MuSK-targeting oligonucleotides

Abstract

The present disclosure provides, inter alia, MuSK-targeted oligonucleotide compositions that alter the splicing of MuSK transcripts through exon skipping, and methods of using such compositions to treat disease.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 301,416, filed January 20, 2022, the contents of which are incorporated herein by reference in their entirety. [Background technology]

[0002] Neurodegenerative diseases are a major public health challenge, affecting one in five people in their lifetime. Alzheimer's disease (AD) is the most common cause of age-related dementia. There is a significant and urgent need for therapeutic agents to prevent and treat AD and other neurodegenerative conditions.

[0003] 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 comprise a small, scattered population of mitotic and physiologically quiescent cells. Satellite cells also participate in normal muscle growth and maintenance throughout life, suggesting they may be utilized to treat muscle-wasting conditions.

[0004] 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 in the majority of cancer patients, elderly patients, and many other patients without a history of neuromuscular dysfunction. Furthermore, muscular dystrophies are a group of often fatal genetic disorders that lead to severe muscle loss, including Duchenne muscular dystrophy, which affects children.

[0005] Neurogenesis and muscle regeneration depend on neural stem cells (NSCs) and muscle satellite cells. An obstacle to developing treatments for neurodegeneration and muscle wasting is a lack of understanding of neural stem cells and satellite cells and the signaling that regulates their regeneration. Thus, there is a need for compositions and methods for promoting neurogenesis and muscle regeneration. Summary of the Invention

[0006] Among other things, the present disclosure provides insight that the presence and / or activity of particular forms of muscle-specific tyrosine kinase (MuSK) proteins, particularly those lacking a functional Ig3 domain, can achieve or contribute to beneficial biological events including, for example, neurogenesis and / or muscle regeneration.

[0007] The present disclosure provides certain techniques for enhancing neurogenesis, particularly in adult humans. In some embodiments, the techniques provided herein may be useful in medicine, particularly including the treatment of diseases, disorders, or conditions associated with neurodegeneration or otherwise associated with decreased or reduced neuronal activity (e.g., neuronal activity in the adult hippocampus and / or subventricular zone). For example, in some embodiments, the techniques provided herein may be useful in the treatment of 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, diabetes-mediated hippocampal neuron loss, brain injury (e.g., traumatic and / or anoxic brain injury), and Huntington's disease.

[0008] Bone morphogenetic protein (BMP) signaling regulates at least two key NSC decision points: 1) quiescence, where proliferating stem cells exit the cell cycle and return to replenish reservoir pools that can provide fresh stem cells, and 2) differentiation into mature progeny (Mira et al., 2010). The present disclosure contemplates that manipulating the BMP pathway in NSCs is an attractive target for modulating neurogenesis in the adult brain. Furthermore, BMP signaling regulates skeletal muscle stem cell activity under both normal and pathological conditions. The present disclosure provides techniques for increasing the levels and / or activity of MuSK forms functionally involved in neurogenesis and / or muscle regeneration, which in some embodiments involves reducing alternative splicing that would generate non-involved MuSK forms. 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.

[0009] Among other things, the present disclosure provides insight that the presence and / or activity of certain forms of muscle-specific tyrosine kinase (MuSK) proteins, particularly those lacking a functional Ig3 domain, can achieve or contribute to neurogenesis in adult humans or otherwise provide neurological benefits. MuSK transcripts can be alternatively spliced, including generating at least one form lacking the Ig3 domain (i.e., ΔIg3-MuSK). The present disclosure recognizes that increasing the presence and / or levels of ΔIg3-MuSK and / or other functional forms in which its Ig3 domain is altered (e.g., mutated, blocked, etc.) or removed can provide the benefits described herein.

[0010] In some embodiments, the present disclosure provides techniques for increasing the level and / or activity of one or more forms of MuSK whose Ig3 domain is altered (e.g., mutated, blocked, removed, etc.) so that it is not effectively involved in interacting with BMPs, for example. In some embodiments, the present disclosure provides techniques for increasing the level and / or activity of one or more forms of MuSK whose Ig3 domain is altered (e.g., mutated, blocked, removed, etc.) so that it is not effectively involved in interacting with BMPs, for example, by reducing the level and / or activity of one or more forms of MuSK whose Ig3 domain is effectively involved in interacting with BMPs. + Provides techniques for reducing MuSK.

[0011] In some embodiments, agents (e.g., MuSK-targeted oligonucleotides) that target the MuSK Ig3 domain described herein and reduce the level and / or activity of MuSK / BMP complexes are useful in the context of neurogenesis and / or muscle regeneration and / or muscle growth.

[0012] 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 muscle fiber size. In this regard, muscle growth may be characterized by a decrease in satellite cell number and / or an increase in muscle fiber size, indicating that satellite cells are differentiating and fusing with / augmenting existing muscle fibers to form new muscle fibers.

[0013] Embodiments of the present invention provide methods for enhancing neurogenesis and / or muscle regeneration and / or growth, thereby upregulating muscle satellite cells, thereby enhancing muscle regeneration and / or growth, by administering, e.g., in a subject in need thereof, a composition that downregulates MuSK Ig3 domain protein expression, MuSK Ig3 domain gene expression, and / or MuSK Ig3 activation of BMP signaling. In some embodiments, such compositions can comprise and / or deliver MuSK-targeting oligonucleotides (e.g., MuSK Ig3-targeting exon-skipping oligonucleotides).

[0014] In some embodiments, enhanced neurogenesis is used in the context of treating diseases or disorders associated with decreased adult hippocampal neurogenesis (AHN). Because AHN occurs throughout human life and is dramatically decreased in AD (Moreno-Jimenez et al., 2019; Steiner et al., 2019), the present disclosure provides compositions and methods for promoting AHN, enhancing cognitive function, and combating neurodegeneration (e.g., Alzheimer's disease).

[0015] In some embodiments, enhanced neurogenesis is used in the context of treating diseases or disorders associated with reduced subventricular zone neurogenesis. NSCs reside in the subventricular zone (SVZ), lining the lateral ventricles, and generate astrocytes and oligodendrocytes that support existing circuits, as well as neurons in the olfactory bulb, which are important for olfactory discrimination. The present disclosure provides compositions and methods for compensating for neuronal degeneration in the SVZ through enhanced endogenous neurogenesis. In some embodiments, the present disclosure provides compositions and methods for treating diseases specifically associated with striatal neurogenesis, such as Parkinson's disease (which can benefit from both increased AHN and striatal neurogenesis in the SVZ; Pitcher et al. 2012, Sterling et al. 2013) and Huntington's disease (Sassone et al., 2018). In some embodiments, the present disclosure provides compositions and methods for treating other diseases, including addictions (e.g., chronic cocaine use and lifelong smoking).

[0016] 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 disorders (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.

[0017] 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-Dreyfus muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, myotonic muscular dystrophy, and oculopharyngeal muscular dystrophy.

[0018] In some embodiments, enhanced muscle growth is used in the context of treating diseases or disorders associated with muscle atrophy or muscle wasting. Muscle atrophy or muscle wasting may be observed in association with various diseases and conditions described herein, such as neuromuscular disorders, or may 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).

[0019] The length of hospital stay and the type and severity of illness can influence the degree of muscle wasting in a subject, and muscle wasting is common in patients suffering from sepsis, organ failure, hyperglycemia, and diseases associated with chronic and systemic inflammation or oxidative stress. Furthermore, hospitalization requiring complete immobilization / bed rest significantly contributes to muscle wasting.

[0020] Additional disorders associated with muscle atrophy / wasting include disorders associated with reduced mobility, such as rheumatoid arthritis, osteoarthritis, and injury. (2016 Powers, Scott K., et al. Medicine and science in sports and exercise 48(11):2307) Thus, in some embodiments, the present disclosure provides therapies for preventing / treating muscle wasting or atrophy associated with or resulting from many of the diseases or conditions described herein.

[0021] In some embodiments, the methods of the invention can also be used when a subject needs enhanced muscle regeneration and growth after surgery, trauma, and / or prolonged immobilization (e.g., due to bed rest or a cast). Because muscle stem cell activity is known to decline with age, the methods of the invention can also be used to prevent or reverse sarcopenia in otherwise healthy patients, potentially leading to significant improvements in quality of life and autonomy.

[0022]

[0013] Embodiments of the present invention also provide methods for preventing or treating neurodegenerative diseases (e.g., AD) and / or muscle fibrosis in a subject in need thereof, e.g., by administering a composition that downregulates MuSK Ig3 domain protein expression, MuSK Ig3 domain gene expression, and / or MuSK Ig3 activation of BMP signaling. The composition may include, for example, a MuSK-targeting oligonucleotide (e.g., a MuSK Ig3-targeting exon-skipping oligonucleotide). The subject may be at risk for or suffering from a variety of neurodegenerative diseases, such as 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, diabetes-mediated hippocampal neuron loss, brain injury (e.g., traumatic and / or anoxic brain injury), Huntington's disease or muscle fibrosis resulting from a disease or condition, including, but not limited to, trauma, genetic disorders, muscle disorders, and aging. Trauma may result, for example, from radiation therapy, crush injury, laceration, and amputation. Genetic diseases or muscle disorders include, but are not limited to, congenital muscular dystrophy, Duchenne muscular dystrophy, Becker muscular dystrophy, amyotrophic lateral sclerosis (ALS), and age-related sarcopenia.

[0023] A feature of the present invention is, inter alia, an oligonucleotide composition comprising a plurality of oligonucleotides, wherein when the oligonucleotide composition is contacted with MuSK transcripts in a transcription splicing system, the relative amounts of transcripts that include and do not include Ig3 domain-encoding sequences are altered compared to the relative amounts observed under such reference conditions selected from the group consisting of the absence of the composition, the presence of a reference composition, and combinations thereof.

[0024] In some embodiments, the oligonucleotide mediates skipping of at least one exon of the MuSK gene. In some embodiments, the exon skipping reduces the level of mRNA encoding a MuSK protein form involved in BMP signaling compared to the level observed without exon skipping. In some embodiments, the MuSK protein form involved in BMP signaling is or comprises a MuSK protein form that forms a MuSK / BMP complex.

[0025] In some embodiments, exon skipping reduces the level and / or activity of MuSK / BMP complexes. In some embodiments, the at least one skipped exon is selected from the group consisting of exons 3, 4, 6, and 7. In some embodiments, the relative amount is the amount of a transcript that includes exons 6 and 7 relative to the amount of a transcript that lacks exons 6 and 7. In some embodiments, the relative amount is the amount of a transcript that includes exons 3 and 4 relative to the amount of a transcript that lacks exons 6 and 7.

[0026] In some embodiments, the alteration comprises skipping one or more of exons 6 and 7 of MuSK. In some embodiments, the alteration comprises skipping one or more of exons 3 and 4 of MuSK. In some embodiments, the alteration comprises skipping one or more of exons 6 and 7 of MuSK, but not skipping exons 3 and 4 of MuSK.

[0027] In some embodiments, MuSK splicing is altered in that the level of a MuSK transcript comprising exons 6 and 7 is decreased, or the level of a MuSK protein form comprising a sequence encoded by exons 6 and 7 is decreased, or both. In some embodiments, MuSK splicing is altered in that the level of a MuSK transcript comprising exons 3 and 4 is decreased, or the level of a MuSK protein form comprising a sequence encoded by exons 3 and 4 is decreased, or both. In some embodiments, MuSK splicing is altered in that the level of a MuSK transcript comprising exons 6 and 7 is increased, or the level of a MuSK protein form comprising a sequence encoded by exons 6 and 7 is increased, or both. In some embodiments, MuSK splicing is altered in that the level of a MuSK transcript comprising exons 3 and 4 is increased, or the level of a MuSK protein form comprising a sequence encoded by exons 3 and 4 is increased, or both. In some embodiments, MuSK splicing is altered in that the level of MuSK transcripts comprising exons 3 and 4 remains substantially unchanged, while the level of MuSK transcripts comprising exons 6 and 7 is decreased. In some embodiments, MuSK splicing is altered in that the level of MuSK protein forms comprising sequences encoded by exons 3 and 4 remains substantially unchanged, while the level of MuSK protein forms comprising sequences encoded by exons 6 and 7 is decreased. In some embodiments, MuSK splicing is altered in that the total level of MuSK transcripts remains substantially unchanged, while the level of MuSK transcripts comprising exons 6 and 7 is decreased.

[0028] In some embodiments, MuSK splicing is altered in that the ratio of MuSK transcripts comprising exons 3 and 4 to MuSK transcripts comprising exons 6 and 7 is increased. In some embodiments, MuSK splicing is altered in that the ratio of MuSK transcripts comprising exons 3 and 4 to MuSK transcripts comprising exons 6 and 7 is increased by about 10%. In some embodiments, MuSK splicing is altered in that the ratio of MuSK transcripts comprising exons 3 and 4 to MuSK transcripts comprising exons 6 and 7 is increased by about 50%. In some embodiments, MuSK splicing is altered in that the ratio of MuSK transcripts comprising exons 3 and 4 to MuSK transcripts comprising exons 6 and 7 is increased by about 80%. In some embodiments, MuSK splicing is altered in that the ratio of MuSK transcripts comprising exons 3 and 4 to MuSK transcripts comprising exons 6 and 7 is increased by about 100%. In some embodiments, MuSK splicing is altered in that the ratio of MuSK transcripts comprising exons 3 and 4 to MuSK transcripts comprising exons 6 and 7 is increased by about 120%. In some embodiments, MuSK splicing is altered in that the ratio of MuSK transcripts comprising exons 3 and 4 to MuSK transcripts comprising exons 6 and 7 is increased by about 150%. In some embodiments, MuSK splicing is altered in that the ratio of MuSK transcripts comprising exons 3 and 4 to MuSK transcripts comprising exons 6 and 7 is increased by about 180%. In some embodiments, MuSK splicing is altered in that the ratio of MuSK transcripts comprising exons 3 and 4 to MuSK transcripts comprising exons 6 and 7 is increased by about 200%. In some embodiments, MuSK splicing is altered in that the ratio of MuSK transcripts comprising exons 3 and 4 to MuSK transcripts comprising exons 6 and 7 is increased by about 250%. In some embodiments, MuSK splicing is altered in that the ratio of MuSK transcripts containing exons 3 and 4 to MuSK transcripts containing exons 6 and 7 is increased by about 300%.In some embodiments, MuSK splicing is altered in that the ratio of MuSK transcripts comprising exons 3 and 4 to MuSK transcripts comprising exons 6 and 7 is increased by about 350%. In some embodiments, MuSK splicing is altered in that the ratio of MuSK transcripts comprising exons 3 and 4 to MuSK transcripts comprising exons 6 and 7 is increased by about 400%. In some embodiments, MuSK splicing is altered in that the ratio of MuSK transcripts comprising exons 3 and 4 to MuSK transcripts comprising exons 6 and 7 is increased by about 450%. In some embodiments, MuSK splicing is altered in that the ratio of MuSK transcripts comprising exons 3 and 4 to MuSK transcripts comprising exons 6 and 7 is increased by about 500%. In some embodiments, MuSK splicing is altered in that the ratio of MuSK transcripts comprising exons 3 and 4 to MuSK transcripts comprising exons 6 and 7 is increased by about 600%. In some embodiments, MuSK splicing is altered in that the ratio of MuSK transcripts comprising exons 3 and 4 to MuSK transcripts comprising exons 6 and 7 is increased by about 700%. In some embodiments, MuSK splicing is altered in that the ratio of MuSK transcripts comprising exons 3 and 4 to MuSK transcripts comprising exons 6 and 7 is increased by about 800%. In some embodiments, MuSK splicing is altered in that the ratio of MuSK transcripts comprising exons 3 and 4 to MuSK transcripts comprising exons 6 and 7 is increased by about 900%. In some embodiments, MuSK splicing is altered in that the ratio of MuSK transcripts comprising exons 3 and 4 to MuSK transcripts comprising exons 6 and 7 is increased by about 1000%. In some embodiments, MuSK splicing is altered in that the ratio of MuSK transcripts comprising exons 3 and 4 to MuSK transcripts comprising exons 6 and 7 is increased by at least 1000%.

[0029] In some embodiments, MuSK splicing is altered in that the ratio of MuSK transcripts comprising exons 3 and 4 to MuSK transcripts comprising exons 6 and 7 is reduced. In some embodiments, MuSK splicing is altered in that the ratio of MuSK transcripts comprising exons 3 and 4 to MuSK transcripts comprising exons 6 and 7 is reduced by about 10%. In some embodiments, MuSK splicing is altered in that the ratio of MuSK transcripts comprising exons 3 and 4 to MuSK transcripts comprising exons 6 and 7 is reduced by about 50%. In some embodiments, MuSK splicing is altered in that the ratio of MuSK transcripts comprising exons 3 and 4 to MuSK transcripts comprising exons 6 and 7 is reduced by about 80%. In some embodiments, MuSK splicing is altered in that the ratio of MuSK transcripts comprising exons 3 and 4 to MuSK transcripts comprising exons 6 and 7 is reduced by about 100%. In some embodiments, MuSK splicing is altered in that the ratio of MuSK transcripts comprising exons 3 and 4 to MuSK transcripts comprising exons 6 and 7 is reduced by about 120%. In some embodiments, MuSK splicing is altered in that the ratio of MuSK transcripts comprising exons 3 and 4 to MuSK transcripts comprising exons 6 and 7 is reduced by about 150%. In some embodiments, MuSK splicing is altered in that the ratio of MuSK transcripts comprising exons 3 and 4 to MuSK transcripts comprising exons 6 and 7 is reduced by about 180%. In some embodiments, MuSK splicing is altered in that the ratio of MuSK transcripts comprising exons 3 and 4 to MuSK transcripts comprising exons 6 and 7 is reduced by about 200%. In some embodiments, MuSK splicing is altered in that the ratio of MuSK transcripts comprising exons 3 and 4 to MuSK transcripts comprising exons 6 and 7 is reduced by about 250%. In some embodiments, MuSK splicing is altered in that the ratio of MuSK transcripts containing exons 3 and 4 to MuSK transcripts containing exons 6 and 7 is reduced by about 300%.In some embodiments, MuSK splicing is altered in that the ratio of MuSK transcripts comprising exons 3 and 4 to MuSK transcripts comprising exons 6 and 7 is reduced by about 350%. In some embodiments, MuSK splicing is altered in that the ratio of MuSK transcripts comprising exons 3 and 4 to MuSK transcripts comprising exons 6 and 7 is reduced by about 400%. In some embodiments, MuSK splicing is altered in that the ratio of MuSK transcripts comprising exons 3 and 4 to MuSK transcripts comprising exons 6 and 7 is reduced by about 450%. In some embodiments, MuSK splicing is altered in that the ratio of MuSK transcripts comprising exons 3 and 4 to MuSK transcripts comprising exons 6 and 7 is reduced by about 500%. In some embodiments, MuSK splicing is altered in that the ratio of MuSK transcripts comprising exons 3 and 4 to MuSK transcripts comprising exons 6 and 7 is reduced by about 600%. In some embodiments, MuSK splicing is altered in that the ratio of MuSK transcripts comprising exons 3 and 4 to MuSK transcripts comprising exons 6 and 7 is reduced by about 700%. In some embodiments, MuSK splicing is altered in that the ratio of MuSK transcripts comprising exons 3 and 4 to MuSK transcripts comprising exons 6 and 7 is reduced by about 800%. In some embodiments, MuSK splicing is altered in that the ratio of MuSK transcripts comprising exons 3 and 4 to MuSK transcripts comprising exons 6 and 7 is reduced by about 900%. In some embodiments, MuSK splicing is altered in that the ratio of MuSK transcripts comprising exons 3 and 4 to MuSK transcripts comprising exons 6 and 7 is reduced by about 1000%. In some embodiments, MuSK splicing is altered in that the ratio of MuSK transcripts comprising exons 3 and 4 to MuSK transcripts comprising exons 6 and 7 is reduced by at least 1000%.

[0030] In some embodiments, the ratio of MuSK transcripts comprising exons 3 and 4 (i.e., total MuSK transcripts) to MuSK transcripts comprising exons 6 and 7 in a cell or system prior to contact with one or more exon skipping oligonucleotides described herein is about 1:1 to 1:0.1. In some embodiments, the ratio of MuSK transcripts comprising exons 3 and 4 (i.e., total MuSK transcripts) to MuSK transcripts comprising exons 6 and 7 in a cell or system prior to contact with one or more exon skipping oligonucleotides described herein is about 1:1, 1:0.9, 1:0.8, 1:0.7, 1:0.6, 1:0.5, 1:0.4, 1:0.3, 1:0.2, or 1:0.1. In some embodiments, the ratio of MuSK transcripts containing exons 3 and 4 (i.e., total MuSK transcripts) to MuSK transcripts containing exons 6 and 7 in a cell or system prior to contact with one or more exon skipping oligonucleotides described herein is approximately 1:0.8.

[0031] In some embodiments, the ratio of MuSK transcripts comprising exons 3 and 4 (i.e., total MuSK transcripts) to MuSK transcripts comprising exons 6 and 7 in a cell or system after contact with one or more exon skipping oligonucleotides described herein is about 1:0.9 to 1:0.0001. In some embodiments, the ratio of MuSK transcripts comprising exons 3 and 4 (i.e., total MuSK transcripts) to MuSK transcripts comprising exons 6 and 7 in a cell or system after contact with one or more exon skipping oligonucleotides described herein is about 1:0.9, 1:0.8, 1:0.7, 1:0.6, 1:0.5, 1:0.4, 1:0.3, 1:0.2, or 1:0.1, 1:0.05, 1:0.01, 1:0.001, or 1:0001. In some embodiments, the ratio of MuSK transcripts containing exons 3 and 4 (i.e., total MuSK transcripts) to MuSK transcripts containing exons 6 and 7 in a cell or system after contact with one or more exon skipping oligonucleotides described herein is about 1:0.9 to 1:0.8, 1:08 to 1:0.7, 1:0.7 to 1:0.6, 1:0.6 to 1:0.5, 1:0.5 to 1:0.4, 1:0.4 to 1:0.3, 1:0.3 to 1:0.2, 1:0.2 to 1:0.1, 1:0.1 to 1:0.05, 1:0.05 to 1:0.01, 1:0.01 to 1:0.001, or 1:0.001 to 1:0001. In some embodiments, after contact with one or more exon skipping oligonucleotides described herein, the ratio of MuSK transcripts containing exons 3 and 4 (i.e., total MuSK transcripts) to MuSK transcripts containing exons 6 and 7 in a cell or system is about 1:0.9 to 1:0.5.

[0032] In some embodiments, the level of MuSK transcripts comprising exons 6 and 7, or the level of MuSK protein forms comprising sequences encoded by exons 6 and 7, or both, is reduced by at least two-fold greater than the reduction observed in the level of MuSK transcripts comprising exons 3 and 4, or the level of MuSK protein forms comprising sequences encoded by exons 3 and 4, or both.

[0033] In some embodiments, the level of a MuSK transcript comprising exons 6 and 7, or the level of a MuSK protein form comprising a sequence encoded by exons 6 and 7, or both, is reduced by a level that is at least three-fold greater than the reduction observed in the level of a MuSK transcript comprising exons 3 and 4, or the level of a MuSK protein form comprising a sequence encoded by exons 3 and 4, or both. In some embodiments, the level of a MuSK transcript comprising exons 6 and 7, or the level of a MuSK protein form comprising a sequence encoded by exons 6 and 7, or both, is reduced by a level that is at least four-fold greater than the reduction observed in the level of a MuSK transcript comprising exons 3 and 4, or the level of a MuSK protein form comprising a sequence encoded by exons 3 and 4, or both.

[0034] In some embodiments, the level of a MuSK transcript comprising exons 6 and 7, or the level of a MuSK protein form comprising a sequence encoded by exons 6 and 7, or both, is reduced by a level that is at least 5-fold greater than the reduction observed in the level of a MuSK transcript comprising exons 3 and 4, or the level of a MuSK protein form comprising a sequence encoded by exons 3 and 4, or both. In some embodiments, the level of a MuSK transcript comprising exons 6 and 7, or the level of a MuSK protein form comprising a sequence encoded by exons 6 and 7, or both, is reduced by a level that is at least 10-fold greater than the reduction observed in the level of a MuSK transcript comprising exons 3 and 4, or the level of a MuSK protein form comprising a sequence encoded by exons 3 and 4, or both.

[0035] In some embodiments, the level of MuSK transcripts comprising exons 6 and 7, or the level of MuSK protein forms comprising sequences encoded by exons 6 and 7, or both, is reduced by more than 60%, and the level of MuSK transcripts comprising exons 3 and 4, or the level of MuSK protein forms comprising sequences encoded by exons 3 and 4, or both, is reduced by less than 40%.

[0036] In some embodiments, the level of MuSK transcripts comprising exons 6 and 7, or the level of MuSK protein forms comprising sequences encoded by exons 6 and 7, or both, is reduced by more than 70%, and the level of MuSK transcripts comprising exons 3 and 4, or the level of MuSK protein forms comprising sequences encoded by exons 3 and 4, or both, is reduced by less than 40%.

[0037] In some embodiments, the level of MuSK transcripts comprising exons 6 and 7, or the level of MuSK protein forms comprising sequences encoded by exons 6 and 7, or both, is reduced by more than 80%, and the level of MuSK transcripts comprising exons 3 and 4, or the level of MuSK protein forms comprising sequences encoded by exons 3 and 4, or both, is reduced by less than 40%.

[0038] In some embodiments, the level of MuSK transcripts comprising exons 6 and 7, or the level of MuSK protein forms comprising sequences encoded by exons 6 and 7, or both, is reduced by more than 90%, and the level of MuSK transcripts comprising exons 3 and 4, or the level of MuSK protein forms comprising sequences encoded by exons 3 and 4, or both, is reduced by less than 40%.

[0039] In some embodiments, the level of MuSK transcripts comprising exons 6 and 7, or the level of MuSK protein forms comprising sequences encoded by exons 6 and 7, or both, is reduced by more than 60%, and the level of MuSK transcripts comprising exons 3 and 4, or the level of MuSK protein forms comprising sequences encoded by exons 3 and 4, or both, is reduced by less than 30%.

