Muscle regeneration and growth

Abstract

To provide a genetically modified mouse which includes a sequence coding MuSK in its genome.SOLUTION: A genetically modified mouse in which a sequence coding MuSK is included in its genome is provided. In the mouse, the sequence coding MuSK does not include a nucleotide sequence between exon 6 and exon 7 (in order from 5' to 3'), and the genetically modified mouse cannot generate full length MuSK transcription product or full length MuSK protein.SELECTED DRAWING: None

Description

[Technical Field]

[0001] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was developed with the following funding awarded by the National Institutes of Health: NIH Grant: T32 Award 5T32AG041688-07, and U01 Grant: NIH 1U01NS064295-04. The government has certain rights in this invention. [Background technology]

[0002] Skeletal muscle has the ability to regenerate after injury. Muscle regeneration depends on resident stem cells called muscle satellite cells. In mature muscle tissue, satellite cells constitute a small, scattered population of mitotically and physiologically quiescent cells. Satellite cells are also involved in normal muscle growth and maintenance throughout life, suggesting that they may be utilized to treat muscle-wasting conditions.

[0003] Skeletal muscle accounts for approximately 35% of body weight and is essential for metabolism, movement, and respiration, highlighting its importance in human health. Muscle wasting reduces mobility, metabolism, and quality of life for the majority of cancer patients, elderly patients, and many others without a history of neuromuscular dysfunction. In addition, muscular dystrophies are a group of often fatal genetic diseases that lead to severe muscle weakness, including Duchenne muscular dystrophy, which affects children. An obstacle to the development of treatments is that the signaling that regulates satellite cells and muscle regeneration is poorly understood. Therefore, there is a need for compositions and methods for muscle regeneration and muscle growth. Summary of the Invention [Problem to be solved by the invention]

[0004] Bone morphogenetic protein (BMP) signaling regulates skeletal muscle stem cell activity under both normal and pathological conditions. Embodiments of the present invention provide techniques for modulating BMP signaling. Among other things, this disclosure provides insight that the critical interaction between BMP and muscle-specific tyrosine kinase protein (MuSK) is mediated by the third immunoglobulin domain (Ig3) of MuSK. This disclosure demonstrates that BMP signaling is disrupted in the absence of the Ig3 domain. Furthermore, this disclosure teaches that modulating BMP signaling through pharmacological intervention targeting MuSK provides an attractive method for enhancing muscle stem cell activity and increasing muscle regeneration.

[0005] The present disclosure provides techniques for increasing the level and / or activity of MuSK forms that functionally participate in muscle regeneration, including in some embodiments by reducing alternative splicing that would otherwise generate MuSK forms that do not. In some embodiments, such an increase is in relevant tissues, such as muscle. Alternatively or additionally, in some embodiments, such an increase is in tissues such as brain tissue (e.g., hippocampus and / or subventricular) and / or lung tissue.

[0006] In some embodiments, the present disclosure provides methods for inhibiting Ig3 by, for example, increasing the level and / or activity of one or more forms of MuSK in which the Ig3 domain has been altered (e.g., mutated, blocked, removed, etc.), thereby preventing it from effectively participating in interactions with BMPs, for example. - In some embodiments, the present disclosure provides techniques for agonizing MuSK, e.g., by reducing the level and / or activity of one or more forms of MuSK whose Ig3 domain effectively participates in the interaction with BMP. +Techniques for reducing MuSK are provided. Alternatively or additionally, in some embodiments, the present disclosure provides techniques for reducing the level and / or activity of MuSK Ig3 / BMP complexes (e.g., inhibiting the formation of such complexes, disrupting such complexes, and / or otherwise antagonizing such complexes). [Means for solving the problem]

[0007] In some embodiments, the present disclosure provides MuSK muscle regeneration agonizing agents that target the MuSK Ig3 domain and / or BMP, thereby reducing the level and / or activity of MuSK / BMP complexes. In some such embodiments, such MuSK muscle regeneration agonizing agents inhibit the formation of such complexes and / or disrupt such complexes. In some embodiments, such MuSK muscle regeneration agonizing agents compete with BMPs for binding to MuSK Ig3 and / or compete with MuSK Ig3 for binding to BMPs.

[0008] In some embodiments, agents that target the MuSK Ig3 domain and / or BMP described herein, thereby decreasing the level and / or activity of MuSK / BMP complexes (e.g., MuSK muscle regeneration agonists), are useful in the context of muscle regeneration and / or muscle growth. In some embodiments, provided agents may enhance muscle growth. In some embodiments, muscle growth occurs in undamaged tissue. In some embodiments, muscle growth occurs in damaged tissue. In some embodiments, enhanced and / or increased muscle growth is determined by a decrease in satellite cell number and / or an increase in myofiber size. In this regard, muscle growth may be characterized by a decrease in satellite cell number and / or an increase in myofiber size, indicating satellite cell differentiation and fusion / expansion into existing and newly formed myofibers.

[0009]

[0010] Embodiments of the present invention provide methods of enhancing muscle regeneration and / or muscle growth, e.g., in a subject in need thereof, by administering a composition that downregulates MuSK Ig3 domain protein expression, MuSK Ig3 domain gene expression, and / or MuSK Ig3 activation of BMP signaling, thereby upregulating muscle satellite cells, resulting in enhanced muscle regeneration and / or muscle growth. In some embodiments, such compositions may comprise and / or deliver a MuSK Ig3-targeted blocking antibody, a MuSK Ig3-targeted exon-skipping oligonucleotide, a MuSK Ig3-targeted CRISPR / Cas9, a MuSK Ig3-targeted siRNA, a MuSK Ig3-targeted small molecule, and / or a MuSK Ig3-targeted shRNA.

[0010] In some embodiments, the subject of interest may be at risk for or suffering from a disease or disorder, including, but not limited to, neuromuscular dysfunction, neurodegenerative disorders, cardiac dysfunction (e.g., myocardial infarction, cardiomyopathy), or genetic diseases characterized by muscle wasting. Alternatively or additionally, in some embodiments, the subject of interest may be at risk for or suffering from a disease or disorder associated with lung injury, including, for example, idiopathic pulmonary fibrosis (IPF), acute respiratory distress syndrome (ARDS), pneumonia, and / or certain infectious diseases, including viral infections, including coronavirus infections such as COVID-19.

[0011] Exemplary neuromuscular dysfunctions or disorders that can be treated by the techniques of the present invention include, but are not limited to, Becker muscular dystrophy, congenital muscular dystrophy, distal muscular dystrophy, Duchenne muscular dystrophy, Emery-Dreifuss muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, myotonic muscular dystrophy, and oculopharyngeal muscular dystrophy.

[0012] In some embodiments, the enhancement of muscle growth is used in the context of treating diseases or disorders associated with muscle atrophy or muscle wasting. Muscle atrophy or muscle wasting can be observed in association with various diseases and conditions described herein, such as neuromuscular disorders, or can be caused directly or indirectly by prolonged inactivity, bed rest, hospitalization, aging, malnutrition, cancer cachexia, chronic inflammatory diseases, etc. Examples of chronic inflammatory diseases include rheumatoid arthritis, chronic heart failure, and chronic obstructive pulmonary disease (COPD).

[0013] The duration of hospitalization and the type and severity of illness can affect the degree of muscle wasting in subjects, and muscle wasting is common in patients suffering from sepsis, organ failure, hyperglycemia, and diseases associated with chronic and systemic inflammation or oxidative stress. Additionally, hospitalization requiring complete immobilization / bed rest significantly contributes to muscle wasting.

[0014] Additional disorders associated with muscle atrophy / wasting include disorders associated with reduced mobility, such as rheumatoid arthritis, osteoarthritis, and injury. Powers et al. (2016). Thus, in some embodiments, the present disclosure provides techniques for preventing / treating muscle wasting or atrophy associated with or resulting from many of the diseases or conditions described herein.

[0015] In some embodiments, the methods of the present invention can also be used when a subject needs muscle regeneration and enhanced muscle growth after surgery, trauma, and / or prolonged immobilization (e.g., resulting from bed rest or casting). Muscle stem cell activity is known to decrease with age, and the methods of the present invention can also be used to prevent or reverse sarcopenia in otherwise healthy patients, resulting in significant improvements in quality of life and autonomy.

[0016] Embodiments of the present disclosure also provide methods of preventing or treating muscle fibrosis, e.g., in a subject in need thereof, by administering a composition that downregulates MuSK Ig3 domain protein expression, MuSK Ig3 domain gene expression, and / or MuSK Ig3 activation of BMP signaling, thereby preventing or reducing the accumulation of extracellular matrix in the extracellular space of muscle. The composition includes a MuSK Ig3 target blocking antibody, ... These may include Ig3-targeted exon skipping oligonucleotides, MuSK Ig3-targeted CRISPR / Cas9, MuSK Ig3-targeted siRNA, MuSK Ig3-targeted small molecules, and / or MuSK Ig3-targeted shRNA.

[0017] The subject may be at risk of or suffer from muscle fibrosis caused by disease or disorder, including but not limited to trauma, genetic disease, muscle disorder and aging.Trauma may be caused by, for example, radiation therapy, crush, laceration and amputation.Genetic disease or muscle disorder includes but is not limited to congenital muscular dystrophy, Duchenne muscular dystrophy, Becker muscular dystrophy, amyotrophic lateral sclerosis (ALS) and aging-related sarcopenia.

[0018] In some embodiments, the present disclosure provides a model system that can be used to screen, verify, characterize, assess, and / or identify agents that downregulate MuSK Ig3 domain protein expression, MuSK Ig3 domain gene expression, and / or MuSK Ig3 activation of BMP signaling, and prevent or reduce the accumulation of extracellular matrix in the extracellular space of muscle.In some embodiments, the model system is an artificially engineered cell line.In some embodiments, the model system is a genetically engineered mouse model.In some embodiments, the mouse model comprises a ΔIg3-MuSK mouse.

[0019] For illustrative purposes, certain embodiments of the invention are shown in the drawings described below. Like numerals in the drawings represent like elements throughout. It should be understood, however, that the invention is not limited to the precise arrangements, dimensions, and equipment shown. [Brief explanation of the drawings]

[0020] [Figure 1] Figure 1 illustrates MuSK expression in satellite cells. Figure 1A shows protein expression markers for satellite cells. Figure 1B shows the structures of full-length MuSK and ΔIg3-MuSK. Figure 1C shows immunohistochemistry (IHC) of isolated, intact WT myofibrils stained immediately (quiescent) or after 24 hours in culture (activated). These IHC data demonstrate that activated, but not quiescent, satellite cells express detectable MuSK. ΔIg3-MuSK satellite cells have a similar expression pattern (not shown). [Figure 2] Figure 1 shows the structures of full-length MuSK and ΔIg3-MuSK in association with the BMP receptor. The Ig3 domain is required for high-affinity BMP binding. ΔIg3-MuSK reduces MuSK-BMP activity, and muscle regeneration and growth are accelerated in ΔIg3-MuSK-expressing muscles. [Figure 3] Figure 1 shows Wnt11 transcription in primary myotubes from neonatal mice treated with or without 25 ng / mL BMP4 for 2 hours. MuSK-dependent BMP4-induced Wnt11 transcription was reduced in ΔIg3-MuSK myotubes as determined by qRT-PCR. These data represent a 5.8-fold increase compared to a 2.6-fold increase in cultures with n=6 replicates in two separate experiments. T-test, p=0.0007. [Figure 4]Figure 4A illustrates the characterization of satellite cells (SCs) in uninjured muscle and regenerating muscle at 5, 7, and 14 days post-injury (dpi). Figure 4A provides a schematic of the experimental protocol. Five- to eight-month-old female tibialis anterior muscles were injured. 5-ethynyl-2'-deoxyuridine (EdU) was administered via intraperitoneal (ip) injection at 4, 6, or 13 dpi, and muscle tissue was harvested at 5, 7, or 14 dpi, respectively. Figure 4B and Figure 4C show representative images of muscles from uninjured, wild-type, and ΔIg3-MuSK mice at 5 dpi, 7 dpi, or 14 dpi, stained with hematoxylin and eosin (H&E; Figure 4B) or antibodies against the extracellular matrix proteins laminin (green) or Pax7 (red) (Figure 4C). Figures 4B and 4C show that there was an increased density of Pax7+ satellite cells in ΔIg3-MuSK mice at 5 dpi. [Figure 5] Figure 5 illustrates the characterization of satellite cells (SCs) in uninjured muscle and regenerating muscle at 5, 7, and 14 days post-injury (dpi). Figure 5A shows that ΔIg3-MuSK SCs significantly increased satellite cell density at 5 dpi. SC mass per cross-sectional area was calculated blindly in contralateral control and injured TA muscles at 5, 7, and 14 dpi (n = 4 mice, approximately 5,000,000 μm counted per mouse, t-test, p = 0.02). Figure 5B shows that ΔIg3-MuSK SCs significantly increased satellite cell proliferation at 5 dpi. The percentage of EdU+ SCs was calculated blindly in injured tibialis anterior muscles at 5, 7, and 14 dpi (n = 4, t-test, p = 0.04). All analyses were performed blindly. EdU was administered 24 h before harvest. [Figure 6] Figure 1 shows the minimum ferret diameter in wild-type and ΔIg3-MuSK muscles collected 7 days after BaCl2 injury in 5-month-old animals. Mann-Whitney t-test: p<0.0001, n=4 mice, 1000-2000 total fibers analyzed per group. Myofibril area was greater in ΔIg3-MuSK mice compared to wild-type mice at 7 dpi, indicating that muscle regeneration is accelerated in ΔIg3-MuSK mice. [Figure 7] Figure 1 shows the generation of the ΔIg3-MuSK mouse model. A mouse model lacking the MuSK Ig3 domain was generated using CRISPR / Cas9. A plasmid encoding hSpCas9 and a gRNA flanking the locus encoding the Ig3 domain (black triangle) was designed to excise an approximately 11 kb region, thereby generating a novel MuSK allele (MuSKΔIg3) lacking the Ig3 coding domain. Arrows indicate PCR primers designed to amplify either WT or MuSKΔIg3. Founder mice homozygous for MuSKΔIg3 were selected by genotyping and confirmed by DNA sequencing. Amplification of genomic DNA of the WT and MuSKΔIg3 alleles by PCR produces 436- and 400-bp amplicons, respectively. WT mice have the WT MuSK allele but lack MuSKΔIg3. Heterozygous MuSKΔIg3 mice carry both WT and MuSKΔIg3 alleles evidenced by 436 and 400 bp products, respectively, whereas MuSKΔIg3 homozygotes amplify only the 436 bp MuSKΔIg3 allele. [Figure 8] Figure 1 shows histograms of myofibril size, as measured by minimum ferret diameter, in WT and ΔIg3-MuSK mice. Fiber size classification performed on muscles from 3-month-old mice shows no difference in average size or size distribution between WT and ΔIg3-MuSK myofibrils. Fiber size classification performed on 5-month-old mice shows increased myofibril size in ΔIg3-MuSK mice. This indicates increased muscle growth between 3 and 5 months in ΔIg3-MuSK mice. Mann-Whitney t-test: p<0.0001, n=3-4 mice, 500 total fibers analyzed per mouse. [Figure 9]Figure 1 shows the number of satellite cells in uninjured ΔIg3-MuSK and WT mice at 3 and 5 months. Immunohistochemistry on muscle sections was used to determine the number of satellite cells, as measured by Pax7+ cells per region. In 3-month-old animals, the number of satellite cells is comparable between WT and ΔIg3-MuSK muscles. At 5 months, the amount of SCs in WT muscle remains unchanged, while the amount of satellite cells in Ig3-MuSK muscle has declined by approximately half. Each dot represents an animal. This indicates that the number of SCs in ΔIg3-MuSK mice is reduced between 3 and 5 months. Unpaired t-test, n = 8 mice, p = 0.01. DETAILED DESCRIPTION OF THE INVENTION

[0021] definition For convenience, the meanings of some terms and phrases used in the specification, examples, and appended claims are provided below. Unless otherwise stated or implicit from context, the following terms and phrases include the meanings provided below.

[0022] These definitions are provided to aid in describing particular embodiments and are not intended to limit the claimed invention, as the scope of the invention is limited only by the claims.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided herein shall prevail.

[0024] About or approximately: The term "about" or "approximately," when used herein in connection with a value, refers to a value that is comparable in the context of a reference value. Generally, a person skilled in the art who is familiar with the context will understand the relevant degree of variation encompassed by "about" in that context. For example, in some embodiments, the term "about" can encompass a range of values ​​that are within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the reference value.

[0025] Administration: As used herein, the term "administration" typically refers to the administration of a composition to a subject or system to achieve delivery of, for example, an agent (e.g., an agonizing agent) that is, is contained in, or is delivered by the composition. Those of skill in the art will be aware of the various routes that may be utilized for administration to a subject, e.g., a human, in appropriate circumstances. For example, in some embodiments, administration may be ophthalmic, oral, buccal, cutaneous (which may be or include one or more of, e.g., topical to the dermis, intradermal, interdermal, transdermal, etc.), intestinal, intraarterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, intraspecific organ (e.g., intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal instillation), vaginal, intravitreal, etc. In some embodiments, the agent (e.g., an agonizing agent) is delivered to the central nervous system (CNS), e.g., via intraventricular administration. In some embodiments, administration may involve only a single dose. In some embodiments, administration may involve the application of a fixed number of doses. In some embodiments, administration may involve dosing that is intermittent (e.g., multiple doses spaced apart in time) and / or periodic (e.g., individual doses separated by a common period) dosing. In some embodiments, administration may involve continuous dosing (e.g., perfusion) for at least a selected period of time.

[0026] Agent: Generally, the term "agent" as used herein can be used to refer to any chemical class of compound or entity, including, for example, polypeptides, nucleic acids, saccharides, lipids, small molecules, metals, or combinations or complexes thereof. In appropriate circumstances, as will be clear from the context to those skilled in the art, the term can be used to refer to an entity that is or includes a cell or organism, or a fraction, extract, or component thereof. Alternatively or additionally, as will be clear from the context, the term can be used to refer to a natural product in that it is found in nature and / or obtained from nature. In some cases, again as will be clear from the context, the term can be used to refer to one or more entities that are artificial in that they are designed, engineered, and / or produced through the action of human hands and / or are not found in nature. In some embodiments, the agent can be used in an isolated or pure form; in some embodiments, the agent can be used in a crude form. In some embodiments, potential agents can be provided as a collection or library, which can be screened, for example, to identify or characterize active agents therein. In some cases, the term "agent" can refer to a compound or entity that is or includes a polymer; in other cases, the term can refer to a compound or entity that includes one or more polymer moieties. In some embodiments, the term "agent" can refer to a compound or entity that is not a polymer and / or that is substantially free of any polymer and / or one or more specific polymer moieties. In some embodiments, the term can refer to a compound or entity that lacks or is substantially free of any polymer moieties.

[0027] Agonist: Those skilled in the art will understand that the term "agonist" can be used to refer to an agent (i.e., an "agonizing agent"), condition, or event whose presence, level, degree, type, or form correlates with an increase in the level or activity of another agent (i.e., an agonized agent or target agent). Generally, an agonist can be or include any chemical class of agent, including, for example, small molecules, polypeptides, nucleic acids, carbohydrates, lipids, metals, and / or any other entity, that exhibits a related activation activity. In some embodiments, an agonist can be direct (in which case it exerts its effect directly on its target); in some embodiments, an agonist can be indirect (in which case it exerts its effect other than by binding to its target, for example, by interacting with a regulator of the target, thereby altering the level or activity of the target). In some embodiments, an agonist is a binding agent that is a protein (e.g., an antibody) or a nucleic acid (e.g., an antisense oligonucleotide) that binds to a target (e.g., a protein or nucleic acid), thereby altering the level, form, and / or activity of the target. In some embodiments, the altered level, form, and / or activity is an increase in the level of an altered protein expressed from the target nucleic acid sequence. Those skilled in the art will understand, upon reading this disclosure, that in some embodiments, an agonizing agent can bind to (and potentially agonize) a binding target, and that binding causes an increase in the level or activity of an additional agonized target. To provide a specific example, in some embodiments, an agonizing agent that binds to a nucleic acid target can alter the level and / or activity of that target, and in some specific embodiments, agonize the activity of that nucleic acid target (e.g., by increasing its modification, splicing, 5'-capping and / or 3'-end formation, transport, and / or translation, etc., thereby increasing the level of a desired product, e.g., mRNA), and / or agonize a downstream target, such as a polypeptide encoded by such nucleic acid target.To give one particular such example, in some embodiments, an agonizing agent may be or may include an oligonucleotide that binds to a primary transcript and alters its splicing pattern, thereby increasing the level and / or activity of a particular splice form (e.g., mature mRNA), which in turn may achieve an increase in the level of a product (e.g., a polypeptide) that is or is encoded by such a particular splice form.

[0028] Agonist therapy: As used herein, the term "agonist therapy" refers to the administration of an agonist that agonizes a specific target of interest to achieve a desired therapeutic effect. In some embodiments, agonist therapy involves administering a single dose of an agonist. In some embodiments, agonist therapy involves administering multiple doses of an agonist. In some embodiments, agonist therapy involves administering an agonist according to a dosing regimen known or expected to achieve a therapeutic effect because, for example, such results have been established to a specified degree of statistical confidence, for example, through administration to a relevant population. In some embodiments, agonist therapy involves the delivery of an agonizing agent described herein. As noted above, in some embodiments, the agonizing agent may be or may include a binding agent that is a protein (e.g., an antibody) or a nucleic acid (e.g., an antisense oligonucleotide) that binds to a target (e.g., a protein or nucleic acid) and thereby alters the level, form, and / or activity of the target. In some embodiments, an agonizing agent may bind to (and potentially agonize) a binding target, and binding causes an increase in the level or activity of a further agonized target. To provide a specific example, in some embodiments, an agonizing agent that binds to a nucleic acid target may alter the level and / or activity of that target, and in some specific embodiments, may agonize the activity of that nucleic acid target (e.g., by increasing its modification, splicing, 5'-capping and / or 3'-end formation, transport, and / or translation, etc., thereby producing the level of a desired product, e.g., mRNA), and / or agonize a downstream target, such as a polypeptide encoded by such nucleic acid target.To give one particular such example, in some embodiments, an agonizing agent may be or may include an oligonucleotide that binds to a primary transcript and alters its splicing pattern, thereby generating the level and / or activity of a particular splice form (e.g., mature mRNA), which in turn may achieve an increased level of a product (e.g., a polypeptide) that is or is encoded by such a particular splice form.

[0029] Antagonist: Those skilled in the art will understand that the term "antagonist," as used herein, can be used to refer to an agent (i.e., an "antagonizing agent"), condition, or event whose presence, level, degree, type, or form correlates with a decrease in the level or activity of another agent (i.e., an inhibited agent, or target). Generally, an antagonist can be or include any chemical class of agent, including, for example, small molecules, polypeptides, nucleic acids, carbohydrates, lipids, metals, and / or any other entity, that exhibits relevant inhibitory activity. In some embodiments, an antagonist can be direct (in which case it exerts its effect directly on its target); in some embodiments, an antagonist can be indirect (in which case it exerts its effect other than by binding to its target, for example, by interacting with a modulator of the target, thereby altering the level or activity of the target). In some embodiments, an antagonist is a binding agent that is a protein (e.g., an antibody) or a nucleic acid (e.g., an antisense oligonucleotide) that binds to a target (e.g., a protein or nucleic acid), thereby altering the level, form, and / or activity of the target. In some embodiments, the alteration in level, form, and / or activity is a decrease in the level of the altered protein expressed from the target nucleic acid sequence. Those skilled in the art will understand, upon reading this disclosure, that in some embodiments, an antagonistic agent can bind to (and potentially antagonize) a binding target, and that binding causes a decrease in the level or activity of the additional target that is antagonized. To provide a specific example, in some embodiments, an antagonistic agent that binds to a nucleic acid target may alter the level and / or activity of that target, and in some specific embodiments, may antagonize the activity of that nucleic acid target (e.g., by reducing its modification, splicing, 5' capping and / or 3' end formation, transport, and / or translation, etc., thereby suppressing the level of undesired products, e.g., mRNA), and / or antagonize downstream targets, such as polypeptides, encoded by such nucleic acid target.To give one particular such example, in some embodiments, the antagonistic agent may be or may include an oligonucleotide that binds to a primary transcript and alters its splicing pattern, thereby suppressing the level and / or activity of a particular splice form (e.g., mature mRNA), which in turn may achieve a reduction in the level of a product (e.g., a polypeptide) that is or is encoded by such a particular splice form.

[0030] Antibody agent: As used herein, the term "antibody agent" refers to an agent that specifically binds to a particular antigen (e.g., which may be or may include an epitope of a protein of interest, e.g., a MuSK protein). In some embodiments, the term encompasses any polypeptide or polypeptide complex that includes sufficient immunoglobulin structural elements to confer specific binding. Exemplary antibody agents include, but are not limited to, monoclonal or polyclonal antibodies. In some embodiments, an antibody agent may include one or more constant region sequences characteristic of murine, rabbit, primate, or human antibodies. In some embodiments, an antibody agent may include one or more sequence elements that are humanized, primatized, chimeric, etc., as known in the art. In many embodiments, the term "antibody agent" is used to refer to one or more of the constructs or formats known or developed in the art for utilizing the structural and functional attributes of antibodies in alternative presentations.For example, in embodiments, antibody agents utilized in accordance with the present invention include intact IgA, IgG, IgE, or IgM antibodies; bispecific or multispecific (e.g., Zybodies®, etc.); antibody fragments such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, and isolated CDRs or sets thereof; single chain Fv; polypeptide-Fc fusions; single domain antibodies (e.g., shark single domain antibodies or fragments thereof, such as IgNAR); cameloid antibodies; masked antibodies (e.g., Probodies®); small modular immunopharmaceuticals ("SMIPs™"); single chain or in a format selected from, but not limited to, tandem diabodies (TandAb®); VHH; Anticalins®; Nanobodies® minibodies; BiTEs®; ankyrin repeat proteins or DARPINs®; Avimers®; DARTs; TCR-like antibodies; Adnectins®; Affilins®; Trans-bodies®; Affibodies®; TrimerX®; microproteins; Fynomers®, Centyrins®; and KALBITOR®. In some embodiments, the antibody may lack covalent modifications (e.g., glycan attachments) that it would have if produced naturally. In some embodiments, antibodies may contain covalent modifications (e.g., glycan attachments, payloads (e.g., detectable moieties, therapeutic moieties, catalytic moieties, etc.), or other pendant groups (e.g., polyethylene glycol, etc.). In many embodiments, an antibody agent is or comprises a polypeptide whose amino acid sequence includes one or more structural elements recognized by those of skill in the art as complementarity-determining regions (CDRs); in some embodiments, an antibody agent is or comprises a polypeptide whose amino acid sequence includes at least one CDR (e.g., at least one heavy chain CDR and / or at least one light chain CDR) that is substantially identical to that found in a reference antibody.In some embodiments, the included CDR is substantially identical to the reference CDR in that it is sequence identical or contains 1 to 5 amino acid substitutions compared to the reference CDR. In some embodiments, the included CDR is substantially identical to the reference CDR in that it exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments, the included CDR is substantially identical to the reference CDR in that it exhibits at least 96%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that at least one amino acid within the included CDRs has been deleted, added, or substituted relative to the reference CDR, but the included CDRs otherwise have an amino acid sequence identical to that of the reference CDR. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that 1 to 5 amino acids within the included CDRs have been deleted, added, or substituted relative to the reference CDR, but the included CDRs otherwise have an amino acid sequence identical to that of the reference CDR. In some embodiments, the included CDRs are substantially identical to the reference CDRs in that at least one amino acid within the included CDRs has been substituted relative to the reference CDR, but the included CDRs otherwise have an amino acid sequence identical to that of the reference CDR. In some embodiments, the included CDR is substantially identical to the reference CDR in that 1 to 5 amino acids within the included CDR have been deleted, added, or substituted relative to the reference CDR, but the included CDR has an amino acid sequence that is otherwise identical to the reference CDR. In some embodiments, the antibody agent is or comprises a polypeptide whose amino acid sequence includes structural elements recognized by those skilled in the art as an immunoglobulin variable domain.In some embodiments, an antibody agent is a polypeptide protein having a binding domain that is homologous or largely homologous to an immunoglobulin binding domain.

[0031] Antibody: As used herein, the term "antibody" refers to an immunoglobulin or derivative thereof that contains an immunoglobulin domain capable of binding to an antigen (e.g., which may be or may include an epitope of a protein of interest, e.g., a MuSK protein). The antibody may be of any species, e.g., human, rodent, rabbit, goat, chicken, etc. The antibody may be a member of any immunoglobulin class, including the human classes: IgG, IgM, IgA, IgD, and IgE, or any of their subclasses, such as IgG1, IgG2, etc. In various embodiments of the invention, the antibody is a fragment such as Fab', F(ab')2, scFv (single-chain variable), or other fragment that retains the antigen-binding site, or a recombinantly produced scFv fragment, including a recombinantly produced fragment. See, e.g., Allen (2002) and references therein. The antibody may be monovalent, bivalent, or multivalent. An antibody can be a chimeric or "humanized" antibody, for example, in which a variable domain of rodent origin is fused with a constant domain of human origin, thus retaining the specificity of the rodent antibody. A domain of human origin need not be directly human, in the sense that it is first synthesized in humans. Instead, a "human" domain can be generated in a rodent whose genome incorporates human immunoglobulin genes. See, e.g., Vaughan et al. (1998). An antibody can be partially or fully humanized. An antibody can be polyclonal or monoclonal, although for purposes of the present invention, monoclonal antibodies are generally preferred. Methods for producing antibodies that specifically bind to virtually any molecule of interest are known in the art. For example, monoclonal or polyclonal antibodies can be purified from the blood or ascites of an animal that produces the antibody (e.g., after natural exposure to or immunization with the molecule or its antigenic fragment), produced using recombinant techniques in cell culture or transgenic organisms, or made at least in part by chemical synthesis. In some embodiments, antibodies may act as antagonists, for example, by binding to a target antigen, resulting in a decrease in the level or activity of said antigen.In some embodiments, antibodies can act as agonists, for example, by binding to a target antigen, resulting in an increase in the level or activity of said antigen.

