Regulation of satellite cell polarity and asymmetric cell division

Abstract

The present disclosure relates to compositions and methods for enhancing asymmetric division of satellite cells and promoting muscle cell / tissue regeneration for the treatment of muscle tissue injury and muscle diseases (including muscular dystrophy). The present disclosure provides compositions and methods (including dosages and dosing regimens) that increase or promote asymmetric cell division and can be used to treat various diseases and disorders (including muscular dystrophy). In one aspect, the present disclosure is a method for increasing asymmetric cell division of skeletal muscle stem cells, the method comprising contacting the skeletal muscle stem cells with an inhibitor of any one of adaptor-associated kinase 1 (AAK1), cyclin G-associated kinase (GAK), or myristoylated and / or palmitoylated serine / threonine kinase 1 (also known as MPSK1, STK16).

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 345,678, filed May 25, 2022, entitled "Modulation of Satellite Cell Polarity and Asymmetric Cell Division" and U.S. Provisional Patent Application No. 63 / 447,807, filed February 23, 2023, entitled "Modulation of Satellite Cell Polarity and Asymmetric Cell Division", which are hereby incorporated by reference in their entirety.

[0002] Statement Regarding the Sequence Listing The Sequence Listing XML associated with this application is provided in XML file format and is hereby incorporated by reference. The name of the XML file containing the Sequence Listing XML is STLS_016_02WO_ST26.xml. The XML file is 28,436 bytes in size, was created on May 24, 2023, and was electronically filed via the USPTO Patent Center.

[0003] Field of the Invention The present disclosure relates to compositions and methods for enhancing asymmetric division of satellite cells and promoting muscle cell / tissue regeneration, including dosages and dosing regimens, for the treatment of muscle tissue injury and muscle diseases (including muscular dystrophy).

Background Art

[0004] Background Stem cells are undifferentiated or immature cells that can give rise to multiple specialized cell types and ultimately terminally differentiated cells. Most adult stem cells are lineage - restricted and are generally referred to by their tissue origin. Unlike any other cell, stem cells can replicate themselves to generate a substantially unlimited supply of mature cell types when needed over the lifespan of an organism.

[0005] Satellite cells are a heterogeneous population of stem cells and small mononuclear progenitor cells found in mature muscle tissue (Kuang et al. Cell, 2007. 129(5): p. 999-1010). Satellite cells play a central regulatory role in satellite cell function and survival and express many gene markers (including the paired box transcription factor Pax7) that can be used as markers of satellite cells (Kuang et al., 2006. J. Cell Biol. 172(1):103-13; Seale et al, 2000. Cell. 102(6):777-86). The satellite cell population is composed of a subpopulation of stem cells (Pax7+ / Myf-) and committed myogenic progenitor cells (Pax7+ / Myf5+). Pax7+ / Myf5- satellite cells give rise to Pax7+ / Myf5+ satellite cells via basal-apical oriented asymmetric cell division within the satellite cell niche. While Pax7+ / Myf5+ satellite cells preferentially differentiate, Pax7+ / Myf5- satellite cells contribute extensively to the satellite cell compartment. This asymmetric satellite cell division helps skeletal muscle maintain its stem cell pool while simultaneously contributing to muscle regeneration.

[0006] Satellite cells are involved in normal muscle growth and the regeneration of injured or diseased muscle tissue. In uninjured muscle, most satellite cells are in a quiescent state and do not differentiate or undergo cell division. However, upon muscle injury (e.g., physical trauma or strain, repeated exercise) or in disease, satellite cells become activated, proliferate, and give rise to a transiently amplified population of progenitor cells that are myogenic progenitor cells (myoblasts) expressing myogenic regulatory factors (MRFs) (e.g., MyoD and Myf5). During muscle regeneration, myoblasts undergo multiple rounds of division before committing to terminal differentiation and fuse with host fibers or generate new muscle fibers to reconstruct the damaged tissue (Charge and Rudnicki, 2004. Physiol. Rev. 84(1):209-38). Skeletal muscle has the ability to regenerate, but this ability is significantly impaired in certain muscle diseases (e.g., Duchenne muscular dystrophy (DMD)). Specifically, skeletal muscle stem cells (also known as satellite cells) have cell-intrinsic defects in DMD, which results in a significantly reduced number of asymmetric divisions. There is clearly a need in the art for muscle disease treatments that address the problem of reduced asymmetric division of satellite cells and methods for generating cells that can produce functional muscle.

Prior Art Documents

Non-Patent Documents

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Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Means for Solving the Problems

[0008] Gist of the Invention The present disclosure provides compositions and methods (including dosages and dosing regimens) that increase or promote asymmetric cell division and can be used to treat various diseases and disorders, including muscular dystrophy.

[0009] In one aspect, the present disclosure provides a method for increasing asymmetric cell division of skeletal muscle stem cells, the method comprising contacting a skeletal muscle stem cell with an inhibitor of any one of adaptor-associated kinase 1 (AAK1), cyclin G-associated kinase (GAK), or myristoylated and / or palmitoylated serine / threonine kinase 1 (also known as MPSK1, STK16). In certain embodiments, the skeletal muscle stem cell is a damaged or injured skeletal muscle stem cell or is present within damaged or injured skeletal muscle tissue. In some embodiments, the muscle tissue is damaged or injured as a result of physical injury or accident, disease, genetic mutation, infection, overuse, loss of blood circulation, muscle atrophy, muscle wasting, dystrophic muscle, or aging. In some embodiments, the skeletal muscle stem cell is a diseased skeletal muscle stem cell comprising mutations associated with muscular dystrophy, optionally Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), Emery-Dreifuss muscular dystrophy, Landouzy-Dejerine muscular dystrophy, facioscapulohumeral muscular dystrophy (FSH), limb-girdle muscular dystrophy, von Graefe-Fuchs muscular dystrophy, oculopharyngeal muscular dystrophy (OPMD), myotonic dystrophy (Steinert's disease), or congenital muscular dystrophy.

[0010] In some embodiments, the damaged or injured muscle stem cell comprises a mutation in the dystrophin gene. In some embodiments, the skeletal muscle stem cell is present within injured muscle tissue. In some embodiments, the skeletal muscle stem cell has reduced asymmetric cell division compared to a normal healthy skeletal muscle stem cell.

[0011] In another aspect, the present disclosure provides a method for increasing skeletal muscle tissue growth or regeneration in a subject, the method comprising administering to the subject an inhibitor of AAK1, GAK, or MPSK1. In some embodiments, the subject has damaged or injured skeletal muscle tissue. In some embodiments, the skeletal muscle tissue is damaged or injured as a result of physical injury or accident, disease, genetic mutation, infection, overuse, loss of blood circulation, muscle atrophy, muscle wasting, dystrophic muscle, or aging. In some embodiments, the subject has a muscle dystrophy, optionally Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), Emery-Dreifuss muscular dystrophy, Landouzy-Dejerine muscular dystrophy, facioscapulohumeral muscular dystrophy (FSH), limb-girdle muscular dystrophy, von Graefe-Fuchs muscular dystrophy, oculopharyngeal muscular dystrophy (OPMD), myotonic dystrophy (Steinert's disease) or congenital muscular dystrophy. In some embodiments, the subject has a mutation in the dystrophin gene. In some embodiments, the skeletal muscle stem cells within the skeletal muscle tissue have reduced asymmetric cell division compared to normal healthy skeletal muscle stem cells. In some embodiments, the inhibitor of AAK1, GAK, or MPSK1 does not substantially inhibit the proliferation or cell cycle progression of the subject's skeletal muscle stem cells. In some embodiments, the method increases skeletal muscle tissue regeneration in the subject. In some embodiments, the subject is a mammal, optionally a human. In some embodiments, the inhibitor of AAK1, GAK, or MPSK1 is administered to the subject systemically or locally, optionally at the site of tissue damage or injury.

[0012] In another aspect, the present disclosure provides a method for treating muscular dystrophy, the method comprising administering to a subject in need thereof an inhibitor of AAK1, GAK, or MPSK1. In some embodiments, the subject has a muscular dystrophy selected from the group consisting of Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), Emery-Dreifuss muscular dystrophy, Landouzy-Dejerine muscular dystrophy, facioscapulohumeral muscular dystrophy (FSH), limb-girdle muscular dystrophy, von Graefe-Fuchs muscular dystrophy, oculopharyngeal muscular dystrophy (OPMD), myotonic dystrophy (Steinert's disease), and congenital muscular dystrophy. In some embodiments, the subject has a mutation in the dystrophin gene. In some embodiments, the skeletal muscle stem cells in the subject have reduced asymmetric cell division compared to normal healthy skeletal muscle stem cells. In some embodiments, the inhibitor of AAK1, GAK, or MPSK1 does not substantially inhibit the proliferation or cell cycle progression of the subject's skeletal muscle stem cells. In some embodiments, the method increases skeletal muscle tissue regeneration in the subject. In some embodiments, the subject is a mammal, optionally a human. In some embodiments, the inhibitor of AAK1, GAK, or MPSK1 is administered to the subject systemically or locally, optionally at the site of tissue damage or injury.

[0013] In certain embodiments of any of the methods disclosed herein, the inhibitor inhibits the expression of AAK1, GAK, or MPSK1, optionally by inhibiting transcription, translation, post-translational modification, or the stability of the protein component, or the gene encoding the protein component. In some embodiments, the inhibitor binds to a polynucleotide sequence that regulates the expression of AAK1, GAK, or MPSK1, where optionally the nucleotide sequence is present within the gene for AAK1, GAK, or MPSK1. In some embodiments, the inhibitor binds to a polynucleotide sequence encoding AAK1, GAK, or MPSK1, or a polynucleotide sequence complementary to the polynucleotide sequence encoding AAK1, GAK, or MPSK1, where optionally the polynucleotide sequence is present within the AAK1 gene or mRNA. In some embodiments, the polynucleotide sequence is DNA or RNA. In some embodiments, the inhibitor comprises a polynucleotide sequence. In some embodiments, the inhibitor comprises a DNA polynucleotide sequence and / or an RNA polynucleotide sequence. In some embodiments, the inhibitor comprises shRNA, microRNA, gRNA, siRNA, an aptamer, or an antisense oligonucleotide. In some embodiments, the inhibitor comprises a guide RNA targeting the AAK1 gene and a polynucleotide sequence encoding a CRISPR-Cas protein.

