Characterization of the binding interaction between MUSK and BMP receptors

Abstract

Provided herein are methods and compositions for increasing neurogenesis or muscle regeneration and preventing or treating diseases, disorders or conditions associated with neurodegeneration or muscle regeneration.

Description

[Technical field]

[0001] The present disclosure generally relates to drugs for nervous system disorders, such as nootropics, cognition-improving drugs, drugs for treating Alzheimer's disease or other forms of dementia, for treating neurodegenerative disorders of the central nervous system.The present disclosure relates to the regulation of the specific interaction between MuSK and BMP or BMP receptor I.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with Government support under Grant No. R21 NS112743 awarded by the National Institutes of Health. The Government has certain rights in this invention. [Background technology]

[0003] Bone morphogenetic proteins (BMPs) are well known to have important functions in every part of the human body. Wang et al.(2014). BMP signaling regulates various cellular events including neural and muscle stem cell activation, neuroplasticity, bone growth, sarcopenia, and tumorigenesis. Some BMP functions include bone and cartilage formation, vascular development, homeostasis, cancer transformation, and intestinal epithelial formation. There is still a question of how BMP signals are regulated in a tissue-specific manner. There are about 20 types of BMPs available for signaling. These 20 BMPs are thought to signal through the same three type 1 BMP receptors. Yadin, Knaus, & Muller(2016). How these signals and functions are regulated in a tissue-specific manner remains to be characterized.

[0004] BMP signaling has been difficult to exploit therapeutically because it is used in many situations, making targeting specificity difficult. BMP2 was developed by the Genetics Institute as a therapeutic promoter of bone growth. See, for example, Burkus et al., J.Spinal Disord.Tech., 15, 337 (2002). The use of BMP2 was limited due to undesirable side effects such as stimulation of tumor progression. See, for example, James et al., Tissue Eng.Part B,Rev.22, 284 (2016).

[0005] MuSK is a receptor tyrosine kinase. The ectodomain of MuSK contains three Ig-like domains and a CRD / Frizzled domain, whereas the intracellular domain contains a tyrosine kinase domain. See Hubbard & Gnanasambandan (2013). MuSK is expressed in skeletal muscle and other mammalian tissues. Garcia-Osta et al. (2006). MuSK plays a vital role in the formation of neuromuscular junctions. When MuSK is absent in mice, these mice are unable to form neuromuscular junctions. They die at birth. See Hubbard & Gnanasambandan (2013).

[0006] MuSK is a BMP coreceptor and binds to BMPs at the BMP Ig3 domain. An important subset of BMP signaling is mediated by the "MuSK-BMP pathway." See Yilmaz et al. (2016); Fish & Fallon (2020). MuSK not only binds to BMPs, but also to type 1 BMP receptors (BMPR1A and BMPR1B). Because MuSK is expressed only in a subset of tissues and cells, the MuSK-BMP pathway is inherently much more selective than general BMP signaling. MuSK binds to BMPs and type I BMP receptors BMPR1a, BMR1b and Acvr1b (also known as ALK 3, 6 and 4, respectively). ALK is an anaplastic lymphoma kinase. Engineering the MuSK Ig3 domain required for high affinity BMP4 binding promotes adult hippocampal neurogenesis and muscle regeneration. See WO 2020 / 214987.

[0007] However, there remains a need in the biomedical field for a better understanding of all MuSK interactions and binding partners. Successful development of drugs that target (e.g., modulate) the BMP pathway will be facilitated by an improved understanding of MuSK interactions and binding partners and the role they play in biological events. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] International Publication No. 2020 / 214987 [Non-patent literature]

[0009] [Non-Patent Document 1] Wang et al. (2014) [Non-Patent Document 2] Yadin, Knaus, & Muller (2016) [Non-Patent Document 3] Burkus et al., J. Spinal Disord. Tech., 15, 337 (2002) [Non-Patent Document 4] James et al.,Tissue Eng.Part B,Rev.22,284(2016) [Non-Patent Document 5] Hubbard & Gnanasambandan (2013) [Non-Patent Document 6] Garcia-Osta et al. (2006) [Non-Patent Document 7] Yilmaz et al. (2016) [Non-Patent Document 8] Fish & Fallon (2020) Summary of the Invention

[0010] [Problem to be solved by the invention] The present invention provides compositions and methods for selective manipulation of bone morphogenetic protein (BMP) pathway.The present invention is based on the observation that BMP receptor (ALK) binds to the Ig2 domain of muscle specific kinase (MuSK).The present specification identifies and characterizes the interaction between BMP receptor and BMP coreceptor muscle specific kinase (MuSK).

[0011] In one aspect, the present invention provides some elements to the observation that the BMP receptor (ALK) binds to the Ig2 domain of MuSK.

[0012] We identified a BMP receptor that binds MuSK. We mapped the MuSK domain required for binding to BMPR1. MuSK-BMPR1 binding may be an important regulatory mechanism for BMP signaling.

[0013] We have mapped the MuSK domains required for binding to BMP receptor (ALK). We have designed and generated various His-tagged deletion constructs of MuSK ectodomain and isolated domains. We have used solid-phase binding assays to quantify the binding of BMPR1A and BMPR1b to different MuSK constructs and generated binding curves for each construct.

[0014] BMPR1a is also known as ALK3, and BMPR1b is also known as ALK6. BMPR1a and BMPR1b are BMP receptors that bind to MuSK, but the binding domains were previously unknown. We identified a single domain (Ig2) required for MuSK-BMPR1 binding. This domain is necessary and sufficient for binding to BMPR1. Thus, this MuSK Ig2 domain is necessary and sufficient for binding to BMP receptors. We designed and expressed deletion constructs of MuSK. We then assayed the binding between these deletion constructs and ALK6 using an ELISA-like solid-phase binding assay. ΔIg2 MuSK showed little or no binding. This result indicates the requirement of the Ig2 domain for MuSK binding to ALK6.

[0015] We have identified where such binding occurs in human cells. We assayed MuSK binding to BMP receptors under various pH conditions. By assaying MuSK binding to BMP receptors under various pH conditions, we showed that MuSK binding to BMPR1 is pH dependent. This characterization of MuSK-BMPR1 interaction is useful to support therapeutic development programs targeting the MuSK-BMP pathway for Alzheimer's disease.

[0016] The present inventors have identified a previously unrecognized splicing variant of MuSK. The present inventors have also shown that alternative splicing of two "small" 30 bp exons (5a and 5b) can regulate both the level of BMP receptor binding and the selectivity of MuSK for different BMP receptors. The proteins encoded by these small exons, the Ig2 region, bind to MuSK. Manipulation of the small exons can increase the selectivity of therapeutic modulation of BMP signaling.

[0017] [Problem Solved] In some embodiments, the disclosure provides methods of treating Alzheimer's disease, muscle-specific kinase myasthenia gravis (MuSK-MG), and other neurological disorders based on modulation of the MuSK Ig2 domain.

[0018] In some embodiments, the present disclosure provides methods of treating muscle disorders (e.g., myocardial infarction, cardiomyopathy) or genetic diseases characterized by muscle wasting. In some embodiments, the subject being treated may be at risk for or suffering from a disease or disorder associated with lung injury, including idiopathic pulmonary fibrosis (IPF), acute respiratory distress syndrome (ARDS), pneumonia, or certain infections, including viral infections, including coronavirus infections such as COVID19.

[0019] Exemplary muscular disorders that can be treated by the techniques of the present invention include, but are not limited to, Becker muscular dystrophy, congenital muscular dystrophy, distal muscular dystrophy, Duchenne muscular dystrophy, Emery-Dreifuss muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, myotonic muscular dystrophy, and oculopharyngeal muscular dystrophy.

[0020] In some embodiments, the enhancement of muscle growth is used to treat diseases or disorders related to muscle atrophy or muscle wasting.Muscle atrophy or muscle wasting can be observed in various diseases and conditions described, such as neuromuscular disorders, or various diseases and conditions caused directly or indirectly by long-term 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).

[0021] In some embodiments, the methods of the invention may also be used when a subject requires muscle regeneration and enhanced muscle growth after surgery, trauma, or prolonged immobilization (e.g., from bed rest or casting). Because muscle stem cell activity declines with age, the methods of the invention may also prevent or reverse sarcopenia in otherwise healthy patients, resulting in significant improvements in quality of life and independence.

[0022] In some embodiments, the disclosure provides methods of treating Alzheimer's disease, MuSK-MG and other neurological or muscular disorders based on modulation of alternative splicing of the 5a and 5b MuSK exons.

[0023] In some embodiments, the disclosure provides a diagnosis of MuSK-MG based on autoantibody binding to the MuSK Ig2 domain.

[0024] In some embodiments, the disclosure provides a diagnosis of MuSK-MG based on autoantibody binding to MuSK exons 5a and 5b.

[0025] In some embodiments, the disclosure provides methods of treating sarcopenia, cachexia and other muscle disorders based on modulation of the MuSK Ig2 domain.

[0026] In some embodiments, the disclosure provides methods of treating sarcopenia, cachexia and other muscle disorders based on the regulation of MuSK exons 5a and 5b.

[0027] In some embodiments, the disclosure provides a method of treating a subject suffering from one or more characteristics of neurodegeneration, cognitive impairment, or muscular disorder, comprising: (a) increasing the level or activity of a MuSK polypeptide lacking a functional Ig2 domain, or (b) decreasing the level or activity of a BMP receptor (ALK)-MuSK polypeptide complex, wherein MuSK comprises a functional Ig2 domain.

[0028] In some embodiments, the disclosure provides a method for increasing neurogenesis, muscle regeneration and muscle growth comprising: (a) increasing the level or activity of a MuSK polypeptide that lacks a functional Ig2 domain; or (b) decreasing the level or activity of a BMP-MuSK polypeptide complex, wherein MuSK comprises a functional Ig2 domain.

[0029] In some embodiments, the method further comprises administering a pharmaceutical composition that contains or delivers a MuSK Ig2 modulator agent.

[0030] In some embodiments, the method further comprises administering a pharmaceutical composition that increases the altered splicing of MuSK transcripts, and the alteration comprises skipping one or more exons in MuSK Ig2 domain, and the skipped exon is exon 5a of MuSK Ig2 domain, or the skipped exon is exon 5b of MuSK Ig2 domain, or the skipped exon is exon 5a and 5b of MuSK Ig2 domain. MuSK 5a and 5b exons are suitable for modulation by both therapeutic antibodies and antisense oligonucleotides. In another embodiment, the modulation is by administration of antisense oligonucleotides to induce exon skipping.

[0031] In some embodiments, the disclosure provides methods for inducing MuSK exon skipping by contacting a system containing a population of MuSK primary transcripts with an oligonucleotide that binds to such primary transcripts, such that skipping of one or both of exons 5a and 5b is increased.

[0032] In some embodiments, the disclosure provides methods for characterizing a MuSK Ig2 modulator agent comprising one or more of the following steps: (a) assessing the ability to decrease MuSK-Ig2-BMP receptor (ALK) complex formation; (b) assessing the ability to alter the splicing pattern of a primary MuSK transcript; (c) assessing the ability to inhibit expression of the transcript; (d) assessing the ability to increase expression of a MuSK transcript lacking a sequence encoding an Ig2 domain; (e) assessing the ability to increase levels of a MuSK polypeptide lacking functional Ig2; and (f) assessing the ability to affect a property of a cell in a population.

[0033] In some embodiments, the disclosure provides a genetically modified mouse comprising a sequence encoding MuSK within its genome, wherein the sequence encoding MuSK does not include nucleotides within the region from exon 5a to exon 5b (in 5' to 3' order), and wherein the genetically modified mouse is incapable of expressing a full-length MuSK transcript or producing a full-length MuSK protein.

[0034] For purposes of illustration, certain embodiments of the invention are shown in the drawings described below. Like reference numerals in the drawings refer to like elements throughout. The invention is not limited to the precise arrangements, dimensions and equipment shown. [Brief description of the drawings]

[0035] [Figure 1] FIG. 1 is a schematic diagram showing the process of BMP binding within cells. [Diagram 2]

[0023] Figure 1 is a schematic diagram showing the full-length ectodomain and endodomain of MuSK. The ectodomain of MuSK contains three Ig-like domains and a cysteine-rich CRD / Frizzled domain, whereas the intracellular domain contains a tyrosine kinase domain. [Diagram 3] FIG. 1 is a schematic diagram showing the deletion constructs we generated. [Figure 4] Electrophoretic gel confirming the presence of full length, ΔIg1, ΔIg2 and ΔIg1Ig2 constructs. [Diagram 5] Electrophoresis gel confirming the Ig1, Ig2, Ig1-Ig2 and ΔCRD / Fz constructs. [Figure 6] Electrophoresis gel confirming the presence and expected sizes of the CRD / Fz, Δ5a5a′ and L83R constructs. [Figure 7] 1 is a line graph showing data from a solid phase binding assay in which MuSK was immobilized and incubated with ALK6. [Figure 8] 1 is a line graph showing that the Ig1-Ig2 construct has high affinity binding to ALK6, with the Ig2 domain required for binding, and that both the Ig1 and Ig2 domains are required for MuSK to bind ALK6. [Figure 9] 1 is a line graph using isolated Ig2 and Ig1 domains showing that the Ig2 domain is sufficient to bind ALK6. [Figure 10] 1 is a line graph showing that MuSK binds strongly and with high affinity to ALK6. Comparison of the ΔIg1 and ΔIg2 curves indicated that the Ig1 domain is not required for MuSK binding to ALK6. [Figure 11] FIG. 1 is a schematic diagram showing an Ig1-Ig2 only construct MuSK ectodomain in which only the Ig1 and Ig2 domains are present and His-tagged for purification. [Figure 12]1 is a line graph showing that MuSK binds significantly to ALK6 under mildly acidic conditions using a solid-phase binding assay. The interaction between MuSK and ALK6 at pH 6 is highly favorable in this condition compared to pH 7.0. [Figure 13] 1 is a line graph showing that MuSK does not exhibit pH dependence when the Ig1 domain is deleted, suggesting that the Ig1 domain is the reason MuSK binds differently to ALK6 at pH 6 and pH 7. [Figure 14] Schematic model of MuSK-ALK6 binding at the cell surface (pH 7.4) and endosomal compartments (pH 6.0). The first top left panel shows MuSK as a dimer and the BMP receptor canonically bound to BMP. An intracellular event then triggers clathrin-mediated endocytosis (top right panel). Accumulation of PIP2 and adaptor proteins is required for cells to initiate clathrin-mediated endocytosis. Clathrin-mediated endocytosis leads to the formation of endosomes (bottom right panel). Endosomes have a slightly acidic pH compared to the extracellular environment of the cell. BMP receptor type 1 promotes binding of MuSK in these conditions (bottom left panel). As the pH drops, BMP dissociates from the BMP receptor and binds to the BMP receptor. [Figure 15] Electrophoresis gel showing detection of novel small exons (5a and 5b) in MuSK, thus demonstrating alternative splicing within the MuSK region containing alternatively spliced ​​exons 5a and 5b. PCR of muscle (center) and satellite cell (right) cDNA using primers (forward, exon 5; reverse exon 8). Note that three forms are detected (5a only; 5a+5b; no small exon). [Figure 16] The amino acid sequence of MuSK, containing alternatively spliced ​​exons 5a and 5b, is provided. [Figure 17] 1 shows the alternative splicing patterns of exons 5a and 5b from mouse soleus muscle. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036] Industrial Applicability More than 20 BMPs signal through only seven BMP receptors. The functions of BMPs are well defined. So how can the same combination of BMP and BMP receptor lead to so many different functions? Such questions make it interesting for researchers to identify all the coreceptors and regulators of the BMP signaling pathway.

[0037] MuSK. MuSK is a receptor tyrosine kinase composed extracellularly of three Ig and one CRD / Fz domains and an intracellular tyrosine domain (TK; Figure 1). The best understood function of MuSK is at the neuromuscular junction (NMJ) where binding of agrin-LRP4 to the Ig1 domain induces MuSK TK activity and synaptic differentiation (Kim et al., 2008; Zhang et al., 2008a).

[0038] MuSK-BMP pathway. The brain has 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) the quiescent state where proliferating stem cells exit the cell cycle and return to replenish the reserve pool that can supply 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 regulating neurogenesis in the adult brain.

[0039] MuSK is also a BMP coreceptor that binds to BMP and its receptors ALK3, 4 and 6, upregulating BMP signaling and shaping the composition of transcriptional responses in myogenic cells. See Yilmaz et al., Sci. Signal., 9, ra87 (2016). This BMP signaling pathway does not regulate or require MuSK TK activity, nor is it activated by agrin-LRP4. The MuSK Ig3 domain is required for high affinity BMP binding, but is not critical for agrin-LRP4 TK activation. The Ig3 domain is endogenously alternatively spliced, including in the brain. Garcia-Osta et al. (2006); Hesser et al. (1999). BMP signaling can induce neural stem cell (NSC) quiescence and inhibit the integration of newborn neurons. We found that suppressing BMP drive by decreasing MuSK-BMP signaling can increase neurogenesis.

[0040] Yilmaz et al., Sci. Signal., 9, ra87 (2016) disclose 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 called Δg3MuSK. This splicing involves the coordinated removal of exons 6 and 7 from the MuSK pre-mRNA. Modulation of MuSK alternative splicing is a strategy to increase neurogenesis in Alzheimer's disease. See WO 2020 / 214987 (Brown University). MuSK is expressed in muscle and upregulated during muscle regeneration. Data suggest that MuSK is involved in BMP signaling during myogenesis. MuSK can act as a BMP coreceptor that binds BMP2, BMP4 and BMP7 as well as type I BMP receptors ALK3 and ALK6. See, for example, Yilmaz et al., Sci. Signal., 9, ra87 (2016), which is incorporated herein by reference. The Ig3 domain of MuSK is required for high affinity binding to BMP. MuSK upregulates BMP signaling as measured by BMP4-dependent phosphorylation of SMAD1 / 5 / 8. MuSK-BMP signaling shapes the size and composition of the BMP-induced transcriptome in myoblasts and myotubes, a role that is independent of 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.

[0041] Activated satellite cells express MuSK protein, and disruption of MuSK-BMP signaling alters satellite cell proliferation in regenerating muscle in vivo. The included data provide information about the role of the MuSK-BMP pathway in satellite cells and muscle regeneration. These data suggest that the MuSK-BMP pathway plays an important role, and the present disclosure provides insight that targeting the MuSK-BMP pathway is more selective than targeting BMP-wide activity, at least because MuSK expression is much more restricted than that of the BMP system. The provided data also teach that targeting the MuSK-BMP pathway enhances muscle growth. In some embodiments, muscle growth occurs, for example, in undamaged tissue.

[0042] The MuSK ectodomain is exposed on the surface of the cell and is therefore suitable for modulation by therapeutic antibodies or other agents acting extracellularly.

[0043] The MuSK Ig2 domain and two small exons have emerged as targets for modulating the MuSK-BMP pathway to treat disorders such as Alzheimer's disease, stroke, PTSD and treatment-resistant depression.

[0044] MuSK Ig2 domain may also be the target of autoantibodies that mediate MuSK-dependent myasthenia gravis. Myasthenia gravis (MG) is a chronic autoimmune neuromuscular disease characterized by skeletal muscle weakness. Jayam Trouth et al. (2012). Clinical studies have shown that myasthenia gravis patients may occasionally have antibodies against MuSK. Guptill, Sanders, & Evoli A (2011). These anti-MuSK antibodies are present in many patients in whom antibodies against acetylcholine receptors are absent. Guptill, Sanders, & Evoli A (2011). MuSK-dependent myasthenia gravis (MuSK-MG) may be caused by autoantibodies against MuSK Ig1 domain, which inhibit agrin-LRP4 signaling. The present disclosure teaches that the autoantibodies present in these patients are against MuSK Ig2 domain and may inhibit MuSK-BMP signaling, and thus may be responsible for the muscle atrophy observed in these patients. The findings provided by the present disclosure have potential value for both diagnosis and treatment.For example, disrupting the interaction between such autoantibodies and MuSK Ig2 may be an effective therapeutic strategy.Detecting autoantibodies that bind to MuSK Ig2 may be useful for diagnosing myasthenia gravis.

[0045] definition For convenience, the meanings of some terms and phrases used in the specification, examples, and the appended claims are listed below. Unless otherwise indicated or implied from the context, these terms and phrases shall have the following meanings. These definitions are useful for describing certain embodiments, but are not intended to limit the claimed invention. Unless otherwise defined, all technical and scientific terms have the same meaning as commonly understood by those skilled in the art to which the invention belongs. In the event of an apparent discrepancy between the meaning of the definitions provided herein and the use of the term in the biomedical field, the meaning of the term provided herein shall prevail.

[0046] About, when used in reference to a value, refers to a similar value relative to the referenced value. Generally, one of ordinary skill in the biomedical arts will understand the degree of dispersion encompassed by about in that context.

[0047] Administration refers to the administration of a pharmaceutical composition to a subject or system to achieve delivery of an agent, e.g., an agonizing agent, that is, or is contained in, or otherwise delivered by the composition. Those skilled in the art of biomedical sciences are aware of the various routes that may be used for administration to a subject, e.g., a human. Administration may be or include one or more of ophthalmic, oral, buccal, topical to the dermis, intradermal, interdermal, transdermal, etc., enteral, intraarterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, into a specific organ, e.g., into the liver, intramucosal, intranasal, oral, intrarectal, subcutaneous, sublingual, topical, e.g., into the trachea by intratracheal instillation, vaginal, vitreous, etc. The agent, e.g., an agonizing agent, is delivered to the central nervous system (CNS), e.g., via intraventricular administration. Administration may involve only a single dose. Administration can involve application of a constant dose. Administration can involve intermittent administration, e.g., multiple doses separated by time or periodic administration, e.g., individual doses separated by administration of a common period. Administration can involve continuous administration, e.g., perfusion over at least a selected period of time.

[0048] Agent is used by those skilled in the biomedical field to refer to any chemical class of compound or entity, including polypeptides, nucleic acids, sugars, lipids, small molecules, metals, or combinations or complexes thereof. Those skilled in the biomedical field know that the term agent refers to an entity that is or contains a cell or organism, or a fraction, extract, or component thereof. The term can refer to a natural product, since it is found in nature or obtained from nature. The term can refer to one or more entities that are artificial, in that they are designed, engineered, or produced by the action of the human hand, or are not found in nature. Agents can be used in isolated or pure form. Agents can be used in crude form. Potential agents can be provided as collections or libraries that can be screened to identify or characterize the active agents therein. The term agent can refer to a compound or entity that is or contains a polymer. The term can refer to a compound or entity that contains one or more polymeric moieties. The term agent can refer to a compound or entity that is not a polymer, or that is substantially free of any polymer or one or more specific polymeric moieties. The term may refer to a compound or entity that is devoid of or substantially free of any polymeric moieties.

[0049] Agonist is used by those skilled in the biomedical field to refer to an agent (i.e., agonizing agent), condition, or event whose presence, level, degree, type, or form correlates with an increase in the activity level of another agent, i.e., an agonized agent or target agent. Agonists can be or include any chemical class of agent, including small molecules, polypeptides, nucleic acids, carbohydrates, lipids, metals, or any other entity that exhibits activation activity. Agonists can be direct (in which case, the agonist directly affects its target). An agonist can be indirect (in which case the agonist exerts its effect other than by binding to its target, e.g., by interacting with a regulator of the target, such that the level or activity of the target is altered). 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, such that the level, morphology or activity of the target is altered. The altered level, morphology or activity is an increased level of an altered protein expressed from the target nucleic acid sequence. One of ordinary skill in the biomedical arts will appreciate that an agonizing agent can bind to a binding target and can induce binding. Knowing that it can potentially agonize a target, and its binding causes an increase in the level or activity of the further agonized target.Agonizing agents that bind to nucleic acid targets can change the level or activity of the target.Agonizing agents can agonize the activity of the nucleic acid target, for example, by increasing its modification, splicing, 5' cap formation or 3' end formation, transport or translation, etc., so that the level of desired products, for example, mRNA, is increased, or downstream targets, such as the polypeptides encoded by such nucleic acid targets, can be agonized.An agonizing agent may be or may include an oligonucleotide that can bind to a primary transcript and alter its splicing pattern to increase the level or activity of a particular spliced ​​form, e.g., mature mRNA, thereby achieving an increase in the level of a product, e.g., a polypeptide that is or is encoded by such a particular spliced ​​form.

[0050] Agonist therapy is used by those skilled in the biomedical field to refer to the administration of an agonist that agonizes a specific target of interest to achieve a desired therapeutic effect. Agonist therapy involves administering a single dose of agonist. Agonist therapy involves administering several doses of agonist. Agonist therapy involves administering an agonist according to a dosing regimen that is known or predicted to achieve a therapeutic effect because such results have been established to a specified degree of statistical confidence, for example, by administration to a relevant population. Agonist therapy involves the delivery of an agonizing agent as described herein. Agonizing agents can be or include binding agents that are proteins, for example, antibodies or nucleic acids, for example, antisense oligonucleotides, that bind to targets, for example, proteins or nucleic acids, such that the level, form or activity of the target is changed. Agonizing agents can bind to binding targets and potentially agonize the binding targets, which binding causes an increase in the level or activity of the further agonized targets. Agonizing agents that bind to nucleic acid targets can change the level or activity of the target.Agonizing agents can agonize the activity of the nucleic acid targets, for example, by increasing its modification, splicing, 5' cap formation or 3' end formation, transport or translation, etc., resulting in the desired product, for example, the level of mRNA, or agonizing downstream targets, such as the polypeptides encoded by such nucleic acid targets.Agonizing agents can be or include oligonucleotides that can bind to primary transcripts and change their splicing patterns to result in specific spliced ​​forms, for example, mature mRNA levels or activities, thereby achieving an increase in the level of products, for example, the polypeptides that are or are encoded by such specific spliced ​​forms.