[0040] In some embodiments, the level of MuSK transcripts comprising exons 6 and 7, or the level of MuSK protein forms comprising sequences encoded by exons 6 and 7, or both, is reduced by more than 60%, and the level of MuSK transcripts comprising exons 3 and 4, or the level of MuSK protein forms comprising sequences encoded by exons 3 and 4, or both, is reduced by less than 20%. In some embodiments, the level of MuSK transcripts comprising exons 6 and 7, or the level of MuSK protein forms comprising sequences encoded by exons 6 and 7, or both, is reduced by more than 60%, and the level of MuSK transcripts comprising exons 3 and 4, or the level of MuSK protein forms comprising sequences encoded by exons 3 and 4, or both, is reduced by less than 10%. In some embodiments, the base sequence of the oligonucleotide comprises a sequence having five or fewer mismatches from an 18-25 base portion of the MuSK gene or its complement.

[0041] In some embodiments, the oligonucleotides correspond to positions 83776 to 83800 and 83854 to 83878 of the MuSK gene sequence represented by SEQ ID NO:77.

[0042] In some embodiments, the oligonucleotides described herein target a region on the MuSK genomic sequence within the region defined by nucleotides 83841-83905 and 83962-84032 on the MuSK gene sequence represented by SEQ ID NO:77.

[0043] In some embodiments, the oligonucleotide targets a region on the MuSK genomic sequence within, or including at least a portion of, the sequence ACCTGTCAGGTTTCTTCTGGGTCCATTCAAGAGAGTGTGAAAGACCGAGTGATTGACTCAAGAC (Region 1, SEQ ID NO: 126). In some embodiments, the oligonucleotide targets a portion of the MuSK transcript that includes a sequence at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% identical to Region 1, SEQ ID NO: 126. In some embodiments, the oligonucleotide targets a portion of the MuSK transcript that includes a sequence identical to at least 10, 15, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consecutive bases of Region 1, SEQ ID NO: 126. In some embodiments, the oligonucleotide targets a portion of the MuSK transcript that includes a sequence identical to 30 or fewer consecutive bases of Region 1, SEQ ID NO: 126. In some embodiments, the oligonucleotide targets a portion of the MuSK transcript comprising a sequence identical to at least 15 contiguous bases of Region 1, SEQ ID NO: 126. In some embodiments, the oligonucleotide targets a portion of the MuSK transcript comprising a sequence identical to 15-30 contiguous bases of Region 1, SEQ ID NO: 126. In some embodiments, the oligonucleotide targets a portion of the MuSK transcript comprising a sequence identical to 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous bases of Region 1, SEQ ID NO: 126. In some embodiments, the oligonucleotide targets a portion of the MuSK transcript comprising a sequence identical to 15 contiguous bases of Region 1, SEQ ID NO: 126. In some embodiments, the oligonucleotide targets a portion of the MuSK transcript comprising a sequence identical to 16 contiguous bases of Region 1, SEQ ID NO: 126. In some embodiments, the oligonucleotide targets a portion of the MuSK transcript that includes a sequence identical to 17 contiguous bases of region 1, SEQ ID NO: 126. In some embodiments, the oligonucleotide targets a portion of the MuSK transcript that includes a sequence identical to 18 contiguous bases of region 1, SEQ ID NO: 126.In some embodiments, the oligonucleotide targets a portion of the MuSK transcript comprising a sequence identical to 19 contiguous bases of region 1, SEQ ID NO: 126. In some embodiments, the oligonucleotide targets a portion of the MuSK transcript comprising a sequence identical to 20 contiguous bases of region 1, SEQ ID NO: 126. In some embodiments, the oligonucleotide targets a portion of the MuSK transcript comprising a sequence identical to 21 contiguous bases of region 1, SEQ ID NO: 126. In some embodiments, the oligonucleotide targets a portion of the MuSK transcript comprising a sequence identical to 22 contiguous bases of region 1, SEQ ID NO: 126. In some embodiments, the oligonucleotide targets a portion of the MuSK transcript comprising a sequence identical to 23 contiguous bases of region 1, SEQ ID NO: 126. In some embodiments, the oligonucleotide targets a portion of the MuSK transcript comprising a sequence identical to 24 contiguous bases of region 1, SEQ ID NO: 126. In some embodiments, the oligonucleotide targets a portion of the MuSK transcript comprising a sequence identical to 25 contiguous bases of region 1, SEQ ID NO: 126. In some embodiments, the oligonucleotide targets a portion of the MuSK transcript that includes a sequence identical to 26 contiguous bases of region 1, SEQ ID NO: 126. In some embodiments, the oligonucleotide targets a portion of the MuSK transcript that includes a sequence identical to 27 contiguous bases of region 1, SEQ ID NO: 126. In some embodiments, the oligonucleotide targets a portion of the MuSK transcript that includes a sequence identical to 28 contiguous bases of region 1, SEQ ID NO: 126. In some embodiments, the oligonucleotide targets a portion of the MuSK transcript that includes a sequence identical to 29 contiguous bases of region 1, SEQ ID NO: 126. In some embodiments, the oligonucleotide targets a portion of the MuSK transcript that includes a sequence identical to 30 contiguous bases of region 1, SEQ ID NO: 126.

[0044] In some embodiments, the oligonucleotide targets a region on the MuSK genomic sequence within, or including at least a portion of, the sequence GGGGAGAAGTTCAGTACTGCCAAGGCTGCAGCCACCATCAGCATAGCAGGTAGGATGCCCCTTCACATTTG (Region 2, SEQ ID NO:211). In some embodiments, the oligonucleotide targets a portion of the MUSK transcript comprising a sequence at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% identical to Region 2, SEQ ID NO:211. In some embodiments, the oligonucleotide targets a portion of the MUSK transcript comprising a sequence identical to at least 10, 15, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consecutive bases of Region 2, SEQ ID NO:211. In some embodiments, the oligonucleotide targets a portion of the MUSK transcript comprising a sequence identical to 30 or fewer consecutive bases of Region 2, SEQ ID NO:211. In some embodiments, the oligonucleotide targets a portion of the MUSK transcript comprising a sequence identical to at least 15 contiguous bases of region 2, SEQ ID NO:211. In some embodiments, the oligonucleotide targets a portion of the MUSK transcript comprising a sequence identical to 15-30 contiguous bases of region 2, SEQ ID NO:211. In some embodiments, the oligonucleotide targets a portion of the MUSK transcript comprising a sequence identical to 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous bases of region 2, SEQ ID NO:211. In some embodiments, the oligonucleotide targets a portion of the MuSK transcript comprising a sequence identical to 15 contiguous bases of region 2, SEQ ID NO:211. In some embodiments, the oligonucleotide targets a portion of the MuSK transcript comprising a sequence identical to 16 contiguous bases of region 2, SEQ ID NO:211. In some embodiments, the oligonucleotide targets a portion of the MuSK transcript that includes a sequence identical to 17 contiguous bases of SEQ ID NO:211 in region 2.In some embodiments, the oligonucleotide targets a portion of the MuSK transcript comprising a sequence identical to 18 contiguous bases of SEQ ID NO:211 in region 2. In some embodiments, the oligonucleotide targets a portion of the MuSK transcript comprising a sequence identical to 19 contiguous bases of SEQ ID NO:211 in region 2. In some embodiments, the oligonucleotide targets a portion of the MuSK transcript comprising a sequence identical to 20 contiguous bases of SEQ ID NO:211 in region 2. In some embodiments, the oligonucleotide targets a portion of the MuSK transcript comprising a sequence identical to 21 contiguous bases of SEQ ID NO:211 in region 2. In some embodiments, the oligonucleotide targets a portion of the MuSK transcript comprising a sequence identical to 22 contiguous bases of SEQ ID NO:211 in region 2. In some embodiments, the oligonucleotide targets a portion of the MuSK transcript comprising a sequence identical to 23 contiguous bases of SEQ ID NO:211 in region 2. In some embodiments, the oligonucleotide targets a portion of the MuSK transcript comprising a sequence identical to 24 contiguous bases of SEQ ID NO:211 in region 2. In some embodiments, the oligonucleotide targets a portion of the MuSK transcript comprising a sequence identical to 25 contiguous bases of SEQ ID NO:211 in region 2. In some embodiments, the oligonucleotide targets a portion of the MuSK transcript comprising a sequence identical to 26 contiguous bases of SEQ ID NO:211 in region 2. In some embodiments, the oligonucleotide targets a portion of the MuSK transcript comprising a sequence identical to 27 contiguous bases of SEQ ID NO:211 in region 2. In some embodiments, the oligonucleotide targets a portion of the MuSK transcript comprising a sequence identical to 28 contiguous bases of SEQ ID NO:211 in region 2. In some embodiments, the oligonucleotide targets a portion of the MuSK transcript comprising a sequence identical to 29 contiguous bases of SEQ ID NO:211 in region 2. In some embodiments, the oligonucleotide targets a portion of the MuSK transcript comprising a sequence identical to 30 contiguous bases of SEQ ID NO:211 in region 2.

[0045] In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to a sequence comprising, or at least a portion of, a sequence within Region 1 (SEQ ID NO: 126), ACTCTGTCAGGTTTCTTCTGGGTCCATTCAAGAGAGTGTGAAAGACCGAGTGATTGACTCAAGAC (Region 1, SEQ ID NO: 126). In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to a sequence at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% identical to Region 1 (SEQ ID NO: 126). In some embodiments, the oligonucleotide comprises a sequence at least 90% identical to a portion of Region 1 (SEQ ID NO: 126), comprising at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consecutive bases of Region 1 (SEQ ID NO: 126). In some embodiments, the oligonucleotide comprises a sequence at least 95% identical to a portion of Region 1 (SEQ ID NO: 126) comprising at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous bases of Region 1 (SEQ ID NO: 126). In some embodiments, the oligonucleotide comprises a sequence at least 90% identical to a portion of Region 1 (SEQ ID NO: 126) comprising at least 10 contiguous bases of Region 1 (SEQ ID NO: 126). In some embodiments, the oligonucleotide comprises a sequence at least 90% identical to a portion of Region 1 (SEQ ID NO: 126) comprising at least 11 contiguous bases of Region 1 (SEQ ID NO: 126). In some embodiments, the oligonucleotide comprises a sequence at least 90% identical to a portion of Region 1 (SEQ ID NO: 126) comprising at least 12 contiguous bases of Region 1 (SEQ ID NO: 126). In some embodiments, the oligonucleotide comprises a sequence at least 90% identical to a portion of Region 1 (SEQ ID NO: 126) comprising at least 13 contiguous bases of Region 1 (SEQ ID NO: 126). In some embodiments, the oligonucleotide comprises a sequence at least 90% identical to a portion of Region 1 (SEQ ID NO: 126) comprising at least 14 contiguous bases of Region 1 (SEQ ID NO: 126).In some embodiments, the oligonucleotide comprises a sequence at least 90% identical to a portion of Region 1 (SEQ ID NO: 126) comprising at least 15 contiguous bases of Region 1 (SEQ ID NO: 126). In some embodiments, the oligonucleotide comprises a sequence at least 90% identical to a portion of Region 1 (SEQ ID NO: 126) comprising at least 16 contiguous bases of Region 1 (SEQ ID NO: 126). In some embodiments, the oligonucleotide comprises a sequence at least 90% identical to a portion of Region 1 (SEQ ID NO: 126) comprising at least 17 contiguous bases of Region 1 (SEQ ID NO: 126). In some embodiments, the oligonucleotide comprises a sequence at least 90% identical to a portion of Region 1 (SEQ ID NO: 126) comprising at least 18 contiguous bases of Region 1 (SEQ ID NO: 126). In some embodiments, the oligonucleotide comprises a sequence at least 90% identical to a portion of Region 1 (SEQ ID NO: 126) comprising at least 19 contiguous bases of Region 1 (SEQ ID NO: 126). In some embodiments, the oligonucleotide comprises a sequence at least 90% identical to a portion of Region 1 (SEQ ID NO: 126) comprising at least 20 contiguous bases of Region 1 (SEQ ID NO: 126). In some embodiments, the oligonucleotide comprises a sequence at least 90% identical to a portion of Region 1 (SEQ ID NO: 126) comprising at least 21 contiguous bases of Region 1 (SEQ ID NO: 126). In some embodiments, the oligonucleotide comprises a sequence at least 90% identical to a portion of Region 1 (SEQ ID NO: 126) comprising at least 22 contiguous bases of Region 1 (SEQ ID NO: 126). In some embodiments, the oligonucleotide comprises a sequence at least 90% identical to a portion of Region 1 (SEQ ID NO: 126) comprising at least 23 contiguous bases of Region 1 (SEQ ID NO: 126). In some embodiments, the oligonucleotide comprises a sequence at least 90% identical to a portion of Region 1 (SEQ ID NO: 126) comprising at least 24 contiguous bases of Region 1 (SEQ ID NO: 126). In some embodiments, the oligonucleotide comprises a sequence that is at least 90% identical to a portion of Region 1 (SEQ ID NO: 126), comprising at least 25 contiguous bases of Region 1 (SEQ ID NO: 126).In some embodiments, the oligonucleotide comprises a sequence at least 90% identical to a portion of Region 1 (SEQ ID NO: 126) comprising at least 26 contiguous bases of Region 1 (SEQ ID NO: 126). In some embodiments, the oligonucleotide comprises a sequence at least 90% identical to a portion of Region 1 (SEQ ID NO: 126) comprising at least 27 contiguous bases of Region 1 (SEQ ID NO: 126). In some embodiments, the oligonucleotide comprises a sequence at least 90% identical to a portion of Region 1 (SEQ ID NO: 126) comprising at least 28 contiguous bases of Region 1 (SEQ ID NO: 126). In some embodiments, the oligonucleotide comprises a sequence at least 90% identical to a portion of Region 1 (SEQ ID NO: 126) comprising at least 29 contiguous bases of Region 1 (SEQ ID NO: 126). In some embodiments, the oligonucleotide comprises a sequence at least 90% identical to a portion of Region 1 (SEQ ID NO: 126) comprising at least 30 contiguous bases of Region 1 (SEQ ID NO: 126).

[0046] In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to at least 10, 15, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous bases of Region 1, SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to 30 or fewer contiguous bases of Region 1, SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to 15-30 contiguous bases of Region 1, SEQ ID NO: 126.

[0047] In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous bases of Region 1, SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to 15 contiguous bases of Region 1, SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to 16 contiguous bases of Region 1, SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to 17 contiguous bases of Region 1, SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to 18 contiguous bases of Region 1, SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to 19 contiguous bases of Region 1, SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to 20 contiguous bases of Region 1, SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to 21 contiguous bases of Region 1, SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to 22 contiguous bases of Region 1, SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to 23 contiguous bases of Region 1, SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to 24 contiguous bases of Region 1, SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to 25 contiguous bases of Region 1, SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to 26 contiguous bases of Region 1, SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to 27 contiguous bases of Region 1, SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to 28 contiguous bases of Region 1, SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to 29 contiguous bases of Region 1, SEQ ID NO:126.In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to 30 contiguous bases of Region 1, SEQ ID NO:126.

[0048] In some embodiments, the oligonucleotide has a length of 30 bases. In some embodiments, the oligonucleotide has a length of 29 bases. In some embodiments, the oligonucleotide has a length of 28 bases. In some embodiments, the oligonucleotide has a length of 27 bases. In some embodiments, the oligonucleotide has a length of 26 bases. In some embodiments, the oligonucleotide has a length of 25 bases. In some embodiments, the oligonucleotide has a length of 24 bases. In some embodiments, the oligonucleotide has a length of 23 bases. In some embodiments, the oligonucleotide has a length of 22 bases. In some embodiments, the oligonucleotide has a length of 21 bases. In some embodiments, the oligonucleotide has a length of 20 bases. In some embodiments, the oligonucleotide has a length of 19 bases. In some embodiments, the oligonucleotide has a length of 18 bases. In some embodiments, the oligonucleotide has a length of 17 bases. In some embodiments, the oligonucleotide has a length of 16 bases. In some embodiments, the oligonucleotide has a length of 15 bases.

[0049] In some embodiments, the oligonucleotide has a length of less than about 50 bases. In some embodiments, the oligonucleotide has a length of less than about 40 bases. In some embodiments, the oligonucleotide has a length of less than about 30 bases. In some embodiments, the oligonucleotide has a length of more than about 10 bases. In some embodiments, the oligonucleotide has a length of more than about 15 bases. In some embodiments, the oligonucleotide has a length of more than about 20 bases. In some embodiments, the base sequence of the oligonucleotide comprises, from 5' to 3', the following: [Table 1-1-1] [Table 1-1-2] [Table 1-1-3]

[0050] In some embodiments, the oligonucleotide is complementary to a nucleotide sequence that is at least 90% identical to any one of SEQ ID NOs: 39-76 and 212-253.

[0051] In some embodiments, the oligonucleotide comprises one or more types of base modifications, sugar modifications, and internucleotide linkage modifications, hi some embodiments, the oligonucleotide comprises a non-natural sugar moiety, a non-natural internucleotide linkage, or both.

[0052] In some embodiments, the oligonucleotide comprises an internucleotide bond modification. In some embodiments, the internucleotide bond of the oligonucleotide comprises a natural phosphate bond, a phosphorothioate bond, or a phosphodithioate bond. In some embodiments, each internucleotide bond of the oligonucleotide is a phosphorothioate bond. In some embodiments, each internucleotide bond of the oligonucleotide is a natural phosphate bond. In some embodiments, the oligonucleotide comprises at least one natural phosphate bond and at least one phosphodithioate bond. In some embodiments, at least 50%, 60%, 70%, 80%, 90%, 94%, or 95% of the internucleotide bond of the oligonucleotide are phosphodithioate bond. In some embodiments, at least 50%, 60%, 70%, 80%, 90%, 94%, or 95% of the internucleotide bond of the oligonucleotide are natural phosphate bond.

[0053] In some embodiments, the oligonucleotide comprises a sugar modification. In some embodiments, the modified sugar moiety has a 2'-modification. In some embodiments, the modified sugar moiety comprises a bicyclic sugar modification. In some embodiments, the modified sugar moiety comprises a 2'-modification, wherein the 2'-modification is a 2'-OR 1 and R 1 is an optionally substituted C 1-6 In some embodiments, the modified sugar moiety comprises a 2'-modification, and the 2'-modification is 2'-MOE. In some embodiments, the modified sugar moiety comprises a 2'-modification, and the 2'-modification is 2'-OMe. In some embodiments, each sugar of the oligonucleotide is a 2'-MOE modified sugar. In some embodiments, the oligonucleotide comprises a 2'-OH sugar (RNA sugar). In some embodiments, the oligonucleotide comprises a 2'-H sugar (DNA sugar). In some embodiments, the oligonucleotide comprises a 2'-MOE sugar. In some embodiments, the oligonucleotide comprises a 2'-OMe sugar. In some embodiments, the oligonucleotide comprises a 2'-MOE, 2'-OMe, 2'-OH, 2'-H sugar, or any combination thereof. In some embodiments, the oligonucleotide comprises at least one 2'-MOE sugar and at least one 2'-OH sugar (RNA sugar). In some embodiments, the oligonucleotide comprises at least one 2'-MOE sugar and at least one 2'-H sugar (DNA sugar).

[0054] In some embodiments, the oligonucleotide has the following structure in the 5' to 3' direction: [Table 2-1-1] [Table 2-1-2] [Table 2-1-3] In the table, * represents a phosphorothioate bond, and each sugar in the oligonucleotide is a 2'-MOE modified sugar.

[0055] In some embodiments, an oligonucleotide composition comprising a mixture of two or more oligonucleotides according to any one of the embodiments.

[0056] In some embodiments, the composition comprises oligonucleotides targeted to regions of the MuSK gene corresponding to positions 83776-83800 and 83854-83878 of SEQ ID NO:77.

[0057] In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of an oligonucleotide and at least one pharmaceutically acceptable inactive ingredient selected from a pharmaceutically acceptable diluent, a pharmaceutically acceptable excipient, and a pharmaceutically acceptable carrier, wherein the oligonucleotide is an oligonucleotide of any one of the embodiments.

[0058] In some embodiments, the oligonucleotide is formulated in a nanocarrier. In some embodiments, the oligonucleotide is formulated in a lipid nanoparticle (LNP). In some embodiments, the oligonucleotide is covalently conjugated to an additional moiety selected from a lipid (e.g., cholesterol), a peptide, an aptamer, an antibody, and a sugar (e.g., N-acetylgalactosamine (GalNAc)). In some embodiments, the oligonucleotide is covalently conjugated to N-acetylgalactosamine (GalNAc).

[0059] In another aspect, the disclosure features a method of altering the relative amounts of MuSK splicing transcripts, the method including administering to a subject a pharmaceutical composition that includes or delivers the composition of any one of the preceding embodiments.

[0060] In some embodiments, the disclosure features an alteration in MuSK splicing transcripts characterized by an increase in the ratio of MuSK transcripts containing Ig3 domain-encoding sequences to MuSK transcripts that do not contain Ig3 domain-encoding sequences. In some embodiments, the alteration in MuSK splicing transcripts characterized by a decrease in the ratio of MuSK transcripts containing Ig3 domain-encoding sequences to MuSK transcripts that do not contain Ig3 domain-encoding sequences. In some embodiments, the alteration in MuSK splicing transcripts characterized by a decrease in MuSK transcripts containing Ig3 domain-encoding sequences while the level of total MuSK transcripts remains substantially the same.

[0061] In some embodiments, the alteration in MuSK splicing transcripts is characterized by a decrease in the level of MuSK transcripts containing exons 6 and 7, or a decrease in the level of MuSK protein forms containing sequences encoded by exons 6 and 7, or both.

[0062] In some embodiments, the alteration in MuSK splicing transcripts is characterized by an increased level of MuSK transcripts comprising exons 6 and 7, or an increased level of MuSK protein forms comprising sequences encoded by exons 6 and 7, or both. In some embodiments, the alteration in MuSK splicing transcripts is characterized by a decreased level of MuSK transcripts comprising exons 3 and 4, or a decreased level of MuSK protein forms comprising sequences encoded by exons 3 and 4, or both. In some embodiments, the alteration in MuSK splicing transcripts is characterized by an increased level of MuSK transcripts comprising exons 3 and 4, or an increased level of MuSK protein forms comprising sequences encoded by exons 3 and 4, or both.

[0063] In some embodiments, the alteration in MuSK splicing transcripts is characterized by the level of MuSK transcripts comprising exons 3 and 4 remaining substantially the same, or the level of MuSK protein forms comprising sequences encoded by exons 3 and 4 remaining substantially the same, or both. In some embodiments, the alteration in MuSK splicing transcripts is characterized by the level of MuSK transcripts comprising exons 3 and 4 remaining substantially the same, or the level of MuSK protein forms comprising sequences encoded by exons 3 and 4 remaining substantially the same, or both, and the level of MuSK transcripts comprising exons 6 and 7 decreasing, or the level of MuSK protein forms comprising sequences encoded by exons 6 and 7 decreasing, or both.

[0064] In another aspect, the disclosure features a method of treating a subject suffering from one or more characteristics of a neurodegenerative disease, the method including administering to the subject a pharmaceutical composition including or delivering the composition of any one of the preceding embodiments.

[0065] In another aspect, the disclosure features a method of increasing neurogenesis, the method including administering to a subject a pharmaceutical composition including or delivering the composition of any one of the preceding embodiments.

[0066] In another aspect, the disclosure features a method of treating a subject suffering from one or more characteristics of neuromuscular dysfunction or muscular dystrophy, the method including administering to the subject a pharmaceutical composition comprising or delivering the composition of any one of the preceding embodiments.

[0067] In another aspect, the disclosure features a method of increasing muscle regeneration and / or muscle growth, the method including administering to a subject a pharmaceutical composition including or delivering the composition of any one of the preceding embodiments.

[0068] In another aspect, the disclosure features a method of treating muscle fibrosis, the method including administering to a subject a pharmaceutical composition including or delivering the composition of any one of the preceding embodiments.

[0069] In some embodiments, the subject is at risk for or suffering from a disease or disorder selected from the group consisting of neuromuscular dysfunction, neurodegenerative disorders, cardiac disorders, and diseases characterized by muscle wasting. In some embodiments, the neurodegenerative disorder is selected from the group consisting 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, diabetes-mediated hippocampal neuron loss, brain injury (e.g., traumatic and / or anoxic brain injury), and Huntington's disease. In some embodiments, the neurodegenerative disease is Alzheimer's disease (AD).

[0070] In some embodiments, the neuromuscular dysfunction is a muscular dystrophy selected from the group consisting of: Becker, congenital, distal, Duchenne, Emery-Dreyfus, facioscapulohumeral, limb-girdle, myotonic, and oculopharyngeal muscular dystrophy.

[0071] In some embodiments, the cardiac disorder is myocardial infarction or cardiomyopathy. In some embodiments, the subject is in need of enhanced muscle regeneration and / or growth after a condition selected from the group consisting of surgery, trauma, and prolonged immobilization. In some embodiments, the prolonged immobilization results from bed rest or a cast. In some embodiments, the subject is at risk for or suffers from sarcopenia. In some embodiments, the subject is at risk for or suffers from muscle fibrosis resulting from a disease or condition selected from the group consisting of trauma, a genetic disease, a muscle disorder, and aging. In some embodiments, the trauma is the result of a condition selected from the group consisting of radiation therapy, a crush injury, a laceration, and an amputation.

[0072] In some embodiments, 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.

[0073] In some embodiments, the composition is delivered to the CNS. In some embodiments, the composition is delivered to the cerebrospinal fluid. In some embodiments, the composition is delivered to muscle. In some embodiments, the composition is delivered to the liver. In some embodiments, the composition may be formulated for systemic or local administration. In some embodiments, 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, ocular administration, or otic administration.

