasting is also observed in a variety of pathologies such as cancer, chronic kidney disease, heart failure, chronic obstructive pulmonary disease (COPD), obesity, as well as after prolonged inactivity or during aging.
  • COPD chronic obstructive pulmonary disease
  • the present disclosure provides methods of treating and/or preventing a muscle wasting disorder in a subject.
  • methods of treating or preventing a muscle wasting disorder comprising administering to a subject one or more agents that reduce the level of or inhibits the activity of PHD3.
  • a muscle wasting disorder is or includes a muscular dystrophy, cachexia, and/or sarcopenia.
  • the present disclosure provides methods of treating and/or preventing a muscular dystrophy.
  • a muscular dystrophy is selected from Becker’s muscular dystrophy, congenital muscular dystrophy, distal muscular dystrophy, Emery-Dreifuss muscular dystrophy, facioscapulohumeral muscular dystrophy, limb- girdle muscular dystrophy, myotonic muscular dystrophy, and oculopharyngeal muscular dystrophy.
  • DMD Duchenne muscular dystrophy
  • DMD is a fatal muscle-wasting disorder caused by a mutation in the dystrophin gene, which affects primarily males (Hoffman et al., 1987; Mendell et al., 2010; Bulfield et al., 1984).
  • the loss of functional dystrophin protein leads to the disruption of the dystrophin-associated protein complex, rendering muscle fibers fragile and prone to damage during contraction (Deconinck et al., 1997; Cole et al., 2002).
  • the disease is characterized by progressive muscle weakness and degeneration (Hoffman et al., 1987).
  • the present disclosure provides methods of treating and/or preventing cachexia.
  • Cachexia is a wasting syndrome that leads to loss of skeletal muscle and fat.
  • Cachexia is estimated to occur in up to 80% of people with advanced cancer, depending on the cancer type and how well they respond to cancer treatment. And it’s thought to directly cause up to FoleyHoagUS12471059.1 Attorney Docket No. HMV-32325 30% of cancer deaths, often because of heart or respiratory failure related to muscle loss.
  • a cachexia is obesity-related.
  • a cachexia is related to cancer, heart disease, kidney disease, or HIV.
  • the present disclosure provides methods of treating and/or preventing sarcopenia.
  • Sarcopenia is the age-related progressive loss of muscle mass and strength.
  • a sarcopenia is aging-related.
  • sarcopenia is obesity- related.
  • Loss of PHD3 has been associated with exercise capacity as well as several pathological conditions, including cancer (German et al., 2016; Chen et al., 2015; Luo et al., 2014).
  • provided methods increase exercise capacity in a subject having a muscle wasting disorder.
  • provided methods increase exercise capacity in a subject having a muscular dystrophy.
  • provided methods increase exercise capacity in a subject having DMD.
  • provided methods increase exercise capacity in a subject having cachexia.
  • provided methods increase exercise capacity in a subject having sarcopenia.
  • provided methods increase exercise capacity in a subject having aging-related sarcopenia. In some embodiments, provided methods increase exercise capacity in a subject having obesity-related sarcopenia. [0106] In some embodiments, exercise capacity is increased 10%-300% relative to exercise capacity prior to administering an agent that reduces the levels of or inhibits the activity of PHD3. In some embodiments, exercise capacity is increased 1.1-fold to 3-fold relative to exercise capacity prior to administering an agent that reduces the levels of or inhibits the activity of PHD3.
  • exercise capacity is increased at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, or more than 100% increase relative to exercise capacity prior to administering an agent that reduces the levels of or inhibits the activity of PHD3.
  • exercise capacity is increased 20%-60% relative to exercise capacity prior to administering an agent that reduces the levels of or inhibits the activity of PHD3.
  • Skeletal Muscle Differentiation [0109] The disclosure reveals significant alterations in gene expression profiles related to skeletal muscle differentiation upon PHD3 loss.
  • a method for increasing skeletal muscle differentiation and/or decreasing skeletal muscle atrophy in a subject in need thereof comprising administering to the subject an agent that reduces the levels of or inhibits the activity of PHD3.
  • provided methods increase expression of one or more genes associated with skeletal muscle differentiation selected from: myoglobin, MyoD, MyoG, Myf5, MyHCI, and MyHcIIa.
  • provided methods increase skeletal muscle differentiation in a subject having a muscle wasting disorder.
  • provided methods increase skeletal muscle differentiation in a subject having a muscular dystrophy.
  • provided methods increase skeletal muscle differentiation in a subject having DMD.
  • provided methods increase skeletal muscle differentiation in a subject having cachexia.
  • provided methods increase skeletal muscle differentiation in a subject having sarcopenia.
  • provided methods increase skeletal muscle differentiation in a subject having aging-related sarcopenia. In some embodiments, provided methods increase skeletal muscle differentiation in a subject having obesity-related sarcopenia. [0113] In some embodiments, provided methods decrease skeletal muscle atrophy in a subject having a muscle wasting disorder. In some embodiments, provided methods decrease skeletal muscle atrophy in a subject having a muscular dystrophy. In some embodiments, provided methods decrease skeletal muscle atrophy in a subject having DMD. In some embodiments, provided methods decrease skeletal muscle atrophy in a subject having cachexia. In some embodiments, provided methods decrease skeletal muscle atrophy in a subject having sarcopenia.
  • provided methods decrease skeletal muscle atrophy in a subject having aging-related sarcopenia. In some embodiments, provided methods decrease skeletal muscle atrophy in a subject having obesity-related sarcopenia.
  • FoleyHoagUS12471059.1 Attorney Docket No. HMV-32325 Sarcolemmal Integrity [0114]
  • the sarcolemma is the plasma membrane of the muscle cell and is surrounded by basement membrane and endomysial connective tissue. Mechanical integrity of the sarcolemma is further supported by key cytoskeletal proteins, including dystrophin, spectrin, and F-actin. Electron microscopy analysis of dystrophic muscle directly shows disruptions in the muscle membrane, termed delta lesions.
  • a method improving sarcolemmal integrity in a subject in need thereof, comprising administering to the subject an agent that reduces the levels of or inhibits the activity of PHD3.
  • provided methods improve sarcolemmal integrity in a subject having a muscle wasting disorder.
  • provided methods improve sarcolemmal integrity in a subject having a muscular dystrophy.
  • provided methods improve sarcolemmal integrity in a subject having DMD.
  • provided methods improve sarcolemmal integrity in a subject having cachexia. In some embodiments, provided methods improve sarcolemmal integrity in a subject having sarcopenia.
  • Fatty Acid Oxidation [0117] The present disclosure provides the insight that loss of PHD3 has a significantly enhances mitochondrial fatty acid oxidation and improves muscle function in DMD mice.
  • a method is provided for increasing fatty acid oxidation and/or decreasing hydroxylation of ACC2 in a subject in need thereof comprising administering to the subject an agent that reduces the levels of or inhibits the activity of PHD3.
  • method increases fatty acid oxidation in muscle cells of the subject and/or decreases hydroxylation of ACC2 in muscle cells of the subject.
  • a method decreases creating activity in a subject.
  • a method restores mitochondrial size and/or morphology in a subject.
  • a subject is afflicted with DMD.
  • a subject is afflicted with a muscular dystrophy selected from Becker’s muscular dystrophy, congenital muscular dystrophy, distal muscular dystrophy, Emery-Dreifuss muscular dystrophy, FoleyHoagUS12471059.1 Attorney Docket No.
  • a subject is afflicted with a muscle wasting disorder.
  • a muscle wasting disorder is cachexia.
  • a cachexia is obesity- related.
  • a cachexia is related to cancer, heart disease, kidney disease, or HIV.
  • a muscle wasting disorder is sarcopenia.
  • a sarcopenia is ageing-related.
  • sarcopenia is obesity-related.
  • the present disclosure provides methods of treating and/or preventing weight gain in a subject. There is evidence of higher body mass index (BMI) and higher fat mass being associates with metabolic risk factors in DMD.
  • BMI body mass index
  • a method is provided for preventing weight gain in a subject in need thereof, comprising administering to the subject an agent that reduces the levels of or inhibits the activity of PHD3.
  • an agent further increases Akt signaling.
  • a method is provided for increasing Akt signaling in an obese or overweight subject, comprising administering to the subject an agent that reduces the levels of or inhibits the activity of PHD3.
  • Akt signaling is increased in the muscle cells of a subject.
  • Akt signaling is increased in the liver of a subject.
  • a subject consumes a high fat or high calorie diet.
  • PHD3 Modulating Agents [0126] In some embodiments, methods of the present disclosure include administering to a subject an agent that modulates PHD3. In some embodiments, a subject is administered one or more agents that reduce the level of or inhibits the activity of PHD3. [0127] Any suitable agents for modulating PHD3 are contemplated by the present disclosure, including, but not limited to, inhibitory polynucleotides, antibodies, antibody conjugates, polypeptide inhibitors, small molecules, and gene therapies.
  • an agent that reduces the levels of or inhibits the activity of PHD3 is an inhibitory polynucleotide.
  • Polynucleotide inhibitors also referred to as nucleic acid inhibitors
  • PHD3 a gene that encodes a protein that regulates PHD3.
  • An FoleyHoagUS12471059.1 Attorney Docket No.
  • HMV-32325 inhibitory polynucleotide can be, e.g., an siRNA, a shRNA, a dsRNA, a ribozyme, a triple-helix former, an aptamer, or an antisense nucleic acid.
  • an inhibitory polynucleotide is selected from the group consisting of siRNA, shRNA, and an antisense oligonucleotide, or a polynucleotide that encodes a molecule selected from the group consisting of siRNA, shRNA, and/or an antisense oligonucleotide.
  • siRNAs are small double stranded RNAs (dsRNAs) that optionally include overhangs.
  • the duplex region of an siRNA is about 18 to 25 nucleotides in length, e.g., about 19, 20, 21, 22, 23, or 24 nucleotides in length.
  • the siRNA sequences can be, in some embodiments, exactly complementary to the target mRNA.
  • dsRNAs and siRNAs in particular can be used to silence gene expression in mammalian cells (e.g., human cells). See, e.g., Clemens et al. (2000) Proc Natl Acad Sci USA 97:6499- 6503; Billy et al.
  • an inhibitory polynucleotide is an siRNA.
  • an siRNA is specific for PHD3.
  • an siRNA is 18-25 nucleotides in length.
  • an siRNA is 18 nucleotides in length.
  • an siRNA is 19 nucleotides in length.
  • an siRNA is 20 nucleotides in length. In some embodiments, an siRNA is 21 nucleotides in length. In some embodiments, an siRNA is 22 nucleotides in length. In some embodiments, an siRNA is 23 nucleotides in length. In some embodiments, an siRNA is 24 nucleotides in length. In some embodiments, an siRNA is 25 nucleotides in length. [0132] In some embodiments, an siRNA is a double stranded RNA (dsRNA). In some embodiments, a dsRNA comprises overhangs. In some embodiments, a dsRNA comprises blunt ends.
  • dsRNA double stranded RNA
  • siRNA molecules can be prepared by chemical synthesis, in vitro transcription, or digestion of long dsRNA by Rnase III or Dicer. These can be introduced into cells by transfection, electroporation, intracellular infection or other methods known in the art. See, for example, each of which is expressly incorporated by reference: Hannon, G J, 2002, RNA Interference, Nature 418: FoleyHoagUS12471059.1 Attorney Docket No. HMV-32325 244-251; Bernstein E et al., 2002, The rest is silence. RNA 7: 1509-1521; Hutvagner G et al., RNAi: Nature abhors a double-strand. Cur. Open.
  • an agent is an antibody-siRNA conjugate.
  • an antibody-siRNA conjugate is specific for muscle tissue.
  • shRNAs [0135] Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes & Dev. 16:948-958; Paul C P, Good P D, Winer I, and Engelke D R. (2002). Effective expression of small interfering RNA in human cells.
  • an inhibitory polynucleotide is an shRNA.
  • a target sequence for an shRNA is set forth in Table 2.
  • Table 2 Exemplary shRNA sequences FoleyHoagUS12471059.1 Attorney Docket No. HMV-32325
  • an shRNA is specific for PHD3.
  • a target sequence for PHD3 is as follows: shPHD3 #1 (TRCN0000001048): CACCTGCATCTACTATCTGAA (SEQ ID NO: 5) shPHD3 #2 (TRCN0000001050): GTGGCTTGCTATCCGGGAAAT (SEQ ID NO: 6) [0141]
  • an shRNA target sequence comprises a sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO: 5.
  • an shRNA target sequence comprises a sequence that differs by no more that 3 nucleotides, no more than 2 nucleotides, no more than 1 nucleotide relative to SEQ ID NO: 5.
  • an shRNA target sequence comprises a sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO: 6. In some embodiments, an shRNA target sequence comprises a sequence that differs by no more that 3 nucleotides, no more than 2 nucleotides, no more than 1 nucleotide relative to SEQ ID NO: 6. FoleyHoagUS12471059.1 Attorney Docket No. HMV-32325 [0142] In some embodiments, an shRNA targets SEQ ID NO:5 or SEQ ID NO: 6. In some embodiments, an shRNA targets SEQ ID NO: 5. In some embodiments, an shRNA targets SEQ ID NO: 6.
  • an shRNA comprises a sense strand set forth in SEQ ID NO: 5. In some embodiments, an shRNA comprises a sense strand set forth in SEQ ID NO: 6. [0144] In some embodiments, an shRNA comprises a sense strand set forth in SEQ ID NO: 5. In some embodiments, an shRNA comprises a sense strand set forth in SEQ ID NO: 6. Antisense oligonucleotides [0145] In some embodiments, an inhibitory polynucleotide is an antisense oligonucleotide (ASO). In some embodiments, an ASO is specific for PHD3.
  • ASO antisense oligonucleotide
  • Antisense oligonucleotide agents can include, for example, from about 8 to about 80 nucleobases (i.e. from about 8 to about 80 nucleotides), e.g., about 8 to about 50 nucleobases, or about 12 to about 30 nucleobases.
  • an ASO is 13 to 30 nucleotides in length.
  • an ASO is 15 nucleotides in length.
  • an ASO is 16 nucleotides in length.
  • an ASO is 17 nucleotides in length.
  • an ASO is 18 nucleotides in length.
  • an ASO is 19 nucleotides in length.
  • an ASO is 20 nucleotides in length. In some embodiments, an ASO is 21 nucleotides in length. In some embodiments, an ASO is 22 nucleotides in length. In some embodiments, an ASO is 23 nucleotides in length. In some embodiments an ASO is 24 nucleotides in length. In some embodiments, an ASO is 25 nucleotides in length.
  • Antisense compounds include ribozymes, external guide sequence (EGS) oligonucleotides (oligozymes), and other short catalytic RNAs or catalytic oligonucleotides which hybridize to the target nucleic acid and modulate its expression.
  • Anti-sense compounds can include a stretch of at least eight consecutive nucleobases that are complementary to a sequence in the target gene.
  • An oligonucleotide need not be 100% complementary to its target nucleic acid sequence to be specifically hybridizable.
  • An oligonucleotide is specifically hybridizable when binding of the oligonucleotide to the target interferes with the normal function of the target molecule to cause a loss of utility, and there is a sufficient degree of complementarity to avoid non-specific binding of the oligonucleotide to non-target sequences under conditions in which specific binding is desired, FoleyHoagUS12471059.1 Attorney Docket No.
  • Hybridization of antisense oligonucleotides with mRNA can interfere with one or more of the normal functions of mRNA.
  • the functions of mRNA to be interfered with include all key functions such as, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity which may be engaged in by the RNA.
  • binding of specific protein(s) to the RNA may also be interfered with by antisense oligonucleotide hybridization to the RNA.
  • exemplary antisense compounds include DNA or RNA sequences that specifically hybridize to the target nucleic acid, e.g., the mRNA encoding one of the gene products described herein.
  • the complementary region can extend for between about 8 to about 80 nucleobases.
  • the compounds can include one or more modified nucleobases.
  • Modified nucleobases may include, e.g., 5- substituted pyrimidines such as 5- iodouracil, 5-iodocytosine, and C5- propynyl pyrimidines such as Cs-propynylcytosine and C 5 -propynyluracil.
  • Other suitable modified nucleobases include, e.g., 7- substituted- 8-aza-7-deazapurines and 7-substituted-7-deazapurines such as, for example, 7-iodo-7- deazapurines, 7-cyano-7-deazapurines, 7-aminocarbonyl-7- deazapurines.
  • 6-amino-7-iodo-7-deazapurines examples include 6-amino-7-iodo-7-deazapurines, 6-amino-7- cyano-7-deazapurines, 6- amino-7- aminocarbonyl-7-deazapurines, 2-amino-6- hydroxy-7-iodo-7-deazapurines, 2- amino-6-hydroxy-7- cyano-7-deazapurines, and 2- amino-6-hydroxy-7-aminocarbonyl-7-deazapurines.
  • an agent is an antibody-ASO conjugate.
  • an antibody-ASO conjugate is specific for muscle tissue.
  • Aptamers are short oligonucleotide sequences that can be used to recognize and specifically bind almost any molecule, including cell surface proteins.
  • RNA interference molecules can be delivered in vitro to cells or in vivo. Typical delivery means known in the art can be used.
  • Any mode of delivery can be used without limitation, including: intravenous, intramuscular, intraperitoneal, intraarterial, local delivery during surgery, endoscopic, or subcutaneous.
  • Vectors can be selected for desirable properties for any particular application.
  • Vectors can be viral, bacterial or plasmid.
  • Adenoviral vectors are useful in this regard.
  • Tissue-specific, cell-type specific, or otherwise regulatable promoters can be used to control the transcription of the inhibitory polynucleotide molecules.
  • Non-viral carriers such as liposomes or nanospheres can also be used.
  • a RNA interference molecule or an RNA interference encoding oligonucleotide can be administered to the subject, for example, as naked RNA, in combination with a delivery reagent, and/or as a nucleic acid comprising sequences that express the siRNA or shRNA molecules.
  • the nucleic acid comprising sequences that express the siRNA or shRNA molecules are delivered within vectors, e.g. plasmid, viral and bacterial vectors. Any nucleic acid delivery method known in the art can be used in the present invention.
  • Suitable delivery reagents include, but are not limited to, e.g., the Minis Transit TKO lipophilic reagent; lipofectin; lipofectamine; cellfectin; polycations (e.g., polylysine), atelocollagen, nanoplexes and liposomes.
  • Small Molecules [0153]
  • an agent that modulates PHD3 is a small molecule.
  • “Small molecule” as used herein, is meant to refer to an agent, which has a molecular weight of less than about 6 kDa and most preferably less than about 2.5 kDa.
  • a small molecule includes molecules that are less than about 1000 molecular weight or less than about 500 molecular weight.
  • Exemplary small molecule compounds include, but are not limited to, peptides, peptidomimetics, nucleic acids, carbohydrates, organic molecules, and biosynthetic molecules.
  • exemplary small molecules can be screened for activity for modulating PHD3.
  • FoleyHoagUS12471059.1 Attorney Docket No. HMV-32325
  • a small molecule is specific for PHD3.