[0051] Antagonist is used by those skilled in the biomedical arts to refer to an agent (i.e., 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 inhibited agent, or target). Antagonists can be or include any chemical class of agent, including small molecules, polypeptides, nucleic acids, carbohydrates, lipids, metals, or any other entity that exhibits relevant inhibitory activity. Antagonists can be direct (where the antagonist directly affects its target). Antagonists can be indirect (where the antagonist exerts its effect other than by binding to its target, e.g., by interacting with a regulator of the target, such that the level or activity of the target is altered). Antagonists are binding agents that are proteins, e.g., antibodies, or nucleic acids, e.g., antisense oligonucleotides, that bind to a target, e.g., a protein or nucleic acid, such that the level, form, or activity of the target is altered. The altered level, form, or activity can be a decrease in the level of an altered protein expressed from a target nucleic acid sequence. Those skilled in the art of biomedical sciences know that antagonists can bind to and potentially antagonize binding targets, and that the binding causes a decrease in the level or activity of further antagonized targets. Antagonists that bind to nucleic acid targets can change the level or activity of the targets. Antagonists can antagonize the activity of the nucleic acid targets, for example, by reducing their modification, splicing, 5'-capping or 3'-end formation, transport or translation, etc., so that the level of undesirable products, for example, mRNA, is suppressed, or downstream targets, such as polypeptides, encoded by such nucleic acid targets, can be antagonized.The antagonist may be or may include an oligonucleotide that can bind to the primary transcript and alter its splicing pattern to suppress the level or activity of a particular spliced ​​form, e.g., mature mRNA, thereby achieving a reduction in the level of a product, e.g., a polypeptide that is or is encoded by such a particular spliced ​​form.

[0052] An antibody agent is used by those skilled in the biomedical field to refer to an agent that specifically binds to a particular antigen, for example, may be or may contain an epitope of a protein of interest, for example, MuSK protein. The term encompasses any polypeptide or polypeptide complex that contains sufficient immunoglobulin structural elements to confer specific binding. Exemplary antibody agents include monoclonal or polyclonal antibodies. An antibody agent may include one or more constant region sequences characteristic of mouse, rabbit, primate or human antibodies. An antibody agent may include one or more sequence elements that are humanized, primatized, chimeric, etc., as known in the biomedical field. In alternative presentations, the term antibody agent may refer to one or more of the constructs or formats known or developed by the biomedical field to utilize the structural and functional features of antibodies.Antibody agents for use under the present invention include, but are not limited to, intact IgA, IgG, IgE or IgM antibodies; bispecific or multispecific antibodies, such as 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 Fv; polypeptide-Fc fusions; single domain antibodies, such as shark single domain antibodies or fragments thereof, such as IgNAR; camelid antibodies; masked antibodies, such as Probodies®; Small Modular ImmunoPharmaceuticals (SMIPs™); single chain or tandem diabodies (TandAb®); VHH; Anticalins®; Nanobodies® minibodies; BiTE®; ankyrin repeat proteins or DARPINs®; Avimers®; DARTs; TCR-like antibodies;, Adnectins®; Affilins®; Trans-bodies®; Affibodies®; TrimerX®; MicroProteins; Fynomers®, Centyrins®; and KALBITOR®. The antibody may lack a covalent modification, e.g., the attachment of a glycan, which it would have if it were produced naturally. The antibody may contain a covalent modification, e.g., the attachment of a glycan, a payload, e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc., or other pendant group, e.g., polyethylene glycol, etc. An antibody agent may be or may include a polypeptide whose amino acid sequence includes one or more structural elements recognized by those skilled in the biomedical arts as complementarity determining regions (CDRs). An antibody agent may be or may include a polypeptide whose amino acid sequence includes at least one CDR substantially identical to that found in a reference antibody, e.g., at least one heavy chain CDR or at least one light chain CDR.The included CDRs may be substantially identical to the reference CDRs because they are sequence identical or contain 1-5 amino acid substitutions compared to the reference CDRs. The included CDRs may be substantially identical to the reference CDRs because they show at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100% sequence identity with the reference CDRs. The included CDRs may be substantially identical to the reference CDRs because they show at least 96%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100% sequence identity with the reference CDRs. The included CDRs may be substantially identical to the reference CDRs in that at least one amino acid in the included CDRs has been deleted, added, or substituted compared to the reference CDR, but the included CDRs have an amino acid sequence that is otherwise identical to the amino acid sequence of the reference CDR. The included CDRs may be substantially identical to the reference CDRs in that 1-5 amino acids in the included CDRs have been deleted, added, or substituted compared to the reference CDR, but the included CDRs have an amino acid sequence that is otherwise identical to the amino acid sequence of the reference CDR. The included CDRs may be substantially identical to the reference CDRs in that at least one amino acid in the included CDRs has been substituted compared to the reference CDR, but the included CDRs have an amino acid sequence that is otherwise identical to the amino acid sequence of the reference CDR. The included CDRs may be substantially identical to the reference CDRs in that one to five amino acids within the included CDRs have been deleted, added, or substituted compared to the reference CDR, but the included CDRs have an amino acid sequence that is otherwise identical to the reference CDR.An antibody agent is, or comprises, a polypeptide whose amino acid sequence includes structural elements recognized by those skilled in the biomedical arts as an immunoglobulin variable domain.An antibody agent is a polypeptide protein having a binding domain which is homologous or largely homologous to an immunoglobulin binding domain.

[0053] Antibody is used by those skilled in the biomedical field to refer to an immunoglobulin or derivative thereof that contains an immunoglobulin domain capable of binding to an antigen, e.g., may be or may contain an epitope of a protein of interest, e.g., MuSK protein. Antibodies can be of any species, e.g., human, rodent, rabbit, goat, chicken, etc. Antibodies can be members of any immunoglobulin class, including the human classes: IgG, IgM, IgA, IgD and IgE, or any of its subclasses, e.g., IgG1, IgG2, etc. Antibodies can be fragments such as Fab', F(ab')2, scFv (single chain variable), or other fragments that retain the antigen binding site, or recombinantly produced scFv fragments, including recombinantly produced fragments. See, e.g., Allen (2002) and references therein. Antibodies can be monovalent, bivalent or multivalent. The antibody may be a chimeric or humanized antibody in which a variable domain of rodent origin is fused to a constant domain of human origin, thus retaining the specificity of rodent antibodies. The domain of human origin need not be directly derived from humans, in the sense that it is first synthesized in humans. Instead, the human domain may be generated in rodents whose genome incorporates human immunoglobulin genes. See, for example, Vaughan et al. (1998). The antibody may be partially or fully humanized. The antibody may be polyclonal or monoclonal, although in 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 biomedical arts. Monoclonal or polyclonal antibodies may be purified from the blood or ascites of an animal that produces the antibody, for example, after natural exposure to or immunization with the molecule or an antigenic fragment thereof, or may be produced using recombinant techniques on cell culture or transgenic organisms, or may be made by chemical synthesis. Antibodies can act as antagonists, for example, by binding to a target antigen, reducing the level or activity of the antigen.Antibodies can act as agonists, for example, by binding to a target antigen, resulting in increased levels or activity of the antigen.

[0054] Antisense is used by those skilled in the biomedical field to refer to a nucleic acid whose nucleotide sequence is complementary to part or all of a sequence found in a coding strand nucleic acid. Typically, a coding strand nucleic acid is one whose sequence includes part or all of an open reading frame or other stretch of residues that encodes part or all of a polypeptide. The term antisense may specifically refer to an oligonucleotide that specifically binds to a coding strand (i.e., to a target sequence within such a coding strand). A coding strand may include both coding and non-coding sequences, and may be, for example, a transcript, such as a primary transcript, that includes both intronic and exonic sequences, to name just a few. Those skilled in the biomedical field will know upon reading this disclosure that an oligonucleotide may be considered or referred to as an antisense oligonucleotide if part or all of its sequence is complementary to a non-coding portion of its target strand. Antisense oligonucleotides bind to coding sequences within a target sense strand. Antisense oligonucleotides bind to non-coding sequences in the target coding strand. Antisense oligonucleotides bind to both coding and non-coding sequences in the target coding strand. Antisense oligonucleotides, when bound to their target sequences in coding strands, such as transcripts, are characterized by altering one or more of the following post-transcriptional processing, such as modification, splicing, 5' capping or 3' end formation, 5' capping or 3' end formation, transport or translation of such coding strands. Antisense oligonucleotides can alter the splicing of their target coding strands, and the antisense coding strand complex is degraded or can be degraded, for example, by RNase H.

[0055] Approximately or about, when used in reference to a number, includes numbers that fall within a range of 5%, 10%, 15% or 20% in either direction (greater or less than that number unless otherwise indicated or clear from the context).

[0056] Binder is used by those skilled in the biomedical field to refer to any entity that binds to a target of interest as described herein. A binder of interest can specifically bind to its target in a particular interaction context to distinguish it from other potential binding partners. A binder can be or include any chemical class of entity, such as polymeric, non-polymeric, small molecule, polypeptide, carbohydrate, lipid, nucleic acid, etc. A binder is a single chemical entity. A binder is a complex of two or more distinct chemical entities that associate with each other under conditions by non-covalent interactions. Those skilled in the biomedical field know that a binder can include a general binding moiety, such as one of biotin / avidin / streptavidin or class-specific antibodies, and a specific binding moiety, such as an antibody or aptamer with a specific molecular target bound to the general binding moiety partner. Such an approach can allow for modular assembly of several binders through binding of different specific binding moieties to the same general binding moiety partner. A binder can be or include, for example, a polypeptide, including an antibody or antibody fragment. The binding agent is or includes a small molecule. The binding agent is or includes a nucleic acid, e.g., an antisense oligonucleotide. The binding agent is an aptamer. The binding agent is a polymer. The binding agent is not a polymer. The binding agent is non-polymeric, since it lacks a polymeric portion. The binding agent is or includes a carbohydrate. The binding agent is or includes a lectin. The binding agent is or includes a peptidomimetic. The binding agent is or includes a scaffold protein. The binding agent is or includes a mimeotope. The binding agent can be or include a stapled peptide. The binding agent can be or include a nucleic acid, such as DNA or RNA, e.g., an antisense oligonucleotide.

[0057] BMP receptors are known to those skilled in the biomedical field. BMP receptors (BMPRs) are serine / threonine kinase receptors composed of a short extracellular domain with 10-12 cysteine ​​residues, a single transmembrane domain, and an intracellular serine / threonine kinase domain. Bragdon et al. (2011) BMP receptors have two types, namely, BMP receptor type 1 (BMPR1) and BMP receptor type 2 (BMPR2). See Sanchez-Duffhues et al. (2020). The number of BMP receptors is limited. Researchers have identified five BMP type I receptors (BRI), namely, ALK1 (Acvrl1), ALK2 (ActRI), ALK3 (BRIa), ALK4 (ActRIb) and ALK6 (BRIb), and three type II receptors, namely, BRII, ActRIIa and ActRIIb. See Bragdon et al. (2011). BMPs bind to a combination of BMP receptors type 1 and type 2. BMPs have canonical and non-canonical signaling pathways. In the canonical pathway, BMPs initiate signaling by interacting with BMP receptors to form a heterotetrameric complex composed of two dimers, type I and type II. See Wang et al. (2014). After this complex is formed, the type II receptor phosphorylates the type I receptor in the GS domain. After the type I receptor is phosphorylated, the type I receptor can phosphorylate downstream SMAD molecules known as R-SMADs. The R-SMADs associated with BMP signaling are Smad1, Smad5 and Smad8. Wang et al. (2014). These Smads then bind to Smad4 and the entire complex is translocated into the nucleus. Wang et al. (2014). Smad complexes act as transcription factors that can activate or inactivate gene regulation Wang et al. (2014). Figure 1 shows the process of BMP binding.

[0058] Bone morphogenetic proteins (BMPs) are known to those skilled in the biomedical arts as essential molecules in the mammalian body, including the human body. BMPs are a group of signaling molecules that belong to the transforming growth factor beta (TGF-β) superfamily of proteins. See Bragdon et al. (2011). Over 20 BMPs have been discovered, making BMPS a member of the transforming growth factor beta (TGF-β) superfamily of proteins. See Bragdon et al. (2011). BMPs were first identified as molecules that support bone development, but BMPs have later been shown to play a role in many developmental functions of the body. See Wang et al. (2014).

[0059] Characteristic sequence elements are used by those skilled in the biomedical field to refer to sequence elements found in a polymer, e.g., a polypeptide or a nucleic acid, that are characteristic parts of the polymer. The presence of a characteristic sequence element correlates with the presence or level of a particular activity or property of the polymer. The presence or absence of a characteristic sequence element defines a particular polymer as a member of a particular family or group of such polymers, or as not being a member of a particular family or group of such polymers. A characteristic sequence element typically comprises at least two monomers, e.g., amino acids or nucleotides. A characteristic sequence element comprises adjacently linked monomers. A characteristic sequence element comprises at least a first and a second stretch of consecutive monomers separated by one or more spacer regions, the length of which may vary across the polymers that share the sequence element.

[0060] Complementary is used by those skilled in the biomedical arts to refer to the ability to form precise pairing between specific bases, nucleosides, nucleotides or nucleic acids. Adenine (A) and uridine (U) are complementary, adenine (A) and thymidine (T) are complementary, and guanine (G) and cytosine (C) are complementary, referred to in the biomedical arts as Watson-Crick base pairing. When the strands are aligned in antiparallel orientation, 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, the nucleotides (nt) form complementary base pairs and the nucleic acids are complementary at that position. The percent complementarity of a first nucleic acid to a second nucleic acid can be evaluated by aligning them in antiparallel orientation for maximum complementarity over an evaluation window, determining the total number of nts of both strands that form complementary base pairs within the window, dividing by the total number of nucleotides in the window, and multiplying by 100. AAAAAAAA and TTTGTTAT are 75% complementary because 12 of the 16 nucleotides are in complementary base pairs. When calculating the number of complementary nts required to achieve a particular % complementarity, round the fraction to the nearest whole number. A position occupied by a non-complementary nucleotide constitutes a mismatch, i.e., the position is occupied by a non-complementary base pair. The evaluation window may have a length as described herein for the duplex or the 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 are fully complementary (having 100% complementarity) with respect to each other. Nucleic acids that are at least 70% complementary over an evaluation window are considered to be substantially complementary over that window. Complementary nucleic acids may be at least 75%, at least 80%, at least 85%, at least 90% or at least 95% complementary over an evaluation window.When a first sequence is referred to as substantially complementary with respect to a second sequence as described herein, the two sequences may be completely complementary, or they may contain one or more mismatched bases upon hybridization, for example, up to about 5%, up to about 10%, up to about 15%, up to about 20% or up to about 25% mismatched bases upon hybridization, for example, 1, 2, 3, 4, 5 or 6 mismatched bases upon hybridization to a duplex of up to 30 base pairs, but retain the ability to hybridize under the conditions most relevant to their intended use.When two oligonucleotides form one or more single-stranded overhangs upon hybridization, such overhangs are not considered as mismatched or unpaired nucleotides for determining the percentage of complementarity. A dsRNA duplex containing one oligonucleotide 21 nucleotides long and another 23 nucleotides long, where the longer oligonucleotide contains a 21 nucleotide sequence that is completely complementary to the shorter oligonucleotide and a two nucleotide overhang, can be called fully complementary. Complementary sequences can contain one or more non-Watson-Crick base pairs or base pairs formed from non-natural and other modified nucleotides, as long as the requirements for hybridization are met. Such non-Watson-Crick base pairs include G:U Wobble base pairing or Hoogsteen base pairing. Those skilled in the biomedical field know that guanine, cytosine, adenine and uracil can be substituted by other bases according to the wobble rules without substantially changing the base pairing properties of polynucleotides containing nucleotides with such bases. See, for example, Murphy & Ramakrishnan (2004). Nucleotides that contain inosine as their base can base pair with nucleotides that contain adenine, cytosine, or uracil. Nucleotides that contain uracil, guanine, or adenine can be substituted, for example, by nucleotides that contain inosine in the nucleotide sequences of the inhibitory RNAs described herein.The terms complementary, completely complementary and substantially complementary can be used for the base matching between any two nucleic acids, for example, the base matching between the sense strand and antisense strand of double-stranded nucleic acid or its part.Those skilled in the biomedical field know that the term hybridize refers to the interaction between two nucleic acid sequences, and the two nucleic acid sequences can be part of the same nucleic acid molecule, or can be part of or comprise different nucleic acid molecules that contain or consist of complementary parts, so that stable duplex structure, i.e., intramolecular duplex or intermolecular duplex, is formed under the specific condition of interest.

[0061] Combination therapy is used by those skilled in the biomedical field to refer to the situation where a subject is exposed to two or more treatment regimens, for example, two or more therapeutic agents, at the same time. Two or more regimens can be administered simultaneously. Such regimens can be administered consecutively, for example, the entire dose of the first regimen is administered before any dose of the second regimen is administered. Such agents are administered in overlapping administration regimens. The administration of combination therapy can involve the administration of one or more agents or modalities to a subject that is administered the other agent or modality in combination. To clarify, combination therapy does not require that each agent is administered together in a single composition or necessarily at the same time, but two or more agents or their active parts can be administered together in a combination composition or even as a combination compound, for example, as part of a single chemical complex or covalent entity.

[0062] Equivalent is used by those skilled in the biomedical field to refer to two or more agents, entities, situations, sets of conditions, etc. that may not be identical to each other, but are sufficiently similar to allow comparisons between them, so that those skilled in the biomedical field know that conclusions can be reasonably drawn based on observed differences or similarities. Equivalent sets of conditions, situations, individuals, or populations are characterized by multiple substantially identical characteristics and one or a few altered characteristics. Those skilled in the biomedical field know what identity is required in any given situation in order for two or more such agents, entities, situations, sets of conditions, etc. to be considered equivalent, depending on the situation. Those skilled in the biomedical field know that sets of situations, individuals, or populations are equivalent to each other if they are characterized by a sufficient number and type of substantially identical characteristics to warrant a reasonable conclusion that differences in the results or phenomena obtained under or in different sets of situations, individuals, or populations are caused by or indicate changes in those characteristics that have been altered.

[0063] "Comprises" and "comprising" refer to elements, components, or steps in a non-exclusive manner, indicating that the referenced element, component, or step may be present, used, or combined with other elements, components, or steps. The singular terms a, an, and the include plural referents unless the context dictates otherwise. Similarly, the inclusive term or should encompass the terms and, unless the context dictates otherwise. The abbreviation eg means a non-limiting example and is synonymous with the term for example.

[0064] Domain is used by those skilled in the biomedical field to refer to a part or portion of an entity. A domain is associated with a particular structural or functional feature of an entity such that when the domain is physically separated from the rest of its parent entity, it substantially or completely retains the particular structural or functional feature. A domain may be or may comprise a part of an entity that, when separated from its (parent) entity and combined with a different (recipient) entity, substantially retains or imparts to the recipient entity one or more structural or functional features that characterized it in the parent entity. A domain is a part or portion of a molecule, such as a small molecule, carbohydrate, lipid, nucleic acid, or polypeptide. A domain is a part of a polypeptide, such as the Ig2 domain of the MuSK protein. A domain may be characterized by a particular structural element, such as a particular amino acid sequence or sequence motif, alpha-helical properties, beta-sheet properties, coiled coil properties, random coil properties, etc., or a particular functional feature, such as binding activity, enzymatic activity, folding activity, signaling activity, etc.

[0065] A dosing regimen is used by those skilled in the biomedical field to refer to a series of unit doses (typically multiple) that are administered individually to a subject and are typically separated by a period of time. A given therapeutic agent has a recommended dosing regimen that may involve one or more doses. A dosing regimen includes multiple doses, each of which is separated in time from the other doses. The individual doses are separated from each other by periods of the same length. A dosing regimen includes multiple doses and at least two different periods that separate the individual doses. All doses within a dosing regimen are the same unit dose amount. Different doses within a dosing regimen are different amounts. A dosing regimen includes a first dose of a first dose amount, followed by one or more additional doses of a second dose amount that is different from the first dose amount. A dosing regimen includes a first dose of a first dose amount, followed by one or more additional doses of a second dose amount that is the same as the first dose amount. A dosing regimen correlates with a desired or beneficial outcome when administered across a relevant population, i.e., it is a therapeutic dosing regimen.

[0066] Engineered refers to the aspect of being manipulated by the hand of man. An antibody, antibody reagent, its antigen-binding portion, CAR or bispecific antibody is considered to be engineered when the sequence of the antibody, antibody reagent, its antigen-binding portion, CAR or bispecific antibody is manipulated by the hand of man so that it differs from the sequence of an antibody that exists in nature. In the biomedical field, the descendants and copies of engineered polynucleotides or polypeptides are typically still referred to as engineered, even if the actual manipulation was performed on the previous entity.

[0067] Expression of a nucleic acid sequence is used by those skilled in the biomedical arts to refer to one or more of these events: (1) production of an RNA template from a DNA sequence, e.g., by transcription; (2) processing of the RNA transcript, e.g., by splicing, editing, 5' capping or 3' end formation; (3) transport of the RNA transcript, e.g., from the nucleus to the cytoplasm; or (4) translation of the RNA into a polypeptide or protein; or (4) post-translational modification of a polypeptide or protein.

[0068] A fragment of a material or entity is used by those skilled in the biomedical arts to refer to a structure that contains distinct parts of the whole but lacks one or more parts found in the whole. A fragment consists of such distinct parts. A fragment consists of or comprises characteristic structural elements or parts found in the whole. A polymeric fragment comprises or consists of monomeric units, i.e., residues, found in the whole polymer. A polymeric fragment comprises or consists of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 25%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more of the monomeric units, i.e., residues, found in the whole polymer. The entire material or entity may be referred to as the parent of the fragment.

[0069] Gene is used by those skilled in the biomedical arts to refer to a DNA sequence in a chromosome that codes for a product, e.g., an RNA product or a polypeptide product. A gene includes coding sequences (i.e., sequences that code for a particular product. A gene includes non-coding sequences. A gene can include both coding sequences, i.e., exon sequences, and non-coding sequences, i.e., intron sequences. A gene can include one or more regulatory elements that can control or affect one or more aspects of gene expression, e.g., cell type specific expression, inducible expression, etc.

[0070] Gene product or expression product is used by those skilled in the biomedical arts to refer to the RNA transcribed from a gene (pre- or post-processing, or the polypeptide encoded by the RNA transcribed from a gene (pre- or post-modification). A gene product can be or include a particular processed form of an RNA transcript, e.g., a particular edited form, a particular spliced ​​form, a particular capped form, etc.

[0071] Homology is used by those skilled in the biomedical field to refer to the overall relatedness between polymer molecules, for example between nucleic acid molecules, for example between DNA molecules or RNA molecules, or between polypeptide molecules. Polymer molecules are considered to be homologous to each other if their sequences are 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%, at least 90%, at least 95% or at least 99% identical. Polymer molecules are considered to be homologous to each other if their sequences are 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%, at least 90%, at least 95% or at least 99% similar.

[0072] Identity is used by those skilled in the biomedical field to refer to the overall relatedness between polymer molecules, for example, between nucleic acid molecules, for example, between DNA molecules or RNA molecules, or between polypeptide molecules. Polymer molecules are considered to be substantially identical to each other if their sequences are 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%, at least 90%, at least 95% or at least 99% identical. The calculation of the percent identity of two nucleic acid or polypeptide sequences can be performed by aligning the two sequences for optimal comparison purposes, for example, gaps can be introduced into one or both of the first and second sequences for optimal alignment, and non-identical sequences can be ignored for comparison. The length of the sequence aligned for comparison can be at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or substantially 100% of the length of the reference sequence. The nucleotides at the corresponding positions are then compared. If a position in the first sequence is occupied by the same residue, e.g., nucleotide or amino acid, as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap, which must be introduced for optimal alignment of the two sequences. The comparison of sequences between two sequences and the determination of the percent identity can be achieved using a mathematical algorithm. The percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller (1989) incorporated in the ALIGN program (version 2.0). Nucleic acid sequence comparisons can be performed using the ALIGN program, using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4.The percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package using the NWSgapdna.CMP matrix.

[0073] Improvement, increase, inhibition, reduction or grammatical equivalents are used by those skilled in the biomedical field to refer to a value compared to baseline or other reference measurement. Suitable reference measurements can be or include measurements on a particular system, such as a single individual, a single cell, or a cell population, for example, under otherwise equivalent conditions not present before or after a particular agent or treatment, or in the presence of an appropriate reference agent, such as a positive or negative control agent. Suitable reference measurements can be or include measurements on an equivalent system that is known or predicted to respond in a particular manner in the presence of an agent or treatment. Those skilled in the biomedical field know that improvement, increase, reduction, etc. typically refer to statistically significant changes. Those skilled in the biomedical field know from the context what degree of change may be relevant. The change may be a fold change, i.e., a change in which the value changed is 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 integer and decimal differences between and above 1, e.g., 1.5, 1.6, 1.7, 1.8 times, etc., relative to the relevant standard. The change may be a percentage change. Thus, The changed value may be an increase or decrease of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% relative to the relevant standard, including all integers and decimal points in between.

[0074] Internucleotidic linkage is used by those skilled in the biomedical field to refer to the phosphorus-containing linkage between the nucleotide units of an oligonucleotide, and is interchangeable with inter-sugar linkage and phosphorus atom bridge. Internucleotidic linkage is a phosphodiester linkage as found in naturally occurring DNA and RNA molecules. Internucleotidic linkage is a modified internucleotidic linkage, in which each oxygen atom of the phosphodiester linkage may be independently replaced by an organic or inorganic moiety. 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 defined and described. Internucleotidic linkage is a phosphotriester linkage, a phosphorothioate diester linkage, or a modified phosphorothioate triester linkage. Those skilled in the biomedical arts know that internucleotide linkages can exist as anions or cations at a given pH due to the presence of acidic or basic moieties within the linkage. Internucleotide linkages can be chiral linkages.

[0075] Linked, when used in reference to two or more moieties, is used by those skilled in the biomedical arts to mean that the moieties are physically associated or connected to each other to form a molecular structure that is sufficiently stable so that the moieties remain associated under the conditions under which the bond is formed and under the conditions under which the new molecular structure is used, e.g., under physiological conditions. The bond can be covalent. The bond can be non-covalent. The moieties can be directly or indirectly linked. When two moieties are directly linked, they are covalently linked to each other or are in sufficient proximity that the 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. 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, typically with each of the linking moieties being covalently linked to the linker. The linker can be any suitable moiety that will react with the two moieties to be joined in a reasonable period of time, under conditions consistent with the stability of the moieties (which can be conditionally protected), and in an amount sufficient to result in a reasonable yield.

[0076] Long-term administration means that the therapeutic agent or treatment agent is administered for a period of at least 12 weeks. This includes that the therapeutic agent or treatment agent is administered to be effective over a period of at least 12 weeks or for a period of at least 12 weeks, and does not necessarily mean that the administration itself is carried out for 12 weeks, for example, when a sustained release composition or a long-acting therapeutic agent or long-acting therapeutic agent is used. Thus, the subject is treated for a period of at least 12 weeks. In many cases, long-term administration lasts for at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months or more, or at least 1 year, at least 2 years, at least 3 years, at least 5 years, at least 7 years, or at least 10 years or more.