[0074] In some embodiments, the composition is formulated for delivery by intramuscular administration. In some embodiments, the composition is formulated for delivery by intravenous administration. In some embodiments, the composition is formulated for delivery by oral administration. [Brief explanation of the drawings]

[0075] For illustrative purposes, specific embodiments of the invention are shown in the drawings described below. Like numerals in the drawings refer to like elements throughout. It should be understood, however, that the invention is not limited to the precise arrangements, dimensions, and apparatus shown. In the drawings:

[0076] [Figure 1] FIG. 1 is a schematic diagram of the interaction between the BMP and Ig3 domains of MuSK and its implications in BMP signaling and neurogenesis and cognition. [Figure 2] FIG. 1 is a schematic diagram of exemplary primer designs for selective detection of full-length and MuSKΔIg3 transcripts and encoded proteins, as well as total MuSK and full-length MuSK transcripts. [Figure 3] Relative MuSK expression measured by qPCR using Taqman or SYBR green technology in LHCN-M2 cells is shown. [Figure 4] FIG. 1 is a schematic diagram of "Region 1" and "Region 2" of exon 7 of the MuSK transcript. [Figure 5] Alignment of ASOs Bld1 to Bld18 on the MuSK gene sequence is shown. [Figure 6] Microscopic acquisition of LHCN-M2 cells 24 h after transfection with 100 nM ASOs Bld1–Bld4, Bld6, Bld7, Bld9, Bld11, Bld12, Bld13, Bld14, Bld15, Bld16, and Bld17 and untreated controls is shown (scale 100 nm). [Figure 7] Relative MuSK expression measured by qPCR in LHCN-M2 cells transfected with 50 and 100 nM ASOs Bld1 to Bld18 is shown. Panel A shows total MuSK expression (i.e., using MuSK34, a primer spanning exon / exon junctions 3-4), and panel B shows expression of MuSK exons 6-7 (i.e., using MuSK67, a primer spanning exon / exon junctions 6-7). All samples are normalized to the housekeeping genes GAPDH and YWHAZ, as well as controls. [Figure 8] Alignment of ASOs Bld1 to Bld18 on the MuSK gene sequence and their effects on MuSK expression are shown. Green: No effect or increase in MuSK expression (MuSK expression was 90-300% of the untreated state). Orange: Moderate decrease in MuSK expression (MuSK expression was 40-70% of the untreated state). Red: High decrease in MuSK expression (MuSK expression was less than 40% of the untreated state). The red area indicates the region important for MuSK expression, where ASOs induced a decrease in MuSK expression of >80%. [Figure 9] Alignment of ASOs Bld19 to Bld38 on the MuSK gene sequence is shown. [Figure 10]Microscopic observations of LHCN-M2 cells 24 hours after transfection with 100 nM ASO Bld 19-22, Bld 29-34 and untreated controls are shown (scale 100 nm). [Figure 11] Relative MuSK expression measured by qPCR in LHCN-M2 cells transfected with ASOs (control, Bld19-Bld28) at 50 and 100 nM. Panel A shows MuSK34 expression, and panel B shows MuSK67 expression. All samples are normalized to the housekeeping genes GAPDH and YWHAZ, as well as controls. [Figure 12] Alignment of ASOs Bld19-Bld38 to the gene sequence and their effect on MuSK expression are shown. Green: No effect or increase in MuSK expression (MuSK expression was 90-300% of the untreated state). Orange: Moderate decrease in MuSK expression (MuSK expression was 40-70% of the untreated state). Red: High decrease in MuSK expression (MuSK expression was less than 40% of the untreated state). Red areas indicate regions important for MuSK expression, where ASOs induced a >80% decrease in MuSK67. Purple areas indicate regions that can be targeted to selectively inhibit MuSK67 expression (with less effect on other exons). [Figure 13] Relative MuSK expression in response to various doses of ASO (Bld25 (Panel A), Bld26 (Panel B), Bld27 (Panel C), Bld28 (Panel D), Bld35 (Panel E), and Bld38 (Panel F)) is shown. MuSK34 (blue) and MuSK67 (red) expression was measured by qPCR and normalized to housekeeping genes and controls. Estimated IC is shown on each graph. The five doses tested were 2.5, 5, 25, 125, and 400 nM. [Figure 14]Relative MuSK expression in response to various doses of ASO (Bld25 (Panel A), Bld26 (Panel B)) is shown. MuSK34 (blue) and MuSK67 (red) expression was measured by qPCR and normalized to housekeeping genes and controls. The four doses tested were 5, 7.5, 12.5, and 25 nM. [Figure 15] Relative MuSK expression in response to a combination of ASOs Bld25 and Bld26 at a final concentration of 12.5 nM compared to an untreated control is shown. MuSK34 (blue) and MuSK67 (red) expression was measured by qPCR and normalized to housekeeping genes and controls. [Figure 16] Gel electrophoresis shows the migration of PCR products from exon 3 to exon 9. cDNA from ASO-treated cells (Bld25, i.e., "hu7-10" or Bld26, i.e., "hu73") was amplified by PCR and loaded onto gel electrophoresis (Panel A). The expected products are shown in Panels B and B'. [Figure 17] An alignment of the sequences of different target portions of "region 1" is shown. ASOs Bld25, Bld26, and Bld51 to Bld66 target different portions of region 1. [Figure 18] The relative changes in gene expression of total MuSK (MuSK34) and Ig3 domain-containing MuSK (MuSK67) are shown for ASO Bld51-Bld66 at a concentration of 12.5 nM (Panel A) and 100 nM (Panel B). [Figure 19] Alignment of ASOs Bld25-1, Bld25-2, Bld25, Bld25-3, Bld25-4, BLd25-5, Bld26-1, Bld26-2, Bld26, Bld26-3, Bld26-4, Bld27, Bld28, Bld35, Bld38 on various target portions of "region 1" and "region 2" of exon 7 of human MuSK is shown. [Figure 20] Figure 1 shows MuSK gene expression analyzed by qPCR. Panel A shows the relative gene expression of MuSKIg3 (MuSK67), and panel B shows the relative gene expression of total MuSK (MuSK34). [Figure 21] MuSK gene expression from ASO alone showed less than 50% relative expression of MuSK67 and more than 60% relative expression of total MuSK (MuSK34) compared to controls. Panel A shows relative gene expression of MuSKIg3 (MuSK67), and panel B shows relative gene expression of total MuSK (MuSK34). [Figure 22] A comparison of Bld25-5 and Bld25 is shown 24 hours (Panels A and B) and 48 hours (Panels C and D) after transfection with ASO. [Figure 23] Alignment of ASOs Bld25, Bld25-A, Bld25-B, Bld25-C, Bld25-D, Bld25-E, Bld25-5, Bld25-5-A, Bld25-5-B, Bld25-5-C, Bld25-5-D, Bld25-5-E, Bld26-2, Bld26-2-A, Bld26-2-B, Bld26-2-C, Bld26-2-D, Bld26, Bld26-B, Bld26-C, and Bld26-D at various target portions of "region 1" of exon 7 of human MuSK is shown. [Figure 24] Relative gene expression of ASOs Bld25-A, Bld25-B, Bld25-C, Bld25-D, and Bld25-E (MuSK34 and MuSK67) is shown. [Figure 25] Relative gene expression of ASOs Bld25-5-A, Bld25-5-B, Bld25-5-C, Bld25-5-D, and Bld25-5-E (MuSK34 and MuSK67) is shown. [Figure 26] A graphical representation of three variants of MuSK RNA is shown. Full-length and ΔIg3 indicate the lengths of the full-length sequence and the sequence containing the deletion of exons 6 and 7, respectively, amplified by PCR from exon 3 to exon 9. [Figure 27] Gel migration of PCR products from cells treated with Bld25 and Bld25-5 and the intensity of the bands from Bld25 and Bld25-5 products are shown. [Figure 28]The sequence of the band below the band at the predicted position of Δ6,7 variant 1 (687 bases) indicates that it was indeed the sequence of this splice variant. [Figure 29] Shown are images of HCN-M2 cells treated with 10 nM siRNA against MuSK for 48 hours and then stained for MuSK protein at 2, 3, 4, and 5 days after siRNA treatment. DETAILED DESCRIPTION OF THE INVENTION

[0077] 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 implicitly indicated from context, the following terms and phrases include the meanings provided below.

[0078] These definitions are provided to aid in the description of particular embodiments and are not intended to limit the claimed invention, since the scope of the invention is limited only by the claims.

[0079] 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.

[0080] About or Approximately: When used herein with reference to a value, the term "about" or "approximately" refers to a similar value in relation to the referenced value. Generally, one of ordinary skill in the art will be familiar with the context and 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 ​​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 referenced value.

[0081] Administration: As used herein, the term "administration" typically refers to the administration of a composition to a subject or system, e.g., to effect delivery of 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 recognize 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 (e.g., may be or include one or more of topical application 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, intratracheal (e.g., by intratracheal instillation), intravaginal, vitreous, 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 include only a single administration. In some embodiments, administration may include administration of a set number of doses. In some embodiments, administration may include intermittent administration (e.g., multiple administrations separated by a period of time) and / or periodic administration (e.g., individual administrations separated by a common period of time). In some embodiments, administration may include continuous administration (e.g., perfusion) for at least a selected period of time.

[0082] Agent: Generally, as used herein, the term "agent" may be used to refer to any chemical class of compound or substance, including, for example, polypeptides, nucleic acids, monosaccharides, lipids, small molecules, metals, or combinations or complexes thereof. In appropriate circumstances, as will be clear to one of skill in the art from the context, the term may be used to refer to a substance that is or includes a cell or organism, or a fragment, extract, or component thereof. Alternatively, or additionally, as will be clear from the context, the term may be used to refer to a natural product found in and / or obtained from nature. In some cases, as will be further clear from the context, the term may be used to refer to one or more substances that are man-made in that they have been designed, engineered, and / or produced by human action and / or are not found in nature. In some embodiments, an agent may be utilized in isolated or pure form, and in some embodiments, an agent may be utilized in crude form. In some embodiments, candidate agents may be provided as a collection or library, among which screening may be performed, e.g., to identify or characterize active agents. In some cases, the term "drug" may refer to a compound or substance that is or includes a polymer, and in some cases, the term may refer to a compound or substance that includes one or more polymer moieties. In some embodiments, the term "drug" may refer to a compound or substance that is not a polymer and / or that is substantially free of any polymer and / or that does not include one or more specific polymer moieties. In some embodiments, the term may refer to a compound or substance that lacks or is substantially free of any polymer moieties.

[0083] Agonist: Those skilled in the art will understand that the term "agonist" can be used to refer to an agent (i.e., an "stimulator"), condition, or event whose presence, level, degree, type, or form correlates with an increase in the level or activity of another agent (i.e., a stimulated or target agent). Generally, an agonist can be or include an agent of any chemical class, including, for example, small molecules, polypeptides, nucleic acids, carbohydrates, lipids, metals, and / or any other substance that exhibits related activating activity. In some embodiments, an agonist can be direct (where the agonist exerts its effect directly on its target), while in some embodiments, an agonist can be indirect (where the agonist exerts its effect other than binding to its target, e.g., by interacting with a modulator of the target, such that the level or activity of the target is altered). 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) and alters the level, formation, 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, a stimulator may bind to (and potentially stimulate) a binding target, which binding in turn causes an increase in the level or activity of the stimulated target. By way of specific example, in some embodiments, a stimulator that binds to a nucleic acid target may alter the level and / or activity of that target, and in some particular embodiments, may stimulate 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, thereby increasing the level of a desired product, e.g., mRNA) and / or a downstream target, e.g., a polypeptide encoded by such nucleic acid target.To take one particular such example, in some embodiments, the stimulatory agent may be or include an oligonucleotide that binds to a primary transcript and alters its splicing pattern, increasing the level and / or activity of a particular spliced ​​form (e.g., mature mRNA), which may then achieve increased levels of a product (e.g., a polypeptide) that is or is encoded by such a particular spliced ​​form.

[0084] Antagonist: As used herein, those skilled in the art will understand that the term "antagonist" can be used to refer to an agent (i.e., an "antagonist"), 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 inhibitor or target). Generally, an antagonist can be or include an agent of any chemical class, including, for example, small molecules, polypeptides, nucleic acids, carbohydrates, lipids, metals, and / or any other substance that exhibits related inhibitory activity. In some embodiments, an antagonist can be direct (in which case the antagonist exerts its effect directly on its target), while in some embodiments, the antagonist can be indirect (in which case the antagonist exerts its effect other than by binding to its target, e.g., by interacting with a modulator of the target, such that the level or activity of the target is altered). 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) and alters the level, formation, and / or activity of the target. In some embodiments, the altered level, formation, and / or activity is a decrease in the level of the altered protein expressed from the target nucleic acid sequence. Those of skill in the art will understand, upon reading this disclosure, that in some embodiments, an antagonist may bind to (and potentially antagonize) a binding target, which binding may also cause a decrease in the level or activity of the antagonized target. By way of specific example, in some embodiments, an antagonist that binds to a nucleic acid target may alter the level and / or activity of that target, and in some particular 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, thereby suppressing the level of an undesired product, e.g., mRNA) and / or antagonize a downstream target, e.g., a polypeptide encoded by such a nucleic acid target.To take one particular such example, in some embodiments, the antagonist may be or include an oligonucleotide that binds to the primary transcript and alters its splicing pattern, suppressing the level and / or activity of a particular spliced ​​form (e.g., mature mRNA), which may then achieve reduced levels of a product (e.g., a polypeptide) that is or is encoded by such a particular spliced ​​form.

[0085] Antibody agent: As used herein, the term antibody agent refers to an agent that specifically binds to a particular antigen (e.g., may be or may include an epitope of a protein of interest, e.g., 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 a mouse antibody, a rabbit antibody, a primate antibody, or a human antibody. In some embodiments, an antibody agent may include one or more sequence elements that are humanized, primatized, or chimerized, as known in the art. In many embodiments, the term "antibody agent" is used to refer to one or more constructs or formats known or developed in the art for utilizing the structural and functional characteristics of antibodies in alternative presentations. For example, embodiments of antibody agents utilized in accordance with the present disclosure include intact IgA, IgG, IgE, or IgM antibodies, bispecific or multispecific antibodies (such as, for example, Zybodies®), antibody fragments such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, and isolated CDRs, or sets thereof, single chain Fvs, polypeptide-Fc fusions, single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof), cameloid antibodies, masked antibodies (e.g., Probodies®), S mallM odular I mmuno PThe antibody may be of a format selected from, but not limited to, harmaceuticals ("SMIPs™"), single-chain or tandem diabodies (TandAb®), VHH, Anticalins®, Nanobodies® minibodies, BiTEs®, ankyrin repeat proteins or DARPINs®, Avimers®, DART, 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 in nature. In some embodiments, antibodies may contain covalent modifications (e.g., attachment of glycans, payloads (e.g., detectable moieties, therapeutic moieties, catalytic moieties, etc.), or other pendant groups (e.g., polyethylene glycol, etc.). In many embodiments, antibody agents are or comprise polypeptides whose amino acid sequences contain one or more structural elements recognized by those skilled in the art as complementarity-determining regions (CDRs), and in some embodiments, antibody agents contain at least one CDR (e.g., at least one complementary region) whose amino acid sequence is substantially identical to that found in a reference antibody. The polypeptide is or comprises at least one light chain CDR (a primary chain CDR and / or at least one light chain CDR). In some embodiments, the contained CDRs are substantially identical to the reference CDRs in that they are either sequence identical or contain 1 to 5 amino acid substitutions compared to the reference CDR. In some embodiments, the contained CDRs are substantially identical to the reference CDRs in that they exhibit 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 contained 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 contained CDR is substantially identical to the reference CDR in that at least one amino acid in the contained CDR has been deleted, added, or substituted compared to the reference CDR, but the contained CDR otherwise has an amino acid sequence that is identical to the amino acid sequence of the reference CDR. In some embodiments, the contained CDR is substantially identical to the reference CDR in that 1 to 5 amino acids in the contained CDR have been deleted, added, or substituted compared to the reference CDR, but the contained CDR otherwise has an amino acid sequence that is identical to the amino acid sequence of the reference CDR. In some embodiments, the contained CDR is substantially identical to a reference CDR in that at least one amino acid within the contained CDR has been substituted relative to the reference CDR, but the contained CDR otherwise has an amino acid sequence that is identical to the amino acid sequence of the reference CDR. In some embodiments, the contained CDR is substantially identical to a reference CDR in that 1 to 5 amino acids within the contained CDR have been deleted, added, or substituted relative to the reference CDR, but the contained CDR otherwise has an amino acid sequence that is identical to the amino acid sequence of the reference CDR. In some embodiments, the antibody agent is or contains a polypeptide whose amino acid sequence contains structural elements recognized by those skilled in the art as an immunoglobulin variable domain. In some embodiments, the antibody agent is a polypeptide protein having a binding domain that is homologous or largely homologous to an immunoglobulin binding domain.

[0086] Antibody: As used herein, the term "antibody" refers to an immunoglobulin or derivative thereof containing an immunoglobulin domain capable of binding to an antigen (e.g., it may be or 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 any of the following human classes: IgG, IgM, IgA, IgD, and IgE, or subclasses thereof, such as IgG1 and IgG2. In various embodiments of the invention, the antibody is a fragment such as a 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, T., Nature Reviews Cancer, Vol. 2, 750-765, 2002, and references therein. The antibody may be monovalent, bivalent, or multivalent. The antibody may be, for example, a chimeric or humanized antibody in which variable domains of rodent origin are fused to constant domains of human origin, thus retaining the specificity of the rodent antibody. Domains of human origin need not originate directly from humans, in the sense of being first synthesized in humans. Instead, "human" domains can be generated in rodents whose genomes incorporate human immunoglobulin genes. See, e.g., Vaughan, et al., (1998), Nature Biotechnology, 16:535-539. Antibodies may be partially or fully humanized. Antibodies may 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 fluid of an animal that produces the antibody (e.g., after natural exposure or after immunization with the molecule or an antigenic fragment thereof), can be produced using recombinant techniques in cell culture or transgenic organisms, or can be made at least in part by chemical synthesis.In some embodiments, an antibody can act as an antagonist, e.g., by binding to a target antigen, resulting in a decrease in the level or activity of that antigen. In some embodiments, an antibody can act as an agonist, e.g., by binding to a target antigen, resulting in an increase in the level or activity of that antigen.

[0087] Antisense: As used herein, the term "antisense" refers 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 a nucleic acid whose sequence includes part or all of an open reading frame or other contiguous 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, a coding strand may include both coding and non-coding sequences (e.g., a transcript, such as a primary transcript, including both intronic and exon sequences, to name just a few). 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 when 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 within a target sense strand, and in some embodiments, an antisense oligonucleotide binds to a non-coding sequence within a target coding strand. In some embodiments, an antisense oligonucleotide binds to both coding and non-coding sequences within a target coding strand. In some embodiments, an antisense oligonucleotide is characterized in that when bound to its target sequence within a coding strand (e.g., a transcript), it alters post-transcriptional processing of such coding strand (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). In some specific embodiments, the antisense oligonucleotide alters the splicing of its target coding strand; alternatively or additionally, in some embodiments, the antisense coding strand complex is or can be degraded, e.g., by RNase H.

[0088] Approximately: As used herein, the term "approximately" or "about" in reference to a number is generally interpreted as including the number within 5%, 10%, 15%, or 20% in either direction (greater or lesser), unless otherwise stated or otherwise clear from the context (except where such number would be less than 0% or more than 100% of a possible value).

[0089] Binding Agent: Generally, the term "binding agent" is used herein to refer to any substance that binds to a target of interest as described herein. In many embodiments, binding agents of interest specifically bind to their target in that they distinguish that target from other potential binding partners in the context of a particular interaction. Generally, binding agents can be or include any chemical class of substance (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 distinct chemical entities that associate with each other through non-covalent interactions under the appropriate conditions. For example, one of skill in the art will recognize that in some embodiments, a binding agent can include a "generic" binding moiety (e.g., 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) that links to the generic binding moiety's partner. In some embodiments, such an approach may allow for the modular assembly of multiple binding agents through linkage of different specific binding moieties to partners of the same generic binding moiety. In some embodiments, the binding agent is or comprises a polypeptide (including, for example, 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, and in some embodiments, the binding agent is not a polymer. In some embodiments, the binding agent is non-polymeric in that it lacks 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 mimeotope. In some embodiments, the binding agent is or comprises a stapled peptide. In some embodiments, the binding agent is or comprises a nucleic acid such as DNA or RNA (e.g., an antisense oligonucleotide).

[0090] Complementary: As used herein, "complementary," in accordance with its art-accepted 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, a technique known in the art as Watson-Crick base pairing. If a nucleotide at a particular position in a first nucleic acid sequence is complementary to a nucleotide located on the opposite side of a second nucleic acid sequence when the strands are aligned in antiparallel orientation, the nucleotides form a complementary base pair and the nucleic acids are complementary at that position. The percentage of complementarity between a first nucleic acid and a second nucleic acid can be evaluated by aligning them in antiparallel orientation for maximum complementarity across an evaluation window, determining the total number of nts on both strands that form complementary base pairs within the window, dividing by the total number of nts within the window, and multiplying by 100. For example, AAAAAAAA and TTTGTTAT are 75% complementary because 12 of the 16 total nt are complementary base pairs. When calculating the number of complementary nts required to achieve a particular percent complementarity, fractions are rounded to the nearest integer. Positions occupied by non-complementary nucleotides constitute mismatches, i.e., positions are occupied by non-complementary base pairs. In certain embodiments, the evaluation window has a length as described herein for the double-stranded portion or target portion. Complementary sequences include base pairing between a polynucleotide comprising a first nucleotide sequence and a polynucleotide comprising a second nucleotide sequence over the entire length of both nucleotide sequences (if they are the same length) or over the entire length of the shorter sequence (if they are different lengths). Such sequences may be referred to herein as "fully 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, the complementary nucleic acids are at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% complementary over the evaluation window.As used herein, when a first sequence is referred to as "substantially complementary" with respect to a second sequence, the two sequences may be perfectly complementary, or they may contain one or more mismatched bases upon hybridization while maintaining the ability to hybridize under conditions most relevant to the intended application, 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 of a duplex of up to 30 base pairs. It should be understood that when two oligonucleotides are designed to form one or more single-stranded overhangs upon hybridization, such overhangs are not considered mismatched or unpaired nucleotides for purposes of determining percent complementarity. For example, a dsRNA duplex containing one oligonucleotide 21 nucleotides in length and another 23 nucleotides in length, where the longer oligonucleotide contains a 21-nucleotide sequence that is perfectly complementary to the shorter oligonucleotide and a 2-nucleotide overhang, may be referred to herein as "fully complementary." As used herein, a "complementary" sequence 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 requirement for hybridization is met. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobble or Hoogstein base pairs. Those skilled in the art recognize that guanine, cytosine, adenine, and uracil can be substituted by other bases without substantially changing the base-pairing properties of polynucleotides containing nucleotides having such bases, according to the so-called "wobble" rules (see, e.g., Murphy, FV IV & V Ramakrishnan, V., Nature Structural and Molecular Biology 11:1251-1252 (2004)). For example, a nucleotide containing inosine as its base can base pair with a nucleotide containing adenine, cytosine, or uracil.Thus, nucleotides containing uracil, guanine, or adenine can be substituted, for example, by nucleotides containing inosine in the nucleotide sequences of the inhibitory RNAs described herein. It will be understood that the terms "complementary," "fully complementary," and "substantially complementary" can be used in reference to base matches between any two nucleic acids, for example, base matches between the sense and antisense strands of a double-stranded nucleic acid, or portions thereof. As used herein, "hybridizing," as understood by one skilled in the art, 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, are part of or comprise different nucleic acid molecules) that contain or consist of complementary portions, resulting in the formation of a stable double-stranded structure (i.e., an intramolecular or intermolecular duplex) under the particular conditions of interest.

[0091] Comprising: The term "comprising" means that other elements may be present in addition to the defined elements presented. The use of "comprising" indicates inclusion rather than limitation.

[0092] Consisting of: The term "consisting of" refers to the compositions, methods, and respective components described herein that do not include any elements not recited in that description of the embodiment. As used herein, the term "consisting essentially of" refers to elements required for a given embodiment. This term allows for the presence of additional elements that do not materially affect the basic and novel or functional characteristics of that embodiment of the invention.

[0093] Combination therapy: As used herein, the term "combination therapy" refers to a situation in which a subject is exposed to two or more therapeutic regimens (e.g., two or more therapeutic agents) simultaneously. In some embodiments, the two or more regimens may be administered simultaneously; in some embodiments, such regimens may be administered sequentially (e.g., all "doses" of a first regimen are administered before any dose of a second regimen); and in some embodiments, such agents are administered in overlapping dosing regimens. In some embodiments, "administration" of a combination therapy may involve administration of one or more agents or modalities to a subject receiving other agents or modalities in combination. For clarity, combination therapy does not require that individual agents be administered together in a single composition (or necessarily simultaneously), although in some embodiments, two or more agents, or active portions thereof, may be administered together in a combined composition or even a combined compound (e.g., as part of a single chemical complex or covalently bonded entity).

[0094] Comparable: As used herein, the term "equivalent" refers to two or more agents, substances, circumstances, sets of conditions, etc. that may not be identical to one another, but that are sufficiently similar to permit comparison, such that one of skill in the art would recognize that one could reasonably conclude based on the observed differences or similarities. In some embodiments, equivalent sets of conditions, circumstances, individuals, or populations are characterized by multiple substantially identical characteristics and one or a few altered characteristics. One of skill in the art will understand the degree of identity required in any given environment for two or more such agents, substances, circumstances, sets of conditions, etc. to be considered equivalent in context. For example, one of skill in the art will recognize that sets of circumstances, individuals, or populations are equivalent to one another when they are characterized by a sufficient number and type of substantially identical characteristics to warrant a reasonable conclusion that differences in results obtained under or with different sets of circumstances, individuals, or populations, or differences in observed phenomena, are caused by or are indicative of variations in those altered characteristics.

[0095] Domain: As used herein, the term domain refers to a section or portion of a substance. In some embodiments, a domain is associated with a particular structural and / or functional characteristic of a substance such that the domain substantially or completely retains the particular structural and / or functional characteristic when physically separated from the remainder of its parent substance. Alternatively or additionally, a domain may be or include a portion of a substance that, when separated from its (parent) substance and linked to a different (recipient) substance, substantially retains and / or confers on the recipient substance one or more structural and / or functional characteristics characterized by the parent substance. 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), and in some such embodiments, a domain is characterized by particular structural elements (e.g., particular amino acid sequences or sequence motifs, a-helical properties, b-sheet properties, coiled-coil properties, random coil properties, etc.) and / or particular functional characteristics (e.g., binding activity, enzymatic activity, folding activity, signaling activity, etc.).