  • a small molecule targets PHD1, PHD2 or PHD3.
  • a small molecule inhibits PHD3.
  • a small molecule is a PHD3 inhibitor, such as, but not limited to, those described in International Patent Application Publication Nos.
  • Compounds useful in the methods of the present invention may be obtained from any available source, including systematic libraries of natural and/or synthetic compounds. Compounds may also be obtained by any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; peptoid libraries (libraries of molecules having the functionalities of peptides, but with a novel, non-peptide backbone which are resistant to enzymatic degradation but which nevertheless remain bioactive; see, e.g., Zuckermann et al., 1994, J. Med. Chem.
  • Libraries of agents may be presented in solution (e.g., Houghten, 1992, Biotechniques 13:412-421), or on beads (Lam, 1991, Nature 354:82-84), chips (Fodor, 1993, Nature 364:555-556), bacteria and/or spores, (Ladner, U.S. Patent No. 5,223,409), plasmids (Cull et al, 1992, Proc Natl Acad Sci USA 89:1865-1869) or on phage (Scott and Smith, 1990, Science 249:386-390; Devlin, 1990, Science 249:404-406; Cwirla et al, 1990, Proc. Natl. Acad. Sci.
  • an agent is a sgRNA specific for a nucleic acid sequence encoding PHD3.
  • a sgRNA comprises a spacer that hybridizes to the target nucleic acid sequence and a scaffold that recruits the Cas9 enzyme. Cas9 will produce a double strand break at the target site.
  • a sgRNA spacer is 15-25 nucleotides in length. In some embodiments, a sgRNA spacer is 17 nucleotides in length.
  • a sgRNA spacer is 18 nucleotides in length. In some embodiments, a sgRNA spacer is 19 nucleotides in length. In some embodiments, a sgRNA spacer is 20 nucleotides in length. In some embodiments, a sgRNA spacer is 21 nucleotides in length. In some embodiments, a sgRNA spacer is 22 nucleotides in length. In some embodiments, a sgRNA spacer is 23 nucleotides in length. In some embodiments, a sgRNA spacer is 24 nucleotides in length. [0162] In some embodiments, a target nucleic acid is immediately adjacent to a protospacer adjacent motif (PAM).
  • PAM protospacer adjacent motif
  • a target nucleic acid is a double stranded DNA (dsDNA). In some embodiments, a target nucleic acid is a single stranded RNA (ssRNA).
  • dsDNA double stranded DNA
  • ssRNA single stranded RNA
  • NHEJ non-homologous end joining
  • HDR homology directed repair
  • an agent that modulates PHD3 is an antibody. In some embodiments, an agent that modulates PHD3 is an anti-PHD3 antibody. [0165] Exemplary antibodies that modulate PHD3 are commercially available.
  • Antibodies also include antigen-binding fragments (referred to herein as “antibody fragment” and “antigen-binding fragment,” or similar terms) which are fragments of an antibody that retain the ability to bind to an target antigen.
  • Such fragments include, e.g., a single chain antibody, a single chain Fv fragment (scFv), an Fd fragment, an Fab fragment, an Fab’ fragment, or an F(ab’)2 fragment.
  • An scFv fragment is a single polypeptide chain that includes both the heavy and light chain variable regions of the antibody from which the scFv is derived.
  • intrabodies, minibodies, triabodies, and diabodies are also included in the definition of antibody and are compatible for use in the methods described herein. See, e.g., Todorovska et al.
  • Bispecific antibodies are also embraced by the term “antibody.” Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens.
  • the agents can be modified, e.g., with a moiety that improves the stabilization and/or retention of the antibodies in circulation, e.g., in blood, serum, or other tissues.
  • a polypeptide described herein can be PEGylated as described in, e.g., Lee et al. (1999) Bioconjug Chem 10(6): 973-8; Kinstler et al. (2002) Advanced Drug Deliveries Reviews 54:477-485; and Roberts et al. (2002) Advanced Drug Delivery Reviews 54:459-476 or HESylated (Fresenius Kabi, Germany; see, e.g., Pavisi ⁇ et al.
  • PHD3 modulating agents can improve the stability, or retention of, the polypeptide by at least 1.5 (e.g., at least 2, 5, 10, 15, 20, 25, 30, 40, or 50 or more) fold.
  • Administration of PHD3 modulating agents [0168] PHD3 modulating agents disclosed herein may be administered over any period of time effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • the period of time may be at least 1 day, at least 10 days, at least 20 days, at least 30, days, at least 60 days, at least three months, at least six months, at least a year, at least three years, at least five years, or at least ten years.
  • the dose may be administered when needed, sporadically, or at regular intervals. For example, the dose may be administered monthly, weekly, biweekly, triweekly, once a day, or twice a day.
  • a dose of the composition is administered at regular intervals over a period of time.
  • a dose of the composition is administered at least once a week.
  • a dose of the composition is administered at least twice a week.
  • a dose of the composition is administered at least three times a week. In some embodiments, a dose of the composition is administered at least once a day. In some embodiments, a dose of the composition is administered at least twice a day. In some embodiments, doses of the composition are administered for at least 1 week, for at least 2 weeks, for at least 3 weeks, for at least 4 weeks, for at least 1 month, for at least 2 months, for at least 3 months, for at least 4 months, for at least 5 months, for at least 6 months, for at least 1 year, for at least two years, at least three years, or at least five years. [0169] In some embodiments, a PHD3 modulating agent is administered intravenously.
  • an agent is administered at least once a week or at least once a month. In some embodiments, an agent is administered for at least 7 days, for at least 30 days, for at least 60 days, or for at least 90 days. [0170] The selected dosage level will depend upon a variety of factors including the activity of the particular agent employed, the route of administration, the time of administration, the rate of excretion or metabolism of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
  • FoleyHoagUS12471059.1 Attorney Docket No.
  • Suitable human doses of any of the compounds described herein can further be evaluated in, e.g., Phase I dose escalation studies. See, e.g., van Gurp et al. (2008) Am J Transplantation 8(8):1711-1718; Hanouska et al. (2007) Clin Cancer Res 13(2, part 1):523-531; and Hetherington et al. (2006) Antimicrobial Agents and Chemotherapy 50(10): 3499-3500. [0172] A physician having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required.
  • EXEMPLIFICATION Example 1 PHD3 Deletion Improves Muscle Function in DMD Mice
  • the present example describes characterization of PHD3 in DMD patients and a DMD model mouse. Loss of dystrophin leads to a disconnection between the cytoskeleton and the extracellular matrix of muscle cells. This results in mitochondrial damage, reduced fat oxidation, and intramuscular lipid accumulation, which ultimately leads to muscle wasting and loss of function (Timpani et al., 2015; Allen et al., 2010; Alderton and Steinhardt, 2000).
  • the present disclosure encompasses a recognition that PHD3 may have a role in DMD. To assess this, the expression of PHD3 in DMD patients was examined.
  • the BL10-mdx mouse strain was specifically chosen due to its higher serum creatine kinase levels and inflammation in muscle, making them an ideal model for assessing standard outcomes and identifying skeletal muscle phenotypes (Grounds et al., 2008).
  • PHD3 flox-CMV-Cre (phd3 -/- ) mice were generated by crossing PHD3 flox mice (phd3 +/+ ) with CMV-Cre (Yoon et al., 2020). These mice were then bred with BL10-mdx mice to obtain the PHD3 KO MDX mouse (phd3 -/- mdx) (FIG. 1B and FIG. 2A).
  • Creatine kinase is an enzyme that catalyzes the conversion of creatine phosphate to creatine, and its levels increase in the blood during muscle damage (Apple and Rhodes, 1985; Yu et al., 2020). Higher levels of CK activity are a well-known marker for the DMD phenotype (Cohn et al., 2002; Sharp et al., 1992). CK activity was not changed in phd3 +/+ and phd3 -/- mice, but phd3 +/+ mdx mice had higher CK activity (FIG. 1F). Interestingly, PHD3 loss in MDX mice (phd3 -/- mdx) resulted in reduced CK activity (FIG. 1F).
  • Example 2 PHD3 Deletion Upregulates Genes Involved in Mitochondrial FAO and Skeletal Muscle Differentiation
  • RNA sequencing analysis of skeletal muscle from phd3 +/+ , phd3 -/- , phd3 +/+ mdx, and phd3 -/- mdx mice was conducted (FIG.3A and FIG. 4A).
  • Principal component analysis (PCA) was used to identify the impact of PHD3 loss on gene expression profiles in the quadricep tissues of these mice (FIG. 3B).
  • PHD3 has limited effect on the mdx model, whereas it exerts a more pronounced effect on the gene expression profiles of the WT (phd3 +/+ ) mice (FIG. 3B-3C and FIG. 4A-4B).
  • Differentially-expressed (DE) genes were further analyzed using pathway enrichment, and it was observed that upregulated genes in the phd3 -/- condition were enriched in Gene Ontology (GO) lipid metabolic pathways (FIG. 3C).
  • GO Gene Ontology
  • RNAs were significantly altered in the phd3 -/- mdx group compared to those in phd3 +/+ mdx mice (FIG. 4F).
  • Muscle contractile genes encode the proteins that make up the muscle fibers, which are altered in various forms of muscular dystrophy (Boscolo Sesillo et al., 2019; Megeney et al., 1996). For example, in Duchenne muscular dystrophy, the expression of MyoD is decreased, which may impair muscle regeneration (Gnocchi et al., 2009). Conversely, the expression of Myogenin and Myf5 is increased in some forms of muscular dystrophy, potentially as a compensatory response to muscle fiber damage (Ustanina et al., 2007; Yamamoto et al., 2018).
  • Skeletal myogenesis is a crucial step in the formation of functional and mature muscles (Hernández-Hernández et al., 2017; Bentzinger et al., 2012). Impairment in this process can directly affect the phenotype of DMD.
  • the gene set for contractile proteins and muscle differentiation markers was measured in a cell system.
  • PHD3-depleted C2C12 was for myogenesis and found that the loss of PHD3 increases the expression levels of Ankrd2, with or without differentiation (FIG. 6A and 6D).
  • this gene expression pattern is not the same as that in vivo.
  • DMD progressive muscular dystrophy
  • myofiber degeneration with fibrosis myofiber degeneration with fibrosis
  • metabolic alterations such as fatty acid metabolism.
  • lipid metabolism changes in Duchenne patient cells are poorly understood.
  • PCA was used to identify the impact of PHD3 loss on metabolite profiles in quadricep tissues from phd3 +/+ , phd3 -/- , phd3 +/+ mdx, and phd3 -/- mdx mice (FIG.
  • the long-chain acyl-carnitines were lower in WT mice compared to MDX mice (FIG.7F). Moreover, these long-chain acyl-carnitines were even lower in MDX mice with PHD3 depletion compared to MDX mice (FIG. 7G). It was reasoned that these fuels might have a higher oxidation rate during PHD3 depletion in vivo, preventing acyl-carnitines from accumulating. Thus, phd3 -/- mdx mice utilize long-chain acyl-carnitines more than phd3 +/+ mdx mice.
  • MDX mice have decreased grip strength compared to WT mice due to their weak and fragile muscle phenotype.
  • the phd3 -/- mdx mice exhibited restored muscle strength compared to phd3 +/+ mdx mice (FIG. 9A).
  • exercise endurance was assessed in phd3 +/+ mdx and phd3 -/- mdx mice using a metabolic treadmill (FIG. 9B).
  • the results showed that phd3 -/- mdx mice ran 20% longer and >30% further distance than phd3 +/+ mdx mice (FIG.9C and 9D).
  • Example 6 Loss of PHD3 prevents weight gain and fat accumulation on a high fat diet
  • HFD high fat diet
  • PHD3 knockout mice on a low fat diet (LFD) did not have any changes in body weight or lean body mass, serum free fatty acids (FFA) or triglycerides (TG) relative to wild-type (WT) mice (data not shown).
  • FFA serum free fatty acids
  • TG triglycerides
  • WT wild-type mice
  • mice 8 week old WT or PHD3 KO mice were challenged with a HFD for 10 weeks, as illustrated in FIG.10A. During that time, mice were weighed every week and glucose tolerance (GTT) was measured in 8weeks or insulin tolerance (ITT) in 10 weeks. Surprisingly, PHD3 KO mice prevent weight gain under a HFD, as shown in FIG. 10B. Usually HFD drive obesity or type 2 diabetes. The glucose tolerance test can be used to screen for type 2 diabetes. To measure their physiological response to glucose, mice were fasted 16 h and the blood glucose level was measured after high glucose injection, over time by time. PHD3 KO mice are also sensitive to glucose, as shown in FIG. 10D.
  • the fasted blood glucose levels were measured in mice under both a high fat and low fat diet (FIG. 12). No change was observed in blood glucose levels between WT and PHD3 KO mice under low fat diet. The blood glucose level was increased in WT mice under HFD, however, PHD3 KO mice were protected from this increase in blood glucose on a HFD. Interestingly, serum insulin levels were dramatically lower in PHD3 KO mice even in LFD condition. This insulin result is consistent with a report by Taniguchi, C., et al. (2013) Nat Med 19, 1325–1330 that mice with PHD3 silencing in the liver have decreased insulin levels.
  • Example 7 Loss of PHD3 alters metabolite profile in muscle and liver [0190]
  • the present example describes the characterization of the metabolite profile of WT and PHD3 KO mice on a HFD. Metabolomics were performed on muscle (quadricep) tissue and liver dissected from WT mice and PHD3 KO mice under HFD condition. Results are shown in FIGs. 13A-E. In muscle, there was a large impact on metabolites associated with arginine and proline metabolism, as well as nicotinate and nicotinamide metabolism (FIG. 13B). In liver, glycerophospholipid metabolism, glycine, serine, and threonine metabolism, and biotin metabolism were the most significantly affected pathways.
  • Example 8 AKT signaling is increased in PHD3 KO mice under HFD [0191]
  • Many molecular sensors detect and response to nutrient availability. For example, nutrient availability determines the levels of AMP/ATP, which directly affects AMPK activity.
  • AMPK is a major regulator of metabolism. It senses glucose, insulin, drugs and stress to regulate energy dependent signaling pathways.
  • AMPK is activated by phosphorylation and coordinates with other enzymes to alter cellular metabolism. For example, AMPK is activated through phosphorylation by LKB1, and inactivated by dephosphorylation by protein phosphatases.
  • AMPK controls catabolic pathways and anabolic pathways, such as fatty acid oxidation by inhibiting ACC2 through phosphorylation.
  • AMPK signaling was assessed on the livers of PHD3 KO mice on HFD. Specifically, it was found that livers from PHD3 KO mice on a high fat diet had decreased p-ACC2 and increased p-Akt at both positions T308 and S473. And that subsequently fasting these mice led to a dramatic increase in p-ACC and decrease of pAkt (both T308 and S473). See FIG.14A and FIG. 14B. Similarly, AKT signaling was found to be increased in the quadriceps of PHD3 KO mice under HFD, as shown in FIG. 15A and FIG. 15B.
  • the disclosure features a method for culturing human cells.
  • HEK293T human embryonic kidney, female
  • C2C12 mouse mesenchymal precursor, sex of cell lines was unknown
  • ATCC American Type Culture Collection
  • Cells were cultured at 37 degree, 5% CO 2 in 4.5 g/L (25 mM) glucose Dulbecco’s modified Eagle's medium, supplemented with 10% fetal bovine serum and penicillin/streptomycin.
  • the disclosure features a method to generate knockout mice.
  • Egln2 tm2Fong Egln1 tm2Fong Egln3 tm2Fong /J mice PHD3-CMV-Cre mice
  • PHD3-CMV-Cre mice Egln2 tm2Fong Egln1 tm2Fong Egln3 tm2Fong /J mice (PHD1 FL /PHD2 FL /PHD3 FL triple floxed strain # 028097) (Takeda et al., 2006) were obtained from The Jackson Laboratory and housed in the New Research Building Animal Facility at Harvard Medical School.
  • PHD3 FL mouse were obtained by breeding between Egln2 tm2Fong Egln1 tm2Fong Egln3 tm2Fong /J mice and C57BL/6J. PHD3 FL mouse were crossed with B6.C-Tg(CMV-cre)1Cgn/J mice (CMV-Cre mice # 006054) to generate PHD3 FL :CMV-Cre knockout mice . Only male mice were used for experiment.
  • the disclosure features a method for preparing and transfecting agents into human cells.
  • pcDNA3.1-HA-PHD3 or HA-pBabe PHD3 were previously described (Lee et al., 2005).
  • Synthetic oligos for shRNAs (sequences in Table 2) were digested with Age I and EcoRI (NEB), and inserted into a pLKO-puromycin vector using T4 DNA ligase (NEB).
  • This shRNA construct was co-transfected into HEK293T cells with pRSV-Rev, pMD2-VSVG, and pMDLg/pRRE plasmids to prepare lentiviral particles.
  • lentiviruses were collected from the supernatant of HEK293T cells.
  • Target cells were infected with virus for 24 hours and selected using puromycin (2 ug/ml) for 48 hours to establish stable cell lines.
  • the disclosure features a method for quantitative RT-PCR analysis.
  • RNA was isolated by extraction with Trizol according to manufacturer instructions (Invitrogen) or with the RNA Clean & Concentrator Kit (Zymo Research).
  • cDNA was synthesized using iScript cDNA synthesis kit (BioRad).
  • Quantitative real-time PCR was performed with SYBR Green Fast Mix (Quanta Biosciences) on a Roche Lightcycler 480 and analyzed by using ⁇ Ct calculations. qPCR analyses in human cell lines are relative to the reference gene B2M. qPCR analyses in mouse cell line or tissue are relative to ⁇ -Actin.
  • the primer sequences are described in Table 3 below.
  • FoleyHoagUS12471059.1 Attorney Docket No. HMV-32325 [0197] Table 3 – Exemplary RT-PCR primer sequences [0198]
  • the disclosure features methods for analyzing protein expression.
  • the disclosure features methods for measuring fatty acid oxidation (FAO).
  • FEO fatty acid oxidation
  • reaction were transferred into tubes containing 200 uL of 1 M perchloric acid to quench reactions and discs of Whatman filter paper (pre-treated with 20 uL of 1 M NaOH) placed in the caps to capture 14 CO 2 . After 1 hour incubation to allow 14 CO 2 capture, the paper disc or the remaining acid solution were transferred to a scintillation vial. Vials were mixed with scintillation fluid, and the average counts per min over 3 min were measured with a standard scintillation counter. Data were normalized to protein content. [0201] In some embodiments, the disclosure features methods for measuring respiration. Respiration was assessed using the Seahorse XFe-96 Analyzer (Seahorse Bioscience).
  • the disclosure features methods for profiling metabolites with mass spectrometry. Metabolites were extracted from flash frozen tissues ( ⁇ 5 mg/ml) in 80% MeOH and analyzed on two distinct methods of hydrophilic interaction liquid chromatography coupled to mass spectrometry (HILIC-MS).
  • electrospray ionization was tailored to negative-ion mode, and in the second method to positive-ion mode.