[0077] A modulator is used by those skilled in the biomedical field to refer to an agent, condition or event whose presence, level, degree, type or form correlates with the increase, decrease or other change in the level or activity of another agent, i.e., the modulated agent or target agent.A modulator can be or include any chemical class of agent, including small molecules, polypeptides, nucleic acids, carbohydrates, lipids, metals, or any other entity that exhibits related activation activity.A modulator can be direct (in this case, the modulator directly affects its target). A modulator agent may be indirect (in which case the modulator agent exerts its effect other than by binding to its target, e.g., by interacting with a regulator of the target, such that the level or activity of the target is altered). A modulator 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 a nucleic acid, such that the level, form or activity of the target is altered. The change in level, form or activity is an increase or decrease in the level of an altered mRNA, protein expressed from a target nucleic acid sequence. Those skilled in the art of biomedical sciences know that a modulator agent can bind to a binding target and potentially regulate the binding target, the binding causing an increase or decrease in the level or activity of a further regulated target. A modulator agent that binds to a nucleic acid target can alter the level or activity of that target. A modulator agent can regulate the activity of that nucleic acid target, e.g., by altering its modification, splicing, 5'- or 3'-capping, transport or translation, etc., resulting in an increase or decrease in the level of a desired product, e.g., an mRNA, or regulating a downstream target, such as a polypeptide encoded by such a nucleic acid target.A modulator agent may be or may include an oligonucleotide that can bind to a primary transcript and alter its splicing pattern to increase, decrease or alter the level or activity of a particular spliced ​​form, e.g., mature mRNA, thereby achieving an increase or decrease in the level of a product, e.g., a polypeptide that is or is encoded by such a particular spliced ​​form.

[0078] Modulator therapy is used by those skilled in the biomedical field to refer to the administration of a modulator agent that modulates a particular target of interest to achieve a desired therapeutic effect. Modulator therapy involves administering a single dose of a modulator. Modulator therapy involves administering multiple doses of a modulator. Modulator therapy involves administering a modulator according to a dosing regimen that is known or predicted to achieve a therapeutic effect because such results have been established to a specified degree of statistical confidence, for example, by administration to a relevant population. Modulator therapy involves the delivery of a modulator agent as described herein. The modulator agent can be or include a binding agent that is a protein, for example, an antibody or a nucleic acid, for example, an antisense oligonucleotide, that binds to a target, for example, a protein or a nucleic acid, such that the level, form or activity of the target is changed. The modulator agent can bind to the binding target and potentially modulate the binding target, which binding causes an increase or decrease in the level or activity of an additional modulator target. The modulator agent that binds to a nucleic acid target can change the level or activity of that target. A modulator agent can modulate the activity of its nucleic acid target, for example, by increasing or decreasing its modification, splicing, 5'-capping or 3'-end formation, transport or translation, etc., so that some level of a desired product, e.g., mRNA, is produced or a downstream target, such as a polypeptide encoded by such a nucleic acid target, is modulated. A modulator agent can be or include an oligonucleotide that can bind to a primary transcript and alter its splicing pattern to result in the level or activity of a particular spliced ​​form, e.g., mature mRNA, thereby achieving an increase or decrease in the level of a product, e.g., a polypeptide that is or is encoded by such a particular spliced ​​form.

[0079] Moiety is used by those skilled in the biomedical arts to refer to a defined chemical group or entity having a particular structure or activity.

[0080] MuSK is known to those skilled in the biomedical field as a receptor tyrosine kinase that is extracellularly composed of three Ig and one CRD / Fz domains and an intracellular tyrosine domain (TK). The best understood function of MuSK is at the neuromuscular junction (NMJ), where binding of agrin-LRP4 to the Ig1 domain induces MuSK TK activity and synaptic differentiation (Kim et al., 2008; Zhang et al., 2008a). MuSK is also a BMP coreceptor that binds to BMP and its receptors ALK3 and 6, upregulating BMP signaling and shaping the composition of the transcriptional response in myogenic cells. See Yilmaz et al. (2016). This BMP signaling pathway does not regulate, require, or is activated by MuSK TK activity by agrin-LRP4. The MuSK Ig3 domain is required for high affinity BMP binding but is not critical for agrin-LRP4 TK activation. The Ig3 domain is endogenously alternatively spliced, including in the brain (see Garcia-Osta et al., 2006; Hesser et al., 1999). Because BMP signaling can induce quiescence in NSCs and inhibit the integration of newborn neurons, suppressing the BMP drive by decreasing MuSK-BMP signaling can increase neurogenesis.

[0081] The MuSK-BMP pathway is known to those skilled in the biomedical field. The brain has 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 important neural stem cell decision points: (1) the quiescent state where proliferating stem cells exit the cell cycle and return to replenish the reserve pool that can supply fresh stem cells, and (2) differentiation into mature progeny. See Mira et al. (2010). Manipulating the BMP pathway in neural stem cells is an attractive target for regulating neurogenesis in the adult brain.

[0082] Nanoparticles are used by those skilled in the biomedical field to refer to particles with a diameter of less than 1000 nanometers (nm). Nanoparticles have a diameter of less than 300 nm as defined by the National Science Foundation. Nanoparticles have a diameter of less than 100 nm as defined by the National Institutes of Health. Nanoparticles are micelles because they contain a closed compartment separated from the bulk solution by a micellar membrane, typically composed of amphiphilic entities that surround and seal a space or compartment to define a lumen. The micellar membrane contains at least one polymer, for example, a biocompatible or biodegradable polymer.

[0083] Nucleic acid, in its broadest sense, is used by those skilled in the biomedical field to refer to any compound or substance that is or can be incorporated into an oligonucleotide chain. Nucleic acid is a compound or substance that is or can be incorporated into an oligonucleotide chain via a phosphodiester bond. Nucleic acid can refer to individual nucleic acid residues, such as nucleotides or nucleosides. Nucleic acid can refer to an oligonucleotide chain that includes individual nucleic acid residues. Nucleic acid is or includes RNA. Nucleic acid is or includes DNA. Nucleic acid is or includes one or more naturally occurring nucleic acid residues, or consists of one or more naturally occurring nucleic acid residues. Nucleic acid is or includes one or more nucleic acid analogs, or consists of one or more nucleic acid analogs. Nucleic acid analogs differ from nucleic acids because they do not utilize a phosphodiester backbone. Nucleic acids are known in the biomedical field and can be or include or consist of one or more peptide nucleic acids, which have peptide bonds instead of phosphodiester bonds in the backbone and are considered within the scope of the present invention. A nucleic acid can have one or more phosphorothioate or 5'-N-phosphoramidite bonds rather than phosphodiester bonds. A nucleic acid can be, comprise, or consist of one or more natural nucleosides, such as adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine. The nucleic acid may be, comprise, or consist of one or more nucleoside analogs, such as 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3 methyladenosine, 5-methylcytidine, C5 propynyl-cytidine, C5 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, (6)-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof.The nucleic acid contains one or more modified sugars, such as 2'-fluororibose, ribose, 2'deoxyribose, arabinose and hexose, compared to those in natural nucleic acids. The nucleic acid has a nucleotide sequence that encodes a functional gene product, such as RNA or a protein. The nucleic acid contains one or more introns. The nucleic acid is prepared by one or more of isolation from a natural source, enzymatic synthesis by polymerization based on a complementary template (in vivo or in vitro), regeneration in a recombinant cell or system, and chemical synthesis. The nucleic acid may contain at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, 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, at least 90, at least 95, at least 10 ... The nucleic acid may be 10, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 20, at least 225, at least 250, at least 275, at least 300, at least 325, at least 350, at least 375, at least 400, at least 425, at least 450, at least 475, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1500, at least 2000, at least 2500, at least 3000, at least 3500, at least 4000, at least 4500, at least 5000 or more residues in length. The nucleic acid may be partially or entirely single-stranded. The nucleic acid may be partially or entirely double-stranded. The nucleic acid may have a nucleotide sequence that includes at least one element that encodes a polypeptide or is the complement of a sequence that encodes a polypeptide. The nucleic acid has enzymatic activity.

[0084] Prodrugs are used by those skilled in the biomedical field to refer to entities that are metabolized in the body when administered to an organism to deliver the desired active, e.g., therapeutic or diagnostic agent. Typically, such metabolism involves the removal of at least one prodrug moiety to form an active agent. Various forms of prodrugs are known to those skilled in the biomedical field. As with other compounds described herein, prodrugs can be provided in any of a variety of forms, e.g., crystalline forms, salt forms, etc. Prodrugs are provided as their pharmaceutically acceptable salts.

[0085] Operably linked is used by those skilled in the biomedical arts to refer to a juxtaposition in a relationship that allows the described components to function in the intended manner. A control element operably linked to a functional element is associated such that expression or activity of the functional element is achieved under conditions compatible with the control element. An operably linked control element, e.g., promoter, enhancer, etc., is contiguous, e.g., covalently linked, with the coding element of interest. The control element acts in trans or cis with the coding functional element of interest.

[0086] or has the inclusive meaning of the term, unless the context clearly indicates a disjunctive meaning of the term Patient is used by those skilled in the biomedical field to refer to any organism to which the provided compositions, e.g., agonizing agents such as ASOs, are or can be administered, e.g., for experimental, diagnostic, preventive, cosmetic or therapeutic purposes. Typical patients include mammals, such as animals, e.g., mice, rats, rabbits, non-human primates or humans. The patient is a human. The patient suffers from or is susceptible to one or more disorders or conditions. The patient exhibits one or more symptoms of the disorder or condition. The patient has been diagnosed with one or more disorders or conditions. The disorder or condition is Alzheimer's disease (AD) or other disease characterized by neurodegeneration. The patient is undergoing or has undergone a particular treatment to diagnose or treat the disease, disorder or condition.

[0087] Pharmaceutical composition is used by those skilled in the biomedical field to refer to an active agent, e.g., an agonizing agent, formulated with one or more pharma- ceutically acceptable carriers. The active agent is present in a unit dose suitable for administration in a treatment regimen that exhibits a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. The pharmaceutical composition may be specially formulated for administration in solid or liquid form, including those adapted for oral administration, drenches, aqueous or non-aqueous solutions or suspensions, tablets, e.g., those targeted for buccal, sublingual and systemic absorption, boluses, powders, granules, pastes for application to the tongue. Parenteral administration, e.g., subcutaneous, intramuscular, intravenous, intraperitoneal, intrathecal, intravenous, intraventricular or epidural injection as a sterile solution or suspension, or sustained release formulation; topical application as a cream, ointment or controlled release patch or spray applied to the skin, lung or oral cavity; vaginally or rectally, e.g., as a pessary, cream or foam; sublingually; ophthalmically; transdermally; or intranasally, to the lungs, and other mucosal surfaces.

[0088] Pharmaceutically acceptable is used by those skilled in the biomedical arts to refer to those compounds, materials, compositions or dosage forms that are, within the scope of sound medical judgment, suitable for contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response or other problem or complication and are commensurate with a reasonable benefit / risk ratio.

[0089] A pharma- ceutically acceptable carrier is used by those skilled in the biomedical arts to mean a pharma- ceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient or solvent encapsulating material, involved in the transport or transfer of a compound 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 harmful to the patient. Pharmaceutically acceptable carriers include sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository wax; 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 buffers; polyesters, polycarbonates or polyanhydrides; and other non-toxic compatible substances used in pharmaceutical formulations.

[0090] Pharmaceutically acceptable salts are used by those skilled in the biomedical field to refer to salts of such compounds that are suitable for use in pharmaceutical situations, i.e., salts that are suitable for contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic reactions, etc., within the scope of sound medical judgment, and commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the biomedical field. Berge et al. (1977) describes pharmaceutically acceptable salts in detail. Pharmaceutically acceptable salts include non-toxic acid addition salts that are salts of amino groups, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or with organic acids such as acetic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or by using other methods used in the biomedical field, such as ion exchange. 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, 2-hydroxy-ethanesulfonate, and 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, etc. Provided compounds contain one or more acidic groups, e.g., oligonucleotides, and pharma- ceutically acceptable salts are alkali metal, alkaline earth metal, or ammonium, e.g., ammonium salts of N(R), where each R is independently defined and described in the salts of the present disclosure.Representative alkali metal salts or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. A pharma- ceutically acceptable salt is a sodium salt. A pharma- ceutically acceptable salt is a potassium salt. A pharma- ceutically acceptable salt is a calcium salt. Pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium, and amine cations formed, where appropriate, using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, alkyls having 1-6 carbon atoms, sulfonates, and arylsulfonates. The compounds provided contain multiple acid groups. Oligonucleotides can contain two or more acid groups, for example, in natural phosphate linkages, or modified internucleotide linkages. Pharmaceutically acceptable salts or salts in general of such compounds contain two or more cations, which may be the same or different. In pharmaceutically acceptable salts (or salts in general), for example, in an aqueous solution with a pKa of about 11 or less, about 10 or less, about 9 or less, about 8 or less, about 7 or less, about 6 or less, about 5 or less, about 4 or less, about 3 or less, or about 2 or less; about 7 or less; about 6 or less; about 5 or less; about 4 or less; about 3 or less, all ionizable hydrogens are replaced by cations in the acidic group. Each internucleotide bond, for example, phosphate group, is independently present in its salt form, for example, in the case of sodium salt, -OP(O)(ONa)-O-). A pharmaceutically acceptable salt is the sodium salt of an oligonucleotide. A pharmaceutically acceptable salt is the sodium salt of an oligonucleotide, in which each acidic phosphate group and modified phosphate group are present as salt form (both sodium salts).

[0091] Polypeptide, which is used interchangeably herein with the term protein, is used by those skilled in the biomedical arts to refer to a polymer of at least three amino acid residues. A polypeptide comprises one or more or any naturally occurring amino acid. A polypeptide comprises one or more or any unnatural amino acid. A polypeptide comprises one or more or any D-amino acid. A polypeptide comprises one or more or any L-amino acid. A polypeptide comprises one or more or any pendant group or other modification, e.g., modifying or attaching 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. 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. A polypeptide can participate in one or more intramolecular or intermolecular disulfide bonds. A polypeptide may be cyclic or may include a cyclic portion. A polypeptide is not cyclic or does not include a cyclic portion. A polypeptide is linear. A polypeptide may include stapled polypeptides. A polypeptide is involved in non-covalent or covalent complex formation with one or more other polypeptides, for example, in an antibody. A polypeptide has a naturally occurring amino acid sequence. A polypeptide has an amino acid sequence that does not occur in nature. A polypeptide has an engineered amino acid sequence because it is designed or produced by the action of the hand of man. The term polypeptide may be added to the name of a reference polypeptide, activity, or structure, and may 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 sequence or function is known, or one of ordinary skill in the biomedical arts will know exemplary polypeptides within the class whose amino acid sequence or function is known.Such an exemplary polypeptide is a reference polypeptide for a class or family of polypeptides. A member of a class or family of polypeptides exhibits significant sequence homology or sequence identity, shares a common sequence motif, e.g., a characteristic sequence element, or shares a common activity, optionally at a comparable level or within a specified range, with the reference polypeptide of the class or every polypeptide in the class. A member polypeptide exhibits an overall sequence homology or sequence identity with the reference polypeptide that is at least about 30-40%, and often exceeds about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more, or includes at least one region, e.g., a conserved region that can contain a characteristic sequence element that exhibits high sequence identity, often exceeding 90% or even 95%, 96%, 97%, 98% or 99%. Such conserved regions usually encompass at least 3-4, and often up to 20 or more amino acids. A conserved region encompasses at least one stretch of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15 or more contiguous amino acids. A useful polypeptide can include a fragment of a parent polypeptide. A useful polypeptide can include multiple fragments, each of which is found in the same parent polypeptide in a spatial arrangement relative to each other that is different from that found in the polypeptide of interest, e.g., fragments that are directly linked in the parent may be spatially separated in the polypeptide of interest or vice versa, or fragments may be present in the polypeptide of interest in a different order than in the parent, such that the polypeptide of interest is a derivative of its parent polypeptide.

[0092] When used in relation to the occurrence of a disease, disorder or condition, prevent or prevention is used by those skilled in the biomedical arts to refer to reducing the risk of developing a disease, disorder or condition, or delaying the onset of one or more characteristics or symptoms of a disease, disorder or condition. Prevention can be considered complete if the onset of a disease, disorder or condition has been delayed for a predetermined period of time.

[0093] Recombinant is used by those skilled in the biomedical arts to refer to a polypeptide that is designed, engineered, prepared, expressed, created, manufactured, or isolated by recombinant means, e.g., 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 is transgenic or otherwise engineered to express a gene or genes or genetic components that code for or direct the expression of a polypeptide or one or more components, parts, elements, or domains thereof; or a polypeptide prepared, expressed, created, or isolated by any other means that involves splicing or ligating selected nucleic acid sequence elements together, chemically synthesizing selected sequence elements, or otherwise generating a nucleic acid that codes for or directs the expression of a polypeptide or one or more components, parts, elements, or domains thereof. One or more of such selected sequence elements are found in nature. One or more of such selected sequence elements are designed in silico. One or more such selected sequence elements may arise, for example, from in vivo or in vitro mutagenesis of known sequence elements, e.g., from natural or synthetic sources in the germline of a source organism of interest, e.g., human, mouse, etc.

[0094] Small molecule is used by those skilled in the biomedical field to mean organic or inorganic compounds with low molecular weight. Small molecules are less than about 5 kilodaltons (kDa) in size. Small molecules are less than about 4 kDa, less than 3 kDa, less than about 2 kDa, or less than about 1 kDa. Small molecules are less than about 800 Daltons (Da), less than about 600 Da, less than about 500 Da, less than about 400 Da, less than about 300 Da, less than about 200 Da, or less than about 100 Da. Small molecules are 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. Small molecules are not polymeric. Small molecules do not include polymeric moieties. Small molecules are not or do not include proteins or polypeptides, e.g., are not oligopeptides or peptides. A small molecule is not or does not include a polynucleotide, e.g., is not an oligonucleotide. A small molecule is not or does not include a polysaccharide. A small molecule is not a glycoprotein, a proteoglycan, a glycolipid, etc. A small molecule is not a lipid. A small molecule is a modulator, e.g., an inhibitor or an activator. A small molecule is biologically active. A small molecule is detectable, e.g., includes at least one detectable moiety. A small molecule is a therapeutic agent. Those skilled in the biomedical field will know that the small molecule compounds described herein can be provided or used in any of a variety of forms, such as crystalline forms, salt forms, protected forms, prodrug forms, ester forms, isomeric forms, e.g., optical or structural isomers, isotopic forms, etc. Those skilled in the biomedical field will know that some small molecule compounds have structures that can exist in one or more stereoisomeric forms. Such small molecules can be used under the present disclosure in the form of individual enantiomers, diastereomers, or geometric isomers, or in the form of a mixture of stereoisomers. Such small molecules can be used under the present disclosure in racemic mixture form.Those skilled in the art of biomedical sciences know that some small molecule compounds have structures that can exist in one or more tautomeric forms.Such small molecules can be used under the present disclosure in the form of individual tautomers or in the form that interconverts between tautomeric forms.Those skilled in the biomedical field know that some small molecule compounds have structures that allow for isotopic substitution. Such small molecules can be used according to the present disclosure in one or more isotopically modified forms or mixtures thereof. Reference to a particular small molecule compound can refer to a particular form of that compound. A particular small molecule compound can be provided or used in a salt form, for example, an acid addition salt form or a base addition salt form, depending on the compound. The salt form can be a pharmaceutically acceptable salt form. If a small molecule compound exists or is found in nature, the compound can be provided or used under this specification in a form that is different from the form in which it exists or is found in nature. Those skilled in the biomedical field know that in some cases, preparing a particular small molecule compound containing an absolute or relative amount of a compound or an absolute or relative amount of a particular form of the compound that is different from the absolute or relative amount of the compound with respect to another component of the preparation, including another form of the compound or form of the compound that is present in a reference preparation of interest, for example, a primary sample from a source of interest, such as a biological source or an environmental source, is different from the compound present in the reference preparation or reference source. A preparation of a single stereoisomer of a small molecule compound can be considered a different form of the compound than a racemic mixture of the compound. A particular salt of a small molecule compound can be considered a different form of the compound than another salt form of the compound. A preparation that contains only one form of the compound that contains one conformer of the double bond ((Z) or (E)) can be considered a different form of the compound than a form of the compound that contains the other conformer of the double bond ((E) or (Z)). A preparation in which one or more atoms are isotopes different than those present in a reference preparation can be considered a different form, etc.

[0095] Specific binding is used by those skilled in the biomedical field to refer to the ability to distinguish between binding partners that may be present in the environment in which binding occurs. A binder that interacts with one specific target in the presence of other potential targets is said to specifically bind to the target, e.g., the target amino acid sequence or the target nucleic acid sequence on the target protein / gene of interest with which it interacts. Specific binding is assessed by detecting or determining the association between the binder and its partner. Specific binding is assessed by detecting or determining the dissociation of the binder-partner complex. Specific binding is assessed by detecting or determining the ability of the binder to compete for alternative interactions between its partner and another entity. Specific binding is assessed by performing such detection or determination over a range of concentrations.

[0096] Specificity is used by those skilled in the biomedical arts to refer to a measure of the ability of a particular ligand to distinguish its binding partner from other potential binding partners.

[0097] Subject is used by those skilled in the biomedical field to refer to an organism, typically a mammal, for example, a human, including prenatal human forms. The subject suffers from a relevant disease, disorder or condition, for example, Alzheimer's disease (AD), or other disease characterized by neurodegeneration. The subject is susceptible to the disease, disorder or condition. The subject exhibits one or more symptoms or characteristics of the disease, disorder or condition. The subject does not have to exhibit symptoms or characteristics of the disease, disorder or condition. The subject can be a person who has one or more characteristics characteristic of susceptibility to a disease, disorder or condition, or risk of a disease, disorder or condition. The subject can be a patient.

[0098] Substantially is used by those skilled in the biomedical arts to refer to a qualitative state that exhibits the entire or nearly entire extent or degree of a feature or characteristic of interest. Those skilled in the biological arts know that biological and chemical phenomena rarely proceed to completion or perfection, or achieve or avoid absolute results. Thus, the term substantially is used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0099] Substantial identity is used by those skilled in the biomedical field to refer to the comparison between amino acid sequences or nucleic acid sequences.Two sequences are generally considered to be substantially identical in biomedical sciences if they contain the same residues at corresponding positions.As is well known in the art, amino acid sequences 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. Exemplary 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, these programs indicate identity. Two sequences are considered to be substantially identical if at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more of the corresponding residues are identical over the relevant stretch of residues, which may be the complete sequence.Relevant stretches are 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, at least 90, at least 95, at least 100, at least 125, at least 150, at least 175, at least 200, at least 225, at least 250, at least 275, at least 300, at least 325, at least 350, at least 375, at least 400, at least 425, at least 450, at least 475, at least 500 or more residues.

[0100] Suffering from is used by those skilled in the biomedical arts to refer to an individual having a disease, disorder, or condition, e.g., Alzheimer's disease (AD), or other disease characterized by neurodegeneration, being diagnosed with or exhibiting one or more symptoms of the disease, disorder, or condition.

[0101] Susceptible to is used by those skilled in the biomedical field to refer to an individual who is susceptible to a disease, disorder or condition, such as Alzheimer's disease (AD), or other disease characterized by neurodegeneration, being an individual who is at a higher risk of developing the disease, disorder or condition than the general population. An individual who is susceptible to a disease, disorder or condition may not have been diagnosed with the disease, disorder or condition. An individual who is susceptible to a disease, disorder or condition may show symptoms of the disease, disorder or condition. An individual who is susceptible to a disease, disorder or condition may not show symptoms of the disease, disorder or condition. An individual who is susceptible to a disease, disorder or condition will develop the disease, disorder or condition. An individual who is susceptible to a disease, disorder or condition will not develop the disease, disorder or condition.

[0102] Symptoms are reduced, as used by those skilled in the biomedical field, to refer to when the magnitude, e.g., intensity, severity, etc., or frequency of one or more symptoms of a particular disease, disorder or condition, such as Alzheimer's disease (AD), or other diseases characterized by neurodegeneration, is reduced.For clarity, the delay in the onset of a particular symptom is considered to be one form of reducing the frequency of that symptom.

[0103] A target gene is used by those skilled in the biomedical field to refer to a gene whose expression is regulated, for example, by modifying splice activity, for example, by inducing exon skipping. The term target portion or target region is used by those skilled in the biomedical field to refer to a contiguous portion of the nucleotide sequence of a target gene. A target portion or target region is one or more exons within the target gene sequence. A target portion can be about 8-36 nucleotides long, for example, about 10-20 or about 15-30 nucleotides long. The length of the target portion can have a specific value or a subrange within the aforementioned ranges. The targeting moiety may be about 15-29, about 15-28, about 15-27, about 15-26, about 15-25, about 15-24, about 15-23, about 15-22, about 15-21, about 15-20, about 15-19, about 15-18, about 15-17, about 18-30, about 18-29, about 18-28, about 18-27, about 18-26, about 18-25, about 18-24, about 18-23, about 18-22, about 18-21, about 18-20, about 19-30, about 19-29, about 19-28, about 19-27, about 19 26, about 19-25, about 19-24, about 19-23, about 19-22, about 19-21, about 19-20, about 20-30, about 20-29, about 20-28, about 20-27, about 20-26, about 20-25, about 20-24, about 20-23, about 20-22, about 20-21, about 21-30, about 21-29, about 21-28, about 21-27, about 21-26, about 21-25, about 21-24, about 21-23 or about 21-22 nucleotides in length.

[0104] Therapeutic agent is used by those skilled in the biomedical field to refer to any agent that has a therapeutic effect or induces desired biological or pharmacological effects when administered to a subject.Therapeutic agent is any substance that can alleviate, improve, alleviate, inhibit, prevent, delay the onset, reduce the severity, or reduce the incidence of one or more symptoms or characteristics of disease, disorder, or condition, such as one or more symptoms or characteristics of Alzheimer's disease (AD) or other diseases characterized by neurodegeneration.

[0105] Therapeutically effective amount is used by those skilled in the biomedical field to mean an amount of a substance, e.g., a therapeutic agent, composition, or formulation, that induces a desired biological response when administered as part of a therapeutic dosing regimen. A therapeutically effective amount of a substance is an amount that is sufficient to treat, diagnose, prevent, or delay the onset of a disease, disorder, or condition when administered to a subject suffering from or susceptible to the disease, disorder, or condition. Those skilled in the art know that the effective amount of a substance can vary depending on factors such as the desired biological endpoint, the substance delivered, the target cell or tissue, and the like. An effective amount of a compound in a formulation for treating a disease, disorder, or condition is an amount that alleviates, improves, relieves, inhibits, prevents, delays the onset, reduces the severity, or reduces the incidence of one or more symptoms or characteristics of the disease, disorder, or condition, e.g., Alzheimer's disease (AD), or another disease characterized by neurodegeneration. A therapeutically effective amount is administered in a single dose. Several unit doses are required to deliver a therapeutically effective amount.