[0032] Antisense: The term "antisense" is used herein to refer to a nucleic acid whose nucleotide sequence is complementary to part or all of a sequence found in a coding strand nucleic acid. Typically, a "coding strand" nucleic acid is one whose sequence includes part or all of an open reading frame or other stretch of residues that encodes part or all of a polypeptide. In some embodiments, the term "antisense" may be used herein specifically with reference to an oligonucleotide that specifically binds to a coding strand (i.e., to a target sequence within such a coding strand). In some embodiments, the coding strand may include both coding and non-coding sequences (e.g., may be a transcription product, such as a primary transcript containing both intron and exon sequences, to give just one example). Those skilled in the art will understand, upon reading this disclosure, that in some embodiments, an oligonucleotide may be considered or referred to as an "antisense" oligonucleotide if part or all of its sequence is complementary to a non-coding portion of its target strand. In some embodiments, an antisense oligonucleotide binds to a coding sequence in a target sense strand; in some embodiments, an antisense oligonucleotide binds to a non-coding sequence in a target coding sequence. In some embodiments, antisense oligonucleotides bind to both coding and non-coding sequences in the target coding strand. In some embodiments, antisense oligonucleotides are characterized by altering the post-transcriptional processing (e.g., one or more of modification, splicing, 5'-capping and / or 3'-end formation, 5'-capping and / or 3'-end formation, transport, and / or translation) of the coding strand when bound to its target sequence in the coding strand (e.g., transcript). In certain embodiments, antisense oligonucleotides alter the splicing of their target coding strand. Alternatively or additionally, in some embodiments, the antisense-coding strand complex is degraded or can be degraded, for example, by RNase H.

[0033] Approximately: As used herein, the term "approximately" or "about" in connection with a number is generally taken to include numbers that fall within 5%, 10%, 15%, or 20% in either direction (greater or lesser) of the number (except in cases where such number would be less than 0% or greater than 100% of the possible value) unless otherwise stated or otherwise clear from the context.

[0034] Binding agent: In general, the term "binding agent" is used herein to refer to any entity that binds to a target of interest as described herein. In many embodiments, a binding agent of interest is one that specifically binds to its target, in that it distinguishes between that target and other potential binding partners in a particular interaction context. Generally, a binding agent can be or include any chemical class of entity (e.g., polymers, non-polymers, small molecules, polypeptides, carbohydrates, lipids, nucleic acids, etc.). In some embodiments, a binding agent is a single chemical entity. In some embodiments, a binding agent is a complex of two or more individual chemical entities associated with each other under relevant conditions through non-covalent interactions. For example, one of skill in the art will understand that in some embodiments, a binding agent can include a "generic" binding moiety (e.g., one of biotin / avidin / streptavidin and / or a class-specific antibody) and a "specific" binding moiety (e.g., an antibody or aptamer with a specific molecular target) linked to a partner of the generic binding moiety. In some embodiments, such approaches may allow for modular assembly of multiple binding agents through linkage of different specific binding moieties with the same generic binding moiety partner. In some embodiments, the binding agent is or comprises a polypeptide (e.g., including an antibody or antibody fragment). In some embodiments, the binding agent is or comprises a small molecule. In some embodiments, the binding agent is or comprises a nucleic acid (e.g., an antisense oligonucleotide). In some embodiments, the binding agent is an aptamer. In some embodiments, the binding agent is a polymer; in some embodiments, the binding agent is not a polymer. In some embodiments, the binding agent is non-polymeric in that they lack a polymer moiety. In some embodiments, the binding agent is or comprises a carbohydrate. In some embodiments, the binding agent is or comprises a lectin. In some embodiments, the binding agent is or comprises a peptidomimetic.In some embodiments, the binding agent is or comprises a scaffold protein. In some embodiments, the binding agent is or comprises a mimotope. In some embodiments, the binding agent is or comprises a staple peptide. In certain embodiments, the binding agent is or comprises a nucleic acid such as DNA or RNA (e.g., an antisense oligonucleotide).

[0035] Characteristic sequence element: As used herein, the phrase "characteristic sequence element" refers to a sequence element found in a polymer (e.g., a polypeptide or nucleic acid) that represents a characteristic portion of that polymer. In some embodiments, the presence of a characteristic sequence element correlates with the presence or level of a particular activity or property of the polymer. In some embodiments, the presence (or absence) of a characteristic sequence element defines a particular polymer as a member (or not) of a particular family or group of such polymers. A characteristic sequence element typically comprises at least two monomers (e.g., amino acids or nucleotides). In some embodiments, a characteristic sequence element comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, or more monomers (e.g., sequentially linked monomers). In some embodiments, a characteristic sequence element comprises at least first and second stretches of adjacent monomers spaced apart by one or more spacer regions, which may or may not vary in length among polymers that share the sequence element.

[0036] Complementary: As used herein, "complementary," according to its art-recognized meaning, refers to the ability for precise pairing between specific bases, nucleosides, nucleotides, or nucleic acids. For example, adenine (A) and uridine (U) are complementary; adenine (A) and thymidine (T) are complementary; and guanine (G) and cytosine (C) are complementary, referred to in the art as Watson-Crick base pairing. If a nucleotide at a particular location in a first nucleic acid sequence is complementary to a nucleotide located opposite it in a second nucleic acid sequence when the strands are aligned in an antiparallel orientation, the nucleotides form a complementary base pair and the nucleic acids are complementary at that location. The percent complementarity of a first nucleic acid to a second nucleic acid can be estimated by aligning them in an antiparallel orientation for maximum complementarity over a window of evaluation, determining the total number of nts in both strands that form complementary base pairs within the window, dividing by the total number of nts in the window, and multiplying by 100. For example, AAAAAAAA and TTTGTTAT are 75% complementary because 12 of the 16 total nt are in complementary base pairs. When calculating the number of complementary nts required to achieve a specific percentage, the fraction is rounded to the nearest integer. A position occupied by a non-complementary nucleotide constitutes a mismatch, i.e., the position is occupied by a non-complementary base pair. In certain embodiments, the evaluation window has a length as described herein for the duplex portion or target portion. A complementary sequence involves base pairing of a polynucleotide comprising a first nucleotide sequence to a polynucleotide comprising a second nucleotide sequence over the entire length of both nucleotide sequences (if the sequences are the same length) or over the entire length of the shorter sequence (if the sequences are different lengths). Such sequences may be referred to herein as "perfectly complementary" (100% complementary) to each other. Nucleic acids that are at least 70% complementary over an evaluation window are considered "substantially complementary" over that window.In certain embodiments, complementary nucleic acids are at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% complementary throughout the evaluation window.When a first sequence is referred to herein as "substantially complementary" to a second sequence, the two sequences can be perfectly complementary, or they can contain one or more mismatched bases upon hybridization, for example, up to about 5%, 10%, 15%, 20%, or 25% mismatched bases upon hybridization, for example, 1, 2, 3, 4, 5, or 6 mismatched base pairs upon hybridization for a duplex of up to 30 base pairs, while retaining the ability to hybridize under the conditions most relevant to their intended use.When two oligonucleotides are designed to form one or more single-stranded overhangs upon hybridization, it should be understood that such overhangs are not considered as mismatched or unpaired nucleotides when determining the percentage of complementarity. For example, two strands of dsRNA, one 21-nucleotide long oligonucleotide and the other 23-nucleotide long oligonucleotide, in which the longer oligonucleotide is perfectly complementary to the shorter oligonucleotide and the two strands contain a two-nucleotide overhang, may be referred to herein as "perfectly complementary." As used herein, "complementary" sequences may contain one or more non-Watson-Crick base pairs and / or base pairs formed from unnatural and other modified nucleotides, so long as the requirements for their ability to hybridize are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobble or Hoogsteen base pairing. Those skilled in the art know that guanine, cytosine, adenine, and uracil can be substituted by other bases according to the so-called "wobble" rules without substantially altering the base-pairing properties of polynucleotides containing nucleotides with such bases. See, for example, Murphy and Ramakrishnan (2004).For example, a nucleotide containing inosine as its base can base pair with a nucleotide containing adenine, cytosine, or uracil. Thus, a nucleotide containing uracil, guanine, or adenine can be replaced by a nucleotide containing inosine, for example, in the nucleotide sequence of the inhibitory RNA described herein. It will be understood that the terms "complementary," "perfectly complementary," and "substantially complementary" can be used to refer to the base matching between any two nucleic acids, for example, the base matching between the sense strand and the antisense strand of a double-stranded nucleic acid, or between parts thereof. As used herein, "hybridizing" refers to the interaction between two nucleic acid sequences (which in some embodiments may be part of the same nucleic acid molecule, and in other embodiments may be part of or comprise different nucleic acid molecules) that contain or consist of complementary portions, so as to form a duplex structure (i.e., an intramolecular or intermolecular duplex) that is stable under specific conditions of interest, as will be understood by those skilled in the art.

[0037] Comprises: The term "comprises" means that other elements may be present in addition to the specified elements that are present. The use of "comprises" indicates inclusion rather than limitation.

[0038] Consisting of: The term "consisting of" refers to the compositions, methods, and their respective components described herein, excluding any elements not recited in that description of an embodiment. As used herein, the term "consisting essentially of" refers to those elements required for a given embodiment. This term permits the presence of additional elements that do not materially affect the basic and novel characteristics or functional characteristics of the embodiment of the invention.

[0039] Combination therapy: As used herein, the term "combination therapy" refers to a condition in which a subject is simultaneously exposed to two or more therapeutic regimens (e.g., two or more therapeutic agents). In some embodiments, two or more regimens can be administered simultaneously; in some embodiments, such regimens can be administered sequentially (e.g., all "doses" of a first regimen are administered before any doses of a second regimen are administered); in some embodiments, such agents are administered in overlapping dosing regimens. In some embodiments, the "administration" of a combination therapy can involve the administration of one or more agents or modalities in combination to a subject receiving another agent or modality. For clarity, combination therapy does not require that individual agents be administered together in a single composition (or even necessarily at the same time), although in some embodiments, two or more agents or active portions thereof can be administered together in a combination composition or even a combination compound (e.g., as part of a single chemical complex or covalent entity).

[0040] Comparable: As used herein, the term "comparable" refers to two or more agents, entities, states, sets of conditions, etc. that are not necessarily identical to one another but are sufficiently similar to permit a comparison between them, such that a person of ordinary skill in the art would understand that conclusions could be reasonably drawn based on observed differences or similarities. In some embodiments, comparable sets of conditions, circumstances, individuals, or populations are characterized by multiple substantially identical traits and one or a few variable traits. A person of ordinary skill in the art will understand the degree of identity required in any given situation for two or more such agents, entities, states, sets of conditions, etc. to be considered comparable in context. For example, a person of ordinary skill in the art will understand that sets of circumstances, individuals, or populations are comparable to one another when they are characterized by a sufficient number and type of substantially identical traits to justify a reasonable conclusion that differences in results obtained or phenomena observed under or with the various sets of circumstances, individuals, or populations are caused by or indicate variations in those variable traits.

[0041] Domain: As used herein, the term "domain" refers to a section or portion of an entity. In some embodiments, a "domain" is associated with a particular structural and / or functional attribute of an entity, such that when the domain is physically separated from the remainder of its parent entity, it substantially or completely retains the particular structural and / or functional attribute. Alternatively or additionally, a domain may be or comprise a portion of an entity that, when separated from its (parent) entity and linked to a different (recipient) entity, substantially retains and / or imparts to the recipient entity one or more structural and / or functional attributes that characterized it in the parent entity. In some embodiments, a domain is a section or portion of a molecule (e.g., a small molecule, carbohydrate, lipid, nucleic acid, or polypeptide). In some embodiments, a domain is a section of a polypeptide (e.g., the Ig3 domain of a MuSK protein); in some such embodiments, a domain is characterized by particular structural elements (e.g., particular amino acid sequences or sequence motifs, a-helical characteristics, b-sheet characteristics, coiled-coil characteristics, random coil characteristics, etc.) and / or by particular functional attributes (e.g., binding activity, enzymatic activity, folding activity, signaling activity, etc.).

[0042] Dosage regimen: Those skilled in the art will understand that the term "dosage regimen" can be used to refer to a set of unit doses (typically more than one) administered individually to a subject, typically separated by a period of time. In some embodiments, a given therapeutic agent has a recommended dosing regimen that can involve one or more doses. In some embodiments, a dosing regimen includes multiple doses, each separated in time from the other doses. In some embodiments, the individual doses are separated from each other by the same length of time; in some embodiments, a dosing regimen includes multiple doses and at least two different periods separating the individual doses. In some embodiments, all doses within a dosing regimen are of the same unit dosage amount. In some embodiments, different doses within a dosing regimen are of different amounts. In some embodiments, a dosing regimen includes a first dose at a first dosage amount, followed by one or more additional doses at a second dosage amount different from the first dosage amount. In some embodiments, the dosing regimen comprises a first dosing at a first dosage amount, followed by one or more additional dosings at a second dosage amount that is the same as the first dosage amount. In some embodiments, the dosing regimen correlates with a desired or beneficial outcome when administered across a relevant population (i.e., is a therapeutic dosing regimen).

[0043] Expression: As used herein, "expression" of a nucleic acid sequence refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of the RNA transcript (e.g., by splicing, editing, 5' capping, and / or 3' end formation); (3) transport of the RNA transcript (e.g., from the nucleus to the cytoplasm); and / or (4) translation of the RNA into a polypeptide or protein; and / or (4) post-translational modification of the polypeptide or protein.

[0044] Isolated or partially purified: As used herein, the term "isolated" or "partially purified," in the case of a nucleic acid or polynucleotide, refers to a nucleic acid or polypeptide that has been separated from at least one other component (e.g., nucleic acid or polypeptide) that is present with the nucleic acid or polypeptide in its natural source and / or that would be present with the nucleic acid or polypeptide when expressed or, in the case of a secreted polypeptide, secreted by a cell. Chemically synthesized nucleic acids or polypeptides, or those synthesized using in vitro transcription / translation, are considered "isolated." The term "purified" or "substantially purified" refers to an isolated nucleic acid or polypeptide that is at least 95% by weight of the nucleic acid or polypeptide of interest, including, for example, at least 96%, at least 97%, at least 98%, at least 99%, or more of the nucleic acid or polypeptide of interest. In some embodiments, the antibodies, antigen-binding portions thereof, or chimeric antigen receptors (CARs) described herein are isolated. In some embodiments, the antibodies, antibody reagents, antigen-binding portions thereof, or CARs described herein are purified.

[0045] Engineered: As used herein, "engineered" refers to an aspect that has been manipulated by the hand of man. For example, an antibody, antibody reagent, its antigen-binding portion, CAR, or bispecific antibody is considered to be "engineered" if the sequence of the antibody, antibody reagent, its antigen-binding portion, CAR, or bispecific antibody has been manipulated by the hand of man so that it differs from the sequence of the antibody as it exists in nature. As is common practice and understood by those skilled in the art, progeny and copies of engineered polynucleotides and / or polypeptides are typically still referred to as "engineered," even if the actual manipulation was performed on a previous entity.

[0046] Fragment: A "fragment" of a material or entity described herein comprises a discrete portion of the whole, but has a structure that lacks one or more portions found in the whole. In some embodiments, a fragment consists of such a discrete portion. In some embodiments, a fragment consists of or comprises a characteristic structural element or portion found in the whole. In some embodiments, a polymer fragment comprises or consists of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, or more monomer units (e.g., residues) found throughout the polymer. In some embodiments, a polymer fragment comprises or consists of at least about 5%, 10%, 15%, 20%, 25%, 30%, 25%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more of the monomer units (e.g., residues) found in the whole polymer. The whole material or entity may, in some embodiments, be referred to as the "parent" of the fragment.

[0047] Gene: As used herein, the term "gene" refers to a DNA sequence in a chromosome that encodes a product (e.g., an RNA product and / or a polypeptide product). In some embodiments, a gene comprises coding sequence (i.e., a sequence that encodes a specific product); in some embodiments, a gene comprises non-coding sequence. In certain embodiments, a gene may comprise both coding (e.g., exons) and non-coding (e.g., introns) sequences. In some embodiments, a gene may comprise one or more regulatory elements that may, for example, control or influence one or more aspects of gene expression (e.g., cell-type specific expression, inducible expression, etc.).

[0048] Gene product or expression product: As used herein, the term "gene product" or "expression product" generally refers to the RNA transcribed from a gene (before and / or after processing) or the polypeptide (before and / or after modification) encoded by the RNA transcribed from a gene. In some embodiments, a gene product can be or include a particular processed form of an RNA transcript (e.g., a particular edited form, a particular spliced ​​form, a particular capped form, etc.).

[0049] Homology: As used herein, the term "homology" refers to the overall relatedness between polymer molecules, for example, between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. In some embodiments, polymer molecules are considered to be "homologous" to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. In some embodiments, polymer molecules are considered to be "homologous" to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% similar.

[0050] Identity: As used herein, the term "identity" refers to the overall relatedness between polymer molecules, for example, between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. In some embodiments, polymer molecules are considered to be "substantially identical" to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. Calculation of the percent identity of two nucleic acid or polypeptide sequences can be performed, for example, by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced into one or both of the first and second sequences for optimal alignment, and non-identical sequences can be disregarded for comparison purposes). In certain embodiments, the length of the sequence to be aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of the length of the reference sequence.The nucleotides at corresponding positions are then compared.If a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, the molecules are identical at that position.The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap that needs to be introduced for optimal alignment of the two sequences.The comparison of sequences and the determination of the percent identity between two sequences can be performed using a mathematical algorithm.For example, the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller (1989), which is incorporated into the ALIGN program (version 2.0). In some exemplary embodiments, nucleic acid sequence comparisons performed with the ALIGN program use a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4.The percent identity between two nucleotide sequences can alternatively be determined using the GAP program in the GCG software package using the NWSgapdna.CMP matrix.

[0051] "Improve," "increase," "inhibit," or "reduce": As used herein, the terms "improve," "increase," "inhibit," or "reduce" or grammatical equivalents refer to a value that is relative to a baseline or other reference measurement. In some embodiments, a suitable reference measurement can be or include a measurement in a particular system (e.g., in a single individual, a single cell, or a cell population) under otherwise comparable conditions in the absence of a particular agent or treatment (e.g., before and / or after), or in the presence of a suitable reference agent (e.g., a positive control agent or a negative control agent). In some embodiments, a suitable reference measurement can be or include a measurement in a comparable system known or expected to respond in a particular manner in the presence of a relevant agent or treatment. One of ordinary skill in the art will understand that "improvement," "increase," "reduction," etc. typically refer to a statistically significant change. Moreover, one of ordinary skill in the art will understand from the context what magnitude of change may be relevant. For example, in some embodiments, the change may be a "fold" change, i.e., whereby the "changed" value represents a difference of 1.1, 1.2, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50 or more fold (e.g., 500, 1000 fold) (including all integers and decimals therebetween and above 1, e.g., 1.5, 1.6, 1.7, 1.8, etc.) compared to the relevant reference. Alternatively or additionally, in some embodiments, the "change" may be a "percentage" change, whereby a "changed" value represents an increase or decrease of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% (including all integers and decimal points in between) compared to the relevant reference.

[0052] Linked: As used herein, the term "linked," when used in reference to two or more moieties, means that the moieties are physically associated or connected to one another to form a molecular structure that is sufficiently stable so that the moieties remain associated under the conditions under which the linkage is formed and, preferably, under the conditions under which the new molecular structure is used, e.g., physiological conditions. In certain preferred embodiments of the invention, the linkage is a covalent linkage. In other embodiments, the linkage is non-covalent. Moieties can be linked directly or indirectly. When two moieties are directly linked, they are covalently bonded to one another or are in sufficient proximity that intermolecular forces between the two moieties maintain their association. When two moieties are indirectly linked, they are each covalently or non-covalently linked to a third moiety that maintains the association between the two moieties. Generally, when two moieties are referred to as being linked by a "linker" or "linking moiety" or "linking part," the connection between the two linked moieties is indirect, and typically, each of the linked moieties is covalently bonded to the linker. The linker can be any suitable moiety that will react with and link two moieties within a reasonable period of time, under conditions consistent with the stability of the moieties (which can, depending on the conditions, be protected as appropriate), and in sufficient amounts to result in reasonable yields.

[0053] Internucleotide linkage: As used herein, the phrase "internucleotide linkage" generally refers to a phosphorus-containing linkage between nucleotide units of an oligonucleotide, and is interchangeable with "intersaccharide linkage" and "phosphorus atom bridge" as used above and herein. In some embodiments, the internucleotide linkage is a phosphodiester linkage found in naturally occurring DNA and RNA molecules. In some embodiments, the internucleotide linkage is a "modified internucleotide linkage" in which each oxygen atom of the phosphodiester linkage is optionally and independently replaced by an organic or inorganic moiety. In some embodiments, such organic or inorganic moieties are ═S, ═Se, ═NR′, —SR′, —SeR′, —N(R′), B(R′). 3、In some embodiments, the internucleotide linkages are selected from, but not limited to, -S-, -Se-, and -N(R')-, where each R' is independently as defined and described below. In some embodiments, the internucleotide linkages are selected from, but not limited to, phosphotriester linkages, phosphorothioate diester linkages (

[0054] [ka]

[0055] ), or modified phosphorothioate triester linkages. It will be understood by those skilled in the art that internucleotide linkages can exist as anions or cations at a given pH due to the presence of acidic or basic moieties in the linkage. In some embodiments, the internucleotide linkage can be a chiral linkage.

[0056] Long-term administration: As used herein, the term "long-term" administration means that a therapeutic agent or drug is administered for a period of at least 12 weeks. This includes that a therapeutic agent or drug is administered for a period of at least 12 weeks or to be effective for that period, and does not necessarily mean that the administration itself is carried out for 12 weeks when, for example, a sustained-release composition or a long-acting therapeutic agent or drug is used. Thus, the subject is treated for a period of at least 12 weeks. In many cases, long-term administration is for at least 4, 5, 6, 8, 9 months or more, or for at least 1, 2, 3, 5, 7, or 10 years or more.

[0057] Moiety: Those skilled in the art will understand a "moiety" to be a defined chemical group or entity having a particular structure and / or activity, as described herein.

[0058] Nanoparticle: As used herein, the term "nanoparticle" refers to a particle having a diameter of less than 1000 nanometers (nm). In some embodiments, a nanoparticle has a diameter of less than 300 nm, as defined by the National Science Foundation. In some embodiments, a nanoparticle has a diameter of less than 100 nm, as defined by the National Institutes of Health. In some embodiments, nanoparticles are micelles in that they contain an encapsulated compartment separated from the bulk solution by a micellar membrane, typically composed of amphiphilic entities that surround and enclose a space or compartment (e.g., define a lumen). In some embodiments, the micellar membrane is composed of at least one polymer, such as, for example, a biocompatible and / or biodegradable polymer.

[0059] Nucleic acid: As used herein, in its broadest sense, refers to any compound and / or substance that is or can be incorporated into an oligonucleotide chain. In some embodiments, nucleic acids are compounds and / or substances that are or can be incorporated into an oligonucleotide chain via phosphodiester linkages. As will be clear from the context, in some embodiments, "nucleic acid" refers to individual nucleic acid residues (e.g., nucleotides and / or nucleosides); in some embodiments, "nucleic acid" refers to an oligonucleotide chain comprising individual nucleic acid residues. In some embodiments, "nucleic acid" is or comprises RNA; in some embodiments, "nucleic acid" is or comprises DNA. In some embodiments, a nucleic acid is, comprises, or consists of one or more naturally occurring nucleic acid residues. In some embodiments, a nucleic acid is, comprises, or consists of one or more nucleic acid analogs. In some embodiments, a nucleic acid analog differs from a nucleic acid in that it does not utilize a phosphodiester backbone. For example, in some embodiments, a nucleic acid is, comprises, or consists of one or more "peptide nucleic acids," which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone and are considered within the scope of the present invention. Alternatively or additionally, in some embodiments, the nucleic acid has one or more phosphorothioate and / or 5'-N-phosphoramidite linkages rather than phosphodiester linkages. In some embodiments, the nucleic acid is, comprises, or consists of one or more naturally occurring nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine).In some embodiments, the nucleic acid is, comprises, or consists of one or more nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof). In some embodiments, the nucleic acid comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) compared to those in naturally occurring nucleic acids. In some embodiments, the nucleic acid has a nucleotide sequence that encodes a functional gene product such as RNA or a protein. In some embodiments, the nucleic acid comprises one or more introns. In some embodiments, the nucleic acid is prepared by one or more of isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template (in vivo or in vitro), replication in a recombinant cell or system, and chemical synthesis. In some embodiments, the nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues in length. In some embodiments, the nucleic acid is partially or entirely single-stranded; in some embodiments, the nucleic acid is partially or entirely double-stranded.In some embodiments, the nucleic acid has a nucleotide sequence comprising at least one element that encodes a polypeptide or is the complement of a sequence that encodes a polypeptide. In some embodiments, the nucleic acid has enzymatic activity.

[0060] Prodrug: Generally, a "prodrug," as the term is used herein and as understood in the art, is an entity that, when administered to an organism, is metabolized in the body to deliver an active agent of interest (e.g., a therapeutic or diagnostic agent). Typically, such metabolism involves the removal of at least one "prodrug moiety," thereby forming an active agent. Various forms of "prodrugs" are known in the art. Examples of such prodrug moieties include: a) Design of Prodrugs, edited by H. Bundgaard (Elsevier, 1985) and Methods in Enzymology, 42:309-396, edited by K. Widder et al. (Academic Press, 1985); b)Prodrugs and Targeted Delivery, edited by J. Rautio (Wiley, 2011); c) A Textbook of Drug Design and Development, edited by Krogsgaard-Larsen; d) Bundgaard, Chapter 5, "Design and Application of Prodrugs", H. Bundgaard, pp. 113-191 (1991); e) Bundgaard, Advanced Drug Delivery Reviews, 8:1~38 (1992); f) Bundgaard et al., Journal of Pharmaceutical Sciences, 77:285 (1988); and g) Kakeya et al., Chem. Pharm. Bull., 32:692 (1984) See, e.g., 1999. As with other compounds described herein, the prodrug may be provided in any of a variety of forms, e.g., crystalline form, salt form, etc. In some embodiments, the prodrug is provided as a pharmaceutically acceptable salt thereof.

[0061] Operably linked: As used herein, the term "operably linked" refers to a juxtaposition wherein the described components are in a relationship permitting them to function in their intended manner. A control element "operably linked" to a functional element is associated such that expression and / or activity of the functional element is achieved under conditions compatible with the control element. In some embodiments, an "operably linked" control element (e.g., promoter, enhancer, etc.) is contiguous (e.g., covalently linked) with a coding element of interest; in some embodiments, the control element acts in trans or cis with the functional coding element of interest.

[0062] Patient: As used herein, the term "patient" refers to any organism to which a provided composition (e.g., an agonizing agent such as an ASO) is or can be administered, for example, experimental, diagnostic, prophylactic, cosmetic, and / or therapeutic purposes. Typical patients include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and / or humans). In some embodiments, the patient is human. In some embodiments, the patient is suffering from or prone to one or more disorders or conditions. In some embodiments, the patient exhibits one or more symptoms of a disorder or condition. In some embodiments, the patient has been diagnosed with one or more disorders or conditions. In some embodiments, the disorder or condition is muscular dystrophy or other disease characterized by neuromuscular dysfunction. In some embodiments, the patient is undergoing or is undergoing a particular therapy to diagnose and / or treat the disease, disorder, or condition.

[0063] Pharmaceutical composition: As used herein, the term "pharmaceutical composition" refers to an active agent (e.g., an agonizing agent) formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a unit dosage appropriate for administration in a therapeutic regimen that exhibits a statistically significant likelihood of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, the pharmaceutical composition may be specially formulated for administration in solid or liquid form, including for oral administration, e.g., drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., buccal, sublingual, and those targeted for systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, e.g., by subcutaneous, intramuscular, intravenous, intraperitoneal, intrathecal, intravenous, intracerebroventricular, or epidural injection, e.g., as a sterile solution or suspension, or sustained release formulation; topical application, e.g., as a cream, ointment, or controlled-release patch or spray applied to the skin, lungs, or mouth; vaginally or rectally, e.g., as a pessary, cream, or foam; sublingually; ophthalmically; transdermally; or adapted for nasal, pulmonary, and other mucosal surfaces.

[0064] Pharmaceutically acceptable: As used herein, the phrase "pharmaceutically acceptable" refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0065] Pharmaceutically acceptable carrier: As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent, that encapsulates a material, or is involved in carrying or transporting a compound of interest from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; pH buffer solutions; polyesters, polycarbonates, and / or polyanhydrides; and other non-toxic, compatible substances employed in pharmaceutical formulations.