  • negative-ion mode analytes were eluted in buffer A (20 mM ammonium acetate, 20 mM ammonium hydroxide) and buffer B (10 mM ammonium hydroxide in 75:25 acetonitrile:methanol).
  • Samples were run on a HILIC silica (3 um, 2.1 x 150 mm) column (Waters) with a binary flow rate of 0.4 mL/min for 10 min on linear gradient (95% buffer B to 0% buffer B) followed by 2 min with (0% buffer B) and ending with a 2-minute linear gradient (0% buffer B to 95% buffer B) and holding (95% buffer B) for 13 min.
  • samples were dried down and reconstituted in a 20:70:10: acetonitrile:MeOH:water mixture.
  • the buffers were: buffer A (10 mM ammonium formate, 0.1% formic acid in water) and buffer B (acetonitrile, 0.1% formic acid).
  • a master mix of reference standards for metabolites in the targeted method were run immediately prior to each set of samples, such that their retention times were associated with peaks in the unknown samples run over that same column. Peaks were integrated in FoleyHoagUS12471059.1 Attorney Docket No. HMV-32325 Tracefinder 3.3. Metabolite levels were normalized to cell number in parallel plates or protein concentration in the same amount of tissue powder samples. [0203] In some embodiments, the disclosure features methods for analysis creatine kinase levels. Creatine kinase (CK) levels were assessed on a weekly basis until the age of 8 wk and on a biweekly basis from 10 until 32 wk.
  • Creatine kinase (CK) levels were assessed on a weekly basis until the age of 8 wk and on a biweekly basis from 10 until 32 wk.
  • the disclosure features methods for evaluating exercise performance. 12-week-old mice were acclimated to the treadmill 2 days prior to the experiments by running for 5 min/day at 5 m/min and 10 m/min followed by 15 m/min for 1 min.
  • the disclosure features methods for analysis grip strength. Grip strength of the forelimbs was assessed using a grid attached to an isometric force transducer (Columbus Instruments). The force transducer recorded the maximum force required to break the mouse's grip from the mesh surface. In total, five strength measurements were recorded, each consisting of three pulls. The three highest values were averaged and normalized to the body weight.
  • paraffin quadriceps sections were rinsed with xylene (three times for 5 min each). Slides were incubated twice in 100% ethanol (10 min each) for rehydration. After rinsing with water, slides were boiled (10 min) with 10 mM sodium citrate buffer (pH 6.0) for antigen unmasking. The sections were washed with PBS with 0.4% Triton X-100 and blocked for 1 hour at 4°C, with PBS FoleyHoagUS12471059.1 Attorney Docket No. HMV-32325 containing 5% normal goat serum. Slides were incubated with primary antibodies in PBS overnight at 4°C.
  • EBD Evans blue dye
  • the EBD solution was then intravenously injected (50 ⁇ l/10 g body weight) into 12-week- old mice. 48 h after the EBD injection, the quadricep muscles were harvested and frozen with liquid nitrogen. Consecutive sections were made from each frozen heart using a cryostat (Microm) with a chamber temperature of -225°C. Several pairs of cross-sectional 10- ⁇ m-thick adjacent sections were randomly selected from the consecutive sections. EBD in this section was observed under a fluorescent microscope (Nikon) using 620 nm excitation/680 nm emission after an immunofluorescence experiment with anti-laminin and DAPI.
  • Tissues were fixed in 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1Mcacodylate buffer, pH 7.4, for at least 1 h at room temperature.
  • tissues were washed in 0.1 M cacodylate buffer and fixed secondarily in 1% osmium tetroxide for 1 h at 4°C.
  • Tissues were then washed in deionized (DI) water before being immersed in 2% aqueous uranyl acetate to be contrast fixed overnight at 4°C.
  • DI deionized
  • tissues were washed and dehydrated. Infiltration proceeded with 1:1 propylene oxide and LX112 Epon resin. Tissues were then embedded and cured at 60°C over 48 h.
  • Confounding factors including gender, age, batch, and disease, as well as hidden factors that could cause gene expression variability, were estimated and removed using probabilistic estimation of expression residuals (PEER) (PMID: 22343431).
  • PEER probabilistic estimation of expression residuals
  • GSEA gene set enrichment analysis
  • fgsea package https://www.biorxiv.org/content/early/2017/06/20/060012
  • genes were ranked based on the Pearson correlation coefficient against PHD3 expression, and enrichment analysis was performed to determine the enriched gene sets co-expressed with PHD3 in control or DMD patient group (GSE38417).
  • FoleyHoagUS12471059.1 Attorney Docket No. HMV-32325 REFERENCES Apple, F.S., and Rhodes, M. (1985). Enzymatic estimation of skeletal muscle damage by analysis of changes in serum creatine kinase. J Appl Physiol.65, 2598-2600. Alderton, J.M., and Steinhardt, R.A. (2000). Calcium influx through calcium leak channels is responsible for the elevated levels of calcium-dependent proteolysis in dystrophic myotubes. J Biol Chem. 275, 9452-60. Allen, D.G., Gervasio, O.L., Yeung, E.W., and Whitehead, N.P. (2010). Calcium and the damage pathways in muscular dystrophy. Can.
  • Dystrophin the protein product of the Duchenne muscular dystrophy locus. Cell 51, 919–928. FoleyHoagUS12471059.1 Attorney Docket No. HMV-32325 Jones, A.E., Sheng, L., Acevedo, A., Veliova, M., Shirihai, O.S., Stiles, L., and Divakaruni, A.S. (2021). Forces, fluxes, and fuels: tracking mitochondrial metabolism by integrating measurements of membrane potential, respiration, and metabolites. Am. J. Physiol. Cell Physiol.320, C80-C91.
  • IFN- ⁇ promotes muscle damage in the mdx mouse model of Duchenne muscular dystrophy by suppressing M2 macrophage activation and inhibiting muscle cell proliferation. J. Immunol.187, 5419. Yamamoto, M., Legendre, N.P., Biswas, A.A., Lawton, A., Yamamoto, S., Tajbakhsh, S., Kardon, G., and Goldhamer, D.J. (2018).

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Abstract

The disclosure provides methods useful for treating and/or preventing muscular dystrophies, such as Duchenne's muscular dystrophy (DMD), and/or other muscle wasting disorders by modulating prolyl hydroxylase 3 (PHD3).

Description

Attorney Docket No. HMV-32325 METHODS FOR TREATING MUSCLE-RELATED DISORDERS BY MODULATING PROLYL HYDROXYLASE 3 CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of priority to U.S Provisional Application No. 63/535,193, filed on August 29, 2023, and U.S Provisional Application No.63/545,878, filed on October 26, 2023, the contents of which are hereby incorporated by reference in their entireties for all purposes. BACKGROUND [0002] Duchenne muscular dystrophy (DMD) is a debilitating genetic disorder characterized by progressive muscle weakness and degeneration, primarily attributed to a mutation in the dystrophin gene. DMD is one of a myriad of pathological conditions that cause progressive muscle wasting. Muscle wasting is a debilitating and life-threatening disease state that presents a serious public health concern. Although there have been advances in treating DMD, there remains an ongoing need for new therapies to treat and prevent DMD and other muscle wasting disorders. SUMMARY [0003] Provided herein are methods and compositions useful for treating and/or preventing muscle wasting and/or increasing skeletal muscle differentiation by modulating prolyl hydroxylase 3 (PHD3). The present disclosure is based, at least in part, on the discovery that PHD3 has a role in muscle wasting disorders, including DMD. [0004] In some embodiments, provided are methods of treating or preventing a muscular dystrophy in a subject comprising administering to a subject an agent that reduces the levels of or inhibits the activity of PHD3. In some embodiments, a muscular dystrophy is DMD. In some embodiments, a muscular dystrophy is selected from Becker’s muscular dystrophy, congenital muscular dystrophy, distal muscular dystrophy, Emery-Dreifuss muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, myotonic muscular dystrophy, and oculopharyngeal muscular dystrophy. FoleyHoagUS12471059.1 Attorney Docket No. HMV-32325 [0005] In some embodiments, provided are methods of treating or preventing a muscle wasting disorder in a subject comprising administering to a subject an agent that reduces the levels of or inhibits the activity of PHD3. [0006] In some embodiments, a muscle wasting disorder is cachexia. In some embodiments, cachexia is an obesity-related cachexia. In some embodiments, a muscle wasting disorder is sarcopenia. In some embodiments, the sarcopenia is ageing-related sarcopenia. [0007] In some embodiments, provided are methods of increasing skeletal muscle differentiation and/or decreasing skeletal muscle atrophy in a subject in need thereof comprising administering to a subject an agent that reduces the levels of or inhibits the activity of PHD3. [0008] In some embodiments, provided are methods of improving sarcolemmal integrity and/or increasing exercise capacity in a subject in need thereof, comprising administering to the subject an agent that reduces the levels of or inhibits the activity of PHD3. [0009] In some embodiments, provided are methods of increasing fatty acid oxidation and/or decreasing hydroxylation of acetyl CoA carboxylase 2 (ACC2) in a subject in need thereof comprising administering to the subject an agent that reduces the levels of or inhibits the activity of PHD3. In some embodiments, the method increases fatty acid oxidation in muscle cells of the subject and/or decreases hydroxylation of ACC2 in muscle cells of a subject. [0010] In some embodiments, a subject is afflicted with a muscular dystrophy. In some embodiments, a subject is afflicted with DMD. In some embodiments, a subject is afflicted with a muscular dystrophy that is selected from Becker’s muscular dystrophy, congenital muscular dystrophy, distal muscular dystrophy, Emery-Dreifuss muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, myotonic muscular dystrophy, and oculopharyngeal muscular dystrophy. [0011] In some embodiments, a subject is afflicted with a muscle wasting disorder. In some embodiments, a muscle wasting disorder is cachexia. In some embodiments, cachexia is an obesity- related cachexia. In some embodiments, a muscle wasting disorder is sarcopenia. In some embodiments, a sarcopenia is ageing-related sarcopenia. [0012] In some embodiments, provided methods decrease creatine kinase activity in a subject. [0013] In some embodiments, provided methods restore mitochondrial size and/or morphology in a subject. FoleyHoagUS12471059.1 Attorney Docket No. HMV-32325 [0014] In some embodiments, provided are methods of preventing weight gain in a subject in need thereof, comprising administering to the subject an agent that reduces the levels of or inhibits the activity of PHD3ments, a subject consumes a high fat or high calorie diet. In some embodiments, an agent further increases Akt signaling. [0015] In some embodiments, provided are methods of increasing Akt signaling in an obese or overweight subject, comprising administering to the subject an agent that reduces the levels of or inhibits the activity of PHD3. [0016] In some embodiments, a subject consumes a high fat or high calorie diet. [0017] In some embodiments, Akt signaling is increase in muscle cells of a subject. [0018] In some embodiments of any of the methods described herein, an agent is a small molecule. [0019] In some embodiments of any of the methods described herein, an agent is a sgRNA specific for a nucleic acid sequence encoding PHD3. [0020] In some embodiments of any of the methods described herein, an agent is an inhibitory polynucleotide. In some embodiments, an inhibitory polynucleotide is selected from the group consisting of siRNA, shRNA, and an antisense oligonucleotide, or a polynucleotide that encodes a molecule selected from the group consisting of siRNA, shRNA, and/or an antisense oligonucleotide. [0021] In some embodiments, an inhibitory polynucleotide is an shRNA. In some embodiments, shRNA targets a sequence listed in Table 2. In some embodiments, an shRNA is specific for PHD3. [0022] In some embodiments, an shRNA targets a sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, an shRNA comprises a sense strand comprising a sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, an shRNA comprises a sense strand comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or more identical to a 40-80 nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. [0023] In some embodiments, an shRNA targets SEQ ID NO: 5 or SEQ ID NO: 6. In some embodiments, an shRNA comprises a sense strand set forth in SEQ ID NO: 5 or SEQ ID NO: 6. In some embodiments, an shRNA comprises a sense strand comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or more identical to SEQ ID NO: 5 or SEQ ID NO: 6. FoleyHoagUS12471059.1 Attorney Docket No. HMV-32325 [0024] In some embodiments, an inhibitory polynucleotide is an siRNA. In some embodiments, an siRNA is specific for PHD3. In some embodiments, an siRNA is about 18 to 25 nucleotides in length. [0025] In some embodiments, an siRNA is a small double stranded RNA (dsRNA). In some embodiments, a dsRNA comprises overhangs. In some embodiment, a dsRNA comprises blunt ends. [0026] In some embodiments, an inhibitory polynucleotide is an antisense oligonucleotide (ASO). In some embodiments, an ASO is specific for PHD3. In some embodiments, an ASP is about 13 to 30 nucleotides in length. [0027] In some embodiments, an agent is an antibody-siRNA conjugate. In some embodiments, an antibody-siRNA conjugate is specific for muscle tissue. [0028] In some embodiments, an agent is an antibody-ASO conjugate. In some embodiments, an antibody-ASO conjugate is specific for muscle tissue. [0029] In some embodiments, an agent is administered intravenously. In some embodiments, an agent is administered at least once a week or at least once a month. In some embodiments an agent is administered for at least 7 days, for at least 30 days, for at least 60 days, or for at least 90 days. [0030] In some embodiments, a subject is a human. [0031] In some embodiments, provided are methods of evaluating the responsiveness of a subject to an agent that reduces the levels of or inhibits the activity of PHD3, the method comprising measuring the hydroxylation of ACC2 in a subject that has received the agent that reduces the levels of or inhibits the activity of PHD3, wherein a decrease in the levels of hydroxylation of ACC2 compared to the levels of hydroxylation of ACC2 in the subject prior to receiving the agent indicates that the subject is responding to the agent that reduces the levels of or inhibits the activity of PHD3. BRIEF DESCRIPTION OF THE DRAWINGS [0032] The drawings included herein, which are composed of the following Figures, are for illustration purposes only and not for limitation. [0033] FIG. 1A shows PHD3 expression levels in human muscle biopsies in control or DMD patient group (GSE38417). Data represent means ± SEM. FoleyHoagUS12471059.1 Attorney Docket No. HMV-32325 [0034] FIG. 1B shows Gene targeting strategy for Phd3 in muscular dystrophy mouse (MDX) model. Phd3+/+ were bred with CMV-Cre for phd3-/- mice generation. phd3-/- and mdx mice for phd3-/- mdx generation. [0035] FIG. 1C shows immunofluorescence of laminin (green), Evans blue (EBD; red), and DAPI (blue) in phd3+/+, mdx, phd3-/- mdx mice quadriceps. Scale is 500 μm. [0036] FIG. 1D-E show the relative intensity of laminin (d) and EBD (e) were normalized by the relative intensity of DAPI for each condition. [0037] FIG. 1F shows the level of creatine kinase (CK) in blood from phd3+/+, mdx, phd3-/- mdx. Values represent mean ± SD, n=4 mice per condition. [0038] FIG. 1G shows the relative muscle mass using body weight as a normalization factor in phd3+/+, phd3-/-, mdx, phd3-/- mdx mice (n=4). [0039] FIG. 2A shows qPCR analyses to confirm the lack of WT mRNA transcripts in pdh3-/- and phd3-/- mdx mouse quadriceps. [0040] FIG. 2B shows body weight were assessed in 12-week-old male phd3+/+, phd3-/-, mdx, phd3-/- mdx mice (n=4). [0041] FIG. 2C shows H&E and immunohistochemistry staining of CD68 in phd3+/+, mdx, phd3-/- mdx mice quadriceps muscles. Scale is 500 µm. [0042] FIG. 3A shows a schematic of the bulk RNA sequencing analysis in the quadricep tissues from phd3+/+, phd3-/-, mdx, phd3-/- mdx (n=3). [0043] FIG. 3B shows principal components analysis (PCA) plot showing clusters of samples based on similarity. The first two components (PC1 and PC2) of gene expression variance are displayed. Each dot represents a sample color-coded by phd3+/+, phd3-/-, mdx, phd3-/- mdx group. [0044] FIG. 3C shows enrichment of KEGG metabolic signature scores in all RNA transcriptomes for phd3-/- versus phd3+/+. [0045] FIG. 3D shows a heatmap of the differentially expressed genes enriched in the quadricep tissues from phd3+/+, phd3-/-, mdx, phd3-/- mdx animals. [0046] FIG. 3E-J show Ankrd2 expression (e), Csrp3 expression (f), Grin2b expression (g), Myom3 expression (h), Myoz2 expression (i), or Smtnl1 expression (j) in the quadricep tissues from phd3+/+, phd3-/-, mdx, phd3-/- mdx (n=3). FoleyHoagUS12471059.1 Attorney Docket No. HMV-32325 [0047] FIG. 3K-P show relative Ankrd2 expression (k), Csrp3 expression (l), Grin2b expression (m), Myom3 expression (n), Myoz2 expression (o), or Smtnl1 expression (p) using mouse β-actin as a reference gene in the quadricep tissues from phd3+/+, phd3-/-, mdx, phd3-/- mdx (n=4). [0048] FIG. 4A shows significantly enriched gene numbers of the bulk RNA sequencing analysis in the quadricep tissues from phd3+/+, phd3-/-, mdx, phd3-/- mdx (n=3). [0049] FIG. 4B shows a Venn diagram depicting overlap between significantly altered genes comparing phd3+/+ vs. phd3-/- and mdx vs. phd3-/- mdx. [0050] FIG. 4C shows the RNA transcriptomes from quadriceps in muscular dystrophy model was segregated into nine clusters based on gene expression level following KEGG pathway analysis for the specific functional pathways enriched within each cluster. [0051] FIG. 4D-E shows enrichment of Gene Ontology term enrichment scores that are decreased (d) or increased (e) by PHD3 loss in mdx skeletal muscles. [0052] FIG. 4F shows the significant gene alteration of positive (blue) and negative (red) directions by PHD3 loss in RNA transcriptomes from quadriceps in muscular dystrophy model. [0053] FIG. 5A-H shows the loss of PHD3 increases the level of mycogenic differentiation genes using mouse β-actin as a reference gene in the quadricep tissues from phd3+/+, phd3-/-, phd3+/+ mdx, phd3-/- mdx (n=4). Relative Myoglobin expression is shown in FIG. 5A. MyoD expression is shown in FIG. 5B., MyoG expression is shown in FIG.5C. Myf5 expression is shown in FIG. 5D., MyHCl expression is shown in FIG. 5E. MyHClla expression is shown in FIG.5F. MyHCllx expression is shown in FIG. 5G. MyHCllb expression is shown in FIG. 5H. [0054] FIG. 5I shows relative PHD3 expression using mouse β-actin as a reference gene in C2Cl2s expressing either control, PHD3, shRNA control, or PHD3 shRNA (n = 4, p value <0.05). [0055] FIG. 6A shows relative Phd3 expression using mouse β-actin as a reference gene in C2C12s expressing shRNA against Phd3 or shRNA control (n = 4). mRNA levels were assessed after 2 days of incubation in DMEM with or without 2% horse serum and Insulin-Transferrin- Selenium liquid media supplement for myoblast differentiation. [0056] FIG. 6B shows immunoblots of ACC2 hydroxylation from C2C12s expressing either control, Phd3, shRNA control, or Phd3 shRNA, and incubated with high glucose DMEM containing 2% horse serum and ITS Liquid Media Supplement for two days. FoleyHoagUS12471059.1 Attorney Docket No. HMV-32325 [0057] FIG. 6C shows C2C12s expressing shRNA against Phd3 or shRNA control, after then cells were in myogenic differentiation for 2 days. Proteins in cell lysates were immunoblotted