[0014] In any particular embodiment of the methods disclosed herein, the inhibitor inhibits the activity of AAK1, GAK, or MPSK1. In some embodiments, the inhibitor binds to AAK1, GAK, or MPSK1. In some embodiments, the inhibitor comprises a polypeptide. In some embodiments, the inhibitor comprises an antibody that binds to AAK1, GAK, or MPSK1, or a functional fragment thereof. In some embodiments, the inhibitor is an organic molecule, for example, a small organic molecule. In some embodiments, the inhibitor is selected from the group consisting of SGC-AAK1-1, LP-935509, LP-922761, BMT-090605, BMT-124110, LP-927443, and BMS-901715. In some embodiments, the inhibitor inhibits AAK1, GAK, or MPSK1 kinase activity or AAK1, GAK, or MPSK1 ATP-binding activity. In some embodiments, the above inhibitor of AAK1, GAK, or MPSK1 does not substantially inhibit the proliferation or cell cycle progression of the above skeletal muscle stem cells. In some embodiments, the inhibitor inhibits AAK1. In some embodiments, the inhibitor inhibits GAK. In some embodiments, the inhibitor inhibits MPSK1.

[0015] In any particular embodiment of any of the methods disclosed herein, the step of contacting the inhibitor with the cell is performed in vitro, in vivo, ex vivo, or in situ. In some embodiments, the cell is of mammalian origin, optionally human.

[0016] In certain embodiments of any of the methods disclosed herein, the inhibitor is administered once daily, every two days, every three days, every four days, every five days, every six days, or every seven days. In some embodiments, the inhibitor is administered once every about three days. In some embodiments, the inhibitor is administered one, two, three, four, five, six, or seven times per week. In some embodiments, the inhibitor is administered twice per week.

[0017] In certain embodiments of any of the methods disclosed herein, the inhibitor is administered at a dose of about 0.01 mg / kg to about 300 mg / kg. In some embodiments, the inhibitor is administered at a dose of about 0.1 mg / kg to about 20 mg / kg. In some embodiments, the inhibitor is administered at a dose of about 0.1 mg / kg, about 0.3 mg / kg, about 0.7 mg / kg, about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, about 8 mg / kg, about 9 mg / kg, about 10 mg / kg, about 11 mg / kg, about 12 mg / kg, about 13 mg / kg, about 14 mg / kg, about 15 mg / kg, about 16 mg / kg, about 17 mg / kg, about 18 mg / kg, about 19 mg / kg or about 20 mg / kg. In some embodiments, the inhibitor is administered at a dose of about 1 mg / kg.

[0018] In other aspects, any of the methods disclosed herein are performed using an inhibitor of STK38 or STK38L. BRIEF DESCRIPTION OF THE DRAWINGS

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Mode for Carrying Out the Invention

[0037] Detailed Description The present disclosure provides compositions and methods for modulating cell signaling pathways and increasing asymmetric division of satellite cells, for example, to increase or enhance muscle regeneration or as a therapeutic strategy for various muscle - wasting diseases (such as, but not limited to, Duchenne muscular dystrophy (DMD)). The methods disclosed herein can similarly be used to increase or stimulate muscle fiber and / or muscle tissue regeneration.

[0038] Adult skeletal muscle has the ability to regenerate. For example, after acute muscle injury, new muscle fibers form within about one week as a result of the expansion and differentiation of self - replicating muscle satellite cells. During regeneration, normally quiescent satellite cells are activated to generate daughter myogenic progenitor cells, which then form new muscle fibers. However, the number of satellite cells decreases during aging, which results in a reduced muscle regeneration capacity. Furthermore, in certain muscle diseases (such as DMD), the reduced satellite cell regenerative capacity and number result in impaired regeneration and accelerated disease progression.

[0039] Satellite cells are a heterogeneous population mainly composed of committed progenitor cells, together with a small population of muscle stem cells that are capable of long-term self-renewal. Satellite cells undergo two forms of cell division: asymmetric division (where a large subpopulation of cells generates daughter cells committed to myogenic differentiation while a small subpopulation of cells gives rise to daughter cells that self-renew); and symmetric division (where one stem cell population generates two identical daughter stem cells). In regenerating muscle, symmetric division of satellite cells generally occurs in a planar orientation (parallel to the muscle fiber), whereas asymmetric division occurs in an apicobasal orientation (perpendicular to the muscle fiber). Thus, in the context of acute muscle injury, asymmetric satellite cell division generates a transient amplifying progenitor cell that can generate stem cells and a cohort of myogenic progenitor cells that divide multiple times and differentiate either by fusing with existing muscle fibers or by forming new muscle fibers, while symmetric satellite cell division promotes the expansion of satellite stem cells and maintains the homeostasis of the stem cell compartment.

[0040] Satellite cells are juxtaposed to the myofiber sheath within a niche that forms beneath the basement membrane. Quiescent satellite cells are polarized and express different adhesion proteins basally versus apically on the cell surface, which influence quiescence and cell polarity. In healthy satellite cells, dystrophin acts as a scaffolding protein during mitosis to bind Par1b, resulting in the asymmetric segregation of Pard3 and the PAR complex, as well as the apicobasal orientation of the centrosome prior to mitosis. After apicobasally oriented asymmetric division, the committed daughter cells are no longer in contact with the basement membrane, while the stem cells that remain in contact with the basement membrane maintain niche interactions and promote reentry into the quiescent state.

[0041] Abbreviations As used in this specification and the appended claims, the singular forms "a", "an", and "the" include references to the plural unless the context clearly dictates otherwise.

[0042] As used herein, the term "and / or" is used in this disclosure to indicate either "and" or "or" where not otherwise indicated.

[0043] Throughout this specification, unless the context requires otherwise, variations such as the words "comprise", "comprises" or "comprising" are to be construed as including the stated element or integer or group of elements or integers but not necessarily excluding any other element or integer or group of elements or integers.

[0044] As used in this application, the terms "about" and "approximately" are used equivalently. Any numerical value used in this application, whether or not accompanied by about / approximately, is meant to cover any normal variations recognized by one of ordinary skill in the relevant art. In certain embodiments, the term "approximately" or "about" refers to a value within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less than this in either direction (greater or less) of the stated reference value, unless otherwise stated or otherwise apparent from the context (except where such a number exceeds 100% of the possible value).

[0045] "Decrease" or "inhibit" refers to, for example, at least a 5% decrease or inhibition, such as 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 99% or 100% decrease or inhibition compared to a reference or control level, for example, in control cells or tissues.

[0046] "Increase" refers to, for example, at least a 5% increase, such as 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 99% or at least 100% increase compared to a reference level or the level in control cells or tissues. Increase also means, for example, at least a 1-fold increase, such as at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, at least 200-fold, at least 500-fold, at least 1000-fold or greater increase compared to a reference level or the level in control cells or tissues.

[0047] The term "inhibitor" can refer to any agent that inhibits the expression or activity of a target gene, mRNA, and / or protein in a cell, tissue, organ, or subject. The expression level or activity of a target mRNA and / or protein in a cell can be reduced via various means, including, but not limited to, reducing the total amount of the target protein or inhibiting one or more activities of the target protein. In various embodiments, an inhibitor can inhibit the expression of a target gene, target mRNA, or target protein, and / or an inhibitor can inhibit the biological activity of a target protein. In certain embodiments, the biological activity is kinase activity. For example, an inhibitor can competitively bind to the ATP binding site of a kinase and inhibit its kinase activity, or it can allosterically block the kinase activity. In certain embodiments, an inhibitor causes increased degradation of a target protein. Methods for determining the expression level or activity of a target gene or polypeptide are known in the art and include, for example, RT-PCR and FACS. Methods for determining kinase activity are known in the art and include, for example, those described in Nat Methods (2005), 2(1):17-25.doi: 10.1038 / nmeth731.

[0048] "Subject" includes animals such as mammals (e.g., humans, pigs, fish, birds, insects, etc.). In some embodiments, the subject is a mammal, particularly a primate, particularly a human. In some embodiments, the subject is a domestic animal (e.g., cattle, sheep, goats, cows, pigs, etc.); poultry (e.g., chickens, ducks, geese, turkeys, etc.); and domesticated animals (e.g., dogs and cats). In some embodiments (e.g., particularly in a research context), the subject is a rodent (e.g., mice, rats, hamsters), rabbit, primate, or pig (e.g., an inbred pig, etc.). The terms "subject" and "patient" are used interchangeably herein.

[0049] "Tissue" is an ensemble of similar cells of the same origin that perform a specific function together (e.g., smooth muscle tissue or skeletal muscle tissue).

[0050] "Antibody" is an immunoglobulin (Ig) molecule that can specifically bind to a target (e.g., carbohydrate, polynucleotide, lipid, or polypeptide) via at least one epitope recognition site located in the variable region of the Ig molecule. As used herein, the term includes not only intact polyclonal or monoclonal antibodies, but also fragments thereof (e.g., dAb, Fab, Fab’, F(ab’) 2 , Fv, single-chain (scFv), synthetic variants thereof, naturally occurring variants, fusion proteins containing antibody portions with the required specificity antigen-binding fragments, chimeric antibodies, nanobodies, and any other modified constructs of immunoglobulin molecules containing the required specificity antigen-binding sites or fragments.

[0051] "Fragment" refers to a portion of a polypeptide or polynucleotide molecule. This portion preferably comprises at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the full length of the reference nucleic acid molecule or polypeptide. Fragments can contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids. A "functional fragment" of an antibody is a fragment that maintains one or more of the activities of the above antibody. For example, it binds to the same epitope of the above antibody and / or has biological activity. In certain embodiments, the functional fragment comprises the 6 CDRs present in the above antibody.

[0052] "Pharmaceutical composition" includes a composition of one or more inhibitors and one or more pharmaceutically acceptable carriers, additives or diluents disclosed herein.