[0066] Pharmaceutically acceptable salts: As used herein, the term "pharmaceutically acceptable salts" refers to salts of such compounds that are suitable for use in pharmaceutical contexts, i.e., salts that, within the scope of sound medical judgment, are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, etc., commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al. (1977) describes pharmaceutically acceptable salts in detail. In some embodiments, pharmaceutically acceptable salts include, but are not limited to, non-toxic acid addition salts of amino groups formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or with organic acids such as acetic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or by using other methods used in the art, such as ion exchange. In some embodiments, pharmaceutically acceptable salts include those of adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, bisulfate, boric acid, butyric acid, camphor, camphorsulfonic acid, citric acid, cyclopentanepropionic acid, digluconic acid, dodecylsulfate, ethanesulfonic acid, formic acid, fumaric acid, glucoheptonic acid, glycerophosphate, gluconic acid, hemisulfate, heptanoic acid, hexanoic acid, hydroiodic acid, 2-hydroxyethanesulfonic acid, lactobionic acid, These include, but are not limited to, salts of lactic acid, lauric acid, lauryl sulfate, malic acid, maleic acid, malonic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, nicotinic acid, nitric acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, pectinate, persulfate, 3-phenylpropionic acid, phosphoric acid, picric acid, pivalic acid, propionic acid, stearic acid, succinic acid, sulfuric acid, tartaric acid, thiocyanate, p-toluenesulfonic acid, undecanoic acid, valeric acid, and the like. In some embodiments, provided compounds contain one or more acidic groups, e.g., oligonucleotides, and the pharmaceutically acceptable salt is an alkali, alkaline earth metal, or ammonium salt (e.g., an ammonium salt of N(R)3, where each R is independently as defined and described in this disclosure).Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. In some embodiments, a pharmaceutically acceptable salt is a sodium salt. In some embodiments, a pharmaceutically acceptable salt is a potassium salt. In some embodiments, a pharmaceutically acceptable salt is a calcium salt. In some embodiments, pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium, and amine cations, formed, where appropriate, using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, alkyls having 1 to 6 carbon atoms, sulfonates, and arylsulfonates. In some embodiments, provided compounds contain more than one acidic group; for example, oligonucleotides may contain two or more acidic groups (e.g., in natural phosphate linkages and / or modified internucleotide linkages). In some embodiments, pharmaceutically acceptable salts, or salts in general, of such compounds contain two or more cations, which may be the same or different. In some embodiments, in a pharmaceutically acceptable salt (or salt in general), all ionized hydrogens in acidic groups (e.g., in an aqueous solution having a pKa of no more than about 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2; in some embodiments, no more than about 7; in some embodiments, no more than about 6; in some embodiments, no more than about 5; in some embodiments, no more than about 4; in some embodiments, no more than about 3) are replaced with cations. In some embodiments, each internucleotide linkage, e.g., phosphate group, is independently present in its salt form (e.g., in the case of a sodium salt, -OP(O)(ONa)-O-). In some embodiments, the pharmaceutically acceptable salt is a sodium salt of an oligonucleotide. In some embodiments, the pharmaceutically acceptable salt is a sodium salt of an oligonucleotide, and each acidic phosphate group and modified phosphate group, if any, is present in salt form (all sodium salts).

[0067] Polypeptide: As used herein, the term "polypeptide," which is used interchangeably with the term "protein," refers to a polymer of at least three amino acid residues. In some embodiments, a polypeptide comprises one or more, or all, naturally occurring amino acids. In some embodiments, a polypeptide comprises one or more, or all, unnatural amino acids. In some embodiments, a polypeptide comprises one or more, or all, D-amino acids. In some embodiments, a polypeptide comprises one or more, or all, L-amino acids. In some embodiments, a polypeptide comprises one or more pendant groups or other modifications modifying or attached to one or more amino acid side chains, for example, at the N-terminus of the polypeptide, at the C-terminus of the polypeptide, or any combination thereof. In some embodiments, a polypeptide comprises one or more modifications, such as acetylation, amidation, aminoethylation, biotinylation, carbamylation, carbonylation, citrullination, deamidation, deimination, eliminylation, glycosylation, lipidation, methylation, pegylation, phosphorylation, sumoylation, or a combination thereof. In some embodiments, a polypeptide may participate in one or more intramolecular or intermolecular disulfide bonds. In some embodiments, a polypeptide may be cyclic and / or may contain a cyclic portion. In some embodiments, a polypeptide is not cyclic and / or does not contain any cyclic portions. In some embodiments, a polypeptide is linear. In some embodiments, a polypeptide may include a stapled polypeptide. In some embodiments, a polypeptide participates in noncovalent complex formation by noncovalent or covalent association with one or more other polypeptides (e.g., as found in antibodies). In some embodiments, a polypeptide has a naturally occurring amino acid sequence. In some embodiments, a polypeptide has a non-naturally occurring amino acid sequence.In some embodiments, a polypeptide has an amino acid sequence that is engineered in that it is designed and / or produced through the work of human hands. In some embodiments, the term "polypeptide" can be added to the name of a reference polypeptide, activity, or structure; in such cases, it is used herein to refer to polypeptides that share a related activity or structure and can therefore be considered members of the same class or family of polypeptides. For each such class, this specification provides exemplary polypeptides within the class whose amino acid sequence and / or function are known and / or would be known to one of skill in the art; in some embodiments, such exemplary polypeptides are reference polypeptides for the polypeptide class or family. In some embodiments, members of a polypeptide class or family exhibit significant sequence homology or identity with the reference polypeptide of the class; in some embodiments, with all polypeptides in the class; share common sequence motifs (e.g., characteristic sequence elements); and / or share common activities (in some embodiments, at comparable levels or within a specified range). For example, in some embodiments, member polypeptides exhibit an overall degree of sequence homology or identity with a reference polypeptide of at least about 30-40%, and often about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, and / or contain at least one region (e.g., a conserved region which, in some embodiments, may contain a distinctive sequence element) that exhibits very high sequence identity, often greater than 90%, or even 95%, 96%, 97%, 98%, or 99%. Such conserved regions typically encompass at least 3-4, and often up to 20 or more, amino acids; in some embodiments, a conserved region encompasses at least one stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more contiguous amino acids.In some embodiments, a useful polypeptide may comprise a fragment of a parent polypeptide. In some embodiments, a useful polypeptide may comprise multiple fragments, each of which is found in the same parent polypeptide in a different spatial arrangement relative to each other than that found in the polypeptide of interest (e.g., fragments that are directly linked in the parent may be spatially separated in the polypeptide of interest, or vice versa, and / or fragments may be present in a different order in the polypeptide of interest than in the parent), thereby making the polypeptide of interest a derivative of its parent polypeptide. In some embodiments, a polypeptide described herein (or a nucleic acid encoding such a polypeptide) may be a functional fragment of one of the amino acid sequences described herein. As used herein, a "functional fragment" is a peptide fragment or segment that retains at least 50% of the activity of a wild-type reference polypeptide according to the assays described herein below. Functional fragments may comprise conservative substitutions of the sequences disclosed herein. In some embodiments, the polypeptides described herein may be variants of the sequences described herein. In some embodiments, the variants are conservatively modified variants. Conservative substitution variants may be obtained, for example, by mutation of a native nucleotide sequence. As used herein, a "variant" is a polypeptide that is substantially homologous to a native or reference polypeptide but has an amino acid sequence that differs from the amino acid sequence of the native or reference polypeptide due to one or more deletions, insertions, or substitutions. A variant polypeptide-encoding DNA sequence includes a sequence that encodes a variant protein or fragment thereof that contains one or more additions, deletions, or substitutions when compared to a native or reference DNA sequence, but retains activity. A wide variety of PCR-based site-directed mutagenesis techniques are known in the art and can be applied by those skilled in the art.In the various embodiments described herein, it is further intended to encompass variants (naturally occurring or not), alleles, homologs, conservatively modified variants, and / or conservatively substituted variants of any of the specific polypeptides described. With respect to amino acid sequences, those skilled in the art will recognize that individual substitutions, deletions, or additions to nucleic acid, peptide, polypeptide, or protein sequences in which a single amino acid or a small percentage of amino acids in the encoded sequence is altered are "conservatively modified variants," where the alteration results in the replacement of an amino acid with a chemically similar amino acid and retains the desired activity of the polypeptide. Such conservatively modified variants are in addition to, and do not exclude, polymorphic variants, interspecies homologs, and alleles consistent with the present disclosure.

[0068] Prevent or prophylaxis: As used herein in reference to the occurrence of a disease, disorder, and / or condition, refers to reducing the risk of developing the disease, disorder, and / or condition and / or delaying the onset of one or more characteristics or symptoms of the disease, disorder, or condition. Prevention may be considered complete if the onset of the disease, disorder, or condition is delayed for a predetermined period of time.

[0069] Recombinant: As used herein, the term "recombinant" is intended to refer to polypeptides that are designed, engineered, prepared, expressed, created, manufactured, and / or isolated by recombinant means, such as polypeptides expressed using a recombinant expression vector transfected into a host cell; polypeptides isolated from a recombinant combinatorial human polypeptide library; polypeptides isolated from an animal (e.g., mouse, rabbit, sheep, fish, etc.) that is transgenic for or otherwise engineered to express one or more genes or genetic elements that encode and / or direct the expression of a polypeptide or one or more components, portions, elements, or domains thereof; and / or polypeptides that are prepared, expressed, created, or isolated by any other means involving splicing or ligating selected nucleic acid sequence elements together, chemically synthesizing selected sequence elements, and / or otherwise generating nucleic acids that encode and / or direct the expression of a polypeptide or one or more components, portions, elements, or domains thereof. In some embodiments, one or more of such selected sequence elements are found in nature. In some embodiments, one or more of such selected sequence elements are designed in silico. In some embodiments, one or more such selected sequence elements arise by mutagenesis (e.g., in vivo or in vitro) of known sequence elements, e.g., from natural or synthetic sources, such as in the germline (e.g., human, mouse, etc.) of a source organism of interest.

[0070] Small molecule: As used herein, the term "small molecule" refers to a low molecular weight organic and / or inorganic compound. Generally, a "small molecule" is a molecule less than about 5 kilodaltons (kD) in size. In some embodiments, a small molecule is less than about 4 kD, 3 kD, 2 kD, or 1 kD. In some embodiments, a small molecule is less than about 800 daltons (D), 600 D, 500 D, 400 D, 300 D, 200 D, or 100 D. In some embodiments, a small molecule is less than about 2000 g / mol, less than about 1500 g / mol, less than about 1000 g / mol, less than about 800 g / mol, or less than about 500 g / mol. In some embodiments, a small molecule is not a polymer. In some embodiments, a small molecule does not include polymeric molecules. In some embodiments, a small molecule is not and / or does not include a protein or polypeptide (e.g., is not an oligopeptide or peptide). In some embodiments, a small molecule is not and / or does not comprise a polynucleotide (e.g., is not an oligonucleotide). In some embodiments, a small molecule is not and / or does not comprise a polysaccharide; for example, in some embodiments, a small molecule is not a glycoprotein, proteoglycan, glycolipid, etc. In some embodiments, a small molecule is not a lipid. In some embodiments, a small molecule is a modulator (e.g., an inhibitor or activator). In some embodiments, a small molecule is biologically active. In some embodiments, a small molecule is detectable (e.g., comprises at least one detectable moiety). In some embodiments, a small molecule is a therapeutic agent. Those of skill in the art will understand, upon reading this disclosure, that certain small molecule compounds described herein may be provided and / or utilized in any of a variety of forms, such as, for example, crystalline forms, salt forms, protected forms, prodrug forms, ester forms, isomeric forms (e.g., optical and / or structural isomers), isotopic forms, etc. Those of skill in the art will understand that certain small molecule compounds have structures that can exist in one or more stereoisomeric forms.In some embodiments, such small molecules may be utilized in accordance with the present disclosure in the form of individual enantiomers, diastereomers, or geometric isomers, or in the form of a mixture of stereoisomers; in some embodiments, such small molecules may be utilized in accordance with the present disclosure in the form of a racemic mixture. Those of skill in the art will recognize that certain small molecule compounds have structures that can exist in one or more tautomeric forms. In some embodiments, such small molecules may be utilized in accordance with the present disclosure in the form of individual tautomers, or in forms that interconvert between tautomeric forms. Those of skill in the art will recognize that certain small molecule compounds may contain isotopic substitutions (e.g., H to H). 2 H or 3 H; 12 for C 11 C. 13 C, or 14 C; 14 For N 13 N or 15 N; 16 against O 17 O or 18 O; 35 for C 36 Cl; 19 against F 18 F; 127 against I 131 I or 125It will be understood that the small molecule has a structure that allows for isotopically modified forms (e.g., I, etc.). In some embodiments, such small molecules may be utilized in accordance with the present disclosure in one or more isotopically modified forms or mixtures thereof. In some embodiments, a reference to a particular small molecule compound may refer to a specific form of that compound. In some embodiments, a particular small molecule compound may be provided and / or utilized in a salt form (e.g., an acid addition or base addition form, depending on the compound); in some such embodiments, the salt form may be a pharmaceutically acceptable salt form. In some embodiments, if a small molecule compound is naturally occurring or found in nature, the compound may be provided and / or utilized in accordance with the present disclosure in a form that is different from that in which it naturally occurs or is found in nature. Those skilled in the art will understand that in some embodiments, a preparation of a particular small molecule compound that contains an absolute or relative amount of the compound or a particular form thereof (e.g., relative to other components of the preparation, including other forms of the compound) that is different from the absolute or relative amount of the compound or form present in a reference preparation of interest (e.g., in a primary sample from a source of interest, such as a biological or environmental source) is different from the compound present in the reference preparation or source. Thus, in some embodiments, for example, a preparation of a single stereoisomer of a small molecule compound can be considered a different form of the compound from a racemic mixture of the compound; a particular salt of a small molecule compound can be considered a different form from other salt forms of the compound; a preparation containing only one form of the compound that contains one conformer of the double bond ((Z) or (E)) can be considered a different form of the compound from one that contains the other conformer of the double bond ((E) or (Z)); a preparation in which one or more atoms are isotopes different from those present in the reference preparation can be considered a different form, etc.

[0071] Specific binding: As used herein, the term "specific binding" refers to the ability to distinguish between possible binding partners in an environment in which binding can occur. A binding agent that interacts with one specific target is said to "specifically bind" to the target with which it interacts (e.g., a target amino acid or nucleic acid sequence on a target protein / gene of interest) in the presence of other potential targets. In some embodiments, specific binding is assessed by detecting or determining the degree of association between the binding agent and its partner; in some embodiments, specific binding is assessed by detecting or determining the degree of dissociation of the binding agent-partner complex; in some embodiments, specific binding is assessed by detecting or determining the ability of the binding agent to compete for alternative interactions between its partner and another entity. In some embodiments, specific binding is assessed by performing such detection or determination over a range of concentrations.

[0072] Specificity: As known in the art, "specificity" is the degree to which a particular ligand is able to distinguish its binding partner from other potential binding partners.

[0073] Subject: As used herein, the term "subject" refers to an organism, typically a mammal (e.g., a human, including in some embodiments prenatal human forms). In some embodiments, the subject is afflicted with a relevant disease, disorder, or condition (e.g., muscular dystrophy or other disease characterized by neuromuscular dysfunction). In some embodiments, the subject is predisposed to a disease, disorder, or condition. In some embodiments, the subject exhibits one or more symptoms or characteristics of a disease, disorder, or condition. In some embodiments, the subject does not exhibit any symptoms or characteristics of a disease, disorder, or condition. In some embodiments, the subject is someone with one or more traits characteristic of susceptibility to or risk for a disease, disorder, or condition. In some embodiments, the subject is a patient. In some embodiments, the subject is an individual to whom and / or to whom a diagnosis and / or therapy is administered.

[0074] Substantially: As used herein, the term "substantially" refers to the qualitative aspect of exhibiting the full or nearly full extent or degree of a characteristic or property of interest. Those skilled in the art of biology will understand that biological and chemical phenomena rarely, if ever, proceed to completion and / or perfection or achieve or avoid absolute results. Thus, the term "substantially" is used herein to capture the potential lack of perfection inherent in many biological and chemical phenomena.

[0075] Substantial identity: As used herein, refers to a comparison between amino acid or nucleic acid sequences. As will be understood by those skilled in the art, two sequences are generally considered to be "substantially identical" if they contain identical residues at corresponding positions. As is well known in the art, amino acid or nucleic acid sequences can be compared using any of a variety of algorithms, including those available in commercial computer programs, such as BLASTN for nucleotide sequences, and BLASTP, gapped BLAST, and PSI-BLAST for amino acid sequences. Exemplary such programs are described in Altschul et al. (1990 and 1997); Baxevanis et al. (1998); and Misener et al. (1999). In addition to identifying identical sequences, the programs described above typically provide an indication of the degree of identity. In some embodiments, two sequences are considered to be substantially identical if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of their corresponding residues are identical over the relevant stretch of residues, which in some embodiments is the complete sequence. In some embodiments, the relevant stretch is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more residues.

[0076] Suffering from: An individual who is "suffering from" a disease, disorder, and / or condition (e.g., muscular dystrophy or other disease characterized by neuromuscular dysfunction) has been diagnosed with and / or exhibits one or more symptoms of the disease, disorder, and / or condition.

[0077] Susceptible: An individual who is "susceptible" to a disease, disorder, and / or condition (e.g., muscular dystrophy or other disease characterized by neuromuscular dysfunction) is one who has a higher risk of developing the disease, disorder, and / or condition than members of the general public. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition may not have been diagnosed with the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition may exhibit symptoms of the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition may not exhibit symptoms of the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition will develop the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition will not develop the disease, disorder, and / or condition.

[0078] Symptoms are reduced: According to the present invention, a "symptom is reduced" when one or more symptoms of a particular disease, disorder, or condition (e.g., muscular dystrophy or other disease characterized by neuromuscular dysfunction) are reduced in magnitude (e.g., intensity, severity, etc.) and / or frequency. For purposes of clarity, delaying the onset of a particular symptom is considered a form of reducing the frequency of that symptom.

[0079] Target gene: As used herein, "target gene" refers to a gene whose expression is to be modulated, for example, by altering splice activity (e.g., by inducing exon skipping). As used herein, the term "target portion" or "target region" refers to a continuous portion of the nucleotide sequence of a target gene. In some embodiments, the target portion or target region is one or more exons within the target gene sequence. The target portion can be about 8 to 36 nucleotides in length, for example, about 10 to 20 or about 15 to 30 nucleotides in length. The length of the target portion can have a specific value or subrange within the aforementioned range. For example, in certain embodiments, the targeting portion may be about 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, It may be 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides.

[0080] Therapeutic Agent: As used herein, the phrase "therapeutic agent" refers to any agent that has a therapeutic effect and / or elicits a desired biological and / or pharmacological effect when administered to a subject. In some embodiments, a therapeutic agent is any substance that can be used to alleviate, ameliorate, relieve, inhibit, prevent, delay the onset of, reduce the severity of, and / or reduce the occurrence of one or more symptoms or characteristics of a disease, disorder, and / or condition (e.g., one or more symptoms or characteristics of muscular dystrophy or other diseases characterized by neuromuscular dysfunction).

[0081] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" refers to an amount of a substance (e.g., a therapeutic agent, composition, and / or formulation) that elicits a desired biological response when administered as part of a therapeutic dosing regimen. In some embodiments, a therapeutically effective amount of a substance is an amount that, when administered to a subject suffering from or susceptible to a disease, disorder, and / or condition, is sufficient to treat, diagnose, prevent, and / or delay the onset of the disease, disorder, and / or condition. As will be understood by those skilled in the art, the effective amount of a substance can vary depending on factors such as the desired biological endpoint, the substance to be delivered, the target cell or tissue, etc. It will be understood that there will be many methods known in the art for determining the effective amount for a given application. For example, pharmacological methods for determining dosage can be used in the context of treatment. In the context of therapeutic or prophylactic applications, the amount of a composition administered to a subject will depend on the type and severity of the disease, as well as individual characteristics such as general health, age, sex, weight, and tolerance to drugs. This will also depend on the degree, severity, and type of disease. Those skilled in the art will be able to determine the appropriate dosage, which depends on these and other factors. For example, an effective amount of a compound in a formulation for treating a disease, disorder, and / or condition is an amount that relieves, improves, alleviates, inhibits, prevents, delays the onset of, reduces the severity of, and / or reduces the occurrence of one or more symptoms or characteristics of the disease, disorder, and / or condition. As used herein, the terms "effective amount" and "therapeutically effective amount" include an amount sufficient to prevent or ameliorate symptoms of a disease or condition, such as decreased mobility, metabolism, and quality of life resulting from muscle wasting, in muscular dystrophies such as Becker muscular dystrophy, congenital muscular dystrophy, distal muscular dystrophy, Duchenne muscular dystrophy, Emery-Dreifuss muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, myotonic muscular dystrophy, and oculopharyngeal muscular dystrophy, as well as in cancer patients, elderly patients, and many others without a history of neuromuscular dysfunction.It will be understood that there are many methods known in the art for determining the effective amount for a given application. For example, pharmacological methods for determining dosage can be used in the context of treatment. In the context of therapeutic or prophylactic application, the amount of the composition administered to a subject will depend on the type and severity of the disease, as well as individual characteristics such as general health, age, sex, weight, and tolerance to drugs. This will also depend on the degree, severity, and type of disease. Those skilled in the art will be able to determine the appropriate dosage depending on these and other factors. The composition can also be administered in combination with one or more additional therapeutic compounds. In some embodiments, a therapeutically effective amount is administered in a single dose; in some embodiments, multiple unit doses are required to deliver a therapeutically effective amount.

[0082] Treating: As used herein, the term "treating" refers to providing treatment, i.e., providing any type of medical or surgical management of a subject. Treatment may be provided to ameliorate, alleviate, inhibit the progression of, prevent or reduce the likelihood of, a disease, disorder, or condition, or to ameliorate, alleviate, inhibit the progression of, or prevent, or reduce the likelihood of, one or more symptoms or signs of a disease, disorder, or condition. Beneficial or desired clinical results include, but are not limited to, relief of one or more symptoms, a decrease in the degree of impairment, a stable (i.e., non-worsening) state of muscular dystrophy, a delay or slowing of muscle wasting, and an increase in lifespan compared to that expected in the absence of treatment. Treating can include administering an active substance to a subject after the onset of one or more symptoms or signs indicating muscular dystrophy or other diseases characterized by neuromuscular dysfunction, for example, to ameliorate, alleviate, reduce the severity of, and / or inhibit or prevent the progression of, and / or to ameliorate, alleviate, reduce the severity of, and / or inhibit one or more symptoms or signs of, the disease.The compositions of the present disclosure can be administered to a subject who has developed muscular dystrophy or other diseases characterized by neuromuscular dysfunction, or who is at an increased risk of developing such disorders compared with members of the general population.The compositions of the present disclosure can be administered prophylactically, i.e., before the onset of any symptoms or signs of the disease.Typically, in this case, the subject will be at risk of developing the disease.

[0083] Variant (mutant): As used herein in the context of a molecule, such as a nucleic acid (e.g., ASO), protein, or small molecule, the term "variant" refers to a molecule that exhibits significant structural identity with a reference molecule, but is structurally different from the reference molecule, for example, in terms of the presence or absence or level of one or more chemical moieties compared to the reference entity. In some embodiments, a variant is also functionally different from its reference molecule. Generally, whether a particular molecule is properly considered to be a "variant" of a reference molecule is based on its degree of structural identity with the reference molecule. As will be understood by those skilled in the art, any biological or chemical reference molecule has certain characteristic structural elements. A variant by definition is an individual molecule that shares one or more such characteristic structural elements but differs from the reference molecule in at least one aspect. To give just a few examples, a polypeptide may have characteristic structural elements composed of multiple amino acids that have designated locations relative to each other in linear or three-dimensional space and / or contribute to a particular structural motif and / or biological function; a nucleic acid may have characteristic structural elements composed of multiple nucleotide residues that have designated locations relative to each other in linear or three-dimensional space. In some embodiments, a variant polypeptide or nucleic acid may differ from a reference polypeptide or nucleic acid as a result of one or more differences in amino acid or nucleotide sequence and / or one or more differences in chemical moieties (e.g., carbohydrates, lipids, phosphate groups) that are covalent components of the polypeptide or nucleic acid (e.g., attached to the polypeptide or nucleic acid backbone). In some embodiments, a variant polypeptide or nucleic acid exhibits an overall sequence identity with a reference polypeptide or nucleic acid that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%. In some embodiments, a variant polypeptide or nucleic acid does not share at least one characteristic structural element with the reference polypeptide or nucleic acid.In some embodiments, the reference polypeptide or nucleic acid has one or more biological activities. In some embodiments, the variant polypeptide or nucleic acid shares one or more of the biological activities of the reference polypeptide or nucleic acid. In some embodiments, the variant polypeptide or nucleic acid lacks one or more of the biological activities of the reference polypeptide or nucleic acid. In some embodiments, the variant polypeptide or nucleic acid exhibits a reduced level of one or more biological activities compared to the reference polypeptide or nucleic acid. In some embodiments, the polypeptide or nucleic acid of interest is considered to be a "variant" of the reference polypeptide or nucleic acid if it has an amino acid or nucleotide sequence that is identical to that of the reference except for a small number of sequence changes in specific positions. Typically, less than about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, or about 2% of the residues in the variant are substituted, inserted, or deleted compared to the reference. In some embodiments, a variant polypeptide or nucleic acid contains about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1 substituted residues compared to the reference. Often, a variant polypeptide or nucleic acid contains a very small number (e.g., less than about 5, about 4, about 3, about 2, or about 1) of substituted, inserted, or deleted functional residues (i.e., residues that participate in a particular biological activity) compared to the reference. In some embodiments, a variant polypeptide or nucleic acid contains at most about 5, about 4, about 3, about 2, or about 1 additions or deletions compared to the reference, and in some embodiments, no additions or deletions. In some embodiments, the variant polypeptide or nucleic acid comprises less than about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 10, about 9, about 8, about 7, about 6, generally less than about 5, about 4, about 3, or about 2 additions or deletions compared to the reference. In some embodiments, the reference polypeptide or nucleic acid is one found in nature. In some embodiments, the reference polypeptide or nucleic acid is a human polypeptide or nucleic acid.

[0084] Vector: As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors can direct the expression of genes to which they are operably linked. Such vectors are referred to herein as "expression vectors." Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques can be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures may generally be performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989).

[0085] Detailed Description of Certain Embodiments MuSK and muscle regeneration and / or growth Satellite cells account for approximately 5% of myonuclei and are normally quiescent and distributed along mature, multinucleated myofibrils. Following muscle injury, satellite cells typically proliferate before either returning to quiescence or differentiating (see Figure 1A). During differentiation, satellite cells become committed myoblasts that fuse into myotubes and ultimately form mature myofibrils in a process called myogenesis. Bone morphogenetic protein (BMP) signaling regulates satellite cell dynamics and muscle regeneration both in vivo and in vitro by modulating transcriptional production. BMP signaling is undetectable in quiescent satellite cells, is upregulated in proliferating satellite cells, and is downregulated during differentiation. However, the mediators that regulate the balance between satellite cell proliferation and differentiation are unknown.

[0086] Muscle-specific kinase (MuSK), also known as muscle-associated receptor tyrosine kinase, is a transmembrane protein first recognized for its essential role in the formation and maintenance of neuromuscular junctions (NMJs). MuSK possesses three extracellular immunoglobulin (Ig)-like and cysteine-rich frizzled (CRD / Fz) domains, as well as an intracellular tyrosine kinase (TK) domain. The Ig1, TK, and potential CRD / Fz domains are required for NMJ formation and maintenance. The Ig1 and TK domains are essential for agrin-LRP4 signaling, which directs synaptic differentiation. For this reason, mice lacking MuSK are neonatally lethal. Two isoforms of MuSK exist in vivo: full-length (FL) MuSK and a naturally occurring splice variant (ΔIg3-MuSK) lacking the Ig3 domain (Figure 1B). FL MuSK mRNA levels are 10-fold higher than that of ΔIg3-MuSK, but both are coordinately expressed.

[0087] Exemplary amino acid sequences of human and mouse MuSK Ig3 domains (ie, MuSK Ig3 domain polypeptides) are as shown below. MuSK human Ig3 domain: ARILRAPESHNVTFGSFVTLHCTATGIPVPTITWIENGNAVSSGSIQESVKDRVIDSRLQLFITKPGLYTCIATNKHGEKFSTAKAAATIS (SEQ ID NO: 1) MuSK mouse Ig3 domain ARILRAPESHNVTFGSFVTLRCTAIGIPVPTISWIENGNAVSSGSIQESVKDRVIDSRLQLFITKPGLYTCIATNKHGEKFSTAKAAATVS (SEQ ID NO: 2) Among other things, this disclosure teaches that modulating MuSK alternative splicing is a strategy for increasing muscle regeneration.

[0088] MuSK is activated by a neurally derived proteoglycan called agrin. Agrin has been characterized for its role in the development of neuromuscular junctions during embryogenesis. Agrin is named for its involvement in the aggregation of acetylcholine receptors during synaptogenesis. In humans, this protein is encoded by the AGRN gene. The agrin protein contains nine domains that are homologous to protease inhibitors.