using PHD3, dystrophin, myosin heavy chain (MHC), or actin. [0058] FIG. 6D-I show relative expression of various genes using mouse β-actin as a reference gene from C2C12s expressing shRNA against PHD3 or shRNA control either with or without myogenic differentiation (n=4). Specifically, FIG. 6D shows relative Ankrd2 expression, FIG. 6E shows relative Smtnl1 expression, FIG. 6F shows relative Myoz2 expression, FIG. 6G shows relative Grin2b expression, FIG. 6H shows relative Csrp3 expression, and FIG. 6I shows relative Myom3 expression. [0059] FIG. 7A shows transmission electron microscopy (TEM) images of mitochondria in quadricep muscles from phd3+/+, mdx, phd3-/- mdx. [0060] FIG. 7B shows quantitation of mitochondrial size and cristae density in five independent TEM images as shown in FIG.7A. [0061] FIG. 7C shows a PCA plot showing clusters of samples based on similarity. The first two components (PC1 and PC2) of metabolite levels variance are displayed. Each dot represents a sample color-coded by phd3+/+, phd3-/-, mdx, phd3-/- mdx group. [0062] FIG. 7D-E show relative abundance of DG(32:0) (d), or DG(34:0) in non-targeted metabolite profiling from phd3+/+, phd3-/-, mdx, phd3-/- mdx mouse quadricep muscles (n = 3). [0063] FIG. 7F shows the relative abundance of acylcarnitines in mouse quadriceps of mdx (phd3+/+ mdx) compared to WT (phd3+/+) group. For all comparisons, a two-tailed t-test was used. N = 4, P < 0.05. [0064] FIG. 7G shows the relative abundance of acylcarnitines in mouse quadriceps MDX with PHD3 depletion (phd3-/- mdx) compared to mdx (phd3+/+ mdx) group. For all comparisons, a two-tailed t-test was used. n = 4, P < 0.05. [0065] FIG. 7H shows palmitate oxidation in quadriceps from phd3+/+, phd3-/-, mdx, phd3-/- mdx using [14C]-palmitate as a substrate and measuring 14CO2 capture on filter membrane as readout for FAO (n = 4). [0066] FIG. 7I shows basal palmitate-driven oxygen consumption rate (OCR) of myogenic differentiated shControl or shPhd3 C2C12 cells was measured using a Seahorse Bioscience XF96 Extracellular Flux Analyzer (n = 15 wells). FoleyHoagUS12471059.1 Attorney Docket No. HMV-32325 [0067] FIG. 7J shows palmitate oxidation in C2C12 cell lines expressing shRNA against PHD3 (yellow and red) or shRNA control (light grey) with or without myogenic differentiation for 2 days. Cells were treated with complete media or serum-free media with 3H-palmitate for 2 h prior to FAO analysis (n = 3). P < 0.05. [0068] FIG. 8A-G show relative abundance of each metabolite in phd3+/+, phd3-/-, mdx, phd3-/- mdx mouse quadricep muscles (n = 3). PC(30:0) (a), PE(32:0) (b), LPC(20:0) (c), LPC(20:4) (d), LPC(20:5) (e), ceramides (f), or amino acids (g) are identified by untargeted metabolomics method. [0069] FIG. 8H shows volcano plots demonstrating that mdx have numerous statistically significant metabolic changes compared to control group. [0070] FIG. 8I shows volcano plots demonstrating that PHD3 depleted mdx quadricep muscles have numerous statistically significant metabolic changes compared to mdx group. [0071] FIG. 8J-M show relative Phd3 expression (j), Acc2 expression (k), Acc1 expression (l), or Cpt1 expression (m) using mouse β-actin as a reference gene from C2Cl2s expressing shRNA against ACC1, ACC2, CPT1, or shRNA control either with myogenic differentiation (n = 4). [0072] FIG. 9A shows age-matched mice (12 weeks old) were allowed to acclimate for 48 h before experiments beginning with mice at rest (n = 6 mice per group). The grip strength was calculated from the individual performances in phd3+/+, mdx, and phd3-/- mdx mice. [0073] FIG. 9B shows a flowchart of the endurance exercise experiments. phd3-/- mdx mice demonstrated increased exercise tolerance compared to mdx mice as a control after repetitive treadmill running (n = 8 mice per group). [0074] FIG. 9C-F show the individual exhaustion time (FIG. 9C), total running distance (FIG. 9D), time to maximum oxygen consumption rate (FIG. 9E), and maximum oxygen uptake (FIG. 9F) of both genotypes were calculated from the individual performances during treadmill running. Data represent mean ± SEM. * P < 0.05. [0075] FIG. 10A shows a schematic of a protocol for generating mice and treating them with a high fat diet condition). [0076] FIG. 10B-C show a relative ratio of weight gain in WT and PHD3 KO mice on a high fat diet (FIG.10B) and low fat diet (FIG. 10C). FoleyHoagUS12471059.1 Attorney Docket No. HMV-32325 [0077] FIG. 10D-E show a glucose as a percent of basal glucose in WT and PHD3 KO mice over time after a high glucose injection. Mice on a high fat diet are shown in FIG. 10D and on low fat diet in FIG. 10E. [0078] FIG. 11A-D show that loss of PHD3 prevents accumulating fat in organ under HFD. Epidiymal and inguinal white adipose tissue from WT and PHD3 KO mice on a high fat diet are shown in FIG. 11A, and quantified in FIG. 11B; livers tissue and H&E stained liver tissue from these mice are shown in FIG.11C and FIG.11D, respectively. [0079] FIG. 12 shows blood glucose and serum insulin levels in WT and PHD3 KO mice on both low fat diet and high fat diet. [0080] FIG. 13A-E show metabolic profiling of PHD3 KO mice under high fat diet. [0081] FIG. 14A-B show analysis of Akt signaling pathway in the liver of PHD3 KO mice on a high fat diet. [0082] FIG. 15A-B show analysis of Akt signaling pathway in the muscle of PHD3 KO mice on a high fat diet. CERTAIN DEFINITIONS [0083] In order for the present invention to be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms are set forth throughout the specification. The publications and other reference materials referenced herein to describe the background of the invention and to provide additional detail regarding its practice are hereby incorporated by reference. [0084] In this application, unless otherwise clear from context, (i) the terms “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article; (ii) the term “or” may be understood to mean “and/or”; (iii) the terms “comprising” and "including" may be understood to encompass itemized components or steps whether presented by themselves or together with one or more additional components or steps; and (iv) where ranges are provided, endpoints are included. [0085] The term “administering" means providing a pharmaceutical agent or composition to a subject, and includes, but is not limited to, administration by a medical professional and self- administration. This involves the physical introduction of a composition comprising a therapeutic agent to a subject, using any of the various methods and delivery systems known to those skilled in FoleyHoagUS12471059.1 Attorney Docket No. HMV-32325 the art. In some embodiments, routes of administration of agents that modulate PHD3 described herein include topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically. In some embodiments, routes of administration for agents that modulate PHD3 described herein include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion. Administering may be performed, for example, once, a plurality of times, and/or over one or more extended periods. [0086] The phrase “pharmaceutically-acceptable carrier” as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. [0087] The terms “prevent,” “preventing,” “prevention,” and the like refer to reducing the probability of developing a disease, disorder, or condition in a subject, who does not have, but is at risk of or susceptible to developing a disease, disorder, or condition. [0088] As used herein, the term “subject” means a human or non-human animal selected for treatment or therapy. A subject encompasses, but is not limited to, a mammal.. For example, a subject can be a human, a non-human primate (e.g., monkey, baboon, or chimpanzee), a domestic animal or a livestock (e.g., a horse, a cow, a pig, a sheep, a goat, a dog, a cat, a rabbit, a guinea pig, a gerbil, a hamster, a rat, and a mouse). In some embodiments, the subject is an infant (e.g., a human infant). [0089] The terms “therapeutically-effective amount” and “effective amount” refers to an amount necessary (for example, at dosages and for periods of time and for the means of enteral or oral administration) to achieve the desired therapeutic result. An effective amount of an agent that modulates PHD3 may vary according to factors such as the disease state, age, sex, and weight of the individual. An effective amount is also one in which medical provider, e.g., the attending physician, determines that any toxic or detrimental effects of the agonist are outweighed by the therapeutically beneficial effects. [0090] As used hererin, “treating” a disease, disorder or condition in a subject or “treating” a subject having a disease, disorder or condition refers to subjecting the subject to a pharmaceutical treatment, e.g., administration of a composition, such that at least one symptom of the disease, disorder, or condition is decreased or prevented from worsening. FoleyHoagUS12471059.1 Attorney Docket No. HMV-32325 DETAILED DESCRIPTION [0091] The present disclosure provides, among other things agents and methods useful for treating a number of conditions including, but not limited to, muscular dystrophies, muscle wasting disorders, obesity, and metabolic disorders. While in no way intended to be limiting, exemplary compositions, kits, and applications are elaborated on below. PHD3 [0092] The present disclosure provides methods that include administration of an agent that modulates prolyl hydroxylase domain 3 (PHD3), as well as compositions and uses encompassing the same. [0093] PHD3, also known as EGLN3, egl-9 family hypoxia inducible factor 3, HIFPH3, and HIFP4H3, is an alpha-ketoglutarate-dependent dioxygenase (Gorres and Raines, 2010; Chan et al., 2002). Mitochondrial fatty acid oxidation (FAO) is negatively regulated by PHD3 (German et al., 2016). PHD3 plays a significant role in muscle metabolism as it has been found to hydroxylate the acetyl CoA carboxylase 2 (ACC2) enzyme, leading to the inhibition of fatty acid oxidation (Yoon et al., 2020). Intriguingly, loss of PHD3 improves metabolic health, promotes fat oxidation, and results in a remarkable 30% increase in exercise capacity by de-repressing the hydroxylation of ACC2 (Yoon et al., 2020). Despite these promising findings, the physiological implications of PHD3- dependent FAO in muscle disease, particularly in the context of DMD, remain unexplored. Consequently, the precise role of PHD3 in the development of DMD, as well as its potential as a therapeutic target for DMD remains elusive. [0094] The present disclosure demonstrates that the deletion of PHD3 leads to notable improvements in both mitochondrial and muscle function in a mouse model of DMD. As described in detail in the examples below, a significant upregulation of genes associated with mitochondrial biogenesis, fatty acid oxidation (FAO), and skeletal muscle structure was observed upon PHD3 deletion. These findings indicate that agents that modulate PHD3 may be useful for enhancing mitochondrial function in individuals affected by DMD. [0095] The disclosure reveals that the loss of PHD3 in an MDX model increases exercise capacity and protects against characteristic muscle damage observed in DMD. In particular, the role of PHD3 in regulating mitochondrial FAO and the expression of genes involved in muscle differentiation and structure were examined, demonstrating that PHD3 plays a critical role in FoleyHoagUS12471059.1 Attorney Docket No. HMV-32325 regulating skeletal muscle mass. By using whole-body PHD3 null mice in an MDX model, it is demonstrated that PHD3 loss is enough to increase FAO by de-repressing ACC2 in MDX mice, effectively slowing muscle deterioration. These findings hold promise for the development of targeted therapies in DMD and shed light onto the molecular mechanisms underlying the regulation of muscle metabolism in DMD. [0096] Exemplary PHD3 nucleic acid and amino acid sequences are available to the public at the GenBank database. Exemplary human PHD3 nucleic acid sequences are available to the public at the GenBank database under NM_001308103.2 and NM_022073.4. Exemplary human PHD3 amino acid sequences are available to the public at the GenBank database under NP_001295032.1 and NP_071356.1. Exemplary murine PHD3 nucleic acid and amino acid sequences are available to the public at the GenBank database, for example, under NM_028133.2 and NP_082409.2 In addition, nucleic acid and polypeptide sequences of PHD3 orthologs in other organisms are known in the art and any of these are also included within the scope of the disclosure. [0097] Exemplary polynucleotide sequences encoding PHD3 and PHD3 polypeptide sequences are set forth in Table 1 below.
Figure imgf000013_0001
12 FoleyHoagUS12471059.1 Attorney Docket No. HMV-32325
Figure imgf000014_0001
13 FoleyHoagUS12471059.1 Attorney Docket No. HMV-32325
Figure imgf000015_0001
Methods of Treating and/or Preventing [0098] The present disclosure provides the insight inhibition of PHD3 may be useful for treating and/or preventing a number of conditions including, but not limited to, muscular dystrophies, muscle wasting disorders, obesity, and metabolic disorders. Provided herein are methods of treating and/or preventing a disease, condition, or disorder, that includes administration of an agent that modulates PHD3. In some embodiments, provided methods include administering one or more agents that reduce the level of or inhibits the activity of PHD3. Muscle Wasting and DMD [0099] In some embodiments, the present disclosure provides methods of treating and/or preventing muscle wasting in a subject. Muscle wasting is characterized by weakness and/or wasting away of muscle tissue, which may occur with or without the breakdown of nerve tissue. There are a number of different types of muscle wasting disorders, including muscular dystrophies. Muscular dystrophies are all associated an eventual loss of strength, increasing disability, and possible deformity. Muscle wasting is also observed in a variety of pathologies such as cancer, chronic kidney disease, heart failure, chronic obstructive pulmonary disease (COPD), obesity, as well as after prolonged inactivity or during aging. Reduced muscle mass and function is associated with a higher morbidity and mortality as well as reduced quality of life. FoleyHoagUS12471059.1 Attorney Docket No. HMV-32325 [0100] In some embodiments, the present disclosure provides methods of treating and/or preventing a muscle wasting disorder in a subject. In some embodiments, provided are methods of treating or preventing a muscle wasting disorder comprising administering to a subject one or more agents that reduce the level of or inhibits the activity of PHD3. In some embodiments, a muscle wasting disorder is or includes a muscular dystrophy, cachexia, and/or sarcopenia. [0101] In some embodiments, the present disclosure provides methods of treating and/or preventing a muscular dystrophy. In some embodiments, provided are methods of treating or preventing a muscular dystrophy comprising administering to a subject one or more agents that reduce the level of or inhibits the activity of PHD3. In some embodiments, a muscular dystrophy is selected from Becker’s muscular dystrophy, congenital muscular dystrophy, distal muscular dystrophy, Emery-Dreifuss muscular dystrophy, facioscapulohumeral muscular dystrophy, limb- girdle muscular dystrophy, myotonic muscular dystrophy, and oculopharyngeal muscular dystrophy. [0102] In some embodiments, the present disclosure provides methods of treating and/or preventing Duchenne muscular dystrophy (DMD). In some embodiments, provided are methods of treating or preventing a DMD comprising administering to a subject one or more agents that reduce the level of or inhibits the activity of PHD3. DMD is a fatal muscle-wasting disorder caused by a mutation in the dystrophin gene, which affects primarily males (Hoffman et al., 1987; Mendell et al., 2010; Bulfield et al., 1984). The loss of functional dystrophin protein leads to the disruption of the dystrophin-associated protein complex, rendering muscle fibers fragile and prone to damage during contraction (Deconinck et al., 1997; Cole et al., 2002). As a result, the disease is characterized by progressive muscle weakness and degeneration (Hoffman et al., 1987). Despite being primarily classified as a muscular disorder, DMD is intricately connected with mitochondrial dysfunction, exacerbating both muscle damage and oxidative stress (Rybalka et al., 2014; Timpani et al., 2015; Terrill et al., 2013). Moreover, dysfunction of mitochondrial fatty acid oxidation (FAO) in muscle cells has been linked to the progression of DMD (Gosselin et al., 2022). However, the role of mitochondrial factors in DMD development and pathogenesis remains unclear. [0103] In some embodiments, the present disclosure provides methods of treating and/or preventing cachexia. Cachexia is a wasting syndrome that leads to loss of skeletal muscle and fat. Cachexia is estimated to occur in up to 80% of people with advanced cancer, depending on the cancer type and how well they respond to cancer treatment. And it’s thought to directly cause up to FoleyHoagUS12471059.1 Attorney Docket No. HMV-32325 30% of cancer deaths, often because of heart or respiratory failure related to muscle loss. In some embodiments, a cachexia is obesity-related. In some embodiments, a cachexia is related to cancer, heart disease, kidney disease, or HIV. [0104] In some embodiments, the present disclosure provides methods of treating and/or preventing sarcopenia. Sarcopenia is the age-related progressive loss of muscle mass and strength. In some embodiments, a sarcopenia is aging-related. In some embodiments, sarcopenia is obesity- related. [0105] Loss of PHD3 has been associated with exercise capacity as well as several pathological conditions, including cancer (German et al., 2016; Chen et al., 2015; Luo et al., 2014). In some embodiments, provided methods increase exercise capacity in a subject having a muscle wasting disorder. In some embodiments, provided methods increase exercise capacity in a subject having a muscular dystrophy. In some embodiments, provided methods increase exercise capacity in a subject having DMD. In some embodiments, provided methods increase exercise capacity in a subject having cachexia. In some embodiments, provided methods increase exercise capacity in a subject having sarcopenia. In some embodiments, provided methods increase exercise capacity in a subject having aging-related sarcopenia. In some embodiments, provided methods increase exercise capacity in a subject having obesity-related sarcopenia. [0106] In some embodiments, exercise capacity is increased 10%-300% relative to exercise capacity prior to administering an agent that reduces the levels of or inhibits the activity of PHD3. In some embodiments, exercise capacity is increased 1.1-fold to 3-fold relative to exercise capacity prior to administering an agent that reduces the levels of or inhibits the activity of PHD3. [0107] In some embodiments, exercise capacity is increased at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, or more than 100% increase relative to exercise capacity prior to administering an agent that reduces the levels of or inhibits the activity of PHD3. [0108] In some embodiments, exercise capacity is increased 20%-60% relative to exercise capacity prior to administering an agent that reduces the levels of or inhibits the activity of PHD3. Skeletal Muscle Differentiation [0109] The disclosure reveals significant alterations in gene expression profiles related to skeletal muscle differentiation upon PHD3 loss. Notably, the analysis described herein revealed the FoleyHoagUS12471059.1 Attorney Docket No. HMV-32325 potential involvement of PHD3 in regulating metabolic pathways, muscle development, and contraction processes during skeletal muscle differentiation. Moreover, the disclosure provides novel