[0053] "Pharmaceutically acceptable" is used herein to refer to those compounds, substances, compositions, and / or dosage forms that are suitable for use in contact with the tissues of humans and animals within the scope of sound medical judgment, without undue toxicity, irritation, allergic response, or other problems or complications, and that are commensurate with a reasonable benefit / risk ratio.

[0054] "Pharmaceutically acceptable carrier" refers to any adjuvant, carrier, additive, glidant, sweetening agent, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizing agent, isotonic agent, solvent, surfactant, and / or emulsifying agent that is acceptable for use in humans and / or domesticated animals and has been approved by the US Food and Drug Administration, but is not limited thereto. Exemplary pharmaceutically acceptable carriers include, but are not limited to: sugars (e.g., lactose, glucose, and sucrose); starches (e.g., corn starch and potato starch); cellulose and its derivatives (e.g., sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate); tragacanth; malt; gelatin; talc; cocoa butter, waxes, animal and vegetable fats, paraffin, silicone, bentonite, silicic acid, zinc oxide; oils (e.g., peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil); glycols (e.g., propylene glycol); polyols (e.g., glycerin, sorbitol, mannitol, and polyethylene glycol); esters (e.g., ethyl oleate and ethyl laurate); agar; buffering agents (e.g., magnesium hydroxide and aluminum hydroxide); alginic acid; pyrogen-free water; isotonic saline solutions; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and any other compatible substances used in pharmaceutical formulations. The use of any conventional medium and / or agent is contemplated in the therapeutic composition, except where it is incompatible with the agents of the present disclosure. Auxiliary active ingredients may also be incorporated into the above compositions.

[0055] "Dosage" means a specific amount of a pharmaceutical agent provided in a single administration or over a specific period. In certain embodiments, the dosage can be administered in 2 or more boluses, tablets, or injections. In certain embodiments, the dosage can be administered in 2 or more injections to minimize injection site reactions in an individual. The dosage can be described as the amount of the pharmaceutical agent per hour, per day, per week, or per month. "Dosage amount" can be used interchangeably with "dosage".

[0056] "Dosage regimen" means a schedule according to which dosages of a pharmaceutical agent are provided, e.g., daily, weekly, or other schedules that can be developed by one of ordinary skill in the art.

[0057] "Effective amount", as used herein, refers to the amount of an agent effective in achieving a particular effect (e.g., increasing asymmetric cell division or tissue regeneration in a cell, tissue, organ, or subject). In certain embodiments, the increase is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70% compared to the amount before treatment or without treatment. In the context of treating a subject, an effective amount can be, for example, an amount effective or sufficient to reduce one or more disease symptoms in the subject (e.g., a subject having muscular dystrophy).

[0058] "Effective concentration", as used herein, refers to the minimum concentration (mass / volume) of an agent and / or composition required to produce a particular physiological effect. As used herein, effective concentration typically refers to the concentration of an agent required to increase, activate, and / or enhance a particular physiological effect.

[0059] Method As described in the accompanying examples, the present disclosure identifies targets useful in increasing asymmetric division and promoting muscle tissue regeneration. Thus, the present disclosure provides methods and compositions for, for example, increasing asymmetric cell division of satellite cells, promoting or increasing muscle cell and tissue growth and regeneration, and treating diseases, disorders and injuries that benefit from muscle tissue generation.

[0060] Asymmetric cell division is a type of cell division that produces two different non-identical daughter cells, typically with different characteristics or cell fates, via unequal inheritance or distribution of cell fate determinants (e.g., cellular proteins and RNAs). Asymmetric satellite cell division (and its regulation) can be determined or measured according to methods known in the art and according to the methods disclosed herein (e.g., using cultured muscle fibers).

[0061] Muscle fiber and tissue regeneration typically refers to the generation of new muscle fibers and tissue as a result of the expansion and differentiation of self-renewing muscle satellite cells. During the regeneration process, normally quiescent satellite cells are activated to produce daughter myogenic progenitor cells, which can then form new muscle fibers that can fuse with existing muscle fibers to generate new muscle tissue. Muscle fiber and tissue generation can be determined or measured according to methods known in the art and according to the methods disclosed herein (e.g., prevalence and / or density or mass of MyoG+ progenitor cells, muscle fiber area, muscle fiber Feret diameter, and / or animal models for determining muscle strength).

[0062] In one embodiment, the present disclosure provides a method for increasing asymmetric cell division of stem cells or any other cells undergoing asymmetric cell division (e.g., skeletal muscle stem cells, or satellite cells), the method comprising contacting the cells with an inhibitor of AAK1, GAK, or MPSK1. In certain embodiments, the inhibitor inhibits AAK1. In certain embodiments, the stem cells are muscle stem cells, retinal stem cells, neural stem cells, hematopoietic stem cells, intestinal stem cells, epidermal stem cells, or cancer or tumor stem cells. In certain specific embodiments, the stem cells are muscle stem cells or satellite cells.

[0063] In another embodiment, the present disclosure provides a method for increasing skeletal muscle tissue growth or regeneration, the method comprising contacting skeletal muscle stem cells with an inhibitor of AAK1, GAK, or MPSK1. In certain embodiments, the inhibitor inhibits AAK1.

[0064] The methods disclosed herein can be performed in vitro, ex vivo, or in vivo. For example, the methods can be used to promote the growth and proliferation of satellite cells in vitro to generate tissue (e.g., muscle tissue) or to treat a subject in need of increased muscle tissue generation.

[0065] In certain embodiments, the method is performed in vitro or ex vivo, for example, to promote or increase asymmetric cell division of stem cells. Such methods can be used, for example, to generate tissue models or organoids. Further, such methods can be used, for example, to generate progenitor cells. In vitro and ex vivo tissues, organoids, and progenitor cells have various uses, including, for example, use in research and use in screening potential therapeutic drug candidates.

[0066] In certain embodiments of any of the methods disclosed herein, the stem cells, such as skeletal muscle stem cells, are damaged or injured stem cells or are present within damaged or injured tissue (e.g., skeletal muscle tissue). In certain embodiments, the stem cells or tissue are damaged or injured as a result of physical injury or accident, disease, genetic mutation, infection, overuse, loss of blood circulation, muscle atrophy, cachexia, muscle wasting, dystrophic muscle, or hematopenia or aging. However, the stem cells, such as skeletal muscle stem cells, can also be healthy or can be present within healthy tissue. In certain embodiments, the stem cells have a reduced asymmetric cell division compared to normal healthy stem cells. However, the stem cells, such as skeletal muscle stem cells, can have comparable asymmetric cell division compared to normal healthy stem cells.

[0067] In many diseases and conditions that affect muscle, there is a reduced number of satellite cells and a reduction in muscle mass associated with a reduced ability of satellite cells to repair, regenerate, and grow skeletal muscle. Exemplary diseases and conditions that affect muscle include wasting diseases (e.g., cachexia, muscular attenuation or atrophy (including sarcopenia)), ICU-induced weakness, surgery-induced weakness (e.g., after knee or hip replacement), and muscle degenerative diseases (e.g., muscular dystrophy), and any of these diseases and conditions can be treated according to the methods of the present disclosure. In certain embodiments, the stem cells or tissue are muscle stem cells (e.g., satellite cells) or muscle tissue damaged or injured due to muscle wasting or atrophy (e.g., cancer-related cachexia).

[0068] In certain embodiments, the stem cells are diseased skeletal muscle stem cells that contain mutations associated with muscular dystrophy, optionally Duchenne muscular dystrophy or Becker muscular dystrophy. In certain embodiments, the damaged or injured muscle stem cells contain mutations in the dystrophin gene.

[0069] In certain embodiments, the present disclosure provides a method of treating muscular dystrophy, the method comprising administering to a subject diagnosed with or suspected of having muscular dystrophy an inhibitor disclosed herein, such as an AAK1 inhibitor. Muscular dystrophy is a hereditary disease characterized by progressive weakness and degeneration of the skeletal or voluntary muscles that control movement. The muscles of the heart and some other involuntary muscles are also affected in some forms of muscular dystrophy. Often, histological pictures show variation in fiber size, muscle cell necrosis and regeneration, and often proliferation of connective and adipose tissue. Progressive muscular dystrophies include at least Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), Emery-Dreifuss muscular dystrophy, Landouzy-Dejerine muscular dystrophy, facioscapulohumeral muscular dystrophy (FSH), limb-girdle muscular dystrophy, von Graefe-Fuchs muscular dystrophy, oculopharyngeal muscular dystrophy (OPMD), myotonic dystrophy (Steinert's disease), and congenital muscular dystrophy. Any of these diseases and / or related symptoms can be treated or ameliorated according to the methods disclosed herein.

[0070] Generally, the cell / tissue / subject is contacted with or administered an effective amount of an inhibitor of AAK1, GAK, or MPSK1 (e.g., an AAK1 inhibitor). The effective amount can be an amount or concentration effective to increase asymmetric cell division, promote cell growth or tissue regeneration, or treat a disease or disorder (including any of those disclosed herein).

[0071] In some embodiments, the inhibitor can be administered at a dosage of about 0.01 mg / kg to about 300 mg / kg. As used herein, mg / kg refers to the amount of inhibitor administered (in mg) per body weight of the subject being administered (in kg). In another embodiment, the inhibitor can be administered at a dosage of about 0.1 mg / kg to about 20 mg / kg. For example, the inhibitor can be administered to a subject at a dosage of about 0.01 mg / kg, 0.03 mg / kg, 0.07 mg / kg, 0.1 mg / kg, 0.3 mg / kg, 0.7 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg or 20 mg / kg, or within the range between any of the foregoing values, for example, between about 10 mg / kg and about 15 mg / kg, between about 6 mg / kg and about 12 mg / kg, between 0.1 and 2 mg / kg, etc. In another embodiment, the inhibitor is administered at a dosage of ≦15 mg / kg. In a preferred embodiment, the inhibitor is administered at a dosage of about 1 mg / kg. For example, the inhibitor can be administered at 1 mg / kg per day for 7 days for a total of 7 mg / kg per week. For example, the compound can be administered twice daily at 10 mg / kg per day for 7 days for a total of 140 mg / kg per week.