[0096] Dosage regimen: Those skilled in the art will understand that the term "dosage regimen" can be used to refer to a series of unit doses (typically two or more) 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 include one or more administrations. In some embodiments, a dosing regimen includes multiple administrations, each of which is separated in time from the other administrations. In some embodiments, the individual administrations are separated from each other by the same length of time, and in some embodiments, a dosing regimen includes multiple administrations and includes at least two different time periods separating the individual administrations. In some embodiments, all administrations within a dosing regimen are the same unit dose. In some embodiments, different administrations within a dosing regimen are different amounts. In some embodiments, a dosing regimen includes a first administration of a first dose, followed by one or more additional administrations of a second dose that is different from the first dose. In some embodiments, a dosing regimen includes a first administration of a first dose, followed by one or more additional administrations of a second dose that is the same as the first dose. In some embodiments, the dosing regimen correlates with a desired or beneficial outcome when administered across a relevant population (ie, is a therapeutic dosing regimen).

[0097] Expression: As used herein, "expression" of a nucleic acid sequence refers to one or more of: (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., transport 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.

[0098] Isolated or Partially Purified: As used herein, the terms "isolated" or "partially purified," in the case of a nucleic acid or polypeptide, refer 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 in the nucleic acid or polypeptide in its natural source and / or that is present in the nucleic acid or polypeptide when expressed by a cell, or that is secreted in the case of a secreted polypeptide. Chemically synthesized nucleic acids or polypeptides, or nucleic acids or polypeptides synthesized using in vitro transcription / translation, are considered "isolated." The terms purified or substantially purified refer to isolated nucleic acids or polypeptides that contain 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 by weight. In some embodiments, an antibody, antigen-binding portion thereof, or chimeric antigen receptor (CAR) described herein is isolated. In some embodiments, an antibody, antibody reagent, antigen-binding portion thereof, or CAR described herein is purified.

[0099] Genetically engineered: As used herein, "genetically engineered" refers to aspects that have been manipulated by the hand of man. For example, an antibody, antibody reagent, antigen-binding portion thereof, CAR, or bispecific antibody is considered "genetically engineered" if the sequence of the antibody, antibody reagent, antigen-binding portion thereof, CAR, or bispecific antibody has been manipulated by the hand of man and differs from the sequence of a naturally occurring antibody. As is common practice and understood by those skilled in the art, progeny and copies of genetically engineered polynucleotides or polypeptides are typically still referred to as "genetically engineered," even if actual manipulation was performed on a previous entity.

[0100] Fragment: A "fragment" of a material or substance described herein has a structure that includes a discrete portion of the whole, but lacks one or more portions found in the whole. In some embodiments, the fragment consists of such a discrete portion. In some embodiments, the fragment consists of or includes a characteristic structural element or portion present in the whole. In some embodiments, the 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). 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) present in the whole polymer. The whole material or substance may, in some embodiments, be referred to as the "parent" of the fragment.

[0101] 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 particular product), and in some embodiments, a gene comprises non-coding sequence. In certain embodiments, a gene can comprise both coding sequence (e.g., exon sequence) and non-coding sequence (e.g., intron sequence). In some embodiments, a gene can comprise one or more regulatory elements that can, for example, control or influence one or more aspects of gene expression (e.g., cell-type specific expression, inducible expression, etc.).

[0102] 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 may 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.).

[0103] Homology: As used herein, the term "homology" refers to the overall relatedness between polymer molecules, e.g., 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.

[0104] Identity: As used herein, the term "identity" refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. In some embodiments, polymeric 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 in 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 sequences 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. Nucleotides at corresponding positions are then compared. When 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 to be introduced for optimal comparison of the two sequences. Sequence comparison and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4:11-17) incorporated into the ALIGN program (version 2.0). In some exemplary embodiments, nucleic acid sequence comparisons performed using the ALIGN program use a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4.Alternatively, the percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package using the NWSgapdna.CMP matrix.

[0105] Improve, "increase," "inhibit," or "reduce": As used herein, the terms "improve," "increase," "suppress," "reduce," or their grammatical equivalents refer to a value relative to a baseline or other reference measurement. In some embodiments, a suitable reference measurement may be or include a measurement in a particular system (e.g., a single individual, a single cell, or a cell population) under otherwise comparable conditions in the absence of (e.g., before and / or after) a particular agent or treatment, or in the presence of an appropriate reference agent (e.g., a positive or negative control agent). In some embodiments, a suitable reference measurement may be or include a measurement in an equivalent system known or expected to respond in a particular way in the presence of the relevant agent or treatment. One of skill in the art will understand that "improvement," "increase," "reduction," etc. typically refer to a statistically significant change. Furthermore, one of 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., 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 times (e.g., 500, 1000 times) (including all integers and decimal points greater than or equal to 1), e.g., 1.5, 1.6, 1.7, 1.8, etc.) relative to the relevant reference. Alternatively or additionally, in some embodiments, the "change" may be a "percent" change, such that the "change" value represents a 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% increase or decrease, including all integers and decimal points in between, relative to the relevant reference.

[0106] 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 sufficiently stable molecular structure so that the moieties remain associated under the conditions under which the bond 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 bond is covalent. In other embodiments, the bond is non-covalent. Moieties may be linked either 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 linked, either covalently or non-covalently, 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 portion," the bond between the two linking moieties is indirect, and typically each of the linked moieties is covalently bonded to the linker. The linker can be any suitable moiety that reacts with the two moieties to be joined under conditions consistent with the stability of the moieties (which can be protected as necessary depending on the conditions), within a reasonable period of time, and in an amount sufficient to produce a reasonable yield.

[0107] Internucleotide linkage: As used herein, the phrase "internucleotide linkage" generally refers to a phosphorus-containing bond between nucleotide units of an oligonucleotide, and is interchangeable with "intersugar linkage" and "phosphorus atom bridge" as used above and herein. In some embodiments, the internucleotide linkage is a phosphodiester linkage found in natural DNA and RNA molecules. In some embodiments, the internucleotide linkage is a "modified internucleotide linkage" in which each oxygen atom of the phosphodiester bond is optionally and independently replaced by an organic or inorganic moiety. In some embodiments, such organic or inorganic moieties are selected from, but are not limited to, =S, =Se, =NR', -SR', -SeR', -N(R')2, B(R')3, -S-, -Se-, and -N(R')-, where each R' is independently as defined and described below. In some embodiments, the internucleotide linkage is a phosphotriester linkage, a phosphorothioate diester linkage, or a phosphodiester diester linkage. [ka] or modified phosphorothioate triester linkages. One skilled in the art will appreciate that internucleotide linkages may exist as anions or cations at a given pH due to the presence of acid or base moieties in the linkage. In some embodiments, the internucleotide linkage may be chiral.

[0108] 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 administering a therapeutic agent or drug to be effective for at least 12 weeks, or for at least 12 weeks, and does not necessarily mean that the administration itself is for 12 weeks, for example, when a sustained-release composition or a long-acting therapeutic agent or drug is used. Thus, a subject is treated for a period of at least 12 weeks. Often, long-term administration is for at least 4, 5, 6, 7, 8, 9 months or more, or at least 1, 2, 3, 5, 7, or 10 years or more.

[0109] Moiety: Those skilled in the art will understand that a "moiety" is a defined chemical group or chemical entity having a particular structure and / or activity as described herein.

[0110] 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, a nanoparticle is a micelle in that it contains an enclosed compartment separated from the bulk solution by a micellar membrane, and is typically composed of an amphiphile that surrounds and encapsulates a space or compartment (e.g., defines a lumen). In some embodiments, the micellar membrane is composed of at least one polymer (e.g., a biocompatible and / or biodegradable polymer).

[0111] Nucleic acid: As used herein, in the 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 a phosphodiester bond. As is clear from the context, in some embodiments, "nucleic acid" refers to individual nucleic acid residues (e.g., nucleotides and / or nucleosides), and in some embodiments, "nucleic acid" refers to an oligonucleotide chain containing individual nucleic acid residues. In some embodiments, "nucleic acid" is or includes RNA, and in some embodiments, "nucleic acid" is or includes DNA. In some embodiments, a nucleic acid is, includes, or consists of one or more naturally occurring nucleic acid residues. In some embodiments, a nucleic acid is, includes, 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 use a phosphodiester backbone. For example, in some embodiments, the 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, etc.).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 contains one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) compared to those of 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 (in vivo or in vitro) by polymerization based on a complementary template, 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 completely single-stranded, and in some embodiments, the nucleic acid is partially or completely double-stranded.In some embodiments, the nucleic acid has a nucleotide sequence that includes at least one element that encodes a polypeptide or the complement of a sequence that encodes a polypeptide, hi some embodiments, the nucleic acid has enzymatic activity.

[0112] Oligonucleotide: As used herein, the term "oligonucleotide" refers to a polymer or oligomer of nucleotide monomers containing any combination of nucleobases, modified nucleobases, sugars, modified sugars, phosphate bridges, or modified phosphorus atom bridges (also referred to herein as internucleotide linkages and further defined herein). Oligonucleotides may be single-stranded or double-stranded. Single-stranded oligonucleotides can have double-stranded regions, and double-stranded oligonucleotides can have single-stranded regions. Examples of oligonucleotides include, but are not limited to, structural genes, genes including regulatory and termination regions, self-replicating systems such as viral DNA or plasmid DNA, single-stranded and double-stranded siRNAs and other RNA interference reagents (RNAi agents or iRNA agents), shRNA, antisense oligonucleotides, ribozymes, microRNAs, microRNA mimics, supermirs, aptamers, antimirs, antagomirs, Ul adaptors, triplex-forming oligonucleotides, G-quadruplex oligonucleotides, RNA activators, immunostimulatory oligonucleotides, and decoy oligonucleotides. The double-stranded and single-stranded oligonucleotides that are effective in inducing RNA interference are also referred to herein as siRNA, RNAi agent or iRNA agent.In some embodiments, these RNA interference-inducing oligonucleotides associate with a cytoplasmic multiprotein complex known as RNAi-induced silencing complex (RISC).In many embodiments, single-stranded and double-stranded RNAi agents are long enough to be cut by endogenous molecules such as Dicer, and can enter RISC machinery to produce smaller oligonucleotides that can participate in RISC-mediated cleavage of target sequences such as target mRNA.

[0113] 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 elements. In some embodiments, an "operably linked" control element (e.g., promoter, enhancer, etc.) is contiguous (e.g., covalently linked) with the coding element of interest. In some embodiments, the control element acts in trans or cis with the coding functional element of interest.

[0114] Patient: As used herein, the term "patient" refers to any organism to which a provided composition (e.g., a stimulatory agent such as an ASO) is or may be administered, for example, experimental, diagnostic, preventative, 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 susceptible 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 Alzheimer's disease or other disease characterized by neurodegeneration. 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 has undergone a particular treatment to diagnose and / or treat the disease, disorder, or condition.

[0115] Pharmaceutical composition: As used herein, the term "pharmaceutical composition" refers to an active agent (e.g., a MuSK-targeting oligonucleotide) formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a unit dosage suitable for administration in a treatment regimen that provides a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, the pharmaceutical compositions may be specially formulated for administration in solid or liquid form, including those adapted for oral administration, e.g., drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those intended for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, e.g., subcutaneous, intramuscular, intravenous, or epidural injections as sterile solutions or suspensions, or sustained release formulations; topical application, e.g., creams, ointments, or controlled-release patches or sprays applied to the skin, lungs, or oral cavity; vaginal or rectal administration, e.g., vaginal suppositories, creams, or foams; sublingual administration; intraocular administration; transdermal administration; or nasal, pulmonary, and other mucosal surface administration.

[0116] Pharmaceutically acceptable: As used herein, the term "pharmaceutically acceptable" refers to compounds, substances, compositions and / or dosage forms that 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.

[0117] Pharmaceutically acceptable carrier: As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable substance, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent, that encapsulates a substance and is involved in the transport or transportation of a compound of interest from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of substances 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 cellulose 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.

[0118] Pharmaceutically acceptable salts: As used herein, the term "pharmaceutically acceptable salts" refers to salts of compounds that are suitable for use in pharmaceutical situations, i.e., salts that are suitable for use in contact with the tissues of humans and lower animals, within the scope of sound medical judgment, without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al. describe pharmaceutically acceptable salts in J. Pharmaceutical Sciences, 66:1-19 (1977). In some embodiments, pharmaceutically acceptable salts include, but are not limited to, non-toxic acid addition salts, such as salts of amino groups formed with inorganic acids, such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or organic acids, such as acetic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or formed by other methods used in the art, such as ion exchange. In some embodiments, other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, and 2-hydroxy-ethanesulfonate. , lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, and the like.In some embodiments, provided compounds contain one or more acidic groups, e.g., oligonucleotides, and pharmaceutically acceptable salts are alkali, alkaline earth metal, or ammonium (e.g., ammonium salts of N(R)3, where each R is independently defined and described in this disclosure) salts. 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 using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, alkyls having 1 to 6 carbon atoms, sulfonates, arylsulfonates, and the like, where appropriate. In some embodiments, provided compounds contain two or more acidic groups; e.g., oligonucleotides may contain two or more acidic groups (e.g., in native 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 ionizable hydrogens in acidic groups (e.g., in an aqueous solution having a pKa of about 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 or less, in some embodiments, about 7 or less, in some embodiments, about 6 or less, in some embodiments, about 5 or less, in some embodiments, about 4 or less, in some embodiments, about 3 or less) are replaced with cations. In some embodiments, each internucleotide bond, 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 the oligonucleotide, wherein each acidic phosphate group and modified phosphate group, if present, is present as a salt form (all sodium salts).

[0119] Polypeptide: As used herein, the term "polypeptide" is used interchangeably with the term "protein" and 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, such as modifications to or attached to one or more amino acid side chains, at the N-terminus of the polypeptide, 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 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 comprise a stapled polypeptide. In some embodiments, a polypeptide participates in non-covalent or covalent complex formation with one or more other polypeptides (e.g., in the case of antibodies). In some embodiments, a polypeptide has an amino acid sequence that occurs in nature. In some embodiments, a polypeptide has an amino acid sequence that does not occur in nature. In some embodiments, a polypeptide has an amino acid sequence that is engineered, in that it has been designed and / or generated by the act of man.In some embodiments, the term "polypeptide" may be added to the name of a reference polypeptide, activity, or structure. In such instances, it is used herein to refer to polypeptides that share a related activity or structure and therefore may be considered members of the same class or family of polypeptides. For each such class, the specification provides exemplary polypeptides within the class whose amino acid sequences and / or functions are known and / or recognized by those of skill in the art. In some embodiments, such exemplary polypeptides are the reference polypeptides for that class or family of polypeptides. In some embodiments, members of a class or family of polypeptides exhibit high sequence homology or identity with the reference polypeptide of the class (and in some embodiments with all polypeptides within the class), share common sequence motifs (e.g., characteristic sequence elements), and / or share a common activity (in some embodiments, at a similar level or within a specified range). For example, in some embodiments, a member polypeptide exhibits a degree of overall sequence homology or identity with a reference polypeptide of at least about 30%-40%, and often greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more, and / or comprises at least one region (e.g., a conserved region which, in some embodiments, may contain characteristic sequence elements) that exhibits very high sequence identity, often greater than 90%, or even greater than 95%, 96%, 97%, 98%, or 99%. Such conserved regions typically encompass at least 3-4, and often up to 20 or more amino acids, and in some embodiments, the conserved region encompasses at least one contiguous sequence of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more consecutive amino acids. In some embodiments, useful polypeptides may include fragments of a parent polypeptide.In some embodiments, a useful polypeptide may comprise multiple fragments, each of which is found in a different spatial arrangement from one another in the same parent polypeptide than is found in the polypeptide of interest (e.g., fragments that are directly linked in the parent polypeptide 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 polypeptide), thus 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 fragment or segment of a peptide that retains at least 50% of the activity of a wild-type reference polypeptide according to the assays described herein below. Functional fragments may include 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 can be obtained, for example, by mutation of a naturally occurring nucleotide sequence. As referred to 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. DNA sequences encoding variant polypeptides include sequences that encode variant proteins or fragments thereof that contain one or more additions, deletions, or substitutions of nucleotides compared to the native or reference DNA sequence but retain activity. A wide variety of PCR-based site-directed mutagenesis approaches are known in the art and can be applied by those skilled in the art. It is further intended that the various embodiments described herein encompass variants (native or otherwise), 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 a nucleic acid, peptide, polypeptide, or protein sequence that alter a single amino acid or a small percentage of amino acids in the encoded sequence are "conservatively modified variants," which changes result in the substitution of an amino acid with a chemically similar amino acid and retain 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.

[0120] Prevent or prevention: As used herein in connection with the occurrence of a disease, disorder, and / or condition, refers to reducing the risk of developing the disease, disorder, and / or condition, as well as delaying the onset of one or more of the characteristics or symptoms of the disease, disorder, and / or condition. Prevention may be considered complete when the onset of the disease, disorder, or condition has been delayed for a predetermined period of time.

[0121] Recombinant: As used herein, the term "recombinant" is intended to refer to a polypeptide that has been designed, engineered, prepared, expressed, created, manufactured, and / or isolated by recombinant means, such as a polypeptide expressed using a recombinant expression vector transfected into a host cell; a polypeptide isolated from a recombinant, combinatorial human polypeptide library; a polypeptide isolated from an animal (e.g., mouse, rabbit, sheep, fish, etc.) that has been transgenic or otherwise engineered to express a gene or genetic element that encodes and / or directs the expression of a polypeptide or one or more components, portions, elements, or domains thereof; and / or a polypeptide prepared, expressed, created, or isolated by other means, including splicing or ligating selected nucleic acid sequence elements together, chemically synthesizing selected sequence elements, and / or otherwise generating a nucleic acid that encodes and / or directs 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 occur in nature. In some embodiments, one or more of such selected sequence elements are designed in silico. In some embodiments, one or more of such selected sequence elements are the result of mutagenesis (e.g., in vivo or in vitro) of known sequence elements, e.g., originating from a natural or synthetic source, such as the germline of a source organism of interest (e.g., human, mouse, etc.).

[0122] 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 comprise a polymeric moiety. In some embodiments, a small molecule is not a protein or polypeptide and / or does not comprise a protein or polypeptide (e.g., is not an oligopeptide or peptide). In some embodiments, a small molecule is not a polynucleotide and / or does not comprise a polynucleotide (e.g., is not an oligonucleotide). In some embodiments, a small molecule is not a polysaccharide and / or does not comprise a polysaccharide (e.g., 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., is 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. One of ordinary 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 tautomers. One of ordinary skill in the art will recognize that certain small molecule compounds have structures that allow for isotopic substitution (e.g., for H). 2 H or 3 Regarding H;12C 11 C. 13 C or 14 Regarding C;14N 13 N or 15 For N;16O 17 O or 18 Regarding O;XXC 36 Regarding Cl;XXF 18F; 131I for XXXI). 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, reference to a particular small molecule compound may refer to a particular 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 salt form or a base addition salt form, depending on the compound), and in some embodiments, the salt form may be a pharmaceutically acceptable salt form. In some embodiments, if the small molecule compound is a compound that occurs in nature or is 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 which occurs in nature or is found in nature. Those skilled in the art will understand that in some embodiments, a preparation of a particular small molecule compound containing an absolute or relative amount (with respect to another component of the preparation, e.g., another form of the compound) of the compound or a particular form thereof that differs 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 distinguished 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 to be a different form of the compound from a racemic mixture of the compound; a particular salt of a small molecule compound can be considered to be a different form of the compound from other salt forms of the compound; a preparation containing only a form of the compound containing one conformational isomer of the double bond ((Z) or (E)) can be considered to be a different form of the compound from one containing the other conformational isomer 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 to be a different form; and so forth.

[0123] Specific binding: As used herein, the term "specific binding" refers to the ability to distinguish between potential binding partners in the context in which the binding occurs. A binding agent that interacts with one specific target in the presence of other potential targets is said to "specifically bind" to the interacting target (e.g., a target amino acid or nucleic acid sequence on a target protein / gene of interest). 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 with alternative interactions between its partner and other substances. In some embodiments, specific binding is assessed by performing such detection or determination over a range of concentrations.

[0124] Specificity: As known in the art, "specificity" is a measure of the ability of a particular ligand to distinguish its binding partner from other potential binding partners.

[0125] 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., Alzheimer's disease (AD), muscular dystrophy, or other diseases characterized by neurodegeneration or neuromuscular dysfunction). In some embodiments, the subject is susceptible to a disease, disorder, or condition. In some embodiments, the subject exhibits one or more symptoms or characteristics of a 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 a person with one or more characteristics that characterize a 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 who is being and / or has been administered a diagnosis and / or therapy.

[0126] Substantially: As used herein, the term "substantially" refers to the qualitative state of exhibiting all or nearly all extent or degree of a characteristic or property of interest. Those skilled in the biology arts will understand that biological and chemical phenomena rarely, if ever, proceed to completion and / or perfection, or achieve or avoid a perfect result. Thus, the term "substantially" is used herein to capture the potential lack of perfection inherent in many biological and chemical phenomena.

[0127] Substantial identity: As used herein, refers to a comparison between amino acid or nucleic acid sequences. As recognized by those skilled in the art, two sequences are generally considered to be "substantially identical" if they have 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 commercially available computer programs, such as BLASTN for nucleotide sequences, and BLASTP, gapped BLAST, and PSI-BLAST for amino acid sequences. Examples of such programs are described in Altschul et al., Basic local alignment search tool, J. Mol. Biol., 215(3):403-410, 1990, Altschul et al., Methods in Enzymology, Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997, Baxevanis et al., Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins, Wiley, 1998, and Misener, et al. (eds.), Bioinformatics Methods and Protocols (Methods in Molecular Biology, Vol. 132), Humana Press, 1999. In addition to identifying identical sequences, the above-mentioned programs usually also 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 contiguous sequence of related residues. In some embodiments, the related contiguous sequence is the entire sequence.In some embodiments, the related contiguous sequence 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 in length.

[0128] Suffering from: An individual who is "suffering from" a disease, disorder, and / or condition (e.g., muscular dystrophy or other diseases characterized by neuromuscular dysfunction) has been diagnosed with and / or exhibits one or more symptoms of the disease, disorder, and / or condition.

[0129] Susceptible: An individual who is "susceptible" to a disease, disorder, and / or condition (e.g., Alzheimer's disease (AD), muscular dystrophy, or other disease characterized by neurodegeneration or neuromuscular dysfunction) is an individual who is at a higher risk of developing the disease, disorder, and / or condition than a member 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 develops the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition does not develop the disease, disorder, and / or condition.

[0130] 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., Alzheimer's disease (AD), muscular dystrophy, or other disease characterized by neurodegeneration or neuromuscular dysfunction) are reduced in degree (e.g., intensity, severity, etc.) and / or frequency. For clarity, delaying the onset of a particular symptom is considered a form for reducing the frequency of that symptom.

[0131] Target gene: As used herein, a target gene refers to a gene whose expression is regulated, for example, through alteration of splice activity (e.g., by inducing exon skipping). As used herein, the term "target portion" or "target region" refers to a contiguous 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 may be about 8 to 36 nucleotides in length, e.g., about 10 to 20 or about 15 to 30 nucleotides in length. The length of the target portion may have a specific value or subrange within the aforementioned ranges. For example, in certain embodiments, the targeting moiety 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, 1 It may be 9 to 28, 19 to 27, 19 to 26, 19 to 25, 19 to 24, 19 to 23, 19 to 22, 19 to 21, 19 to 20, 20 to 30, 20 to 29, 20 to 28, 20 to 27, 20 to 26, 20 to 25, 20 to 24, 20 to 23, 20 to 22, 20 to 21, 21 to 30, 21 to 29, 21 to 28, 21 to 27, 21 to 26, 21 to 25, 21 to 24, 21 to 23, or 21 to 22 nucleotides.

[0132] Therapeutic Agent: As used herein, the phrase "therapeutic agent" refers to any agent that has a therapeutic effect and / or induces a desired biological and / or pharmacological effect when administered to a subject. In some embodiments, a therapeutic agent is a substance that can be used to alleviate, ameliorate, relieve, suppress, prevent, delay the onset of, reduce the severity of, and / or reduce the incidence of one or more symptoms or characteristics of a disease, disorder, and / or condition (e.g., one or more symptoms or characteristics of Alzheimer's disease (AD), muscular dystrophy, or other diseases characterized by neurodegeneration or neuromuscular dysfunction).

[0133] 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, when administered as part of a therapeutic dosing regimen, elicits a desired biological response. In some embodiments, a therapeutically effective amount of a substance is an amount sufficient to treat, diagnose, prevent, and / or delay the onset of a disease, disorder, and / or condition when administered to a subject suffering from or susceptible to 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 being delivered, and the target cell or tissue. It will be appreciated that numerous methods are known in the art for determining an effective amount for a given application. For example, pharmacological methods for determining dosage can be used in therapeutic settings. In terms of therapeutic or prophylactic applications, the amount of a composition administered to a subject will depend on the type and severity of the disease and individual characteristics such as general health, age, sex, weight, and tolerance to drugs. It will also depend on the extent, severity, and type of disease. One skilled in the art will be able to determine the appropriate dosage depending 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, ameliorates, alleviates, inhibits, prevents, delays the onset of, reduces the severity of, and / or reduces the incidence 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 the signs or symptoms of a disease, such as muscular dystrophies, such as Becker, congenital, distal Duchenne, Emery-Dreyfus, facioscapulohumeral, limb-girdle, myotonic, and oculopharyngeal muscular dystrophies, as well as Alzheimer's disease (AD), Parkinson's disease, or other diseases characterized by decreased mobility, metabolism, and quality of life resulting from neurodegeneration and muscle wasting in cancer patients, elderly patients, and many other patients without a history of neuromuscular dysfunction. It will be appreciated that there are numerous methods known in the art for determining the effective amount for a given application. For example, pharmacological methods for determining dosage may be used in therapeutic situations.For therapeutic or prophylactic use, the amount of the composition administered to a subject depends on the type and severity of the disease, as well as individual characteristics such as general health, age, sex, weight, and tolerance to drugs. It also depends on the extent, 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, while in some embodiments, multiple unit doses are required to deliver a therapeutically effective amount.