insights into the potential role of PHD3 in regulating muscle structure genes, which may have important implications for therapies targeting muscle wasting disorders. [0110] In some embodiments, a method is provided for increasing skeletal muscle differentiation and/or decreasing skeletal muscle atrophy in a subject in need thereof comprising administering to the subject an agent that reduces the levels of or inhibits the activity of PHD3. [0111] In some embodiments, provided methods increase expression of one or more genes associated with skeletal muscle differentiation selected from: myoglobin, MyoD, MyoG, Myf5, MyHCI, and MyHcIIa. [0112] In some embodiments, provided methods increase skeletal muscle differentiation in a subject having a muscle wasting disorder. In some embodiments, provided methods increase skeletal muscle differentiation in a subject having a muscular dystrophy. In some embodiments, provided methods increase skeletal muscle differentiation in a subject having DMD. In some embodiments, provided methods increase skeletal muscle differentiation in a subject having cachexia. In some embodiments, provided methods increase skeletal muscle differentiation in a subject having sarcopenia. In some embodiments, provided methods increase skeletal muscle differentiation in a subject having aging-related sarcopenia. In some embodiments, provided methods increase skeletal muscle differentiation in a subject having obesity-related sarcopenia. [0113] In some embodiments, provided methods decrease skeletal muscle atrophy in a subject having a muscle wasting disorder. In some embodiments, provided methods decrease skeletal muscle atrophy in a subject having a muscular dystrophy. In some embodiments, provided methods decrease skeletal muscle atrophy in a subject having DMD. In some embodiments, provided methods decrease skeletal muscle atrophy in a subject having cachexia. In some embodiments, provided methods decrease skeletal muscle atrophy in a subject having sarcopenia. In some embodiments, provided methods decrease skeletal muscle atrophy in a subject having aging-related sarcopenia. In some embodiments, provided methods decrease skeletal muscle atrophy in a subject having obesity-related sarcopenia. FoleyHoagUS12471059.1 Attorney Docket No. HMV-32325 Sarcolemmal Integrity [0114] The sarcolemma is the plasma membrane of the muscle cell and is surrounded by basement membrane and endomysial connective tissue. Mechanical integrity of the sarcolemma is further supported by key cytoskeletal proteins, including dystrophin, spectrin, and F-actin. Electron microscopy analysis of dystrophic muscle directly shows disruptions in the muscle membrane, termed delta lesions. This discovery led to the theory that the loss of dystrophin and associated proteins at the sarcolemma renders the membrane leaky and the muscle susceptible to contraction- induced injury. [0115] In some embodiments, a method is provided improving sarcolemmal integrity in a subject in need thereof, comprising administering to the subject an agent that reduces the levels of or inhibits the activity of PHD3. [0116] In some embodiments, provided methods improve sarcolemmal integrity in a subject having a muscle wasting disorder. In some embodiments, provided methods improve sarcolemmal integrity in a subject having a muscular dystrophy. In some embodiments, provided methods improve sarcolemmal integrity in a subject having DMD. In some embodiments, provided methods improve sarcolemmal integrity in a subject having cachexia. In some embodiments, provided methods improve sarcolemmal integrity in a subject having sarcopenia. Fatty Acid Oxidation [0117] The present disclosure provides the insight that loss of PHD3 has a significantly enhances mitochondrial fatty acid oxidation and improves muscle function in DMD mice. [0118] In some embodiments, a method is provided for increasing fatty acid oxidation and/or decreasing hydroxylation of ACC2 in a subject in need thereof comprising administering to the subject an agent that reduces the levels of or inhibits the activity of PHD3. In some embodiments, method increases fatty acid oxidation in muscle cells of the subject and/or decreases hydroxylation of ACC2 in muscle cells of the subject. [0119] In some embodiments, a method decreases creating activity in a subject. In some embodiments, a method restores mitochondrial size and/or morphology in a subject. [0120] In some embodiments, a subject is afflicted with DMD. In some embodiments, a subject is afflicted with a muscular dystrophy selected from Becker’s muscular dystrophy, congenital muscular dystrophy, distal muscular dystrophy, Emery-Dreifuss muscular dystrophy, FoleyHoagUS12471059.1 Attorney Docket No. HMV-32325 facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, myotonic muscular dystrophy, and oculopharyngeal muscular dystrophy. [0121] In some embodiments, a subject is afflicted with a muscle wasting disorder. In some embodiments, a muscle wasting disorder is cachexia. In some embodiments, a cachexia is obesity- related. In some embodiments, a cachexia is related to cancer, heart disease, kidney disease, or HIV. [0122] In some embodiments, a muscle wasting disorder is sarcopenia. In some embodiments, a sarcopenia is ageing-related. In some embodiments, sarcopenia is obesity-related. Weight Gain [0123] In some embodiments, the present disclosure provides methods of treating and/or preventing weight gain in a subject. There is evidence of higher body mass index (BMI) and higher fat mass being associates with metabolic risk factors in DMD. In some embodiments, a method is provided for preventing weight gain in a subject in need thereof, comprising administering to the subject an agent that reduces the levels of or inhibits the activity of PHD3. [0124] In some embodiments, an agent further increases Akt signaling. In some embodiments, a method is provided for increasing Akt signaling in an obese or overweight subject, comprising administering to the subject an agent that reduces the levels of or inhibits the activity of PHD3. In some embodiments, Akt signaling is increased in the muscle cells of a subject. In some embodiments, Akt signaling is increased in the liver of a subject. [0125] In some embodiments, a subject consumes a high fat or high calorie diet. PHD3 Modulating Agents [0126] In some embodiments, methods of the present disclosure include administering to a subject an agent that modulates PHD3. In some embodiments, a subject is administered one or more agents that reduce the level of or inhibits the activity of PHD3. [0127] Any suitable agents for modulating PHD3 are contemplated by the present disclosure, including, but not limited to, inhibitory polynucleotides, antibodies, antibody conjugates, polypeptide inhibitors, small molecules, and gene therapies. [0128] In some embodiments, an agent that reduces the levels of or inhibits the activity of PHD3 is an inhibitory polynucleotide. Polynucleotide inhibitors (also referred to as nucleic acid inhibitors) can be used to decrease expression of an endogenous gene encoding one of the gene products described herein, such as PHD3, or a gene that encodes a protein that regulates PHD3. An FoleyHoagUS12471059.1 Attorney Docket No. HMV-32325 inhibitory polynucleotide can be, e.g., an siRNA, a shRNA, a dsRNA, a ribozyme, a triple-helix former, an aptamer, or an antisense nucleic acid. [0129] In some embodiments, an inhibitory polynucleotide is selected from the group consisting of siRNA, shRNA, and an antisense oligonucleotide, or a polynucleotide that encodes a molecule selected from the group consisting of siRNA, shRNA, and/or an antisense oligonucleotide. siRNAs [0130] siRNAs are small double stranded RNAs (dsRNAs) that optionally include overhangs. For example, the duplex region of an siRNA is about 18 to 25 nucleotides in length, e.g., about 19, 20, 21, 22, 23, or 24 nucleotides in length. The siRNA sequences can be, in some embodiments, exactly complementary to the target mRNA. dsRNAs and siRNAs in particular can be used to silence gene expression in mammalian cells (e.g., human cells). See, e.g., Clemens et al. (2000) Proc Natl Acad Sci USA 97:6499- 6503; Billy et al. (2001) Proc Natl Acad Sci USA 98:14428-14433; Elbashir et al. (2001) Nature 411:494-8; Yang et al. (2002) Proc Natl Acad Sci USA 99:9942-9947, and U.S. Patent Application Publication Nos. 20030166282, 20030143204, 20040038278, and 20030224432. [0131] In some embodiments, an inhibitory polynucleotide is an siRNA. In some embodiments, an siRNA is specific for PHD3. In some embodiments, an siRNA is 18-25 nucleotides in length. In some embodiments, an siRNA is 18 nucleotides in length. In some embodiments, an siRNA is 19 nucleotides in length. In some embodiments, an siRNA is 20 nucleotides in length. In some embodiments, an siRNA is 21 nucleotides in length. In some embodiments, an siRNA is 22 nucleotides in length. In some embodiments, an siRNA is 23 nucleotides in length. In some embodiments, an siRNA is 24 nucleotides in length. In some embodiments, an siRNA is 25 nucleotides in length. [0132] In some embodiments, an siRNA is a double stranded RNA (dsRNA). In some embodiments, a dsRNA comprises overhangs. In some embodiments, a dsRNA comprises blunt ends. [0133] siRNA molecules can be prepared by chemical synthesis, in vitro transcription, or digestion of long dsRNA by Rnase III or Dicer. These can be introduced into cells by transfection, electroporation, intracellular infection or other methods known in the art. See, for example, each of which is expressly incorporated by reference: Hannon, G J, 2002, RNA Interference, Nature 418: FoleyHoagUS12471059.1 Attorney Docket No. HMV-32325 244-251; Bernstein E et al., 2002, The rest is silence. RNA 7: 1509-1521; Hutvagner G et al., RNAi: Nature abhors a double-strand. Cur. Open. Genetics & Development 12: 225-232; Brummelkamp, 2002, A system for stable expression of short interfering RNAs in mammalian cells. Science 296: 550-553; Lee N S, Dohjima T, Bauer G, Li H, Li M-J, Ehsani A, Salvaterra P, and Rossi J. (2002). Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells. Nature Biotechnol.20:500-505; Miyagishi M, and Taira K. (2002). U6-promoter- driven siRNAs with four uridine 3′ overhangs efficiently suppress targeted gene expression in mammalian cells. Nature Biotechnol. 20:497-500; Paddison P J, Caudy A A, Bernstein E, Hannon G J, and Conklin D S. (2002). [0134] In some embodiments, an agent is an antibody-siRNA conjugate. In some embodiments, an antibody-siRNA conjugate is specific for muscle tissue. shRNAs [0135] Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes & Dev. 16:948-958; Paul C P, Good P D, Winer I, and Engelke D R. (2002). Effective expression of small interfering RNA in human cells. Nature Biotechnol.20:505-508; Sui G, Soohoo C, Affar E-B, Gay F, Shi Y, Forrester W C, and Shi Y. (2002). A DNA vector-based RNAi technology to suppress gene expression in mammalian cells. Proc. Natl. Acad. Sci. USA 99(6):5515-5520; Yu J-Y, DeRuiter S L, and Turner D L. (2002). RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells. Proc. Natl. Acad. Sci. USA 99(9):6047-6052, PCT publications WO2006/066048 and WO2009/029688, U.S. published application U.S.2009/0123426, each of which is incorporated by reference in its entirety. [0136] In some embodiments, an inhibitory polynucleotide is an shRNA. [0137] In some embodiments, a target sequence for an shRNA is set forth in Table 2. [0138] Table 2: Exemplary shRNA sequences
Figure imgf000022_0001
FoleyHoagUS12471059.1 Attorney Docket No. HMV-32325
Figure imgf000023_0001
[0139] In some embodiments, an shRNA is specific for PHD3. [0140] In some embodiments, a target sequence for PHD3 (direct shRNA sequence) is as follows: shPHD3 #1 (TRCN0000001048): CACCTGCATCTACTATCTGAA (SEQ ID NO: 5) shPHD3 #2 (TRCN0000001050): GTGGCTTGCTATCCGGGAAAT (SEQ ID NO: 6) [0141] In some embodiments, an shRNA target sequence comprises a sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO: 5. In some embodiments, an shRNA target sequence comprises a sequence that differs by no more that 3 nucleotides, no more than 2 nucleotides, no more than 1 nucleotide relative to SEQ ID NO: 5. In some embodiments, an shRNA target sequence comprises a sequence that is at least 85%, 90%, or 95% identical to SEQ ID NO: 6. In some embodiments, an shRNA target sequence comprises a sequence that differs by no more that 3 nucleotides, no more than 2 nucleotides, no more than 1 nucleotide relative to SEQ ID NO: 6. FoleyHoagUS12471059.1 Attorney Docket No. HMV-32325 [0142] In some embodiments, an shRNA targets SEQ ID NO:5 or SEQ ID NO: 6. In some embodiments, an shRNA targets SEQ ID NO: 5. In some embodiments, an shRNA targets SEQ ID NO: 6. [0143] In some embodiments, an shRNA comprises a sense strand set forth in SEQ ID NO: 5. In some embodiments, an shRNA comprises a sense strand set forth in SEQ ID NO: 6. [0144] In some embodiments, an shRNA comprises a sense strand set forth in SEQ ID NO: 5. In some embodiments, an shRNA comprises a sense strand set forth in SEQ ID NO: 6. Antisense oligonucleotides [0145] In some embodiments, an inhibitory polynucleotide is an antisense oligonucleotide (ASO). In some embodiments, an ASO is specific for PHD3. [0146] Antisense oligonucleotide agents can include, for example, from about 8 to about 80 nucleobases (i.e. from about 8 to about 80 nucleotides), e.g., about 8 to about 50 nucleobases, or about 12 to about 30 nucleobases. In some embodiments, an ASO is 13 to 30 nucleotides in length. In some embodiments, an ASO is 15 nucleotides in length. In some embodiments, an ASO is 16 nucleotides in length. In some embodiments, an ASO is 17 nucleotides in length. In some embodiments, an ASO is 18 nucleotides in length. In some embodiments, an ASO is 19 nucleotides in length. In some embodiments, an ASO is 20 nucleotides in length. In some embodiments, an ASO is 21 nucleotides in length. In some embodiments, an ASO is 22 nucleotides in length. In some embodiments, an ASO is 23 nucleotides in length. In some embodiments an ASO is 24 nucleotides in length. In some embodiments, an ASO is 25 nucleotides in length. [0147] Antisense compounds include ribozymes, external guide sequence (EGS) oligonucleotides (oligozymes), and other short catalytic RNAs or catalytic oligonucleotides which hybridize to the target nucleic acid and modulate its expression. Anti-sense compounds can include a stretch of at least eight consecutive nucleobases that are complementary to a sequence in the target gene. An oligonucleotide need not be 100% complementary to its target nucleic acid sequence to be specifically hybridizable. An oligonucleotide is specifically hybridizable when binding of the oligonucleotide to the target interferes with the normal function of the target molecule to cause a loss of utility, and there is a sufficient degree of complementarity to avoid non-specific binding of the oligonucleotide to non-target sequences under conditions in which specific binding is desired, FoleyHoagUS12471059.1 Attorney Docket No. HMV-32325 i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment or, in the case of in vitro assays, under conditions in which the assays are conducted. [0148] Hybridization of antisense oligonucleotides with mRNA can interfere with one or more of the normal functions of mRNA. The functions of mRNA to be interfered with include all key functions such as, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity which may be engaged in by the RNA. Binding of specific protein(s) to the RNA may also be interfered with by antisense oligonucleotide hybridization to the RNA. Exemplary antisense compounds include DNA or RNA sequences that specifically hybridize to the target nucleic acid, e.g., the mRNA encoding one of the gene products described herein. The complementary region can extend for between about 8 to about 80 nucleobases. The compounds can include one or more modified nucleobases. Modified nucleobases may include, e.g., 5- substituted pyrimidines such as 5- iodouracil, 5-iodocytosine, and C5- propynyl pyrimidines such as Cs-propynylcytosine and C5-propynyluracil. Other suitable modified nucleobases include, e.g., 7- substituted- 8-aza-7-deazapurines and 7-substituted-7-deazapurines such as, for example, 7-iodo-7- deazapurines, 7-cyano-7-deazapurines, 7-aminocarbonyl-7- deazapurines. Examples of these include 6-amino-7-iodo-7-deazapurines, 6-amino-7- cyano-7-deazapurines, 6- amino-7- aminocarbonyl-7-deazapurines, 2-amino-6- hydroxy-7-iodo-7-deazapurines, 2- amino-6-hydroxy-7- cyano-7-deazapurines, and 2- amino-6-hydroxy-7-aminocarbonyl-7-deazapurines. See, e.g., U.S. Patent Nos. 4,987,071; 5,116,742; and 5,093,246; “Antisense RNA and DNA,” D.A. Melton, Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1988); Haselhoff and Gerlach (1988) Nature 334:585-59; Helene, C. (1991) Anticancer Drug D 6:569-84; Helene (1992) Ann NY Acad Sci 660:27-36; and Maher (1992) Bioassays 14:807- 15. [0149] In some embodiments, an agent is an antibody-ASO conjugate. In some embodiments, an antibody-ASO conjugate is specific for muscle tissue. Aptamers [0150] Aptamers are short oligonucleotide sequences that can be used to recognize and specifically bind almost any molecule, including cell surface proteins. The systematic evolution of ligands by exponential enrichment (SELEX) process is powerful and can be used to readily identify such aptamers. Aptamers can be made for a wide range of proteins of importance for therapy and FoleyHoagUS12471059.1 Attorney Docket No. HMV-32325 diagnostics, such as growth factors and cell surface antigens. These oligonucleotides bind their targets with similar affinities and specificities as antibodies do (see, e.g., Ulrich (2006) Handb Exp Pharmacol 173:305-326). [0151] Antisense or RNA interference molecules can be delivered in vitro to cells or in vivo. Typical delivery means known in the art can be used. Any mode of delivery can be used without limitation, including: intravenous, intramuscular, intraperitoneal, intraarterial, local delivery during surgery, endoscopic, or subcutaneous. Vectors can be selected for desirable properties for any particular application. Vectors can be viral, bacterial or plasmid. Adenoviral vectors are useful in this regard. Tissue-specific, cell-type specific, or otherwise regulatable promoters can be used to control the transcription of the inhibitory polynucleotide molecules. Non-viral carriers such as liposomes or nanospheres can also be used. [0152] In the present methods, a RNA interference molecule or an RNA interference encoding oligonucleotide can be administered to the subject, for example, as naked RNA, in combination with a delivery reagent, and/or as a nucleic acid comprising sequences that express the siRNA or shRNA molecules. In some embodiments the nucleic acid comprising sequences that express the siRNA or shRNA molecules are delivered within vectors, e.g. plasmid, viral and bacterial vectors. Any nucleic acid delivery method known in the art can be used in the present invention. Suitable delivery reagents include, but are not limited to, e.g., the Minis Transit TKO lipophilic reagent; lipofectin; lipofectamine; cellfectin; polycations (e.g., polylysine), atelocollagen, nanoplexes and liposomes. Small Molecules [0153] In some embodiments, an agent that modulates PHD3 is a small molecule. “Small molecule” as used herein, is meant to refer to an agent, which has a molecular weight of less than about 6 kDa and most preferably less than about 2.5 kDa. For example, a small molecule includes molecules that are less than about 1000 molecular weight or less than about 500 molecular weight. Exemplary small molecule compounds include, but are not limited to, peptides, peptidomimetics, nucleic acids, carbohydrates, organic molecules, and biosynthetic molecules. In some embodiments, exemplary small molecules can be screened for