[0072] In many embodiments, the dosages described herein may refer to a single dosage, a daily dosage, or a weekly dosage. In one embodiment, the inhibitor may be administered once a day. In another embodiment, the compound may be administered twice a day. In some embodiments, the inhibitor may be administered three times a day. In some embodiments, the inhibitor may be administered four times a day. In some embodiments, the inhibitor may be administered once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, eleven times, twelve times, thirteen times, fourteen times, fifteen times, sixteen times, seventeen times, eighteen times, nineteen times, twenty times, twenty-one times, twenty-two times, twenty-three times, or twenty-four times per week. In a preferred embodiment, the inhibitor may be administered twice a week. In other embodiments, the inhibitor is administered once every two weeks. In a further embodiment, the inhibitor is administered once every day, every two days, every three days, every four days, every five days, every six days, or every seven days. In a preferred embodiment, the inhibitor is administered once every about three days. In a preferred embodiment, the inhibitor is administered once every about four days.

[0073] In some embodiments, the period during which the inhibitor is administered (the "treatment period") may be one week, two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, eight weeks, nine weeks, ten weeks, twelve weeks, thirteen weeks, fourteen weeks, fifteen weeks, sixteen weeks, seventeen weeks, eighteen weeks, nineteen weeks, twenty weeks, twenty-one weeks, twenty-two weeks, twenty-three weeks, or twenty-four weeks. In some embodiments, the treatment period may be up to four weeks. In a preferred embodiment, the treatment period may be two weeks. In some embodiments, the treatment period may be up to two weeks. In some embodiments, the treatment period may be up to three weeks.

[0074] In some embodiments, the inhibitor can be administered up to two or three times per week for up to four weeks. In a further preferred embodiment, the inhibitor can be administered twice per week for two weeks. In a preferred embodiment, the inhibitor can be administered once every about three days for two weeks.

[0075] In some embodiments, after the treatment period, a period during which the inhibitor is not administered ("non-treatment period") can follow. In some embodiments, after the non-treatment period, a period follows during which the treatment is resumed. In other embodiments, the treatment may not be resumed after the non-treatment period.

[0076] A wide variety of administration methods can be used with the inhibitor according to the methods disclosed herein. For example, the inhibitor can be administered locally, orally, intraperitoneally, intravenously, intraarterially, transdermally, sublingually, intramuscularly, rectally, transbuccally, intranasally, by liposomes, via inhalation, vaginally, intravitreally, via local delivery (e.g., by catheter or stent), subcutaneously, intraadiposally, intraarticularly, intrathecally, transmucosally, to the lungs, or parenterally, e.g., by injection (including subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subepidermal, intraarticular, subdural, and intrasternal); by depot or reservoir implantation (e.g., subcutaneously or intramuscularly), administered or co-administered. In some embodiments, the inhibitor can be administered orally or parenterally, e.g., intravenously or subcutaneously. In some embodiments, the inhibitor can be administered orally once per day at a dose of ≤ 15 mg / kg.

[0077] The actual dosage used can be varied according to the requirements of the subject and the severity of the condition being treated. The dosing regimen can be selected according to various factors, including the type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal or hepatic function of the patient; and the particular compound used. A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the inhibitor required to prevent, counteract or arrest the progression of the above-mentioned condition. For convenience, the total daily dosage, if required, can be administered in divided doses throughout the day.

[0078] Inhibitor The accompanying examples describe the inhibition of AAK1, GAK, or MPSK1 for use as a target for the treatment of DMD and similar muscle wasting diseases and for promoting asymmetric cell division and tissue regeneration and treating conditions that would benefit therefrom. As shown in the accompanying examples, AAK1, GAK, or MPSK1 can be inhibited to increase asymmetric cell division. The methods disclosed herein can target AAK1, GAK, or MPSK1 for inhibition. In certain embodiments, the method can target AAK1 for inhibition. On the other hand, the method can indirectly inhibit the targets identified herein by increasing the expression or activity of inhibitors of the targets. In each case, the agent used can be referred to herein as an "inhibitor" because it ultimately inhibits the target. In certain embodiments, the methods disclosed herein result in a decreased activity of AAK1, GAK, or MPSK1.

[0079] In certain embodiments, the methods disclosed herein are performed using an inhibitor of AAK1. AAK1 is a serine / threonine kinase enzyme and is one of four kinases in the Numb-associated kinase (NAK) family of proteins in mammals. AAK1, adapter-associated kinase 1, interacts directly with the membrane-tethered active form of Notch released by metalloprotease cleavage. Active AAK1 acts upstream of γ-secretase cleavage by stabilizing both the membrane-tethered activated form of Notch and its monoubiquitinated counterpart. AAK1 is thought to act as an adapter for Notch interactions with components of the clathrin-mediated pathway such as Eps15b.

[0080] In certain embodiments, the methods disclosed herein are performed using inhibitors of another member of the NAK family. The Numb-associated kinases (NAKs) of the protein kinase family constitute a diverse family with respect to Ser / Thr kinases in both their function and structure, and share little conservation outside the kinase domain. Humans have four known homologs: AAK1 (adapter-associated kinase 1), BIKE / BMP2K (BMP-2-inducible kinase), GAK (cyclin G-associated kinase), and MPSK1 (myristoylated and palmitoylated serine / threonine kinase 1, also known as STK16). BIKE is structurally related to AAK1, plays a role in osteoblast differentiation, and has recently been identified as a clathrin-coated vesicle-associated protein. GAK is a known association partner of cyclin G and CDK5, and some of its known functions are shared with AAK1. It is essential for clathrin transport, mediates binding to the plasma membrane and the trans-Golgi network, and is also required for maintenance of centrosome maturation and progression through mitosis. MPSK1 is the most distantly related to the above family members, and its physiological function is poorly understood, but it is known to be a Golgi-associated kinase that plays a role in the regulation of secretion in the constitutive secretory pathway in the trans-Golgi network. Furthermore, MPSK1 is also associated with mammary gland development in mice. In certain embodiments, the methods disclosed herein are performed using an inhibitor of any one or more of AAK1, GAK, and MPSK1.

[0081] In certain embodiments, the human AAK1 protein has the following sequence of SEQ ID NO: 1 (however, other isoforms are known in the art and are shown below):

Chemical Structure

[0082] Other isoforms of the human AAK1 protein known in the art include those of SEQ ID NO: 2 and SEQ ID NO: 3:

Chemical formula

[0083] In certain embodiments, the human BIKE / BMP2K protein has the following sequence of SEQ ID NO: 4 (however, other isoforms are known in the art):

Chemical formula

[0084] In certain embodiments, the human GAK protein has the following sequence of SEQ ID NO: 5 (however, other isoforms are known in the art):

Chemical formula

[0085] In certain embodiments, the human MPSK1 protein has the following sequence of SEQ ID NO: 6 (however, other isoforms are known in the art):

Chemical formula

[0086] In certain embodiments, the methods disclosed herein are performed using an inhibitor of STK38 or STK38L. Serine / threonine kinase 38 (STK38) protein is a member of the AGC serine / threonine kinase family of proteins. The kinase activity of this protein is regulated by autophosphorylation and phosphorylation by other upstream kinases. This protein has been shown to function in the cell cycle and apoptosis. This protein has also been found to regulate protein stability and the transcriptional activity of the MYC oncogene. Further, STK38 and STK38L have been shown to phosphorylate AAK1. Alternative splicing results in multiple transcript variants of STK38. Serine / threonine kinase 38-like protein has been shown to enable ATP binding activity; magnesium ion binding activity; and protein serine / threonine kinase activity. It is involved in intracellular signaling and acts upstream of or within protein phosphorylation.

[0087] In certain embodiments, the human STK38 protein has the following sequence of SEQ ID NO: 7 (however other isoforms are known in the art):

Chemical formula

[0088] In certain embodiments, the human STK38L protein has the following sequence of SEQ ID NO: 8 (however other isoforms are known in the art):

Chemical formula

[0089] In certain embodiments, the methods disclosed herein can be performed with any agent that can inhibit the expression or activity of a target gene, mRNA or protein, such as an inhibitor of a gene, mRNA or protein, complex or pathway disclosed herein, such as an inhibitor of the AAK1, GAK, or MPSK1 gene, mRNA, or protein. In certain embodiments, the methods disclosed herein result in a decrease in the expression level or activity of a target gene, mRNA or protein, such as AAK1, in one or more cells or tissues (e.g., in a subject), as compared to the expression level or activity in control cells or tissues not contacted with the inhibitor, or a reference level that can be predetermined.

[0090] In certain embodiments, the methods disclosed herein result in increased asymmetric cell division or increased cell polarity in one or more cell types (e.g., satellite cells) or in one or more tissues (e.g., skeletal muscle tissue) (e.g., in a subject), as compared to the expression level or activity in control cells or tissues not contacted with the inhibitor, or a reference level.

[0091] The methods described herein can be performed using any type of inhibitor that results in a reduced amount or level of a target gene, mRNA, or protein, for example, in a cell or tissue, such as a cell or tissue in a subject. In certain embodiments, the inhibitor causes, for example, a reduction in the active target protein, a reduction in the total target protein, a reduction in the target mRNA level, and / or a reduction in the target protein activity in a cell or tissue contacted with the inhibitor. Methods for measuring total protein or mRNA levels, or activity, in a cell are known in the art. In certain embodiments, the inhibitor inhibits or reduces the activity or expression of the target protein, such as mRNA and / or protein expression. In certain embodiments, the inhibitor causes an increase in the degradation of the target protein, resulting in a small amount of the target protein in the cell or tissue.

[0092] As used herein, an inhibitor refers to an agent that can block or inhibit the expression and / or activity of a target protein identified herein. Examples of various types of inhibitors include, but are not limited to, RNA interference agents that target the target protein, blocking antibodies against the target protein, small molecules and peptides that interfere with the expression, function, or activity of any target protein. Another approach is the systemic or local delivery of a DNA plasmid encoding an inhibitor or dominant negative form of the target protein. Further, inhibitors include agents that inhibit or modulate the expression, function, or activity of any target protein.