[0134] Treatment: As used herein, the term "treatment" refers to providing therapy, i.e., providing any type of medical or surgical management for a subject. Treatment may be provided to reverse, alleviate, inhibit the progression of, prevent or reduce the likelihood of a disease, disorder, or condition, or to reverse, alleviate, inhibit or prevent the progression of, 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, alleviation of one or more symptoms, a decrease in the degree of deficit, a stabilized (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 agent to a subject after the onset of one or more symptoms or signs indicative of Alzheimer's disease (AD), muscular dystrophy, or other diseases characterized by neurodegeneration or neuromuscular dysfunction, for example, to reverse, alleviate, or reduce the severity of the condition, and / or inhibit or prevent the progression of the condition, and / or to reverse, alleviate, or reduce the severity of one or more symptoms or signs of the condition, and / or to inhibit or prevent one or more symptoms or signs of the condition. The compositions of the present disclosure can be administered to subjects who have developed Alzheimer's disease, muscular dystrophy, or other diseases characterized by neurodegeneration or neuromuscular dysfunction, or to subjects who are at increased risk for developing such disorders compared to 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 condition. Typically, in this case, the subject is at risk for developing the condition.

[0135] Variant: As used herein in the context of a molecule, e.g., a nucleic acid (e.g., an ASO), a protein, or a small molecule, the term "variant" refers to a molecule that exhibits significant structural identity with a reference molecule but that differs structurally from the reference molecule, e.g., in the presence or absence, or level, of one or more chemical moieties compared to the reference molecule. In some embodiments, a variant is also functionally distinct from its reference molecule. Generally, whether a particular molecule is properly considered a "variant" of a reference molecule is based on the degree of structural identity with the reference molecule. As recognized by those skilled in the art, any biological or chemical reference molecule possesses certain characteristic structural elements. By definition, a variant is a distinct molecule that shares one or more such characteristic structural elements but differs from the reference molecule in at least one aspect. To give a few examples, a polypeptide may have characteristic sequence elements composed of multiple amino acids that have designated positions relative to each other in linear or three-dimensional space and / or contribute to a particular structural motif and / or biological function, and a nucleic acid may have characteristic sequence elements composed of multiple nucleotide residues that have designated positions 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 at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% overall sequence identity with a reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid does not share at least one characteristic sequence element with a reference polypeptide or nucleic acid. In some embodiments, a reference polypeptide or nucleic acid has one or more biological activities.In some embodiments, a variant polypeptide or nucleic acid shares one or more biological activities of a reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid lacks one or more biological activities of a reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid has a reduced level of one or more biological activities compared to a reference polypeptide or nucleic acid. In some embodiments, a polypeptide or nucleic acid of interest is considered a "variant" of a reference polypeptide or nucleic acid if it has an amino acid or nucleotide sequence identical to the reference amino acid or nucleic acid, except for minor sequence changes at specific positions. Typically, less than about 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, or 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, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substituted residue compared to the reference. In many cases, a variant polypeptide or nucleic acid contains very few (e.g., less than about 5, about 4, about 3, about 2, or about 1) substituted, inserted, or deleted functional residues (i.e., residues responsible for a particular biological activity) compared to the reference. In some embodiments, the variant polypeptide or nucleic acid contains no more than 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 contains 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 additions or deletions compared to the reference, typically less than about 5, about 4, about 3, or about 2 additions or deletions. 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.

[0136] 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, wherein 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 are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors." Standard techniques may 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 practiced in the art or as described herein. The foregoing techniques and procedures can generally be performed according to conventional methods 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)), which is incorporated herein by reference for any purpose. (Mode for Carrying Out the Invention)

[0137] Neurogenesis Neurogenesis occurs in distinct regions of the adult mammalian brain. Neural stem cells (NSCs) are an endogenous source of new neurons and are active throughout life in virtually all mammals, including humans (Eriksson et al., 1998; Ernst et al., 2014; Moreno-Jimenez et al., 2019; Spalding et al., 2013). Extensive research in rodent models has shown that neurogenesis supports learning and memory, sensory function, and mood regulation (Enwere et al., 2004; Gage, 2019; Imayoshi et al., 2008; Zhang et al., 2008b). NSCs reside within two neurogenic niches: the subgranular zone (SGZ) of the dentate gyrus of the hippocampus and the subventricular zone (SVZ), which lines the lateral ventricles. NSCs in the SVZ generate astrocytes and oligodendrocytes that support existing circuits, as well as neurons in the olfactory bulb, which are important for olfactory discrimination. NSCs in the dentate gyrus give rise to granule neurons, which are important for learning and memory. The majority of NSCs in the human brain are located within the hippocampus. Most hippocampal NSCs exist in a resting state called quiescence. For neurogenesis to occur, quiescent NSCs must be activated in response to exogenous or endogenous cues. Newborn neurons functionally integrate into local circuits within the hippocampus and contribute to cognitive function. The activation capacity of quiescent NSCs declines during healthy and pathological aging, and this decline precedes cognitive decline (Enwere et al. 2004; Giachino et al. 2014; Capilla-Gonzalez et al. 2014).

[0138] Recent studies indicate that endogenous or exogenous NSCs may be a valuable source of new neurons for millions of individuals suffering from cognitive decline or brain injury. Activating endogenous NSCs through exercise, refeeding, or young blood ameliorates age-related cognitive impairment in mice (Brandhorst et al., 2015; van Praag et al., 2005; Villeda et al., 2011, 2014). The accumulation of negative signals that degrade the neurogenic niche may contribute to the reduction in newborn neurons in aging and AD. However, the lack of specific therapeutic targets capable of overcoming inhibitory signals has made it difficult to harness the neurogenic potential of NSCs. This disclosure recognizes that recent mechanistic studies suggest that BMP signaling may represent a promising pathway to target in the context of AD and other diseases characterized by neurodegeneration. BMPs negatively regulate NSC activation (Mira et al., 2010) and are upregulated in AD and APP transgenic mice (Crews et al., 2010). The present disclosure provides techniques for specifically regulating BMP signaling in the neurogenic niche.

[0139] Adult hippocampal neurogenesis Adult hippocampal neurogenesis (AHN) is important for normal learning and memory. AHN is abundant in healthy aging humans but declines from the early stages of Alzheimer's disease (AD). AHN develops throughout life in humans but declines dramatically in AD (Moreno-Jimenez et al., 2019; Steiner et al., 2019). Studies in animal models highlight the role of AHN in improving cognition relative to AD pathology. Therefore, restoring AHN may be an attractive target for AD therapy. Interventions that promote adult hippocampal neurogenesis may enhance cognitive function and combat neurodegeneration.

[0140] AHN is important for learning and memory. Newly born dentate granule cells are hyperexcitable and exhibit robust synaptic plasticity. Therefore, dysregulation of quiescence and / or failure to integrate into mature circuits may contribute to age-related declines in neurogenesis and cognitive decline in aging and dementia.

[0141] AHN in humans. Although AHN has been established for decades in rodents and other species, the existence of this process in humans remained controversial until very recently. BrdU incorporation (Eriksson et al., 1998), 14 Reports using C dating (Ernst et al., 2014; Spalding et al., 2013) and immature neuronal markers (Boldrini et al., 2018; Moreno-Jimenez et al., 2019; Tobin et al., 2019) have provided independent lines of support from multiple laboratories for human AHN. Mathematical modeling of radiocarbon birth dating data has estimated that 35% of hippocampal neurons are replaced by newborn neurons during adulthood, at a rate of 1.75% per year (Spalding et al., 2013). However, another recent report using immature neuronal markers failed to detect significant levels of AHN in adult humans (Sorrells et al., 2018). A detailed comparison of these reports reveals several methodological and sample differences that may explain why Sorrells et al. failed to detect adult neurogenesis in their paper (Kempermann et al., 2018; Lucassen et al., 2019).

[0142] Alzheimer's disease and Alzheimer's disease are devastating disorders. They are progressive, fatal, and incur significant social and economic costs. 5.8 million Americans are living with AD. By 2050, this number is projected to increase to 14 million. In 2019, AD and other dementias cost the United States $290 billion. By 2050, these costs could rise to $110 million. There are no effective treatments. Several high-profile clinical trials have recently been conducted. Nearly all of these trials are based on the "amyloid hypothesis." There is a significant unmet need for innovative and effective treatments for AD. Alzheimer's disease destroys the hippocampus, a brain region necessary for memory encoding. The hippocampus is one of two sites of adult neurogenesis in the brain. Numerous animal studies have shown that these adult-born neurons are necessary for learning and memory. Recent important studies have provided compelling evidence of robust neurogenesis in the adult human brain. Importantly, levels of adult neurogenesis are significantly reduced in AD brains compared with age-matched controls. (See E.P. Moreno-Jimenez et al. Nature Med. https: / / doi.org / 10.I038 / s41591-019-0375-9; 2019; see also the related editorial Nature 567:433; 28 March 2019.) Therefore, promoting adult neurogenesis has emerged as a highly attractive target for the treatment of AD.

[0143] The hippocampus is one of the earliest and most affected brain regions in AD, and its atrophy is a hallmark of disease progression (Allison et al., 2019). Furthermore, both rodent and human studies have demonstrated that hippocampal-dependent learning is impaired in the setting of Alzheimer's disease (Crews et al., 2010). Notably, AHN levels in AD patients are only 30% of those observed in age-matched controls (Moreno-Jimenez et al., 2019). Importantly, a recent mouse study using a genetically diverse AD mouse model showed that the total number of hippocampal neurons (NeuN+ cells) correlates with cognition (Neuner Neuron 2019). Finally, a recent study found that exercise-mediated rescue of pathology in AD mice requires AHN, and AHN ablation alone exacerbates cognitive deficits in these mice (Choi et al., 2018). Therefore, strategies to compensate for the degeneration of hippocampal neurons through enhanced endogenous neurogenesis may open new pathways for treating Alzheimer's disease.

[0144] Other diseases associated with AHN include diseases and disorders associated with progressive memory loss, such as frontotemporal dementia (Terreros-Roncal et al., 2019) and stroke (Lindvall et al., 2015). AHN has also been associated with psychiatric disorders such as major depressive disorder (MDD), bipolar disorder, schizophrenia, post-traumatic stress disorder (PTSD), substance-related and addictive disorders (Yun et al., 2016), as well as temporal lobe epilepsy (Cook et al., 1992), hippocampal sclerosis (Tai et al., 2018), Niemann-Pick disease type C (Hong et al., 2015), and diabetes-mediated hippocampal neuronal loss (Ho et al., 2013; Gold et al., 2007).

[0145] Subventricular zone neurogenesis In addition to the hippocampus (i.e., the subgranular zone (SGZ) of the dentate gyrus of the hippocampus), NSCs reside in the subventricular zone (SVZ), which lines the lateral ventricles. NSCs within the SVZ generate astrocytes and oligodendrocytes that support existing circuits, as well as neurons in the olfactory bulb, which are important for olfactory discrimination. Recent evidence suggests that SVZ NSCs may give rise to well-differentiated neurons in the striatum in response to ischemic stroke or neurodegenerative diseases (Arvidsson et al. 2002; Parent et al. 2002; Thored et al. 2006; Ernst et al. 2014).

[0146] The present disclosure recognizes that strategies to compensate for neuronal degeneration within the SVZ through enhanced endogenous neurogenesis have the potential to open new pathways for treating diseases specifically associated with striatal neurogenesis, such as Parkinson's disease (which may benefit from increased both AHN and striatal neurogenesis in the SVZ; Pitcher et al. 2012, Sterling et al. 2013) and Huntington's disease (Sassone et al., 2018). Other diseases, including addiction (e.g., chronic cocaine use and lifetime smoking), are also associated with reduced striatal volume (Barros-Loscertales et al. 2011, Das et al. 2012) and may be treated via enhanced endogenous neurogenesis in the SVZ.

[0147] MuSK and Neurogenesis MuSK is a receptor tyrosine kinase (TK) composed of three Ig domains, one CRD / Fz domain, and an intracellular tyrosine domain on the extracellular side (Figure 1). The best understood function of MuSK is at the neuromuscular junction (NMJ), where agrin-LRP4 binding to the Ig1 domain induces MuSK TK activity and synaptic differentiation (Kim et al., 2008; Zhang et al., 2008a).

[0148] MuSK-BMP Pathway. The brain harbors neural stem cells (NSCs) that generate neurons and glial cells throughout life (Moreno-Jimenez et al., 2019; Steiner et al., 2019). BMPs regulate at least two key NSC decision points: 1) quiescence, where proliferating stem cells exit the cell cycle and return to replenish reservoir pools that can provide fresh stem cells, and 2) differentiation into mature progeny (Mira et al., 2010). The present disclosure contemplates that manipulating the BMP pathway in NSCs is an attractive target for modulating neurogenesis in the adult brain.

[0149] MuSK is also a BMP coreceptor and has recently been found to upregulate BMP signaling, which binds to BMP and its receptors ALK3 and 6, shaping the transcriptional response in myogenic cells (Yilmaz et al., 2016). This BMP signaling pathway neither regulates nor requires MuSK TK activity, nor is it activated by agrin-LRP4. Importantly, the MuSK Ig3 domain is required for high-affinity BMP binding but not agrin-LRP4 TK activation. Furthermore, the Ig3 domain is endogenously alternatively spliced, including in the brain (Garcia-Osta et al., 2006; Hesser et al., 1999). Because BMP signaling can induce NSC quiescence and inhibit the integration of newborn neurons, we found that suppressing BMP drive by reducing MuSK-BMP signaling could increase neurogenesis (Figure 1).

[0150] Yilmaz et al. (2016) reported that the "Ig3" domain of MuSK is required for high-affinity binding of BMPs. The predominant species of endogenously expressed MuSK is full-length. This Ig3 domain can be endogenously alternatively spliced ​​to generate an isoform termed "Δg3MuSK." This splicing involves the coordinate removal of exons 6 and 7 from the MuSK pre-mRNA.

[0151] Exemplary amino acid sequences of human and mouse MuSK Ig3 domains (ie, MuSK Ig3 domain polypeptides) are set forth below. MuSK human Ig3 domain: ARILRAPESHNVTFGSFVTLHCTATGIPVPTITWIENGNAVSSGSIQESVKDRVIDSRLQLFITKPGLYTCIATNKHGEKFSTAKAAATIS (SEQ ID NO: 116) MuSK mouse Ig3 domain ARILRAPESHNVTFGSFVTLRCTAIGIPVPTISWIENGNAVSSGSIQESVKDRVIDSRLQLFITKPGLYTCIATNKHGEKFSTAKAAATVS (SEQ ID NO: 117)

[0152] In particular, the present disclosure provides compositions such as MuSK-targeted oligonucleotides that modulate MuSK alternative splicing as a strategy to increase AHN in AD.

[0153] MuSK and muscle regeneration and / or growth Satellite cells account for approximately 5% of myonuclei, are distributed along mature multinucleated muscle fibers, and are normally quiescent. Following muscle injury, satellite cells typically proliferate before either returning to quiescence or differentiating. Upon differentiation, satellite cells become committed myoblasts that fuse into myotubes and ultimately form mature muscle fibers in a process called myogenesis. Bone morphogenetic protein (BMP) signaling regulates satellite cell dynamics and muscle regeneration both in vivo and in vitro by regulating transcriptional production. BMP signaling is undetectable in quiescent satellite cells, upregulated in proliferating satellite cells, and downregulated during differentiation. However, the mediators that regulate the balance between satellite cell proliferation and differentiation remain unknown.

[0154] 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 domains, a cysteine-rich frizzled (CRD / Fz) domain, and an intracellular tyrosine kinase (TK) domain. The Ig1 domain, TK domain, and potentially the CRD / Fz domain 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. FL MuSK mRNA levels are 10× higher than that of ΔIg3-MuSK, but the two are coordinately expressed.

[0155] In particular, the present disclosure provides compositions such as MuSK-targeted oligonucleotides that modulate MuSK alternative splicing as a strategy to increase muscle regeneration.

[0156] MuSK is activated by a neurally derived proteoglycan called agrin, which 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 share homology with protease inhibitors.

[0157] 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., Sci. Signal. 9:ra87, doi:10.1126 / scisignal.aaf0890, 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 organization 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.

[0158] Activated satellite cells express MuSK protein, and inhibition of MuSK-BMP signaling alters satellite cell proliferation in regenerating muscle in vivo. Furthermore, previous studies suggest a role for the MuSK-BMP pathway in satellite cells and muscle regeneration, and that targeting the MuSK-BMP pathway enhances muscle growth (see, e.g., PCT Publication No. 2021 / 076883, incorporated herein by reference).

[0159] MuSK-targeting oligonucleotides As described herein, strategies for modulating the MuSK-BMP pathway include MuSK-targeting oligonucleotides (e.g., MuSK Ig3-targeting oligonucleotides). Specifically, the present disclosure provides, inter alia, oligonucleotides and compositions thereof that target the region spanning exon 6 and / or exon 7 of MuSK to induce exon skipping of exon 6 and / or exon 7. Such alternative splicing activity results in increased expression of ΔIg3-MuSK.

[0160] The present disclosure also describes regions within the MuSK transcript at or near exon 6 and / or exon 7 that are particularly useful as target sequences for oligonucleotides that induce exon skipping of exon 6 and / or exon 7, thereby generating the ΔIg3-MuSK transcript.

[0161] In some embodiments, the present disclosure also provides specific oligonucleotides and combinations thereof that induce the alternative splicing activity of MuSK to generate ΔIg3-MuSK transcripts.

[0162] The present disclosure further provides compositions comprising one or more MuSK-targeting oligonucleotides that can be administered to a subject in a therapeutically effective amount to induce exon skipping of MuSK exons 6 and / or 7 and increase neurogenesis and / or muscle regeneration in the subject.

[0163] The present disclosure includes compositions and methods related to one or more nucleotide sequences that are, comprise, or encode an oligonucleotide that binds to and inhibits expression of messenger RNA (mRNA) produced by a target gene (e.g., MuSK). The oligonucleotide may be a single-stranded (e.g., antisense oligonucleotide) or a double-stranded nucleic acid. In some embodiments, the oligonucleotide comprises a double-stranded RNA duplex, such as a microRNA (miRNA) or a small interfering RNA (siRNA). In some embodiments, the oligonucleotide is an siRNA or miRNA, or a vector containing a nucleotide sequence encoding the siRNA or miRNA. In some embodiments, the oligonucleotide is an antisense oligonucleotide (ASO), or a vector containing a nucleotide sequence encoding the ASO.

[0164] In some embodiments, the oligonucleotides are capable of inhibiting expression of the full-length MuSK sequence, which includes three extracellular immunoglobulin (Ig)-like domains (Ig1, Ig2, and Ig3), a cysteine-rich frizzle domain (CRD / Fz), and an intracellular tyrosine kinase (TK) domain.

[0165] As described herein, the oligonucleotides can target MuSK sequences of human MuSK or one or more non-human species, e.g., non-human primate MuSK, e.g., cynomolgus monkey (Macaca fascicularis) MuSK (Gene ID 102127677), or, e.g., green monkey (Chlorocebus sabaeus) (Gene ID: 103219025), or mouse MuSK (Gene ID: 18198). In some embodiments, the MuSK-targeting oligonucleotide comprises an antisense strand that is complementary to the same target portion in a human and / or mouse MuSK transcript. In some embodiments, the oligonucleotide comprises a sequence that is complementary to a target portion of a human MuSK transcript that differs by 1, 2, or 3 nucleotides from the sequence of the mouse or human MuSK transcript. It will be understood that oligonucleotides that alter the splicing of human MuSK can also alter the splicing (i.e., induce exon skipping) of non-primate MuSK, such as rat or mouse MuSK, particularly when conserved regions of the MuSK transcript are targeted.

[0166] The amino acid and nucleotide sequences of human MuSK are known in the art and can be found in publicly available databases, for example, the National Center for Biotechnology Information (NCBI) Reference Sequence (RefSeq) database; the genomic nucleotide sequence is set forth in RefSeq accession number NG_016016.2 (SEQ ID NO: 77) and the mRNA / protein sequence is set forth in NM_005592.4 / NP_005583.1 (muscle, skeletal receptor tyrosine- NM_001166280.2 / NP_001159752.1 (muscle, skeletal receptor tyrosine-protein kinase isoform 2), NM_001166281.2 / NP_001159753.1 (muscle, skeletal receptor tyrosine-protein kinase isoform 3), and NM_001369398.1 / NP_001356327.1 (muscle, skeletal receptor tyrosine-protein kinase isoform 4). See www.ncbi.nlm.nih.gov / gene?Db=gene& Cmd=Details Search&Term=4593, which is incorporated herein by reference.

[0167] In some embodiments, the oligonucleotide comprises a nucleic acid strand that is complementary to a target portion of a MuSK transcript, e.g., MuSK mRNA (e.g., complementary to a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a target portion of SEQ ID NO: 77, e.g., Bld25 / hu7-10 (SEQ ID NO: 63), Bld26 / hu73 (SEQ ID NO: 64), etc.). In some embodiments, the target portion comprises a region corresponding to positions 83776-83800 and / or 83854-83878 of SEQ ID NO: 77, or corresponding regions in different versions of the genomic MuSK sequence. The target portion may be 15 to 30 nucleotides in length, e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length, although shorter and longer target portions are also contemplated.

[0168] In some embodiments, the target portion of a MuSK transcript, e.g., MuSK mRNA, comprises the sequence ACCTGTCAGGTTTCTTCTGGGTCCATTCAAGAGAGTGTGAAAGACCGAGTGATTGACTCAAGAC (Region 1, SEQ ID NO: 126), or a region or portion thereof. In some embodiments, the target portion comprises a sequence that is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% identical to Region 1, SEQ ID NO: 126. In some embodiments, the target portion comprises a sequence identical to at least 10 contiguous bases of Region 1, SEQ ID NO: 126. In some embodiments, the target portion comprises a sequence identical to at least 15 contiguous bases of Region 1, SEQ ID NO: 126. In some embodiments, the target portion comprises a sequence identical to at least 18 contiguous bases of Region 1, SEQ ID NO: 126. In some embodiments, the target portion comprises a sequence identical to at least 19 contiguous bases of Region 1, SEQ ID NO: 126. In some embodiments, the target portion comprises a sequence identical to at least 20 contiguous bases of Region 1, SEQ ID NO:126. In some embodiments, the target portion comprises a sequence identical to at least 21 contiguous bases of Region 1, SEQ ID NO:126. In some embodiments, the target portion comprises a sequence identical to at least 22 contiguous bases of Region 1, SEQ ID NO:126. In some embodiments, the target portion comprises a sequence identical to at least 23 contiguous bases of Region 1, SEQ ID NO:126. In some embodiments, the target portion comprises a sequence identical to at least 24 contiguous bases of Region 1, SEQ ID NO:126. In some embodiments, the target portion comprises a sequence identical to at least 25 contiguous bases of Region 1, SEQ ID NO:126. In some embodiments, the target portion comprises a sequence identical to at least 30 contiguous bases of Region 1, SEQ ID NO:126. In some embodiments, the target portion comprises a sequence identical to at least 35 contiguous bases of Region 1, SEQ ID NO:126. In some embodiments, the target portion comprises a sequence identical to at least 15 contiguous bases and no more than 30 contiguous bases of Region 1, SEQ ID NO:126.

[0169] In some embodiments, the target portion comprises a sequence identical to 10 contiguous bases of Region 1, SEQ ID NO:126. In some embodiments, the target portion comprises a sequence identical to 11 contiguous bases of Region 1, SEQ ID NO:126. In some embodiments, the target portion comprises a sequence identical to 12 contiguous bases of Region 1, SEQ ID NO:126. In some embodiments, the target portion comprises a sequence identical to 13 contiguous bases of Region 1, SEQ ID NO:126. In some embodiments, the target portion comprises a sequence identical to 14 contiguous bases of Region 1, SEQ ID NO:126. In some embodiments, the target portion comprises a sequence identical to 15 contiguous bases of Region 1, SEQ ID NO:126. In some embodiments, the target portion comprises a sequence identical to 16 contiguous bases of Region 1, SEQ ID NO:126. In some embodiments, the target portion comprises a sequence identical to 17 contiguous bases of Region 1, SEQ ID NO:126. In some embodiments, the target portion comprises a sequence identical to 18 contiguous bases of Region 1, SEQ ID NO:126. In some embodiments, the target portion comprises a sequence identical to 19 contiguous bases of Region 1, SEQ ID NO:126. In some embodiments, the target portion comprises a sequence identical to 20 contiguous bases of Region 1, SEQ ID NO:126. In some embodiments, the target portion comprises a sequence identical to 21 contiguous bases of Region 1, SEQ ID NO:126. In some embodiments, the target portion comprises a sequence identical to 22 contiguous bases of Region 1, SEQ ID NO:126. In some embodiments, the target portion comprises a sequence identical to 23 contiguous bases of Region 1, SEQ ID NO:126. In some embodiments, the target portion comprises a sequence identical to 24 contiguous bases of Region 1, SEQ ID NO:126. In some embodiments, the target portion comprises a sequence identical to 25 contiguous bases of Region 1, SEQ ID NO:126. In some embodiments, the target portion comprises a sequence identical to 26 contiguous bases of Region 1, SEQ ID NO:126. In some embodiments, the target portion comprises a sequence identical to 27 contiguous bases of Region 1, SEQ ID NO:126. In some embodiments, the target portion comprises a sequence identical to 28 contiguous bases of Region 1, SEQ ID NO: 126. In some embodiments, the target portion comprises a sequence identical to 29 contiguous bases of Region 1, SEQ ID NO: 126.In some embodiments, the target portion comprises a sequence identical to 30 contiguous bases of region 1, SEQ ID NO:126.