activity for modulating PHD3. FoleyHoagUS12471059.1 Attorney Docket No. HMV-32325 [0154] In some embodiments, a small molecule is specific for PHD3. In some embodiments, a small molecule targets PHD1, PHD2 or PHD3. In some embodiments, a small molecule inhibits PHD3. [0155] In some embodiments, a small molecule is a PHD3 inhibitor, such as, but not limited to, those described in International Patent Application Publication Nos. WO 2008/135639, WO 2013063221, and WO 2013/032893, and U.S. Patent Application Publication No. US 20140256722. [0156] In some embodiments, a small molecule is a salidroside. [0157] In some embodiments, a small molecule that modulates PHD3 is identified by screening a library. Libraries of chemical and/or biological mixtures comprising arrays of small molecules, often fungal, bacterial, or algal extracts, can be screened for their ability to reduce the activity of PHD3 and/or reduce the expression of PHD3. This application contemplates using, among other things, small chemical libraries, peptide libraries, or collections of natural products. Tan et al. described a library with over two million synthetic compounds that is compatible with miniaturized cell-based assays (J Am Chem Soc (1998) 120:8565-8566). It is within the scope of this application that such a library may be used to screen for inhibitors (e.g., hydroxylase inhibitors, kinase inhibitors) of any one of the gene products described herein, e.g., cyclin dependent kinases. There are numerous commercially available compound libraries, such as the Chembridge DIVERSet. Libraries are also available from academic investigators, such as the Diversity set from the NCI developmental therapeutics program. Rational drug design may also be employed. [0158] Compounds useful in the methods of the present invention may be obtained from any available source, including systematic libraries of natural and/or synthetic compounds. Compounds may also be obtained by any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; peptoid libraries (libraries of molecules having the functionalities of peptides, but with a novel, non-peptide backbone which are resistant to enzymatic degradation but which nevertheless remain bioactive; see, e.g., Zuckermann et al., 1994, J. Med. Chem. 37:2678-85, which is expressly incorporated by reference); spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the ‘one- bead one-compound’ library method; and synthetic library methods using affinity chromatography selection. The biological library and peptoid library approaches are limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule FoleyHoagUS12471059.1 Attorney Docket No. HMV-32325 libraries of compounds (Lam, 1997, Anticancer Drug Des. 12:145, which is expressly incorporated by reference). [0159] Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91:11422; Zuckermann et al. (1994). J. Med. Chem.37:2678; Cho et al. (1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Engl.33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl.33:2061; and in Gallop et al. (1994) J. Med. Chem. 37:1233, each of which is expressly incorporated by reference. [0160] Libraries of agents may be presented in solution (e.g., Houghten, 1992, Biotechniques 13:412-421), or on beads (Lam, 1991, Nature 354:82-84), chips (Fodor, 1993, Nature 364:555-556), bacteria and/or spores, (Ladner, U.S. Patent No. 5,223,409), plasmids (Cull et al, 1992, Proc Natl Acad Sci USA 89:1865-1869) or on phage (Scott and Smith, 1990, Science 249:386-390; Devlin, 1990, Science 249:404-406; Cwirla et al, 1990, Proc. Natl. Acad. Sci. 87:6378-6382; Felici, 1991, J. Mol. Biol. 222:301-310; Ladner, supra., each of which is expressly incorporated by reference). Single Guide RNAs (sgRNAs) [0161] In some embodiments, an agent is a sgRNA specific for a nucleic acid sequence encoding PHD3. A sgRNA comprises a spacer that hybridizes to the target nucleic acid sequence and a scaffold that recruits the Cas9 enzyme. Cas9 will produce a double strand break at the target site. In some embodiments, a sgRNA spacer is 15-25 nucleotides in length. In some embodiments, a sgRNA spacer is 17 nucleotides in length. In some embodiments, a sgRNA spacer is 18 nucleotides in length. In some embodiments, a sgRNA spacer is 19 nucleotides in length. In some embodiments, a sgRNA spacer is 20 nucleotides in length. In some embodiments, a sgRNA spacer is 21 nucleotides in length. In some embodiments, a sgRNA spacer is 22 nucleotides in length. In some embodiments, a sgRNA spacer is 23 nucleotides in length. In some embodiments, a sgRNA spacer is 24 nucleotides in length. [0162] In some embodiments, a target nucleic acid is immediately adjacent to a protospacer adjacent motif (PAM). In some embodiments, a target nucleic acid is a double stranded DNA (dsDNA). In some embodiments, a target nucleic acid is a single stranded RNA (ssRNA). FoleyHoagUS12471059.1 Attorney Docket No. HMV-32325 [0163] In some embodiments, a double strand break is repaired by non-homologous end joining (NHEJ). In some embodiments, a double strand break is repaired by homology directed repair (HDR). Antibodies [0164] In some embodiments, an agent that modulates PHD3 is an antibody. In some embodiments, an agent that modulates PHD3 is an anti-PHD3 antibody. [0165] Exemplary antibodies that modulate PHD3 are commercially available. For example, EPR17869 (ab225327)(Abcam), EG188e/d5 (Invitrogen), MAB6954 (R&D Systems) are commercially available monoclonal anti-PHD3 antibodies. NB100-139 (Novus biologics) and PA1- 16526 (Invitrogen) are commercially available polyclonal anti-PHD3 antibodies. [0166] Antibodies also include antigen-binding fragments (referred to herein as “antibody fragment” and “antigen-binding fragment,” or similar terms) which are fragments of an antibody that retain the ability to bind to an target antigen. Such fragments include, e.g., a single chain antibody, a single chain Fv fragment (scFv), an Fd fragment, an Fab fragment, an Fab’ fragment, or an F(ab’)2 fragment. An scFv fragment is a single polypeptide chain that includes both the heavy and light chain variable regions of the antibody from which the scFv is derived. In addition, intrabodies, minibodies, triabodies, and diabodies are also included in the definition of antibody and are compatible for use in the methods described herein. See, e.g., Todorovska et al. (2001) J Immunol Methods 248(1):47-66; Hudson and Kortt (1999) J Immunol Methods 231(1):177-189; Poljak (1994) Structure 2(12):1121-1123; Rondon and Marasco (1997) Annual Review of Microbiology 51:257-283, the disclosures of each of which are incorporated herein by reference in their entirety. Bispecific antibodies (including DVD-Ig antibodies; see below) are also embraced by the term “antibody.” Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. [0167] In some embodiments, the agents can be modified, e.g., with a moiety that improves the stabilization and/or retention of the antibodies in circulation, e.g., in blood, serum, or other tissues. For example, a polypeptide described herein can be PEGylated as described in, e.g., Lee et al. (1999) Bioconjug Chem 10(6): 973-8; Kinstler et al. (2002) Advanced Drug Deliveries Reviews 54:477-485; and Roberts et al. (2002) Advanced Drug Delivery Reviews 54:459-476 or HESylated (Fresenius Kabi, Germany; see, e.g., Pavisić et al. (2010) Int J Pharm 387(1-2):110-119). The FoleyHoagUS12471059.1 Attorney Docket No. HMV-32325 stabilization moiety can improve the stability, or retention of, the polypeptide by at least 1.5 (e.g., at least 2, 5, 10, 15, 20, 25, 30, 40, or 50 or more) fold. Administration of PHD3 modulating agents [0168] PHD3 modulating agents disclosed herein may be administered over any period of time effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The period of time may be at least 1 day, at least 10 days, at least 20 days, at least 30, days, at least 60 days, at least three months, at least six months, at least a year, at least three years, at least five years, or at least ten years. The dose may be administered when needed, sporadically, or at regular intervals. For example, the dose may be administered monthly, weekly, biweekly, triweekly, once a day, or twice a day. In certain embodiments, a dose of the composition is administered at regular intervals over a period of time. In some embodiments, a dose of the composition is administered at least once a week. In some embodiments, a dose of the composition is administered at least twice a week. In certain embodiments, a dose of the composition is administered at least three times a week. In some embodiments, a dose of the composition is administered at least once a day. In some embodiments, a dose of the composition is administered at least twice a day. In some embodiments, doses of the composition are administered for at least 1 week, for at least 2 weeks, for at least 3 weeks, for at least 4 weeks, for at least 1 month, for at least 2 months, for at least 3 months, for at least 4 months, for at least 5 months, for at least 6 months, for at least 1 year, for at least two years, at least three years, or at least five years. [0169] In some embodiments, a PHD3 modulating agent is administered intravenously. In some embodiments, an agent is administered at least once a week or at least once a month. In some embodiments, an agent is administered for at least 7 days, for at least 30 days, for at least 60 days, or for at least 90 days. [0170] The selected dosage level will depend upon a variety of factors including the activity of the particular agent employed, the route of administration, the time of administration, the rate of excretion or metabolism of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts. FoleyHoagUS12471059.1 Attorney Docket No. HMV-32325 [0171] Suitable human doses of any of the compounds described herein can further be evaluated in, e.g., Phase I dose escalation studies. See, e.g., van Gurp et al. (2008) Am J Transplantation 8(8):1711-1718; Hanouska et al. (2007) Clin Cancer Res 13(2, part 1):523-531; and Hetherington et al. (2006) Antimicrobial Agents and Chemotherapy 50(10): 3499-3500. [0172] A physician having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician could prescribe and/or administer doses of the compounds employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. [0173] In some embodiments, a method is provided for evaluating the responsiveness of a subject to an agent that reduces the levels of or inhibits the activity of PHD3, the method comprising measuring the hydroxylation of ACC2 in a subject that has received the agent that reduces the levels of or inhibits the activity of PHD3, wherein a decrease in the levels of hydroxylation of ACC2 compared to the levels of hydroxylation of ACC2 in the subject prior to receiving the agent indicates that the subject is responding to the agent that reduces the levels of or inhibits the activity of PHD3. EXEMPLIFICATION Example 1: PHD3 Deletion Improves Muscle Function in DMD Mice [0174] The present example describes characterization of PHD3 in DMD patients and a DMD model mouse. Loss of dystrophin leads to a disconnection between the cytoskeleton and the extracellular matrix of muscle cells. This results in mitochondrial damage, reduced fat oxidation, and intramuscular lipid accumulation, which ultimately leads to muscle wasting and loss of function (Timpani et al., 2015; Allen et al., 2010; Alderton and Steinhardt, 2000). [0175] The present disclosure encompasses a recognition that PHD3 may have a role in DMD. To assess this, the expression of PHD3 in DMD patients was examined. PHD3 expression was higher in the muscles of DMD patients compared to controls (FIG. 1A), as shown by human muscle gene expression databases (GSE38417), suggesting a potential implication of PHD3 in the DMD phenotype. [0176] To investigate the role of PHD3 in DMD, a new mouse model was developed by knocking out PHD3 in the MDX mouse. The MDX mouse, which carries a nonfunctional FoleyHoagUS12471059.1 Attorney Docket No. HMV-32325 dystrophin gene due to a nonsense mutation in exon 23, was selected as the DMD model. The BL10-mdx mouse strain was specifically chosen due to its higher serum creatine kinase levels and inflammation in muscle, making them an ideal model for assessing standard outcomes and identifying skeletal muscle phenotypes (Grounds et al., 2008). First, PHD3 flox-CMV-Cre (phd3-/-) mice were generated by crossing PHD3 flox mice (phd3+/+) with CMV-Cre (Yoon et al., 2020). These mice were then bred with BL10-mdx mice to obtain the PHD3 KO MDX mouse (phd3-/-mdx) (FIG. 1B and FIG. 2A). Loss of dystrophin in mdx mice and DMD patients leads to sarcolemmal disruption and instability during muscle contraction (Gibbs et al., 2021). To investigate the molecular organization of skeletal muscle fibers, laminin staining was performed in quadricep tissue sections from phd3+/+, phd3+/+mdx, and phd3-/-mdx mice. The levels of laminin protein were similar in all three groups, indicating no changes in muscle structure (FIG. 1C-1E). Sarcolemmal integrity was assessed in mdx mice using the Evan’s Blue Dye (EBD) tracer assay (Straub et al., 1997), which revealed increased blood serum albumin infiltration in phd3+/+ mdx mice due to sarcolemmal fragility (FIG. 1C). However, the loss of PHD3 improved sarcolemmal damage in phd3-/-mdx mice to levels similar to phd3+/+ controls (FIG 1C-1E). To further understand the impact of PHD3 loss on muscle function, creatine kinase levels were measured in mouse blood. Creatine kinase is an enzyme that catalyzes the conversion of creatine phosphate to creatine, and its levels increase in the blood during muscle damage (Apple and Rhodes, 1985; Yu et al., 2020). Higher levels of CK activity are a well-known marker for the DMD phenotype (Cohn et al., 2002; Sharp et al., 1992). CK activity was not changed in phd3+/+ and phd3-/- mice, but phd3+/+ mdx mice had higher CK activity (FIG. 1F). Interestingly, PHD3 loss in MDX mice (phd3-/-mdx) resulted in reduced CK activity (FIG. 1F). These alterations appeared in the absence of significant changes in body weight in 12-week-old mice (FIG. 2B). Skeletal muscle mass is a critical indicator of muscle health and function, both in healthy individuals and those with muscle disorders. In MDX mice, the increase in muscle mass is a result of compensatory hypertrophy, a process in which the remaining functional muscle fibers grow in size to compensate for the loss of damaged fibers. Recent studies have suggested that fibrosis, or excessive connective tissue accumulation, in dystrophic muscle, may also contribute to increased muscle mass (Malecova et al., 2018). In MDX mice (phd3+/+ mdx), there was a significant increase in skeletal muscle mass, while in phd3-/- mdx mice, the muscle mass was dramatically decreased (FIG. 1G). These findings suggest that PHD3 plays an essential role in the regulation of muscle mass and function, particularly in the context of muscular FoleyHoagUS12471059.1 Attorney Docket No. HMV-32325 dystrophy. Moreover, inflammation in skeletal muscle is a crucial component of the pathophysiology of DMD, contributing to progressive weakness and muscle damage (Rosenberg et al., 2015). Dysregulated immune responses, as evidenced by the infiltration of immune cells such as neutrophils, macrophages, and T-cells, have been observed in DMD muscle biopsies (Tidball et al., 2018; Dort et al., 2019; Villalta et al., 2011). In the mouse model, there was an increase in CD68(+) neutrophil infiltration in the skeletal muscle of mdx mice, while there was a decrease in phd3-/- mdx mice (FIG. 2C). These results suggest that the loss of PHD3 protects against muscle damage in a mouse model of muscular dystrophy. Example 2: PHD3 Deletion Upregulates Genes Involved in Mitochondrial FAO and Skeletal Muscle Differentiation [0177] To understand the molecular mechanisms underlying the effects of PHD3 deletion in DMD mice, RNA sequencing analysis of skeletal muscle from phd3+/+, phd3-/-, phd3+/+mdx, and phd3-/-mdx mice was conducted (FIG.3A and FIG. 4A). Principal component analysis (PCA) was used to identify the impact of PHD3 loss on gene expression profiles in the quadricep tissues of these mice (FIG. 3B). PHD3 has limited effect on the mdx model, whereas it exerts a more pronounced effect on the gene expression profiles of the WT (phd3+/+) mice (FIG. 3B-3C and FIG. 4A-4B). Differentially-expressed (DE) genes were further analyzed using pathway enrichment, and it was observed that upregulated genes in the phd3-/- condition were enriched in Gene Ontology (GO) lipid metabolic pathways (FIG. 3C). The significant alterations in RNAs involved in lipid metabolism pathways indicate the potential role of PHD3 in the regulation of metabolism during skeletal muscle differentiation. Functional annotation analysis of the differentially expressed genes were categorized 10 groups based on their own expression pattern between phd3-/-, phd3+/+, phd3-/- mdx, and phd3+/+mdx mice (FIG. 4C). This analysis revealed that several gene sets involved in muscle development and function, as well as energy metabolism (cluster 2 and 7), were significantly altered in PHD3 knockout mice in the mdx model. [0178] Analysis of gene expression in the phd3-/-mdx and mdx mice identified 52 genes that were differentially-expressed between the two groups. Visualization of these genes in a heatmap highlights the similarity in gene expression between the phd3-/- and phd3-/-mdx conditions for these 52 genes, while the phd3-/-mdx and mdx show clear differences in gene expression (FIG. 3D). Further, these 52 differentially-expressed genes were analyzed using Gene Ontology term FoleyHoagUS12471059.1 Attorney Docket No. HMV-32325 enrichment scores, which revealed a decrease in expression of genes associated with mannosyltransferase activity, fibroblast growth factor binding, and phospholipase binding (FIG. 4D), while an increase in expression of genes associated with muscle contraction, muscle development, and muscle system associated with PHD3 loss in mdx skeletal muscles (FIG.4E). Although DMD is a genetic disease caused by dystrophin depletion, 52 RNAs were significantly altered in the phd3-/-mdx group compared to those in phd3+/+mdx mice (FIG. 4F). Among them, it was found that genes encoding contractile proteins and muscle differentiation (Czosnek et al., 1982; Sutherland et al., 1993), such as Ankrd2, Csrp3, Grin2b, Myom3, Myoz2, and Smtnl1, were upregulated in phd3-/- and phd3-/-mdx mice compared to those in PHD wildtype group, respectively (FIG. 3E-3J), indicating a potential role of PHD3 in regulating gene expression during skeletal muscle differentiation. Consistent with the bulk RNA sequencing data, Ankrd2, Csrp3, Grin2b, Myom3, Myoz2, and Smtnl1 were also upregulated in phd3+/+mdx mice and further increased in phd3-/-mdx mice, as confirmed by qPCR (FIG. 3K-3P). The results suggest that the loss of PHD3 in mdx mice enhances muscle function by upregulating genes involved in contractile proteins and skeletal muscle differentiation. Example 3: PHD3 Modulation Influences Muscle Contractile Genes and Enhances Myogenic Differentiation [0179] Muscle contractile genes encode the proteins that make up the muscle fibers, which are altered in various forms of muscular dystrophy (Boscolo Sesillo et al., 2019; Megeney et al., 1996). For example, in Duchenne muscular dystrophy, the expression of MyoD is decreased, which may impair muscle regeneration (Gnocchi et al., 2009). Conversely, the expression of Myogenin and Myf5 is increased in some forms of muscular dystrophy, potentially as a compensatory response to muscle fiber damage (Ustanina et al., 2007; Yamamoto et al., 2018). Interestingly, genes encoding contractile proteins, including myoglobin, MyoD, MyoG, and Myf5, were also induced by PHD3 loss (FIG. 5A-5D). However, there was no alteration in the expression of muscle fiber genes, including MyHCl, MyHClla, MyHCllx, and