[0093] Inhibitors that can be used to practice the methods of the present disclosure include, but are not limited to, agents that inhibit, reduce, or decrease the expression or activity of a biomolecule (such as, but not limited to, a target gene, mRNA, or protein (such as AAK1)). In certain embodiments, the inhibitor can cause increased degradation of the biomolecule. In certain embodiments, the inhibitor can inhibit the biomolecule by competitive, non-competitive, or uncompetitive means. Exemplary inhibitors include nucleic acids, DNA, RNA, guide RNA (gRNA), short hairpin RNA (shRNA), small interfering RNA (siRNA), modified mRNA (mRNA), microRNA (miRNA), antisense RNA, proteins, protein mimetics, peptides, peptide mimetics, antibodies, small molecules, organic small molecules, inorganic molecules, chemicals, enzymes, receptors, or analogs (such as those involved in signal transduction) that mimic the binding site of another protein, therapeutic agents, pharmaceutical compositions, drugs, and combinations thereof, but are not limited thereto. In some embodiments, the inhibitor can be a nucleic acid molecule (including, but not limited to, siRNA) that reduces the amount of a functional protein in a cell. Thus, a compound or agent said to "be able to inhibit" a particular target protein includes any type of inhibitor.

[0094] In certain embodiments, the inhibitor comprises a nucleic acid that binds to a target gene or mRNA. Thus, the nucleic acid inhibitor can comprise a sequence complementary to the target polynucleotide sequence or a region or its antisense thereof. In certain embodiments, the nucleic acid inhibitor comprises a sequence of at least 8, at least 10, at least 12, at least 14, at least 16, at least 20, at least 24, or at least 30 nucleotides corresponding to or complementary to the target polynucleotide sequence or its antisense. In various embodiments, the nucleic acid inhibitor comprises a region corresponding to or complementary to any nucleic acid target.

[0095] In certain embodiments, the target gene or mRNA is the Aak1 (e.g., human Aak1) gene or mRNA. In certain embodiments, the AAK1 is human AAK1 isoform 1, human AAK1 isoform 2, or human AAK1 isoform 3.

[0096] In certain embodiments, the target gene or mRNA is the GAK 1 (e.g., human GAK) gene or mRNA.

[0097] In certain embodiments, the target gene or mRNA is the MPSK1 (e.g., human MPSK1) gene or mRNA.

[0098] In certain embodiments, the nucleic acid inhibitor, when administered to a cell, is an RNA interference or antisense RNA agent or a portion or mimetic thereof, or a morpholino, that reduces the expression of the target gene. Typically, the nucleic acid inhibitor comprises at least a portion of the target nucleic acid molecule, or an ortholog thereof, or at least a portion of the complementary strand of the target nucleic acid molecule. In some embodiments, the expression of the target gene is reduced by at least about 10%, at least about 25%, at least about 50%, at least about 75%, or even 90 - 100%.

[0099] A "complementary" nucleic acid sequence is a nucleic acid sequence that can hybridize with another nucleic acid sequence composed of complementary nucleotide base pairs. By "hybridize" is meant pairs that form double-stranded molecules between complementary nucleotide bases under appropriate stringency conditions (e.g., in DNA, adenine (A) forms a base pair with thymine (T), and similarly, guanine (G) and cytosine (C) form base pairs) (see, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507).

[0100] "Antisense" refers to a nucleic acid sequence that is complementary to a certain nucleic acid sequence, regardless of its length. In certain embodiments, antisense RNA refers to a single-stranded RNA molecule that can be introduced into individual cells, tissues, or subjects, and through a mechanism that does not rely on the endogenous gene silencing pathway, results in reduced expression of the target gene. Antisense nucleic acids can include a modified backbone, such as phosphorothioate, phosphorodithioate, or others known in the art, or can include non-natural internucleoside linkages. Antisense nucleic acids can include, for example, locked nucleic acid (LNA).

[0101] "RNA interference", as used herein, refers to the use of agents (such as Dicer and RNA-induced silencing complex (RISC)) that reduce the expression of a target gene by degradation of the target mRNA through the endogenous gene silencing pathway. RNA interference can be achieved using various agents, including short hairpin RNA (shRNA) and small interfering RNA (siRNA). "Short hairpin RNA" or "shRNA" refers to a double-stranded artificial RNA molecule having a hairpin turn that can be used to silence target gene expression via RNA interference (RNAi). Expression of shRNA in cells is typically achieved by plasmid delivery or via viral or bacterial vectors. shRNA is an advantageous mediator of RNAi in that it has relatively slow degradation and metabolic turnover. Small interfering RNA (siRNA) is typically a class of double-stranded RNA molecules that are 20-25 base pairs in length, are similar to miRNA, and operate within the RNA interference (RNAi) pathway. It interferes with the expression of a specific gene having a complementary nucleotide sequence by degrading the mRNA post-transcriptionally and preventing translation. In certain embodiments, siRNA is 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, or 24 nucleotides in length and has a 2-base overhang at its 3' end. siRNA can be introduced into individual cells and / or culture systems and can result in degradation of the target mRNA sequence.

[0102] As used herein, "morpholino" refers to a modified nucleic acid oligomer, where the standard nucleobases are attached to a morpholine ring and linked through phosphorodiamidate linkages. Similar to siRNA and shRNA, morpholinos bind to complementary mRNA sequences. However, morpholinos function through steric inhibition of mRNA translation and alteration of mRNA splicing rather than targeting the complementary mRNA sequence for degradation.

[0103] In certain embodiments, the nucleic acid inhibitor is a messenger RNA that can be introduced into a cell, where it encodes a target polypeptide inhibitor disclosed herein. In certain embodiments, the mRNA is modified, for example, by incorporation of one or more modified nucleosides, to increase its stability or reduce its immunogenicity. Suitable modifications are known in the art.

[0104] In certain embodiments, the inhibitor comprises an expression cassette encoding a target polynucleotide or polypeptide inhibitor disclosed herein. In certain embodiments, the expression cassette is present in a gene therapy vector, such as a viral gene therapy vector. A variety of gene therapy vectors, including viral gene therapy vectors, are known in the art, including, for example, AAV-based gene therapy vectors.

[0105] In some embodiments, the inhibitor is a polypeptide inhibitor. In certain embodiments, the polypeptide inhibitor binds to the target polypeptide and thus inhibits its activity, e.g., kinase activity. Examples of polypeptide inhibitors include any type of polypeptide such as antibodies and fragments thereof (e.g., peptides and proteins). In certain specific embodiments, the inhibitor is a dominant negative form of the target protein (e.g., a fragment of the target protein that binds the substrate but lacks enzymatic activity). In certain embodiments, the inhibitor is an antibody that binds to the target protein, e.g., an antibody that inhibits the activity of the target protein when bound thereto. Antibodies that specifically bind to AAK1, GAK, or MPSK1 are available in the art and can be readily generated.

[0106] In certain embodiments, the inhibitor induces the degradation of the target polypeptide. For example, the inhibitor includes proteolysis targeting chimeras (PROTACs) that induce the selective intracellular proteolysis of the target protein. PROTACs contain functional domains that can be covalently linked protein-binding molecules: one can engage an E3 ubiquitin ligase and the other binds to the target protein intended for degradation. The recruitment of the E3 ligase to the target protein results in ubiquitination and subsequent degradation of the target protein by the proteasome. In certain embodiments, the inhibitor is a PROTAC that targets any of the targets disclosed herein.

[0107] In certain embodiments, the inhibitor directly inhibits the expression or activity of a target gene, mRNA, or protein; for example, it can directly bind to the target gene, mRNA, or protein. In some embodiments, the inhibitor indirectly inhibits the expression or activity of a target gene, mRNA, or protein; for example, it can bind to and inhibit a protein (e.g., a transcription factor) that mediates the expression of the target gene, mRNA, or protein, or it can bind to and inhibit another protein (e.g., another protein present in a complex with the target protein) involved in the activity of the target protein.

[0108] The effect / action of the inhibitor can be determined, for example, by the expression of the target protein in a cell and / or an indicator of one or more activities.

[0109] In certain embodiments, the inhibitor comprises one or more components of a gene editing system. As used herein, the term "gene editing system" refers to a protein, nucleic acid, or combination thereof that, when introduced into a cell, can modify a target locus of an endogenous DNA sequence. Many gene editing systems suitable for use in the methods of the present invention are known in the art (including, but not limited to, zinc finger nuclease systems, transcription activator-like effector nuclease (TALEN) systems, meganucleases or Argonaute-based systems (Nat Biotechnol. 2016 July; 34(7):768-73) or base editors (Komor et al., Nature 533, 420-424, doi:10.1038 / nature17946), and CRISPR / Cas systems). The present disclosure encompasses the use of any of these alternative means for site-specific DNA editing (e.g., introduction of an inhibitory mutation into a target gene (e.g., Aak1)).

[0110] In some embodiments, the gene editing system used in the methods described herein is a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) / Cas (CRISPR-associated) nuclease system, which is an engineered nuclease system based on a bacterial system that can be used for mammalian genome manipulation. Generally, the system includes a CRISPR-associated endonuclease (e.g., a Cas endonuclease) and a guide RNA (gRNA). The gRNA is composed of two parts: a crispr-RNA (crRNA) specific to a target genomic DNA sequence, and a trans-activating RNA (tracrRNA) that facilitates the endonuclease binding to the DNA at the targeted insertion site. In some embodiments, the crRNA and tracrRNA may be present in the same RNA oligonucleotide (referred to as a single guide RNA (sgRNA)). In some embodiments, the crRNA and tracrRNA may be present as separate RNA oligonucleotides. In such embodiments, the gRNA is composed of a crRNA oligonucleotide and a tracrRNA oligonucleotide that associate to form a crRNA:tracrRNA duplex. As used herein, the term "guide RNA" or "gRNA" refers to a combination of tracrRNA and crRNA that exists as either an sgRNA or a crRNA:tracrRNA duplex.