[0089] MuSK is expressed in muscle and is upregulated during muscle regeneration. Data suggest that MuSK is involved in BMP signaling in myogenesis. MuSK may act as a BMP coreceptor, binding to BMP2, BMP4, and BMP7, as well as the type I BMP receptors ALK3 and ALK6. See, for example, Yilmaz et al. (2016). The Ig3 domain of MuSK is required for high-affinity binding to BMPs. MuSK upregulates BMP signaling, as measured by BMP4-dependent phosphorylation of SMAD1 / 5 / 8. Importantly, MuSK-BMP signaling shapes the size and composition of the BMP-induced transcriptome in myoblasts and myotubes, and this role is independent of any MuSK tyrosine kinase activity. MuSK is a BMP coreceptor that enhances BMP signaling and regulates myogenic factors such as myogenic factor 5 (Myf5) in immortalized myogenic cells.

[0090] As described herein, activated satellite cells express MuSK protein, and disruption of MuSK-BMP signaling alters satellite cell differentiation during in vivo muscle regeneration. The data contained herein provide information regarding the role of the MuSK-BMP pathway in satellite cells and muscle regeneration. These data suggest that the MuSK-BMP pathway plays an important role, and the present disclosure provides insight that targeting the MuSK-BMP pathway is more selective than targeting the broad activity of BMPs, at least because MuSK expression is much more restricted than that of the BMP pathway. The data provided herein also teach that targeting the MuSK-BMP pathway enhances muscle growth. In some embodiments, muscle growth occurs, for example, in undamaged tissue.

[0091] MuSK Muscle Regeneration (MR) Agonizer In some embodiments, the present disclosure provides techniques for achieving (e.g., inducing, enhancing, etc.) muscle regeneration in a subject by administering an agent in the presence of which MuSK muscle regeneration levels and / or activity are increased (i.e., a MuSK muscle regeneration (MuSK MR) agonizing agent). As described herein, muscle regeneration (MR) may also include or contribute to increased muscle growth. For example, in some embodiments, MuSK An MR agonizing agent is an agent that increases the level or activity of one or more MuSK polypeptides that lack an effective Ig3 domain (e.g., ΔIg3-MuSK) because, for example, such domain has been mutated, removed, or otherwise inactivated (e.g., by blocking, modification, etc.). Alternatively or additionally, in some embodiments, a MuSK MR agonizing agent blocks, inactivates, mutates, or removes a functional Ig3 domain from MuSK, or achieves, supports, or contributes to such blocking, inactivation, mutation, or removal.

[0092] In some embodiments, the present disclosure provides technology relating to MuSK MR agonizing agents, including, for example, providing such agents themselves, and / or providing methods and / or reagents for identifying, characterizing, producing, and / or using them, and / or compositions that contain and / or deliver them.

[0093] In some embodiments, the MuSK MR agonizing agent may directly interact with a MuSK polypeptide (e.g., with full-length MuSK and / or with ΔIg3-MuSK). In some embodiments, the MuSK MR agonizing agent may not directly interact with a MuSK polypeptide, but rather affects the level and / or activity of ΔIg3-MuSK through some other interaction (e.g., with a precursor or regulator or downstream product of MuSK).

[0094] In principle, the ΔIg3-MuSK MR agonizing agent can be of any chemical class (e.g., small molecule, polypeptide [e.g., antibody], nucleic acid, etc.). In some embodiments, the MuSK MR agonizing agent is an agent that downregulates MuSK Ig3 domain protein expression, MuSK Ig3 domain gene expression, and / or MuSK Ig3 activation of BMP signaling, thereby inducing muscle regeneration. In some embodiments, the MuSK MR agonizing agent is an agonizing agent that increases MuSK ΔIg3 expression. In certain embodiments described in more detail herein, the MuSK MR agonizing agent can be or comprise a small molecule. In certain embodiments described in more detail herein, the MuSK MR agonizing agent can be or comprise an antibody that binds to a MuSK polypeptide (e.g., an antibody that blocks MuSK Ig3 and / or sequester one or more MuSK polypeptide forms that contain functional Ig3). In certain embodiments described in more detail herein, the MuSK MR agonizing agent may be or may include a nucleic acid agent. For example, in some embodiments, the MuSK MR agonizing agent may be or may include a nucleic acid (e.g., a gene therapy vector, or an RNA therapeutic such as mRNA) encoding a MuSk form lacking a functional Ig3 domain (e.g., ΔIg3-MuSK). Alternatively or additionally, in some embodiments, the nucleic acid MuSK MR agonizing agent may be or may include an oligonucleotide such as a MuSK Ig3-targeted exon skipping oligonucleotide, a MuSK Ig3-targeted CRISPR / Cas9 gRNA (e.g., that modifies and / or removes Ig3), a MuSK Ig3-targeted siRNA (e.g., that inhibits the production / expression of MuSK Ig3 from the transcript encoding it), and / or a MuSK Ig3-targeted shRNA.

[0095] small molecule In some embodiments, the MuSK MR agonizing agent can be or can include a small molecule compound.

[0096] In some embodiments, small molecule MuSK MR agonizing agents target therapeutic agents that target MuSK splicing; for example, in some embodiments, such small molecule compounds enhance splicing events that generate messages encoding ΔIg3-MuSK and / or inhibit splicing events that generate messages encoding other MuSK splice variants. In some embodiments, small molecule MuSK MR agonizing agents alter the BMP signaling pathway. In some embodiments, small molecule MuSK MR agonizing agents alter the BMP signaling pathway, which further induces muscle regeneration.

[0097] In some embodiments, the small molecule MuSK MR agonizing agent targets one or more of the type I BMP receptors ALK3 (ALK is anaplastic lymphoma kinase) and ALK6, and the type I activin receptor ALK4. In some embodiments, the small molecule MuSK MR agonizing agent is an ALK inhibitor. In some embodiments, the small molecule MuSK MR agonizing agent is an ALK inhibitor selected from the group consisting of crizotinib, ceritinib, alectinib, brigatinib, and lorlatinib.

[0098] In some embodiments, small molecule MuSK MR agonizing agents target the MuSK Ig3 domain and / or BMP, thereby reducing the level and / or activity of MuSK / BMP complexes. In some such embodiments, small molecule MuSK MR agonizing agents inhibit the formation of such complexes and / or disrupt such complexes. In some embodiments, such MuSK MR agonizing agents inhibit the formation of MuSK Ig3 domains and / or BMPs, thereby reducing the level and / or activity of MuSK / BMP complexes. Competes with BMP for binding to Ig3 and / or competes with MuSK Ig3 for binding to BMP.

[0099] antibody drugs In some embodiments, the MuSK MR agonizing agent is an antibody agent.

[0100] In some embodiments, such antibody agents specifically bind to a MuSK polypeptide.In some embodiments, antibody agents that target MuSK specifically bind to the Ig3 domain of a MuSK polypeptide.

[0101] In some embodiments, antibodies that target the Ig3 domain of a MuSK protein may specifically bind to the Ig3 domain relative to the Ig1 or Ig2 domains of MuSK.

[0102] In some embodiments, the anti-MuSK antibody agent targets the MuSK Ig3 domain and / or BMP, thereby reducing the level and / or activity of the MuSK / BMP complex. In some such embodiments, the anti-MuSK antibody agent inhibits the formation of such a complex and / or disrupts such a complex. In some embodiments, such an anti-MuSK antibody agent competes with BMP for binding to MuSK Ig3 and / or competes with MuSK Ig3 for binding to BMP.

[0103] In some embodiments, the anti-MUSK antibody agent is internalized by a cell (e.g., a satellite cell). In some embodiments, the antibody agent described herein (e.g., is or comprises an antibody or antigen-binding fragment thereof) can be or comprise an immunoglobulin, a heavy chain antibody, a light chain antibody, or other protein scaffold with antibody-like properties, as well as other immunological binding moieties known in the art, including Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, disulfide-linked Fv fragments, scFv fragments, diabodies, triabodies, tetrabodies, minibodies, maxibodies, tandabs, BiTe, and any combination thereof.

[0104] In some embodiments, anti-MuSK antibody agents target, for example, the Ig3 domain of MUSK. In some embodiments, such antibody agents can inhibit or substantially prevent binding of BMP to the MuSK Ig3 domain.

[0105] In some embodiments, an antibody agent may be or may comprise, for example, an antibody, which may be, for example, an immunoglobulin molecule of four polypeptide chains, for example, two heavy (H) chains and two light (L) chains. The heavy chain may comprise a heavy chain variable domain and a heavy chain constant domain. The heavy chain constant domain may comprise a CH1, hinge, CH2, CH3, and, in some cases, a CH4 region. The light chain may comprise a light chain variable domain and a light chain constant domain. The light chain constant domain may comprise a CL. The heavy chain variable domain of the heavy chain and the light chain variable domain of the light chain may be further divided into regions of variability called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Such heavy and light chain variable domains may each comprise three CDRs and four framework regions arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, one or more of which may be engineered as described herein.

[0106] In some embodiments, an antibody agent (e.g., an anti-MUSK antibody) may comprise various heavy and light chains described herein. In some embodiments, an antibody may comprise two heavy and light chains. In various embodiments, the present disclosure encompasses antibodies comprising at least one heavy and / or light chain disclosed herein, at least one heavy and / or light chain framework domain disclosed herein, at least one heavy and / or light chain CDR domain disclosed herein, and / or any heavy and / or light chain constant domain disclosed herein.

[0107] In some embodiments, the antibody agent is or comprises a monoclonal antibody. Typically, a monoclonal antibody is obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are substantially identical except for possible naturally occurring mutations that may be present in minor amounts. Thus, the modifier "monoclonal" as used herein indicates the character of the antibody as not being a mixture of individual antibodies. In some embodiments, a monoclonal antibody directed to a specific epitope is derived from a single cell line (e.g., a B cell line).

[0108] In some embodiments, an antibody agent (e.g., an anti-MUSK antibody) can be or comprise a polyclonal antibody. In contrast to monoclonal antibodies, polyclonal antibodies are typically heterogeneous antibody populations, i.e., antibodies in a particular population contain structural variations, such as affinities for different epitopes on a particular antigen (e.g., the Ig3 domain of MuSK, or regions within the Ig3 domain). Several methods of producing polyclonal antibodies are known in the art, including the use of multiple subcutaneous and / or intraperitoneal injections of the relevant antigen into animals, optionally with co-administration of one or more adjuvants.

[0109] Oligonucleotides In some embodiments, the MuSK MR agonizing agents described herein are or comprise oligonucleotides.

[0110] Synthetic oligonucleotides provide useful molecular tools in a wide variety of applications. For example, oligonucleotides are useful in therapeutic, diagnostic, research, and new nanomaterial applications. The use of naturally occurring nucleic acids (e.g., unmodified DNA or RNA) is limited by their susceptibility to, for example, endonucleases and exonucleases. Therefore, to circumvent these shortcomings, various synthetic counterparts have been developed. These include synthetic oligonucleotides containing chemical modifications, such as base modifications, sugar modifications, backbone modifications, etc., that make these molecules less susceptible to degradation and improve other properties of the oligonucleotides, among others. Chemical modifications can also lead to certain undesirable effects, such as increased toxicity.

[0111] Among other things, the present disclosure encompasses the recognition that structural elements of oligonucleotides, such as base sequence, chemical modifications (e.g., modifications of sugars, bases, and / or internucleotide linkages and their pattern), and / or stereochemistry (e.g., stereochemistry of backbone chiral centers (chiral internucleotide linkages) and / or their pattern), can have a significant impact on properties, such as stability, splicing alteration ability, etc. In some embodiments, oligonucleotide properties can be tailored by optimizing chemical modifications (modifications of bases, sugars, and / or internucleotide linkages) and / or stereochemistry (pattern of backbone chiral centers).

[0112] In some embodiments, the present disclosure demonstrates that oligonucleotide compositions comprising oligonucleotides with controlled structural elements, such as controlled chemical modifications, provide unexpected properties, including, but not limited to, those described herein. In some embodiments, provided compositions comprising oligonucleotides with chemical modifications (e.g., base modifications, sugar modifications, internucleotide linkage modifications, etc.) have improved properties, such as improved splicing alteration ability, improved protein binding profile, and / or improved delivery. In particular, in some embodiments, the present disclosure provides compositions and methods for altering transcript splicing. In some embodiments, the present disclosure provides compositions and methods for improving transcript splicing. In some embodiments, altering transcript splicing using the provided compositions and methods includes producing products with desired and / or improved biological functions and / or knocking down undesired products, for example, by modifying splicing products so that undesired biological functions can be suppressed or eliminated.

[0113] In some embodiments, the splicing product is mRNA. In some embodiments, the modification comprises skipping one or more exons. In some embodiments, the splicing of transcript is improved in that exon skipping increases the level of mRNA and protein with improved beneficial activity compared to the absence of exon skipping.

[0114] In some embodiments, exon skipping improves splicing of transcripts in that it reduces the levels of mRNA and protein with undesired activity compared to the absence of exon skipping. In some embodiments, targets are knocked down through exon skipping, which causes premature stop codons and / or frameshift mutations by skipping one or more exons.

[0115] In some embodiments, the oligonucleotide of the present disclosure comprises one or more natural nucleobases, and / or one or more modified nucleobases derived from natural nucleobases.Examples include, but are not limited to, uracil, thymine, adenine, cytosine, and guanine, each of which has its amino group protected by an acyl protecting group; pyrimidine analogs such as 2-fluorouracil, 2-fluorocytosine, 5-bromouracil, 5-iodouracil, 2,6-diaminopurine, azacytosine, pseudoisocytosine, and pseudouracil; and other modified nucleobases such as 8-substituted purine, xanthine, or hypoxanthine (the latter two being natural degradation products).

[0116] Modified nucleobases also include extended size nucleobases to which one or more aryl rings, such as phenyl rings, have been added.

[0117] In some embodiments, the modified nucleobase is of any one of the following structures, optionally substituted:

[0118] [ka]

[0119] In some embodiments, modified nucleobase is not substituted.In some embodiments, modified nucleobase is substituted.In some embodiments, modified nucleobase is substituted, for example, to contain heteroatom, alkyl group, or a linking moiety that is connected to fluorescent moiety, biotin or avidin moiety, or other protein or peptide.In some embodiments, modified nucleobase is not a nucleobase in the most classical sense, but is a "universal base" that functions similarly to nucleobase.One representative example of such universal base is 3-nitropyrrole.

[0120] In some embodiments, the oligonucleotides described herein comprise nucleosides incorporating modified nucleobases and / or nucleobases covalently linked to modified sugars. Some examples of nucleosides incorporating modified nucleobases include 4-acetylcytidine; 5-(carboxyhydroxymethyl)uridine; 2'-O-methylcytidine; 5-carboxymethylaminomethyl-2-thiouridine; 5-carboxymethylaminomethyluridine; dihydrouridine; 2'-O-methylpseudouridine; beta,D-galactosyl euosin; 2'-O-methylguanosine; N-acetylcytidine; 6 -Isopentenyl adenosine; 1-methyl adenosine; 1-methyl pseudouridine; 1-methyl guanosine; l-methyl inosine; 2,2-dimethyl guanosine; 2-methyl adenosine; 2-methyl guanosine; N 7 -Methylguanosine; 3-methylcytidine; 5-methylcytidine; 5-hydroxymethylcytidine; 5-methylcytosine, 5-formylcytosine; 5-carboxylcytosine; N 6 -Methyladenosine; 7-methylguanosine; 5-methylaminoethyluridine; 5-methoxyaminomethyl-2-thiouridine; beta, D-mannosyl eosin; 5-methoxycarbonylmethyluridine; 5-methoxyuridine; 2-methylthio-N 6 -isopentenyladenosine; N-((9-beta,D-ribofuranosyl-2-methylthiopurin-6-yl)carbamoyl)threonine; N-((9-beta,D-ribofuranosylpurin-6-yl)-N-methylcarbamoyl)threonine; uridine-5-oxyacetic acid methyl ester; uridine-5-oxyacetic acid(v); pseudouridine; queosine; 2-thiocytidine; 5-methyl-2-thiouridine; 2-thiouridine; 4-thiouridine; 5-methyluridine; 2'-O-methyl-5-methyluridine; and 2'-O-methyluridine.

[0121] In some embodiments, the nucleoside includes a 6'-modified bicyclic nucleoside analog having either (R) or (S) chirality at the 6'-position, including the analogs described in U.S. Patent No. 7,399,845. In other embodiments, the nucleoside includes a 5'-modified bicyclic nucleoside analog having either (R) or (S) chirality at the 5'-position, including the analogs described in U.S. Publication No. 20070287831. In some embodiments, the nucleobase or modified nucleobase is 5-bromouracil, 5-iodouracil, or 2,6-diaminopurine. In some embodiments, the nucleobase or modified nucleobase is modified by substitution with a fluorescent moiety.

[0122] In some embodiments, the oligonucleotides described herein contain one or more modified nucleotides in which the phosphate group or the linking phosphorus in the nucleotide is linked to various positions on the sugar or modified sugar.By way of non-limiting example, the phosphate group or the linking phosphorus can be linked to the 2', 3', 4', or 5' hydroxyl moiety of the sugar or modified sugar.Nucleotides incorporating the modified nucleobases described herein are also contemplated in this context.

[0123] Other modified sugars can also be incorporated into oligonucleotide molecules. In some embodiments, modified sugars include the following: -F; -CF, -CN, -N, -NO, -NO, -OR', -SR', or -N(R'), where each R' is independently as defined above and described herein; -O-(C 10 alkyl), -S-(C1-C 10 alkyl), -NH-(C1-C 10 alkyl), or -N(C1-C 10 alkyl)2; -O-(C2-C 10 alkenyl), -S-(C2-C 10 alkenyl), -NH-(C2-C 10 alkenyl), or -N(C2-C 10 alkenyl)2; -O-(C2-C 10alkynyl), -S-(C2-C 10 alkynyl), -NH-(C2-C 10 alkynyl), or -N(C2-C 10 alkynyl)2; or -O-(C1-C 10 alkylene)-O-(C1-C 10 alkyl), -O-(C1-C 10 Alkylene)-NH-(C1-C 10 alkyl) or -O-(C1-C 10 Alkylene)-NH(C1-C 10 alkyl)2, -NH-(C1-C 10 alkylene)-O-(C1-C 10 alkyl), or -N(C1-C 10 Alkyl)-(C1-C 10 alkylene)-O-(C1-C 10 alkyl), where alkyl, alkylene, alkenyl, and alkynyl may be substituted or unsubstituted. Examples of substituents include -O(CH) n OCH3 and -O(CH2) n Examples include, but are not limited to, NH2, where n is 1 to about 10, MOE, DMAOE, DMAEOE.

[0124] In some embodiments, the 2'-OH of ribose is one of the following: -H, -F; -CF, -CN, -N, -NO, -NO, -OR', -SR', or -N(R'), where each R' is independently as defined above and described herein; -O-(C 10 alkyl), -S-(C1-C 10 alkyl), -NH-(C1-C 10 alkyl), or -N(C1-C 10 alkyl)2; -O-(C2-C 10 alkenyl), -S-(C2-C 10 alkenyl), -NH-(C2-C 10 alkenyl), or -N(C2-C 10 alkenyl)2; -O-(C2-C 10alkynyl), -S-(C2-C 10 alkynyl), -NH-(C2-C 10 alkynyl), or -N(C2-C 10 alkynyl)2; or -O-(C1-C 10 alkylene)-O-(C1-C 10 alkyl), -O-(C1-C 10 Alkylene)-NH-(C1-C 10 alkyl) or -O-(C1-C 10 Alkylene)-NH(C1-C 10 alkyl)2, -NH-(C1-C 10 alkylene)-O-(C1-C 10 alkyl), or -N(C1-C 10 Alkyl)-(C1-C 10 alkylene)-O-(C1-C 10 In some embodiments, 2'-OH is replaced with -H (deoxyribose). In some embodiments, 2'-OH is replaced with -F. In some embodiments, 2'-OH is replaced with -OR'. In some embodiments, 2'-OH is replaced with -OMe. In some embodiments, 2'-OH is replaced with -OCHCHOMe (MOE).

[0125] Modified sugars also include locked nucleic acids (LNAs). In some embodiments, locked nucleic acids have the structure shown below: A locked nucleic acid of the structure below is shown, where Ba represents a nucleobase or modified nucleobase described herein, and where R 2s is -OCH2C4'-.

[0126] [ka]

[0127] In some embodiments, the invention provides oligonucleotides comprising one or more modified internucleotide linkages independently having the structure of Formula I:

[0128] [ka]

[0129] During the ceremony, P * is an asymmetric phosphorus atom, Rp or Sp; W is O, S, or Se; Each of X, Y, and Z is independently -O-, -S-, -N(-LR 1 )-, or L; L is a covalent bond or an optionally substituted straight or branched C1-C 10 alkylene, and one or more methylene units of L are optionally and independently selected from optionally substituted C1-C6 alkylene, C1-C6 alkenylene, -C≡C-, -C(R')2-, -Cy-, -O-, -S-, -SS-, -N(R')-, -C(O)-, -C(S)-, -C(NR')-, -C(O)N(R') replaced by -, -N(R')C(O)N(R')-, -N(R')C(O)-, -N(R')C(O)O-, -OC(O)N(R')-, -S(O)-, -S(O)2-, -S(O)2N(R')-, -N(R')S(O)2-, -SC(O)-, -C(O)S-, -OC(O)-, or -C(O)O-; R 1 is halogen, R, or optionally substituted C1-C 50an aliphatic compound, wherein one or more methylene units are optionally and independently selected from optionally substituted C1-C6 alkylene, C1-C6 alkenylene, -C≡C-, -C(R')2-, -Cy-, -O-, -S-, -SS-, -N(R')-, -C(O)-, -C(S)-, -C(NR')-, -C(O)N(R') replaced by -, -N(R')C(O)N(R')-, -N(R')C(O)-, -N(R')C(O)O-, -OC(O)N(R')-, -S(O)-, -S(O)2-, -S(O)2N(R')-, -N(R')S(O)2-, -SC(O)-, -C(O)S-, -OC(O)-, or -C(O)O-; each R' is independently -R, -C(O)R, -CO2R, or -SO2R; or two R' on the same nitrogen, together with their intervening atoms, form an optionally substituted heterocyclic or heteroaryl ring; or two R' on the same carbon together with their intervening atoms form an optionally substituted aryl, carbocyclic, heterocyclic, or heteroaryl ring; -Cy- is an optionally substituted divalent ring selected from phenylene, carbocyclylene, arylene, heteroarylene, or heterocyclylene; each R is independently hydrogen or an optionally substituted group selected from C1-C6 aliphatic, phenyl, carbocyclyl, aryl, heteroaryl, or heterocyclyl; and each

[0130] [ka]

[0131] independently represents the connection to the nucleoside.

[0132] In some embodiments, the internucleotide linkage having the structure of Formula I is

[0133] [ka]

[0134] is.

[0135] Among other things, the present disclosure provides variously designed oligonucleotides, which may comprise various nucleobases and their patterns, sugar groups and their patterns, internucleotide linkages and their patterns, and / or additional chemical moieties and their patterns, as described herein. In some embodiments, the provided oligonucleotides can downregulate MuSK Ig3 domain protein expression, MuSK Ig3 domain gene expression, and / or MuSK Ig3 activation of BMP signaling levels, thereby increasing muscle regeneration. In some embodiments, the provided oligonucleotides can lead to a decrease in the expression, level, and / or activity of one or more MuSK Ig3 domains and / or their products in cells of a subject or patient. In some embodiments, the cells normally express or produce proteins encoded by MuSK Ig3 domains. In some embodiments, the provided oligonucleotides have a base sequence that consists of, comprises, or includes a portion (e.g., a span of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more contiguous bases) of the base sequence of an oligonucleotide disclosed herein, which can lead to a decrease in the expression, level, and / or activity of a MuSK Ig3 domain gene or gene product, wherein each T can be independently replaced with a U, or vice versa, and wherein the oligonucleotide includes at least one non-naturally occurring modification of the base, sugar, and / or internucleotide linkage.

[0136] As described herein, highly abundant full-length MuSK possesses a BMP-binding Ig3 domain, enhances BMP signaling, and influences muscle regeneration. In contrast, ΔIg3-MuSK has lower BMP signaling, promotes muscle regeneration, and / or prevents muscle fibrosis. In some embodiments, the present disclosure provides exon-skipping ASOs that switch MuSK from full-length MuSK to the muscle-promoting ΔIg3-MuSK splice form.

[0137] In some embodiments, the one or more skipped exons are selected from exons 6 and 7 or the MuSK gene. In some embodiments, exon 6 of MuSK is skipped. In some embodiments, exon 7 of MuSK is skipped. In some embodiments, both exons 6 and 7 of MuSK are skipped.

[0138] In various embodiments, the active compound is an oligonucleotide that induces the skipping of one or more exons in the MuSK gene. In various embodiments, the active compound is an oligonucleotide that induces the skipping of multiple exons in the MuSK gene. In some embodiments, the active compound is an oligonucleotide that induces the skipping of exon 6, exon 7, or both in the MuSK gene. In some embodiments, the active compound is an oligonucleotide that induces the skipping of exon 6 in the MuSK gene. In some embodiments, the active compound is an oligonucleotide that induces the skipping of exon 7 in the MuSK gene. In some embodiments, the active compound is an oligonucleotide that induces the skipping of exons 6 and 7 in the MuSK gene. In some embodiments, multiple oligonucleotides can be used together. In some such embodiments, two or more different exon skipping oligonucleotides (e.g., at least one that induces the skipping of exon 6 and one that induces the skipping of exon 7) can be used in combination. Alternatively or additionally, in some embodiments, at least one exon skipping oligonucleotide may be used in combination with at least one degradative oligonucleotide that can target, for example, a MuSK transcript containing a functional Ig3 domain or a portion thereof (e.g., targeting the transcript for RNase H degradation).

[0139] In some embodiments, oligonucleotides are provided and / or utilized in salt form. In some embodiments, oligonucleotides are provided as salts that include negatively charged internucleotide linkages (e.g., phosphorothioate internucleotide linkages, natural phosphate linkages, etc.) present in their salt form. In some embodiments, oligonucleotides are provided as pharmaceutically acceptable salts. In some embodiments, oligonucleotides are provided as metal salts. In some embodiments, oligonucleotides are provided as sodium salts. In some embodiments, oligonucleotides are provided as metal salts, e.g., sodium salts, where each negatively charged internucleotide linkage is independently in salt form (e.g., -OP(O)(SNa)-O- for phosphorothioate internucleotide linkages, -OP(O)(ONa)-O- for natural phosphate linkages, etc., for sodium salts).

[0140] Characterization of MuSK MR agonizing agents The MuSK muscle regeneration (MR) agonizing agents provided herein may be identified, assessed, and / or characterized for one or more of their physical / chemical properties and / or biological activities. Those skilled in the art will be aware of a variety of techniques, including specific assays, that may be utilized for such identification, assessment, and / or characterization.

[0141] In some embodiments, small molecule MuSK MR agonizing agents can interfere with the interaction between MuSK Ig3 and BMP, for example, by directly binding to MuSK Ig3 or to BMP. In some such embodiments, such agents can be characterized by direct binding assays to MuSK Ig3 and / or BMP (e.g., assessing their affinity for, specificity for, and / or one or more kinetic or thermodynamic properties of their interaction with their target) and / or by competitive binding assays (e.g., assessing their ability to disrupt or over-disrupt preformed complexes between MuSK Ig3 and BMP and / or reduce complex formation). In some embodiments, such binding assays are desirably performed at multiple concentrations; in some embodiments, such binding assays can be performed with full-length MuSK or with some other polypeptide or agent that is or includes MuSK Ig3.

[0142] In some embodiments, a MuSK MR agonizing agent (e.g., an antibody or small molecule that binds to the Ig3 domain of MuSK) will compete with BMP for binding of the Ig3 domain when contacted with a cell expressing MuSK. In some embodiments, such an antibody agent specifically binds to an epitope of MuSK that is expressed in a particular cell type (e.g., satellite cells). In some embodiments, such an antibody agent ... epitope is at least about 10 -4 M, at least about 10 -5 M, at least about 10 -6 M, at least about 10 -7 M, at least about 10 -8 M, at least about 10 -9The ligand may have a binding affinity (e.g., as measured by a dissociation constant) for a MuSK protein, e.g., the Ig3 domain of a MuSK protein, of or below M. Those skilled in the art will understand that in some cases, binding affinity (e.g., as measured by a dissociation constant) may be influenced by non-covalent intermolecular interactions, such as hydrogen bonding, electrostatic interactions, hydrophobicity, and van der Waals forces, between two molecules. Alternatively, or additionally, the binding affinity between a ligand and its target molecule may be influenced by the presence of other molecules. Those skilled in the art will be familiar with a variety of techniques for measuring binding affinity and / or dissociation constants in accordance with the present disclosure, including, but not limited to, ELISA, gel shift assays, pull-down assays, equilibrium dialysis, analytical ultracentrifugation, surface plasmon resonance (SPR), biolayer interferometry, grating-binding interferometry, and spectroscopic assays.

[0143] In some embodiments, a competitive assay can be used to identify antibodies that compete with the anti-MuSK antibody agents described herein for binding to the Ig3 domain of MuSK. In some embodiments, such competing antibodies bind to the same epitope within the Ig3 domain of MuSK that is bound by the anti-MuSK antibodies described herein. Exemplary epitope mapping methods are known. See, for example, Morris (1996).