MyHCllb, which are directly involved in oxidative metabolism (FIG. 5E-5H). To investigate the effects of PHD3 on muscle structure gene expression, PHD3 expression was measured during the differentiation of myoblast cells. It was found that that PHD3 expression significantly decreases during the differentiation of skeletal myoblasts into myotubes (FIG. 6A). This indicates that PHD3 modulation is crucial for skeletal cell FoleyHoagUS12471059.1 Attorney Docket No. HMV-32325 differentiation and maturation. To induce myogenic differentiation, C2C12 cells must slow down their proliferation and switch to a differentiation state. It is worth noting that PHD3 activity is acutely sensitive to nutrient levels, and energetic inputs affect ACC2 hydroxylation. To examine whether PHD3 activity is sensitive to myogenic differentiation with different nutrient levels, PHD3- depleted or PHD3-overexpressing C2C12 cell lines were generated (FIG. 5I) and probed ACC2 hydroxylation during myogenic differentiation using these cell lines (FIG. 6B). As expected, ACC2 hydroxylation was increased by PHD3 overexpression compared to control (pBABE plasmid expression), whereas it was decreased in PHD3-depleted compared to shControl cell lines (shCon). Interestingly, the results show that PHD3-dependent ACC2 hydroxylation was decreased during myogenic differentiation (FIG. 6B). This suggests a negative correlation between PHD3-mediated hydroxylation of ACC2 and myogenic differentiation. Since PHD3 deletion improves muscle function in DMD mice, it was investigated whether it affects the expression of dystrophin, the protein whose loss causes DMD. PHD3 deletion had no significant effect on dystrophin expression in PHD3 depletion (FIG. 6C), suggesting that its effects are independent of dystrophin expression. [0180] Skeletal myogenesis is a crucial step in the formation of functional and mature muscles (Hernández-Hernández et al., 2017; Bentzinger et al., 2012). Impairment in this process can directly affect the phenotype of DMD. To determine whether differentiation can be promoted by PHD3 depletion to create functional muscles, the gene set for contractile proteins and muscle differentiation markers was measured in a cell system. PHD3-depleted C2C12 was for myogenesis and found that the loss of PHD3 increases the expression levels of Ankrd2, with or without differentiation (FIG. 6A and 6D). However, this gene expression pattern is not the same as that in vivo. During differentiation, the expression of Smtnl1 and Myoz2 was induced by shControl- expressing cells, yet shPHD3-expressing cells had decreased levels of these genes (Figures 3E and 3F). Additionally, some of the genes induced upon myogenesis were induced by PHD3 depletion (FIG. 6G-6I) suggesting that PHD3 modulation in skeletal muscle cells increases the levels of muscle structure genes. Example 4: PHD3 Deletion Improves Mitochondrial Fatty Acid Oxidation in DMD Mice [0181] Next, it was investigated whether PHD3 deletion affects mitochondrial function and oxidative metabolism in DMD mice. The cellular flux of mitochondrial metabolism is a crucial metabolic indicator of cellular energy status, and its disruption leads to metabolic abnormalities FoleyHoagUS12471059.1 Attorney Docket No. HMV-32325 (Spinelli and Haigis, 2018; Jones et al., 2021). Previous research has demonstrated that muscle cell mitochondria from DMD patients and animal models exhibit abnormal shapes, including smaller or rounded mitochondria (Moore et al., 2020). In the mice model, mitochondria in MDX mice were smaller and appeared more damaged compared to control mice (phd3+/+) (FIG. 7A). Interestingly, the mitochondrial size was restored to levels similar to control mice in phd3-/-mdx mice (FIG. 7A- 7B). Moreover, the most distinctive features of DMD are progressive muscular dystrophy, myofiber degeneration with fibrosis, and metabolic alterations such as fatty acid metabolism. However, lipid metabolism changes in Duchenne patient cells are poorly understood. First the effects of PHD3 depletion on oxidative metabolism in a genetic mouse model of muscular dystrophy using non- targeted metabolic profiling analysis were investigated in vivo. PCA was used to identify the impact of PHD3 loss on metabolite profiles in quadricep tissues from phd3+/+, phd3-/-, phd3+/+mdx, and phd3-/-mdx mice (FIG. 7C) and found that profiles of phd3-/- mdx mice were clearly distinct from those of the other three groups (phd3+/+, phd3-/-, and phd3+/+mdx mice). [0182] Moreover, PHD3 depletion resulted in increased lipid accumulation in muscle tissue compared to mdx mice, as demonstrated by distinct lipids profiles (FIGs. 7D, 7E, 8A-8F). Interestingly, PHD3 loss in mdx mice resulted in an increase in fatty acid-driven lipids (FIGs. 7D, 7E, 8C-8E). Some phospholipids, including PC(30:0) and PE(32:0), were decreased by PHD3 depletion in both normal and MDX mice (FIG.8A-8B). However, other metabolites including amino acids remained unchanged in these mice (FIG. 8F-8G). It was hypothesized that the lipid metabolism regulated by PHD3 affects the muscle of MDX mice, given the significant increase in long-chain acylcarnitines and fatty acid-driven lipid metabolism in phd3-/-mdx mice. When the data was separated to compare phd3+/+ and phd3+/+mdx mice, the TCA cycle metabolites were increased in phd3+/+mdx mice, while long-chain acylcarnitines were decreased in phd3-/-mdx mice (FIG. 8H- 8I). To further understand the metabolic adaptations driven by PHD3 in vivo, the levels of long- chain acyl-carnitines in quadriceps from phd3-/-, phd3+/+, phd3-/-mdx, and phd3+/+mdx mice were analyzed using LC-MS/MS. The long-chain acyl-carnitines were lower in WT mice compared to MDX mice (FIG.7F). Moreover, these long-chain acyl-carnitines were even lower in MDX mice with PHD3 depletion compared to MDX mice (FIG. 7G). It was reasoned that these fuels might have a higher oxidation rate during PHD3 depletion in vivo, preventing acyl-carnitines from accumulating. Thus, phd3-/-mdx mice utilize long-chain acyl-carnitines more than phd3+/+mdx mice. To investigate the metabolic alterations resulting from FAO activity, was measured using labeled FoleyHoagUS12471059.1 Attorney Docket No. HMV-32325 palmitate in the quadriceps muscle (FIG. 7H). The findings showed that palmitate oxidation was increased in phd3-/- mice compared to control mice tissue. Interestingly, FAO was decreased in phd3+/+mdx mice, consistent with the higher expression level of PHD3 in patients (FIG. 1A). Additionally, it was observed that a significant increase in mitochondrial fatty acid oxidation in phd3-/- mice compared to phd3+/+mdx (FIG.7H). Therefore, the loss of PHD3 is crucial in regulating fatty acid oxidation, even in the context of mdx disease. [0183] During the early stages of myogenesis, FAO is induced in skeletal muscle and plays a critical role in regulating the expression of genes involved in myogenesis (Fritzen et al., 2020). To examine whether PHD3 regulates glucose oxidation in addition to fat oxidation, the basal oxygen consumption rate (OCR) resulting from fat oxidation were measured in WT or PHD3-KD C2C12 cell lines under myogenesis conditions using a Seahorse flux analyzer (FIG.7I). The loss of PHD3 increased basal fat oxidation-driven respiratory activity during myogenic differentiation, consistent with increased FAO in mouse skeletal muscle (FIG. 7I). To test whether PHD3 regulates FAO through ACC2, C2C12 cell lines expressing shRNA against ACC1, ACC2, CPT1, or shRNA control were generated (FIG.8K-8M), and FAO activity was measured in these cell lines after myogenic differentiation (FIG. 7J). Control cells showed induced FAO activity during myogenesis, which was further increased by PHD3 depletion (shown in orange and red), while ACC2 depletion also resulted in higher FAO activity, and the FAO activity in ACC2 and PHD3 double knock-out cells remained unchanged, with no FAO activity observed in CPT1 depletion due to the inability to import long-chain acyl-carnitines into mitochondria (FIG. 7J), indicating that PHD3 loss increases FAO in cells and in mouse quadriceps. Example 5: PHD3 Deletion Improves Exercise Capacity in DMD Mice [0184] To investigate whether the loss of PHD3 improves skeletal muscle function in MDX mice, a mouse limb grip strength test on 12-week-old mice was performed. MDX mice have decreased grip strength compared to WT mice due to their weak and fragile muscle phenotype. However, the phd3-/-mdx mice exhibited restored muscle strength compared to phd3+/+mdx mice (FIG. 9A). To evaluate the effect of PHD3 loss on skeletal muscle function in a DMD model, exercise endurance was assessed in phd3+/+mdx and phd3-/-mdx mice using a metabolic treadmill (FIG. 9B). The results showed that phd3-/-mdx mice ran 20% longer and >30% further distance than phd3+/+mdx mice (FIG.9C and 9D). Furthermore, the maximal oxygen consumption rate (VO2 FoleyHoagUS12471059.1 Attorney Docket No. HMV-32325 peak) was increased in phd3-/-mdx animals (FIG. 9E). phd3+/+mdx mice reached VO2 peak sooner than phd3-/-mdx animals, which is consistent with the finding that phd3-/-mdx mice had more overall exercise endurance (FIG. 9F). Thus, the loss of PHD3 function improves muscle function and exercise capacity in the mdx model of DMD phenotype. These results demonstrate that PHD3 loss improves exercise capacity in DMD disease. Example 6: Loss of PHD3 prevents weight gain and fat accumulation on a high fat diet [0185] The present example examines the physiological relevance of PHD3 on high fat diet (HFD) condition. PHD3 knockout mice on a low fat diet (LFD) did not have any changes in body weight or lean body mass, serum free fatty acids (FFA) or triglycerides (TG) relative to wild-type (WT) mice (data not shown). In view of the role of PHD3 in improving mitochondrial fatty acid oxidation, such as described above, the role PHD3 in fat metabolism was characterized under a stress condition of high fat diet. [0186] To understand the role of PHD3 in HFD challenge, 8 week old WT or PHD3 KO mice were challenged with a HFD for 10 weeks, as illustrated in FIG.10A. During that time, mice were weighed every week and glucose tolerance (GTT) was measured in 8weeks or insulin tolerance (ITT) in 10 weeks. Surprisingly, PHD3 KO mice prevent weight gain under a HFD, as shown in FIG. 10B. Usually HFD drive obesity or type 2 diabetes. The glucose tolerance test can be used to screen for type 2 diabetes. To measure their physiological response to glucose, mice were fasted 16 h and the blood glucose level was measured after high glucose injection, over time by time. PHD3 KO mice are also sensitive to glucose, as shown in FIG. 10D. Similar measurements of the mouse body weight and GTT for PHD3 KO mice and WT mice again showed that there was no change in either body weight or glucose tolerance in PHD3 KO mice on a LFD. Therefore, loss of PHD3 confers specific protection against gaining weight under HFD. [0187] Next, to understand whether PHD3 KO mice on a HFD have lower accumulation of fat in metabolic organ, the weight of white adipose tissue was dissected and measured. As shown in FIG. 11A and FIG.11B, both epididymal and inguinal fat was smaller in PHD3 KO mice compared to WT. This corresponded to a large reduction in the percent weight of adipose tissue relative to total bodyweight in PHD3 KO mice compared to WT. [0188] An even more dramatic effect was observed in the livers of these mice. WT mice have fatty liver during HFD challenge, in contrast, PHD3 KO mice on a HFD have a healthy 37 FoleyHoagUS12471059.1 Attorney Docket No. HMV-32325 appearing liver, both in gross organ appearance and with H&E staining, FIG.11C and FIG.11D. As shown in FIG.11D, it is apparent in H&E stained liver tissue from these mice, there is a large accumulation of lipids in WT mouse liver, while, the PHD3 KO mouse liver looks very dense and there is very little lipid visible. [0189] Basal insulin levels were also found to be decreased in PHD3 KO mice. The fasted blood glucose levels were measured in mice under both a high fat and low fat diet (FIG. 12). No change was observed in blood glucose levels between WT and PHD3 KO mice under low fat diet. The blood glucose level was increased in WT mice under HFD, however, PHD3 KO mice were protected from this increase in blood glucose on a HFD. Interestingly, serum insulin levels were dramatically lower in PHD3 KO mice even in LFD condition. This insulin result is consistent with a report by Taniguchi, C., et al. (2013) Nat Med 19, 1325–1330 that mice with PHD3 silencing in the liver have decreased insulin levels. Example 7: Loss of PHD3 alters metabolite profile in muscle and liver [0190] The present example describes the characterization of the metabolite profile of WT and PHD3 KO mice on a HFD. Metabolomics were performed on muscle (quadricep) tissue and liver dissected from WT mice and PHD3 KO mice under HFD condition. Results are shown in FIGs. 13A-E. In muscle, there was a large impact on metabolites associated with arginine and proline metabolism, as well as nicotinate and nicotinamide metabolism (FIG. 13B). In liver, glycerophospholipid metabolism, glycine, serine, and threonine metabolism, and biotin metabolism were the most significantly affected pathways. Example 8: AKT signaling is increased in PHD3 KO mice under HFD [0191] Many molecular sensors detect and response to nutrient availability. For example, nutrient availability determines the levels of AMP/ATP, which directly affects AMPK activity. AMPK is a major regulator of metabolism. It senses glucose, insulin, drugs and stress to regulate energy dependent signaling pathways. AMPK is activated by phosphorylation and coordinates with other enzymes to alter cellular metabolism. For example, AMPK is activated through phosphorylation by LKB1, and inactivated by dephosphorylation by protein phosphatases. AMPK controls catabolic pathways and anabolic pathways, such as fatty acid oxidation by inhibiting ACC2 through phosphorylation. FoleyHoagUS12471059.1 Attorney Docket No. HMV-32325 [0192] AMPK signaling was assessed on the livers of PHD3 KO mice on HFD. Specifically, it was found that livers from PHD3 KO mice on a high fat diet had decreased p-ACC2 and increased p-Akt at both positions T308 and S473. And that subsequently fasting these mice led to a dramatic increase in p-ACC and decrease of pAkt (both T308 and S473). See FIG.14A and FIG. 14B. Similarly, AKT signaling was found to be increased in the quadriceps of PHD3 KO mice under HFD, as shown in FIG. 15A and FIG. 15B. Moreover, these effects were found to be strongly responsive to fasting conditions. Together, these data support a role for PHD3 in regulating AKT metabolism in mice. Example 9: Methods [0193] In some embodiments, the disclosure features a method for culturing human cells. HEK293T (human embryonic kidney, female), and C2C12 (mouse mesenchymal precursor, sex of cell lines was unknown) cell-lines were obtained from American Type Culture Collection (ATCC). Cells were cultured at 37 degree, 5% CO2 in 4.5 g/L (25 mM) glucose Dulbecco’s modified Eagle's medium, supplemented with 10% fetal bovine serum and penicillin/streptomycin. C2C12 cell differentiation toward myoblasts was induced by 2% horse serum (Gibco) and Insulin-Transferrin- Selenium Liquid Media Supplement (Sigma) for two days. [0194] In some embodiments, the disclosure features a method to generate knockout mice. For PHD3-CMV-Cre mice, Egln2tm2Fong Egln1tm2Fong Egln3tm2Fong/J mice (PHD1FL/PHD2FL/PHD3FLtriple floxed strain # 028097) (Takeda et al., 2006) were obtained from The Jackson Laboratory and housed in the New Research Building Animal Facility at Harvard Medical School. All protocols were approved by the IACUC of Harvard Medical School and were in accordance with NIH guidelines. Mice were housed at 20-22 degrees on a 12 h light/dark cycle with ad libitum access to food (LabDiet 5053) and water. PHD3FL mouse were obtained by breeding between Egln2tm2Fong Egln1tm2Fong Egln3tm2Fong/J mice and C57BL/6J. PHD3FL mouse were crossed with B6.C-Tg(CMV-cre)1Cgn/J mice (CMV-Cre mice # 006054) to generate PHD3FL:CMV-Cre knockout mice
Figure imgf000040_0001
. Only male mice were used for experiment. To generate MDX:PHD3FL:CMV-Cre knockout mice (phd3-/-mdx), PHD3FL:CMV-Cre knockout mice (phd3-/-) were crossed with C57BL/10ScSn-Dmdmdx/J (mdx strain #001801). Only male mice were used for experiment. Genotyping was performed by PCR using genomic DNA obtained from the tails. All FoleyHoagUS12471059.1 Attorney Docket No. HMV-32325 animal studies were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee, the Standing Committee on Animals at Harvard Medical School. [0195] In some embodiments, the disclosure features a method for preparing and transfecting agents into human cells. pcDNA3.1-HA-PHD3 or HA-pBabe PHD3 were previously described (Lee et al., 2005). Synthetic oligos for shRNAs (sequences in Table 2) were digested with Age I and EcoRI (NEB), and inserted into a pLKO-puromycin vector using T4 DNA ligase (NEB). This shRNA construct was co-transfected into HEK293T cells with pRSV-Rev, pMD2-VSVG, and pMDLg/pRRE plasmids to prepare lentiviral particles. On the 3rd day after transfection, lentiviruses were collected from the supernatant of HEK293T cells. Target cells were infected with virus for 24 hours and selected using puromycin (2 ug/ml) for 48 hours to establish stable cell lines. [0196] In some embodiments, the disclosure features a method for quantitative RT-PCR analysis. RNA was isolated by extraction with Trizol according to manufacturer instructions (Invitrogen) or with the RNA Clean & Concentrator Kit (Zymo Research). cDNA was synthesized using iScript cDNA synthesis kit (BioRad). Quantitative real-time PCR was performed with SYBR Green Fast Mix (Quanta Biosciences) on a Roche Lightcycler 480 and analyzed by using ΔΔCt calculations. qPCR analyses in human cell lines are relative to the reference gene B2M. qPCR analyses in mouse cell line or tissue are relative to β-Actin. The primer sequences are described in Table 3 below. FoleyHoagUS12471059.1 Attorney Docket No. HMV-32325 [0197] Table 3 – Exemplary RT-PCR primer sequences
Figure imgf000042_0001