[0111] In some embodiments, the CRISPR / Cas system includes a Cas protein, a crRNA, and a tracrRNA. In some embodiments, the crRNA and tracrRNA are combined as a double-stranded RNA molecule to form a gRNA. In some embodiments, the crRNA:tracrRNA double-strand is formed in vitro prior to introduction into the cell. In some embodiments, the crRNA and tracrRNA are introduced into the cell as separate RNA molecules, and then the crRNA:tracrRNA double-strand is formed intracellularly. In some embodiments, polynucleotides encoding the crRNA and tracrRNA are provided. In such embodiments, the polynucleotides encoding the crRNA and tracrRNA are introduced into the cell, and then the crRNA and tracrRNA molecules are transcribed intracellularly. In some embodiments, the crRNA and tracrRNA are encoded by a single polynucleotide. In some embodiments, the crRNA and tracrRNA are encoded by separate polynucleotides.

[0112] In some embodiments, the Cas endonuclease is directed to a target insertion site by the sequence specificity of the crRNA portion of the gRNA, which may include a protospacer motif (PAM) sequence near the target insertion site. Various PAM sequences suitable for use with particular endonucleases (e.g., Cas9 endonuclease) are known in the art (see, e.g., Nat Methods. 2013 Nov; 10(11): 1116-1121 and Sci Rep. 2014; 4: 5405).

[0113] The specificity of the gRNA for the target locus is mediated by a crRNA sequence that is complementary to the DNA sequence at the target locus, for example, a sequence of about 20 nucleotides that is complementary to the target DNA sequence. In some embodiments, the crRNA used in the methods of the invention is at least 90% complementary to the DNA sequence of the target locus. In some embodiments, the crRNA sequence used in the methods of the invention is at least 95%, 96%, 97%, 98%, or 99% complementary to the DNA sequence of the target locus. In some embodiments, the crRNA sequence used in the methods of the invention is 100% complementary to the DNA sequence of the target locus. In some embodiments, the crRNA sequences described herein are designed to minimize off-target binding using algorithms known in the art (e.g., Cas-OFFinder) to identify target sequences specific to a particular target locus or target gene.

[0114] In some embodiments, the endonuclease is a Cas protein or ortholog. In some embodiments, the endonuclease is a Cas9 protein. In some embodiments, the Cas9 protein is derived from Streptococcus pyogenes (e.g., SpCas9), Staphylococcus aureus (e.g., SaCas9), or Neisseria meningitides (NmeCas9). In some embodiments, the Cas endonuclease is a Cas9 protein or Cas9 ortholog, and is selected from the group consisting of SpCas9, SpCas9-HF1, SpCas9-HF2, SpCas9-HF3, SpCas9-HF4, SaCas9, FnCpf, FnCas9, eSpCas9, and NmeCas9. In some embodiments, the endonuclease is selected from the group consisting of C2C1, C2C3, Cpf1 (also referred to as Cas12a), Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csx12), Cas10, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, Csx10, Csx16, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, and Csf4. In some embodiments, the Cas9 is a Cas9 nickase variant. The Cas9 nickase variant contains only one catalytically active domain (neither the HNH domain nor the RuvC domain).

[0115] In certain embodiments, the inhibitor is a small molecule inhibitor, or a stereoisomer, enantiomer, diastereomer, isotopically enriched form, prodrug, or pharmaceutically acceptable salt thereof. Various small molecule inhibitors of the targets disclosed herein that include AAK1 are known and available.

[0116] In certain embodiments, the AAK1 inhibitor inhibits or reduces AAK1 enzyme activity, such as the phosphorylation of the AP2M1 / mu2 subunit of adapter protein complex 2 (AP-2) and / or NUMB.

[0117] In certain embodiments, AAK1 inhibitors include, but are not limited to, LX9211, SGC-AAK1-1, LP-935509, LP-922761, BMT-090605, BMT-124110, LP-927443, and BMS-901715. The structure of exemplary AAK1 inhibitors is shown in FIG. 4.

[0118] LX9211 is described in PCT Application Publication No. WO2015153720. LX9211 is used interchangeably with SAT3003 herein. The AAK1 inhibitor is (S)-1-((2’,6-bis(difluoromethyl)-[2,4’-bipyridin]-5-yl)oxy)-2,4-dimethylpentan-2-amine and its pharmaceutically acceptable salts and solvates (including LX9211 and its salts):

Chemical formula

[0119] SGC-AAK1-1 (N-(6-(3-(N,N-diethylsulfamoylamino)phenyl)-1H-indazol-3-yl)cyclopropanecarboxamide) is an ATP-competitive kinase inhibitor against AP2-associated protein kinase 1 / AAK1 and BMP-2-inducible protein kinase / BIKE / BMP2K (Ki = 9.1 and 17 nM, respectively, by ATP-site fluorescent tracer displacement assay; AAK1 IC50 = 270 nM by coupled enzyme assay). SGC-AAK1-1 downregulates the cellular AP2M1 Thr156 phosphorylation level in a dose-dependent manner (ECmax ~12.5 μM) without detectable cytotoxicity.

[0120] LP-935509 is a selective ATP-competitive and brain-penetrant inhibitor of adapter protein-2 associated kinase 1 (AAK1) with IC 50 and K i values of 3.3 nM and 0.9 nM, respectively.

[0121] LP-922761 is a potent, selective and orally active adapter protein-2 associated kinase 1 (AAK1) inhibitor with IC 50 values of 4.8 nM and 7.6 nM in enzyme and cell assays, respectively. LP-922761 also inhibits BMP-2-inducible protein kinase (BIKE) with an IC 50 value of 24 nM.

[0122] BMT-090605 is a potent, selective AAK1 inhibitor with an IC 50 value of 0.6 nM. BMT-090605 exhibits anti-nociceptive activity. BMT-090605 inhibits BMP-2-inducible protein kinase (BIKE) with an IC 50 value of 45 nM.

[0123] BMT-124110 is a potent, selective AAK1 inhibitor with an IC 50It is a potent and selective AAK1 inhibitor having. BMT-124110 exhibits anti-invasive receptive activity. BMT-090605 inhibits BMP-2-inducible protein kinase (BIKE) at an IC 50 of 17 nM.

[0124] BMS-901715 is a potent and selective adapter protein-2 associated kinase 1 (AAK1) inhibitor.

[0125] Additional AAK1 inhibitors that can be used in accordance with the present disclosure include, but are not limited to, those described in the following references: Hartz, R. A. et al. Discovery, Structure-Activity Relationships, and In Vivo Evaluation of Novel Aryl Amides as Brain Penetrant Adaptor Protein 2-Associated Kinase 1 (AAK1) Inhibitors for the Treatment of Neuropathic Pain. J Med Chem 64, 11090-11128 (2021); Verdonck, S. et al. Synthesis and Structure-Activity Relationships of 3,5-Disubstituted-pyrrolo[2,3-b]pyridines as Inhibitors of Adaptor-Associated Kinase 1 with Antiviral Activity. J Med Chem 62, 5810-5831 (2019); Wells, C. et al. SGC-AAK1-1: A Chemical Probe Targeting AAK1 and BMP2K. Acs Med Chem Lett 11, 340-345 (2020); Kostich, W. et al. Inhibition of AAK1 Kinase as a Novel Therapeutic Approach to Treat Neuropathic Pain. J Pharmacol Exp Ther 358, 371-386 (2016); Hesselink. LX9211 A Selective Inhibitor of AAK1 (Adapter- Associated Kinase) for Neuropathic Pain? Some Thoughts on Selectivity and Specificity. Austin Neurology 3, 1013- (2018); Martinez-Gualda, B., Schols, D. & Jonghe, S. D.A patent review of adaptor associated kinase 1 (AAK1) Inhibitors (2013-present). Expert Opin Ther Pat 31, 1-26 (2021). Further AAK1 inhibitors include, but are not limited to, 3-methyloxetan-3-yl-4-(3-(2-methoxypyridin-3-yl)pyrazolo[1,5-a]pyrimidin-5-yl)piperazine-1-carboxylate, and pharmaceutically acceptable salts thereof (described in US Patent Publication No. 20160039824).

[0126] Other examples of AAK1 inhibitors include, but are not limited to, those disclosed in PCT Application Publication Nos. WO2013134219 (2013), WO2015035167 (2015), WO2015026574 (2015), WO2013134336 (2013), WO2013134228 (2013), WO2015142714 (2015), WO2015035117 (2015), WO2015153720 (2015), WO2017059085 (2017), WO2015006100 (2015), WO2013134036 (2013), WO2015038112 (2015), WO2014130258 (2014), WO2016164295 (2016), WO2014022167 (2014), WO2015116060 (2015), WO2015054358 (2015), WO2015116492 (2015), WO2016022312 (2016), and WO2015142714.

[0127] In certain embodiments, GAK (cyclin G-associated kinase) inhibitors include, but are not limited to, SGC-AAK1-1 as described in Wells, C. et al. SGC-AAK1-1: A Chemical Probe Targeting AAK1 and BMP2K. Acs Med Chem Lett 11, 340-345 (2020).

[0128] In certain embodiments, examples of MPSK1 (myristoylated and palmitoylated serine / threonine kinase 1) inhibitors include, but are not limited to, SGC-AAK1-1 as described in Wells, C. et al. SGC-AAK1-1: A Chemical Probe Targeting AAK1 and BMP2K. Acs Med Chem Lett 11, 340-345 (2020).

[0129] Pharmaceutical composition In certain aspects, the present disclosure encompasses compositions, e.g., pharmaceutical compositions, comprising an inhibitor disclosed herein, e.g., an AAK1 inhibitor (including any of the various classes of inhibitors described herein). The invention encompasses pharmaceutical compositions comprising an inhibitor and a pharmaceutically acceptable carrier, diluent, or additive. Any inert additive commonly used as a carrier or diluent can be used in the compositions of the invention (e.g., sugars, polyhydric alcohols, soluble polymers, salts, and lipids). Examples of sugars and polyhydric alcohols that can be used include, but are not limited to, lactose, sucrose, mannitol, and sorbitol. Illustrative examples of soluble polymers that can be used are polyoxyethylene, poloxamer, polyvinylpyrrolidone, and dextran. Useful salts include, but are not limited to, sodium chloride, magnesium chloride, and calcium chloride. Examples of lipids that can be used include, but are not limited to, fatty acids, glycerol fatty acid esters, glycolipids, and phospholipids.