[0170] In some embodiments, the target portion of a MuSK transcript, e.g., MuSK mRNA, comprises the sequence GGGGAGAAGTTCAGTACTGCCAAGGCTGCAGCCACCATCAGCATAGCAGGTAGGATGCCCCTTCACATTTG (Region 2, SEQ ID NO:211), or a region or portion thereof. In some embodiments, the target portion comprises a sequence at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% identical to Region 2, SEQ ID NO:211. In some embodiments, the target portion comprises a sequence identical to at least 10 contiguous bases of Region 2, SEQ ID NO:211. In some embodiments, the target portion comprises a sequence identical to at least 15 contiguous bases of Region 2, SEQ ID NO:211. In some embodiments, the target portion comprises a sequence identical to at least 18 contiguous bases of Region 2, SEQ ID NO:211. In some embodiments, the target portion comprises a sequence identical to at least 19 contiguous bases of Region 2, SEQ ID NO:211. In some embodiments, the target portion comprises a sequence identical to at least 20 contiguous bases of region 2, SEQ ID NO:211. In some embodiments, the target portion comprises a sequence identical to at least 21 contiguous bases of region 2, SEQ ID NO:211. In some embodiments, the target portion comprises a sequence identical to at least 22 contiguous bases of region 2, SEQ ID NO:211. In some embodiments, the target portion comprises a sequence identical to at least 23 contiguous bases of region 2, SEQ ID NO:211. In some embodiments, the target portion comprises a sequence identical to at least 24 contiguous bases of region 2, SEQ ID NO:211. In some embodiments, the target portion comprises a sequence identical to at least 25 contiguous bases of region 2, SEQ ID NO:211. In some embodiments, the target portion comprises a sequence identical to at least 30 contiguous bases of region 2, SEQ ID NO:211. In some embodiments, the target portion comprises a sequence identical to at least 35 contiguous bases of region 2, SEQ ID NO:211.

[0171] In some embodiments, the target portion comprises a sequence identical to 10 contiguous bases of region 2, SEQ ID NO:211. In some embodiments, the target portion comprises a sequence identical to 11 contiguous bases of region 2, SEQ ID NO:211. In some embodiments, the target portion comprises a sequence identical to 12 contiguous bases of region 2, SEQ ID NO:211. In some embodiments, the target portion comprises a sequence identical to 13 contiguous bases of region 2, SEQ ID NO:211. In some embodiments, the target portion comprises a sequence identical to 14 contiguous bases of region 2, SEQ ID NO:211. In some embodiments, the target portion comprises a sequence identical to 15 contiguous bases of region 2, SEQ ID NO:211. In some embodiments, the target portion comprises a sequence identical to 16 contiguous bases of region 2, SEQ ID NO:211. In some embodiments, the target portion comprises a sequence identical to 17 contiguous bases of region 2, SEQ ID NO:211. In some embodiments, the target portion comprises a sequence identical to 18 contiguous bases of region 2, SEQ ID NO:211. In some embodiments, the target portion comprises a sequence identical to 19 contiguous bases of region 2, SEQ ID NO:211. In some embodiments, the target portion comprises a sequence identical to 20 contiguous bases of region 2, SEQ ID NO:211. In some embodiments, the target portion comprises a sequence identical to 21 contiguous bases of region 2, SEQ ID NO:211. In some embodiments, the target portion comprises a sequence identical to 22 contiguous bases of region 2, SEQ ID NO:211. In some embodiments, the target portion comprises a sequence identical to 23 contiguous bases of region 2, SEQ ID NO:211. In some embodiments, the target portion comprises a sequence identical to 24 contiguous bases of region 2, SEQ ID NO:211. In some embodiments, the target portion comprises a sequence identical to 25 contiguous bases of region 2, SEQ ID NO:211. In some embodiments, the target portion comprises a sequence identical to 26 contiguous bases of region 2, SEQ ID NO:211. In some embodiments, the target portion comprises a sequence identical to 27 contiguous bases of region 2, SEQ ID NO:211. In some embodiments, the target portion comprises a sequence identical to 28 contiguous bases of Region 2, SEQ ID NO: 211. In some embodiments, the target portion comprises a sequence identical to 29 contiguous bases of Region 2, SEQ ID NO: 211.In some embodiments, the target portion comprises a sequence identical to 30 contiguous bases of region 2, SEQ ID NO:211.

[0172] In some embodiments, the target portion comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of the sequences listed in Table 1 below. [Table 1-2-1] [Table 1-2-2] [Table 1-2-3]

[0173] Administration of a MuSK-targeting oligonucleotide described herein can reduce the level of full-length MuSK transcript or full-length MuSK protein in a subject or biological sample (e.g., a blood, serum, or plasma sample, or a sample containing liver cells) compared to the level before administration of the composition. In some embodiments, the level of full-length MuSK transcript or full-length MuSK protein is reduced by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% compared to the level before administration.

[0174] In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to a target portion of a MuSK transcript, e.g., MuSK mRNA, hi some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a target portion of a MuSK transcript, e.g., MuSK mRNA.

[0175] In some embodiments, the oligonucleotide comprises a sequence at least 90% identical to a portion of ACTCTGTCAGGTTTCTTCTGGGTCCATTCAAGAGAGTGTGAAAGACCGAGTGATTGACTCAAGAC (Region 1, SEQ ID NO: 126), comprising at least 10, 15, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous bases of Region 1, SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a sequence at least 95% identical to a portion of SEQ ID NO: 126 comprising at least 10, 15, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous bases of Region 1, SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a sequence at least 90% identical to a portion of SEQ ID NO: 126 comprising at least 10 contiguous bases of Region 1, SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a sequence at least 90% identical to Region 1, a portion of SEQ ID NO: 126 comprising at least 15 contiguous bases of SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a sequence at least 90% identical to Region 1, a portion of SEQ ID NO: 126 comprising at least 18 contiguous bases of SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a sequence at least 90% identical to Region 1, a portion of SEQ ID NO: 126 comprising at least 19 contiguous bases of SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a sequence at least 90% identical to Region 1, a portion of SEQ ID NO: 126 comprising at least 20 contiguous bases of SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a sequence at least 90% identical to Region 1, a portion of SEQ ID NO: 126 comprising at least 21 contiguous bases of SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a sequence at least 90% identical to Region 1, a portion of SEQ ID NO: 126 comprising at least 22 contiguous bases of SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a sequence at least 90% identical to a portion of SEQ ID NO:126, comprising region 1, at least 23 contiguous bases of SEQ ID NO:126.In some embodiments, the oligonucleotide comprises a sequence at least 90% identical to Region 1, a portion of SEQ ID NO: 126 comprising at least 24 contiguous bases of SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a sequence at least 90% identical to Region 1, a portion of SEQ ID NO: 126 comprising at least 25 contiguous bases of SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a sequence at least 90% identical to Region 1, a portion of SEQ ID NO: 126 comprising at least 26 contiguous bases of SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a sequence at least 90% identical to Region 1, a portion of SEQ ID NO: 126 comprising at least 27 contiguous bases of SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a sequence at least 90% identical to Region 1, a portion of SEQ ID NO: 126 comprising at least 28 contiguous bases of SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a sequence at least 90% identical to Region 1, a portion of SEQ ID NO: 126 comprising at least 29 contiguous bases of SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a sequence at least 90% identical to a portion of SEQ ID NO:126, comprising region 1, at least 30 contiguous bases of SEQ ID NO:126.

[0176] In some embodiments, the oligonucleotide comprises a sequence that is at least 90% identical to a portion of ACTCTGTCAGGTTTCTTCTGGGTCCATTCAAGAGAGTGTGAAAGACCGAGTGATTGACTCAAGAC (Region 1, SEQ ID NO: 126), comprising 10, 15, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consecutive bases of Region 1, SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a sequence that is at least 95% identical to a portion of SEQ ID NO: 126 comprising 10, 15, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consecutive bases of Region 1, SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a sequence that is at least 90% identical to a portion of SEQ ID NO: 126 comprising 10 consecutive bases of Region 1, SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a sequence at least 90% identical to region 1, a portion of SEQ ID NO: 126 comprising 15 contiguous bases of SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a sequence at least 90% identical to region 1, a portion of SEQ ID NO: 126 comprising 18 contiguous bases of SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a sequence at least 90% identical to region 1, a portion of SEQ ID NO: 126 comprising 19 contiguous bases of SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a sequence at least 90% identical to region 1, a portion of SEQ ID NO: 126 comprising 20 contiguous bases of SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a sequence at least 90% identical to region 1, a portion of SEQ ID NO: 126 comprising 21 contiguous bases of SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a sequence at least 90% identical to region 1, a portion of SEQ ID NO: 126 comprising 22 contiguous bases of SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a sequence at least 90% identical to a portion of SEQ ID NO:126, including region 1, 23 contiguous bases of SEQ ID NO:126.In some embodiments, the oligonucleotide comprises a sequence at least 90% identical to region 1, a portion of SEQ ID NO: 126 comprising 24 contiguous bases of SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a sequence at least 90% identical to region 1, a portion of SEQ ID NO: 126 comprising 25 contiguous bases of SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a sequence at least 90% identical to region 1, a portion of SEQ ID NO: 126 comprising 26 contiguous bases of SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a sequence at least 90% identical to region 1, a portion of SEQ ID NO: 126 comprising 27 contiguous bases of SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a sequence at least 90% identical to region 1, a portion of SEQ ID NO: 126 comprising 28 contiguous bases of SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a sequence at least 90% identical to region 1, a portion of SEQ ID NO: 126 comprising 29 contiguous bases of SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a sequence at least 90% identical to a portion of SEQ ID NO:126, including region 1, 30 contiguous bases of SEQ ID NO:126.

[0177] In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to the sequence ACCTGTCAGGTTTCTTCTGGGTCCATTCAAGAGAGTGTGAAAGACCGAGTGATTGACTCAAGAC (Region 1, SEQ ID NO: 126). In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to a sequence that is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% identical to Region 1, SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to at least 10 contiguous bases of Region 1, SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to at least 15 contiguous bases of Region 1, SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to at least 18 contiguous bases of Region 1, SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to at least 19 contiguous bases of Region 1, SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to at least 20 contiguous bases of Region 1, SEQ ID NO:126. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to at least 21 contiguous bases of Region 1, SEQ ID NO:126. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to at least 22 contiguous bases of Region 1, SEQ ID NO:126. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to at least 23 contiguous bases of Region 1, SEQ ID NO:126. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to at least 24 contiguous bases of Region 1, SEQ ID NO:126. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to at least 25 contiguous bases of Region 1, SEQ ID NO:126. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to at least 26 contiguous bases of Region 1, SEQ ID NO:126. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to at least 27 contiguous bases of Region 1, SEQ ID NO:126.In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to at least 28 contiguous bases of Region 1, SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to at least 29 contiguous bases of Region 1, SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to at least 30 contiguous bases of Region 1, SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to at least 35 contiguous bases of Region 1, SEQ ID NO: 126.

[0178] In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to 10 contiguous bases of Region 1, SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to 11 contiguous bases of Region 1, SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to 12 contiguous bases of Region 1, SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to 13 contiguous bases of Region 1, SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to 14 contiguous bases of Region 1, SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to 15 contiguous bases of Region 1, SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to 16 contiguous bases of Region 1, SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to 17 contiguous bases of Region 1, SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to 18 contiguous bases of Region 1, SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to 19 contiguous bases of Region 1, SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to 20 contiguous bases of Region 1, SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to 21 contiguous bases of Region 1, SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to 22 contiguous bases of Region 1, SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to 23 contiguous bases of Region 1, SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to 24 contiguous bases of Region 1, SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to 25 contiguous bases of Region 1, SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to 26 contiguous bases of Region 1, SEQ ID NO:126.In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to 27 contiguous bases of Region 1, SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to 28 contiguous bases of Region 1, SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to 29 contiguous bases of Region 1, SEQ ID NO: 126. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to 30 contiguous bases of Region 1, SEQ ID NO: 126.

[0179] In some embodiments, the oligonucleotide comprises the subsequence GGGGAGAAGTTCAGTACTGCCAAGGCTGCAGCCACCATCAGCATAGCAGGTAGGATGCCCCTTCACATTTG (Region 2, SEQ ID NO:211), a sequence that is at least 90% identical to a subsequence comprising at least 10, 15, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous bases of Region 2, SEQ ID NO: 211. In some embodiments, the oligonucleotide comprises a sequence that is at least 95% identical to a portion of SEQ ID NO:211 comprising at least 10, 15, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous bases of Region 2, SEQ ID NO:211. In some embodiments, the oligonucleotide comprises a sequence that is at least 90% identical to a portion of SEQ ID NO:211 comprising region 2, 10, 15, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous bases of SEQ ID NO:211. In some embodiments, the oligonucleotide comprises a sequence that is at least 95% identical to a portion of SEQ ID NO:211 comprising region 2, 10, 15, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous bases of SEQ ID NO:211.

[0180] In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to a portion of the sequence GGGGAGAAGTTCAGTACTGCCAAGGCTGCAGCCACCATCAGCATAGCAGGTAGGATGCCCCTTCACATTTG (Region 2, SEQ ID NO:211). In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to at least 10 contiguous bases of Region 2, SEQ ID NO:211. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to at least 15 contiguous bases of Region 2, SEQ ID NO:211. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to at least 18 contiguous bases of Region 2, SEQ ID NO:211. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to at least 19 contiguous bases of Region 2, SEQ ID NO:211. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to at least 20 contiguous bases of Region 2, SEQ ID NO:211. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to at least 21 contiguous bases of Region 2, SEQ ID NO:211. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to at least 22 contiguous bases of Region 2, SEQ ID NO:211. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to at least 23 contiguous bases of Region 2, SEQ ID NO:211. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to at least 24 contiguous bases of Region 2, SEQ ID NO:211. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to at least 25 contiguous bases of Region 2, SEQ ID NO:211. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to at least 30 contiguous bases of Region 2, SEQ ID NO:211. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to at least 35 contiguous bases of Region 2, SEQ ID NO:211.

[0181] In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to 10 contiguous bases of Region 2, SEQ ID NO:211. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to 11 contiguous bases of Region 2, SEQ ID NO:211. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to 12 contiguous bases of Region 2, SEQ ID NO:211. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to 13 contiguous bases of Region 2, SEQ ID NO:211. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to 14 contiguous bases of Region 2, SEQ ID NO:211. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to 15 contiguous bases of Region 2, SEQ ID NO:211. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to 16 contiguous bases of Region 2, SEQ ID NO:211. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to 17 contiguous bases of Region 2, SEQ ID NO:211. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to 18 contiguous bases of Region 2, SEQ ID NO:211. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to 19 contiguous bases of Region 2, SEQ ID NO:211. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to 20 contiguous bases of Region 2, SEQ ID NO:211. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to 21 contiguous bases of Region 2, SEQ ID NO:211. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to 22 contiguous bases of Region 2, SEQ ID NO:211. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to 23 contiguous bases of Region 2, SEQ ID NO:211. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to 24 contiguous bases of Region 2, SEQ ID NO:211. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to 25 contiguous bases of Region 2, SEQ ID NO:211. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to 26 contiguous bases of region 2, SEQ ID NO:211.In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to 27 contiguous bases of Region 2, SEQ ID NO: 211. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to 28 contiguous bases of Region 2, SEQ ID NO: 211. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to 29 contiguous bases of Region 2, SEQ ID NO: 211. In some embodiments, the oligonucleotide comprises a nucleic acid strand complementary to 30 contiguous bases of Region 2, SEQ ID NO: 211.

[0182] In some embodiments, the MuSK-targeting oligonucleotides of the present disclosure are antisense oligonucleotides comprising a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 1-38, 127-142, 159-167, and 177-193 in Table 2 below. [Table 2-2-1] [Table 2-2-2] [Table 2-2-3]

[0183] In some embodiments, oligonucleotides have sequences that differ from those explicitly set forth in Table 2, e.g., by substituting one or more residues, or residue types, with alternative residues or residue types, e.g., analogs or other corresponding residue types. For example, in some embodiments, one or more "T" residues (or all "T" residues) in the sequences presented in Table 2 are "U" residues or analogs thereof.

[0184] In some embodiments, the oligonucleotide contains a mismatch with the target. Base pairs can be ranked based on their tendency to promote dissociation or melting (e.g., based on the free energy of association or dissociation of a particular pair; the simplest approach is to examine pairs on an individual pair-by-pair basis, but adjacent or similar analyses can also be used). In terms of promoting dissociation, A:U is preferred over G:C, G:U is preferred over G:C, and I:C is preferred over G:C (I = inosine).

[0185] In some embodiments, an oligonucleotide can include one or more (eg, 2, 3, 4, or 5) nucleotides at the 3' and / or 5' end that are not complementary to the target sequence.

[0186] Chemical structure of MuSK-targeting oligonucleotides Synthetic oligonucleotides provide useful molecular tools in a wide variety of applications. For example, oligonucleotides are useful in therapeutic, diagnostic, research, and novel nanomaterial applications. The use of natural nucleic acids (e.g., unmodified DNA or RNA) is limited, for example, by their susceptibility to endonucleases and exonucleases. Therefore, to circumvent these drawbacks, various synthetic counterparts have been developed. These include synthetic oligonucleotides containing chemical modifications, such as base modifications, sugar modifications, backbone modifications, etc., which, among other things, make these molecules less susceptible to degradation and improve other properties of the oligonucleotides.

[0187] Among other things, the present disclosure encompasses the recognition that structural elements of oligonucleotides, such as base sequence, chemical modifications (e.g., sugar, base, and / or internucleotide linkage modifications and their pattern), and / or stereochemistry (e.g., the stereochemistry of backbone chiral centers (chiral internucleotide linkages) and / or their pattern), can significantly affect properties such as stability, splice alteration ability, etc. In some embodiments, oligonucleotide properties can be tailored by optimizing chemical modifications (base, sugar, and / or internucleotide linkage modifications) and / or stereochemistry (pattern of backbone chiral centers).

[0188] In some embodiments, the present disclosure demonstrates that oligonucleotide compositions comprising oligonucleotides with controlled structural elements, e.g., 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, or improved protein binding profiles, and / or improved delivery. In particular, in some embodiments, the present disclosure provides compositions and methods for altering the splicing of transcripts (e.g., MuSK transcripts). In some embodiments, the present disclosure provides compositions and methods for improving transcript splicing. In some embodiments, altered transcript splicing by the provided compositions and methods includes the production of products with desired and / or improved biological functions and / or the knockdown of undesired products, e.g., by modifying the splicing product so that the undesired biological function can be suppressed or eliminated.

[0189] In some embodiments, the splicing product is mRNA. In some embodiments, the alteration comprises skipping one or more exons. In some embodiments, splicing of the transcript is improved in that exon skipping increases the levels of mRNA and protein with improved beneficial activity compared to the absence of exon skipping.

[0190] In some embodiments, splicing of a transcript is improved in that exon skipping reduces the levels of mRNA and protein with undesired activity compared to the absence of exon skipping. In some embodiments, a target is knocked down via exon skipping by skipping one or more exons that cause premature stop codons and / or frameshift mutations.

[0191] 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; 2-fluorouracil, 2-fluorocytosine, 5-bromouracil, 5-iodouracil, 2,6-diaminopurine, azacytosine; pyrimidine analogs such as pseudoisocytosine and pseudouracil; and other modified nucleobases such as 8-substituted purine, xanthine, or hypoxanthine (the latter two are natural degradation products).

[0192] Modified nucleobases also include extended size nucleobases to which one or more aryl rings, such as phenyl rings, have been added.

[0193] In some embodiments, the modified nucleobase is of any one of the following structures, optionally substituted: [ka]

[0194] In some embodiments, the modified nucleobase is unsubstituted. In some embodiments, the modified nucleobase is substituted. In some embodiments, the modified nucleobase is substituted to contain, for example, a heteroatom, alkyl group, or linking moiety connected to a fluorescent moiety, a biotin or avidin moiety, or other protein or peptide. In some embodiments, the modified nucleobase is a "universal base" that is not a nucleobase in the most classical sense, but functions similarly to a nucleobase. A representative example of such a universal base is 3-nitropyrrole.

[0195] 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-galactosylqueosine, 2'-O-methylguanosine, N-acetylcytidine, 5-(carboxyhydroxymethyl)uridine ... 6 -Isopentenyl adenosine, 1-methyl adenosine, 1-methylpseudouridine, 1-methylguanosine, l-methylinosine, 2,2-dimethylguanosine, 2-methyladenosine, 2-methylguanosine, N 7 -methylguanosine, 3-methyl-cytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5-methylcytosine, 5-formylcytosine, 5-carboxylcytosine, N 6 -methyladenosine, 7-methylguanosine, 5-methylaminoethyluridine, 5-methoxyaminomethyl-2-thiouridine, beta, D-mannosylqueosine, 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.

[0196] In some embodiments, the nucleoside comprises 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 comprises 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. 2007 / 0287831. 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.

[0197] In some embodiments, the oligonucleotides described herein contain one or more modified nucleotides in which the phosphate group or linked phosphorus in the nucleotide is linked to various positions on the sugar or modified sugar. As a non-limiting example, the phosphate group or linked phosphorus can be linked to the 2', 3', 4', or 5' hydroxyl moiety of the sugar or modified sugar. Nucleotides incorporating modified nucleobases described herein are also contemplated in this regard.

[0198] Other modified sugars can also be incorporated into oligonucleotide molecules. In some embodiments, the modified sugar contains one or more substituents at the 2' position, including one of the following: -F; -CF, -CN, -N, -NO, -NO, -OR', -SR', or -N(R') (wherein each R' is independently as defined above and described herein); -O-(C-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 10 alkynyl), -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), or salts thereof, wherein the alkyl, alkylene, alkenyl, and alkynyl may be substituted or unsubstituted. Examples of substituents include -O(CH2) n OCH3 and -O(CH2) nNH2 or a salt thereof (wherein n is 1 to about 10), including, but not limited to, -OCH2CH2OMe(MOE) or a salt thereof, -OCH2CH2N(CH3)2(DMAOE) or a salt thereof, -OCH2CH2OCH2CH2N(CH3)2(DMAEOE) or a salt thereof.

[0199] In some embodiments, the 2'-OH of the ribose is substituted with a substituent that includes one of the following: -H, -F; -CF, -CN, -N, -NO, -NO, -OR', -SR', or -N(R') (wherein each R' is independently as defined above and described herein); -O-(C-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 10 alkynyl), -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 10In some embodiments, the 2'-OH is substituted with -H (deoxyribose). In some embodiments, the 2'-OH is substituted with -F. In some embodiments, the 2'-OH is substituted with -OR'. In some embodiments, the 2'-OH is substituted with -OMe. In some embodiments, the 2'-OH is substituted with -OCHCHOMe (MOE).

[0200] 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 following structure is shown, where Ba represents a nucleobase or modified nucleobase described herein, and R 2s is -OCH2C4'-. [ka]

[0201] In some embodiments, each sugar of the oligonucleotide is or comprises a modified sugar moiety. In some embodiments, each sugar of the oligonucleotide is or comprises a 2'-MOE-modified sugar. In some embodiments, each sugar of the oligonucleotide is or comprises a 2'-OMe-modified sugar. In some embodiments, each sugar of the oligonucleotide is or comprises a 2'-OH-modified sugar. In some embodiments, each sugar of the oligonucleotide is or comprises a 2'-H-modified sugar.

[0202] In some embodiments, the present disclosure provides oligonucleotides comprising 2'-MOE modified sugars, 2'-OMe modified sugars, 2'-OH modified sugars, 2'-H modified sugars, or combinations thereof. In some embodiments, provided oligonucleotides comprise at least one 2'-MOE sugar and at least one 2'-OH sugar (RNA sugar). In some embodiments, provided oligonucleotides comprise at least one 2'-MOE sugar and at least one 2'-H sugar (DNA sugar).

[0203] In some embodiments, the present invention provides oligonucleotides comprising one or more modified internucleotide linkages independently having the structure of Formula I: [ka] During the ceremony, P* is an asymmetric phosphorus atom, in either the Rp or Sp configuration; W is O, S, or Se; Each of X, Y and Z independently represents -O-, -S-, -N(-LR 1 )- or L, L is a covalent bond or an optionally substituted linear or branched C-C 10 alkylene, wherein one or more methylene units of L are optionally and independently selected from optionally substituted C1-C6 alkylene, C1-C6 alkenylene, [ka] substituted by -C(R')2-, -Cy-, -O-, -S-, -SS-, -N(R')-, -C(O)-, -C(S)-, -C(NR')-, -C(O)N(R')-, -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 C-C50 aliphatic, wherein one or more methylene units are optionally and independently selected from optionally substituted C1-C6 alkylene, C1-C6 alkenylene, [ka] substituted by -C(R')2-, -Cy-, -O-, -S-, -SS-, -N(R')-, -C(O)-, -C(S)-, -C(NR')-, -C(O)N(R')-, -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; 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; each [ka] independently represent the connection to the nucleoside.

[0204] In some embodiments, the internucleotide linkage having the structure of Formula I is [ka] is.

[0205] In some embodiments, the oligonucleotide comprises both a non-natural internucleotide linkage as described herein and a natural phosphate linkage. In some embodiments, each internucleotide linkage of the oligonucleotide is a non-natural internucleotide linkage. In some embodiments, each internucleotide linkage of the oligonucleotide is a chiral internucleotide linkage. In some embodiments, each internucleotide linkage of the oligonucleotide is a phosphorothioate linkage. [ka] .

[0206] In some embodiments, each internucleotide linkage of the oligonucleotide is a native phosphate linkage. In some embodiments, the oligonucleotide comprises at least one native phosphate linkage and at least one phosphodithioate linkage. In some embodiments, at least 50% of the internucleotide linkages of the oligonucleotide are phosphodithioate linkages. In some embodiments, at least 60% of the internucleotide linkages of the oligonucleotide are phosphodithioate linkages. In some embodiments, at least 70% of the internucleotide linkages of the oligonucleotide are phosphodithioate linkages. In some embodiments, at least 80% of the internucleotide linkages of the oligonucleotide are phosphodithioate linkages. In some embodiments, at least 90% of the internucleotide linkages of the oligonucleotide are phosphodithioate linkages. In some embodiments, at least 94% of the internucleotide linkages of the oligonucleotide are phosphodithioate linkages. In some embodiments, at least 95% of the internucleotide linkages of the oligonucleotide are phosphodithioate linkages.