[0144] In some embodiments, an assay may be provided to identify anti-MuSK antibody agents having biological activity. In some embodiments, an assay may be provided to identify anti-MuSK antibody agents having neutralizing activity against MuSK. Antibody agents having such biological activity in vivo and / or in vitro may also be provided. In some embodiments, the antibodies of the present disclosure may be tested for such biological activity.

[0145] The "biological activity" of an anti-MuSK antibody agent can include, for example, binding affinity for a particular MuSK epitope (e.g., within the Ig3 domain), neutralization or inhibition of MuSK binding to BMP, neutralization or inhibition of MuSK activity in vivo (e.g., IC 50 ), pharmacokinetics, and cross-reactivity (e.g., with non-human homologs or orthologs of MUSK protein, or with other proteins or tissues). Other biological properties or characteristics of an antigen-binding agent recognized in the art may include, for example, avidity, selectivity, solubility, folding, immunotoxicity, expression, and formulation. The foregoing properties or characteristics may be observed, measured, and / or assessed using standard techniques, including, but not limited to, ELISA, competitive ELISA, surface plasmon resonance analysis (BIACORE™), or kinetic exclusion assays (KINEXA™), in vitro or in vivo neutralization assays, receptor-ligand binding assays, cytokine or growth factor production and / or secretion assays, and signal transduction and immunohistochemistry assays.

[0146] In some embodiments, the MuSK MR agonizing agents described herein are characterized in that, for example, when the MuSK MR agonizing agent (e.g., an agonizing oligonucleotide) contacts a cell expressing MuSK, it will increase the level or activity of MuSK ΔIg3 mRNA and / or protein.

[0147] In some embodiments, MuSK MR agonizing oligonucleotide is characterized by its ability to change the splicing activity of MuSK pre-mRNA in cells.For example, MuSK MR agonizing oligonucleotide can be transfected into cells, and after a period of incubation, the expression of alternative processed forms of MuSK RNA transcripts (for example, exons 6 and 7 skipped) can be measured by RT-PCR.For example, the efficiency of MuSK exon skipping in cultured cells is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or greater than 95%.

[0148] In some embodiments, the MuSK MR agonizing oligonucleotide increases MuSK ΔIg3 mRNA. In some embodiments, the MuSK MR agonizing oligonucleotide alters the splicing of MuSK pre-mRNA. In some embodiments, the MuSK MR agonizing oligonucleotide promotes the skipping of exon 6 and / or exon 7.

[0149] The modulation of MuSK ΔIg3 expression can be measured in the body fluids of subjects treated with MuSK MR agonizing oligonucleotides, which may or may not contain animal cells, tissues, or organs. Methods for obtaining samples for analysis, such as body fluids (e.g., sputum, serum, CSF), tissues (e.g., biopsies), or organs, and methods for preparing samples to enable analysis are well known to those skilled in the art. The effect of treatment on a subject can be assessed by measuring biomarkers associated with target gene expression in one or more biological fluids, tissues, or organs collected from animals that have been contacted with one or more compositions described in the present application.

[0150] In some embodiments, an increase in MuSK ΔIg3 mRNA means that the intracellular level of MuSK ΔIg3 mRNA is higher than a reference level, such as the level of MuSK ΔIg3 mRNA in a control (e.g., in a subject that has not been administered a MuSK MR agonizing oligonucleotide). An increase in intracellular MuSK ΔIg3 mRNA can be measured as an increase in the level of produced MuSK ΔIg3 protein and / or mRNA. In some embodiments, an increase in MuSK ΔIg3 mRNA can be measured, for example, by the methods described in the Examples below, and / or by RNA solution hybridization, nuclease protection, Northern hybridization, reverse transcription, gene expression monitoring using microarrays, antibody binding, enzyme-linked immunosorbent assay (ELISA), nucleic acid sequencing, Western blotting, radioimmunoassay (RIA), other immunoassays, fluorescence-activated cell analysis (FACS), or other methods. It may be determined by assay techniques, such as any other technique or combination of techniques that can detect the presence of mRNA or protein (eg, in a subject or in a sample obtained from a subject).

[0151] In some embodiments, the level of MuSK ΔIg3 mRNA in the sample obtained from the subject who has undergone MuSK MR agonizing oligonucleotide treatment can be compared with the level of MuSK ΔIg3 mRNA in the subject who has not been treated with MuSK MR agonizing oligonucleotide, thereby determining the extent to which MuSK ΔIg3 mRNA is increased by MuSK MR agonizing oligonucleotide treatment.In some embodiments, the reference level of MuSK ΔIg3 mRNA is obtained from the same subject before undergoing MuSK MR agonizing oligonucleotide treatment.In some embodiments, the reference level of MuSK ΔIg3 mRNA is a range determined by a group of subjects who have not undergone MuSK MR agonizing oligonucleotide treatment.In some embodiments, the level of full-length MuSK mRNA is compared with the level of MuSK ΔIg3 mRNA. In some embodiments, the ratio of MuSK ΔIg3 mRNA (e.g., MuSK mRNA without exons 6 and 7) to full-length MuSK mRNA in a subject treated with a MuSK MR agonizing oligonucleotide is, for example, 1-fold, 1.5-5-fold, 5-10-fold, 10-50-fold, 50-100-fold, greater than about 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100-fold, or higher than a reference ratio.

[0152] In some embodiments, the increased level of MuSK ΔIg3 mRNA is, for example, 1-fold, 1.5-5-fold, 5-10-fold, 10-50-fold, 50-100-fold, about 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100-fold or higher than a reference value.

[0153] In some embodiments, an increase in MuSK ΔIg3 mRNA in a subject can be indicated by an increase in MuSK ΔIg3 protein compared to a reference level. In some embodiments, the reference level of MuSK ΔIg3 protein is the MuSK ΔIg3 protein level obtained from a subject who has or is at risk of having, for example, a neuromuscular dysfunction, a neurodegenerative disorder, a cardiac dysfunction (e.g., myocardial infarction, cardiomyopathy), or a genetic disease characterized by muscle wasting before treatment. Also contemplated are methods of contacting a body fluid, organ, or tissue with an effective amount of one or more compositions described herein. The body fluid, organ, or tissue can be contacted with one or more compositions comprising a MuSK MR agonizing oligonucleotide, resulting in the expression of MuSK ΔIg3 and modulation of MuSK expression in cells of the body fluid, organ, or tissue. The effective amount can be determined by monitoring the effect of the MuSK MR agonizing oligonucleotide administered to the subject or contacted with the cells on functional MuSK ΔIg3 protein expression.

[0154] In some embodiments, a MuSK MR agonizing agent, when administered to a population of cells (e.g., comprising satellite cells (SCs), myoblasts, myogenic progenitor cells (MPCs)), increases the number of cells in an activated state (e.g., actively proliferating). Cells within a population can be assessed for their activated state by methods known in the art, including, for example, an EdU assay, which compares EdU+ cycling cells to the total cell count. In some embodiments, a MuSK MR agonizing agent, when administered to a population of cells comprising satellite cells, decreases the number of quiescent satellite cells and / or increases the number of activated satellite cells in the population.

[0155] In some embodiments, a MuSK MR agonizing agent, when administered to a population of cells comprising SCs, MPCs, and / or myoblasts, increases the number of cells expressing genes or myogenic factors (e.g., Pax7, MyoD, myogenin, and MERGE) and / or decreases the number of cells expressing genes associated with the MuSK-BMP signaling pathway (e.g., RGS4, Msx2, Myf5, Ptx3, Id1). In some embodiments, a MuSK MR agonizing agent, when administered to a population of cells comprising satellite cells and / or myoblasts, increases the level of expression of genes associated with myogenic factors (e.g., Pax7, MyoD, myogenin, and MERGE) and / or decreases the level of expression of genes associated with the MuSK-BMP signaling pathway (e.g., RGS4, Msx2, Myf5, Ptx3, Id1) in the population of cells.

[0156] In some embodiments, the population of cells comprises satellite cells and / or myoblasts that have been induced to be satellite cells and / or myoblasts (e.g., from stem cells such as embryonic stem cells or pluripotent stem cells).

[0157] In some embodiments, the population of cells is obtained from a healthy subject. In some embodiments, the population of cells is obtained from a subject suffering from a disease or disorder, such as neuromuscular dysfunction, neurodegenerative disorder, cardiac dysfunction (e.g., myocardial infarction, cardiomyopathy), or a genetic disease characterized by muscle wasting.

[0158] In some embodiments, the MuSK MR agonizing agent increases muscle regeneration in a subject when contacted with a population of cells derived from the subject. In some embodiments, the MuSK MR agonizing agent is contacted with the population of cells in vivo, for example, by injection into the subject. In some embodiments, the MuSK MR agonizing agent is contacted with the population of cells ex vivo by obtaining a population of cells derived from the subject, and muscle regeneration is increased when the treated cells are reintroduced into the subject.

[0159] In some embodiments, a MuSK MR agonizing agent, when administered to a subject, will increase muscle regeneration and / or muscle growth, and / or neuromuscular function, and / or myogenesis. Exemplary methods for assessing these biological effects are detailed, for example, in the Examples below.

[0160] Models Among other things, the present disclosure provides useful model systems as described herein. For example, in some embodiments, the provided model systems can be used to screen, validate, characterize, assess and / or identify one or more MuSK MR agonizing agents.

[0161] In some embodiments, the model systems provided herein are or comprise artificially engineered cell lines. In some embodiments, the engineered cell lines are immortalized MuSK - / - It is a myogenic cell line.

[0162] In some embodiments, the model systems provided herein are or comprise engineered mice described herein, hi some embodiments, the mice provided are or comprise ΔIg3-MuSK mice.

[0163] In some embodiments, the provided model systems (e.g., cell lines and / or mice) are used to screen, validate, characterize, assess, and / or identify agents described herein, including, for example, small molecule agents, antibody agents, oligonucleotide agents, etc., and combinations thereof. In some embodiments, the activity of such agents is compared to an appropriate reference (e.g., a positive control and / or a negative control). In some embodiments, a suitable reference can be or include the absence of any agent or the presence of an agent whose activity or performance in the model system is known. In some embodiments, a suitable reference can be a historical reference. In some embodiments, a suitable reference can be an age-matched or contemporaneous reference.

[0164] Agonizing agent production antibody The antibodies and antigen-binding fragments of the present invention may be prepared and / or purified by any technique known in the art that allows for the subsequent formation of a stable antibody or antibody fragment.

[0165] Nucleic acid encoding an anti-MuSK antibody agent of the present disclosure can be readily isolated and sequenced by conventional procedures.

[0166] In some embodiments, expressed antibodies of the present disclosure can be purified to homogeneity after isolation from host cells. Isolation and / or purification of antibodies of the present disclosure can be carried out by conventional methods for isolating and purifying proteins. For example, without wishing to be bound by theory, MuSK antibody agents of the present disclosure can be recovered and / or purified from recombinant cell cultures by well-known methods, including, but not limited to, protein A purification, protein G purification, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography. High-performance liquid chromatography ("HPLC") can also be employed for purification. See, e.g., Colligan (1997-2001). In some embodiments, antibodies of the present disclosure can be isolated and / or purified by additional combinations of filtration, ultrafiltration, salting out, dialysis, and the like.

[0167] Purified anti-MuSK agents of the present disclosure can be characterized, for example, by ELISA, ELISPOT, flow cytometry, immunocytology, BIACORE™ analysis, SAPIDYNE KINEXA™ equilibrium exclusion assay, SDS-PAGE, and Western blot, or by HPLC analysis, as well as by numerous other functional assays disclosed herein.

[0168] Oligonucleotides Agonizing agent, for example, the agonizing oligonucleotide described herein, can be synthesized by standard methods known in the art, for example, by using an automatic synthesizer.After chemical synthesis (for example, solid-phase synthesis using phosphoramidite method), agonizing oligonucleotide molecule can be deprotected, annealed to ds molecule, and purified (for example, by gel electrophoresis or HPLC).The protocol for preparing agonizing oligonucleotide is known in the art.

[0169] Agonizing oligonucleotides can also be formed intracellularly by transcription of RNA from an expression construct introduced into cells. See, for example, Yu et al. (2002). Expression constructs for the in vivo production of agonizing oligonucleotide molecules can contain one or more antisense coding sequences operably linked to elements necessary for proper transcription of the antisense coding sequence, including, for example, promoter elements and transcription termination signals. Preferred promoters for use in such expression constructs include the polymerase III HI-RNA promoter (see, for example, Brummelkamp et al. (2002)) and the U6 polymerase III promoter (see, for example, Sui et al. (2002); Paul et al. (2002); and Yu et al. (2002)). Agonizing oligonucleotide expression constructs can further include one or more vector sequences that facilitate the cloning of expression constructs. Standard vectors that can be used include, for example, the pSilencer2.0-U6 vector (Ambion Inc., Austin, Tex.).

[0170] Pharmaceutical Compositions The present disclosure provides pharmaceutical compositions that contain and / or deliver the agonizing agents described herein. The present disclosure also provides pharmaceutical compositions that are or contain cell populations that have been exposed to the agonizing agents described herein.

[0171] For example, in some embodiments, provided pharmaceutical compositions may comprise and / or deliver a MuSK MR agonizing agent, such as an antibody or nucleic acid agent, that, when administered, achieves an increase in the level and / or activity of a MuSK polypeptide lacking a functional Ig3 domain for interaction with BMP (e.g., a MuSK ΔIg3 polypeptide, or another MuSK variant polypeptide having a truncated Ig3. Alternatively or additionally, in some embodiments, provided pharmaceutical compositions may comprise and / or deliver a population of cells that have been exposed to a MuSK MR agonizing agent, such that neuronal cell number and / or activity is increased in the population.

[0172] In many embodiments, the pharmaceutical composition will be or include an active agent (e.g., an agonizing agent or precursor thereof described herein) in combination with one or more pharmaceutically acceptable excipients. Those skilled in the art will understand that the components of a particular pharmaceutical composition may be affected by the route of administration of the pharmaceutical composition.

[0173] The compositions of the present disclosure can be formulated for a variety of modes of administration, including systemic and local or regional administration. Techniques and formulations can generally be found in Remington (2000).

[0174] The compositions of the present invention can be prepared and administered in a variety of oral, parenteral, and topical dosage forms.Therefore, the compositions of the present invention can be administered by injection (for example, intravenously, intramuscularly, intradermally, subcutaneously, intraduodenally, or intraperitoneally).The compositions described herein can also be administered by inhalation, for example, intranasally.In addition, the compositions of the present invention can be administered transdermally.It is also envisioned that the compositions of the present invention can be administered using multiple routes of administration (for example, intramuscularly, orally, transdermally).

[0175] In some embodiments, the pharmaceutical compositions described herein may be formulated for delivery by a route selected from intravenous injection, intrathecal administration, oral administration, buccal administration, inhalation, nasal administration, topical administration, ophthalmic administration, or otic administration. In some embodiments, the pharmaceutical composition may be formulated for delivery by intrathecal administration. In some embodiments, the pharmaceutical composition may be formulated for delivery by intravenous administration. In some embodiments, the pharmaceutical composition may be formulated for delivery by oral administration.

[0176] In certain embodiments, oligonucleotide and composition are delivered to CNS.In certain embodiments, oligonucleotide and composition are delivered to cerebrospinal fluid.In certain embodiments, oligonucleotide and composition are administered to brain parenchyma.In certain embodiments, oligonucleotide and composition are delivered to animal / subject by intrathecal administration or intraventricular administration.The wide distribution of the oligonucleotide and composition described herein in the central nervous system can be achieved by intraparenchymal administration, intrathecal administration or intraventricular administration.

[0177] In certain embodiments, parenteral administration is by injection, e.g., by syringe, pump, etc. In certain embodiments, the injection is a bolus injection. In certain embodiments, the injection is administered directly into tissues such as the striatum, caudate, cortex, hippocampus, and cerebellum.

[0178] In certain embodiments, methods of specifically localizing pharmaceutical agents, such as by bolus injection, involve administering a therapeutically effective concentration (EC 50 ) by 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, or 50-fold. In certain embodiments, the pharmaceutical agent in the antisense compound is as further described herein. In certain embodiments, the target tissue is brain tissue. In certain embodiments, the target tissue is hippocampal tissue. In certain embodiments, EC 50Reducing the dose is desirable because it lowers the dose required to achieve a pharmacological result in patients in need thereof.

[0179] In certain embodiments, the antisense oligonucleotides are delivered by injection or infusion once a month, every two months, every 90 days, every three months, every six months, twice a year, or once a year.

[0180] In addition to the active ingredient, these pharmaceutical compositions can contain suitable pharmaceutically acceptable carriers, including excipients and auxiliaries that facilitate the processing of the active compound into preparations that can be used pharmaceutically. Preparations formulated for oral administration can be in the form of tablets, dragees, capsules, or solutions.

[0181] Oral pharmaceutical preparations can be obtained by combining active compound with solid excipients, optionally grinding the resulting mixture, and then processing the granular mixture after adding suitable excipients if necessary to obtain tablets or dragee cores.Suitable excipients are, in particular, sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations, such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose (CMC), and / or fillers such as polyvinylpyrrolidone (PVP: povidone).If desired, disintegrants can be added, such as cross-linked polyvinylpyrrolidone, agar, or alginic acid or its salt, such as sodium alginate.

[0182] Sugar-coated core is provided with suitable coating.For this purpose, can be used concentrated sugar solution, which can optionally contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol (PEG) and / or titanium dioxide, lacquer solution and suitable organic solvent or solvent mixture.For identifying or characterizing various combinations of active compound dosage, dyes or pigments can be added to tablet or sugar-coated coating.

[0183] Orally usable pharmaceutical preparations include push-fit capsules made of gelatin, and soft sealed capsules made of gelatin, and plasticizers such as glycerol or sorbitol.Push-fit capsules can contain active ingredients mixed with fillers such as lactose, binders such as starch, and / or lubricants such as talc or magnesium stearate, and optionally stabilizers.In soft capsules, active compounds can be dissolved or suspended in suitable liquids such as fatty oils, liquid paraffin, or liquid polyethylene glycol (PEG).In addition, stabilizers can be added.

[0184] In some embodiments, the pharmaceutical composition is a tablet, pill, capsule, liquid, inhalant, nasal spray, suppository, suspension, gel, colloid, dispersion, suspension, solution, emulsion, ointment, lotion, eye drops, or ear drops.

[0185] Depending on the specific condition being treated, the pharmaceutical compositions of the present disclosure can be formulated into liquid or solid dosage forms and administered systemically or locally. Pharmaceutical compositions can be delivered, for example, in timed or sustained low-release forms, as known to those skilled in the art. Techniques for formulation and administration can be found in Remington (2000). Suitable routes include oral, buccal, inhalation spray, sublingual, rectal, transdermal, vaginal, transmucosal, nasal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injection, and intrathecal, direct intraventricular, intravenous, intraarticular, intrasternal, intrasynovial, intrahepatic, intralesional, intracranial, intraperitoneal, intranasal, or intraocular injection; or other modes of delivery.

[0186] For injection, the pharmaceutical compositions of the present disclosure may be formulated and diluted in aqueous solutions, such as in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer. For such transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

[0187] The use of pharmaceutically acceptable inert carriers to formulate the compositions disclosed herein for the practice of this disclosure into dosages suitable for systemic administration is within the scope of this disclosure. With proper selection of carriers and appropriate manufacturing practices, compositions of this disclosure, particularly those formulated as solutions, can be administered parenterally, such as by intravenous injection.

[0188] In some embodiments, the compositions described herein can be formulated into dosages suitable for oral administration using pharmaceutically acceptable carriers available in the art. Such carriers allow the compounds of the present disclosure to be formulated as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions, etc. for oral ingestion by a subject (e.g., a patient) to be treated.

[0189] For nasal or inhalation delivery, one or more solubilizing, diluent, or dispersing agents such as saline, preservatives such as benzyl alcohol, absorption enhancers, and fluorocarbons may be employed.

[0190] In some embodiments, provided compositions can include and / or deliver a precursor of an active agent, which becomes or releases an active therapeutic agent upon administration. In some embodiments, for example, the precursor can be or include a prodrug of a small molecule agonizing agent, a nucleic acid encoding a protein agonizing agent, or the like.

[0191] In certain embodiments, provided pharmaceutical compositions comprise or deliver a therapeutically effective amount (e.g., an amount that is effective when administered according to established protocols) of a provided oligonucleotide (which, as described herein, may be provided in a pharmaceutically acceptable salt form, e.g., as a sodium salt, ammonium salt, etc.); in some embodiments, such provided pharmaceutical compositions comprise the relevant oligonucleotide and at least one pharmaceutically acceptable inactive ingredient selected from a pharmaceutically acceptable diluent, a pharmaceutically acceptable excipient, and a pharmaceutically acceptable carrier. In some embodiments, salt forms of provided oligonucleotides contain two or more cations, e.g., in some embodiments, up to the number of negatively charged acidic groups (e.g., phosphate, phosphorothioate, etc.) in the oligonucleotide.

[0192] Pharmaceutically acceptable salts are generally well known to those skilled in the art and include, by way of example and not limitation, acetate, benzenesulfonate, besylate, benzoate, bicarbonate, bitartrate, bromide, calcium edetate, carnsylate, carbonate, citrate, edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexylurea, methyl ... The salts may include sorbate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, malate, maleate, mandelate, mesylate, mucate, napsylate, nitrate, pamoate (embonate), pantothenate, phosphate / diphosphate, polygalacturonate, salicylate, stearate, subacetate, succinate, sulfate, tannate, tartrate, or teoclate. Other pharmaceutically acceptable salts may be found, for example, in Remington, The Science and Practice of Pharmacy (20th ed., 2000). Preferred pharmaceutically acceptable salts include, for example, acetate, benzoate, bromide, carbonate, citrate, gluconate, hydrobromide, hydrochloride, maleate, mesylate, napsylate, pamoate (embonate), phosphate, salicylate, succinate, sulfate, or tartrate salts.

[0193] As will be understood by those skilled in the art, oligonucleotides can be formulated as several salts, for example, for pharmaceutical use. In some embodiments, the salts are metal cation salts and / or ammonium salts. In some embodiments, the salts are metal cation salts of oligonucleotides. In some embodiments, the salts are ammonium salts of oligonucleotides. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc. In some embodiments, the salts are sodium salts of oligonucleotides. In some embodiments, pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium, and amine cations formed with counterions such as hydroxide, carboxylate, sulfate, phosphate, nitrate, sulfonate, phosphorothioate, etc., which may be present in the provided oligonucleotide, as appropriate. As will be understood by those skilled in the art, oligonucleotide salts may contain more than one cation, such as sodium ion, since there may be more than one anion in the oligonucleotide.

[0194] In some embodiments, the provided oligonucleotides and compositions thereof may be effective over a wide dosage range. For example, in the treatment of an adult, dosages of about 0.01 to about 1000 mg, about 0.5 to about 100 mg, about 1 to about 50 mg per day, and about 5 to about 100 mg per day are examples of dosages that may be used. The exact dosage will depend on the route of administration, the form in which the compound is administered, the subject to be treated, the subject's weight, and the preferences and experience of the attending physician.

[0195] In some embodiments, the present disclosure provides techniques (e.g., compositions, methods, etc.) for combination therapy, e.g., with other therapeutic agents and / or medical procedures. In some embodiments, the provided oligonucleotides and / or compositions can be used together with one or more other therapeutic agents. In some embodiments, the provided compositions include the provided oligonucleotides and one or more other therapeutic agents. In some embodiments, the one or more other therapeutic agents may have one or more different targets and / or one or more different mechanisms directed to the target compared to the provided oligonucleotides in the compositions. In some embodiments, the therapeutic agent is an oligonucleotide. In some embodiments, the therapeutic agent is a small molecule drug. In some embodiments, the therapeutic agent is a protein. In some embodiments, the therapeutic agent is an antibody. Several therapeutic agents can be utilized in accordance with the present disclosure. In some embodiments, the provided oligonucleotides or compositions thereof are administered before, simultaneously with, or after one or more other therapeutic agents and / or medical procedures. In some embodiments, the provided oligonucleotides or compositions thereof are administered simultaneously with one or more other therapeutic agents and / or medical procedures. In some embodiments, the provided oligonucleotides or compositions thereof are administered before one or more other therapeutic agents and / or medical procedures. In some embodiments, the provided oligonucleotides or compositions thereof are administered after one or more other therapeutic agents and / or medical procedures. In some embodiments, the provided compositions include one or more other therapeutic agents.

[0196] Production of pharmaceutical compositions For preparing pharmaceutical compositions from the compositions of the present invention, pharmaceutically acceptable carriers can be either solid or liquid.Solid form preparations include powder, tablets, pills, capsules, cachets, suppositories and dispersible granules.Solid carriers can be one or more substances that can also act as diluents, flavoring agents, binders, preservatives, tablet disintegrating agents or encapsulating materials.

[0197] In powders, the carrier is a finely divided solid in admixture with the finely divided active ingredient. In tablets, the active ingredient is mixed with a carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired.

[0198] Powders and tablets preferably contain 5% to 70% of the therapeutic agent. Suitable carriers include magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, low-melting waxes, cocoa butter, and the like. The term "preparation" is intended to include the formulation of an active therapeutic agent with an encapsulating material as a carrier, with or without other carriers, in which the active ingredient is surrounded by the carrier, thus providing a capsule in which it is associated with the carrier. Similarly, cachets and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid dosage forms suitable for oral administration.

[0199] For preparing suppositories, a low melting wax, such as a mixture of fatty acid glycerides or cocoa butter, is first melted and the active ingredient is dispersed homogeneously therein by stirring, etc. The molten homogeneous mixture is then poured into convenient sized molds, allowed to cool, and thereby to solidify.

[0200] Liquid form preparations include solutions, suspensions, and emulsions, for example, water or water / propylene glycol solutions.For parenteral injection, liquid preparations can be formulated in solution in aqueous polyethylene glycol solution.

[0201] When parenteral application is required or desired, particularly suitable admixtures for the compositions of the present invention are injectable sterile solutions, preferably oily or aqueous solutions, as well as suspensions, emulsions, or implants, including suppositories. Particularly, carriers for parenteral administration include aqueous solutions of dextrose, saline, pure water, ethanol, glycerol, propylene glycol, peanut oil, sesame oil, polyethylene block polymers, etc. Ampoules are convenient unit dosages. The compositions of the present invention can also be incorporated into liposomes or administered via transdermal pumps or patches. Pharmaceutical admixtures suitable for use in the present invention include, for example, those described in Pharmaceutical Sciences (17th Edition, Mack Pub. Co., Easton, PA) and WO96 / 05309.

[0202] Aqueous solutions suitable for oral use can be prepared by dissolving the active ingredient in water and adding suitable colorants, flavorings, stabilizers, and thickeners as desired.Aqueous suspensions suitable for oral use can be made by dispersing the finely divided active ingredient in water with viscous materials such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, and other well-known suspending agents.

[0203] Also included are solid form preparations that are intended to be converted into liquid form preparations for oral administration shortly before use.Such liquid forms include solutions, suspensions, and emulsions.These preparations may contain, in addition to the active ingredient, colorants, flavorings, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizers, etc.

[0204] Pharmaceutical preparations are preferably in unit dosage form.In such form, the preparation is divided into unit doses containing appropriate amounts of active ingredients.The unit dosage form can be a packaged preparation, and the package contains individual amounts of preparation, such as tablets, capsules, and powders packed in vials or ampoules.Also, the dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form.

[0205] The quantity of the active component in a unit dose preparation may be varied or adjusted according to the particular application and the potency of the active component. The composition may also contain other compatible therapeutic agents, if desired.

[0206] Patient Populations: In some embodiments, suitable patients or populations are those suffering from and / or susceptible to a disease, disorder, such as neuromuscular dysfunction, cardiac dysfunction (e.g., myocardial infarction, cardiomyopathy), or a genetic disease characterized by muscle wasting, or that would otherwise benefit from increased muscle regeneration.

[0207] In some embodiments, the subject and / or population may additionally or alternatively suffer from and / or be susceptible to a disease, disorder, or condition that is a neurodegenerative disease, disorder, or condition, which may be one or more of Alzheimer's disease (AD), Parkinson's disease, dementia (e.g., frontotemporal dementia), stroke, major depressive disorder (MDD), bipolar disorder, schizophrenia, post-traumatic stress disorder (PTSD), substance-related and addictive disorders (e.g., chronic cocaine use and lifetime smoking), temporal lobe epilepsy, hippocampal sclerosis, Niemann-Pick disease type C, diabetic hippocampal neuronal loss, and Huntington's disease.

[0208] In some embodiments, the population may additionally or alternatively suffer from and / or be susceptible to a pulmonary disease or disorder, hi some embodiments, such disease or disorder is one or more of idiopathic pulmonary fibrosis (IPF), acute respiratory distress syndrome (ARDS), pneumonia, and pulmonary complications resulting from viral infection.

[0209] In some embodiments, the suitable patient or population is a model organism. In some embodiments, the suitable patient or population is a human. In some embodiments, the human has an age in the range of about 0 to about 6 months, about 6 to about 12 months, about 6 to about 18 months, about 18 to about 36 months, about 1 to about 5 years, about 5 to about 10 years, about 10 to about 15 years, about 15 to about 20 years, about 20 to about 25 years, about 25 to about 30 years, about 30 to about 35 years, about 35 to about 40 years, about 40 to about 45 years, about 45 to about 50 years, about 50 to about 55 years, about 55 to about 60 years, about 60 to about 65 years, about 65 to about 70 years, about 70 to about 75 years, about 75 to about 80 years, about 80 to about 85 years, about 85 to about 90 years, about 90 to about 95 years, or about 95 to about 100 years.