[0130] Furthermore, the pharmaceutical composition may further include the following: binders (e.g., gum arabic, corn starch, gelatin, carbomer, ethyl cellulose, guar gum, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, povidone), disintegrants (e.g., corn starch, potato starch, alginic acid, silicon dioxide, croscarmellose sodium, crospovidone, guar gum, sodium starch glycolate, Primogel), buffers of various pH and ionic strengths (e.g., Tris-HCL, acetic acid, phosphoric acid), additives (e.g., albumin or gelatin to prevent adsorption to the surface), detergents (e.g., Tween® 20, Tween® 80, Pluronic® F68, bile salts), protease inhibitors, surfactants (e.g., sodium lauryl sulfate), permeation enhancers, solubilizing agents (e.g., glycerol, polyethylene glycol, cyclodextrin), flow promoters (e.g., colloidal silicon dioxide), antioxidants (e.g., ascorbic acid, sodium pyrosulfite, butylated hydroxyanisole), stabilizers (e.g., hydroxypropyl cellulose, hydroxypropyl methyl cellulose), viscosity increasing agents (e.g., carbomer, colloidal silicon dioxide, ethyl cellulose, guar gum), sweeteners (e.g., sucrose, aspartame, citric acid), flavoring and odor-masking agents (e.g., peppermint, methyl salicylate, or orange flavor), preservatives (e.g., thimerosal, benzyl alcohol, parabens), lubricants (e.g., stearic acid, magnesium stearate, polyethylene glycol, sodium lauryl sulfate), flow-aids (e.g., colloidal silicon dioxide), plasticizers (e.g., diethyl phthalate, triethyl citrate), emulsifiers (e.g., carbomer, hydroxypropyl cellulose, sodium lauryl sulfate, methyl cellulose, hydroxyethyl cellulose, sodium carboxymethyl cellulose), polymer coatings (e.g., poloxamer or poloxamine), coating and film-forming agents (e.g., ethyl cellulose, acrylate, polymethacrylate) and / or adjuvants.

[0131] In one embodiment, the pharmaceutical composition is prepared with a carrier that protects the inhibitor against rapid elimination from the body, including implants and microencapsulation delivery systems (e.g., controlled release formulations). Biodegradable biocompatible polymers can be used (e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid). Methods for the preparation of such formulations are apparent to those skilled in the art. The substances can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposome suspensions (including liposomes targeted to infected cells with monoclonal antibodies against viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

[0132] Furthermore, the present invention encompasses pharmaceutical compositions containing any solid or liquid physical form of the inhibitor. For example, the inhibitor can be in crystalline form, amorphous form, and can have any particle size. The particles can be micronized or aggregated (micronized granules, powders, oils, oily suspensions, or any other form of solid or liquid physical form).

[0133] When the inhibitor exhibits insufficient solubility, methods for solubilizing the compound can be used. Such methods are known to those skilled in the art and include using co-solvents (e.g., ethanol, propylene glycol, polyethylene glycol (PEG) 300, PEG 400, DMA (10 - 30%), DMSO (10 - 20%), NMP (10 - 20%)), surfactants (e.g., polysorbate 80, polysorbate 20 (1 - 10%), cremophor EL, cremophor RH40, cremophor RH60 (5 - 10%), pluronic® F68 / poloxamer 188 (20 - 50%), Solutol HS15 (20 - 50%), vitamin E TPGS, and d-α-tocopheryl PEG 1000 succinate (20 - 50%)), using complexation such as HP β-CD and SBE β-CD (10 - 40%), and using advanced approaches such as micelles, addition of polymers, nanoparticle suspensions, and liposome formation, pH adjustment and salt formation, among others.

[0134] The inhibitor can also be administered or co-administered in a slow release dosage form. The inhibitor can be in a gaseous, liquid, semi-liquid or solid form formulated in a manner suitable for the route of administration to be used. For oral administration, suitable solid oral formulations include tablets, capsules, pills, granules, pellets, sachets and effervescent agents, powders, etc. Suitable liquid oral formulations include solutions, suspensions, dispersions, syrups, emulsions, oils, etc. For parenteral administration, reconstitution of lyophilized powder is typically used.

[0135] The appropriate dosage of an inhibitor for use in treating a disease or disorder described herein can be determined by one of ordinary skill in the relevant art. Therapeutic dosages are generally identified through dosage range studies in humans based on preliminary evidence obtained from animal studies. The dosage should be sufficient to produce the desired therapeutic effect without causing unwanted side effects. The mode of administration, dosage form, and appropriate pharmaceutical additives can also be adequately utilized and adjusted by one of ordinary skill in the art. All changes and modifications are contemplated within the scope of this patent application.

[0136] In certain embodiments, the disclosure includes unit dosage forms of a pharmaceutical composition comprising an agent that inhibits the expression or activity of a target polypeptide (or results in a reduced level of the target protein) and a pharmaceutically acceptable carrier, diluent, or additive, wherein the unit dosage form is effective to increase the expression of hemoglobin γ in one or more tissues in a subject to whom the unit dosage form is administered.

[0137] In certain embodiments, the unit dosage form comprises an effective amount, effective concentration, and / or inhibitory concentration of an inhibitor for treating a disease or disorder (e.g., DMD) disclosed herein.

Examples

[0138] Example 1: AAK1 inhibition enhances asymmetric division of satellite cells The effect of AAK1 inhibition on the asymmetric division of satellite cells was first examined by contacting cultured muscle fibers with small interfering RNA (siRNA) specific for AAK1.

[0139] The Myf5-Cre / R26R-eYFP transgenic mouse, which has a knock-in of Cre recombinase in the coding region of myogenic determination factor Myf5 (Tallquist et al. Development, 2000. 127(23): p. 5059-70) and was mated with a knock-in of Cre-activated yellow fluorescent protein (eYFP) at the ROSA26 locus (Srinivas et al. BMC Dev Biol, 2001. 1: p. 4), was used as a lineage reporter model to distinguish committed satellite myogenic cells (eYFPPos) expressing Myf5-Cre from satellite stem cells (eYFPNeg) that do not express Myf5-Cre at all (Kuang et al. 2007). The Myf5-Cre / R26R-nTnG transgenic mouse has a CMV / β-actin promoter, loxP-flanked nuclear TdTomato (nTdT) and nuclear GFP (nGFP) cassette within the ROSA.26(Sor) locus. When mated with the Myf5-Cre transgenic line, all cells express nTdT except for cells expressing Myf5, which express the nGFP signal. These transgenic models enable visualization and quantification of de novo Myf5 expression in daughter cells committed during asymmetric division (Kuang et al. 2007; Wang et al. Cell Stem Cell, 2019. 24(3): p. 419-432 e6.; Le Grand et al. Cell Stem Cell, 2009. 4(6): p. 535-47).

[0140] Skeletal muscle fiber cultures were performed as previously described (Dumont et al. Nat Med, 2015. 21(12): p. 1455-63). Briefly, the extensor digitorum longus (EDL) muscle was carefully dissected and incubated for 45 minutes at 37 °C in DMEM containing 2% L-glutamine, 4.5% glucose, and 110 mg / mL sodium pyruvate (Gibco), and 0.2% collagenase I (Sigma). Muscle fibers were isolated using gentle trituration with a glass pipette in DMEM+ containing 2% L-glutamine, 4.5% glucose, and 110 mg / ml sodium pyruvate (Gibco). Muscle fibers were cultured at 37 °C for 42 or 72 hours in DMEM+ containing 2% L-glutamine, 4.5% glucose, and 110 mg / ml sodium pyruvate (Gibco), 20% FBS (Wisent), 1% chicken embryo extract (MP Biomedicals), and 2.5 ng / ml bFGF (Cedarlane). Transfection of satellite cells on muscle fibers was performed using Lipofectamine RNAimax (Life Technologies) and validated Smartpool siRNA (Dharmacon) for AAK1 or scramble (SCR) at a final concentration of 50 nM. To ensure maximum efficiency, two transfections were performed 4 and 16 hours after muscle fiber isolation as previously described (Wang et al. Cell Stem Cell, 2019. 24(3): p. 419-432 e6). A pool of four siRNAs targeting AAK1 was used. The siRNA sequences were as follows: GAAGGUGGAUUCGCUCUUG (SEQ ID NO: 9); GGACUCAAAUCUCCUGACA (SEQ ID NO: 10); GCAGAUAUUUGGGCUCUAG (SEQ ID NO: 11); and AAAUGUGCCUUGAAACGUA (SEQ ID NO: 12).

[0141] The percentages of myogenin (MyoG+ cells), total number of satellite cells per fiber (#), number of myogenic cells per fiber, percentage of YFP-satellite stem cells, number of symmetric satellite stem cell divisions, number of asymmetric satellite stem cell divisions, ratio of asymmetric stem cell divisions, ratio of YFP-divisions, and ratio of YFP+ divisions were quantified by counting satellite cells and their progeny in the cultured myofibers. Cells on myofibers were immunostained with anti-Pax7 antibody (42 h) or anti-Pax7 antibody and anti-myogenin antibody (72 h) and counted manually in a blinded fashion, enumerating them through immunohistochemistry.

[0142] Inhibition of AAK1 by siRNA resulted in an approximately three-fold increase in the ratio of asymmetric satellite stem cell divisions in culture after 42 h (Figure 1A) and the ratio of committed myogenic cells in culture after 72 h (Figure 1B). Notably, there was no change in the total number of satellite cells in culture at 42 h (Figure 2A), or the total number of myogenic cells in culture at 72 h (Figure 2B). This suggests that AAK1 inhibition had no effect on cell cycle progression.