[0106] Treating is used by those skilled in the biomedical field to refer to providing treatment, i.e. providing any medical or surgical management of the subject.Treatment can be provided to reverse, alleviate, inhibit the progression of disease, disorder or condition, prevent disease, disorder or condition, or reduce the possibility of disease, disorder or condition, or to reverse, alleviate, inhibit, or prevent the progression of one or more symptoms or signs of disease, disorder or condition, or to prevent one or more symptoms or signs of disease, disorder or condition, or reduce the possibility of one or more symptoms or signs of disease, disorder or condition. 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) or another disease characterized by neurodegeneration, for example, to reverse, alleviate, reduce the severity of, or inhibit the progression of, or prevent, a pathology, or to reverse, alleviate, reduce the severity of, or inhibit, one or more symptoms or signs of, a pathology. The compositions of the present disclosure can be administered to a subject who has developed Alzheimer's disease (AD) or another disease characterized by neurodegeneration, or who is at high risk of developing such a disorder 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, a pathology. A subject is typically at risk of developing a pathology.

[0107] A variant, as used in the context of a biological molecule, e.g., a nucleic acid, e.g., an antisense oligonucleotide (ASO), a protein, or a small molecule, refers to a molecule that exhibits significant structural identity with a reference molecule, but is structurally different from the reference molecule, e.g., in the presence or absence or level of one or more chemical moieties compared to the reference entity. A variant is also functionally different from its reference molecule. Whether a particular molecule is properly considered to be a variant of a reference molecule is based on its degree of structural identity with the reference molecule. Any biological or chemical reference molecule has a characteristic structural element. 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 embodiment. To give just a few examples, a polypeptide can have a characteristic sequence element that is composed of multiple amino acids that have a designated position relative to each other in linear or three-dimensional space, or that contribute to a particular structural motif or biological function. A nucleic acid can have a characteristic sequence element that is composed of multiple nucleotide residues that have a designated position relative to each other in linear or three-dimensional space. A mutant polypeptide or a mutant nucleic acid may differ from a reference polypeptide or a reference nucleic acid due to one or more differences in amino acid sequence or nucleotide sequence, or one or more differences in chemical moieties, such as carbohydrates, lipids, phosphate groups, that are covalent components of the polypeptide or nucleic acid, such as those attached to the polypeptide backbone or the nucleic acid backbone. A mutant polypeptide or a mutant nucleic acid exhibits at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% or at least 99% overall sequence identity with a reference polypeptide or a reference nucleic acid. A mutant polypeptide or a mutant nucleic acid does not share at least one characteristic sequence element with a reference polypeptide or a reference nucleic acid. A reference polypeptide or a reference nucleic acid has one or more biological activities. A mutant polypeptide or a mutant nucleic acid shares one or more biological activities of a reference polypeptide or a reference nucleic acid.A mutant polypeptide or nucleic acid lacks one or more of the biological activities of a reference polypeptide or nucleic acid. A mutant polypeptide or nucleic acid exhibits a reduced level of one or more biological activities compared to a reference polypeptide or nucleic acid. A polypeptide or nucleic acid of interest is considered a mutant of a reference polypeptide or nucleic acid if it has the same amino acid or nucleotide sequence as the reference, but has some sequence changes at certain positions. Typically, less than about 20%, less than about 15%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, or less than about 2% of the residues in the mutant are replaced, inserted, or deleted compared to the reference. A mutant polypeptide or nucleic acid contains about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1 replacement residue compared to the reference. Often, the variant polypeptide or variant nucleic acid contains a small number of substituted, inserted or deleted functional residues (i.e., residues involved in a particular biological activity) compared to the reference, e.g., less than about 5, less than about 4, less than about 3, less than about 2, or less than about 1. The variant polypeptide or variant nucleic acid contains no more than about 5, no more than about 4, no more than about 3, no more than about 2, or no more than about 1 additions or deletions compared to the reference. The variant polypeptide or variant nucleic acid contains no more than about 25, no more than about 20, no more than about 19, no more than about 18, no more than about 17, no more than about 16, no more than about 15, no more than about 14, no more than about 13, no more than about 10, no more than about 9, no more than about 8, no more than about 7, no more than about 6, typically no more than about 5, no more than about 4, no more than about 3, or no more than about 2 additions or deletions compared to the reference. The reference polypeptide or reference nucleic acid is found in nature. The reference polypeptide or reference nucleic acid is a human polypeptide or human nucleic acid.

[0108] A vector is used by those skilled in the biomedical field to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it is 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 into which additional DNA segments can be ligated into the viral genome. Some vectors, e.g., bacterial vectors with a bacterial origin of replication, and episomal mammalian vectors, replicate autonomously in the host cell into which they are introduced. Other vectors, e.g., non-episomal mammalian vectors, can be integrated into the genome of the host cell upon introduction into the host cell and are replicated along with the host genome. Certain vectors can direct the expression of genes to which they are operably linked. Such vectors are known in the biomedical field as expression vectors. Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation, e.g., electroporation, lipofection. Enzymatic reactions and purification techniques can be performed according to manufacturer's specifications or as commonly accomplished in the biomedical field. The techniques and procedures described above may generally be carried out according to conventional methods well known in the biomedical arts, as described in various general and more specific references cited and described throughout this specification. See, for example, Molecular Cloning: A Laboratory Manual (2012).

[0109] Unless otherwise defined herein, scientific and technical terms used in connection with this application shall have the meanings commonly understood by those of ordinary skill in the biomedical arts. The present invention is not limited to the particular methodology, protocols, and reagents, etc. described herein, as such may vary.

[0110] The disclosure described herein does not relate to processes for cloning humans, processes for altering the genetic identity of the human germ line, use of human embryos for industrial or commercial purposes, or processes for altering the genetic identity of animals that may cause human or animal suffering without any substantial medical benefit, nor to the animals resulting from such processes.

[0111] MuSK Ig2 modulator.

[0112] In some embodiments, the present disclosure provides techniques for modulating biological events mediated by the MuSK Ig2 domain. For example, in some embodiments, the present disclosure provides techniques for disrupting or otherwise interfering with one or more binding interactions between the MuSK Ig2 domain and a binding partner (e.g., a BMP receptor or an autoantibody) as described herein.

[0113] In some embodiments, a MuSK Ig2 modulator agent increases the level or activity of one or more MuSK polypeptides (e.g., ΔIg2-MuSK) that lack an effective Ig2 domain, for example because such domain has been mutated, removed, or otherwise inactivated (e.g., by blocking, modification, etc.). Alternatively or additionally, in some embodiments, a MuSK Ig2 modulator agent blocks, inactivates, mutates, or removes a functional Ig2 domain from MuSK, or achieves, assists, or contributes to such blocking, inactivation, mutation, or removal. Thus, in some embodiments, a MuSK Ig2 modulator agent may be considered a ΔIg2-MuSK agonizing agent.

[0114] In some embodiments, the disclosure provides technology relating to MuSK Ig2 modulator agents, including, for example, providing such agents themselves, or providing methods or reagents for identifying, characterizing, making or using them, or compositions that contain or deliver them.

[0115] In some embodiments, a MuSK Ig2 modulator agent may directly interact with a MuSK polypeptide (e.g., with full-length MuSK or with ΔIg2-MuSK). In some embodiments, a MuSK Ig2 modulator agent may not directly interact with a MuSK polypeptide, but rather affect the level or activity of ΔIg2-MuSK through some other interaction (e.g., with a precursor or regulator or downstream product of MuSK).

[0116] In principle, MuSK Ig2 modulator agents can be of any chemical class (eg, small molecules, polypeptides [eg, antibodies], nucleic acids, etc.).

[0117] In some embodiments, the MuSK Ig2 regulator is an agent that downregulates MuSK Ig2 activation of MuSK Ig2 domain protein expression, MuSK Ig2 domain gene expression, or BMP signaling through one or more of BMPR.Without wishing to be bound by any particular theory, the present disclosure proposes that such MuSK Ig2 regulator can thereby induce neurogenesis, or reduce muscle atrophy, or stimulate muscle growth (e.g., myogenesis).

[0118] In some embodiments, the MuSK Ig2 modulator agent is an agonizing agent that increases expression of MuSK ΔIg2.

[0119] In some specific embodiments described in further detail herein, the MuSK Ig2 modulator agent may be or may include a small molecule.

[0120] In some particular embodiments described in further detail herein, a MuSK Ig2 modulator agent may be or include an antibody that binds to a MuSK polypeptide (e.g., an antibody that blocks MuSK Ig2 or an antibody that captures one or more MuSK polypeptide forms that comprise functional Ig2). Alternatively or additionally, in some particular embodiments, a MuSK Ig2 modulator agent may be or include an agent that disrupts (e.g., prevents, competes, etc.) the interaction between MuSK Ig2 and an antibody (e.g., an autoantibody). For example, in some embodiments, a MuSK Ig2 modulator agent may be or include a MuSK Ig2 domain but lacks one or more other MuSK elements (e.g., in some embodiments, a MuSK Ig2 modulator agent may be or include a soluble peptide that is or includes a MuSK Ig2 domain or an autoantibody-binding portion thereof).

[0121] In some specific embodiments described in more detail herein, MuSK Ig2 regulator agent can be or include a nucleic acid agent.For example, in some embodiments, MuSK Ig2 regulator agent can be or include a nucleic acid (e.g., gene therapy vector, or RNA therapeutic agent such as mRNA) that encodes MuSK form lacking functional Ig2 domain (e.g., ΔIg2-MuSK).Alternatively or additionally, in some embodiments, nucleic acid MuSK Ig2 regulator agent can be or include an oligonucleotide, for example, MuSK Ig2 targeting exon skipping oligonucleotide, MuSK Ig2 targeting CRISPR / Cas9 gRNA (e.g., modifying or removing Ig2), MuSK Ig2 targeting siRNA (e.g., inhibiting the production / expression of MuSK Ig2 from the transcript encoding MuSK Ig2), or MuSK Ig2 targeting shRNA.

[0122] Small molecules.

[0123] In some embodiments, a MuSK Ig2 modulator agent may be or may include a small molecule compound.

[0124] In some embodiments, small molecule MuSK Ig2 modulators target therapeutic agents that target MuSK splicing.For example, in some embodiments, such small molecule compounds enhance the splicing events that generate messages that code for ΔI2-MuSK, or inhibit the splicing events that generate messages that code for other MuSK splice variants.In some embodiments, small molecule MuSK Ig2 modulators change the BMP signaling pathway.In some embodiments, small molecule MuSK Ig2 modulators change the BMP signaling pathway by targeting BMP receptors, which can further induce neurogenesis, or reduce muscle atrophy, or stimulate muscle growth (e.g., myogenesis).

[0125] In some embodiments, the small molecule MuSK Ig2 modulator agent targets one or more of the type I BMP receptors, ALK3 (ALK is anaplastic lymphoma kinase) and ALK6, and the type I activin receptor ALK4. In some embodiments, the small molecule MuSK Ig2 modulator agent is an ALK inhibitor. In some embodiments, the small molecule MuSK Ig2 modulator agent is an ALK inhibitor selected from the group consisting of crizotinib, ceritinib, alectinib, brigutinib, and lorlatinib.

[0126] In some embodiments, small molecule MuSK Ig2 modulator agents target MuSK Ig2 domains or BMPs such that the level or activity of MuSK / BMP complexes is reduced. In some such embodiments, small molecule MuSK Ig2 modulator agents inhibit the formation of such complexes or disrupt such complexes. In some embodiments, such MuSK Ig2 modulator agents compete with BMP receptors for binding to MuSK Ig2 or compete with MuSK Ig2 for binding to BMP receptors.

[0127] Antibody agents.

[0128] In some embodiments, the MuSK Ig2 modulator agent is an antibody agent.

[0129] In some embodiments, such antibody agents specifically bind to a MuSK polypeptide, hi some embodiments, antibody agents targeting MuSK specifically bind to the Ig2 domain of a MuSK polypeptide.

[0130] In some embodiments, an antibody that targets the Ig2 domain of a MuSK protein may specifically bind to the Ig2 domain relative to the Ig1 or Ig3 domains of MuSK.

[0131] In some embodiments, the antibody MuSK Ig2 modulator agent targets the MuSK Ig2 domain or the BMP receptor such that the level or activity of the MuSK / BMP receptor complex is reduced. In some such embodiments, the antibody MuSK Ig2 modulator agent inhibits the formation of such a complex or disrupts such a complex. In some embodiments, such an antibody MuSK Ig2 modulator agent competes with the BMP receptor for binding to MuSK Ig2 or competes with MuSK Ig2 for binding to the BMP receptor.

[0132] In some embodiments, the anti-MUSK antibody agent is internalized by cells (e.g., cells having a neuronal cell type). In some embodiments, the antibodies or antigen-binding fragments thereof described herein may be or include immunoglobulins, heavy chain antibodies, light chain antibodies, or other protein scaffolds with antibody-like properties, as well as other immunological binding moieties known in the art, including Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, disulfide-linked Fv fragments, scFv fragments, diabodies, triabodies, tetrabodies, minibodies, maxibodies, tandabs, BiTe, and any combination thereof. In some embodiments, the anti-MUSK antibody or antigen-binding fragment thereof targets, for example, the Ig2 domain of MuSK. In some embodiments, such antibodies or antigen-binding fragments thereof may inhibit or substantially prevent binding of BMP receptors to the MuSK Ig2 domain.

[0133] An antibody may be an immunoglobulin molecule of four polypeptide chains, for example, two heavy (H) chains and two light (L) chains. The heavy chain may comprise a heavy chain variable domain and a heavy chain constant domain. The heavy chain constant domain may comprise a CH1 region, a hinge region, a CH2 region, a CH3 region, and sometimes a CH4 region. The light chain may comprise a light chain variable domain and a light chain constant domain. The light chain constant domain may comprise a CL. The heavy chain variable domain of the heavy chain and the light chain variable domain of the light chain may typically be further subdivided into variable regions called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Such heavy chain variable domains and light chain variable domains may each comprise three CDRs and four framework regions arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (one or more of which may be engineered as described herein).

[0134] In some embodiments, an antibody agent (e.g., an anti-MUSK antibody) can include various heavy and light chains as described herein. In some embodiments, an antibody can include two heavy and light chains. In various embodiments, the disclosure encompasses an antibody that includes at least one heavy or light chain disclosed herein, at least one heavy or light chain framework domain disclosed herein, at least one heavy or light chain CDR domain disclosed herein, or any heavy or light chain constant domain disclosed herein.

[0135] In some embodiments, the antibody agent is or comprises a monoclonal antibody. Typically, a monoclonal antibody is obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies that comprise the population are substantially identical except for natural mutations that may be present in small amounts. Thus, the modifier monoclonal as used herein indicates the characteristic of the antibody that it is not a mixture of individual antibodies. In some embodiments, a monoclonal antibody against a particular epitope is derived from a single cell line (e.g., a B cell line).

[0136] In some embodiments, an antibody agent (e.g., an anti-MuSK antibody) may be or include a polyclonal antibody. In contrast to monoclonal antibodies, polyclonal antibodies typically represent a heterogeneous antibody population, i.e., antibodies within a particular population contain structural variations, such as affinities for different epitopes on a particular antigen (e.g., the Ig2 domain of MuSK, or regions within the Ig2 domain). Several methods of producing polyclonal antibodies are known in the art, including the use of multiple subcutaneous or intraperitoneal injections of the relevant antigen into animals, optionally with the co-administration of one or more adjuvants.

[0137] Oligonucleotides.

[0138] In some embodiments, the MuSK Ig2 modulator agents described herein are or comprise oligonucleotides.

[0139] Synthetic oligonucleotides provide molecular tools that are useful for a wide variety of applications. For example, oligonucleotides are useful for therapeutic, diagnostic, research, and new nanomaterial applications. The use of naturally occurring nucleic acids (e.g., unmodified DNA or unmodified RNA) is limited, for example, by their susceptibility to endonucleases and exonucleases. To avoid these shortcomings, various synthetic counterparts have been developed. These include synthetic oligonucleotides that contain chemical modifications, such as base modifications, sugar modifications, backbone modifications, etc., that make these molecules less susceptible to degradation and improve other properties of the oligonucleotide. Chemical modifications may also result in certain undesirable effects, such as increased toxicity.

[0140] The present disclosure embraces the recognition that structural elements of an oligonucleotide, such as base sequence, chemical modifications (e.g., modifications of the sugar, base or internucleotide linkages and their pattern) or stereochemistry (e.g., the stereochemistry of the backbone chiral centers (chiral internucleotide linkages) or their pattern), can greatly affect properties, such as stability, ability to alter splicing, etc. In some embodiments, oligonucleotide properties can be tailored by optimizing chemical modifications (modifications of the base, sugar or internucleotide linkages) or stereochemistry (pattern of backbone chiral centers).

[0141] 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, the provided compositions comprising oligonucleotides with chemical modifications (e.g., base modifications, sugar modifications, internucleotide linkage modifications, etc.) have improved properties, e.g., improved ability to alter splicing, or improved protein binding profile, or improved delivery, etc. In particular, in some embodiments, the present disclosure provides compositions and methods for altering transcript splicing. In some embodiments, the altered transcript splicing by the provided compositions and methods includes the production of a product with a desired or improved biological function, or the knockdown of an undesired product, e.g., by modifying the splicing product so that the undesired biological function can be suppressed or eliminated.

[0142] In some embodiments, the splicing product is mRNA.In some embodiments, the change comprises skipping one or more exons.In some embodiments, the splicing of transcript is improved in that exon skipping increases the level of mRNA and protein with improved beneficial activity compared to the absence of exon skipping.

[0143] In some embodiments, the splicing of transcripts is improved in that exon skipping reduces the level of mRNA and protein with undesired activity compared to the absence of exon skipping.In some embodiments, target is knocked down by exon skipping, which causes premature stop codon or frameshift mutation by skipping one or more exons.

[0144] In some embodiments, the oligonucleotide of the present disclosure comprises one or more natural nucleobases, or one or more modified nucleobases derived from natural nucleobases.Examples include, but are not limited to, uracil, thymine, adenine, cytosine and guanine, each amino group of which is protected by acyl protecting group, 2-fluorouracil, 2-fluorocytosine, 5-bromouracil, 5-iodouracil, 2,6-diaminopurine, azacytosine, pyrimidine analogues such as pseudoisocytosine and pseudouracil, and other modified nucleobases such as 8-substituted purine, xanthine or hypoxanthine (the latter two are natural decomposition products).

[0145] Modified nucleobases also include extended size nucleobases to which one or more aryl rings, such as a phenyl ring, have been added.

[0146] In some embodiments, a modified nucleobase has any one of the following structures, optionally substituted:

[0147] [ka]

[0148] In some embodiments, modified nucleobase is unsubstituted. In some embodiments, modified nucleobase is substituted. In some embodiments, modified nucleobase is substituted to contain heteroatoms, alkyl groups or binding moieties, such as fluorescent moieties, biotin moieties or avidin moieties, or other proteins or peptides. In some embodiments, modified nucleobase is not a nucleobase in the most classical sense, but is a universal base that functions similarly to a nucleobase. One representative example of such a universal base is 3-nitropyrrole.

[0149] In some embodiments, the oligonucleotides described herein include nucleosides incorporating modified nucleobases 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 ... 6 -Isopentenyl adenosine;1-Methyl adenosine;1-Methyl pseudouridine;1-Methyl guanosine;l-Methyl inosine;2,2-Dimethyl guanosine;2-Methyl adenosine;2-Methyl guanosine;N 7 -Methylguanosine; 3-methyl-cytidine; 5-methylcytidine; 5-hydroxymethylcytidine; 5-methylcytosine, 5-formylcytosine; 5-carboxylcytosine; N 6 -Methyladenosine;7-Methylguanosine;5-Methylaminoethyluridine;5-Methoxyaminomethyl-2-thiouridine;β,D-mannosylqueosine;5-Methoxycarbonylmethyluridine;5-Methoxyuridine;2-Methylthio-N 6 -isopentenyladenosine; N-((9-β,D-ribofuranosyl-2-methylthiopurin-6-yl)carbamoyl)threonine; N-((9-β,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.

[0150] In some embodiments, the nucleoside has either (R)- or (S)-chirality at the 6' position and comprises a 6' modified bicyclic nucleoside analog, including the analogs described in U.S. Patent No. 7,399,845. In other embodiments, the nucleoside has either (R)- or (S)-chirality at the 5' position and comprises a 5' modified bicyclic nucleoside analog, including the analogs described in U.S. Patent Publication No. 20070287831. In some embodiments, the nucleobase or modified nucleobase is 5-bromouracil, 5-iodouracil, or 2,6-diaminopurine. In some embodiments, the nucleobase or modified nucleobase is modified by substitution with a fluorescent moiety.

[0151] In some embodiments, the oligonucleotide described herein comprises one or more modified nucleotides, in which the phosphate group or the linking phosphorus in the nucleotide is linked to various positions of the sugar or modified sugar.As a non-limiting example, the phosphate group or the linking phosphorus can be linked to the 2', 3', 4' or 5' hydroxyl moiety of the sugar or modified sugar.In this context, the nucleotide incorporating the modified nucleobase described herein is also contemplated.

[0152] Other modified sugars can also be incorporated into the oligonucleotide molecules. In some embodiments, the modified sugar is -F; -CF3, -CN, -N3, -NO, -NO2, -OR', -SR', or -N(R')2, where each R' is independently as defined above and described herein; -O-(C1-C 10 Alkyl), -S-(C1-C 10 Alkyl), -NH-(C1-C 10 Alkyl), or -N(C1-C 10 Alkyl)2;-O-(C2-C 10 alkenyl), -S-(C2-C 10 alkenyl), -NH-(C2-C 10 alkenyl), or -N(C2-C 10 alkenyl)2;-O-(C2-C 10alkynyl), -S-(C2-C 10 alkynyl), -NH-(C2-C 10 alkynyl), or -N(C2-C 10 alkynyl)2; or -O--(C1-C 10 Alkylene)-O--(C1-C 10 Alkyl), -O-(C1-C 10 Alkylene)-NH-(C1-C 10 Alkyl), or -O-(C1-C 10 Alkylene)-NH(C1-C 10 Alkyl)2, -NH-(C1-C 10 Alkylene)-O-(C1-C 10 Alkyl), or -N(C1-C 10 Alkyl)-(C1-C 10 Alkylene)-O-(C1-C 10 The 2'-position contains one or more substituents including one of the following: -O(CH2) -alkyl (wherein alkyl, alkylene, alkenyl, and alkynyl may be substituted or unsubstituted). Examples of substituents include, but are not limited to, -O(CH2) n OCH3, and -O(CH2) n Examples include NH2 (wherein n is 1 to about 10), MOE, DMAOE, and DMAEOE.

[0153] In some embodiments, the 2'-OH of ribose is -H, -F; -CF3, -CN, -N3, -NO, -NO2, -OR', -SR', or -N(R')2 (wherein each R' is independently defined above and as described herein); -O-(C1-C2 10 Alkyl), -S-(C1-C 10 Alkyl), -NH-(C1-C 10 Alkyl), or -N(C1-C 10 Alkyl)2;-O-(C2-C 10 alkenyl), -S-(C2-C 10 alkenyl), -NH-(C2-C 10 alkenyl), or -N(C2-C 10 alkenyl)2;-O-(C2-C 10alkynyl), -S-(C2-C 10 alkynyl), -NH-(C2-C 10 alkynyl), or -N(C2-C 10 alkynyl)2; or -O--(C1-C 10 Alkylene)-O--(C1-C 10 Alkyl), -O-(C1-C 10 Alkylene)-NH-(C1-C 10 Alkyl), or -O-(C1-C 10 Alkylene)-NH(C1-C 10 Alkyl)2, -NH-(C1-C 10 Alkylene)-O-(C1-C 10 Alkyl), or -N(C1-C 10 Alkyl)-(C1-C 10 Alkylene)-O-(C1-C 10 In some embodiments, the 2'-OH is substituted by one of the following substituents: -alkyl, -alkylene, -alkenyl, and -alkynyl, where alkyl, alkylene, alkenyl, and alkynyl may be substituted or unsubstituted. In some embodiments, the 2'-OH is substituted by -H (deoxyribose). In some embodiments, the 2'-OH is substituted by -F. In some embodiments, the 2'-OH is substituted by -OR'. In some embodiments, the 2'-OH is substituted by -OMe. In some embodiments, the 2'-OH is substituted by -OCH2CH2OMe (MOE).

[0154] 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'-.

[0155] [ka]

[0156] In some embodiments, the present invention provides oligonucleotides that include one or more modified internucleotide linkages independently having the structure of Formula I:

[0157] [ka]

[0158] During the ceremony, P* is an asymmetric phosphorus atom, either Rp or Sp; W is O, S or Se; Each of X, Y and Z is independently -O-, -S-, -N(-LR 1 )- or L; L is a covalent bond or an optionally substituted linear or branched C1-C 10 alkylene, wherein one or more methylene units of L are optionally substituted C1-C6 alkylene, C1-C6 alkenylene,

[0159] [ka]

[0160] , -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 C1-C 50 aliphatic, wherein one or more methylene units are optionally substituted C1-C6 alkylene, C1-C6 alkenylene,

[0161] [ka]

[0162] , -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

[0163] [ka]

[0164] represents an independent connection to a nucleoside.

[0165] In some embodiments, the internucleotide linkage having the structure of Formula I is:

[0166] [ka]

[0167] In particular, the present disclosure provides oligonucleotides of various designs that may include various nucleobases and patterns thereof, sugars and patterns thereof, internucleotide bonds and patterns thereof, or additional chemical moieties and patterns thereof, as described in the present disclosure. In some embodiments, the provided oligonucleotides can downregulate MuSK Ig2 domain protein expression, MuSK Ig2 domain gene expression, or MuSK Ig2 activation of BMP signaling levels via BMP receptors, thereby increasing adult hippocampal neurogenesis (AHN), improving cognition in AD, or achieving muscle regeneration and muscle growth (e.g., inducing, enhancing, etc.). In some embodiments, the provided oligonucleotides can lead to a decrease in the expression, level, or activity of one or more of the MuSK Ig2 domains, or products thereof, in cells of a subject or patient. In some embodiments, the cells normally express or produce proteins encoded by MuSK Ig2 domains. In some embodiments, oligonucleotides are provided that are capable of directing a decrease in expression, level or activity of a MuSK Ig2 domain gene or a MuSK Ig2 domain gene product, and have a base sequence that consists of, includes, or includes a portion thereof (e.g., a stretch of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more contiguous bases) of the base sequence of an oligonucleotide disclosed herein, where each T can be independently replaced by U, or vice versa, and the oligonucleotide comprises at least one non-naturally occurring modification of the base, sugar or internucleotide linkage.