[0210] In some embodiments, the human is a human infant. In some embodiments, the human is a human toddler. In some embodiments, the human is a human child. In some embodiments, the human is an adult. In yet other embodiments, the human is an elderly human.

[0211] In some embodiments, suitable patients or populations may be defined and characterized by one or more criteria, such as age group, sex, genetic background, pre-existing clinical conditions, prior exposure to therapy, and the like.

[0212] In some embodiments, suitable patients or populations are those suffering from, for example, neuromuscular dysfunction, neurodegenerative disorders, cardiac dysfunction (e.g., myocardial infarction, cardiomyopathy), or genetic diseases characterized by muscle wasting. In some embodiments, suitable patients or populations are those undergoing surgery or experiencing injury, or those suffering from trauma and / or prolonged immobilization (e.g., from bed rest or casting). In some embodiments, suitable patients or populations are those suffering from sarcopenia. In some embodiments, suitable patients or populations are those suffering from or at risk of muscle fibrosis resulting from diseases or conditions, including, but not limited to, trauma, genetic diseases, muscle disorders, and aging. Trauma can result from, for example, radiation therapy, crush injury, laceration, and amputation. In some embodiments, suitable patients or populations are those suffering from or at risk for genetic diseases associated with muscle fibrosis, such as congenital muscular dystrophy, Duchenne muscular dystrophy, Becker muscular dystrophy, amyotrophic lateral sclerosis (ALS), age-related sarcopenia, distal muscular dystrophy, Emery-Dreifuss muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, myotonic muscular dystrophy, and oculopharyngeal muscular dystrophy.

[0213] In some embodiments, suitable patients or populations may be defined by screening tools for diseases or disorders associated with muscle fibrosis and / or muscle wasting. In some embodiments, suitable patients or populations may be defined by screening tools and methods for diagnosing diseases associated with muscle fibrosis and / or muscle wasting.

[0214] In some embodiments, suitable patients or populations may be defined according to the results obtained in structural imaging (e.g., magnetic resonance imaging (MRI), computed tomography (CT), ultrasound, etc.). In some embodiments, suitable patients or populations may be defined according to the results of neurological testing. In some embodiments, cognitive testing involves one or more of the following tests: Motor Screening Task (MOT), Reaction Time (RTI), Paired Associates Learning (PAL), Spatial Working Memory (SWM), Pattern Recognition Memory (PRM), Delayed Matching to Sample (DMS), and Rapid Visual Processing (RVP). Rapid Visual Processing (RVP), Delayed Matching to Sample (DMS), and Match to Sample Visual Search (MTS). In some embodiments, suitable patients or populations may be defined according to the results of assessments of measurements such as muscle enzymes, EMG, muscle biopsy, genetic testing, cardiac tests (e.g., ECG), strength, and respiratory function assessments.

[0215] Administration Those skilled in the art will understand that in some embodiments, the dosage administered to a subject, particularly a human, may vary depending, for example, on the particular therapeutic agent and / or formulation employed, the method of administration, the dosing regimen, one or more characteristics of the particular subject being treated, etc. In some embodiments, a clinician skilled in the art will determine the therapeutically effective amount of a therapeutic agent to be administered to a human or other subject to treat or prevent a particular medical condition. The precise amount of a therapeutic agent required to be therapeutically effective will depend on numerous factors, such as the specific activity of the therapeutic agent and the route of administration, in addition to many subject-specific considerations that are within the skill of the art.

[0216] In some embodiments, administration may be ophthalmic, oral, buccal, cutaneous (e.g., may be or may include one or more of topical to the dermis, intradermal, interdermal, transdermal, etc.), intestinal, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, intraspecific organ (e.g., intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal instillation), vaginal, intravitreal, etc.

[0217] Those skilled in the art will understand, upon reading this disclosure, that in some embodiments it may be desirable to achieve delivery of a MuSK MR agonizing agent to muscle. Alternatively or additionally, in some embodiments it may be desirable to achieve delivery of a MuSK MR agonizing agent to the CNS (e.g., the brain, such as the hippocampus and / or subventricular region) and / or lung.

[0218] In some embodiments, the agent (eg, agonizing agent) is delivered via systemic delivery and / or local delivery to muscle (eg, via intramuscular injection).

[0219] In some embodiments, MuSK MR agonizing agent is administered using a viral vector that effectively delivers MuSK MR agonizing agent in the form of nucleic acid payload.In some embodiments, viral vector targets certain cell types (such as myoblasts, muscle cells, myotubes, satellite cells and myofibrils).AAV1, AAV6 and AAV9 vectors are used to target different muscle cell types.For example, see Arnett et al. (2014) and Riaz et al. (2015).

[0220] Those skilled in the art will understand, upon reading this disclosure, that in some embodiments it may be desirable to achieve delivery of MuSK MR agonizing agents to the CNS, and in some embodiments, to the brain.

[0221] In some embodiments, systemic administration achieves delivery to the CNS (e.g., the brain, e.g., the hippocampus and / or subventricular zone). In some embodiments, an agent (e.g., an agonizing agent) is delivered to the central nervous system (CNS), e.g., via intraventricular administration.

[0222] In addition, certain viral vectors are known to selectively target neurons and effectively deliver genetic payloads to the brain. For example, AAV2 / 1 vectors have been demonstrated to effectively deliver nucleic acid payloads (e.g., gene therapy, encoded RNA, etc.) to neurons in the hippocampus. See, for example, Hammond et al. (2017); Guggenhuber et al. (2010); Lawlor et al. (2007). Similarly, certain AAV vectors (e.g., AAV2 / 1 and / or AAV4 vectors) have been demonstrated to target certain cells in the subventricular zone and effectively deliver nucleic acid payloads thereto. See, for example, Liu et al. (2005); Bockstael et al. (2012).

[0223] For subjects suffering from or susceptible to a disease, disorder or condition associated with neurodegeneration, administration that achieves delivery to the CNS, e.g., to the brain (e.g., to the hippocampus and / or subventricular region), may be desirable.

[0224] In some embodiments, effective delivery can be achieved by systemic administration of the compositions described herein. Alternatively or additionally, in some embodiments, effective delivery can be achieved by local administration to the CNS and / or brain, for example, by intrathecal and / or intracavitary (e.g., intracerebroventricular) delivery.

[0225] Techniques for local administration to the CNS and / or brain have been developed and have proven effective, for example, for various protein therapeutics (see, e.g., Calias et al. (2014)); for small molecules (see, e.g., Dodou (2012)); for cellular compositions (see, e.g., Eftekharzadeh et al. (2015)); and for nucleic acid therapeutics (see, e.g., Otsuka et al. (2011); see also the prescribing information for onasemnogene abeparvovec-xioi (sold under the trade name Zolgensma™) and for nusinersen (sold under the trade name Spinraza™)).

[0226] Those skilled in the art will know that intrathecal delivery can be particularly effective in achieving delivery to the hippocampus, including for cell, protein, and nucleic acid therapeutics.

[0227] Systemic administration techniques (including, e.g., oral, parenteral, mucosal, etc.) are well established for a wide variety of agents. Systemic administration to achieve CNS and / or brain delivery may, in some embodiments, rely on the ability to cross the blood-brain barrier (BBB).

[0228] Certain active agents and / or delivery systems are known to cross the BBB. Recent technologies have been shown to achieve CNS and / or even brain delivery of active substances such as oligonucleotides, which have historically been considered particularly difficult in this regard. As just one example, Min et al. (2020), incorporated herein by reference, describe glucose-coated polymeric nanocarriers that transport oligonucleotides across the BBB.

[0229] It has also been reported that incorporating certain chemical properties into oligonucleotide therapeutics can facilitate their transport across the BBB. For example, Khorkova et al. (2017) state that "2'-modified phosphorothioate oligonucleotides...may be particularly applicable to CNS disorders, given their long half-lives, with effects in the brain lasting up to 6 months after a single injection. In another type of sugar moiety modification, locked nucleic acids (LNAs), a bridge connecting the 2' oxygen and the 4' carbon is introduced. This modification substantially increases the melting temperature of LNA-DNA and LNA-RNA hybrids, thereby enabling the creation of shorter ODN-based compounds with increased bioavailability and reduced manufacturing costs. A recently proposed conformationally constrained oligonucleotide analog, tricyclo-DNA, has three additional C atoms between the C(5') and C(3') of the sugar (Figure 2). This modification increases stability, hydrophobicity, and RNA affinity, improving tissue uptake and BBB permeability." (Citations omitted.)

[0230] For subjects suffering from or susceptible to diseases or disorders such as idiopathic pulmonary fibrosis (IPF), acute respiratory distress syndrome (ARDS), pneumonia, and pulmonary complications resulting from viral infections, administration that achieves pulmonary delivery may be desirable.

[0231] Oligonucleotides In some embodiments, ASOs have been developed to enhance their delivery to target sites. As described in the art, oligos are covalently attached to carriers or ligands, such as lipid particles, liposomes, nanoparticles, and more recently, the sugar N-acetylgalactosamine, to enhance safe delivery to target sites. See Verma (2018).

[0232] Certain technologies have been developed to improve the efficiency of cellular delivery of ASOs to target sites, such as muscle. For example, aminoglycosides (AGs) have been shown to improve the delivery of antisense phosphorodiamidate morpholino oligomers (PMOs) both in vitro and in vivo. See Wang et al. (2019). Short cell-penetrating peptides (CPPs), which can be directly attached to oligonucleotides by covalent bonding or by forming noncovalent nanoparticle complexes, can facilitate cellular uptake. See McClorey and Banerjee (2018). ASO-fatty acid conjugates have also been reported to enhance the functional uptake of antisense oligonucleotides (ASOs) in muscle. See Prakash et al. (2019).

[0233] Those skilled in the art will recognize eteplirsen (ExonDys) an approved treatment for Duchenne muscular dystrophy (DMD), a third-generation phosphorodiamidate morpholino ASO. 51) is likely to be familiar with

[0234] Eteplirsen, sold under the trade name Exondys 51™ (Sarepta Therapeutics'), splices out exon 51 in the pre-mRNA, restoring the reading frame in 13% of patients with acceptable frameshift mutations. See Crudele and Chamberlain (2019).

[0235] Eteplirsen is administered via intravenous infusion over 35 to 60 minutes. The recommended dosage is 30 mg / kg body weight weekly. In single-dose vials, the pharmaceutical composition is formulated as a 100 mg / 2 mL or 500 mg / mL (50 mg / mL) solution.

[0236] In some embodiments, the oligonucleotide therapeutics described herein may be administered intravenously. In some such embodiments, such oligonucleotide therapeutics may be administered according to a regimen reasonably comparable to that used for eteplirsen (sold under the trade name Exondys 51™).

[0237] In some embodiments, the lower dose of the agonizing oligonucleotide described herein is 12 mg. In some embodiments, a total of 5 mg to 60 mg of agonizing oligonucleotide is administered to a subject per dose. In some embodiments, a total of 12 mg to 48 mg of agonizing oligonucleotide is administered to a subject per dose. In some embodiments, a total of 12 mg to 36 mg of agonizing oligonucleotide is administered to a subject per dose. In some embodiments, a total of 12 mg of agonizing oligonucleotide is administered to a subject per dose.

[0238] Those skilled in the art will be familiar with nusinersen (sold under the brand name Spinraza™), an antisense oligonucleotide therapeutic agent that targets gene transcripts directed to survival motor neuron-2 (SMN-2) and is indicated for the treatment of spinal muscular atrophy (SMA) in pediatric and adult patients. Spinraza is administered intrathecally. In particular, its recommended dosage is 12 mg / 5 mL (2.4 mg / mL) per single vial, following a regimen involving four loading doses, the first three of which are administered 14 days apart, with the fourth administered 30 days after the third dose; and maintenance doses administered once every four months thereafter. Platelet counts, coagulation laboratory tests, and quantitative spot urine protein tests are recommended at baseline and before each dose.

[0239] In some embodiments, the oligonucleotide therapeutics described herein may be administered intrathecally. In some such embodiments, such oligonucleotide therapeutics may be administered according to a regimen reasonably comparable to that used for nusinersen (sold under the trade name Spinraza™).

[0240] In some embodiments, the oligonucleotide therapeutics described herein may be administered intrathecally. In some such embodiments, such oligonucleotide therapeutics may be administered according to a regimen reasonably comparable to that used for nusinersen (sold under the trade name Spinraza™).

[0241] cell therapy Given the ability of the MuSK MR agonizing agents described herein to promote muscle regeneration (e.g., from cell populations that are or include SCs, MPCs, and / or myoblasts), one of skill in the art reading this disclosure will understand that, among other things, the present disclosure provides techniques for enhancing the levels of SCs, MPCs, and / or myoblasts present in a cell population. That is, contacting an original cell population with a MuSK MR agonizing agent described herein can produce a resultant population having an increased level and / or percentage of SCs, MPCs, and / or myoblasts compared to that in the original population; administration of such a MuSK MR agonizing agent described herein can achieve such an increase.

[0242] In some embodiments, the original cell population may be or comprise SC, MPC, and / or myoblast. In some embodiments, the original cell population is or comprises embryonic stem cells and / or pluripotent stem cells. In some embodiments, the embryonic stem cells and / or pluripotent stem cells are or have been differentiated into myogenic progenitor cells, for example, using techniques known in the art. See, for example, Miyagoe-Suzuki et al. (2017).

[0243] In some embodiments, as discussed above, such administration delivers a MuSK MR agonizing agent, thereby exposing it to (i.e., contacting) the relevant original cell population in vivo (e.g., in a human, particularly an adult, e.g., in the muscle tissue of such a human).

[0244] In some embodiments, administration according to the present disclosure involves ex vivo contact of a MuSK MR agonizing agent with a population of cells (e.g., a population of original cells) that may be or may contain, for example, SCs, MPCs, and / or myoblasts. For example, in some embodiments, the MuSK MR agonizing agent is administered ex vivo (e.g., in vitro) to a population of cells derived from a subject. In some embodiments, a population of cells obtained from a subject.

[0245] In some embodiments, the MuSK MR agonizing agent of particular use ex vivo may be or may include a small molecule and an antibody or nucleic acid agent, or a combination thereof. In certain such embodiments, one or more agents that are or include nucleic acids (e.g., one or more gene therapies [e.g., nucleic acid vectors and / or transcripts], oligonucleotides, and / or gRNAs) may be particularly useful for ex vivo and / or in vitro administration to cells. CRISPR / Cas modification of cell populations is an established and growing field, and those skilled in the art will be familiar with, for example, MuSK MR agonizing agents. One will understand the applicability of such strategies in accordance with the present disclosure to modify and / or disrupt Ig3 domain sequences. Alternatively or additionally, a nucleic acid encoding (or whose expression product encodes) a MuSK form lacking a functional Ig3 domain can be introduced into cells ex vivo and / or in vitro. Still further alternatively or additionally, oligonucleotides can be utilized that direct exon skipping of MuSK transcripts in favor of forms lacking functional Ig3 and / or that direct degradation of forms containing functional Ig3 (and / or block translation).

[0246] In some embodiments, the population of cells is contacted with, and simultaneously or subsequently stimulated and / or expanded by, a MuSK MR agonizing agent. Alternatively or additionally, the population of cells is enriched and / or selected for cells that exhibit characteristics of activated satellite cells, or for the expression of myogenic factors (e.g., Pax7, MyoD, myogenin, and MERGE), or for reduced / absent expression of genes associated with the MuSK-BMP signaling pathway (e.g., RGS4, Msx2, Myf5, Ptx3, Id1).

[0247] In some embodiments, the resulting cell population achieved by ex vivo contact of the original cell population with the MuSK MR agonizing agent is then administered to a subject.In some embodiments, the resulting cell population is administered to a subject suffering from or prone to a disease or disorder, such as neuromuscular dysfunction, neurodegenerative disorder, cardiac dysfunction (e.g., myocardial infarction, cardiomyopathy), or a genetic disease characterized by muscle wasting.In some embodiments, the resulting cell population is administered to the subject from which the original cell population is obtained.In some embodiments, the resulting cell population is administered to a subject different from the one from which the original cell population is obtained; in some such embodiments, the original cell population is obtained from a healthy subject, and the resulting cell population is administered to a subject suffering from or prone to a disease or disorder, such as neuromuscular dysfunction, neurodegenerative disorder, cardiac dysfunction (e.g., myocardial infarction, cardiomyopathy), or a genetic disease characterized by muscle wasting.

[0248] In some embodiments, administering a population of cells contacted with a MuSK MR agonizing agent effectively treats a disease or disorder in a subject, such as a neuromuscular dysfunction, a neurodegenerative disorder, a cardiac dysfunction (e.g., myocardial infarction, cardiomyopathy), or a genetic disease characterized by muscle wasting.

[0249] In some embodiments, the stimulated and / or expanded population of SCs, MPCs, and / or myoblasts described herein can be formulated into a cell therapy. In some embodiments, the cell therapy comprises a pharmaceutically acceptable carrier, diluent, and / or excipient. The pharmaceutically acceptable carriers described herein, such as vehicles, adjuvants, excipients, and diluents, are well known and readily available to those skilled in the art. Preferably, the pharmaceutically acceptable carrier is chemically inert to the active agent, such as the cell therapy, and does not elicit any adverse side effects or toxicity under the conditions of use.

[0250] In some embodiments, the cellular therapy may be formulated for administration by any suitable route, such as, for example, intravenous, intratumoral, intraarterial, intramuscular, intraperitoneal, intrathecal, epidural, and / or subcutaneous routes of administration. Preferably, the cellular therapy is formulated for a parenteral route of administration. In some embodiments, the cellular therapy is administered to a subject by infusion.

[0251] In some embodiments, cellular therapeutic agents suitable for parenteral administration may be aqueous or non-aqueous isotonic sterile injection solutions, which may contain, for example, antioxidants, buffers, bacteriostats, and solutes that render the composition isotonic with the blood of the intended recipient. Aqueous or non-aqueous sterile suspensions may contain one or more suspending agents, solubilizing agents, thickening agents, stabilizers, and preservatives.

[0252] In some embodiments, a single therapeutic cell described herein may increase and provide a therapeutic benefit. 2 or more, e.g., 10 3 10 or more 4 10 or more 5 or more, or 10 8 Alternatively or additionally, 10 or more therapeutic cells are administered as a cellular therapy. 12 or less, e.g., 10 1110 or less 9 10 or less 7 or less than 10 5 In some embodiments, 10 or fewer therapeutic cells described herein are administered to a subject as a cellular therapy. 2 ~10 5 , 10 4 ~10 7 , 10 3 ~10 9 , or 10 5 ~10 10 The therapeutic cells described herein are administered as a cellular therapy.

[0253] The dose of the cellular therapy agent described herein can be administered to a subject once or in a series of subdoses administered over an appropriate period of time, for example, daily, twice weekly, weekly, biweekly, twice monthly, bimonthly, twice yearly, or yearly, as needed. A dosage unit containing an effective amount of the cellular therapy agent can be administered in a single daily dose, or the total daily dosage can be administered in two, three, four, or more divided doses administered daily as needed. In some embodiments, the cellular therapy agent is administered in combination with another therapy.

[0254] Combination therapy In some embodiments, a MuSK MR agonizing therapy described herein is administered in combination with another therapy, ie, whereby the subject is exposed to both therapies simultaneously.

[0255] The dosage of the MuSK MR agonizing therapy described herein, and the dosage and dosing schedule of another therapy administered in combination, may depend on various parameters, including, but not limited to, the disease being treated (e.g., a neuromuscular dysfunction, neurodegenerative disorder, cardiac dysfunction, or genetic disease characterized by muscle wasting), the general health of the subject, and the discretion of the administering physician.

[0256] The MuSK MR agonizing therapy can be administered to a subject in need thereof prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of the other therapy. In various embodiments, the MuSK MR agonizing therapy and other therapy are administered 1 minute apart, 10 minutes apart, 30 minutes apart, less than 1 hour apart, 1 hour apart, 1-2 hours apart, 2-3 hours apart, 3-4 hours apart, 4-5 hours apart, 5-6 hours apart, 6-7 hours apart, 7-8 hours apart, 8-9 hours apart, 9-10 hours apart, 10-11 hours apart, 11-12 hours apart, less than 24 hours apart, or less than 48 hours apart. In one embodiment, the MuSK MR agonizing therapy and other therapy are administered within 3 hours. In another embodiment, the MuSK MR agonizing therapy and other therapy are administered within 1 minute to 24 hours apart.

[0257] A synergistic combination of MuSK MR agonizing therapy with other therapies may allow for the use of lower dosages of one or both of these agents and / or less frequent administration of therapies in subjects suffering from genetic diseases characterized by neuromuscular dysfunction, neurodegenerative disorders, cardiac dysfunction, or muscle wasting. A synergistic effect may result in improved efficacy of these agents and / or a reduction in any adverse or unwanted side effects associated with the use of either agent alone.

[0258] In some embodiments, MuSK MR agonizing therapy is administered in combination with standard of care treatment for the associated disease, disorder, or condition (e.g., neuromuscular dysfunction, neurodegenerative disorder, cardiac dysfunction, or genetic disease characterized by muscle wasting).

[0259] Therapies for DMD include deflazacort (Emflaza; PTC Therapeutics), eteplirsen (Exondys 51; Sarepta Therapeutics), ataluren (Translarna; PTC Therapeutics), and glucocorticoids such as prednisone. In some embodiments, MuSK MR agonizing therapy is administered in combination with one or more therapies for DMD.

[0260] Approved therapies for ALS include Radicava, Rilutek, Tiglutik, and Nuedexta. In some embodiments, the MuSK MR agonizing therapy is administered in combination with one or more therapies for ALS.

[0261] Approved therapies for cardiomyopathy include, but are not limited to, angiotensin II converting enzyme (ACE) inhibitors, angiotensin II receptor blockers (ARBs), and spironolactone. In some embodiments, MuSK MR agonizing therapy is administered in combination with one or more therapies for cardiomyopathy.

[0262] In some embodiments, MuSK MR agonizing therapy is administered in combination with one or more therapies that alleviate symptoms or characteristics of an associated disease, disorder, or condition, or with a therapy for the same. In some embodiments, MuSK MR agonizing therapy is administered in combination with one or more other therapies that alleviate symptoms or characteristics, thereby reducing side effects associated with the other therapies. In some embodiments, the side effects associated with the therapies are characterized by one or more of muscle cramps and spasms, constipation, fatigue, excessive saliva and phlegm, pain, depression, sleep disturbances, and uncontrolled outbursts of laughing or crying.

[0263] Any therapy known to be useful or that has been used and is currently being considered or used to treat or prevent neuromuscular dysfunction, neurodegenerative disorders, cardiac dysfunction, or genetic disorders characterized by muscle wasting may be used in combination with a MuSK MR agonizing therapy in accordance with the invention described herein. [Example]

[0264] [Example 1] The role of the MuSK-BMP pathway in regulating satellite cell dynamics and muscle regeneration in vivo Regeneration in constitutive ΔIg3-MuSK muscle The BMP signaling pathway promotes muscle regeneration and satellite cell proliferation in vivo. Several lines of evidence support a role for the MuSK-BMP pathway in this process. MuSK levels are upregulated in whole muscle during regeneration. We previously reported that the MuSK-BMP pathway regulates the expression of several transcripts encoding myogenic factors in cultured myoblasts and myotubes. Satellite cells have been reported to express MuSK mRNA, but the presence of MuSK protein in these cells has not been reported.

[0265] To determine whether satellite cells express MuSK protein, we performed immunohistochemistry (IHC) on intact myofibrils isolated from mouse hindlimb muscle and stained for Pax7, a satellite cell-specific protein in muscle, MyoD, expressed in activated satellite cells, and MuSK. As shown in Figure 1C, activated but not quiescent satellite cells express detectable MuSK protein, which resembles the expression pattern of BMPs in satellite cells.

[0266] ΔIg3-MuSK mice To investigate MuSK-BMP signaling in vivo, we used CRISPR / Cas9 to generate mice with a constitutive deletion of the Ig3 domain of MuSK (ΔIg3-MuSK mice), a domain required for high-affinity BMP binding.

[0267] A mouse model lacking the MuSK Ig3 domain was generated using CRISPR / Cas9 (Figure 7). A plasmid encoding hSpCas9 and a gRNA flanking the locus encoding the Ig3 domain (black triangles) was designed to excise an approximately 11 kb region, thereby generating a novel MuSK allele lacking the Ig3 coding domain (MuSK ΔIg3 ) were generated. Arrows indicate WT or MuSK. ΔIg3 Amplify either The gDNA sequence used to target a sequence within the MuSK gene is shown below.

[0268] Musk_sgRNAex6up1: TGCTCATATCTAAATGCGAT (SEQ ID NO: 3) Musk_sgRNAex7dw1: GCACTCCATGGCATCTGGAA (SEQ ID NO: 4) Musk_sgRNAex6up2: GAGCATAAATGTTCTAGACT (SEQ ID NO: 5) Musk_sgRNAex7dw2: CTCCATGGCATCTGGAAGGG (SEQ ID NO: 6) MuSK ΔIg3 Mice homozygous for WT and MuSK were selected by genotyping and confirmed by DNA sequencing. ΔIg3 Amplification of the genomic DNA of the alleles produces amplicons of 436 and 400 bp, respectively. WT mice have the WT MuSK allele, but the MuSK ΔIg3 Heterozygous MuSK ΔIg3 Mice are WT and MuSK, evidenced by products of 436 and 400 bp, respectively. ΔIg3 alleles, while MuSK ΔIg3 Homozygotes have a 436 bp MuSK ΔIg3 Amplify only the allele (Figure 7).

[0269] Our studies demonstrated that MuSK-BMP signaling was disrupted in primary myotubes cultured from ΔIg3-MuSK mice. Significantly reduced levels of MuSK-BMP-regulated transcripts were measured; for example, transcription of Wnt11 was defective (see Figure 3). In vivo studies also demonstrated that MuSK-BMP signaling was disrupted in these mice (see Figures 4B-C and Figures A-B).

[0270] We also used ΔIg3-MuSK mice to examine satellite cell proliferation in uninjured and injured regenerating muscles. As shown in Figure 4A, satellite cell numbers were found to be equivalent in uninjured tibialis anterior (TA) and soleus muscles between ΔIg3-MuSK and WT mice, suggesting that the MuSK-BMP pathway is not required for satellite cell development. In contrast, as shown in Figures 4B-C and 4A-B, satellite cell abundance at 5 dpi was higher in ΔIg3-MuSK compared to WT muscle. To test whether this difference was due to increased proliferation, mice were injected with 5-ethynyl-2'-deoxyuridine (EdU) on day 4 and observed that satellite cell proliferation was significantly higher in ΔIg3-MuSK muscle compared to WT muscle between 4 and 5 dpi (Figure 5B). These results suggest that MuSK-BMP signaling regulates satellite cell proliferation after injury.

[0271] The same regeneration experiments described above were performed, but tissues were harvested from mice at 7 dpi and 14 dpi (when muscle regeneration was nearly complete), and the results are also shown in Figures 4B-C and 5A. As shown in Figures 4D and 5B, satellite cell numbers returned to near baseline at 7 and 14 dpi.

[0272] Together, these data indicate that MuSK-BMP signaling regulates satellite cell activity during muscle regeneration and suggest that constitutive ΔIg3-MuSK mice will have altered muscle regeneration due to abnormal satellite cell proliferation and differentiation.

[0273] method Tibialis anterior muscles from constitutive ΔIg3-MuSK and WT mice were injured using 1.2% BaCl2 (in PBS). The injected BaCl2 disrupts muscle fibers but leaves the basement membrane and satellite cells intact, allowing for regeneration. For uninjured controls of both the TA contralateral to the injured muscle and another TA muscle, uninjured littermate mice were used to control for the effects of systemic factors released by the injured muscle. Mice were used at 5 months of age (when full muscle growth had been achieved). Regeneration was analyzed at four time points: 5 dpi, when satellite cell activation is at its peak; 7 dpi and 14 dpi, when the muscle is nearly regenerated; and 14 dpi, which is sufficient time to allow complete muscle regeneration in WT mice. 21 dpi. Mice are perfusion-fixed as the Pax7 antibody gives optimal staining under these conditions (see Figure 4C).

[0274] To assess muscle regeneration, TA muscles were harvested and weighed. IHC was performed by staining with DAPI, laminin, which indicates the myofibril cell membrane, and embryonic myosin heavy chain (emb-MyHC). The number of centrally nucleated myofibrils and the number of myofibrils expressing emb-MyHC were counted, both of which are markers of regenerated myofibrils. The weight of the injured muscle was compared with that of the control muscle, and the percentage of control muscle weight was calculated. The size of the regenerated myofibrils was also calculated using laminin staining and morphometry with image software.