[0207] In some embodiments, at least 50% of the internucleotide linkages of the oligonucleotide are natural phosphate linkages. In some embodiments, at least 60% of the internucleotide linkages of the oligonucleotide are natural phosphate linkages. In some embodiments, at least 70% of the internucleotide linkages of the oligonucleotide are natural phosphate linkages. In some embodiments, at least 80% of the internucleotide linkages of the oligonucleotide are natural phosphate linkages. In some embodiments, at least 90% of the internucleotide linkages of the oligonucleotide are natural phosphate linkages. In some embodiments, at least 94% of the internucleotide linkages of the oligonucleotide are natural phosphate linkages. In some embodiments, at least 95% of the internucleotide linkages of the oligonucleotide are natural phosphate linkages.

[0208] Among other things, the present disclosure provides oligonucleotides of various designs, which may contain various nucleobases and their patterns, sugars and their patterns, internucleotide linkages and their patterns, and / or additional chemical moieties and their patterns described in the present disclosure. 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 adult hippocampal neurogenesis (AHN) and improving cognition in AD. 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 of the MuSK Ig3 domain and / or its products in the cells of a subject or patient. In some embodiments, provided oligonucleotides can direct a decrease in the expression, level, and / or activity of a MuSK Ig3 domain and / or one or more products thereof in cells of a subject or patient, while the expression, level, and / or activity of all forms of MuSK remains substantially the same. In some embodiments, the cells normally express or produce a protein encoded by the MuSK Ig3 domain. In some embodiments, provided MuSK-targeting oligonucleotides can direct a decrease in the expression, level, and / or activity of a MuSK Ig3 domain gene or gene product, and have a base sequence that consists of, includes, 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 an oligonucleotide sequence disclosed herein, wherein each T may be independently replaced with U, or vice versa, and wherein the oligonucleotide comprises at least one non-natural modification of a base, sugar linkage, and / or internucleotide linkage.

[0209] As described herein, naturally occurring, highly abundant full-length MuSK retains the BMP-binding Ig3 domain, enhances BMP signaling, and thus suppresses neurogenesis. In contrast, ΔIg3-MuSK has lower BMP signaling, promotes AHN, and improves cognition. In some embodiments, the present disclosure provides exon-skipping MuSK-targeting oligonucleotides that switch MuSK from the AHN-restricted full-length MuSK to the AHN-permissive ΔIg3-MuSK splice form.

[0210] As described herein, naturally occurring, highly abundant full-length MuSK retains the BMP-binding Ig3 domain and enhances BMP signaling, thus affecting muscle regeneration. In contrast, ΔIg3-MuSK has lower BMP signaling, promoting muscle regeneration and / or preventing muscle fibrosis. In some embodiments, the present disclosure provides exon-skipping MuSK-targeting oligonucleotides that switch MuSK from full-length MuSK to the pro-myogenic ΔIg3-MuSK splice form.

[0211] In some embodiments, the one or more skipped exons are selected from exons 6 and / or 7 of 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.

[0212] In some embodiments, the MuSK-targeting oligonucleotides described herein can provide exon skipping of exons 6 and / or 7, but not exons 3 and / or 4.

[0213] In some embodiments, the MuSK-targeting oligonucleotides described herein can provide exon skipping of exons 6 and / or 7 at a higher level than they provide exon skipping of exons 3 and / or 4.

[0214] In some embodiments, the MuSK-targeting oligonucleotides described herein provide exon skipping such that the level of MuSK transcripts comprising exons 6 and 7, or the level of MuSK protein forms comprising sequences encoded by exons 6 and 7, or both, is reduced by more than 60%, and the level of MuSK transcripts comprising exons 3 and 4, or the level of MuSK protein forms comprising sequences encoded by exons 3 and 4, or both, is reduced by less than 40%.

[0215] In some embodiments, the MuSK-targeting oligonucleotide alters the splicing of MuSK transcripts such that the level of MuSK transcripts comprising exons 6 and 7, or the level of MuSK protein forms comprising sequences encoded by exons 6 and 7, or both, is reduced by more than 60%, and the level of MuSK transcripts comprising exons 3 and 4, or the level of MuSK protein forms comprising sequences encoded by exons 3 and 4, or both, is reduced by less than 40%.

[0216] In some embodiments, the MuSK-targeting oligonucleotide alters the splicing of MuSK transcripts such that the level of MuSK transcripts comprising exons 6 and 7, or the level of MuSK protein forms comprising sequences encoded by exons 6 and 7, or both, is reduced by more than 70%, and the level of MuSK transcripts comprising exons 3 and 4, or the level of MuSK protein forms comprising sequences encoded by exons 3 and 4, or both, is reduced by less than 40%.

[0217] In some embodiments, the MuSK-targeting oligonucleotide alters the splicing of MuSK transcripts such that the level of MuSK transcripts comprising exons 6 and 7, or the level of MuSK protein forms comprising sequences encoded by exons 6 and 7, or both, is reduced by more than 80%, and the level of MuSK transcripts comprising exons 3 and 4, or the level of MuSK protein forms comprising sequences encoded by exons 3 and 4, or both, is reduced by less than 40%.

[0218] In some embodiments, the MuSK-targeting oligonucleotide alters the splicing of MuSK transcripts such that the level of MuSK transcripts comprising exons 6 and 7, or the level of MuSK protein forms comprising sequences encoded by exons 6 and 7, or both, is reduced by more than 90%, and the level of MuSK transcripts comprising exons 3 and 4, or the level of MuSK protein forms comprising sequences encoded by exons 3 and 4, or both, is reduced by less than 40%.

[0219] In some embodiments, the MuSK-targeting oligonucleotide alters the splicing of MuSK transcripts such that the level of MuSK transcripts comprising exons 6 and 7, or the level of MuSK protein forms comprising sequences encoded by exons 6 and 7, or both, is reduced by more than 60%, and the level of MuSK transcripts comprising exons 3 and 4, or the level of MuSK protein forms comprising sequences encoded by exons 3 and 4, or both, is reduced by less than 30%.

[0220] In some embodiments, the MuSK-targeting oligonucleotide alters the splicing of MuSK transcripts such that the level of MuSK transcripts comprising exons 6 and 7, or the level of MuSK protein forms comprising sequences encoded by exons 6 and 7, or both, is reduced by more than 60%, and the level of MuSK transcripts comprising exons 3 and 4, or the level of MuSK protein forms comprising sequences encoded by exons 3 and 4, or both, is reduced by less than 20%.

[0221] In some embodiments, the MuSK-targeting oligonucleotide alters the splicing of MuSK transcripts such that the level of MuSK transcripts comprising exons 6 and 7, or the level of MuSK protein forms comprising sequences encoded by exons 6 and 7, or both, is reduced by more than 60%, and the level of MuSK transcripts comprising exons 3 and 4, or the level of MuSK protein forms comprising sequences encoded by exons 3 and 4, or both, is reduced by less than 10%.

[0222] In various embodiments, the active compound is an oligonucleotide that directs the skipping of one or more exons in the MuSK gene. In various embodiments, the active compound is an oligonucleotide that directs the skipping of multiple exons in the MuSK gene. In some embodiments, the active compound is an oligonucleotide that directs the skipping of exon 6, exon 7, or both in the MuSK gene. In some embodiments, the active compound is an oligonucleotide that directs the skipping of exon 6 in the MuSK gene. In some embodiments, the active compound is an oligonucleotide that directs the skipping of exon 7 in the MuSK gene. In some embodiments, the active compound is an oligonucleotide that directs the skipping of exons 6 and 7 in the MuSK gene. In some embodiments, multiple oligonucleotides may be used together. In some such embodiments, two or more different exon skipping oligonucleotides (e.g., at least one that directs the skipping of exon 6 and one that directs the skipping of exon 7) may be used in combination. Alternatively or additionally, in some embodiments, at least one exon skipping oligonucleotide can be used in combination with at least one degradative oligonucleotide (e.g., one that targets the transcript for RNase H degradation), which can, for example, target a MuSK transcript that includes a functional Ig3 domain or a portion thereof.

[0223] 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, in which 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).

[0224] In some embodiments, within a composition, individual oligonucleotides may be considered to be of the same composition and / or structure even though at a particular time within the composition (e.g., a liquid composition) a particular such oligonucleotide may be in different salt forms (and may be dissolved, e.g., an anionic form in a liquid composition). For example, one of skill in the art will recognize that at a given pH, individual internucleotide bonds along an oligonucleotide chain may be in the acid (H) form or one of several possible salt forms (e.g., sodium salts, or salts of different cations depending on which ions may be present in the preparation or composition), and will understand that so long as their acid forms (e.g., replacing all cations, if any, with H+) are of the same composition and / or structure, such individual oligonucleotides may be properly considered to be of the same composition and / or structure.

[0225] In some embodiments, the oligonucleotide composition comprises two or more oligonucleotides. In some embodiments, the oligonucleotide composition comprises two or more oligonucleotides, wherein each of the plurality of oligonucleotides is independently a plurality of oligonucleotides described herein. For example, in some embodiments, each of the plurality independently shares the same base sequence and the same internucleotide linkages. In some embodiments, at least two of the plurality, or each of the plurality, independently target the same exon of the same transcript (e.g., exons 6 and / or 7 of MuSK). In some embodiments, at least two of the plurality, or each of the plurality, independently target different exons of the same transcript (e.g., exons 3, 4, 6, and / or 7 of MuSK). In some embodiments, at least two of the plurality, or each of the plurality, independently target different transcripts of the same nucleic acid or different nucleic acids. In some embodiments, at least two of the plurality, or each of the plurality, independently target transcripts of different genes. In some embodiments, at least two of the plurality, or each of the plurality, independently target different regions on a MuSK transcript. Notably, such compositions can be utilized to target two or more targets, in some embodiments, simultaneously within the same system.

[0226] Characterization of MuSK-targeting oligonucleotides The MuSK-targeting oligonucleotides provided herein can be identified, evaluated, and / or characterized for one or more of their physical / chemical properties and / or biological activities. Those skilled in the art will recognize various approaches, including specific assays, that may be utilized for such identification, evaluation, and / or characterization.

[0227] In some embodiments, the MuSK-targeting oligonucleotides described herein are characterized in that, e.g., when contacted with a cell expressing MuSK, they increase the level or activity of MuSK ΔIg3 mRNA and / or protein, e.g., compared to another form of MuSK or other suitable reference. In some embodiments, achieving such an increase may be considered to represent "stimulating" MuSK ΔIg3.

[0228] In some embodiments, the MuSK-targeting oligonucleotide is characterized by its ability to alter the splicing activity of MuSK pre-mRNA in cells. For example, cells may be transfected with the MuSK-targeting oligonucleotide, and after an incubation period, expression of an alternative form of MuSK RNA transcript (e.g., in which exons 6 and 7 have been skipped) may 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%.

[0229] In some embodiments, the MuSK-targeting oligonucleotide increases MuSK ΔIg3 mRNA. In some embodiments, the MuSK-targeting oligonucleotide alters splicing of MuSK pre-mRNA. In some embodiments, the MuSK-targeting oligonucleotide promotes skipping of exon 6 and / or exon 7.

[0230] Modulation of MuSK ΔIg3 expression can be measured in the body fluids of a subject treated with a MuSK MR-targeting oligonucleotide, which may or may not contain cells, tissues, or organs of the animal. 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 related to target gene expression in one or more biological fluids, tissues, or organs taken from an animal contacted with one or more compositions described herein.

[0231] 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., a subject not administered a MuSK-targeting 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 determined, for example, by methods described in the Examples below and / or assay techniques such as 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 techniques or combinations of techniques capable of detecting the presence of MuSK ΔIg3 mRNA or protein (e.g., in a subject or a sample obtained from a subject).

[0232] In some embodiments, the level of MuSK ΔIg3 mRNA in a sample obtained from a subject receiving MuSK-targeting oligonucleotide treatment can be compared with the level of MuSK ΔIg3 mRNA in a subject not treated with MuSK-targeting oligonucleotide to determine the extent to which MuSK-targeting oligonucleotide treatment has increased MuSK ΔIg3 mRNA. In some embodiments, a reference level of MuSK ΔIg3 mRNA is obtained from the same subject before receiving MuSK-targeting oligonucleotide treatment. In some embodiments, the reference level of MuSK ΔIg3 mRNA is a range determined by a population of subjects not receiving MuSK-targeting 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 to full-length MuSK mRNA (e.g., MuSK mRNA that does not contain exons 6 and 7) in a subject receiving MuSK-targeted oligonucleotide therapy is, for example, 1-fold, 1.5-5-fold, 5-10-fold, 10-50-fold, 50-100-fold, about 1.1-fold, 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, more than 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold or more greater than a reference ratio.

[0233] 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 fold, 1.2 fold, 1.5 fold, 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, 100 fold or more higher than the reference value.

[0234] 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, for example, a MuSK ΔIg3 protein level obtained from a subject having or at risk of having AD or a disease characterized by neurodegeneration. In some embodiments, the reference level of MuSK ΔIg3 protein is, for example, a MuSK ΔIg3 protein level obtained from a subject having or at risk of having neuromuscular dysfunction, a neurodegenerative disorder, a cardiac disorder (e.g., myocardial infarction, cardiomyopathy), or a genetic disease characterized by muscle wasting, prior to treatment. Methods of contacting a bodily fluid, organ, or tissue with an effective amount of one or more compositions described herein are also contemplated. Contacting a bodily fluid, organ, or tissue with one or more compositions comprising a MuSK-targeting oligonucleotide can result in expression of MuSK ΔIg3 in cells of the bodily fluid, organ, or tissue and modulation of MuSK expression. An effective amount can be determined by monitoring the effect on functional MuSK ΔIg3 protein expression of a MuSK-targeting oligonucleotide administered to a subject or contacted with a cell.

[0235] In some embodiments, a MuSK-targeting oligonucleotide or a composition comprising the same, when administered to a cell population (e.g., comprising NSCs and / or neural progenitor cells (MPCs)), increases the number of cells in an activated state (e.g., actively proliferating). Cells within the population can be assessed for their activation state by methods known in the art, including, for example, an EdU assay in which EdU+ cycling cells are compared to the total number of cells. In some embodiments, a MuSK-targeting oligonucleotide or a composition comprising the same, when administered to a cell population comprising NSCs, decreases the number of quiescent NSCs and / or increases the number of activated NSCs in the population.

[0236] In some embodiments, a MuSK-targeting oligonucleotide or a composition comprising same, when administered to a cell population (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 activation state by methods known in the art, including, for example, an EdU assay in which EdU+ cycling cells are compared to total cell number. In some embodiments, a MuSK-targeting oligonucleotide or a composition comprising same, when administered to a cell population comprising satellite cells, decreases the number of quiescent satellite cells and / or increases the number of activated satellite cells within the population.

[0237] In some embodiments, a MuSK-targeting oligonucleotide, or a composition comprising the same, when administered to a cell population comprising NSCs and / or NPCs, increases the number of cells expressing genes associated with early neurons (e.g., Dex) and / or decreases the number of cells expressing genes associated with mature neurons (e.g., Map2), astrocytes (e.g., GFAP and S100b), and / or oligodendrocytes (e.g., CNPase and O4). In some embodiments, a MuSK-targeting oligonucleotide, when administered to a cell population comprising NSCs and / or NPCs, increases the expression level of genes associated with early neurons (e.g., Dex) and / or decreases the expression level of genes associated with mature neurons (e.g., Map2), astrocytes (e.g., GFAP and S100b), and / or oligodendrocytes (e.g., CNPase and O4) in the cell population.

[0238] In some embodiments, the cell population comprises NSCs that have been induced to become NSCs (eg, from stem cells such as embryonic stem cells or pluripotent stem cells).

[0239] In some embodiments, a MuSK-targeting oligonucleotide or a composition comprising the same, when administered to a cell population 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-targeting oligonucleotide or a composition comprising the same, when administered to a cell population comprising satellite cells and / or myoblasts, increases the expression levels of genes associated with myogenic factors (e.g., Pax7, MyoD, myogenin, and MERGE) and / or decreases the expression levels of genes associated with the MuSK-BMP signaling pathway (e.g., RGS4, Msx2, Myf5, Ptx3, Id1) in the cell population.

[0240] In some embodiments, the cell population comprises satellite cells and / or myoblasts that have been induced to become satellite cells and / or myoblasts (e.g., from stem cells such as embryonic stem cells or pluripotent stem cells).

[0241] In some embodiments, the cell population is obtained from a healthy subject, hi some embodiments, the cell population is obtained from a subject having or at risk of having AD or a disease characterized by neurodegeneration, or a subject suffering from a disease or disorder such as neuromuscular dysfunction, a neurodegenerative disorder, a cardiac disorder (e.g., myocardial infarction, cardiomyopathy), or a genetic disease characterized by muscle wasting.

[0242] In some embodiments, the MuSK-targeting oligonucleotide or a composition comprising same increases neurogenesis in a subject when contacted with a cell population derived from the subject. In some embodiments, the MuSK-targeting oligonucleotide or a composition comprising same is contacted with the cell population in vivo, for example, by injection into the subject. In some embodiments, the MuSK-targeting oligonucleotide or a composition comprising same is contacted with the cell population ex vivo by obtaining the cell population from the subject, and when the treated cells are reintroduced into the subject, neurogenesis is increased.

[0243] In some embodiments, the MuSK-targeting oligonucleotide or composition comprising same increases muscle regeneration and / or growth in a subject when contacted with a cell population derived from the subject. In some embodiments, the MuSK-targeting oligonucleotide or composition comprising same is contacted with the cell population in vivo, for example, by injection into the subject. In some embodiments, the MuSK-targeting oligonucleotide or composition comprising same is contacted with the cell population by obtaining the cell population from the subject, and when the treated cells are reintroduced into the subject, muscle regeneration is increased.

[0244] In some embodiments, the MuSK-targeting oligonucleotide or a composition comprising same increases neurogenesis and / or growth and / or improves cognition when administered to a subject. Exemplary methods for assessing these biological effects are detailed, for example, in the Examples below.

[0245] In some embodiments, a MuSK-targeting oligonucleotide or a composition comprising the same increases muscle regeneration and / or growth, and / or neuromuscular function and / or myogenesis when administered to a subject. Exemplary methods for assessing these biological effects are detailed, for example, in the Examples below.

[0246] Production of MuSK-targeting oligonucleotides The MuSK-targeting oligonucleotides described herein can be synthesized by standard methods known in the art, for example, by using an automated synthesizer. After chemical synthesis (for example, solid-phase synthesis using the phosphoramidite method), the oligonucleotide molecules can be deprotected, annealed to ds molecules, and purified (for example, by gel electrophoresis or HPLC). Protocols for preparing MuSK-targeting oligonucleotides and oligonucleotides are known in the art.

[0247] In some embodiments, the present disclosure provides techniques for preparing chiral controlled oligonucleotides and compositions thereof. In some embodiments, the present disclosure provides techniques for preparing stereochemically pure oligonucleotides and compositions thereof. In some embodiments, the provided oligonucleotides and compositions thereof are highly pure. In some embodiments, the oligonucleotides of the present disclosure are at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% stereochemically pure at the linkage phosphorus of the chiral internucleotide linkage. In some embodiments, the oligonucleotides of the present disclosure are stereoselectively prepared and are substantially free of stereoisomers. In some embodiments, provided compositions comprise a plurality of oligonucleotides sharing the same base sequence of the same pattern of chiral linkage phosphorus stereochemistry (e.g., comprising one or more of Rp and / or Sp, where each chiral linkage phosphorus is independently Rp or Sp), but at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of all oligonucleotides sharing the same base sequence with the plurality of oligonucleotides in the composition share the same pattern of chiral linkage phosphorus stereochemistry, or are a plurality of oligonucleotides. In some embodiments, provided compositions comprise a plurality of oligonucleotides sharing the same base sequence of the same pattern of chiral linkage phosphorus stereochemistry, but at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of all oligonucleotides sharing the same configuration with the plurality of oligonucleotides in the composition share the same pattern of chiral linkage phosphorus stereochemistry, or are a plurality of oligonucleotides.

[0248] MuSK-targeting oligonucleotides can also be formed intracellularly by transcription of RNA from an expression construct introduced into the cell (e.g., Yu et al., Proc. Natl. Acad. Sci. USA 2002;99:6047-6052). Expression constructs for in vivo production of oligonucleotide molecules can include 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, e.g., Brummelkamp et al., Science 2002;296:550-553) and the U6 polymerase-III promoter (see, e.g., Sui et al., Proc. Natl. Acad. Sci. USA 2002, Paul et al., Nature Biotechnol. 2002;20:505-508, and Yu et al., Proc. Natl. Acad. Sci. USA 2002;99:6047-6052). The MuSK-targeting oligonucleotide expression construct may further comprise one or more vector sequences that facilitate cloning of the expression construct. Standard vectors that can be used include, for example, the pSilencer 2.0-U6 vector (Ambion Inc., Austin, Tex.).

[0249] Pharmaceutical Composition The present disclosure provides pharmaceutical compositions that comprise and / or deliver the MuSK-targeting oligonucleotides described herein. The present disclosure also provides pharmaceutical compositions that are or comprise cell populations that have been exposed to the MuSK-targeting oligonucleotides described herein.

[0250] For example, in some embodiments, provided pharmaceutical compositions may comprise and / or deliver a MuSK-targeting oligonucleotide that, when administered, achieves an increase in the level and / or activity of a MuSK polypeptide lacking an Ig3 domain that functions to interact with BMPs (e.g., a MuSK ΔIg3 polypeptide, or another MuSK variant polypeptide with a disrupted Ig3. Alternatively, or additionally, in some embodiments, provided pharmaceutical compositions may comprise and / or deliver a cell population exposed to a MuSK-targeting oligonucleotide, resulting in an increase in neuronal cell number and / or activity in the population.

[0251] In many embodiments, a pharmaceutical composition is or comprises an active agent (e.g., a MuSK-targeting oligonucleotide or precursor thereof described herein) in combination with one or more pharmaceutically acceptable excipients. One of ordinary skill in the art will appreciate that the components of a particular pharmaceutical composition may be affected by the route of administration of the pharmaceutical composition.

[0252] The compositions of the present disclosure can be formulated for a variety of modes of administration, including systemic and topical or local administration. Techniques and formulations are generally described in Remington, The Science and Practice of Pharmacy, (20th ed. 2000).

[0253] The compositions of the present invention can be prepared and administered in a wide variety of oral, parenteral, and topical dosage forms. Thus, the compositions of the present invention can be administered by injection (e.g., intravenous, intramuscular, intradermal, subcutaneous, intraduodenal, or intraperitoneal). The compositions described herein can also be administered by inhalation, such as intranasally. Furthermore, the compositions of the present invention can be administered transdermally. It is also contemplated that the compositions of the present invention can be administered using multiple routes of administration (e.g., intramuscular, oral, transdermal).

[0254] 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, ocular 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 is formulated for delivery by oral administration.

[0255] In certain embodiments, the oligonucleotides and compositions are delivered to the CNS. In certain embodiments, the oligonucleotides and compositions are delivered to the cerebrospinal fluid. In certain embodiments, the oligonucleotides and compositions are administered to the brain parenchyma. In certain embodiments, the oligonucleotides and compositions are delivered to the animal / subject by intrathecal or intraventricular administration. Widespread distribution of the oligonucleotides and compositions described herein within the central nervous system can be achieved by intraparenchymal, intrathecal, or intraventricular administration.

[0256] 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 nucleus, cortex, hippocampus, and cerebellum.

[0257] In certain embodiments, methods of specifically localizing the pharmaceutical, such as by bolus injection, reduce the median effective concentration (EC50) by 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, or 50-fold. In certain embodiments, the pharmaceutical is an antisense compound, 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, a reduction in EC50 is desirable because it reduces the dose required to achieve a pharmacological result in a patient in need thereof.

[0258] In certain embodiments, the antisense oligonucleotides are delivered by injection or infusion monthly, every two months, every 90 days, every three months, every six months, twice a year, or once a year.

[0259] In addition to the active ingredient, these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers, including excipients and adjuvants, which facilitate the processing of the active compound into pharmaceutically usable preparations. Preparations formulated for oral administration may be in the form of tablets, dragees, capsules, or solutions.

[0260] Oral pharmaceutical preparations can be prepared by combining an active compound with a solid excipient, optionally grinding the resulting mixture, and, if desired, adding suitable additives and then processing the granular mixture to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers, such as 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 polyvinylpyrrolidone (PVP: povidone). If necessary, disintegrants such as cross-linked polyvinylpyrrolidone, agar, or alginic acid or its salts, such as sodium alginate, can be added.

[0261] The dragee cores are provided with a suitable coating. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol (PEG), and / or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyes or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

[0262] Orally usable pharmaceutical preparations include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer such as glycerol or sorbitol. Push-fit capsules can contain the active ingredient mixed with a filler such as lactose, a binder such as starch, and / or a lubricant such as talc or magnesium stearate, and optionally, a stabilizer. In soft capsules, the active compound may be dissolved or suspended in a suitable liquid, such as fatty oils, liquid paraffin, or liquid polyethylene glycol (PEG). In addition, stabilizers may be added.

[0263] In some embodiments, the pharmaceutical composition is a tablet, pill, capsule, liquid, inhalant, nasal spray solution, suppository, suspension, gel, colloid, dispersion, suspension, solution, emulsion, ointment, lotion, eye drops, or ear drops.

[0264] Depending on the specific condition being treated, the pharmaceutical compositions of the present disclosure may be formulated into liquid or solid dosage forms and administered systemically or locally. Pharmaceutical compositions may also be delivered, for example, in timed or sustained-release forms known to those skilled in the art. Formulation and administration techniques can be found in Remington, *The Science and Practice of Pharmacy* (20th ed. 2000). Suitable routes include oral, buccal, inhalation spray, sublingual, rectal, transdermal, vaginal, transmucosal, nasal, or intestinal administration; intramuscular, subcutaneous, or intramedullary injection; and parenteral delivery, including intrathecal, direct intraventricular, intravenous, intraarticular, intrasternal, intrasynovial, intrahepatic, intralesional, intracranial, intraperitoneal, intranasal, or intraocular injection, or other delivery modes.