[0168] As described herein, highly abundant full-length MuSK has a BMP receptor (ALK)-binding Ig2 domain, enhances BMP signaling, and thus inhibits neurogenesis and muscle regeneration / growth (e.g., myogenesis). In contrast, ΔIg2-MuSK reduces BMP signaling through BMP receptor (ALK), promotes AHN, and improves cognition. In some embodiments, the present disclosure provides an exon-skipping ASO that switches MuSK from AHN-restricted full-length MuSK to AHN-permissive ΔIg2-MuSK splice form.

[0169] In some embodiments, the one or more skipped exons are selected from exons 5a and 5b or the MuSK gene. In some embodiments, exon 5a of MuSK is skipped. In some embodiments, exon 5b of MuSK is skipped. In some embodiments, both exons 5a and 5b of MuSK are skipped.

[0170] In various embodiments, the active compound is an oligonucleotide that induces skipping of one or more exons in the MuSK gene. In various embodiments, the active compound is an oligonucleotide that induces skipping of multiple exons in the MuSK gene. In some embodiments, the active compound is an oligonucleotide that induces skipping of exon 5a, exon 5b, or both in the MuSK gene. In some embodiments, the active compound is an oligonucleotide that induces skipping of exon 5a in the MuSK gene. In some embodiments, the active compound is an oligonucleotide that induces skipping of exon 5b in the MuSK gene. In some embodiments, the active compound is an oligonucleotide that induces skipping of exons 5a and 5b in the MuSK gene. In some embodiments, multiple oligonucleotides can be used together. In some such embodiments, two or more different exon skipping oligonucleotides (e.g., at least one that induces skipping of exon 5a and one that induces skipping of exon 5b) can be used in combination. Alternatively or additionally, in some embodiments, at least one exon skipping oligonucleotide may be used in combination with at least one degradative oligonucleotide that may target a MuSK transcript that contains, for example, a functional Ig2 domain or a portion thereof (e.g., targets the transcript for RNase H degradation).

[0171] In some embodiments, oligonucleotides are provided or utilized in salt form. In some embodiments, oligonucleotides are provided as salts that contain negatively charged internucleotide bonds (e.g., phosphorothioate internucleotide bonds, natural phosphate bonds, etc.) present in their salt form. In some embodiments, oligonucleotides are provided as pharma- ceutically acceptable salts. In some embodiments, oligonucleotides are provided as metal salts. In some embodiments, oligonucleotides are provided as sodium salts. In some embodiments, oligonucleotides are provided as metal salts, e.g., sodium salts, where each negatively charged internucleotide bond is independently in salt form (e.g., -OP(O)(SNa)-O- for phosphorothioate internucleotide bonds, -OP(O)(ONa)-O- for natural phosphate bonds, etc., in sodium salts).

[0172] Model Among other things, the present disclosure provides model systems useful as described herein. For example, in some embodiments, the provided model systems can be used to screen, validate, characterize, evaluate or identify one or more MuSK MR agonizing agents.

[0173] In some embodiments, the model systems provided herein are or include an artificially engineered cell line. In some embodiments, the engineered cell line is an immortalized MuSK- / - myogenic cell line.

[0174] In some embodiments, the model system provided herein is or comprises an engineered mouse described herein, hi some embodiments, the mouse provided is or comprises a ΔIg2-MuSK mouse.

[0175] In some embodiments, the model systems provided (e.g., cell lines or mice) are used to screen, validate, characterize, evaluate, or identify agents described herein, including, for example, small molecule agents, antibody agents, oligonucleotide agents, etc., and combinations thereof. In some embodiments, the activity of such agents is compared to a suitable reference (e.g., a positive control or a negative control). In some embodiments, a suitable reference can be or include the absence of any agent, or the presence of an agent of known activity or performance in the model system. In some embodiments, a suitable reference can be a historical reference. In some embodiments, a suitable reference can be a current reference or a contemporaneous reference.

[0176] In some embodiments, the co-crystal structure of model system MuSK and BMP receptor provided herein. In some embodiments, the co-crystal structure can be used to guide the design of small molecule MuSK MR agonizing agents through computer-based simulation. In some embodiments, the co-crystal structure can be used to guide high-throughput screening of small molecule MuSK MR agonizing agents. In some embodiments, the small molecule MuSK MR agonizing agent targets MuSK. In some embodiments, the small molecule MuSK MR agonizing agent targets one or more of type I BMP receptors, ALK3 (ALK is anaplastic lymphoma kinase) and ALK6, and type I activin receptor ALK4, such as ALK inhibitors (e.g., crizotinib, ceritinib, alectinib, brigutinib, lorlatinib).

[0177] Characterization of MuSK Ig2 modulators.

[0178] MuSK Ig2 modulator agents may be identified, evaluated or characterized for one or more of their physical / chemical properties or biological activities.Those skilled in the biomedical arts will be aware of a variety of techniques, including specific assays, that may be used for such identification, evaluation or characterization.

[0179] Small molecule MuSK Ig2 regulator agent can, for example, directly bind to MuSK Ig2 or BMP receptor, thereby preventing the interaction between MuSK Ig2 and BMP receptor.Such agents can be characterized, for example, by direct binding assays that evaluate their affinity, specificity, or one or more kinetic or thermodynamic characteristics of their interaction with their target, MuSK Ig2 or BMP receptor, or, for example, by competitive binding assays that evaluate their ability to disrupt or reduce the excessive preformed complex of MuSK Ig2 and BMP receptor.Such binding assays are desirably carried out at several concentrations.Such binding assays can also be carried out with full-length MuSK, or with any other polypeptide or agent that is or contains MuSK Ig2.

[0180] A MuSK Ig2 modulator agent, e.g., an antibody or small molecule that binds to the Ig2 domain of MuSK, when contacted with a cell expressing MuSK, competes with the BMP receptor for binding of the Ig2 domain. Such an antibody agent specifically binds to an epitope of MuSK that is expressed in a particular cell type, e.g., a neuronal cell type. Such an antibody agent has a binding affinity for a MuSK protein, e.g., the Ig2 domain of a MuSK protein, of at least about 10, e.g., as measured by a dissociation constant. -4 M, at least about 10 -5 M, at least about 10 -6 M, at least about 10 -7 M, at least about 10 -8 M, at least about 10 -9M or less. Those skilled in the biomedical field know that binding affinity (e.g., when measured by dissociation constant) can be influenced by non-covalent intermolecular interactions such as hydrogen bonds, electrostatic interactions, hydrophobicity and van der Waals forces between two molecules. The binding affinity between a ligand and its target molecule can be influenced by other molecules. Those skilled in the biomedical field will be familiar with various techniques for measuring binding affinity or dissociation constant under the present disclosure, including, for example, but not limited to, ELISA, gel shift assay, pull-down assay, equilibrium dialysis, analytical ultracentrifugation, surface plasmon resonance (SPR), biolayer interferometry, grating binding interferometry and spectroscopy assay.

[0181] The competitive assay can identify antibodies that compete with the anti-MuSK antibody agents described herein for binding to the Ig2 domain of MuSK. Such competing antibodies bind to the same epitope in the Ig2 domain of MuSK that is bound by the anti-MuSK antibodies described herein. Exemplary epitope mapping methods are known. See, for example, Morris, Epitope mapping protocols, Methods in Molecular Biology, 66 (1996).

[0182] Assays can be provided to identify anti-MuSK antibody agents having biological activity. Assays can be provided to identify anti-MuSK antibody agents having neutralizing activity against MuSK. Antibody agents having such biological activity in vivo or in vitro can also be provided. Antibodies of the present disclosure can be assayed for such biological activity.

[0183] The biological activity of an anti-MuSK antibody agent can be, for example, binding affinity for a particular MuSK epitope, such as within the Ig2 domain, neutralization or inhibition of MuSK binding to BMP, in vivo MuSK activity, e.g., IC 50The term may refer to neutralization or inhibition of, pharmacokinetics, and cross-reactivity with, for example, non-human homologs or orthologs of MUSK protein, or with other proteins or tissues. Other biological properties or characteristics of antigen-binding agents recognized in the biomedical arts may include, for example, binding activity, selectivity, solubility, folding, immunotoxicity, expression and formulation. These properties or characteristics may be observed, measured or assessed using standard techniques, including, but not limited to, ELISA, competitive ELISA, surface plasmon resonance analysis (BIACORE™), or kinetic exclusion assay (KINEXA™), in vitro or in vivo neutralization assays, receptor-ligand binding assays, cytokine or growth factor production or secretion assays, and signal transduction and immunohistochemistry assays.

[0184] The MuSK Ig2 modulators described herein are characterized in that, for example, when a MuSK Ig2 modulator, e.g., an agonizing oligonucleotide, contacts a cell expressing MuSK, it increases the level or activity of MuSK ΔIg2 mRNA or MuSK ΔIg2 protein.

[0185] MuSK Ig2 regulator oligonucleotide is characterized by its ability to change the splicing activity of MuSK pre-mRNA in cells.For example, cells can be transfected with MuSK Ig2 regulator oligonucleotide, and after a period of incubation, the expression of alternative forms of the processed form of MuSK RNA transcripts, for example, when exons 5a and 5b are skipped, can be measured by RT-PCR.For example, the efficiency of MuSK exon skipping in cultured cells is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more than 95%.

[0186] MuSK Ig2 modulator oligonucleotides can increase MuSK ΔIg2 mRNA. MuSK Ig2 modulator oligonucleotides can alter splicing of MuSK pre-mRNA. MuSK Ig2 modulator oligonucleotides can promote skipping or inclusion of exon 5a or exon 5b.

[0187] The regulation of MuSK ΔIg2 expression can be measured for the body fluids of the subject treated with MuSK Ig2 regulator oligonucleotide, which can contain cells; tissues; or animal organs.The method of obtaining samples for analysis, such as body fluids, for example, sputum, serum or CSF, tissues, for example, biopsies or organs, and the method of preparing samples to enable analysis are well known to those skilled in the biomedical field.The effect of treatment on a subject can be evaluated by measuring the biomarkers related to target gene expression in one or more biological fluids, tissues or organs taken from an animal that has been contacted with one or more compositions described in this application.

[0188] An increase in MuSK ΔIg2 mRNA means that the intracellular level of MuSK ΔIg2 mRNA is higher than a reference level, for example, the level of MuSK ΔIg2 mRNA in a control (e.g., a subject not administered a MuSK Ig2 modulator oligonucleotide). An increase in intracellular MuSK ΔIg2 mRNA can be measured as an increase in the level of MuSK ΔIg2 protein or MuSK ΔIg2 mRNA produced. An increase in MuSK ΔIg2 mRNA can be determined, for example, by the methods described herein, or by assay techniques, such as RNA solution hybridization, nuclease protection, Northern hybridization, reverse transcription, gene expression monitoring by microarray, antibody binding, enzyme-linked immunosorbent assay (ELISA), nucleic acid sequencing, Western blotting, radioimmunoassay (RIA), other immunoassays, fluorescence activated cell analysis (FACS), or any other technique or combination of techniques capable of detecting MuSK ΔIg2 mRNA or MuSK ΔIg2 protein in a subject, or in a sample obtained from a subject.

[0189] By comparing the level of MuSK ΔIg2 mRNA in the sample obtained from the subject receiving MuSK Ig2 modulator oligonucleotide treatment with the level of MuSK ΔIg2 mRNA in the subject not treated with MuSK Ig2 modulator oligonucleotide, the extent to which MuSK Ig2 modulator oligonucleotide treatment increases MuSK ΔIg2 mRNA can be determined.The reference level of MuSK ΔIg2 mRNA is obtained from the same subject before receiving MuSK Ig2 modulator oligonucleotide treatment.The reference level of MuSK ΔIg2 mRNA is a range determined by a population of subjects not receiving MuSK Ig2 modulator oligonucleotide treatment.The level of full-length MuSK mRNA is compared to the level of MuSK ΔIg2 mRNA. For example, a ratio of MuSK ΔIg2 mRNA to full length MuSK mRNA, e.g., MuSK mRNA without exons 5a and 5b, in a subject receiving MuSK Ig2 modulator oligonucleotide therapy that is greater than 1 fold, 1.5-5 fold, 5-10 fold, 10-50 fold, 50-100 fold, about 1.1 fold, about 1.2 fold, about 1.5 fold, about 2 fold, about 3 fold, about 4 fold, about 5 fold, about 6 fold, about 7 fold, about 8 fold, about 9 fold, about 10 fold, or greater than a reference ratio.

[0190] The increased level of MuSK ΔIg2 mRNA may be, for example, greater than 1 fold, 1.5-5 fold, 5-10 fold, 10-50 fold, 50-100 fold, about 1.1 fold, about 1.2 fold, about 1.5 fold, about 2 fold, about 3 fold, about 4 fold, about 5 fold, about 6 fold, about 7 fold, about 8 fold, about 9 fold, about 10 fold, about 15 fold, about 20 fold, about 30 fold, about 40 fold, about 50 fold, about 60 fold, about 70 fold, about 80 fold, about 90 fold, or about 100 fold of the reference value.

[0191] The increase in MuSK ΔIg2 mRNA in a subject can be indicated by an increase in MuSK ΔIg2 protein compared to a reference level. The reference level of MuSK ΔIg2 protein is, for example, the MuSK ΔIg2 protein level obtained from a subject having or at risk of having Alzheimer's disease or a disease characterized by neurodegeneration before treatment. A method is also contemplated in which a body fluid, organ or tissue is contacted with an effective amount of one or more compositions described herein. A body fluid, organ or tissue can be contacted with one or more compositions comprising MuSK Ig2 regulator oligonucleotide to cause the expression of MuSK ΔIg2 in the cells of the body fluid, organ or tissue and the regulation of MuSK expression. An effective amount can be determined by monitoring the effect of the MuSK Ig2 regulator oligonucleotide administered to a subject or contacted to a cell on the functional MuSK ΔIg2 protein expression.

[0192] MuSK Ig2 regulators, when administered to a population of cells, for example, containing neural stem cells or neural progenitor cells (NPCs), increase the number of cells in an activated state, for example, active proliferation.Cells in the population can be evaluated for their activated state by methods known in the biomedical field, including, for example, EdU assay, which compares EdU+ cycling cells with total cell number.MuSK Ig2 regulators, when administered to a population of cells, containing neural stem cells, decrease the number of quiescent neural stem cells in the population, or increase the number of activated neural stem cells.

[0193] MuSK Ig2 modulator agents, when administered to a population of cells comprising neural stem cells or neural progenitor cells, increase the number of cells expressing genes associated with early neurons, e.g., Dex, or decrease the number of cells expressing genes associated with mature neurons, e.g., Map2, astrocytes, e.g., GFAP and S100b, or oligodendrocytes, e.g., CNPase and O4. MuSK Ig2 modulator agents, when administered to a population of cells comprising neural stem cells or neural progenitor cells, increase the level of expression of genes associated with early neurons, e.g., Dex, or decrease the level of expression of genes associated with mature neurons, e.g., Map2, astrocytes, e.g., GFAP and S100b, or oligodendrocytes, e.g., CNPase and O4, within the population of cells.

[0194] The population of cells includes neural stem cells that have been induced to be neural stem cells from stem cells, such as embryonic stem cells or pluripotent stem cells.

[0195] The population of cells is obtained from a healthy subject. The population of cells is obtained from a subject suffering from a disease characterized by neurodegeneration or muscular disorders.

[0196] When the MuSK Ig2 modulator is contacted with a population of cells derived from a subject, it increases neurogenesis or muscle regeneration or muscle growth in the subject. The MuSK Ig2 modulator is contacted with a population of cells in vivo, for example, by injection into the subject. The MuSK Ig2 modulator is contacted with a population of cells ex vivo by obtaining a population of cells from the subject, and when the treated cells are reintroduced into the subject, it increases neurogenesis or muscle regeneration or muscle growth.

[0197] MuSK Ig2 modulator agents, when administered to a subject, increase neurogenesis, improve cognition, or achieve muscle regeneration or growth. Exemplary methods for assessing these biological effects are detailed herein.

[0198] Production of agonizing antibodies Antibodies and antigen-binding fragments can be prepared or purified by any technique known in the biomedical art that allows for the subsequent formation of a stable antibody or antibody fragment.

[0199] Nucleic acid encoding an anti-MuSK antibody agent of the disclosure can be readily isolated and sequenced by conventional procedures.

[0200] The expressed antibody of the present disclosure can be purified to homogeneity after being isolated from host cells. The isolation or purification of the antibody of the present disclosure can be performed by conventional methods for isolating and purifying proteins. For example, without wishing to be bound by theory, the MuSK antibody agent of the present disclosure can be recovered and purified from recombinant cell culture by well-known methods, including, but not limited to, protein purification, protein G purification, ammonium sulfate precipitation or ethanol precipitation, acid extraction, anion exchange chromatography or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. High performance liquid chromatography (HPLC) can also be used for purification. The antibody of the present disclosure can be isolated or purified by further combinations of filtration, ultrafiltration, salting out, dialysis, etc.

[0201] Purified anti-MuSK agents of the present disclosure may be characterized by ELISA, ELISPOT, flow cytometry, immunocytology, BIACORE™ analysis, SAPIDYNE KINEXA™ kinetic exclusion assay, SDS-PAGE and Western blot, or by HPLC analysis, as well as by several other functional assays disclosed herein.

[0202] Production of agonizing oligonucleotides Agonizing agents, such as the agonizing oligonucleotides described herein, can be synthesized by standard methods known in the biomedical field, for example, by using an automatic synthesizer.After chemical synthesis, for example, solid-phase synthesis using phosphoramidite method), agonizing oligonucleotide molecules can be deprotected, annealed to ds molecules, and purified, for example, by gel electrophoresis or HPLC.Protocols for the preparation of agonizing oligonucleotides are known in the biomedical field.

[0203] Agonizing oligonucleotides can also be formed in cells by transcription of RNA from expression constructs introduced into cells.See, for example, Yu et al., Proc.Natl.Acad.Sci.USA, 99, 6047-6052 (2002).The expression construct for the in vivo production of agonizing oligonucleotide molecules can comprise one or more antisense coding sequences operably linked to the elements required for proper transcription of antisense coding sequences, including, for example, promoter elements and transcription termination signals. Preferred promoters for such expression constructs include polymerase III HI-RNA promoter (see, for example, Brummelkamp et al., Science 2002;296:550-553) and U6 polymerase-III promoter (see, for example, Sui et al., Proc. Natl. Acad. Sci. USA 2002; Paul et al., Nature Biotechnol., 20,505-508 (2002); and Yu et al., Proc. Natl. Acad. Sci. USA, 99,6047-6052) (2002). The agonizing oligonucleotide expression construct can further include one or more vector sequences that facilitate cloning of the expression construct. Standard vectors that can be used include, for example, pSilencer 2.0-U6 vector (Ambion Inc., Austin, TX, USA).

[0204] Pharmaceutical compositions.

[0205] The present disclosure provides pharmaceutical compositions that contain or deliver the agonizing agents described herein. The present disclosure also provides pharmaceutical compositions that are or contain cell populations exposed to the agonizing agents described herein.

[0206] For example, provided pharmaceutical compositions can include or deliver a MuSK Ig2 modulator agent, such as an antibody or nucleic acid agent, that upon administration achieves an increase in the level or activity of a MuSK polypeptide, e.g., a MuSK ΔIg2 polypeptide, or another Ig2-disrupted MuSK mutant polypeptide that lacks a functional Ig2 domain for interaction with BMP. Provided pharmaceutical compositions can include or deliver a population of cells exposed to a MuSK Ig2 modulator agent, such that the number or activity of neuronal cells is increased within the population.

[0207] A pharmaceutical composition may be or may contain an active agent, such as an agonizing agent as described herein, or a precursor thereof, in combination with one or more pharma- ceutically acceptable excipients.Those skilled in the biomedical arts will know that the components of a particular pharmaceutical composition may be affected by the route of administration of the pharmaceutical composition.

[0208] The compositions of the present disclosure can be formulated for a variety of modes of administration, including systemic and topical or localized administration. Techniques and formulations can be found generally in Remington, The Science and Practice of Pharmacy (20th ed. 2000).

[0209] The compositions of the present invention can be prepared and administered in several oral, parenteral and topical dosage forms.The compositions of the present invention can be administered by injection, for example, intravenously, intramuscularly, intradermally, subcutaneously, intraduodenally or intraperitoneally.The compositions described herein can be administered by inhalation, for example, intranasally.The compositions of the present invention can be administered transdermally.Several administration routes can be used to administer the compositions of the present invention, for example, intramuscularly, orally, transdermally.

[0210] 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 aural administration.The pharmaceutical compositions may be formulated for delivery by intrathecal administration.The pharmaceutical compositions may be formulated for delivery by intravenous administration.The pharmaceutical compositions may be formulated for delivery by oral administration.

[0211] The oligonucleotides and compositions can be delivered to the central nervous system. The oligonucleotides and compositions can be delivered to the cerebrospinal fluid. In certain embodiments, the oligonucleotides and compositions can be administered to the brain parenchyma. The oligonucleotides and compositions can be delivered to animals / subjects by intrathecal or intraventricular administration. The broad distribution of the oligonucleotides and compositions described herein in the central nervous system can be achieved by intraparenchymal, intrathecal or intraventricular administration.

[0212] Parenteral administration can be by injection, e.g., syringe, pump, etc. The injection can be a bolus injection. The injection can be administered directly into tissues such as the striatum, caudate, cortex, hippocampus, and cerebellum.

[0213] Methods that specifically localize a drug, such as by bolus injection, are used to measure the median effective concentration (EC 50 ) can be reduced by 20, 25, 30, 35, 40, 45 or 50 fold. Pharmaceuticals in antisense compounds as further described herein. The target tissue can be brain tissue. The target tissue can be hippocampal tissue. EC 50A reduction in may be desirable since it would reduce the dose required to achieve a pharmacological result in a patient in need thereof.

[0214] Antisense oligonucleotides can be delivered by injection or infusion once a month, once every two months, once every 90 days, once every three months, once every six months, twice a year, or once a year.

[0215] These pharmaceutical compositions may also contain suitable pharma- ceutically acceptable carriers, including excipients and auxiliaries that facilitate the processing of the active compounds into pharma- ceutically usable preparations. Preparations formulated for oral administration may be tablets, dragees, capsules or solutions.

[0216] The pharmaceutical preparation for oral use can be obtained by combining active compound with solid excipient, optionally grinding the mixture obtained, and processing the mixture of granules after adding suitable auxiliaries if necessary to obtain tablets or dragee cores.Suitable excipients are 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, hydroxypropylmethyl-cellulose, sodium carboxymethyl-cellulose (CMC) or polyvinylpyrrolidone (PVP: povidone).Disintegrants such as crosslinked polyvinylpyrrolidone, agar, or alginic acid or its salts, such as sodium alginate, can be added.

[0217] Dragee cores are provided with suitable coatings.Can use gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol (PEG) or titanium dioxide, lacquer solutions and concentrated sugar solutions that can optionally contain suitable organic solvents or solvent mixtures.Dyes or pigments can be added to tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

[0218] The pharmaceutical preparations that can be used orally include push-fit capsules made of gelatin, and soft sealed capsules made of gelatin, and plasticizers such as glycerol or sorbitol.The push-fit capsules can contain active ingredients mixed with fillers such as lactose, binders such as starch, or lubricants such as talc or magnesium stearate, and optionally stabilizers.In soft capsules, active compounds can be dissolved or suspended in suitable liquids such as fatty oils, liquid paraffin, or liquid polyethylene glycol (PEG).Stabilizers can be added.

[0219] The pharmaceutical composition can be a tablet, pill, capsule, liquid, inhalant, nasal solution, suppository, suspension, gel, colloid, dispersion, suspension, solution, emulsion, ointment, lotion, eye drops or ear drops.

[0220] Depending on the specific condition to be treated, the pharmaceutical composition of the present disclosure may be formulated into a liquid or solid dosage form and administered systemically or locally. The pharmaceutical composition may be delivered, for example, in a timed or sustained low release form, as known to those skilled in the biomedical field. Techniques for formulation and administration may be found in Remington, The Science and Practice of Pharmacy (20th ed.2000). Suitable routes may include oral administration, buccal administration by inhalation spray, sublingual administration, rectal administration, transdermal administration, vaginal administration, transmucosal administration, nasal administration or intestinal administration; parenteral delivery, including intramuscular injection, subcutaneous injection, intramedullary injection, and intrathecal injection, direct intraventricular injection, intravenous injection, intraarticular injection, intrasternal injection, intrasynovial injection, intrahepatic injection, intralesional injection, intracranial injection, intraperitoneal injection, intranasal injection or intraocular injection, or other delivery modes.

[0221] For injection, the pharmaceutical composition of the present disclosure can be formulated and diluted in aqueous solutions, such as physiologically compatible buffers, such as Hanks' solution, Ringer's solution or physiological saline buffer.For such transmucosal administration, a penetrant suitable for the barrier to be permeated is used in the formulation.Such penetrants are well known in the biomedical field.

[0222] The use of pharma- ceutically acceptable inert carriers to formulate the compositions disclosed herein for carrying out the present disclosure into dosages suitable for systemic administration is within the scope of the present disclosure.By appropriate selection of carriers and suitable manufacturing practices, the compositions of the present disclosure formulated as solutions can be administered parenterally, such as by intravenous injection.

[0223] The compositions described herein can be formulated into dosages suitable for oral administration using pharma- ceutically acceptable carriers available in the biomedical field.Such carriers allow the compounds of the present disclosure to be formulated as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions, etc. for oral ingestion by subjects, e.g., patients to be treated.

[0224] For nasal or inhalation delivery, one or more solubilizing, diluent or dispersing substances, such as saline, preservatives, such as benzyl alcohol, absorption enhancers and fluorocarbons, may be used.

[0225] The provided compositions can include or deliver a precursor of an active agent, which upon administration becomes or releases an active therapeutic agent. For example, the precursor can be or include a prodrug of a small molecule agonizing agent, or a nucleic acid encoding a protein agonizing agent, etc.