[0275] Satellite cell dynamics were also compared at each time point, and the total number and percentage of satellite cells were determined at each stage (quiescent, proliferating, and differentiating). EdU (5-ethynyl-2'-deoxyuridine) was injected intraperitoneally 1–3 days before harvest. After harvest, EdU+ cells were detected using the Click-it EdU Cell Proliferation Assay Kit (Thermo Fisher Scientific) to identify satellite cells that were actively proliferating during the EdU administration period. As outlined in Figure 1A, muscle sections were also stained for quiescent and differentiating satellite cells for their individual unique protein expression profiles. All antibodies were commercially available or sourced from the Developmental Studies Hybridoma Bank. Differences in total satellite cell numbers or percentages of quiescent, proliferating, or differentiating satellite cells between ΔIg3-MuSK and WT muscles suggest that MuSK-BMP signaling regulates satellite cell dynamics during muscle regeneration. To confirm that BMP signaling is active, a readout of BMP signaling is obtained by staining for phosphorylated Smad1 / 5 / 8. Potential CRISPR / Cas9 off-target effects are controlled for by repeating key studies in a second independent ΔIg3-MuSK line generated.

[0276] Conditional satellite cell-regeneration in ΔIg3-MuSK muscle In addition to using constitutive ΔIg3-MuSK mice, we also generated mice (SC-ΔIg3-MuSK mice) in which the Ig3 domain of MuSK was conditionally ablated in satellite cells upon tamoxifen injection. MUSK is expressed in intact muscle and is upregulated during regeneration. We used tamoxifen-inducible conditional SC-ΔIg3-MuSK mice to determine whether the results are due to MuSK-BMP satellite cell autonomy, as opposed to contributions from MuSK-BMP signaling from other cell types. This conditional mouse model also provides information on whether MuSK-BMP signaling regulates the functional development of satellite cells, as disrupted MuSK-BMP activity can be induced after the mice reach adulthood. Conditional SC-ΔIg3-MuSK mice exhibit altered muscle regeneration due to abnormal SC proliferation and differentiation after injury.

[0277] method Mice with tamoxifen-induced ablation of the Ig3 domain of MuSK in satellite cells were generated and designated conditional SC-ΔIg3-MuSK mice. LoxP / LoxP We generated mice in which the Ig3 domain of MuSK is flanked by LoxP sites. Pax7CreERT2;nuclear-tdTomato mice were provided by Dr. Bradley Olwin. In conditional SC-ΔIg3-MuSK mice, generated from the cross of these two strains, Pax7 is normally expressed; CreERT2 is expressed under the Pax7 promoter. Upon tamoxifen administration, which induces CreERT2 activity, the Ig3 domain of MuSK is excised in all Pax7+ cells and therefore all satellite cells. Additionally, these mice express nuclear tdTomato under the Pax7 promoter at the safety locus Rosa26. This tdTomato labeling facilitates analysis because Pax antibody staining would be unnecessary and because fibers derived from satellite cells will express tdTomato.

[0278] Using SC-ΔIg3-MuSK mice, muscle injury and muscle regeneration assays and analyses are performed as described above, except that mice are injected with an appropriate dose of tamoxifen daily for 5 days prior to injury to induce Cre recombination.

[0279] statistical analysis T-tests are performed with appropriate post-hoc corrections for multiple comparisons (e.g., Bonferroni). The number of animals required for an experiment is determined using preliminary data and the G * A power analysis was performed using Power (version 3.1). These calculations indicate sufficient power to observe significance (Fig. 5A, 5 dpi, n = 4 mice / group, effect size (d) = 2.13). * Using power, it was determined that 7 animals per group was optimal for observation and an effect size (d) of 2.13 (a=0.05; power=0.95).

[0280] result Both constitutive ΔIg3-MuSK and conditional SC-ΔIg3-MuSK muscles exhibit altered regenerative capacity compared with WT control muscles after injury, with dysregulated satellite cell proliferation and differentiation observed due to disruption of MuSK-BMP signaling. The results described above indicate that these mice have increased satellite cell proliferation during regeneration, indicating that normal MuSK-BMP signaling prevents muscle hypertrophy. Furthermore, these findings indicate that constitutive ΔIg3-MuSK and conditional SC-ΔIg3-MuSK muscles have abnormal regenerated weight, number of regenerated fibers, and / or size of regenerated myofibrils when harvested 14 and 21 days after injury compared with WT muscles. Constitutive ΔIg3-MuSK and conditional ΔIg3-MuSK mice also have abnormal numbers of quiescent, proliferating, and differentiating satellite cells at all time points. The lack of significant differences in outcomes between constitutive and conditional ΔIg3-MuSK mice would indicate that MuSK-BMP signaling during satellite cell development does not contribute to the injury phenotype and would suggest that any defects are satellite cell-dependent and autonomous.

[0281] Alternatively, although there was no difference in final muscle regeneration, the timing and / or satellite cell kinetics in constitutive ΔIg3-MuSK TA muscles and conditional SC-ΔIg3-MuSK TA muscles differed compared to WT. This indicates that MuSK-BMP signaling regulates satellite cell kinetics, but other pathways that compensate for muscle regeneration are properly maintained, demonstrating that MuSK is a regulator of satellite cell kinetics.

[0282] Assessment of fiber size in regenerating wild-type and ΔIg3-MuSK muscle after damage As described above, a phenotype observed in regenerating ΔIg3-MuSK muscle is an increase in satellite cell number and proliferation at 5 dpi. Increased myofiber diameter is an indicator of accelerated regeneration. Therefore, this study compared myofiber size in regenerating WT and ΔIg3-MuSK TA muscles.

[0283] Muscles from WT and ΔIg3-MuSK TA were harvested 7 days after BaCl injury from 5-month-old mice. Muscles were frozen in Optimal Cutting Temperature (OCT) compound and stored at -80°C. Muscles were cryosectioned into 10-micron sections on charged slides and then immunoblotted using rabbit polyclonal anti-laminin (ab11575) from Abcam (Dapi) (4',6-diamidino-2-phenylindole; VECTASHIELD® Antifade with DAPI). Mounting medium) and stained for laminin. Slides were imaged at 20x magnification using a fluorescent microscope. Images were analyzed using the computer program MyoVision to measure fiber size and minimum ferret diameter. Programmatic fiber outlining was performed manually. DAPI staining was used to ensure that only regenerating fibers (i.e., with a central nucleus) were analyzed, as opposed to intact fibers. 1,000-2,000 fibers were analyzed per phenotype from four different animals.

[0284] As shown in Figure 6, the minimum ferret diameter was significantly increased in regenerating ΔIg3-MuSK compared to WT muscle at 7 dpi, this increase in diameter being indicative of accelerated regeneration.

[0285] Uninjured mice In addition, myofibril size and satellite cell number were assessed in uninjured WT and ΔIg3-MuSK mice.

[0286] WT and ΔIg3-MuSK TA muscles were collected from WT and ΔIg3-MuSK mice at 3 and 5 months of age. Muscles were frozen in Optimal Cutting Temperature (OCT) compound and stored at -80°C. Muscles were cryosectioned into 10-micron sections onto charged slides and then stained for Dapi (4',6-diamidino-2-phenylindole; VECTASHIELD® Antifade Mounting Medium with DAPI) and laminin using rabbit polyclonal anti-laminin (ab11575) from Abcam. Slides were imaged at 20x magnification using a fluorescent microscope. Images were analyzed using the computer program MyoVision to measure fiber size and minimum ferret diameter. Programmatic fiber outlining was performed manually. DAPI staining was used to ensure that only regenerating fibers (i.e., those with a central nucleus) were analyzed, as opposed to intact fibers. Between 1000 and 2000 fibers were analyzed for each phenotype from four different animals.

[0287] As shown in Figure 8, no differences in mean myofibril size or size distribution were observed between WT and ΔIg3-MuSK mice at 3 months. Significant differences were observed at 5 months. However, notably, 5-month-old ΔIg3-MuSK mice had increased myofibril size compared to 5-month-old WT mice. This finding indicates that ΔIg3-MuSK mice had increased muscle growth compared to WT mice when they were aged from 3 to 5 months. Mann-Whitney t-test: p<0.0001, n=3–4 mice, 500 total fibers analyzed per mouse.

[0288] Additionally, immunohistochemistry was performed on muscle sections to determine the number of satellite cells, as measured by Pax7+ cells, per region. The results, shown in Figure 9, indicate that in 3-month-old animals, the number of satellite cells was comparable between WT and ΔIg3-MuSK muscles. At 5 months, the amount of SCs in WT muscle remained unchanged, whereas the amount of satellite cells in Ig3-MuSK muscle was reduced by approximately half. Each point represents an animal. Unpaired t-test, n = 8 mice, p = 0.01.

[0289] As described herein, muscle regeneration is characterized by an explosive proliferation of satellite cells, as observed, for example, in the 5 dpi mice described above. In contrast, muscle growth is characterized by a decrease in satellite cell number, as growth occurs when satellite cells differentiate and fuse with each other and / or with existing muscle fibers (resulting in an increase in fiber size, as observed for uninjured ΔIg3-MuSK in Figure 8).

[0290] Thus, the findings described herein demonstrate that MuSK modulators (e.g., MuSK agonists) may be useful both in the context of muscle injury, muscle repair and / or muscle regeneration, and in the context of muscle growth (e.g., in muscle atrophy and / or muscle development).

[0291] [Example 2] The role of the MuSK-BMP pathway in regulating the satellite cell transcriptome Microarray analysis was performed on immortalized WT and MuSK- / - myogenic cell lines, demonstrating that MuSK modulates the magnitude and composition of transcriptional output in these cells. Several MuSK-regulated genes discovered in this study, such as inhibitor of differentiation 1 (Id1) and Myf5, are also known to regulate myogenesis. As shown in Figure 3, we now demonstrate that transcription of key MuSK-BMP-dependent genes is also reduced in primary myotubes derived from neonatal constitutive ΔIg3-MuSK mice. As noted above, BMP signaling promotes muscle regeneration, promotes satellite cell proliferation, and is downregulated during satellite cell differentiation. The data shown in Figures 4C-D and 5A indicate that disrupted MuSK-BMP activity leads to aberrant satellite cell proliferation during muscle regeneration in vivo.

[0292] RNA sequencing (RNA-seq) analysis of satellite cells from conditional SC-ΔIg3-MuSK mice may be used to probe the MuSK-BMP mechanism. In preparation for these investigations, we analyzed an existing RNA-seq dataset from NCBI's Gene Expression Omnibus (GEO) database (GSE121589), which included RNA-seq of satellite cells from uninjured and injured regenerating muscle. Raw data files were reprocessed by trimming adapters and low-quality reads using TrimGalore!, and the data were then aligned to the ENSEMBL GRCm38 mouse genome using HiSat2. StringTie was used to determine transcripts per million (TPM) in each sample, and DESeq2 was used to identify differentially expressed genes (DEGs).

[0293] Key MuSK-BMP-dependent genes were found to be highly regulated in WT satellite cells in regenerating muscle, as indicated by significant changes in TMP from satellite cells in uninjured muscle (see Table 1). Combined with the data described herein (Figures 4-5), these data suggest that conditional SC-ΔIg3-MuSK mice provide valuable insight into genes regulated by the MuSK-BMP pathway in satellite cells.

[0294] [Table 1]

[0295] Using conditional SC-ΔIg3-MuSK mice, we investigated the transcriptomes of primary quiescent satellite cells (Example 2A) and activated proliferating satellite cells (Example 2B) isolated from uninjured and injured muscles. This study revealed the MuSK-BMP-regulated transcriptome in satellite cells. Some of the same MuSK-BMP-regulated genes were identified in immortalized myoblasts and primary myotubes. For example, Id1, a downstream target of BMP, has higher expression in WT compared to Ig3-MuSK- / - satellite cells; Id1 is three-fold more activated in BMP-treated WT compared to MuSK- / - myoblasts; and Id1 is highly expressed in proliferating satellite cells. Id1 is known to negatively regulate MyoD and myogenin, and is therefore downregulated during satellite cell differentiation. DEGs in WT and ΔIg3-MuSK satellite cells were compared at baseline and after injury. The MuSK-BMP pathway is more active in proliferating satellite cells than in quiescent satellite cells and MuSK-BMP transcripts such as Id1 regulate satellite cell activity during regeneration.

[0296] Genes regulated by MuSK-BMP signaling in satellite cells To identify genes regulated by MuSK-BMP signaling in satellite cells, we first performed RNA-Seq on quiescent satellite cells from uninjured muscle. Because satellite cells account for a small percentage of total myonuclei in uninjured muscle, hindlimb muscles from uninjured mice were pooled to ensure sufficient satellite cell yield. Satellite cells were isolated from tamoxifen-induced and non-tamoxifen-induced conditional SC-ΔIg3-MuSK mice. The use of tamoxifen-induced and non-tamoxifen-induced conditional SC-ΔIg3-MuSK mice generated satellite cells with expression of WT MuSK or ΔIg3-MuSK and tdTomato under the Pax7 promoter. For simplicity, these are referred to as WT and ΔIg3-MuSK SC. tdTomato expression in these satellite cells was used for flow cytometric cell sorting using methods optimized for these cells. The Pax7CerERT2 allele was crossed with mice carrying a LoxP-transfected nuclear tdTomato gene, which was successfully used to track live cells. This yielded approximately 100-150K cells per mouse. These mice were then crossed with LoxP-transfected ΔIg3-MuSK mice to investigate the specific effects of reducing MuSK-BMP signaling in satellite cells.

[0297] In consultation with the Computational Biology Core (CBC), we performed RNA sequencing and subsequent bioinformatics to identify MuSK-BMP-dependent genes that are differentially expressed in satellite cells when the MuSK-BMP pathway is disrupted. cDNA libraries are prepared using the TruSeq RNA V2 (Illumina) library preparation kit. RNA integrity is checked prior to library construction. Approximately 50 million reads are sequenced per sample, as genes of interest represent a small fraction of the transcriptome sequenced. Data analysis is performed as previously performed for the publicly available GEO dataset described above, with assistance from the CBC. A list of DEGs between WT and ΔIg3-MuSK SCs is compiled to assess the role of the Ig3 domain of MuSK on gene expression in quiescent satellite cells. The 10 most relevant DEGs are validated by qRT-PCR. Ingenuity Pathway Analysis (Qiagen) is also performed using this data as a means to explore the role of the MuSK-BMP pathway in satellite cells.

[0298] Further dissection of genes regulated in satellite cells of regenerating muscle To further investigate the MuSK-BMP pathway and determine which genes are regulated in satellite cells of regenerating muscle, we performed RNA-seq analysis on WT and ΔIg3-MuSK satellite cells collected from injured, regenerating TA muscles. The same muscle injury protocol described in Example 1 was used, except that TA from both hind limbs was injured to increase satellite cell number. Since satellite cell proliferation has been reported to peak at 5 dpi, as shown in Figure 4E, satellite cells were examined at 5 dpi to demonstrate abnormal satellite cell proliferation in injured Ig3-MuSK muscles. At 5 dpi, satellite cell number increased more than 30-fold compared to that in uninjured muscles (see Figure 5A), indicating that regenerating TA muscles at 5 dpi have sufficient satellite cells for this purpose. Satellite cells were isolated using flow cytometry, and then RNA-seq and subsequent analysis were performed as described in Example 2A.

[0299] A potential hazard to this technique is that quiescent satellite cells are sorted from pooled hindlimb muscles to ensure a sufficiently high yield, while activated satellite cells are only sorted from regenerating TA muscles. To address any concerns about the effect of muscle fiber type, only the predominantly slow-twitch soleus muscles in the mouse hindlimb can be eliminated. While regenerating TA muscles yield sufficient numbers of activated satellite cells, gastrocnemius muscles can be selected instead to further increase cell yield.

[0300] Additionally, satellite cells begin activation immediately after harvest. Therefore, it is possible that cells described as "quiescent" from this study may actually be in the earliest stage of activation. To address this, high-resolution in situ hybridization (ISH) was performed on select MuSk-BMP-regulated transcripts to confirm their presence in quiescent satellite cells. As an alternative approach to this study, primary satellite cell cultures were generated from these mice and RNA-seq was performed with and without BMP4 treatment to probe the MuSK-BMP pathway. However, because satellite cells in culture are not in their niche and MuSK regulation of activation functions differently than in vivo, quiescent satellite cells cannot be examined using this method.

[0301] Statistical power: CBC offers a service to help perform a power analysis to determine an appropriate n. Prior to the RNA-seq experiment described above, consult with CBC to determine an appropriate n to be used for the RNA-seq experiment.

[0302] result The results described above suggest that the Ig3 domain of MuSK regulates the expression of BMP-regulated genes (Figure 2). This includes genes known to regulate satellite cell dynamics and myogenesis, such as Id1 and Myf5, as well as other novel genes not previously identified as involved in muscle regeneration. Furthermore, the transcriptome differences between WT and ΔIg3-MuSK satellite cells are greater in satellite cells from regenerating muscle (i.e., activated, proliferating satellite cells) compared with uninjured muscle (i.e., quiescent satellite cells). This is because the MuSK-BMP pathway regulates satellite cell proliferation and differentiation; however, this pathway is not involved in quiescence, as no detectable MuSK was observed in quiescent satellite cells in muscle fibers (Figure 1C).

[0303] [Example 3] The role of the MuSK-BMP pathway in muscle fibrosis Muscle fibrosis is the disruption of functional parenchyma by interstitial elements and is an often overlooked sequela of traumatic muscle injury, aging, and congenital disorders.

[0304] The remarkable regenerative capacity of skeletal muscle depends on its interaction with the same interstitial connective tissue elements as myogenic precursors, which are responsible for the initiation and propagation of fibrosis. Despite this remarkable capacity for regeneration, fibrotic replacement of functional muscle by stromal elements is well documented in response to trauma, genetic disease, and aging. Indeed, muscle fibrosis poses considerable clinical problems for patients following radiation therapy, crush injuries, lacerations, and amputations, resulting in progressive loss of function and significant morbidity.

[0305] The extracellular matrix (ECM) is an essential component of skeletal muscle. It provides the skeletal structure that holds myofibrils and capillaries, as well as the nerves that supply the muscle. Additionally, it plays a key role in the force transmission, maintenance, and repair of muscle fibers. Excessive accumulation of ECM components, especially collagen, due to excessive ECM production, altered ECM degradative activity, or a combination of both, is defined as fibrosis.

[0306] Recent investigations have begun to elucidate the biomolecular mechanisms underlying the balance between muscle regeneration and muscle fibrosis. Although the deposition of extracellular elements and proliferation of interstitial cells after injury appear to underlie the pathogenesis of fibrosis formation, these same elements have proven essential for successful regeneration after injury, suggesting a critical, well-regulated balance between functional muscle tissue and the surrounding connective tissue.

[0307] Skeletal muscle fibrosis impairs muscle function, negatively impacts muscle regeneration after injury, and increases muscle susceptibility to re-injury. Therefore, it is considered a major cause of muscle weakness. Skeletal muscle fibrosis is a hallmark of muscular dystrophy, aging, and severe muscle damage. Fibrosis also plays a major role in many muscle disorders, including congenital muscular dystrophy, Duchenne muscular dystrophy, Becker muscular dystrophy, age-related sarcopenia, muscle repair after injury, amyotrophic lateral sclerosis (ALS, also known as Lou Gehrig's disease), and other muscle-wasting conditions. Therefore, a better understanding of the mechanisms of muscle fibrosis will advance our understanding of the events occurring in dystrophic muscle diseases and help develop innovative anti-fibrosis therapies that reverse fibrosis in such conditions. Furthermore, agents that alleviate fibrosis will be beneficial in these conditions.

[0308] The MuSK-BMP pathway regulates satellite cell proliferation and differentiation, suggesting that it plays an important role in muscle regeneration. This example explored whether this pathway is also involved in muscle fibrosis.

[0309] Tibialis anterior muscles were injured with BaCl2 injection. At 5, 7, and 14 days post-injury (dpi), samples of wild-type and ΔIg3-MuSK muscles were acquired and stained with either hematoxylin and eosin (H&E) or an antibody against the extracellular matrix protein laminin. As shown in Figure 4B, at 14 dpi, WT myofibrils in H&E-stained muscle are separated by "distinct" spaces, whereas myofibrils in ΔIg3-MuSK muscle are tightly packed with minimal extracellular space. Such spacing in H&E is characteristic of accumulated extracellular matrix. Laminin staining confirms this interpretation. As can be seen in Figure 4C, the "spaces" between myofibrils contain abundant extracellular matrix, as indicated by interstitial immunoreactivity.

[0310] These data suggest that the MuSK-BMP pathway also plays an important role in muscle fibrosis and that the third immunoglobulin domain of MuSK, Ig3, provides a therapeutic target for preventing and / or treating muscle fibrosis.

[0311] [Example 4] MuSK MR agonizing oligonucleotide Design and Synthesis of Exon-Skipping ASOs. Without wishing to be bound by any theory, the MuSK MR agonizing oligonucleotides described herein are designed according to, but not limited to, the following general guidelines (see Aartsma-Rus et al. (2012)): · RNA or DNA is modified for resistance to endonucleases or exonucleases (e.g., 2'MoE, 2'OMe, PMO, phosphorothioates); ·Designed against a target sequence; Typically 12-25 nucleotides, more optimally 17-20; Typically most effective at melting temperatures above 48°C; Typically most effective at 40%-60% GC content to prevent steric hindrance / dimerization and accessibility to the target; ·Typically, they most effectively target open / accessible pre-mRNA structures; Typically, splice regulatory or exon-specific sites are most effectively targeted (e.g., intronic splice enhancers, intronic splice silencers (e.g., Spinraza targeting the ISS of SMN2 exon 7), exonic splice enhancers, exonic splice silencers); Typically, sequence compositions containing no more than two guanine (G) or cytosine (C) nucleotides in direct succession are most effective (e.g., CCC or GGG).

[0312] ASO Chemistry. We plan to develop 2'-O-2-methoxyethyl (2'MOE) ASOs that also contain phosphorothioate linkages in the sugar backbone. Methods for designing and testing such ASOs are well established, including manufacturing, pharmacokinetics, biodistribution, and toxicology in rodents and non-human primates (Bennett and Swayze, 2010; Chiriboga et al., 2016; Hua et al., 2015; Mercuri et al., 2018; Rigo et al., 2014). The MOE group added to the 2' position of the ribose increases the Tm by approximately 2°C per residue, thus enhancing binding affinity and improving nuclease resistance. The phosphorothioate modification confers further nuclease resistance and increases affinity for plasma proteins, resulting in ASOs that are efficiently distributed to tissues and taken up by cells with pharmaceutical needs. This chemistry is off-patent, providing commercial advantages.

[0313] ASO design. ASOs will be synthesized by a commercial facility and provided by Bolden Therapeutics, Inc. to the Fallon and Webb lab at Brown University for screening. The strategy for designing ASOs will involve scanning exon and intron sequences flanking both the 5' and 3' splice sites using overlapping ASOs (1-2 bp shifts per oligo). The optimal length of the ASO is approximately 17-mer, which provides a good balance between target specificity and drug exposure. ASOs will be prescreened in silico for potential off-target effects, as well as compositional bias (GC content) and propensity for unwanted dimer formation.

[0314] ASOs will be designed such that they induce skipping of both exons 6 and 7 in MuSK (Figure 3). Importantly, these two exons are coordinately spliced ​​in vivo (Garcia-Osta et al., 2006; Hesser et al., 1999).

[0315] Screening and selection of optimal exon-skipping ASOs We plan to design RT-qPCR TaqMan assays to specifically quantify the following distinct MuSK splice forms: 1) full-length (FL) MuSK; 2) the desired product Δexon encoding ΔIg3-MuSK; and 3) the Δexon and Δexon of potential "incomplete" skipping isoforms. We plan to perform conventional RT-PCR in parallel to detect any unexpected products. All screening will be performed in mouse C2C12 myoblasts. This cell line endogenously expresses MuSK and is efficiently transfected using standard methods such as Lipofectamine 2000. Cells will be transfected with several concentrations of candidate ASOs ranging from approximately 0.1 to 10 nM. After one day of treatment, RNA will be extracted, and splicing will be measured by RT-qPCR to assess exon skipping efficiency. Our goal is to isolate at least one ASO that induces coordinate splicing of exons 6 and 7 in ≧80% of cases.

[0316] Testing selected ASOs for their ability to inhibit MuSK-BMP signaling in cultured cells We observed that knock-in mice constitutively expressing only ΔIg3-MuSK showed increased satellite cell numbers and increased myofiber size after injury compared with WT mice, indicating enhanced muscle regeneration. However, because in vivo skipping may be less than 100% efficient (Rigo et al., 2014), it is important to establish the relationship between the level of skipping achieved and physiological effects. Therefore, to this end, we plan to measure the level of MuSK-BMP-dependent signaling in ASO-treated cells.

[0317] We plan to use qRT-PCR to measure the levels of MuSK-BMP-dependent transcripts (e.g., Dok7 and Wnt11; Figure 4, or RGS4; Yilmaz et al., 2016). We have extensive experience with this system, gained during the discovery and characterization of MuSK as a BMP coreceptor (Yilmaz et al., 2016). Cells treated with either exon skipping or control ASOs will be stimulated with BMP for 2 hours. Transcript levels will then be measured, and the response to BMP will correlate with the degree of exon skipping.

[0318] The data from ΔIg3-MuSK mice provided herein support that increasing ΔIg3-MuSK expression provides beneficial effects. The present disclosure recognizes that, in some embodiments, skipping exons 6 and 7 may be inefficient with a single ASO. In some embodiments, it may be desirable to prepare one or more ASOs directed against exon 7 for use alone and / or with ASOs directed against the exons.

[0319] Those skilled in the art will understand, upon reading this disclosure, that in some embodiments it may be desirable to replicate studies (e.g., at least three times) and / or analyze the data with appropriate statistical methodology (e.g., by t-test with appropriate correction for multiple comparisons (e.g., Bonferroni)).

[0320] The work described herein provides techniques for the efficient development of ASO-mediated therapies for neuromuscular diseases and disorders to enhance muscle regeneration.

[0321] [Example 5] Immortalized ΔIg3-MuSK cell line This example provides a ΔIg3-MuSK cell line used as a model system for screening, validating, characterizing, assessing and / or identifying one or more MuSK MR agonizing agents described herein in vitro.

[0322] To generate the ΔIg3-MuSK cell line, primary myoblasts were transfected with wild-type and MuSK-Ig3 cells carrying at least one copy of the immortalizing transgene H-2Kb-tsA58 (Pimentel et al., 2017; Morgan et al., 1994). - / - Cells were isolated from the hind limbs of neonatal mice. Cells were cultured in DMEM growth medium containing 20% ​​FBS, 1% penicillin / streptomycin, 2% L-glutamine, 1% chicken embryo extract, and 1% IFN-γ at 33°C and 10% CO2. Cells were subcloned by plating at a density of 0.5 cells / well onto Matrigel-coated 96-well plates. Individual clones were selected for expansion based on morphology and tested for their ability to expand and grow on gelatin substrates. Myogenicity was tested by plating in differentiation medium (DMEM with 5% horse serum, 1% penicillin / streptomycin) at 37°C and 10% CO2, and myogenic clones were expanded.

[0323] This cell line can be used to screen, verify, characterize, assess and / or identify the agents described herein, including, for example, small molecule agents, antibody agents, oligonucleotide agents, etc., and combinations thereof. For example, the immortalized ΔIg3-MuSK myogenic cell line can be used in high-throughput screening of small molecule MuSK MR agonizing agents. Examples of small molecule MuSK MR agonizing agents include small molecules that target one or more of the type I BMP receptors ALK3 (ALK is anaplastic lymphoma kinase) and ALK6, and the type I activin receptor ALK4, such as ALK inhibitors (e.g., crizotinib, ceritinib, alectinib, brigatinib, lorlatinib).

[0324] The gene expression profile of the ΔIg3-MuSK cell line can be observed in response to treatment / exposure with a particular MuSK MR agonizing agent, for example, to determine the effect and characterize the agent on muscle growth and regeneration. Cellular assays measuring proliferation and differentiation markers can be used to characterize MuSK MR agonizing agents.