[0265] For injection, the pharmaceutical compositions of the present disclosure may be formulated and diluted in aqueous solutions, such as Hank's solution, Ringer's solution, or physiologically compatible buffers such as 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.

[0266] For the practice of the present disclosure, the use of pharmaceutically acceptable inert carriers to formulate the compositions disclosed herein into dosages suitable for systemic administration is within the scope of the present disclosure. With appropriate selection of carriers and appropriate manufacturing practices, compositions of the present disclosure, particularly those formulated as solutions, can be administered parenterally, such as by intravenous injection.

[0267] 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 can formulate the compounds of the present disclosure as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions, etc., for oral ingestion by a subject (e.g., a patient) to be treated.

[0268] For nasal or inhalation delivery, one or more solubilizing, diluent, or dispersing substances may be employed, such as saline, a preservative such as benzyl alcohol, an absorption enhancer, and a fluorocarbon.

[0269] In some embodiments, provided compositions may include and / or deliver a precursor of an active agent, which upon administration becomes or releases the active therapeutic agent. In some embodiments, for example, the precursor may be or include a prodrug of a small molecule stimulatory agent, a nucleic acid encoding a protein stimulatory agent, or the like.

[0270] 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 as a pharmaceutically acceptable salt form, e.g., 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, the salt form of a provided oligonucleotide comprises two or more cations, for example, in some embodiments, up to the number of negatively charged acidic groups (e.g., phosphate, phosphorothioate, etc.) in the oligonucleotide.

[0271] Pharmaceutically acceptable salts are generally well known to those skilled in the art and include, but are not limited to, acetate, benzenesulfonate, besylate, benzoate, bicarbonate, bitartrate, bromide, calcium edetate, carnsylate, carbonate, citrate, edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate, Examples of suitable pharmaceutically acceptable salts include hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, malate, maleate, mandelate, mesylate, mucate, napsylate, nitrate, pamoate (embonate), pantothenate, phosphate / diphosphate, polygalacturonate, salicylate, stearate, diacetate, succinate, sulfate, tannate, tartrate, and teoclate. Other pharmaceutically acceptable salts are described, 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.

[0272] As will be understood by those skilled in the art, oligonucleotides may be formulated as several salts, for example, for pharmaceutical use. In some embodiments, the salt is a metal cation salt and / or an ammonium salt. In some embodiments, the salt is a metal cation salt of the oligonucleotide. In some embodiments, the salt is an ammonium salt of the oligonucleotide. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc. In some embodiments, the salt is a sodium salt of the oligonucleotide. In some embodiments, pharmaceutically acceptable salts include non-toxic ammonium cations, quaternary ammonium cations, 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, salts of oligonucleotides may contain more than one cation, for example, sodium ions, so that more than one anion may be present in the oligonucleotide.

[0273] 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 per day, about 0.5 to about 100 mg, about 1 to about 50 mg, and about 5 to about 100 mg per day are exemplary 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 being treated, the subject's weight, and the preference and experience of the attending physician.

[0274] 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 may be used 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 of action on the target compared to the provided oligonucleotides in the composition. 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. Multiple 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, provided oligonucleotides or compositions thereof are administered after one or more other therapeutic agents and / or medical procedures, hi some embodiments, provided compositions include one or more other therapeutic agents.

[0275] Manufacturing 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 powders, tablets, pills, capsules, cachets, suppositories and dispersible granules.Solid carriers can be one or more substances that can also function as diluents, flavoring agents, binders, preservatives, tablet disintegrating agents or encapsulating materials.

[0276] 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 the carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired.

[0277] 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 formulations of an active therapeutic agent with an encapsulating material as a carrier, in which the active ingredient, with or without other carriers, is surrounded by the carrier to provide a capsule in which the carrier is associated with the active ingredient. Similarly, cachets and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid dosage forms suitable for oral administration.

[0278] 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 as by stirring, etc. The molten homogeneous mixture is then poured into convenient sized molds and allowed to cool and solidify.

[0279] 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.

[0280] When parenteral administration is necessary or desired, particularly suitable mixtures 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 suitable carriers for parenteral administration include aqueous solutions of dextrose, saline, purified water, ethanol, glycerol, propylene glycol, peanut oil, sesame oil, polyoxyethylene block polymers, and the like. Ampoules are convenient unit doses. The compositions of the present invention may also be incorporated into liposomes or administered via transdermal pumps or patches. Pharmaceutical mixtures suitable for use in the present invention include, for example, those described in Pharmaceutical Sciences (17th Ed., Mack Pub. Co., Easton, PA) and WO 96 / 05309, which are incorporated herein by reference.

[0281] Aqueous solutions suitable for oral use can be prepared by dissolving the active ingredient in water and adding suitable colorants, flavors, stabilizers, and thickening agents, as desired. Aqueous suspensions suitable for oral use can be made by dispersing the finely divided active ingredient in water together with viscous substances such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, and other well-known suspending agents.

[0282] Also included are solid form preparations intended to be converted immediately before use into liquid form preparations for oral administration. Such liquid forms include solutions, suspensions, and emulsions. These preparations may contain, in addition to the active ingredient, colorants, flavors, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizers, etc.

[0283] Pharmaceutical preparations are preferably in unit dosage form.In this 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 preparations such as packets of tablets, capsules, and powders in vials or ampoules.The unit dosage form can also be a capsule, tablet, cachet, or lozenge itself, or any suitable number of these can be packaged.

[0284] The quantity of active ingredient in a unit dose preparation may be varied or adjusted according to the particular application and the potency of the active ingredient. The composition may also contain other compatible therapeutic agents, if desired.

[0285] Patient population In some embodiments, suitable patients or populations are those suffering from and / or susceptible to a disease, disorder, or condition associated with neurodegeneration (e.g., AD), or who would otherwise benefit from increased neurogenesis. In some embodiments, suitable patients or populations are those suffering from and / or susceptible to a disease, disorder, such as neuromuscular dysfunction, cardiac disorders (e.g., myocardial infarction, cardiomyopathy), or genetic disorders characterized by muscle wasting, or who would otherwise benefit from increased muscle regeneration.

[0286] In some embodiments, the neurodegenerative disease, disorder, or condition is 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, diabetes-mediated hippocampal neuron loss, brain injury (e.g., traumatic and / or anoxic brain injury), and Huntington's disease.

[0287] 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 from viral infections.

[0288] In some embodiments, the suitable patient or population is a model organism, hi some embodiments, the suitable patient or population is a human. In some embodiments, the human has an age ranging from about 0 to about 6 months old, about 6 to about 12 months old, about 6 to about 18 months old, about 18 to about 36 months old, about 1 to about 5 years old, about 5 to about 10 years old, about 10 to about 15 years old, about 15 to about 20 years old, about 20 to about 25 years old, about 25 to about 30 years old, about 30 to about 35 years old, about 35 to about 40 years old, about 40 to about 45 years old, about 45 to about 50 years old, about 50 to about 55 years old, about 55 to about 60 years old, about 60 to about 65 years old, about 65 to about 70 years old, about 70 to about 75 years old, about 75 to about 80 years old, about 80 to about 85 years old, about 85 to about 90 years old, about 90 to about 95 years old, or about 95 to about 100 years old.

[0289] 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 a human adult. In yet other embodiments, the human is an elderly human.

[0290] In some embodiments, suitable patients or populations may be characterized by one or more criteria such as age group, sex, genetic background, pre-existing clinical conditions, previous exposure to therapy, etc.

[0291] In some embodiments, suitable patients or populations are those suffering from, for example, neuromuscular dysfunction, neurodegenerative disorders, cardiac disorders (e.g., myocardial infarction, cardiomyopathy), or genetic disorders characterized by muscle wasting. In some embodiments, suitable patients or populations are those suffering from surgery or have experienced injury, trauma, and / or prolonged immobilization (e.g., due to bed rest or a cast). 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 due to diseases or conditions, including, but not limited to, trauma, genetic disorders, muscle disorders, and aging. Trauma can result, for example, from radiation treatments, crush injuries, lacerations, and amputations. In some embodiments, suitable patients or populations are those suffering from or at risk of 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-Dreyfus muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, myotonic muscular dystrophy, and oculopharyngeal muscular dystrophy.

[0292] In some embodiments, suitable patients or populations may be defined according to screening tools for diseases or disorders related to Alzheimer's disease. In some embodiments, suitable patients or populations may be defined according to screening tools for other diseases characterized by neurodegeneration, such as 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, diabetes-mediated hippocampal neuron loss, brain injury (e.g., traumatic and / or anoxic brain injury), and Huntington's disease.

[0293] In some embodiments, suitable patients or populations may be defined according to screening tools for diseases or disorders associated with muscle fibrosis and / or muscle wasting. In some embodiments, suitable patients or populations may be defined according to screening tools and methods for diagnosing diseases associated with muscle fibrosis and / or muscle wasting.

[0294] In some embodiments, a suitable patient or population may be defined according to the results of structural imaging (e.g., magnetic resonance imaging (MRI), computed tomography (CT), ultrasound, etc.). In some embodiments, a suitable patient or population may be defined according to the results of cognitive testing. In some embodiments, a suitable patient or population may be defined according to the results of neurological testing. In some embodiments, the cognitive testing involves one or more of the following tests: motor screening task (MOT), reaction time (RTI), paired-associate 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 matched-to-sample visual search (MTS). In some embodiments, a suitable patient or population may be defined according to the results of assessments such as muscle enzyme measurements, EMG, muscle biopsy, genetic testing, cardiac testing (e.g., ECG), strength and respiratory function assessments, etc.

[0295] 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 used, 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 administer to a human or other subject to treat or prevent a particular condition. The precise amount of therapeutic agent required to achieve a therapeutic effect 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 known to those of skill in the art.

[0296] In some embodiments, administration may be ophthalmic, oral, buccal, cutaneous (e.g., may be or include one or more of topical application 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, intratracheal (e.g., by intratracheal instillation), intravaginal, intravitreal, etc.

[0297] Those skilled in the art, having read this disclosure, will understand that in some embodiments it may be desirable to achieve delivery of MuSK-targeting oligonucleotide compositions to muscle. Alternatively or additionally, in some embodiments it may be desirable to achieve delivery of MuSK-targeting oligonucleotide compositions to the CNS (e.g., the brain, such as the hippocampus and / or subventricular region) and / or lung.

[0298] In some embodiments, MuSK-targeted oligonucleotide compositions are delivered via systemic delivery and / or local delivery to muscle (eg, via intramuscular injection).

[0299] In some embodiments, the MuSK-targeting oligonucleotide composition is administered using a viral vector, effectively delivering the MuSK-targeting oligonucleotide composition in the form of a nucleic acid payload. In some embodiments, the viral vector targets specific cell types (e.g., myoblasts, myocytes, myotubes, satellite cells, and myofibers). AAV1, AAV6, and AAV9 vectors have been used to target different muscle cell types (see, e.g., Arnett et al., Mol Ther Methods Clin Dev. 1. pii:14038, 2014 and Riaz et al., Skeletal Muscle 5(37) 2015).

[0300] Those skilled in the art, upon reading this disclosure, will understand that in some embodiments it may be desirable to achieve delivery of MuSK-targeting oligonucleotide compositions to the CNS, and in some embodiments, to the brain.

[0301] 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., a stimulant or a MuSK-targeting oligonucleotide) is delivered to the central nervous system (CNS) via intraventricular administration.

[0302] Furthermore, certain viral vectors are known to selectively target neurons and effectively deliver gene payloads to the brain. For example, AAV2 / 1 vectors have been established to effectively deliver nucleic acid payloads (e.g., gene therapy, coding RNA, etc.) to hippocampal neurons. (See, e.g., Hammond et al., PLoS One 12:e0188830, 2017; Guggenhuber et al., PLoS One 5:e15707, 2010; Lawlor et al., Mol. Neurodeg. 2:11, 2007.) Similarly, certain AAV vectors (e.g., AAV2 / 1 and / or AAV4 vectors) have been established to target specific cells in the subventricular zone and effectively deliver nucleic acid payloads. See, e.g., Liu et al Gene Thep 12:1503,2005; Bockstael et al Hum Gene Therap 23:doi.org / 10.1089 / hum.2011.216,2012).

[0303] In the case of 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.

[0304] In some embodiments, effective delivery may be achieved by systemic administration of the compositions described herein. Alternatively, or additionally, in some embodiments, effective delivery may be achieved by local administration to the CNS and / or brain, for example, by intrathecal and / or intracavitary (e.g., intracerebroventricular) delivery.

[0305] Techniques for localized 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., Pharmacol. & Therap. 144:122, 2014), small molecules (see, e.g., Dodou Pharm. J. 289:501, 2012), cellular compositions (see, e.g., Eftekharzadeh et al., Iran J Basic Med Sci 18:520, 2015), and nucleic acid therapeutics (see, e.g., Otsuka et al., J. Neurotrauma 28:1063, 2011; see also the prescribing information for onasemnogene abeparvovec-xioi (sold under the trade name Zolgensma™) and nusinersen (sold under the trade name Spinraza™)).

[0306] Those skilled in the art will recognize that intrathecal delivery can be particularly effective for achieving delivery to the hippocampus, including cell, protein, and nucleic acid therapeutic agents.

[0307] Systemic administration techniques (including, e.g., oral, parenteral, mucosal, etc.) are well established for a wide variety of drugs. In some embodiments, systemic administration to achieve CNS and / or brain delivery may rely on the ability to cross the blood-brain barrier (BBB).

[0308] Certain active agents and / or delivery systems are known to cross the BBB. Recent techniques have shown that even agents such as oligonucleotides, which have historically been considered particularly challenging to deliver to the CNS and / or brain, can be successfully delivered. For example, Min et al., Angew Chem Int Ed Engl doi:10.1002 / anie.201914751, 2020, incorporated herein by reference, describes glucose-coated polymeric nanocarriers that transport oligonucleotides across the BBB.

[0309] It has also been reported that incorporating certain chemicals into oligonucleotide therapeutic agents can facilitate their movement across the BBB. For example, Khorkova et al. (Nature Biotech 35:249, 2017, incorporated herein by reference) report: "2'-modified phosphorothioate oligonucleotides...are potentially particularly suitable for CNS disorders, given their long half-life, with effects in the brain lasting up to 6 months after a single injection." Another type of sugar modification, locked nucleic acid (LNA), introduces a bridge connecting the 2' oxygen and the 4' carbon. This modification significantly increases the melting temperature of LNA-DNA and LNA-RNA hybrids, thus enabling the generation of shorter ODN-based compounds with increased bioavailability and reduced manufacturing costs. The recently proposed tricyclo-DNA, a conformationally constrained oligonucleotide analog, contains three additional C atoms between the C(5') and C(3') sugars (Figure 2). This modification increases stability, hydrophobicity, and RNA affinity, improving tissue uptake and BBB penetration. (Citation omitted).

[0310] In subjects suffering from or susceptible to diseases or disorders such as idiopathic pulmonary fibrosis (IPF), acute respiratory distress syndrome (ARDS), pneumonia, and pulmonary complications due to viral infections, administration that achieves pulmonary delivery may be desirable.

[0311] In some embodiments, oligonucleotides (e.g., antisense oligonucleotides) are developed to enhance their delivery to target sites. As described in the art, oligonucleotides are covalently or non-covalently attached to additional chemical moieties (e.g., carriers or ligands) to enhance delivery. See Thomas C. Roberts et al., Nature Reviews Drug Discovery, volume 19, pages 673-694 (2020), which is incorporated herein by reference in its entirety.

[0312] As will be understood by those skilled in the art, various bioconjugation techniques can be utilized to enhance delivery of oligonucleotides to target sites. For example, oligonucleotides can be covalently conjugated to lipids (e.g., cholesterol, which promotes interaction with circulating lipoprotein particles), peptides (for cell targeting and / or cell penetration), aptamers, antibodies, and sugars (e.g., N-acetylgalactosamine (GalNAc) (see Verma, Ann Indian Acad Neurol. 2018 21(1):3-8. doi:10.4103 / aian.AIAN_298_17) to enhance safer delivery to target sites.

[0313] As will be appreciated by those skilled in the art, lipid conjugates include, for example, oligonucleotides bound to cholesterol, α-tocopherol (vitamin E), long-chain (>C18) fatty acids, lipoprotein particles (e.g., HDL and LDL), and the like.

[0314] As will be appreciated by those skilled in the art, conjugation of N-acetylgalactosamine (GalNAc) can enhance uptake of oligonucleotides into target sites (eg, liver cells).

[0315] As will be appreciated by those skilled in the art, antibody and aptamer conjugates can be used to enhance oligonucleotide delivery. For oligonucleotide delivery, various receptors have been successfully targeted, including, for example, HIV gp160 protein, HER2, CD7 (a T cell marker), CD71 (a transferrin receptor highly expressed in cardiac and skeletal muscle), and TMEFF2. Similarly, oligonucleotides have also been conjugated with antibodies against CD44 (a neural stem cell marker), EPHA2, and EGFR193. Furthermore, aptamers can be conjugated to oligonucleotides to enhance their delivery.

[0316] As will be appreciated by those skilled in the art, various nanocarriers can be used to enhance oligonucleotide delivery. For example, oligonucleotides can form non-covalent complexes with cationic polymers (e.g., polyethyleneimine), dendrimers, CPPs (e.g., MPG-8, PepFect6, RVG-9R228, and Xentry-KALA229), and inorganic methods (e.g., calcium phosphate nanoparticles).

[0317] As will be appreciated by those skilled in the art, various lipoplexes and liposomes (eg, lipid nanoparticles (LNPs)) can be used to enhance oligonucleotide delivery.

[0318] In some embodiments, MuSK-targeting oligonucleotides are modified to form bioconjugates (e.g., conjugated with sugars, peptides, antibodies, aptamers, lipids, etc.) to enhance the...

Claims

1. An oligonucleotide composition comprising multiple oligonucleotides, The oligonucleotide composition is characterized in that, upon contact with a MuSK transcript in a transcription splicing system, the relative amount of the transcript lacking the Ig3 domain coding sequence decreases compared to the relative amount observed under such reference conditions, selected from the group consisting of the absence of the composition, the presence of a reference composition, and combinations thereof. The oligonucleotide mediates the skipping of exon 6 and / or exon 7 of the MuSK gene, and MuSK splicing is altered in such a way that the level of MuSK transcript containing exons 6 and 7 is reduced, or the level of MuSK protein morphology containing the sequence encoded by exons 6 and 7 is reduced, or both. The base sequence of the oligonucleotide includes a sequence having five or fewer mismatches in the 18-25 base length portion of the MuSK gene or its complement. composition.

2. The composition according to claim 1, wherein the exon skipping reduces the level of mRNA encoding the MuSK protein morphology involved in BMP signaling compared to the level observed in the absence of the exon skipping.

3. The composition according to claim 1, wherein the MuSK splicing is altered such that the level of the MuSK transcript containing exons 3 and 4 remains substantially unchanged, while the level of the MuSK transcript containing exons 6 and 7 decreases.

4. The composition according to claim 1, wherein the MuSK splicing is altered in such a way that the total level of the MuSK transfer remains substantially unchanged, while the level of the MuSK transfer containing exons 6 and 7 decreases.

5. The composition according to claim 1, wherein the level of MuSK transcripts containing exons 6 and 7, or the level of MuSK protein morphology containing sequences encoded by exons 6 and 7, or both, is reduced by more than 60%, more than 70%, more than 80%, or more than 90%, and the level of MuSK transcripts containing exons 3 and 4, or the level of MuSK protein morphology containing sequences encoded by exons 3 and 4, or both, is reduced by less than 40%, less than 30%, less than 20%, or less than 10%.

6. The composition according to claim 1, wherein the oligonucleotide targets a region in the MuSK genome sequence corresponding to positions 83776-83800 and / or 83854-83878 of SEQ ID NO: 77, or a region including at least a portion thereof.

7. The composition according to claim 1, wherein the iSCSI targets a region on the MuSK genome sequence that includes, or at least a portion thereof, the sequence ACTCTGTCAGGTTTCCTCTGGGTCCATTCCAAGAGAGAGAGAGAGACCGAGTGATTGAACTCAAGAC (region 1, SEQ ID NO: 126).

8. The composition according to claim 1, wherein the oligonucleotide targets a portion of the MuSK transcript that has the same sequence as region 1, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consecutive bases of sequence number 126.

9. The composition according to claim 1, wherein the oligonucleotide targets a portion of the MuSK transcript that has the same sequence as the 21st to 25th consecutive bases of region 1, sequence number 126.

10. The composition according to claim 1, wherein the oligonucleotide targets a region on the MuSK genome sequence that is in or includes at least a portion of the sequence GGGGAGAGAAGTTCAGTAACTGCCAAGGCCTGCACCATCAGCCATAGCAGGTAGGATGCCCTCTCCACATTTTG (region 2, SEQ ID NO: 211).

11. The composition according to claim 1, wherein the oligonucleotide targets a portion of the MuSK transcript that has the same sequence as region 2, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consecutive bases of SEQ ID NO:

211.

12. The composition according to claim 1, wherein the plurality of oligonucleotides include one or more nucleotide sequences that are at least 90% identical to sequences selected from SEQ ID NOs: 25, 26, 163, 177, 182, 1-24, 27-38, 127-142, 159-162, 164-167 and 178-193.

13. The composition according to claim 1, wherein the plurality of oligonucleotides comprises one or more nucleotide sequences selected from SEQ ID NOs: 25, 26, 163, 177, 182, 1-24, 27-38, 127-142, 159-162, 164-167 and 178-193.

14. The composition according to claim 1, wherein the base sequence of the oligonucleotide contains a nucleotide sequence that is at least 90% identical to any one of SEQ ID NOs. 25, 26, 163, 177, 182, 1-24, 27-38, 127-142, 159-162, 164-167 and 178-193 in the 5' to 3' direction.

15. The composition according to claim 1, wherein the base sequence of the oligonucleotide includes one of sequence numbers 25, 26, 163, 177, 182, 1-24, 27-38, 127-142, 159-162, 164-167, and 178-193 in the direction from 5' to 3'.

16. The composition according to claim 15, wherein the base sequence of the oligonucleotide includes the nucleotide sequence of SEQ ID NO: 25 in the direction from 5' to 3'.

17. The composition according to claim 15, wherein the base sequence of the oligonucleotide consists of the nucleotide sequence of SEQ ID NO: 25 in the direction from 5' to 3'.

18. The composition according to claim 15, wherein the base sequence of the oligonucleotide includes the nucleotide sequence of SEQ ID NO: 26 in the direction from 5' to 3'.

19. The composition according to claim 15, wherein the base sequence of the oligonucleotide consists of the nucleotide sequence of SEQ ID NO: 26 in the direction from 5' to 3'.

20. The composition according to claim 15, wherein the base sequence of the oligonucleotide includes the nucleotide sequence of SEQ ID NO: 163 in the direction from 5' to 3'.

21. The composition according to claim 15, wherein the base sequence of the oligonucleotide consists of the nucleotide sequence of SEQ ID NO: 163 in the direction from 5' to 3'.

22. The composition according to claim 15, wherein the base sequence of the oligonucleotide includes the nucleotide sequence of SEQ ID NO: 177 in the direction from 5' to 3'.

23. The composition according to claim 15, wherein the base sequence of the oligonucleotide consists of the nucleotide sequence of SEQ ID NO: 177 in the direction from 5' to 3'.

24. The composition according to claim 15, wherein the base sequence of the oligonucleotide includes the nucleotide sequence of SEQ ID NO: 182 in the direction from 5' to 3'.

25. The composition according to claim 15, wherein the base sequence of the oligonucleotide consists of the nucleotide sequence of SEQ ID NO: 182 in the direction from 5' to 3'.

26. The composition according to claim 13, wherein the oligonucleotide is complementary to a nucleotide sequence that is at least 90% identical to any one of SEQ ID NOs. 39-76 and 212-253.

27. ​​The composition according to claim 13, wherein the oligonucleotide is complementary to a nucleotide sequence comprising any one of SEQ ID NOs: 39-76 and 212-253.

28. The composition according to claim 1, wherein the oligonucleotide comprises one or more types of modifications: base modification, sugar modification, and internucleotide bond modification.

29. The composition according to claim 28, wherein the internucleotide bonds of the oligonucleotide include a natural phosphate bond, a phosphorothioate bond, or a phosphodithioate bond.

30. The composition according to claim 29, wherein the internucleotide bonds in the oligonucleotide are phosphorothioate bonds.

31. The composition according to claim 28, wherein the oligonucleotide comprises one or more sugar modifications.

32. The composition according to claim 31, wherein the sugar modification is a 2'-modification.

33. The composition according to claim 32, wherein the 2'-modification is a 2'-MOE sugar modification.

34. The composition according to claim 33, wherein each of the oligonucleotide sugars is a 2'-MOE modified sugar.

35. The composition according to any one of claims 28 to 34, wherein the oligonucleotide has one of the following structures in the direction from 5' to 3': Table 18-1 Table 18-2 Table 18-3 In the table, * represents a phosphorothioate bond, and each sugar in the oligonucleotide is a 2'-MOE modified sugar.

36. The oligonucleotide composition according to claim 35, wherein the oligonucleotide has the structure of SEQ ID NO: 102 in the direction from 5' to 3'.

37. The oligonucleotide composition according to claim 35, wherein the oligonucleotide has the structure of SEQ ID NO: 103 in the direction from 5' to 3'.

38. An oligonucleotide composition comprising a mixture of two or more oligonucleotides according to any one of claims 1 to 33.

39. A pharmaceutical composition comprising a therapeutically effective amount of an oligonucleotide composition according to any one of claims 1 to 33, and at least one pharmaceutically acceptable inactive component selected from a pharmaceutically acceptable diluent, a pharmaceutically acceptable excipient, and a pharmaceutically acceptable carrier.

40. A pharmaceutical composition for use in a method of treating a subject suffering from one or more characteristics of a neurodegenerative disease, comprising or delivering the composition according to any one of claims 1 to 33.

41. A pharmaceutical composition for use in a method of treating a subject suffering from one or more characteristics of neuromuscular dysfunction or muscular dystrophy, comprising or delivering the composition described in any one of claims 1 to 33.

42. A pharmaceutical composition for use in a method for treating myofibrosis, comprising or delivering the composition described in any one of claims 1 to 33.

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