[0226] The provided pharmaceutical compositions can include or deliver a therapeutically effective amount (e.g., an amount effective when administered according to an established protocol) of the provided oligonucleotide, which can be provided in a pharma- ceutically acceptable salt form, e.g., as a sodium salt, ammonium salt, etc. Such provided pharmaceutical compositions include the relevant oligonucleotide and at least one pharma- ceutically acceptable inactive ingredient selected from a pharma- ceutically acceptable diluent, a pharma-ceutically acceptable excipient, and a pharma-ceutically acceptable carrier. The salt form of the provided oligonucleotide contains two or more cations, up to the number of negatively charged acidic groups, e.g., phosphates, phosphorothioates, etc., in the oligonucleotide.

[0227] Pharmaceutically acceptable salts are generally well known to those skilled in the biomedical arts and include, for example, 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, ), hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, malate, maleate, mandelate, mesylate, mucate, napsylate, nitrate, pamoate (embonate), pantothenate, phosphate / diphosphate, polygalacturonate, salicylate, stearate, subacetate, succinate, sulfate, tannate, tartrate or teoclate. Other pharma- ceutically acceptable salts can be found, for example, in Remington, The Science and Practice of Pharmacy (20th ed. 2000). Preferred pharma- ceutically acceptable salts include, for example, acetate, benzoate, bromide, carbonate, citrate, gluconate, hydrobromide, hydrochloride, maleate, mesylate, napsylate, pamoate (embonate), phosphate, salicylate, succinate, sulfate or tartrate salts.

[0228] As known to those skilled in the biomedical field, oligonucleotides can be formulated as several salts, for example, for pharmaceutical use. The salt is a metal cation salt or ammonium salt. The salt is a metal cation salt of oligonucleotide. The salt is an ammonium salt of oligonucleotide. Representative alkali metal salts or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc. The salt is a sodium salt of oligonucleotide. Pharmaceutically acceptable salts include, where appropriate, non-toxic ammonium, quaternary ammonium, and amine cations formed by counterions such as hydroxide, carboxylate, sulfate, phosphate, nitrate, sulfonate, phosphorothioate, etc. that may be present in the provided oligonucleotide. As recognized by those skilled in the biomedical field, oligonucleotide salts can contain multiple cations, for example, sodium ions, since multiple anions may be present in the oligonucleotide.

[0229] The provided oligonucleotides and compositions thereof can be effective over a wide dosage range. For example, in the treatment of an adult, dosages of about 0.01 to about 1000 mg, about 0.5 to about 100 mg, about 1 to about 50 mg / day, and about 5 to about 100 mg / day are examples of dosages that can be used. The exact dosage depends on the route of administration, the form in which the compound is administered, the subject being treated, the weight of the subject being treated, and the preference and experience of the attending physician.

[0230] The present disclosure provides techniques, e.g., compositions, methods, etc., for combination therapy with, e.g., other therapeutic agents or medical procedures. The provided oligonucleotides or compositions may be used with one or more other therapeutic agents. The provided compositions include the provided oligonucleotides and one or more other therapeutic agents. The one or more other therapeutic agents may have one or more different targets, or one or more different mechanisms for a target, when compared to the provided oligonucleotides in the composition. The therapeutic agent is an oligonucleotide. The therapeutic agent is a small molecule drug. The therapeutic agent is a protein. The therapeutic agent is an antibody. Several therapeutic agents can be used according to the present disclosure. The provided oligonucleotides or compositions thereof are administered before, simultaneously with, or after one or more other therapeutic agents or medical procedures. The provided oligonucleotides or compositions thereof are administered simultaneously with one or more other therapeutic agents or medical procedures. The provided oligonucleotides or compositions thereof are administered before one or more other therapeutic agents or medical procedures. The provided oligonucleotides or compositions thereof are administered after one or more other therapeutic agents or medical procedures. The provided compositions include one or more other therapeutic agents.

[0231] Production of pharmaceutical compositions.

[0232] For preparing pharmaceutical compositions from the compositions of the present disclosure, pharma-ceutically acceptable carriers can be either solid or liquid.Solid form preparations include powder, tablet, pill, capsule, cachet, suppository and dispersible granule.Solid carriers can be one or more substances that can also act as diluents, flavorings, binders, preservatives, tablet disintegrating agents or encapsulating materials.

[0233] In powders, the carrier is a finely divided solid in admixture with the finely divided active ingredient. In tablets, the active ingredient is mixed with a carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired.

[0234] Powders and tablets can contain 5% to 70% of the therapeutic agent. Suitable carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, low melting wax, cocoa butter, and the like. The term preparation includes formulations of active therapeutic agents that include encapsulating materials as carriers, providing a capsule in which the active ingredient, with or without other carriers, is surrounded by the carrier, thus associating with it. Similarly, cachets and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid dosage forms suitable for oral administration.

[0235] 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 by stirring The molten homogeneous mixture is then poured into convenient sized molds, allowed to cool and solidify.

[0236] 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.

[0237] When parenteral application is required or desired, particularly suitable mixtures for the compositions of the present invention are injectable sterile solutions in oily or aqueous solutions, as well as suspensions, emulsions or implants, including suppositories. In particular, carriers for parenteral administration include aqueous dextrose solutions, saline, pure 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 can 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 23 rdThis includes those described in “Eds. ed. (Elsevier, 2020).”

[0238] Aqueous solutions suitable for oral use can be prepared by dissolving the active ingredient in water and adding suitable colorants, flavors, stabilizers and thickeners, if desired. Aqueous suspensions suitable for oral use can be made by dispersing finely divided active ingredients in water using viscous materials such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose and other well-known suspending agents.

[0239] Also included are solid form preparations that are intended to be converted immediately before use into liquid form preparations for oral administration.Such liquid forms include solutions, suspensions and emulsions.These preparations can contain, in addition to the active ingredient, colorants, flavors, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizers, etc.

[0240] Pharmaceutical preparations can be in unit dosage form.In such form, the preparation is divided into unit doses containing appropriate amounts of active ingredients.The unit dosage form can be a packaged preparation, the package containing individual amounts of preparations, for example, packaged tablets, capsules, and powders in vials or ampoules.The unit dosage form can be a capsule, tablet, cachet or lozenge itself, or any of these in the appropriate number of packaged forms.

[0241] 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 can also contain other compatible therapeutic agents, if desired.

[0242] Administration Those skilled in the art will understand that in some embodiments, the dosage administered to a subject, particularly a human, may vary depending on, for example, the specific therapeutic agent or formulation used, the method of administration, the administration regimen, one or more characteristics of the particular subject being treated, etc. In some embodiments, a clinician in the art will determine the therapeutically effective amount of a therapeutic agent to be administered to a human or other subject to treat or prevent a particular medical condition. The exact amount of a therapeutic agent required to be therapeutically effective will depend on a number of factors, such as the specific activity of the therapeutic agent and the route of administration, in addition to many subject-specific considerations that are within the skill of the art.

[0243] In some embodiments, administration may be ophthalmic, oral, buccal, dermal (which may be or include one or more of, e.g., topical to the dermis, intradermal, interdermal, transdermal, etc.), enteral, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, within a particular organ (e.g., within the liver), intramucosal, intranasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal instillation), vaginal, vitreous, etc.

[0244] Those of skill in the art will appreciate upon reading this disclosure that in some embodiments it may be desirable to achieve delivery of a MuSK Ig2 modulator agent to muscle. Alternatively or additionally, in some embodiments it may be desirable to achieve delivery of a MuSK Ig2 modulator agent to the CNS (e.g., the brain, e.g., the hippocampus or subventricular region) or lung.

[0245] In some embodiments, the agent (eg, an agonizing agent) is delivered via systemic delivery or via local delivery to a muscle (eg, via intramuscular injection).

[0246] In some embodiments, the MuSK Ig2 modulator agent is administered using a viral vector to effectively deliver the MuSK Ig2 modulator agent 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 various muscle cell types (see, for example, Arnett et al., Mol Ther Methods Clin Dev.1.pii:14038,2014, and Riaz et al., Skeletal Muscle 5(37)2015).

[0247] Those of skill in the art will understand upon reading this disclosure that in some embodiments it may be desirable to achieve delivery of a MuSK Ig2 modulator agent to the CNS, and in some embodiments to the brain. Those of skill in the art will understand upon reading this disclosure that in some embodiments it may be desirable to achieve delivery of a MuSK Ig2 modulator agent to muscle.

[0248] In some embodiments, systemic administration achieves delivery to the CNS (e.g., the brain, e.g., the hippocampus or subventricular zone) or muscle. In some embodiments, an agent (e.g., an agonizing agent) is delivered to the central nervous system (CNS) or muscle by intraventricular administration.

[0249] In addition, certain viral vectors are known to selectively target neurons and effectively deliver genetic payloads to the brain. For example, AAV2 / 1 vectors have been established to effectively deliver nucleic acid payloads (e.g., gene therapy, encoded RNA, etc.) to neuronal cells in the hippocampus. See, for example, 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 vectors or AAV4 vectors) have been established to effectively deliver nucleic acid payloads to specific cells in the subventricular zone. See, e.g., Liu et al., Gene Therap. 12, 1503 (2005); Bockstael et al., Hum. Gene Therap. 23, 216 (2012).

[0250] In subjects suffering from or susceptible to a disease, disorder or condition associated with neurodegeneration, administration that achieves delivery to the CNS, for example the brain (eg, the hippocampus or subventricular region), may be desirable.

[0251] In some embodiments, effective delivery can be achieved by systemic administration of the compositions described herein.Alternatively or additionally, in some embodiments, effective delivery can be achieved by local administration to the CNS, brain or muscle, for example, by intrathecal or intracavitary (e.g., intraventricular) delivery.

[0252] Techniques for localized administration to the CNS, brain, or muscle have been developed and have proven effective for, e.g., a variety of protein therapeutics (see, e.g., Calias et al., Pharmacol. & Therap. 144:122, 2014), small molecules (see, e.g., Dodou Pharm 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 trademark name Zolgensma™] and nusinersen [sold under the trademark name Spinraza™]).

[0253] Those skilled in the art will recognize that intrathecal delivery can be particularly effective in achieving delivery of therapeutic agents, including cells, proteins and nucleic acids, to the hippocampus.

[0254] Systemic administration techniques (including, e.g., oral, parenteral, mucosal, etc.) are well established for a wide variety of agents. Systemic administration to achieve CNS, muscle, or brain delivery may, in some embodiments, rely on the ability to cross the blood-brain barrier (BBB).

[0255] Certain active agents or delivery systems are known to cross the BBB. Recent techniques have been shown to achieve CNS or even brain delivery of agents such as oligonucleotides, which were previously considered particularly difficult in this regard. To give one example, Min et al., Angew Chem. Int. Ed. Engl., (2020), incorporated herein by reference, describes glucose-coated polymeric nanocarriers that transport oligonucleotides across the BBB.

[0256] The incorporation of certain chemical entities into oligonucleotide therapeutics can facilitate their transport across the BBB. For example, Khorkova et al., Nature Biotech 35:249, 2017 (incorporated herein by reference) describe that 2'-modified phosphorothioate oligonucleotides...may be particularly applicable to CNS disorders given their long half-life, with effects in the brain lasting up to 6 months after a single injection. In another type of sugar moiety modification, locked nucleic acid (LNA), a bridge is introduced connecting the 2' oxygen and the 4' carbon. This modification substantially increases the melting temperature of LNA-DNA and LNA-RNA hybrids, thus allowing the creation of even shorter ODN-based compounds with increased bioavailability and reduced manufacturing costs. The recently proposed tricyclo-DNA, a conformationally constrained oligonucleotide analog, has three additional C atoms between the C(5') and C(3') of the sugar (Figure 2). This modification increases stability, hydrophobicity and RNA affinity, and improves tissue uptake and BBB permeability.

[0257] Administration that achieves pulmonary delivery may be desirable 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.

[0258] Oligonucleotides In some embodiments, ASOs are developed to enhance delivery to target sites. As described in the art, oligoloads are covalently linked to carriers or ligands, such as lipid particles, liposomes, nanoparticles, and more recently, sugars such as N-acetylgalactosamine, to enhance safer delivery to target sites. See Verma, Ann. Indian Acad. Neurol. 2018 21(1):3-8.

[0259] To improve the efficiency of cellular delivery of ASOs to target sites, e.g., muscles, certain techniques have been developed. For example, aminoglycosides (AGs) have been shown to improve the delivery of antisense phosphorodiamidate morpholino oligomers (PMOs) both in vitro and in vivo. See Wang, et al., Mol. Ther. Nucleic Acids, 2019; 16: 663-674. Short cell-penetrating peptides (CPPs), which can be directly attached to oligonucleotides via covalent bonds or through the formation of non-covalent nanoparticle complexes, can promote cellular uptake. See McClorey et al.; Biomedicines, 6(2), 51 (2018). Also, ASO fatty acid conjugates have been reported to enhance the functional uptake of antisense oligonucleotides (ASOs) in muscles. See Prakash et al.; Nucleic Acids Research, 47, 2019, 6029-6044.

[0260] Those of skill in the art will be familiar with eteplirsen (ExonDys 51), a third generation phosphorodiamidate morpholino ASO, which is an approved treatment for Duchenne muscular dystrophy (DMD).

[0261] Eteplirsen (Sarepta Therapeutics), marketed under the trade name Exondys 51™, splices exon 51 into the pre-mRNA, restoring the reading frame to 13% of patients with the appropriate frameshift mutation (see Crudele et al. Human Molecular Genetics, Volume 28, Issue R1, pp. R102-R107, 2019).

[0262] Eteplirsen is administered by intravenous infusion over 35 to 60 minutes. In particular, its recommended dosage is 30 mg / kg body weight weekly. In single-dose vials, the pharmaceutical composition is formulated as a 100 mg / 2 mL or 500 mg / mL (50 mg / mL) solution.

[0263] In some embodiments, the oligonucleotide therapeutics described herein may be administered intravenously. In some such embodiments, such oligonucleotide therapeutics may be administered according to a regimen reasonably equivalent to that used for eteplirsen (sold under the trade name Exondys 51™).

[0264] In some embodiments, the lower dose of agonizing oligonucleotide described herein is 12 mg. In some embodiments, a total of 5 mg to 60 mg of agonizing oligonucleotide is administered to the subject per dose. In some embodiments, a total of 12 mg to 48 mg of agonizing oligonucleotide is administered to the subject per dose. In some aspects, a total of 12 mg to 36 mg of agonizing oligonucleotide is administered to the subject per dose. In some aspects, a total of 12 mg of agonizing oligonucleotide is administered to the subject per dose.

[0265] Those skilled in the art will be familiar with nusinersen (sold under the trade name Spinraza™), an antisense oligonucleotide therapeutic that targets survival motor neuron-2 (SMN2) specific gene transcripts and is indicated for the treatment of spinal muscular atrophy (SMA) in pediatric and adult patients. Spinraza is administered intrathecally. In particular, its recommended dosage is 12 mg / 5 mL (2.4 mg / mL) in a single-dose vial per administration, following a regimen with four loading doses. The first three of the four loading doses are administered at 14-day intervals, the fourth of the four loading doses is administered 30 days after the third dose, and then a maintenance dose is administered once every four months. Platelet counts, coagulation laboratory tests, and quantitative spot urine protein tests are recommended at baseline and before each administration.

[0266] In some embodiments, the oligonucleotide therapeutics described herein may be administered intrathecally. In some such embodiments, such oligonucleotide therapeutics may be administered according to a regimen reasonably equivalent to that used for nusinersen (sold under the brand name Spinraza™).

[0267] In some embodiments, the oligonucleotide therapeutics described herein may be administered intrathecally. In some such embodiments, such oligonucleotide therapeutics may be administered according to a regimen reasonably equivalent to that used for nusinersen (sold under the brand name Spinraza™).

[0268] cell therapy In light of the ability of the MuSK Ig2 modulator agents described herein to promote muscle regeneration (e.g., derived from a cell population that is or contains SCs, MPCs or myoblasts), one of skill in the art will understand upon reading this disclosure that, among other things, this disclosure provides techniques for enhancing the levels of neurons, SCs, MPCs or myoblasts present within a cell population. That is, contacting an original cell population with a MuSK Ig2 modulator agent described herein can produce a resultant population that has increased levels of neurogenesis or muscle regeneration or growth compared to the levels in the original population. Administration of such a MuSK Ig2 modulator agent described herein can achieve such an increase.

[0269] In some embodiments, the original cell population may be or include neurons, SCs, MPCs, or myoblasts. In some embodiments, the original cell population is or includes embryonic stem cells or pluripotent stem cells. In some embodiments, the embryonic stem cells or pluripotent stem cells are or have been differentiated into neural cells or myogenic progenitor cells, for example, using techniques known in the art (e.g., Miyagoe-Suzuki et al., Stem Cells Int. 7824614 2017).

[0270] In some embodiments, as described above, such administration delivers the MuSK Ig2 modulator agent (e.g., to a human, particularly an adult, e.g., to muscle tissue of such a human) so that it is exposed (i.e., contacted) in vivo to the relevant original cell population.

[0271] In some embodiments, administration according to the present disclosure contacts the MuSK Ig2 modulator agent ex vivo with a population of cells (e.g., the original population of cells) that may be or may include, for example, neural stem cells (NCS), SCs, MPCs, or myoblasts. For example, in some embodiments, the MuSK Ig2 modulator agent is administered ex vivo (e.g., in vitro) to a population of cells derived from a subject. In some embodiments, the population of cells obtained from a subject.

[0272] In some embodiments, the MuSK Ig2 regulator agent used specifically ex vivo may be or include a small molecule, and an antibody, or a nucleic acid agent, or a combination thereof. In some particular such embodiments, one or more agents that are or include a nucleic acid (e.g., one or more gene therapies [e.g., nucleic acid vectors or transcripts], oligonucleotides, or gRNAs) may be particularly useful for ex vivo or in vitro administration to cells. CRISPR / Cas modification of cell populations is an established and growing field, and those skilled in the art will understand the applicability of such strategies according to the present disclosure, for example, to modify or destroy MuSK Ig2 domain sequences. Alternatively or additionally, a nucleic acid encoding a MuSK form that lacks a functional Ig2 domain (or a nucleic acid whose expression product encodes a MuSK form that lacks a functional Ig2 domain) can be introduced into a cell ex vivo or in vitro. Further alternatively or additionally, oligonucleotides can be utilized that direct exon skipping of the MuSK transcript to promote forms lacking functional Ig2, or that direct degradation (or block translation) of forms that contain functional Ig2.

[0273] In some embodiments, a population of cells is contacted with a MuSK Ig2 modulator agent and simultaneously or subsequently stimulated or expanded. Alternatively or additionally, the population of cells is enriched or selected for cells that exhibit characteristics of early neurons, e.g., Dex, or mature neurons, e.g., Map2, astrocytes, e.g., GFAP and S100b, or oligodendrocytes, e.g., CNPase and O4.

[0274] In some embodiments, the obtained population of cells, which is achieved by contacting the original population of cells with a MuSK Ig2 modulator agent ex vivo, is then administered to a subject. In some embodiments, the obtained population of cells is administered to a subject suffering from or susceptible to a disease or disorder, such as a muscle disorder, a neuromuscular dysfunction, a neurodegenerative disorder, a cardiac disorder (e.g., myocardial infarction, cardiomyopathy), or a genetic disease characterized by muscle wasting. In some embodiments, the obtained population of cells is administered to the subject from which the original population of cells was obtained. In some embodiments, the obtained population of cells is administered to a subject different from the subject from which the original population of cells was obtained. In some such embodiments, the original population is obtained from a healthy subject, and the obtained population is administered to a subject suffering from or susceptible to a disease or disorder, such as a muscle disorder, a neuromuscular dysfunction, a neurodegenerative disorder, a cardiac disorder (e.g., myocardial infarction, cardiomyopathy), or a genetic disease characterized by muscle wasting.

[0275] In some embodiments, administering a population of cells contacted with a MuSK Ig2 modulator agent effectively treats a disease or disorder in a subject, such as a muscle disorder, neuromuscular dysfunction, a neurodegenerative disorder, a cardiac disorder (e.g., myocardial infarction, cardiomyopathy), or a genetic disease characterized by muscle wasting.

[0276] In some embodiments, the stimulated or expanded population of NSC, SC, MPC or myoblast cells described herein can be formulated into a cell therapy. In some embodiments, the cell therapy comprises a pharmaceutically acceptable carrier, diluent or excipient. The pharmaceutically acceptable carriers described herein, such as vehicles, adjuvants, excipients and diluents, are well known and readily available to those skilled in the art. Preferably, the pharmaceutically acceptable carrier is chemically inert to the active agent, such as the cell therapy, and does not induce adverse side effects or toxicity under the conditions of use.

[0277] In some embodiments, the cellular therapy may be formulated for administration by any suitable route, such as intravenous, intratumoral, intraarterial, intramuscular, intraperitoneal, intrathecal, epidural, or subcutaneous. Preferably, the cellular therapy is formulated for a parenteral route of administration. In some embodiments, the cellular therapy is administered to a subject by injection.

[0278] In some embodiments, cellular therapeutics suitable for parenteral administration may be aqueous or non-aqueous isotonic sterile injection solutions that may contain, for example, antioxidants, buffers, bacteriostats, and solutes that render the composition isotonic with the blood of the intended recipient. Aqueous or non-aqueous sterile suspensions may contain one or more suspending agents, solubilizing agents, thickening agents, stabilizers, and preservatives.

[0279] In some embodiments, a single therapeutic cell as described herein can be expanded and provide a therapeutic benefit. 2 More than one, e.g., 10 3 More than 10 pieces 4 More than 10 pieces 5 More than 10 8 Alternatively or additionally, one or more therapeutic cells are administered as a cellular therapy. 12 10 or less, for example 11 Less than or equal to 10 9 Less than or equal to 10 7 10 or less 5 No more than 10 therapeutic cells are administered to a subject as a cellular therapy. In some embodiments, no more than 10 therapeutic cells are administered to a subject as a cellular therapy, as described herein. 2 ~10 5 pieces, 10 4 ~10 7 pieces, 10 3 ~10 9 Pieces or 10 5 ~10 10 The therapeutic cells are administered as a cellular therapy.

[0280] Doses of the cellular therapy agents described herein may be administered to a subject in a single dose or in a series of subdoses administered over a suitable period of time, for example, on a daily, twice-weekly, weekly, biweekly, twice-monthly, bimonthly, twice-yearly or annual basis, as needed. A dosage unit containing an effective amount of the cellular therapy agent may be administered in a single daily dose, or the total daily dose may be administered in two, three, four or more subdoses administered daily, as needed. In some embodiments, the cellular therapy agent is administered in combination with another therapy.

[0281] Combination therapy In some embodiments, a MuSK Ig2 modulator therapy described herein is administered in combination with another therapy, ie, such that the subject is exposed to both therapies simultaneously.

[0282] The dosage of a MuSK Ig2 modulator therapy described herein, and the dosage of another therapy administered in combination, as well as the administration schedule, can depend on a variety of parameters, including, but not limited to, the disease being treated (e.g., a muscle disorder, neuromuscular dysfunction, neurodegenerative disorder, cardiac disorder, or genetic disease characterized by muscle wasting), the general health of the subject, and the discretion of the administering physician.

[0283] A MuSK Ig2 modulator therapy may be administered to a subject in need thereof prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) administration of the other therapy. In various embodiments, the MuSK Ig2 modulator therapy and the other therapy are administered 1 minute apart, 10 minutes apart, 30 minutes apart, less than 1 hour apart, 1 hour apart, 1-2 hours apart, 2-3 hours apart, 3-4 hours apart, 4-5 hours apart, 5-6 hours apart, 6-7 hours apart, 7-8 hours apart, 8-9 hours apart, 9-10 hours apart, 10-11 hours apart, 11-12 hours apart, 24 hours or less apart, or 48 hours or less apart. In one embodiment, the MuSK Ig2 modulator therapy and the other therapy are administered within 3 hours. In another embodiment, the MuSK Ig2 modulator therapy and the other therapy are administered 1 minute to 24 hours apart.

[0284] A synergistic combination of MuSK Ig2 modulator therapy with another therapy may allow for the use of lower dosages of one or both of these agents, or less frequent administration of the therapy to subjects suffering from muscle disorders, neuromuscular dysfunction, neurodegenerative disorders, cardiac disorders, or genetic diseases characterized by muscle wasting. A synergistic effect may result in improved efficacy of these agents, or a reduction in adverse or undesirable side effects associated with the use of either agent alone.

[0285] In some embodiments, MuSK Ig2 modulator therapy is administered in combination with standard of care treatment for the relevant disease, disorder or condition (e.g., a muscle disorder, neuromuscular dysfunction, neurodegenerative disorder, cardiac disorder, or genetic disease characterized by muscle wasting).

[0286] Therapies for DMD include glucocorticoids such as deflazacort (Emflaza; PTC Therapeutics), eteplirsen (Exondys 51; Sarepta Therapeutics), ataluren (Translarna; PTC Therapeutics), and prednisone. In some embodiments, the MuSK Ig2 modulator therapy is administered in combination with one or more therapies for DMD.

[0287] Approved therapies for ALS include Radicava, Rilutek, Tiglutik and Nuedexta. In some embodiments, the MuSK Ig2 modulator therapy is administered in combination with one or more therapies for ALS.

[0288] Approved therapies for cardiomyopathies include, but are not limited to, angiotensin II converting enzyme (ACE) inhibitors, angiotensin II receptor blockers (ARBs) and spironolactone. In some embodiments, MuSK Ig2 modulator therapy is administered in combination with one or more therapies for cardiomyopathies.

[0289] In some embodiments, MuSK Ig2 modulator therapy is administered in combination with one or more therapies that alleviate symptoms or characteristics of associated disease, disorder or condition, or for that purpose.In some embodiments, MuSK Ig2 modulator therapy is administered in combination with one or more other therapies that alleviate symptoms or characteristics, thereby alleviating side effects associated with the other therapies.In some embodiments, the side effects associated with the therapies are characterized by one or more of muscle spasms and muscle cramps, constipation, fatigue, excess saliva and phlegm, pain, depression, sleep problems, and uncontrolled bursts of laughing or screaming.

[0290] Any therapy known to be useful or that has been, is being, or is currently being used to treat or prevent a muscle disorder, neuromuscular dysfunction, neurodegenerative disorder, cardiac disorder, or genetic disease characterized by muscle wasting may be used in combination with a MuSK Ig2 modulator therapy according to the invention described herein.

[0291] Plasmids and protein production.

[0292] To prepare the MuSK constructs of the present disclosure, including full-length MuSK and constructs containing deletions of one or more MuSK domains (e.g., Ig1, Ig2 or Ig3), plasmids can be used for packaging the constructs and for expression of the constructs in host cells.