[0325] References Aartsma-Rus, A., et al., (2012). Overview on AON design. Methods Mol. Biol. 867: 117-129. Akins, MR, et al., (2017). Axonal ribosomes and mRNAs associate with fragile X granules in adult rodent and human brains. Human molecular genetics 26: 192-209. Allen, TM, (2002). Ligand-targeted therapeutics in anticancer therapy. Nature Reviews Cancer 2:750-763. Altschul et al., (1990). Basic local alignment search tool. J. Mol. Biol., 215(3): 403-410. Altschul et al., (1997). Methods in Enzymology; Altschul et al., Nucleic Acids Res. 25:3389-3402. Arnett, A.L.H., et al., (2014). Adeno-associated viral vectors do not efficiently target muscle satellite cells. Molecular Therapy - Methods & Clinical Development (2014) 1, 14038. Baxevanis et al., (1998). BIOINFORMATICS: A PRACTICAL GUIDE TO THE ANALYSIS OF GENES AND PROTEINS, Wiley. Bennett, C.F. and Swayze, E.E. (2010). RNA targeting therapeutics: molecular mechanisms of antisense oligonucleotides as a therapeutic platform. Annu. Rev. Pharmacol. Toxicol. 50: 259-293. Bentzinger, C.F., et al., (2012). Building muscle: molecular regulation of myogenesis. Cold Spring Harbor PERSPECTIVES IN BIOLOGY 4. Bockstael, O., et al., (2012). Rapid Transgene Expression in Multiple Precursor Cell Types of Adult Rat Subventricular Zone Mediated by Adeno-Associated Type 1 Vectors. Human Gene Therapy 23: doi.org / 10.1089 / hum.2011.216, Berge, S.M., et al., (1977). Pharmaceutical salts. J. Pharmaceutical Sciences, 66: 1-19. Bowen, D.C., et al., (1998). Localization and regulation of MuSK at the neuromuscular junction. Developmental Biology 199: 309-319. Brummelkamp, T.R., et al., (2002). A system for stable expression of short interfering RNAs in mammalian cells. Science; 296:550-553. Calias, P., et al., (2014). Intrathecal delivery of protein therapeutics to the brain: A critical reassessment. Pharmacology & Therapeutics, 114(2), 114-122. Charville, G.W., et al., (2015). Ex Vivo Expansion and In Vivo Self-Renewal of Human Muscle Stem Cells. Stem Cell Reports 5: 621-632. Chiriboga, C.A., et al., (2016). Results from a phase 1 study of nusinersen (ISIS-SMN(Rx)) in children with spinal muscular atrophy. Neurology 86(10): 890-897. Chothia C. and Lesk, A.M. (1987). Canonical structures for the hypervariable regions of immunoglobulins. J. Mol. Biol. 196(4): 901-917. Chothia C., et al., (1989). Conformations of immunoglobulin hypervariable regions. Nature 342(6252): 877-883. Cohen, S., et al., (2015). Muscle wasting in disease: molecular mechanisms and promising therapies. Nature reviews. Drug discovery 14: 58-74. Collins, C.A., et al., (2005). Stem cell function, self-renewal, and behavioral heterogeneity of cells from the adult muscle satellite cell niche. Cell 122: 289-301. Colligan, (1997-2001). Current Protocols in Immunology, or Current Protocols in Protein Science, John Wiley & Sons, NY, N.Y., e.g., chapters 1, 4, 6, 8, 9, and 10. Crudele, J.M. and Chamberlain, J.S. (2019). AAV-based gene therapies for the muscular dystrophies. Human Molecular Genetics, 28: R102-R107. CURRENT PROTOCOLS IN IMMUNOLOGY (CPI) (2003). John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc. (ISBN 0471142735, 9780471142737). CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (CPMB), (2014). Frederick M. Ausubel (ed.), John Wiley and Sons (ISBN 047150338X, 9780471503385). CURRENT PROTOCOLS IN PROTEIN SCIENCE (CPPS), (2005). John E. Coligan (ed.), John Wiley and Sons, Inc. Davis et al., (2012). BASIC METHODS IN MOLECULAR BIOLOGY, Elsevier Science Publishing, Inc., New York, USA (ISBN 044460149X). DeChiara, T.M., et al., (1996). The receptor tyrosine kinase MuSK is required for neuromuscular junction formation in vivo. Cell 85: 501-512. Dodou, K., (2012). Intrathecal route of drug delivery can save lives or improve quality of life. Pharm J. 289: 501. Eftekharzadeh, M., et al., (2015). The effect of intrathecal delivery of bone marrow stromal cells on hippocampal neurons in rat model of Alzheimer’s disease. Iran J. Basic Med. Sci. 18: 520-525. Egner, I.M., et al., (2016). Satellite cell depletion prevents fiber hypertrophy in skeletal muscle. Development 143: 2898-2906. Faul, F., et al., (2009). Statistical power analyses using G*Power 3.1: tests for correlation and regression analyses. Behavior Research Methods 41: 1149-1160. Garcia-Osta, A., et al., (2006). MuSK Expressed in the Brain Mediates Cholinergic Responses, Synaptic Plasticity, and Memory Formation. Journal of Neuroscience 26 (30): 7919-7932. Gozo, M.C., et al., (2013). Foxc2 induces Wnt4 and Bmp4 expression during muscle regeneration and osteogenesis. Cell death and differentiation 20: 1031-1042. Green, M.R. and Sambrook, J. (2012). MOLECULAR CLONING: A LABORATORY MANUAL, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (ISBN 1936113414). Groffen, A.J., et al., (1998). Primary structure and high expression of human agrin in basement membranes of adult lung and kidney. Eur. J. Biochem. 254(1): 123-128. Guggenhuber, S., et al., (2010). AAV Vector-Mediated Overexpression of CB1 Cannabinoid Receptor in Pyramidal Neurons of the Hippocampus Protects against Seizure-Induced Excitoxicity. PLoS One 5:e15707. Hammond, S.L., et al., (2017). Cellular selectivity of AAV serotypes for gene delivery in neurons and astrocytes by neonatal intracerebroventricular injection. PLoS One 12:e0188830. Harris, J.B. (2003). Myotoxic phospholipases A2 and the regeneration of skeletal muscles. Toxicon: Official Journal of the International Society on Toxinology 42: 933-945. Hesser, B.A., et al., (1999). Identification and characterization of a novel splice variant of MuSK. FEBS Letters 442: 133-137. https: / / vectorlabs.com / vectashield-mounting-medium-with-dapi.html https: / / www.abcam.com / laminin-antibody-ab11575.html https: / / www.myovision.com Hua, Y. et al., (2015). Motor neuron cell-nonautonomous rescue of spinal muscular atrophy phenotypes in mild and severe transgenic mouse models. Genes Dev. 29(3): 288-297. Ibebunjo, C. and Martyn, J. (2001). Disparate dysfunction of skeletal muscles located near and distant from burn site in the rat. Muscle & nerve 24: 1283-1294. IMMUNOLOGY, (2006). Werner Luttmann, published by Elsevier. Isselbacher, et al., (1996). HARRISON’S PRINCIPLES OF INTERNAL MEDICINE, 13 ed., 1814-1882. JANEWAY'S IMMUNOBIOLOGY, (2014). Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), Taylor & Francis Limited, (ISBN 0815345305, 9780815345305). Janssen, I., et al., (2000). Skeletal muscle mass and distribution in 468 men and women aged 18-88 yr. Journal of applied physiology 89: 81-88. Kabat, et al., (1991). SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST. Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91 3242. 18 Id. Khorkova, O. and Wahlestedt, C. (2017). Oligonucleotide therapies for disorders of the nervous system. Nature Biotech 35: 249-263. Kim, D., et al., (2015). HISAT: a fast spliced aligner with low memory requirements. Nature Methods 12: 357-360. Koukourikos, K., et al., (2014). Muscle atrophy in intensive care unit patients. Acta informatica medica : AIM : journal of the Society for Medical Informatics of Bosnia & Herzegovina : casopis Drustva za medicinsku informatiku BiH 22: 406-410. Kroger, S., and Schroder, J.E. (2002). Agrin in the developing CNS: new roles for a synapse organizer. News Physiol. Sci. 17(5): 207-212. Krueger, F. (2015). Trim Galore! [http: / / www.bioinformatics.babraham.ac.uk / projects / trim_galore / ] Kyba, M. (2016). SKELETAL MUSCLE REGENERATION IN THE MOUSE: METHODS AND PROTOCOLS, Humana Press. LABORATORY METHODS IN ENZYMOLOGY: DNA, (2013). Jon Lorsch (ed.) Elsevier (ISBN 0124199542). Lawlor, P.A., et al., (2007). Novel rat Alzheimer's disease models based on AAV-mediated gene transfer to selectively increase hippocampal Aβ levels. Mol. Neurodeg. 2:11. LEWIN'S GENES XI, (2014). published by Jones & Bartlett Publishers (ISBN-1449659055). Liu, G., et al., (2005). Adeno-associated virus type 4 (AAV4) targets ependyma and astrocytes in the subventricular zone and RMS. Gene Therapy 12: 1503-1508. Love, M.I., et al., (2014). Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biology 15: 550. MacCallum, R.M., et al., (1996). Antibody-antigen interactions: contact analysis and binding site topography. J. Mol. Biol. 262(5): 732-745. MacIntosh, B.R., et al., (2006). Skeletal muscle: form and function. Human Kinetics (Champaign, IL) 2nd ed., 423 p. Mazhar, S. and Herbst, R. (2012). The formation of complex acetylcholine receptor clusters requires MuSK kinase activity and structural information from the MuSK extracellular domain. Molecular and Cellular Neurosciences 49: 475-486. McClorey, G. and Banerjee, S., (2018). Cell-Penetrating Peptides to Enhance Delivery of Oligonucleotide-Based Therapeutics. Biomedicines, 6(2): 51. Mercuri, E. et al., (2018). Nusinersen versus sham control in later-onset spinal muscular atrophy. New England Journal of Medicine, 378: 625-635. Messeant, J., et al., (2015). MuSK frizzled-like domain is critical for mammalian neuromuscular junction formation and maintenance. J Neuroscience 35: 4926-4941. Min, H.S., et al., (2020). Systemic Brain Delivery of Antisense Oligonucleotides across the Blood-Brain Barrier with a Glucose-Coated Polymeric Nanocarrier. Angew. Chem. Int. Ed. 59: 8173-8180. Misener, et al, (1999). Bioinformatics Methods and Protocols (Methods in Molecular Biology, Vol. 132), Humana Press. Miyagoe-Suzuki, Y., et al., (2017). Skeletal Muscle Cells Generated from Pluripotent Stem Cells. Stem Cells Int. 7824614. MOLECULAR BIOLOGY AND BIOTECHNOLOGY: A COMPREHENSIVE DESK REFERENCE, (1995). Robert A. Meyers (ed.), published by VCH Publishers, Inc. (ISBN 1-56081-569-8). Morgan, J.E., et al., (1994). Myogenic cell lines derived from transgenic mice carrying a thermolabile T antigen: a model system for the derivation of tissue-specific and mutation-specific cell lines. Dev Biol. 162: 486-498. Morris (1996). “Epitope Mapping Protocols”, Methods in Molecular Biology, vol. 66. Murphy, F.V. and Ramakrishnan, V., (2004). Structure of a purine-purine wobble base pair in the decoding center of the ribosome. Nature Structural and Molecular Biology 11: 1251-1252. Murphy, M.M., et al., (2011). Satellite cells, connective tissue fibroblasts and their interactions are crucial for muscle regeneration. Development 138: 3625-3637. Myers, E. W. and Miller, W. (1988). Optimal alignments in linear space. CABIOS 4, 11-17. Ono, Y., et al., (2011). BMP signaling permits population expansion by preventin g premature myogenic differentiation in muscle satellite cells. Cell death and differentiation 18: 222-234. Otsuka, S., et al, (2011). Delayed intrathecal delivery of RhoA siRNA to the contused spinal cord inhibits allodynia, preserves white matter, and increases serotonergic fiber growth. J Neurotrauma. 28: 1063-1076. Padlan, E.A., et al., (1995). Identification of specificity-determining residues in antibodies. FASEB J. 9(1): 133-139. Paul, C.P., et al., (2002). Effective expression of small interfering RNA in human cells. Nature Biotechnol.; 20:505-508 Pawlikowski, B., et al., (2015). Pervasive satellite cell contribution to uninjured adult muscle fibers. Skeletal muscle 5: 42. Pertea, M., et al., (2016). Transcript-level expression analysis of RNA-seq experiments with HISAT, StringTie and Ballgown. Nature Protocols 11: 1650-1667. Pimentel, H., et al., (2017). Differential analysis of RNA-seq incorporating quantification uncertainty. Nature Methods 14: 687-690. Powers, S.K., et al. (2016). Disease-Induced Skeletal Muscle Atrophy and Fatigue. Med. Sci. Sports Exerc. 48(11): 2307-2319. Prakash, T.P., et al. (2019). Fatty acid conjugation enhances potency of antisense oligonucleotides in muscle. Nucleic Acids Research, 47: 6029-6044. Randolph, M.E. and Pavlath, G.K. (2015). A muscle stem cell for every muscle: variability of satellite cell biology among different muscle groups. Frontiers in aging neuroscience 7: 190. Remedio, L., et al., (2016). Diverging roles for Lrp4 and Wnt signaling in neuromuscular synapse development during evolution. Genes & Development 30: 1058-1069. Remington, (2000). THE SCIENCE AND PRACTICE OF PHARMACY, 20th ed. Riaz, M., et al., (2015). Differential myofiber-type transduction preference of adeno-associated virus serotypes 6 and 9. Skeletal Muscle 5 (37). Rigo, F., et al., (2014). Pharmacology of a Central Nervous System Delivered 2'-O-Methoxyethyl-Modified Survival of Motor Neuron Splicing Oligonucleotide in Mice and Nonhuman Primates. J. Pharmacol. Exp. Ther. 350(1): 46-55. Rodgers, J.T., et al., (2014). mTORC1 controls the adaptive transition of quiescent stem cells from G0 to G(Alert). Nature 510: 393-396. Rupp, F., et al., (1991). Structure and expression of a rat agrin. Neuron. 6 (5): 811-823. Sacco, A., et al., (2008). Self-renewal and expansion of single transplanted muscle stem cells. Nature 456: 502-506. Sambasivan, R., et al., (2011). Pax7-expressing satellite cells are indispensable for adult skeletal muscle regeneration. Development 138: 3647-3656. Sander, J.D. and Joung, J.K. (2014). CRISPR-Cas systems for editing, regulating and targeting genomes. Nature Biotechnology 32: 347-355. Sartori, R., et al., (2013). BMP signaling controls muscle mass. Nature Genetics 45: 1309-1318. Schultz, E., et al., (1978). Satellite cells are mitotically quiescent in mature mouse muscle: an EM and radioautographic study. The Journal of Experimental Zoology 206: 451-456. Seale, P., et al., (2000). Pax7 is required for the specification of myogenic satellite cells. Cell 102: 777-786. Stantzou, A., et al., (2017). BMP signaling regulates satellite cell-dependent postnatal muscle growth. Development 144: 2737-2747. Stiegler, A.L., et al., (2006). Crystal structure of the agrin-responsive immunoglobulin-like domains 1 and 2 of the receptor tyrosine kinase MuSK. Journal of molecular biology 364: 424-433. Stiegler, A.L., et al., (2009). Crystal structure of the frizzled-like cysteine-rich domain of the receptor tyrosine kinase MuSK. J. Mol. Biol. 393: 1-9. Sui, G., et al., (2002). A DNA vector-based RNAi technology to suppress gene expression in mammalian cells. Proc. Natl. Acad. Sci. USA 99(8): 5515-5520. THE ENCYCLOPEDIA OF MOLECULAR CELL BIOLOGY AND MOLECULAR MEDICINE, Robert S. Porter et al., (eds.), published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908). THE MERCK MANUAL OF DIAGNOSIS AND THERAPY, (2011). 19th Edition, published by Merck Sharp & Dohme Corp., (ISBN 978-0-911910-19-3). Till, J.H., et al., (2002). Crystal structure of the MuSK tyrosine kinase: insights into receptor autoregulation. Structure 10: 1187-1196. Tsen, G., et al., (1995). Agrin is a heparan sulfate proteoglycan. J Biol Chem. 270(7): 3392-3399. Valenzuela, D.M., et al., (1995). Receptor tyrosine kinase specific for the skeletal muscle lineage: expression in embryonic muscle, at the neuromuscular junction, and after injury. Neuron 15: 573-584. van Velthoven, C.T.J., et al., (2017). Transcriptional Profiling of Quiescent Muscle Stem Cells In Vivo. Cell reports 21: 1994-2004. Vaughan, T.J., et al., (1998). Human antibodies by design. Nature Biotechnology, 16: 535-539. Verma, A. (2018). Recent Advances in Antisense Oligonucleotide Therapy in Genetic Neuromuscular Diseases. Ann. Indian Acad, Neurol. 21(1): 3-8. Wang, M., et al., (2019). Aminoglycoside Enhances the Delivery of Antisense Morpholino Oligonucleotides In Vitro and in mdx Mice. Mol. Ther. Nucleic Acids, 16: 663-674. Yilmaz, A., et al., (2016). MuSK is a BMP co-receptor that shapes BMP responses and calcium signaling in muscle cells. Science Signaling 9: ra87. Yin, H., et al., (2013). Satellite cells and the muscle stem cell niche. Physiological Reviews 93: 23-67. Yu, J.-Y., et al., (2002). RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells. Proc. Natl. Acad. Sci. USA, 99: 6047-6052. Zammit, PS, et al., (2002). Kinetics of myoblast proliferation show that resident satellite cells are competent to fully regenerate skeletal muscle fibers. Experimental cell research 281: 39-49. Zammit, PS, et al., (2004). Muscle satellite cells adopt divergent fates: a mechanism for self-renewal? The Journal of Cell Biology 166: 347-357. Zhou, X., et al., (2010). Reversal of cancer cachexia and muscle wasting by ActRIIB antagonism leads to prolonged survival. Cell 142: 531-543. equivalent 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. The scope of the present invention is not intended to be limited to the above description, but rather is set forth in the following claims.

Claims

1. 1. A method of treating a subject suffering from one or more hallmarks of a neuromuscular dysfunction or muscular dystrophy, comprising: Increasing the level or activity of a MuSK polypeptide that lacks a functional Ig3 domain; and / or A method of treating a subject comprising decreasing the level or activity of a BMP-MuSK polypeptide complex, wherein said MuSK comprises a functional Ig3 domain.

2. 1. A method for increasing muscle regeneration and / or muscle growth, comprising: Increasing the level or activity of a MuSK polypeptide that lacks a functional Ig3 domain; and / or A method of increasing muscle regeneration and / or muscle growth comprising decreasing the level or activity of a BMP-MuSK polypeptide complex, wherein said MuSK comprises a functional Ig3 domain.

3. 1. A method for preventing or treating muscle fibrosis, comprising: Increasing the level or activity of a MuSK polypeptide that lacks a functional Ig3 domain; and / or A method of preventing or treating muscle fibrosis comprising reducing the level or activity of a BMP-MuSK polypeptide complex, wherein said MuSK comprises a functional Ig3 domain.

4. 4. The method of any one of claims 1 to 3, further comprising administering to the subject a pharmaceutical composition comprising or delivering a MuSK muscle regeneration (MR) agonizing agent.

5. The method of claim 4, wherein the MuSK MR agonizing agent is an agent that downregulates MuSK Ig3 domain protein expression, MuSK Ig3 domain gene expression, and / or MuSK Ig3 activation of BMP signaling, and the composition prevents or reduces the accumulation of extracellular matrix in the extracellular space of muscle.

6. The method of claim 3 , wherein the MuSK MR agonizing agent is or comprises a small molecule.

7. The method of claim 4 , wherein the MuSK MR agonizing agent is or comprises an antibody agent.

8. The method of claim 4 , wherein the MuSK MR agonizing agent is or comprises an oligonucleotide.

9. The method of claim 7 , wherein the antibody agent specifically binds to a MuSK polypeptide.

10. The method of claim 9 , wherein the antibody agent targets MuSK and specifically binds to the Ig3 domain of a MuSK polypeptide.

11. The method of claim 10, wherein the antibody that targets the Ig3 domain of a MuSK protein is capable of specifically binding to the Ig3 domain relative to the Ig1 or Ig2 domains of MuSK.

12. The method of claim 7, wherein the antibody agent is an immunoglobulin molecule comprising four polypeptide chains, e.g., two heavy (H) chains and two light (L) chains.

13. The method of claim 7 , wherein the antibody agent is or comprises a monoclonal antibody.

14. The method of claim 7, wherein the antibody agent can be or comprise a polyclonal antibody.

15. The method of claim 8 , wherein the MuSK MR agonizing agent is an oligonucleotide.

16. 16. The method of claim 15, wherein the oligonucleotide is a MuSK Ig3-targeted CRISPR / Cas9.

17. The method of claim 15, wherein the oligonucleotide is a MuSK Ig3-targeting siRNA.

18. The method of claim 15, wherein the oligonucleotide is a MuSK Ig3-targeting shRNA.

19. 16. The method of claim 15, wherein the step further comprises increasing altered splicing of the transcript.

20. 20. The method of claim 19, wherein said altering splicing of a transcript is or comprises altering MuSK splicing.

21. The method of claim 20, wherein the alteration of MuSK splicing includes the production of a product with a desired and / or improved biological function, and / or the knockdown of an undesired product by modifying the splicing product so that the undesired biological function can be suppressed.

22. 22. The method of claim 21, wherein the alteration in MuSK splicing comprises the production of a transcript lacking a sequence encoding a MuSK Ig3 domain.

23. 23. The method of claim 22, wherein the splicing product is mRNA.

24. 21. The method of claim 20, wherein the alteration comprises skipping one or more exons.

25. 25. The method of claim 24, wherein exon skipping increases the splicing of the transcript in that it increases the levels of mRNA and protein with improved beneficial activity compared to the absence of exon skipping.

26. 25. The method of claim 24, wherein exon skipping increases the splicing of the transcript in that it reduces the levels of mRNA and protein with undesired activity compared to the absence of exon skipping.

27. 27. The method of claim 26, wherein exon skipping increases the splicing of the transcript in that it reduces the levels of MuSK Ig3 domain mRNA and protein.

28. 25. The method of claim 24, wherein the skipped exon or exons are in the MuSK Ig3 domain.

29. 29. The method of claim 28, wherein the skipped exon is exon 6 of the MuSK Ig3 domain.

30. 29. The method of claim 28, wherein the skipped exon is exon 7 of the MuSK Ig3 domain.

31. 29. The method of claim 28, wherein the skipped exons are exons 6 and 7 of the MuSK Ig3 domain.

32. 29. The method of claim 28, wherein the composition comprises oligonucleotides containing controlled structural elements, such as controlled chemical modifications, that provide unexpected properties.

33. 33. The method of claim 32, wherein the oligonucleotide comprises a chemical modification.

34. 34. The method of claim 33, wherein the chemical modifications comprise one or more types of base modifications, sugar modifications, and internucleotide linkage modifications.

35. 35. The method of claim 34, wherein the chemical modification comprises a sugar modification.

36. The method of claim 15, wherein the sugar modification is a 2-MOE modification.

37. A method for inducing MuSK exon skipping by contacting a system containing a population of MuSK primary transcripts with an oligonucleotide that binds to such primary transcripts, such that skipping of one or both of exons 6 and 7 is increased.

38. 38. The method of claim 37, wherein the oligonucleotide comprises controlled structural elements, e.g., controlled chemical modifications, that provide unexpected properties.

39. 39. The method of claim 38, wherein the oligonucleotide comprises a chemical modification.

40. 40. The method of claim 39, wherein the chemical modifications comprise one or more types of base modifications, sugar modifications, and internucleotide linkage modifications.

41. 41. The method of claim 40, wherein the chemical modification comprises a sugar modification.

42. The method of claim 41, wherein the sugar modification is a 2-MOE modification.

43. 38. The method of claim 37, further comprising administering to the subject a pharmaceutically effective amount of a composition that contains and / or delivers the oligonucleotide to the subject.

44. 44. The method of claim 43, wherein the composition is delivered to the CNS.

45. 44. The method of claim 43, wherein the composition is delivered to the cerebrospinal fluid.

46. 44. The method of claim 43, wherein the composition is administered into a muscle.

47. 44. The method of claim 43, wherein the composition can be formulated for systemic or localized administration.

48. 44. The method of claim 43, wherein the composition is formulated for delivery by a route selected from intravenous injection, intravenous infusion, intramuscular injection, intrathecal administration, oral administration, buccal administration, inhalation, nasal administration, topical administration, ophthalmic administration, or otic administration.

49. 49. The method of claim 48, wherein the composition is formulated for delivery by intramuscular administration.

50. 49. The method of claim 48, wherein the composition is formulated for delivery by intravenous administration.

51. 49. The method of claim 48, wherein the composition is formulated for delivery by oral administration.

52. 52. The method of any one of claims 4-51, wherein the subject is at risk for or suffering from a disease or disorder selected from the group consisting of neuromuscular dysfunction, neurodegenerative disorders, cardiac dysfunction, and diseases characterized by muscle wasting.

53. 53. The method of claim 52, wherein the neuromuscular dysfunction is a muscular dystrophy selected from the group consisting of Becker muscular dystrophy, congenital muscular dystrophy, distal muscular dystrophy, Duchenne muscular dystrophy, Emery-Dreifuss muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, myotonic muscular dystrophy, and oculopharyngeal muscular dystrophy.

54. 53. The method of claim 52, wherein the cardiac dysfunction is myocardial infarction or cardiomyopathy.

55. 52. The method of any one of claims 4 to 51, wherein the subject is in need of muscle regeneration and / or enhanced muscle growth following a condition selected from the group consisting of surgery, trauma, and prolonged immobilization.

56. 56. The method of claim 55, wherein the prolonged motor immobilization results from bed rest or casting.

57. 52. The method of any one of claims 4 to 51, wherein the subject is at risk of or suffering from sarcopenia.

58. 52. The method of any one of claims 4-51, wherein the subject is at risk for or suffering from muscle fibrosis resulting from a disease or condition selected from the group consisting of trauma, a genetic disease, a muscle disorder, and aging.

59. 59. The method of claim 58, wherein the trauma is the result of a condition selected from the group consisting of radiation therapy, a crush injury, a laceration, and an amputation.

60. 59. The method of claim 58, wherein the genetic disease or muscle disorder is selected from the group consisting of congenital muscular dystrophy, Duchenne muscular dystrophy, Becker muscular dystrophy, amyotrophic lateral sclerosis (ALS), and age-related sarcopenia.

61. A population of cells exposed to a MuSK muscle regeneration agonizing agent, whereby the level or percentage of cells characterized by myogenic markers is increased within the population compared to that observed without said exposure.

62. 62. The population of claim 61, wherein the myogenic marker is selected from the group consisting of Pax7, MyoD, Myogenin and MERGE, and combinations thereof.

63. 62. The population of claim 61, wherein the increase in level or percentage is at least a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or greater than a 95% increase compared to that observed without the exposure.

64. 62. The population of claim 61, wherein the muscle marker is indicative of activated muscle (e.g., satellite) cells.

65. A method comprising contacting a population of original cells that are or contain muscle progenitor cells with a MuSK muscle regeneration agonizing agent to generate a resultant population, said contacting being carried out under conditions and for a sufficient time such that the level or percentage of cells characterized by muscle markers is significantly higher in the resultant population than in the original population.

66. 66. The method of claim 65, wherein said contacting step occurs in vivo.

67. 67. The method of claim 66, wherein the contacting step occurs in an adult.

68. 68. The method of claim 66 or 67, wherein the contacting step occurs at a site within muscle tissue.

69. 68. The method of claim 66 or 67, wherein the contacting step occurs ex vivo.

70. 70. The method of claim 69, wherein the population of cells is obtained from a subject suffering from a disease characterized by neuromuscular dysfunction, neurodegenerative disorder, cardiac dysfunction, or muscle wasting.

71. 70. The method of claim 69, further comprising administering the resulting population to the subject.

72. 66. The method of claim 65, wherein the neural marker is selected from the group consisting of Pax7, MyoD, Myogenin and MERGE, and combinations thereof.

73. 66. The method of claim 65, wherein the muscle marker is indicative of activated muscle (e.g., satellite) cells.

74. 1. A method for characterizing a MuSK muscle regeneration agonizing agent, comprising: assessing the ability to reduce MuSK-Ig3-BMP complex formation (assessing re-prevent formation, shear formation, direct binding to Ig3 and / or BMP, concentration dependence, etc.); assessing the ability to alter the splicing pattern of the primary MuSK transcript; assessing the ability to inhibit expression of transcripts (including, for example, Ig3) (dependence includes inducing degradation, inhibiting translation, etc.); The ability to increase the expression of MuSK transcripts lacking the sequence encoding the Ig3 domain A step of assessing power; assessing the ability to increase levels of a MuSK polypeptide lacking functional Ig3; and assessing the ability to affect the characteristics of cells in the population A method for characterizing a MuSK muscle regeneration agonizing agent, comprising one or more of:

75. 75. The method of claim 74, wherein the MuSK MR agonizing agent is an oligonucleotide.

76. 76. The method of claim 75, wherein the oligonucleotide comprises at least one modification.

77. The method of claim 75 or claim 76, wherein the oligonucleotide, when administered to a subject, alters the splicing activity of the primary MuSK transcript, thereby increasing the skipping of one or both of exons 6 and 7.

78. A genetically modified mouse comprising in its genome a sequence encoding MuSK, wherein said sequence encoding MuSK does not include the nucleotide distance from exon 6 to exon 7 (in 5' to 3' order); A genetically modified mouse, wherein said genetically modified mouse is incapable of expressing a full-length MuSK transcript or producing a full-length MuSK protein.

79. 79. The genetically modified mouse of claim 78, wherein the mouse is unable to express a MuSK protein comprising the amino acid sequence in SEQ ID NO:

2.

80. 79. The genetically modified mouse of claim 78, wherein the mouse is capable of expressing a MuSK transcript encoding a MuSK protein lacking an Ig3 domain.

81. 79. The genetically modified mouse of claim 78, wherein the mouse exhibits increased muscle regeneration compared to a mouse capable of expressing the full-length MuSK transcript or producing the full-length MuSK protein.

82. 82. The genetically modified mouse of claim 81, wherein the increase in muscle regeneration comprises an increase in motor function.

83. 79. The genetically modified mouse of claim 78, wherein the mouse has been genetically modified by removing the nucleotide distance from exon 6 to exon 7 (in 5' to 3' order) in the sequence encoding MuSK using the CRISPR / Cas9 system.

84. 84. The genetically modified mouse of claim 83, wherein the CRISPR / Cas9 system comprises gDNA targeting a region within exon 6 and / or exon 7 of the MuSK gene sequence.

85. 85. The genetically modified mouse of claim 84, wherein the gDNA-targeted sequence comprises SEQ ID NOs: 3-6.

Images