[0198] In some embodiments, the disclosure features methods for analyzing protein expression. Western blotting was performed using antibodies against ACC (Cell Signaling Technologies (CST), ACC2 isoform (CST), actin (Sigma), myosin heavy chain (MHC) (Abcam), dystrophin (Abcam), MyoD (Abcam), hydroxyproline (Abcam), PHD3 (Novus Biologicals) and tubulin (Sigma). 1% NP40 buffer containing protease and phosphatase inhibitors was used to prepare lysates, unless otherwise indicated. For ACC hydroxylation time course studies, the pan- PHD inhibitor dimethyloxalylglycine (DMOG, Frontier Scientific, 0.05 mM) was added to the lysis buffer to prevent further hydroxylation in the lysate. For immunoprecipitation of endogenous ACC2, cells were lysed with buffer containing 50 mM Tris-HCl (pH 7.8), 150 mM NaCl, 1 % NP40, 0.1 mM DTT, 0.05 mM DMOG, protease inhibitors (Roche) and 100 µl of phosphatase FoleyHoagUS12471059.1 Attorney Docket No. HMV-32325 inhibitor 2/3 (Sigma). Cell lysates were centrifuged for 15 min to remove debris. Then, 2 µg of ACC antibody (CST) or ACC2 antibody (CST), was added to the cleared cell lysate (500 µg of protein) and immunoprecipitated with EZview Red Protein G Affinity resin (Sigma). [0199] In some embodiments, the disclosure features methods for measuring fatty acid oxidation (FAO). For FAO assays, C2C12 cells (plated in 12 well-plates) were pre-incubated for 4 hours in serum-free medium, containing 5 mM glucose and supplemented with 100 μM palmitate or hexanoate and 1 mM carnitine. Cells were switched to 600 μl medium containing 1 μCi [9,10- 3H(N)]-palmitic acid (Perkin Elmer) and 1 mM carnitine for 2 hours. Medium was collected and released 3H2O was eluted in columns packed with DOWEX 1X2-400 ion exchange resin (Sigma). Basal FAO assays were performed in cells not pre-incubated with fatty acids prior to FAO analysis. Counts per minute (CPM) were normalized to protein content in parallel cell plates. [0200] For ex vivo FAO assays, we used radiolabeled palmitate with crude mitochondrial extraction from tissue, as previously described (Hirschey et al., 2010; Huynh et al., 2014). Eppendorf tubes and all buffers were degassed using nitrogen gas.7% BSA/5 mM palmitate (5:1 molar ratio) or 7% BSA/5 mM palmitate/0.01 mCi/mL [14C]palmitate was prepared with water using a 42°C water bath. 0.4 mCi of [14C]palmitate was used per reaction for animal tissues (200 mg). Fresh tissues were homogenized in sucrose–Tris–EDTA (STE) buffer (0.25 M sucrose, 10 mM Tris–HCl and 1 mM EDTA) using a 2 mL Dounce homogenizer. Samples were centrifuged at 420 xg to isolate crude mitochondria and normalized by protein concentration ~100 mg of mitochondria were mixed with 100 mM sucrose, 10 mM Tris–HCl (pH 7.4), 5 mM KH2PO4, 0.2 mM EDTA, 80 mM KCl, 1 mM MgCl2, 2 mM L-carnitine, 0.1 mM malate, 0.05 mM coenzyme A, 2 mM ATP, 1 mM DTT and 0.7% BSA/500 mM palmitate/0.4 mCi [14C]palmitate (pH 8.0), and incubated 37°C for 30 min. Then, reactions were transferred into tubes containing 200 uL of 1 M perchloric acid to quench reactions and discs of Whatman filter paper (pre-treated with 20 uL of 1 M NaOH) placed in the caps to capture 14CO2. After 1 hour incubation to allow 14CO2 capture, the paper disc or the remaining acid solution were transferred to a scintillation vial. Vials were mixed with scintillation fluid, and the average counts per min over 3 min were measured with a standard scintillation counter. Data were normalized to protein content. [0201] In some embodiments, the disclosure features methods for measuring respiration. Respiration was assessed using the Seahorse XFe-96 Analyzer (Seahorse Bioscience). For experiments that measure the basal levels of oxygen consumption rate, 1x105 of shControl or FoleyHoagUS12471059.1 Attorney Docket No. HMV-32325 shPHD3 C2C12 cells were incubated with 2% horse serum (Gibco) and Insulin-Transferrin- Selenium Liquid Media Supplement (Sigma) in DMEM media for 48 h prior to the experiment. Following this incubation, media was changed to a non-buffered, serum-free Seahorse Media (Seahorse Bioscience) supplemented with 10 mM glucose, 2 mM L-glutamine and 1 mM sodium pyruvate. For basal OCR using fat, cells were incubated in serum free media overnight and incubated in serum-free Seahorse Media (Seahorse Bioscience) supplemented with 0.5 mM glucose, 1 mM L-glutamine, 0.5 mM carnitine and XF BSA-palmitate. Values were normalized to cell number. [0202] In some embodiments, the disclosure features methods for profiling metabolites with mass spectrometry. Metabolites were extracted from flash frozen tissues (~5 mg/ml) in 80% MeOH and analyzed on two distinct methods of hydrophilic interaction liquid chromatography coupled to mass spectrometry (HILIC-MS). In one method, electrospray ionization was tailored to negative-ion mode, and in the second method to positive-ion mode. For negative-ion mode, analytes were eluted in buffer A (20 mM ammonium acetate, 20 mM ammonium hydroxide) and buffer B (10 mM ammonium hydroxide in 75:25 acetonitrile:methanol). Samples were run on a HILIC silica (3 um, 2.1 x 150 mm) column (Waters) with a binary flow rate of 0.4 mL/min for 10 min on linear gradient (95% buffer B to 0% buffer B) followed by 2 min with (0% buffer B) and ending with a 2-minute linear gradient (0% buffer B to 95% buffer B) and holding (95% buffer B) for 13 min. For positive- ion mode, samples were dried down and reconstituted in a 20:70:10: acetonitrile:MeOH:water mixture. The buffers were: buffer A (10 mM ammonium formate, 0.1% formic acid in water) and buffer B (acetonitrile, 0.1% formic acid). Samples were run on a HILIC silica (3 um, 2.1 x 150mm) column (waters) with a binary flow rate of 0.25 mL/min for 10 min on linear gradient (95% buffer B to 40% buffer B) followed by 4.5 min with (40% buffer B) and ending with a 2-minute linear gradient (40% buffer B to 95% buffer B) and holding (95% bufferm B) for 13 min. For both ion- modes, a Q Exactive hybrid quadrupole orbitrap mass spectrometer (Thermo Fisher Scientific) with a full-scan analysis over 70–800 m/z and high resolution (70,000) was used for mass detection. A targeted-method developed for 176 compounds (118 on positive and 58 on negative) was used to identify metabolites. A master mix of reference standards for metabolites in the targeted method were run immediately prior to each set of samples, such that their retention times were associated with peaks in the unknown samples run over that same column. Peaks were integrated in FoleyHoagUS12471059.1 Attorney Docket No. HMV-32325 Tracefinder 3.3. Metabolite levels were normalized to cell number in parallel plates or protein concentration in the same amount of tissue powder samples. [0203] In some embodiments, the disclosure features methods for analysis creatine kinase levels. Creatine kinase (CK) levels were assessed on a weekly basis until the age of 8 wk and on a biweekly basis from 10 until 32 wk. To this end, blood was collected via a small angled cut in the tail in a heparin-coated Microvette CB 300 (Sarstedt) and stored on ice for a maximum of 2 h. Subsequently, blood samples were centrifuged at 4°C for 5min at 18,000 g. The obtained plasma was used to measure CK levels according to the CK ELISA kit instructions (Abcam). [0204] In some embodiments, the disclosure features methods for evaluating exercise performance. 12-week-old mice were acclimated to the treadmill 2 days prior to the experiments by running for 5 min/day at 5 m/min and 10 m/min followed by 15 m/min for 1 min. For exercise experiments, speed was increased 5 m/min every 5 min until reaching 20 m/min until exhaustion. Respiratory exchange ratio (RER), VO2, VCO2 and heat were monitored using the Oxymax Modular Treadmill System. [0205] In some embodiments, the disclosure features methods for analysis grip strength. Grip strength of the forelimbs was assessed using a grid attached to an isometric force transducer (Columbus Instruments). The force transducer recorded the maximum force required to break the mouse's grip from the mesh surface. In total, five strength measurements were recorded, each consisting of three pulls. The three highest values were averaged and normalized to the body weight. [0206] Mouse quadriceps were isolated from 12-week-old mice between 9 and 11 am following ad libitum feeding (fed). Quadriceps were fixed in 10% buffered formalin, dehydrated through a series of ethanol solutions of increasing concentration and submitted to the Dana- Farber/Harvard Cancer Center Pathology Cores for embedding in paraffin, sectioning, and hematoxylin and eosin staining. Immunohistostaining was performed using anti-CD68 antibody (Abcam). The fresh frozen quadriceps were sectioned and stained with H&E. Immunofluorescence was performed on the same samples using anti-Laminin (Novus Biologicals) and DAPI. In brief, paraffin quadriceps sections were rinsed with xylene (three times for 5 min each). Slides were incubated twice in 100% ethanol (10 min each) for rehydration. After rinsing with water, slides were boiled (10 min) with 10 mM sodium citrate buffer (pH 6.0) for antigen unmasking. The sections were washed with PBS with 0.4% Triton X-100 and blocked for 1 hour at 4°C, with PBS FoleyHoagUS12471059.1 Attorney Docket No. HMV-32325 containing 5% normal goat serum. Slides were incubated with primary antibodies in PBS overnight at 4°C. After 3 washes with PBS, 5 min at room temperature, slides were incubated for 2 hours with goat anti-mouse IgG conjugated to the fluorescent Alexa 488 dye (1:400) and goat anti-rabbit IgG conjugated to the fluorescent Alexa 594 dye (1:400) in PBS. After 3 washes, nuclei were visualized with 1 mg/ml DAPI. Following 3 washes in PBS and 1 wash with 75% ethanol, the sections were mounted in ProLong Gold Antifade reagent (Life Technologies). Digital images of stained sections were taken using confocal microscope (Nikon TE2000 w/C1 Point Scanning Confocal). [0207] To evaluate changes in sarcolemma membrane permeability of skeletal muscle, the distribution of in vivo Evans blue dye (EBD) (Sigma) was assessed. EBD is a low-molecular- weight, nontoxic dye that binds to serum albumin upon intravenous infusion and recirculation. It cannot cross the sarcolemma of intact myocytes, but if the membrane is ruptured, it enters the intracellular space and binds to intracellular proteins. To prepare the EBD solution, 2% EBD was dissolved in PBS (pH 7.2) and sterilized by passing it through a membrane filter with a pore size of 0.2 µm. The EBD solution was then intravenously injected (50 µl/10 g body weight) into 12-week- old mice. 48 h after the EBD injection, the quadricep muscles were harvested and frozen with liquid nitrogen. Consecutive sections were made from each frozen heart using a cryostat (Microm) with a chamber temperature of -225°C. Several pairs of cross-sectional 10-µm-thick adjacent sections were randomly selected from the consecutive sections. EBD in this section was observed under a fluorescent microscope (Nikon) using 620 nm excitation/680 nm emission after an immunofluorescence experiment with anti-laminin and DAPI. [0208] Tissues were fixed in 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1Mcacodylate buffer, pH 7.4, for at least 1 h at room temperature. Next, tissues were washed in 0.1 M cacodylate buffer and fixed secondarily in 1% osmium tetroxide for 1 h at 4°C. Tissues were then washed in deionized (DI) water before being immersed in 2% aqueous uranyl acetate to be contrast fixed overnight at 4°C. The following day, tissues were washed and dehydrated. Infiltration proceeded with 1:1 propylene oxide and LX112 Epon resin. Tissues were then embedded and cured at 60°C over 48 h. Cured blocks were sectioned at 80-nm thickness and put on coated copper slot grids that had been carbon coated and glow discharged. Grids were contrast stained with 2% uranyl acetate for 10 min and lead citrate for 5 min. Grids were imaged on a JEOL 1400 transmission electron microscope (TEM) equipped with a side mount Gatan Orius SC1000 digital camera. Quantification was performed using Velocity software. FoleyHoagUS12471059.1 Attorney Docket No. HMV-32325 [0209] Human muscle expression datasets were obtained from GEO under the respective accession identifiers and from GTEx (PMID: 23715323). Confounding factors, including gender, age, batch, and disease, as well as hidden factors that could cause gene expression variability, were estimated and removed using probabilistic estimation of expression residuals (PEER) (PMID: 22343431). Expression residuals obtained from PEER were used for further analysis. To identify the enriched gene sets correlated with PHD3 expression, we performed gene set enrichment analysis (GSEA) (PMID: 16199517) using the fgsea package (https://www.biorxiv.org/content/early/2016/06/20/060012). Specifically, genes were ranked based on the Pearson correlation coefficient against PHD3 expression, and enrichment analysis was performed to determine the enriched gene sets co-expressed with PHD3 in control or DMD patient group (GSE38417). [0210] Details regarding the specific statistical tests, definition of center, and number of replicates (n), can be found for each experiment in the figure legends. All experiments were performed at least twice. Data from animal studies are shown as mean ± SEM, while data from in vitro studies are shown as mean ± SD. When comparing two groups, statistical analysis was performed using a two-tailed Student’s t test and statistical significances were considered when P values were less than 0.05. P values were calculated assuming a normal distribution and were corrected for multiple hypothesis using the Benjamini-Hochberg procedure. GraphPad, Prism, and Excel were used for all quantifications and statistical analyses. 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Small-molecule activation of lysosomal TRP channels ameliorates Duchenne muscular dystrophy in mouse models. Sci Adv. 6, eaaz2736. EQUIVALENTS [0213] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims. FoleyHoagUS12471059.1

Claims

Attorney Docket No. HMV-32325 CLAIMS 1. A method of treating or preventing a muscular dystrophy in a subject comprising administering to the subject an agent that reduces the levels of or inhibits the activity of prolyl hydroxylase 3 (PHD3). 2. The method of claim 1, wherein the muscular dystrophy is Duchenne’s muscular dystrophy (DMD). 3. The method of claim 1, wherein the muscular dystrophy is selected from Becker’s muscular dystrophy, congenital muscular dystrophy, distal muscular dystrophy, Emery-Dreifuss muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, myotonic muscular dystrophy, and oculopharyngeal muscular dystrophy. 4. A method of treating or preventing a muscle wasting disorder in a subject comprising administering to the subject an agent that reduces the levels of or inhibits the activity of prolyl hydroxylase 3 (PHD3). 5. The method of claim 4, wherein the muscle wasting disorder is cachexia. 6. The method of claim 5, wherein the cachexia is an obesity-related cachexia. 7. The method of claim 4, wherein the muscle wasting disorder is sarcopenia. 8. The method of claim 7, wherein the sarcopenia is ageing-related sarcopenia. 9. A method of increasing skeletal muscle differentiation and/or decreasing skeletal muscle atrophy in a subject in need thereof comprising administering to the subject an agent that reduces the levels of or inhibits the activity of prolyl hydroxylase 3 (PHD3). FoleyHoagUS12471059.1 Attorney Docket No. HMV-32325 10. A method of improving sarcolemmal integrity and/or increasing exercise capacity in a subject in need thereof, comprising administering to the subject an agent that reduces the levels of or inhibits the activity of prolyl hydroxylase 3 (PHD3). 11. A method of increasing fatty acid oxidation and/or decreasing hydroxylation of acetyl CoA carboxylase 2 (ACC2) in a subject in need thereof comprising administering to the subject an agent that reduces the levels of or inhibits the activity of prolyl hydroxylase 3 (PHD3). 12. The method of claim 11, wherein the method increases fatty acid oxidation in muscle cells of the subject and/or decreases hydroxylation of ACC2 in muscle cells of the subject. 13. The method of any one of claims 9-12, wherein the subject is afflicted with Duchenne’s muscular dystrophy (DMD). 14. The method of claim 13, wherein the method decreases creatine kinase activity in the subject. 15. The method of claim 13 or 14, wherein the method restores mitochondrial size and/or morphology in the subject. 16. The method of any one of claims 9-12, wherein the subject is afflicted with a muscular dystrophy selected from Becker’s muscular dystrophy, congenital muscular dystrophy, distal muscular dystrophy, Emery-Dreifuss muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, myotonic muscular dystrophy, and oculopharyngeal muscular dystrophy. 17. The method of any one of claims 9-12, wherein the subject is afflicted with a muscle wasting disorder. 18. The method of claim 17, wherein the muscle wasting disorder is cachexia. 19. The method of claim 18, wherein the cachexia is an obesity-related cachexia. FoleyHoagUS12471059.1 Attorney Docket No. HMV-32325 20. The method of claim 17, wherein the muscle wasting disorder is sarcopenia. 21. The method of claim 20, wherein the sarcopenia is ageing-related sarcopenia. 22. A method of preventing weight gain in a subject in need thereof, comprising administering to the subject an agent that reduces the levels of or inhibits the activity of prolyl hydroxylase 3 (PHD3). 23. The method of claim 22, wherein the subject consumes a high fat or high calorie diet. 24. The method of claim 22 or claim 23, wherein the agent further increases Akt signaling. 25. A method of increasing Akt signaling in an obese or overweight subject, comprising administering to the subject an agent that reduces the levels of or inhibits the activity of prolyl hydroxylase 3 (PHD3). 26. The method of claim 25, wherein the subject consumes a high fat or high calorie diet. 27. The method of claim 25 or claim 26, wherein Akt signaling is increased in the muscle cells of the subject. 28. The method of any one of claims 1 to 27, wherein the agent is a small molecule. 29. The method of any one of claims 1 to 27, wherein the agent is a sgRNA specific for a nucleic acid sequence encoding PHD3. 30. The method of any one of claims 1 to 27, wherein the agent is an inhibitory polynucleotide. 31. The method of claim 30, wherein the inhibitory polynucleotide is selected from the group consisting of siRNA, shRNA, and an antisense oligonucleotide, or a polynucleotide that encodes a molecule selected from the group consisting of siRNA, shRNA, and/or an antisense oligonucleotide. FoleyHoagUS12471059.1 Attorney Docket No. HMV-32325 32. The method of claim 31, wherein the inhibitory polynucleotide is an shRNA. 33. The method of claim 32, wherein the shRNA is specific for PHD3. 34. The method of claim 32, wherein shRNA targets any one of the sequences listed in Table 1. 35. The method of claim 32, wherein the shRNA targets a sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. 36. The method of claim 32, wherein the shRNA comprises a sense strand set forth in SEQ ID NO: 5. 37. The method of claim 32, wherein the shRNA comprises a sense strand set forth in SEQ ID NO: 6. 38. The method of claim 31, wherein the inhibitory polynucleotide is an siRNA. 39. The method of claim 38, wherein the siRNA is specific for PHD3. 40. The method of claim 38 or claim 39, wherein the siRNA is about 18 to 25 nucleotides in length. 41. The method of any one of claims 38 to 40, wherein the siRNA is a small double stranded RNA (dsRNA). 42. The method of claim 41, wherein the dsRNA comprises overhangs. 43. The method of claim 41, wherein the dsRNA comprises blunt ends. 44. The method of claim 35, wherein the inhibitory polynucleotide is an antisense oligonucleotide (ASO). 45. The method of claim 44, wherein the ASO is specific for PHD3. FoleyHoagUS12471059.1 Attorney Docket No. HMV-32325 46. The method of claim 44 or claim 45, wherein the ASO is about 13 to 30 nucleotides in length. 47. The method of any one of claims 1 to 27, wherein the agent is an antibody-siRNA conjugate. 48. The method of claim 47, wherein the antibody-siRNA conjugate is specific for muscle tissue. 49. The method of any one of claims 1 to 27, wherein the agent is an antibody-ASO conjugate. 50. The method of claim 49, wherein the antibody-ASO conjugate is specific for muscle tissue. 51. The method of any one of claims 1 to 50, wherein the agent is administered intravenously. 52. The method of any one of claims 1 to 50, wherein the agent is administered at least once a week or at least once a month. 53. The method of any one of claims 1 to 50, wherein the agent is administered for at least 7 days, for at least 30 days, for at least 60 days, or for at least 90 days. 54. The method of any one of claims 1 to 53, wherein the subject is a human. 55. A method of evaluating the responsiveness of a subject to an agent that reduces the levels of or inhibits the activity of prolyl hydroxylase 3 (PHD3), the method comprising measuring the hydroxylation of acetyl CoA carboxylase 2 (ACC2) in a subject that has received the agent that reduces the levels of or inhibits the activity of PHD3, wherein a decrease in the levels of hydroxylation of ACC2 compared to the levels of hydroxylation of ACC2 in the subject prior to receiving the agent indicates that the subject is responding to the agent that reduces the levels of or inhibits the activity of PHD3. FoleyHoagUS12471059.1
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