[0293] In an exemplary embodiment, full-length ectodomain MuSK cloned into 6xHis tagged pCEP-Pu mammalian expression vector plasmid to generate His-tagged full-length ectodomain MuSK recombinant protein has been used in previous studies. Yilmaz et al. (2016). ΔIg1, ΔIg2, ΔIg1 / 2, Δ5a / 5b, Ig1 / 2 MuSK ectodomain deletion constructs were synthesized with NheI 5' and NotI 3' restriction enzyme recognition sites and cloned into pUC57 bacterial expression vector (Genewiz). MuSK ectodomain deletion constructs were cloned into pCEP-Pu mammalian expression vector and Sanger sequenced for sequence confirmation. From the full-length ectodomain MuSK pCEP-Pu plasmid, isolated Ig1 domain and isolated Ig2 domain were generated by PCR cloning.

[0294] PCR primers for Ig1: forward GATCTTCTTTCTCCTTTGCCTGGC (SEQ ID NO: 1); reverse TAAGCAGCGGCCGCCATCTTCACTTGCAGGGCACC (SEQ ID NO: 2), and PCR primers for Ig2: forward CCTCAGCTAGCACCTAAAATAACTCGTCCTCCC (SEQ ID NO: 3); reverse AATTAGCGGCCGCCTTCACCAGTTTGGAGTAAGC (SEQ ID NO: 4).

[0295] The isolated Ig1 and Ig2 PCR products were cloned into pCEP-Pu expression vector and Sanger sequenced for confirmation. For production of MuSK ectodomain his-tagged proteins, human embryonic kidney 293 (HEK 293) PEAKrapid cells (ATCC CRL-2828) were transfected with MuSK ectodomain plasmid using Promega FugeneHD® and selected by puromycin treatment. Ectodomain MuSK-his recombinant proteins were secreted into the culture medium and conditioned medium was collected every 2-3 days for 2 weeks. MuSK-his recombinant proteins were purified from the conditioned medium using nickel affinity chromatography. Purity of the purified proteins was assessed by SDS-PAGE and quantified using ELISA and NanoDrop with a calculated extinction coefficient of 0.1%.

[0296] MuSK PCR and sequencing.

[0297] To amplify MuSK exons expressed in vivo, RNA from wild-type soleus mouse muscle was isolated and reverse transcribed.

[0298] Primers were designed to amplify the region of exons 5–8: Forward: ACTGGTGAAGCTGGAAGTGG (SEQ ID NO: 7) Reverse: GCCTTGGCTGTCTTTCTGTG (SEQ ID NO: 8) PCR was performed using DreamTaq Green PCR Master Mix (ThermoFisher), and the products were subjected to agarose gel electrophoresis to ensure sufficient separation of PCR bands for gel extraction of DNA and to prevent cross-contamination of the separated DNA bands. Extracted DNA was Sanger sequenced to identify the DNA sequence of each band.

[0299] RNA sequencing and bioinformatics.

[0300] RNA from wild-type and ΔIg3-MuSK mouse soleus muscles was extracted (Genewiz) using the RNeasy Fibrous Tissue mini kit for RNA unstranded library preparation and sequencing (Qiagen). Samples were sequenced at a depth of approximately 50 million reads per sample. Reads are assessed for adapter sequence quality using Trimmomatic, trimmed, and then aligned to the reference genome using GSNAP. Read count matrices are generated using htseq-count. See Andrews, FastQC (2018); Bolger, Lohse, & Usadel (2014); Wu & Nacu (2010); Anders, Pyl, & Huber (2015). Read count matrices are uploaded to the integrated Differential Expression and Pathway (iDEP) analysis tool for exploratory data analysis, differential gene expression and pathway GO analysis. Ge, Son, & Yao (2018). The read count matrix is ​​used to run LeafCutter to identify and measure known and unknown alternative splicing events, see Li et al. (2018).

[0301] Sequence Listing PCR primer for MuSK Ig1: Forward GATCTTCTTTCTCCTTTGCCTGGC (SEQ ID NO: 1) PCR primer for MuSK Ig1: reverse TAAGCAGCGGCCGCCATCTTCACTTGCAGGGCACC (SEQ ID NO: 2) PCR primer for MuSK Ig2: Forward CCTCAGCTAGCACCTAAAATAACTCGTCCTCCC (SEQ ID NO: 3) PCR primer for MuSK Ig2: reverse AATTAGCGGCCGCCTTCACCAGTTTGGAGTAAGC (SEQ ID NO: 4) MuSK human Ig3 domain: ARILRAPESHNVTFGSFVTLHCTATGIPVPTITWIENGNAVSSGSIQESVKDRVIDSRLQLFITKPGLYTCIATNKHGEKFSTAKAAATIS (SEQ ID NO: 5) MuSK Mouse Ig3 Domain ARILRAPESHNVTFGSFVTLRCTAIGIPVPTISWIENGNAVSSGSIQESVKDRVIDSRLQLFITKPGLYTCIATNKHGEKFSTAKAAATVS (SEQ ID NO: 6) Primers used to amplify MuSK cDNA: forward;

[0302] ACTGGTGAAGCTGGAAGTGG (SEQ ID NO: 7) Primers used to amplify MuSK cDNA: reverse.

[0303] GCCTTGGCTGTCTTTCTGTG (SEQ ID NO: 8) The following examples are provided to illustrate the present invention and are not intended to limit the scope of the invention.

[0304] Example 1 This example provides constructs including full length (FL), MuSK with an Ig1 deletion (ΔIg1), MuSK with an Ig2 deletion (ΔIg2), and MuSK with deletions of both Ig1 and Ig2 (ΔIg1Ig2) that were generated to test and characterize the binding properties of various MuSK domains with BMPs and their receptors.

[0305] material and method Antibodies and Materials. ALK2, ALK3, ALK4, ALK5 and ALK6 were obtained from R&D Systems (Minneapolis, MN, USA). Goat anti-human IgG Fc secondary antibody, HRP (catalog number A18817) was obtained from ThermoFisher Scientific (Waltham, MA, USA). An expression vector containing a His-tagged MuSK ectodomain was a kind gift from Dr. Markus Ruegg. The substrate 3,3',5,5'-tetramethylbenzidine (TMB) (catalog number T0440-100ML) was obtained from Sigma-Aldrich (St. Louis, MO, USA).

[0306] Production of protein constructs. The first step was to generate deletion constructs of MuSK protein. To map the domains that MuSK binds to BMP receptors, we generated deletion constructs of MuSK that lack specific domains. We used these deletion constructs to determine the domains of MuSK that bind to BMP receptors.

[0307] The full length MuSK was sent to GENEWIZ (South Plainfield, NJ, USA), who produced a plasmid containing the sequence and sent it back to us. GENEWIZ provided us with the MuSK sequence in the pUC57 plasmid, a plasmid commonly used for cloning vectors.

[0308] Subcloning Efficiency™ DH5α Competent Cells were used to transform DNA obtained from GENEWIZ. The bacteria were then spread onto an agar plate. The next day, colonies were picked and added to tubes containing broth. Ampicillin was used throughout the process as a form of selection. Colonies picked from the agar plate were left overnight in LB medium.

[0309] The next day, minipreps were performed using a QIAprep Spin Miniprep Kit from QIAGEN (Venlo, Netherlands). Pellet 1–5 ml of bacterial overnight culture by centrifugation at >8000 rpm (6800 × g) for 3 min at room temperature (15–25 °C). Resuspend pelleted bacterial cells in 250 μL of Buffer P1 and transfer to a microcentrifuge tube. Add 250 μL of Buffer P2 and mix thoroughly by inverting the tube 4–6 times until the solution is clear. Do not allow the lysis reaction to proceed for more than 5 min. If LyseBlue reagent is used, the solution will turn blue. Add 350 μL of Buffer N3 and immediately mix thoroughly by inverting the tube 4–6 times. If LyseBlue reagent is used, the solution will turn colorless. Centrifuge for 10 min at 13,000 rpm (approximately 17,900 × g) in a tabletop microcentrifuge. Apply the supernatant from step 5 to the QIAprep spin column by decanting or pipetting. Centrifuge for 30-60 seconds and discard the flow-through or apply vacuum to the manifold to draw solution through the QIAprep spin column and turn off the vacuum source. Recommended: Wash the QIAprep spin column by adding 0.5 ml of Buffer PB. Centrifuge for 30-60 seconds and discard the flow-through or apply vacuum to the manifold to draw solution through the QIAprep spin column and turn off the vacuum source. (This step is only required if using endA+ strains or other bacterial strains with high nuclease activity or carbohydrate content.) Wash the QIAprep spin column by adding 0.75 ml of Buffer PE. Centrifuge for 30-60 seconds and discard the flow-through or apply vacuum to the manifold to draw solution through the QIAprep spin column and turn off the vacuum source. Transfer the QIAprep spin column to a collection tube. Centrifuge for 1 minute to remove residual wash buffer. Place the QIAprep column into a clean 1.5 ml microcentrifuge tube. To elute the DNA, add 50 µL of Buffer EB (10 mM Tris Cl, pH 8.5) or water to the center of the QIAprep spin column, let sit for 1 min, and centrifuge for 1 min.For more details, see the QIAprep Miniprep Handbook, (December 2006).

[0310] The concentration of the DNA obtained from the minipreps was then measured using a NanoDrop 2000 / 2000c from ThermoFisher Scientific.

[0311] The DNA obtained so far has the MuSK insert in a pUC57 vector. The next step was to move the MuSK insert into the pCEP4 mammalian expression vector. Ampicillin is the prokaryotic selection form. For eukaryotic selection, puromycin is used. OriP allows the cells to take up the plasmid. EBNA-1 allows activation of viral DNA replication. BM40 is a secretory peptide that allows the cells to secrete proteins into the medium.

[0312] To complete the subcloning of the MuSK insert into the pCEP4 expression vector, the MuSK insert was cut from the pUC57 vector by restriction enzymes. After adding the restriction enzymes, the DNA was run on an agarose gel to separate the MuSK insert from the rest of the vector. To extract the MuSK insert from the gel, we used a QIAquick extraction kit. To extract the MuSK insert from the gel, we used a QIAquick® Gel Extraction Kit (cat. nos. 28704, 28706, 28506 and 28115). Using a clean sharp scalpel, excise the DNA fragment from the agarose gel. Weigh the gel slice into a colorless tube. Add 3 volumes of Buffer QG to one volume of gel (approximately 100 μL for 100 mg gel). The maximum amount of gel per spin column is 400 mg. For >2% agarose gel, add 6 volumes of Buffer QG. Incubate at 50 °C for 10 min (or until the gel slice is completely dissolved). Vortex the tube every 2-3 min to aid in dissolving the gel. After the gel slice is completely dissolved, ensure that the mixture is yellow in color (similar to Buffer QG without dissolved agarose). If the mixture is orange or purple in color, add 10 μL of 3 M sodium acetate, pH 5.0, and mix. The mixture will turn yellow. Add 1 gel volume of isopropanol to the sample and mix. Place the QIAquick spin column into a prepared 2 ml collection tube, or into a vacuum manifold. To bind the DNA, apply the sample to the QIAquick column and centrifuge for 1 min or apply vacuum to the manifold until the entire sample has passed through the column. Discard the flow-through and return the QIAquick column to the same tube. For sample volumes >800 μL, load and spin / apply vacuum again. If the DNA is to be subsequently used for sequencing, in vitro transcription or microinjection, add 500 µL of Buffer QG to the QIAquick column and centrifuge or apply vacuum for 1 min. Discard the flow-through and place the QIAquick column back into the same tube.To wash, add 750 μL of Buffer PE to the QIAquick column and centrifuge or apply vacuum for 1 min. Discard the flow-through and return the QIAquick column to the same tube. NOTE: If the DNA will be used for salt-sensitive applications (e.g., sequencing, blunt-end ligation), leave the column for 2–5 min after adding Buffer PE. Centrifuge the QIAquick column in the prepared 2 ml collection tube for 1 min to remove residual wash buffer. Place the QIAquick column in a clean 1.5 ml microcentrifuge tube. To elute the DNA, add 50 μL of Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of the QIAquick membrane and centrifuge the column for 1 min. To increase the DNA concentration, add 30 μL of Buffer EB to the center of the QIAquick membrane and leave the column for 1 min, then centrifuge for 1 min. Increasing the incubation time up to 4 min after adding Buffer EB to the QIAquick membrane can increase the yield of purified DNA. If the purified DNA will be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA. Before loading the gel, mix the solution by pipetting up and down.

[0313] After the DNA is extracted from the gel, the MuSK insert is ligated into the pCEP4 vector using ligation enzyme. After subcloning, the vector was transformed using DH5α Competent Cells. The cells were streaked onto LB agar plates. The same process was then repeated with this newly formed vector. Minipreps were also performed and the DNA concentration was measured again. All MuSK deletion constructs generated were sequenced in-house by GENEWIZ.

[0314] Transfection and medium harvest. HEK293 cells were seeded in T75 flasks at 70% confluency. After 24 hours, DNA was transfected into the growth medium using FuGENE® HD. FuGENE® HD was obtained from Promega (Madison, WI, USA). 24 hours after transfection, the growth medium was replaced with high-dose antibiotic selection medium. 48 hours after these medium changes, the supernatant was harvested and the cells were placed into low-dose antibiotic selection medium.

[0315] To collect the medium, the medium was centrifuged in a sterile conical tube to pellet the cells. The supernatant was then transferred to a new sterile tube. The supernatant was frozen using liquid nitrogen and then stored at -80°C until it was time to purify the protein.

[0316] Protein purification. Reagents used for purification were Nalgene Rapid-Flow filter Unit 0.45um SFCA sterile filter (Fisher, Cat. No. 156-0045), Amicon Ultra-15 Centrifugal Filter Devices (Fisher, Cat. No. UFC901024), Amicon Pro Affinity Concentration Kit-Ni-NTA (Fisher, Cat. No. ACK5010NT) and Gibco Distilled Water (Invitrogen, Cat. No. 15230162).

[0317] The harvested media was thawed overnight at 4°C on a rocker. The growth media was then filtered using a Nalgene Rapid-Flow filter Unit 0.45um SFCA sterile filter. The growth media was then concentrated using Amicon Ultra-15 Centrifugal Filter Devices. The concentrated harvested media was then transferred to sterile tubes and stored on ice until it was time to purify them.

[0318] MuSK protein was purified using nickel column affinity purification. Prior to use, dialysis was performed to exchange buffer and remove imidazole from the purified protein. The final storage buffer was phosphate buffered saline, pH 7.4.

[0319] The protocol for Amicon® Pro Affinity Concentration Kit-Ni-NTA (cat. no. ACR5000NT, ACK5003NT, ACK5010NT, ACK5030NT, ACK5050NT, ACK5100NT) metal chelation affinity purification of His-tagged recombinant proteins was provided by EMD Millipore.

[0320] Bead preparation. Vortex Ni-NTA resin thoroughly before adding to device to ensure uniform suspension. Remove collection tube cap and open exchanger cap. Add 200 μL of resin slurry to the base of exchanger. Close exchanger cap. Up to 500 μL of packed resin (1000 μL slurry volume) may be added per device. It is recommended to use a large pore tip (catalog no. 02-707-134, Fisher Scientific) for resin transfer. Centrifuge at 1000 g x 1 min in a swinging bucket rotor to remove storage buffer. Add 500 μL of 1X binding buffer. Centrifuge at 1000 g x 1 min.

[0321] Protein binding. Add 500 μL of sample to the exchanger. Up to 9 mL of sample can be added. The volume loaded is determined by the expression level of the target protein and the binding capacity of the resin. Incubate for 60 min at room temperature with gentle agitation. Upward agitation using a plate shaker at a low setting is recommended. End-over-end mixing, especially with small volumes or for extended periods of time, can cause significant bead loss on the sides of the feeder tube. Centrifuge the device at 1000 g x 1 min in a swinging bucket rotor. Collection of sample flow-through from a 50 mL collection tube is optional. To ensure maximum protein capture, collect all resin in solution before centrifugation. Add 1.5 mL of wash buffer. Centrifuge at 1000 g x 1 min. Collection of wash fraction from a 50 mL collection tube is optional. Due to the large capacity of the exchanger, the volume of the wash can be increased to increase sample purity. Multiple wash steps are not required.

[0322] Sample elution. Samples can be eluted without concentration by adding elution buffer and centrifuging (1000g x 2 min) directly into a clean 50 ml collection tube. Given the limited volume handling capacity of the AU-0.5 device, this protocol is recommended if elution volumes >1.5 ml are required.

[0323] Solid-phase binding assay. To assay the binding between MuSK and ALK, we used an ELISA-like solid-phase binding assay. The plates used for the solid-phase binding assay were from Sigma 96-well plates (catalog number M9410-1CS). The first step of the process was to immobilize the MuSK constructs on the 96-well plates. 50ul of MuSK was added in triplicate to the 96-well plates at a concentration of 16ug / ml. The plates were covered and incubated overnight at 4°C. The next day, the plates were washed using PBS (at the desired pH). The wells were then placed in Block (1% BSA) for 1 hour. After blocking, all washing steps were performed using PBST (0.05% Tween 20 and PBS solution).

[0324] Block was added, followed by serial dilutions of ALK. For all assays, the ALK concentrations added were 3.125, 6.25, 12.5, 25, 50, and 100 nM. To detect the presence of ALK, a goat anti-human IgG Fc secondary antibody, HRP was used at a concentration of 1:250. TMB was then added to detect the presence of HRP. The reaction time was recorded, and when the reaction produced a sufficiently high signal, sulfuric acid was used to quench the reaction.

[0325] To quantify the signal generated from these wells, the signal was measured at 450 nm using a plate reader. The absorbance was then background subtracted and plotted on a curve using Prism8 software. Each data point represented the average of three replicates.

[0326] SDS-PAGE and PNGase F. SDS-PAGE is a method to separate proteins by electrophoresis. Kaulich et al. (2020). SDS (sodium dodecyl sulfate) is used to denature proteins before separating the samples using polyacrylamide gels. Kaulich et al. (2020). SDS provides a negative charge to the sample, which is important when running the gel using electrophoresis. SDS-PAGE confirms the purity of the sample. Samples were treated with PNGase F to assess the glycosylation of the samples. PNGase F is an enzyme that releases N-linked oligosaccharides.

[0327] To assess the purity of the generated MuSK constructs, the constructs were run on an SDS-PAGE gel. 1 ug of protein was added to each well of the gel. Mini-PROTEAN® TGX™ Precast Gels from BioRad (Hercules, CA) were used for these gel assays. To stain the gels, AcquaStain Protein Gel Stain (Bulldog-Bio, Portsmouth, NH, USA) was used to stain the gels overnight.

[0328] To demonstrate that the protein constructs were properly glycosylated, PNGase F was incubated with the proteins and then run side-by-side on a gel with constructs that were not treated with PNGase F. The protocol used for treating samples with PNGase F was that provided by New England Biolabs (Ipswich, MA, USA). Typical reaction conditions are as follows: Denaturation reaction conditions: Combine 1-20 μg glycoprotein with 1 μL of Glycoprotein Denaturation Buffer (10X) and HO (if required) to make a total reaction volume of 10 μL. Denature glycoprotein by heating reaction at 100 °C for 10 min. Cool denatured glycoprotein on ice and centrifuge for 10 s. Make the total reaction volume 20 μL by adding 2 μL of GlycoBuffer 2 (10X), 2 μL of 10% NP-40, and 6 μL of HO. PNGase F is inhibited by SDS, so NP-40 is added to the reaction mixture under denaturing conditions. Failure to include NP-40 in the denaturation protocol will result in loss of enzyme activity. Add 1 μL of PNGase F and mix gently. Incubate the reaction at 37 °C for 1 h. Analyze by any method. Note: The easiest way to assess the extent of deglycosylation is by mobility shift on an SDS-PAGE gel.

[0329] Non-denaturing reaction conditions: When deglycosylating native glycoproteins, it is recommended that an aliquot of glycoprotein be subjected to the denaturing protocol to provide a positive control of fully deglycosylated protein. The non-denaturing reaction can then be compared to the denaturing reaction to determine the extent of reaction completion. Combine 1-20 μg glycoprotein, 2 μL GlycoBuffer 2 (10x), and HO (if needed) to make a total reaction volume of 20 μL. Add 2-5 μL PNGase F and mix gently. Incubate the reaction at 37 °C for 4-24 h.

[0330] Longer incubation times and more enzyme may be required to deglycosylate native glycoproteins. Analyze by any method. The simplest way to assess the degree of deglycosylation is by mobility shift on SDS-PAGE gels.

[0331] result SDS-PAGE of MuSK constructs.

[0332] From FIG. 4, it is confirmed that constructs were produced for each construct. The full-length construct showed a smear since it has several glycosylation sites. The full-length construct had a mass near the 75 kDa marker, but the full-length treated with PNGase F had a slightly lower molecular weight than the untreated one. The band along the 30 kDa marker in the treated lane indicates the PNGase F enzyme used in the treatment. The ΔIg1 construct in FIG. 4 showed a smear since it has several glycosylation sites. The ΔIg1 construct had a mass near the FL, but the ΔIg1 treated with PNGase F had a slightly lower molecular weight than the untreated one. The band along the 30 kDa marker in the treated lane indicates the PNGase F enzyme used in the treatment.

[0333] The ΔIg2 construct in Figure 4 shows a smear because it has several glycosylation sites. The ΔIg2 construct had a mass similar to that of ΔIg1, but lighter than that of FL. The ΔIg2 treated with PNGase F had a slightly lower molecular weight than the untreated one. The band along the 30 kDa marker in the treated lane indicates the PNGase F enzyme used in the treatment. The ΔIg1-Ig2 construct in Figure 4 shows a smear because it has several glycosylation sites. The ΔIg1 construct had a mass almost lighter than FL, but the ΔIg1 treated with PNGase F had a slightly lower molecular weight than the untreated one. The band along the 30 kDa marker in the treated lane indicates the PNGase F enzyme used in the treatment.

[0334] In FIG. 5, the results for Ig1, Ig2, Ig1-Ig2 and ΔCRD / Fz are shown, confirming that the predicted constructs were generated. The Ig1 band formed approximately 15 kDa. The Ig1 band treated with PNGase F appeared at the same marker as the untreated one, indicating that the Ig1 domain does not have a glycosylation site. The band along the 30 kDa marker in the treated lane indicates the PNGase F enzyme used in the treatment. In FIG. 5, the Ig2 band has a mass just above 15 kDa. The Ig2 band treated with PNGase F appeared at the same marker as the untreated one, indicating that the Ig2 domain does not have a glycosylation site. The band along the 30 kDa marker in the treated lane indicates the PNGase F enzyme used in the treatment.

[0335] In Figure 5, the Ig1-Ig2 band appears to have a molecular weight of approximately 30 kDa. The Ig1-Ig2 band does not show a smear and the mass is not affected after treatment with PNGase F. The results showed that the Ig1 and Ig2 domains are not glycosylated and do not have glycosylation sites. The single band along the 30 kDa marker in the treated lane indicates the PNGase F enzyme used in the treatment. In Figure 5, a faint band of ΔCRD / Fz appears around the 70 kDa mark. In Figure 5, the band appears as a smear because ΔCRD / Fz is glycosylated. This is further confirmed by the fact that the mass of the sample treated with PNGase F is smaller than the untreated one. The single band along the 30 kDa marker in the treated lane indicates the PNGase F enzyme used in the treatment.

[0336] Figure 6 shows the results for the CRD / Fz, Δ5a5a' and L83R constructs. The CRD / Fz band in Figure 6 is faint, indicating some glycosylation. PNGase F enzyme overlaps with the CRD / Fz band in the treated sample. 5a5a' is an alternatively spliced ​​exon. The size of the bands shows that there is not much difference between this sample and the full-length sample. The smaller size of the treated sample indicates that the sample has glycosylation sites. See the faint band in Figure 6. Figure 6 also shows the L83R construct. MuSK L83R is important because it was shown that the leucine at position 83 is required for MuSK dimerization. Stiegler, Burden, & Hubbard (2006). The goal was to prevent MuSK from dimerizing by mutating the leucine to an arginine. Figure 6 shows MuSK L83R run on an SDS-PAGE gel. MuSK is glycosylated, so the band is faint. The only band along the 30 kDa marker in the treated lane represents the PNGase F enzyme used in the treatment.

[0337] Example 2 The Ig2 domain is necessary and sufficient for MuSK-BMP receptor binding We used an ELISA-like solid-phase binding assay and MuSK deletion constructs to map the domains of MuSK that bind to BMP receptors (ALKs). Yilmaz et al. (2016) previously showed that MuSK binds to ALK6, but did not determine which MuSK domains bind to ALK6.

[0338] FIG. 7 shows data from a solid phase binding assay in which MuSK was immobilized and incubated with ALK6. ALK concentration is shown on the x-axis and absorbance is shown on the y-axis. The higher the absorbance, the better the binding. FIG. 7 shows that when the Ig2 domain is missing, MuSK shows little to no binding compared to the full length ectodomain of MuSK (FL ECD). At an ALK6 concentration of 100 nM, the FL ECD had an absorbance of 1 and the ΔIg2 MuSK construct had an absorbance of 0.15. In the presence of the Ig2 domain, MuSK increased 6.6-fold. This increase is significant. FIG. 7 shows that the Ig2 domain is required for MuSK binding to ALK6. It is important to note the shape of the curves as it shows that there is not a high affinity for binding for the ΔIg2 curve.

[0339] The next step was to isolate the binding of MuSK ectodomain to the Ig1-Ig2Ig1Ig2 region using the Ig2 domain required for binding. Figure 8 shows the binding assay data from the Ig1-Ig2 and ΔIg1-Ig2 constructs. Figure 8 shows that the Ig1-Ig2 construct has high affinity binding to ALK6. The Ig1-Ig2 curve reaches an absorbance of 3 at a concentration of 100 nM. On the other hand, with the ΔIg1-Ig2 construct, MuSK shows little or no binding. This is even more evident when compared to the Ig1-Ig2 curve. The half-maximum of the Ig1-Ig2 curve is at the 6.25 nM concentration, indicating that the Ig1-Ig2 construct has high affinity binding. Figure 13 Both the Ig1 and Ig2 domains are required for MuSK to bind to ALK6.

[0340] To show that the Ig2 domain is sufficient to bind to ALK6, we used isolated Ig2 and Ig1 domains in Figure 9. Figure 9 shows that the Ig2 curve has an absorbance curve of 1.8 and a half-maximal concentration of 6.25 nM. Full-length MuSK did not saturate in this assay, only having a half-maximal value of about 0.5 nM. The isolated Ig1 domain curve showed little or no binding. These results indicate that the Ig2 domain is sufficient for binding, as it showed a binding curve with high affinity properties. Figures 8 and 9 show that the Ig2 domain is necessary and sufficient for binding of MuSK to ALK6. See also Table 1, which provides half-maximal and saturation levels for the binding assay. Full-length data was taken from Yilmaz et al. (2016). Saturation percentage measured at 100 nM.

[0341] [Table 1]

[0342] Example 3 The Ig1 domain is not required for MuSK binding.

[0343] The Ig1 domain is important as the domain by which MuSK binds to agrin-LRP4. Stiegler, Burden, & Hubbard (2006). The crystal structure also shows that MuSK dimerizes via the Ig1 domain. Therefore, it was important to demonstrate the possible role of the Ig1 domain in the MuSK binding interaction to BMP. The MuSK ΔIg1 construct was immobilized in a solid-phase binding assay. Figure 10 shows that MuSK binds to ALK6 potently and with high affinity. The ΔIg1 curve shows half-maximum at a concentration of 12.5 nM. Compared with the ΔIg2 curve in the same Figure 10, this result shows that the Ig1 domain is not required for MuSK binding to ALK6. Figure 10 shows that MuSK binding to ALK6 occurs at the Ig2 domain position. Although solid phase binding assays do not provide quantitative binding differences between different constructs, the MuSK ΔIg1 construct had the highest absorbance compared to any of the constructs assayed. Across several assays, the absorbance of 100 nM ALK6 was consistently above 3. ΔIg1 consistently produced high affinity curves, as can be seen in FIG. 10.

[0344] Example 4 MuSK-ALK binding is pH dependent BMP co-receptors have been shown to bind to BMPs and BMP receptors in a pH-dependent manner. Yadin, Knaus, & Muller (2016). MuSK is a BMP co-receptor. We have shown herein that MuSK binds to BMP receptors. We further investigated whether MuSK binds to BMP receptors in a pH-dependent manner.

[0345] Structural review articles indicate that BMP receptors and BMPs can bind to their binding partners in a pH-dependent manner. See Yadin, Knaus, & Muller (2016). Repulsive guidance molecules (RGMs) are proteins that bind BMPs better at slightly acidic pH. See Healey et al. (2015). RGMs bind BMP2 better at pH 5.5 compared to pH 7.4. Healey et al. (2015).

[0346] To assay the effect of pH on MuSK binding to BMP receptors, an Ig1-Ig2 only construct was used. As shown in FIG. 11, the Ig1-Ig2 only construct MuSK ectodomain, in which only the Ig1 and Ig2 domains are present. FIG. 11 shows a schematic of this Ig1-Ig2 domain, which was His-tagged for purification. Using a solid-phase binding assay, we assayed whether pH affects MuSK binding to ALK6 (FIG. 12). FIG. 12 shows that MuSK binds significantly to ALK6 under weakly acidic conditions. At pH 6, the half-maximal value was 6.25 nM, whereas at pH 7.4, the half-maximal value was 25 nM. The difference in absorbance was significant, with the pH 6.0 sample reaching an absorbance of 3, whereas the pH 7.4 sample barely reaching an absorbance of 0.75. This difference indicates that MuSK prefers to bind to ALK6 under weakly acidic conditions. Figure 12 also shows that at pH 6.0, MuSK exhibits a high affinity curve with the first part of the curve rising rapidly and then the remainder of the curve plateauing around an absorbance of 3. This high affinity curve indicates that the interaction between MuSK and ALK6 at pH 6 is highly favorable at this condition compared to pH 7.0.

[0347] Figure 13 shows that MuSK does not show pH dependence when the Ig1 domain is deleted. Figure 13 shows that ΔIg1 MuSK binds with high affinity to ALK6 whether the pH is 6.0 or 7.4. This suggests that the Ig1 domain is the reason why MuSK binds to ALK6 differently at pH 6 and pH 7. In Figure 13, both curves reach a maximum absorbance of nearly 4. The half-maximum of both curves is about 6.5 nM. The figure shows a high affinity curve with a rapid initial rise in absorbance curve.

[0348] Example 5 Although there is still no complete structure of the MuSK ectodomain, a crystal structure of MuSK Ig1-Ig2 alone is available. See Stiegler, Burden, & Hubbard (2006). This structure shows that the Ig1 and Ig2 domains interact with each other. Stiegler, Burden, & Hubbard (2006). There are three contact points between the Ig1 and Ig2 domains, two of which are van der Waals contacts, one of which is a hydrogen bond between an asparagine and a valine. Stiegler, Burden, & Hubbard (2006). These Ig2 deletion constructs remove the central domain of the protein. It is understood that the lack of MuSK binding to ALK6 in Figure 7 could be due to the requirement of the Ig2 domain or that the entire protein is misfolded.

[0349] To determine how important Ig2 domain is for the interaction between MuSK and ALK6, we designed and expressed isolated Ig2 domain. Figure 9 shows that isolated Ig2 domain binds to BMP receptor. This result from Figure 7 and Figure 9 shows that Ig2 domain is necessary and sufficient for MuSK binding to BMP receptor. Ig1 domain plays a role in binding to agrin-LRP4, which is the canonical binding partner of MuSK. See Herbst & Burden (2000). Ig3 domain binds to BMP. See Yilmaz et al. (2016). Thus, Ig2 domain binds to BMP receptor.

[0350] BMPs and BMP receptors are known to have binding partners that bind in a pH-dependent manner. See Healey et al. (2015). To show that MuSK and BMP receptors bind in a pH-dependent manner, we used a binding assay at a mildly acidic pH. To assay pH-dependence, we assayed binding affinity at pH 6.0, whereas previous assays were performed at pH 7.4. Figure 12 shows that the Ig1-Ig2 construct binds much better and with higher affinity at lower pHs compared to pH 7.4 at pH 6.0. When the ΔIg1 construct was assayed, there was no difference in binding affinity or absorbance at the two different pHs. See Figure 13. These results show that MuSK binds to BMP receptors in a pH-dependent manner. These results also show that the Ig1 domain is required for this pH-dependence, since deletion of Ig1 eliminated the pH sensitivity for this interaction.

[0351] When a protein interaction has a pH dependency, the responsible residue causing this pH dependency is usually a histidine. See Cui et al. (2020). Therefore, upon discovery of the pH-dependent binding of MuSK to the BMP receptor, it was important to check whether there was a histidine residue present in the MuSK sequence. A histidine residue was found within the MuSK Ig2 domain. Furthermore, we can see from the crystal structure that the histidine is exposed and faces outward. This histidine residue may play a role in controlling how MuSK responds differently under two different pHs.

[0352] In this example, the histidine residues are mutated to alanines and the binding of the mutated constructs is assayed to determine the lack of pH sensitivity or lack of binding together in these constructs.

[0353] MuSK binding to ALK6 is favored at a slightly acidic pH. MuSK binds best to ALK6 when the Ig1 domain is deleted (Figure 10). Several reasons can explain why MuSK binds strongly and with high affinity to ALK6 lacking the Ig1 domain. Considering that MuSK binds to ALK6 via the Ig2 domain, the deletion of the Ig1 domain may eliminate the steric hindrance that the BMP receptor experiences when binding. The Ig1 domain may slightly block the binding interface at the Ig2 domain, and therefore the ΔIg1 construct binds significantly better than the other constructs. The Ig1 domain is required for MuSK dimerization as shown in the crystal structure of the Ig1-Ig2 domain. See Stiegler, Burden, & Hubbard (2006). See Figure 19.

[0354] Example 6 A working model for how MuSK binds to BMP receptors in the context of BMP signaling.

[0355] At the surface of the cell, the pH is 7.4, BMP binds to the BMP receptor, and MuSK dimerizes using the Ig1 domain. See Stiegler, Burden, & Hubbard (2006); Yadin, Knaus, & Muller (2016); and Lardner (2001). The first panel of Figure 14 shows MuSK as a dimer and the BMP receptor canonically binding to BMP. An event within the cell then triggers clathrin-mediated endocytosis, shown in the next panel of Figure 14. For cells to initiate clathrin-mediated endocytosis, accumulation of PIP2 and adaptor proteins is required. Kaksonen & Roux (2018). Clathrin-mediated endocytosis leads to the formation of endosomes. Endosomes are known to have a slightly acidic pH compared to the extracellular environment of the cell. Geisow & Evans (1984) BMP receptor type 1 promotes binding of MuSK in these conditions. In Figure 14, if the pH is lowered, BMP should also dissociate from the BMP receptor and bind to the BMP receptor, but experimental data has not yet shown this. BMP receptors are known to be present in endosomes. Hartung et al. (2006). The MuSK-BMP pathway and the MuSK-BMP receptor pathway may lead to phosphorylation of SMAD proteins. Thus, the endosome and MuSK-BMP pathway create an enhanced SMAD pathway in cells.

[0356] This disclosure shows that the Ig2 domain binds to ALK6. NMR is planned to identify the residues in the Ig2 domain where MuSK binds to the BMP receptor. The pH dependence indicates that histidines in the Ig2 domain are important for the interaction. The Ig2 domain is about 15 kDa. We plan to express the protein in E. Coli and use NMR to further identify what residues are perturbed when the Ig2 domain binds to ALK6. Considering that MuSK ECD binds best when the Ig1 domain is deleted, and further considering that Ig1 has three contact points with the Ig2 domain, the perturbed residues may be those contact points. See Stiegler, Burden, & Hubbard (2006).

[0357] MuSK likely binds to BMP receptors primarily in endosomes. We plan to disrupt clathrin-mediated endocytosis in cells and observe whether the SMAD pathway is altered as a result. We use the antiemetic / antipsychotic drug prochlorperazine, because it has been shown to inhibit in vivo endocytosis of membrane proteins. See Chew et al. (2020). MuSK and BMP receptors signal through endocytosis, as inhibition of endocytosis affects the SMAD pathway and cellular phosphorylation.

[0358] The role of MuSK dimerization or monomerization in binding to BMP receptors. As shown in Figure 10, MuSK binds best to BMP receptors when the Ig1 domain is deleted. It is also evident from the crystal structure that MuSK dimerizes through the Ig1 domain. MuSK likely dimerizes and monomerizes to regulate binding to BMP receptors. We plan to use size-exclusion chromatography to assess whether the Ig1 sample is monomeric and whether the other sample is dimeric. Size-exclusion chromatography can be used to assay whether MuSK is dimeric at pH 6.0. MuSK can dimerize at pH 7.4 and is monomeric at pH 6.0, so MuSK binds well at that pH.

[0359] Example 7 RNA analysis For this example, we used 5-month-old Pax7 mice. CreERT2 ;TdTomato Fl / Fl Mice were used. See Pawlikowski et al. (2015). The transversus abdominis (TA) muscle of mice was injured using an injection of 50 μL of 1.2% BaCl2. Four days after injury, the transversus abdominis muscle was harvested and then digested using 4000 U / mL collagenase II for 90 minutes, minced, followed by a series of cell filtrations. Satellite cells (SCs) were then isolated from the cell suspension by FACS sorting using TdTomato expression and antibodies against integrin α-7 as positive cell selection markers, and antibodies against CD45, CD31, and Ly-6aA / E as negative cell selection markers. RNA was isolated from satellite cells using the Qiagen RNeasy mini kit. cDNA was generated using the AzuraQuant cDNA synthesis kit. The following PCR primers were used to amplify MuSK cDNA: forward: ACTGGTGAAGCTGGAAGTGG (SEQ ID NO: 7) and reverse: GCCTTGGCTGTCTTTCTGTG (SEQ ID NO: 8). PCR products were run on a 3% agarose, 0.004% EtBr gel in TBE.

[0360] The results are shown in Figure 15. The amino acid sequence of MuSK, containing alternatively spliced ​​exons 5a and 5b, is provided in Figure 16. Figure 17 shows the alternative splicing pattern of exons 5a and 5b from mouse soleus muscle.

[0361] Example 8 MuSK Ig2 Modulator Oligonucleotides Design and synthesis of exon-skipping ASOs. Without wishing to be bound by any theory, the MuSK MR agonizing oligonucleotides described herein are designed according to the following general guidelines, without limitation (see Aartsma-Rus et al., Humana Press, 2012, 117-129): · RNA or DNA modified for resistance to endonucleases or exonucleases (e.g., 2'MoE, 2'OMe, PMO, phosphorothioates); · Designed against target sequences; Typically 15-25 nucleotides, more optimally 17-20 nucleotides; Typically most effective at melt temperatures above 48°C; Typically most effective with 40%-60% GC content to prevent steric hindrance / dimerization, availability to access targets; Typically the most effective targeting open / accessible pre-mRNA structures; Typically the most effective targeted splice regulatory sites or exon-defined sites (e.g., intronic splice enhancers, intronic splice silencers (e.g., Spinraza targeted against the ISS of SMN2 exon 7, exon splice enhancers, exon splice silencers); Typically most effective with sequence compositions containing no more than two guanine (G) or cytosine (C) nucleotides in direct succession (e.g., CCC or GGG).

[0362] ASO Chemistry. We develop 2'-O-2-Methoxyethyl (2'MOE) ASOs that also contain phosphorothioate linkages in the sugar backbone. Methods for designing and testing such ASOs are well established, including manufacturing, pharmacokinetics, biodistribution and toxicology in rodents and non-human primates (Bennett and Swayze, 2010; Chiriboga et al., 2016; Hua et al., 2015; Mercuri et al., 2018; Rigo et al., 2014). The MOE group added to the 2' position of the ribose increases the Tm by approximately 2°C per residue, thus increasing binding affinity as well as improving nuclease resistance. The phosphorothioate modification confers additional nuclease resistance as well as increasing affinity for plasma proteins, resulting in ASOs that are efficiently distributed to tissues and taken up by cells in need of formulation. This chemistry is off-patent, offering commercial advantages.

[0363] ASO design. ASOs are synthesized by a commercial facility and provided by Bolden Therapeutics, Inc. to the Fallon and Webb lab at Brown University for screening. The strategy for designing ASOs involves scanning exonic and intronic sequences flanking both 5' and 3' splice sites with overlapping ASOs (1-2 bp shifts / oligo). The optimal length of the ASO is approximately 17-mer, which provides a good balance between target specificity and drug exposure. ASOs are pre-screened in silico for potential off-target effects, as well as compositional bias (GC content) and propensity for undesired dimer formation.

[0364] The ASO is designed to induce skipping of both exons 5a and 5b of MuSK.

[0365] Screening and selection of optimal exon-skipping ASOs.

[0366] We designed a RT-qPCR TaqMan assay to specifically quantify the following different MuSK splice forms: 1) full-length (FL) MuSK; 2) the desired product Δ exon encoding ΔIg2-MuSK. 5a-5b and 3) potential "incomplete" skipping isoform Δ exons. 5a and Δexon 5b We perform conventional RT-PCR in parallel to detect unexpected products. We transfect cells with several concentrations of candidate ASOs ranging from about 0.1 to 10 nM. After one day of treatment, RNA is extracted and splicing is measured by RT-qPCR to evaluate exon skipping efficiency. Our goal is to isolate at least one ASO that induces ≥80% coordinate splicing of exons 5a and 5b.

[0367] Selected ASOs will be tested for their ability to inhibit MuSK-BMP receptor signaling in cultured cells.

[0368] We observed that the ΔIg2 domain of MuSK is required for MuSK-BMP receptor binding. However, since in vivo skipping may be less than 100% efficient (Rigo et al., 2014), it is important to establish a relationship between the level of skipping achieved and physiological effects. Therefore, to this end, we measure the level of MuSK-BMP receptor (ALK)-dependent signaling in ASO-treated cells.

[0369] We use qRT-PCR to measure the level of MuSK-BMP receptor-dependent transcripts. The cells treated with either exon skipping of control ASO are stimulated with ALK for 2 hours. Then, the level of transcripts is measured, and the response to BMP is correlated with the degree of exon skipping.

[0370] Data. The present disclosure recognizes that in some embodiments, the efficiency of skipping exons 5a and 5b may be insufficient with a single ASO. In some embodiments, it may be desirable to prepare one or more ASOs against exon 5b for use alone or together with an exon 5a-specific ASO.

[0371] Those of skill in the art will understand upon reading this disclosure that in some embodiments it may be desirable to replicate the studies (e.g., at least three times) or to analyze the data using appropriate statistical methods (e.g., by t-test with appropriate correction for multiple comparisons (e.g., Bonferroni)).

[0372] The work described herein provides the technology for the efficient development of ASO-mediated therapies for neuromuscular diseases and disorders to enhance muscle regeneration.

[0373] List of references Those of skill in the biomedical arts may use these patents, patent applications and scientific references as guide in making and using the invention.

[0374] Patent documents.

[0375] US Patent No. 9,574,015 (Burden et al.) discloses a method of treating a subject suffering from a neuromuscular disorder, i.e., improving motor function in a subject suffering from muscle-specific receptor kinase myasthenia gravis (MuSK-MG), by administering a portion of a MuSK polypeptide, i.e., muscle-specific receptor kinase (MuSK) first immunoglobulin-like domain 1 (Ig1). Burden does not disclose a method of increasing neurogenesis, including a step or a specific step of increasing the level or activity of a MuSK polypeptide lacking a functional lg3 domain, or a step or a specific step of decreasing the level or activity of a BMP-MuSK polypeptide complex, where MuSK comprises a functional lg3 domain.

[0376] WO 2012 / 109075 (Plexxikon Inc.). This patent publication discloses modulating the activity of MuSK using a kinase and a compound that selectively modulates the kinase, and uses for treatment (paragraph

[0003] ) by modulating kinase activity, for example, for disease indications with neurodegenerative or cognitive impairment characteristics, suitable for neurodegenerative diseases of Alzheimer's disease (paragraph

[0001] ). Compounds include compounds of formula I (disclosed), and any subgeneric formulas, claimed compounds, and compounds described herein that are modulators of protein kinases. Disease indications suitable for treatment by modulating kinase activity with the compounds described. This patent publication does not disclose increasing the level or activity of a MuSK polypeptide lacking a functional lg3 domain, or decreasing the level or activity of a BMP-MuSK polypeptide complex, where MuSK comprises a functional lg3 domain. This patent publication does not disclose its use in methods of increasing neurogenesis, or increasing the level or activity of a MuSK polypeptide lacking a functional lg3 domain, or decreasing the level or activity of a BMP-MuSK polypeptide complex, wherein MuSK comprises a functional lg3 domain.

[0377] WO 2020 / 214987 (Brown University) Neurogenesis, discloses methods and compositions for increasing neurogenesis and preventing or treating diseases, disorders or conditions associated with neurodegeneration.

[0378] Non-patent literature.

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[0381] Yun et al.,Re-evaluating the link between neuropsychiatric disorders and dysregulated adult neurogenesis.Nature Medicine, 22(11), p.1239(2016). Zhang et al.,A role for adult TLX-positive neural stem cells in learning and behavior.Nature,451,1004-1007(2008b). Zhang et al., LRP4 serves as a coreceptor of agrin. Neuron, 60, 285-297 (2008a). Textbooks and technical references Current Protocols in Immunology (CPI) (2003). John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc. (ISBN 0471142735, 9780471142737). Current Protocols in Molecular Biology(CPMB),(2014).Frederick M.Ausubel(ed.),John Wiley and Sons(ISBN 047150338X,9780471503385). Current Protocols in Protein Science(CPPS),(2005).John E.Coligan(ed.),John Wiley and Sons,Inc. Immunology(2006).Werner Luttmann,published by Elsevier. Janeway’s Immunobiology,(2014).Kenneth Murphy,Allan Mowat,Casey Weaver(eds.),Taylor&Francis Limited,(ISBN 0815345305,9780815345305). Laboratory Methods in Enzymology:DNA,(2013).Jon Lorsch(ed.)Elsevier(ISBN 0124199542). Lewin’s Genes XI,(2014).published by Jones&Bartlett Publishers(ISBN-1449659055). Molecular Biology and Biotechnology:a Comprehensive Desk Reference,(1995).Robert A.Meyers(ed.),published by VCH Publishers,Inc.(ISBN 1-56081-569-8). Molecular Cloning:A Laboratory Manual,4th ed.,Michael Richard Green and Joseph Sambrook,(2012).Cold Spring Harbor Laboratory Press,Cold Spring Harbor,N.Y.,USA(ISBN 1936113414). The Encyclopedia of Molecular Cell Biology and Molecular Medicine, Robert S. Porter et al. (eds.), published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908). The Merck Manual of Diagnosis and Therapy,19 th edition(Merck Sharp&Dohme Corp., 2018). Pharmaceutical Sciences 23 rd edition (Elsevier, 2020). [List of embodiments] Specific compositions and methods for characterizing the binding interaction between Musk and Bmp receptors have been described. The scope of the present invention should be defined solely by the claims. Those skilled in the biomedical field will interpret all claim terms in the broadest possible manner consistent with the context and spirit of the present disclosure. The detailed description herein is illustrative and not limiting or exhaustive. The present invention is not limited to the specific methods, protocols, and reagents described herein, which may vary in practice. Where the specification or claims describe ordered steps or functions, alternative embodiments may perform those functions in a different order or substantially simultaneously. As those skilled in the biomedical field will recognize, other equivalents and modifications beyond those already described are possible without departing from the inventive concept described herein.

[0382] All patents and publications cited throughout this specification are incorporated by reference to disclose and describe the materials and methods used in conjunction with the technology described herein. The patents and publications are provided solely for their disclosure prior to the filing date of this specification. All statements as to disclosure and publication dates of patents and publications are from the information and belief of the inventors. The inventors make no admission as to the accuracy of the contents or dates of these documents. In the event of a conflict between the dates provided herein and the actual publication dates, the actual publication dates shall control. The inventors may have antedated such disclosures by reason of prior invention or for another reason. In the event of a conflict between the scientific or technical teachings of the earlier patents or publications and this specification, the teachings of this specification and these claims shall control.

[0383] Where this specification provides a range of values, each intervening value between the upper and lower limits of that range is within the range of values, unless the context dictates otherwise.

Claims

1. 1. A pharmaceutical composition for treating a subject suffering from one or more features of neurodegeneration or cognitive impairment by modulating a MuSK Ig2 domain, comprising a MuSK Ig2 modulator agent, the MuSK Ig2 modulator agent comprising: (a) increasing the level or activity of a MuSK polypeptide that lacks a functional Ig2 domain; and / or (b) A pharmaceutical composition that reduces the level or activity of a BMP receptor (ALK)-MuSK polypeptide complex, wherein said MuSK comprises a functional Ig2 domain.

2. The pharmaceutical composition of claim 1, wherein the MuSK Ig2 modulator increases neurogenesis in the subject.

3. The pharmaceutical composition of claim 1 or 2, wherein the MuSK Ig2 modulator comprises a small molecule, a polypeptide or a nucleic acid.

4. The pharmaceutical composition of claim 1, wherein the MuSK Ig2 modulator comprises a small molecule.

5. The pharmaceutical composition according to claim 1, wherein the MuSK Ig2 modulator binds to an autoantibody against the MuSK Ig2 domain.

6. The pharmaceutical composition of claim 1, wherein the MuSK Ig2 modulator comprises a polypeptide.

7. The pharmaceutical composition of claim 6, wherein the polypeptide comprises an antibody.

8. The pharmaceutical composition of claim 7, wherein the antibody targets the MuSK Ig2 domain and / or the BMP receptor.

9. The pharmaceutical composition of claim 1, wherein the MuSK Ig2 modulator comprises an oligonucleotide.

10. The pharmaceutical composition of claim 9, wherein the oligonucleotide regulates alternative splicing of the 5a and / or 5b MuSK exons.

11. The pharmaceutical composition of claim 10, wherein the oligonucleotide increases exon skipping of the 5a and / or 5b MuSK exons.

12. The oligonucleotide increases exon 5a skipping of the MuSK Ig2 domain; The oligonucleotide increases the skipping of exon 5b of the MuSK Ig2 domain; or The pharmaceutical composition of claim 11 , wherein the oligonucleotide increases skipping of exons 5a and 5b of the MuSK Ig2 domain.

13. A pharmaceutical composition described in any one of claims 9 to 12, wherein the oligonucleotide increases the ratio of MuSK transcripts lacking an Ig2 domain to full-length MuSK transcripts, e.g., the ratio of MuSK mRNA lacking exons 5a and 5b, compared to a reference ratio.

14. The pharmaceutical composition of claim 13, wherein the ratio of MuSK transcript lacking an Ig2 domain to full-length MuSK transcript, e.g., the ratio of MuSK mRNA lacking exons 5a and 5b, is greater than 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 or 10-fold compared to the reference ratio.

15. A pharmaceutical composition described in any one of claims 9 to 14, wherein the oligonucleotide increases the level of a MuSK transcript lacking an Ig2 domain.

16. The pharmaceutical composition of claim 15, wherein the oligonucleotide increases the level of a MuSK transcript lacking an Ig2 domain by more than 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 or 10-fold above a reference level.

17. A pharmaceutical composition described in any one of claims 1 to 16, wherein the BMP receptor (ALK) is ALK6.

18. A method of inducing MuSK exon skipping by contacting a system containing a population of MuSK primary transcripts with an oligonucleotide that binds to such primary transcripts, such that skipping of one or both of exons 5a and 5b is increased.

19. The method of claim 18, wherein the system is a cell.

20. The method of claim 19, wherein the cells are derived from a subject suffering from one or more characteristics of neurodegeneration or cognitive impairment.

21. The method described in claim 19 or 20, wherein the efficiency of MuSK5a and 5b exon skipping in cells is more than 10%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90% or more than 95%.

22. 1. A method for characterizing a MuSK Ig2 modulator agent, comprising: (a) assessing the ability to bind to the Ig2 domain of MuSK; (b) assessing the ability to decrease MuSK-Ig2-BMP receptor (ALK) complex formation; (c) assessing the ability to alter the splicing pattern of a primary MuSK transcript; (d) assessing the ability to inhibit expression of the transcript; (e) assessing the ability to increase expression of a MuSK transcript lacking a sequence encoding an Ig2 domain; (f) assessing the ability to increase levels of a MuSK polypeptide lacking functional Ig2; and (g) assessing the ability to affect a property of cells in the population; The method includes one or more of the following:

23. A genetically modified mouse comprising a sequence encoding MuSK in its genome, wherein the sequence encoding MuSK does not include the range of nucleotides from exon 5a to exon 5b (in 5' to 3' order); A genetically modified mouse that is unable to express the full-length MuSK transcript and is unable to produce the full-length MuSK protein.

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