[0143] The effects of siRNA inhibition of other genes (including Gak, Bmp2k, MPSK1, and Numb) were also examined as described above and compared to siRNA inhibition of Aak1 and / or control siRNA. The sequences of the pool of four siRNAs targeting BMP2K / BIKE included the following: GCAGGUAUCACCCGAGUAU (SEQ ID NO: 13), UAUCCUACUUUGCGUUUAA (SEQ ID NO: 14), GGGAAGUGCUUAUCUUAAU (SEQ ID NO: 15), and GCUCAAGUCCACUAUGUAA (SEQ ID NO: 16). The sequences of the pool of four siRNAs targeting GAK included the following: GAGGGAGGCUGCAGGCUAA (SEQ ID NO: 17), GACCAAACAGCAAGACUUA (SEQ ID NO: 18), UGGCAGAGAGUAUGCAUUA (SEQ ID NO: 19), and CCUGGAUGCUUGUGAUAUU (SEQ ID NO: 20). The sequences of the pool of four siRNAs targeting MPSK1 included the following: GAAAGAACGAGGUGCUAAG (SEQ ID NO: 21), UCAGUCAGUUGGAGGCAUU (SEQ ID NO: 22), ACCCAAAUCUGAUCAAAUC (SEQ ID NO: 23), and GGACUUGGGUUCUAUGAAU (SEQ ID NO: 24).

[0144] siRNA inhibition of Gak had effects similar to siRNA inhibition of Aak1, resulting in an increased number and percentage of asymmetric satellite cell divisions (Figures 6B and 6C), a reduced percentage of YFP-divisions and YFP+ divisions (Figures 7C and 7D), and an increased number of YFP+ satellite-bound cells per fiber (Figure 7E). siRNA inhibition of MPSK1 also increased the number and percentage of asymmetric satellite cell divisions (Figures 8B and 8C). However, siRNA inhibition of Bmp2k had no significant effect on the number and percentage of asymmetric satellite cell divisions, whereas inhibition of Numb resulted in a complete loss of asymmetric division (Figures 8B and 8C).

[0145] The effect of pharmacological inhibition of AAK1 on asymmetric division of satellite cells was also examined by contacting satellite cells cultured on isolated muscle fibers with a small molecule inhibitor of AAK1. Either SGC-AAK1-1 (catalog number HY-117626, MedChemExpress) or LP-935509 (catalog number HY-123940, MedChemExpress) was added to the culture medium at a final concentration of 100 nM, and an equal dilution of DMSO was used as a vehicle control.

[0146] Pharmacological inhibition of AAK1 resulted in no change in the total number of Pax7-expressing satellite cells (Figure 3A), no change in the total number of YFP-negative satellite stem cells (Figure 3B), and no change in the number of symmetric satellite stem cell divisions (Figure 3C). However, treatment with the AAK1 inhibitor caused a significant 6-fold increase in the number of asymmetric satellite stem cell divisions (Figure 3D).

[0147] These results clearly show that AAK1 inhibition enhances asymmetric satellite cell division, supporting the use of AAK1 inhibition to treat diseases and injuries that would benefit from increased asymmetric satellite cell division and tissue regeneration.

[0148] Example 2: AAK1 inhibition stimulates asymmetric division of dystrophin-deficient MDX muscle stem cells The effect of AAK1 inhibition on asymmetric division of satellite cells was examined essentially as described in Example 1, using tissues obtained from Mdx mice. Mdx mice are an established model of Duchenne muscular dystrophy (see, for example, Swiderski, K. and Lynch, G.S., Am J Phsiol Cell Physiol 2021 Aug 1; 321(2):C409-C412, EPub 2021 Jul 14).

[0149] Satellite cells cultured on isolated muscle fibers were contacted with small molecule inhibitors of AAK1. Either SGC-AAK1-1 (Catalog No. HY-117626, MedChemExpress) or LP-935509 (Catalog No. HY-123940, MedChemExpress) was added to the culture medium at a final concentration of 100 nM, and an equal dilution of DMSO was used as a vehicle control.

[0150] The total number of satellite cells per fiber (#), the percentage of YFP-satellite stem cells, the number of symmetric satellite stem cell divisions, and the proportion of asymmetric stem cell divisions were quantified by counting satellite cells and their progeny in the cultured muscle fibers. Cells on muscle fibers were immunostained with anti-Pax7 antibody (42 h) or anti-Pax7 antibody and anti-myogenin antibody (72 h) and counted manually in a blinded fashion for immunohistochemistry.

[0151] Pharmacological inhibition of AAK1 resulted in no change in the total number of Pax7-expressing satellite cells (Figure 5A), no change in the total number of YFP-negative satellite stem cells (Figure 5B), and no change in the number of symmetric satellite stem cell divisions (Figure 5C). However, treatment with AAK1 inhibitors caused a significant increase in the number of asymmetric satellite stem cell divisions (Figure 5D).

[0152] These data support the enhancement of muscle regeneration via AAK1 inhibition by restoring the functional rescue of skeletal muscle satellite cells and the balance between satellite cell self-renewal and myogenic progenitor cell expansion in Duchenne muscular dystrophy (DMD) and other muscle diseases.

[0153] Example 3: Inhibition of AAK1 increases myogenic progenitor cell density and muscle fiber ferret in vivo Since LX9211 (SAT-3003)-mediated inhibition of AAK1 stimulated asymmetric division of mdx satellite stem cells, next, the effect of LX9211 was examined in C57BL / 10ScSn-Dmd of Duchenne muscular dystrophymdx It was examined in vivo using the / J (mdx) mouse model. LX9211 (1 mg / kg) or DMSO (control) was administered simultaneously with intramuscular injection of cardiotoxin (CDX, 50 μl intramuscularly into both bilateral tibialis anterior muscles [TA]) (10 μl per gram of body weight intraperitoneally) (day 0), and again on day 3. Ten days after CDX injection, the left TA was harvested for single-cell assay: it was processed by enzymatic digestion for mononuclear cell isolation, then filtered, plated, and stained for Pax7 and myogenin (MyoG) using anti-Pax7 antibody and anti-MyoG antibody before imaging. The right TA was harvested for histology: it was cryosectioned and then immunostained for Pax7 and MyoG before imaging via high-throughput Opera analysis (Figure 10).

[0154] Treatment with LX9211 (SAT-3003) increased the expansion of MyoG-positive (MyoG+) myogenic progenitor cells, as evidenced by a significant (about 100%) increase in the percentage of MyoG+ cells (Figure 11B and 11C) (but not in the % of Pax7-positive (Pax7+) cells (Figure 11A)). Muscles treated with LX9211 (SAT3003) also had a significant shift in the distribution of minimal myofiber ferrets compared to vehicle-treated controls (Figure 11D).

[0155] These data are consistent with the LX9211-mediated inhibition of AAK1, which induces an increase in asymmetric muscle stem cell division in regenerating mdx muscles and stimulates myofiber regeneration. Thus, these data support the use of AAK1 inhibition (e.g., using the LX9211 inhibitor) to treat diseases and injuries that benefit from increased asymmetric satellite cell division and tissue regeneration.

[0156] Example 4. Differential effects of AAK1 inhibitor administration regimens on myogenic progenitor cell density and myofiber ferrets Next, the effects of LX9211 (SAT3003)-mediated AAK1 inhibition according to different dosing regimens were examined using the C57BL / 10ScSn-Dmd mdx / J (mdx) mouse model. LX9211 (1 mg / kg) or DMSO (control) was administered according to the following dosing regimens (intraperitoneally at 10 μl per gram of body weight): (1) day 0 and day 3 (“2×”, twice a week for 1 week); (2) day 0, day 3, day 7, and day 10 (“4×”, twice a week for 2 weeks); (3) day 0, day 3, day 5, day 7, day 10, day 12, day 14, day 17 (“7×”, four times a week for 2 weeks); or (4) DMSO only (control) on day 0, day 3, day 5, day 7, day 10, day 12, day 14, day 17 (four times a week for 2 weeks). Ten days after CDX injection, the right TA was harvested for single-cell assay: it was processed by enzymatic digestion for mononuclear cell isolation, then filtered, plated, and stained for Pax7 and MyoG prior to imaging. The left TA was harvested for histology: cryosectioned and then immunostained for Pax7 and MyoG prior to imaging with Zeiss software (Figure 12).

[0157] LX9211 (SAT-3003) treatment according to dosing regimen 2 (“4×”) increased muscle mass compared to the vehicle-treated control (Figure 13A). LX9211 treatment according to dosing regimen 2 also increased the expansion of MyoG-positive myogenic progenitor cells, as evidenced by a significant (≈100%) increase in the percentage and number of MyoG+ cells (Figure 13B and 13C) (but not Pax7+ cells (Figure 13D)). Neither dosing regimen 1 (“2×”) nor dosing regimen 3 (“7×”) significantly increased muscle mass or the percentage of MyoG+ cells, but dosing regimen 1 increased the number of both MyoG+ and Pax7+ cells.

[0158] Treatment with LX9211 (SAT3003) according to dosing regimen 2 (“4X”) increased total TA muscle fiber area (Figure 14A) and mean Feret diameter (Figure 14B) compared to vehicle treatment control. Animals treated with LX9211 according to dosing regimen 2 (“4X”) also had a significant shift in the distribution of minimum muscle fiber Ferets compared to vehicle treatment control (Figure 14D). Neither dosing regimen 1 nor dosing regimen 3 significantly increased total TA muscle fiber area (Figure 14A) or mean Feret diameter (Figure 14B), and did not significantly shift the distribution of minimum muscle fiber Ferets (Figure 14C and 14E).

[0159] These data add further support for the use of AAK1 inhibition (e.g., using LX9211) in inducing an increase in asymmetric muscle stem cell division in regenerating mdx muscle and stimulating muscle fiber regeneration. Further, these data introduce an unexpected effect of different dosing regimens in stimulating the effect of LX9211.

[0160] Example 5. Increase in muscle strength with increasing dose of AAK1 inhibitor Next, the effects of LX9211 (SAT3003)-mediated AAK1 inhibition according to various doses were examined in C57BL / 10ScSn-Dmd of Duchenne muscular dystrophy mdxThe / J(mdx) mouse model was used for the investigation. LX9211 (0 mg / kg, 0.1 mg / kg, 0.3 mg / kg, or 1 mg / kg) was administered on days 0, 3, 7, and 10 (intraperitoneally at 10 μl per gram of body weight). On day 17, the right TA was subjected to physiological tests using a Dynamic Muscle Control (DMC) 300C-LR-FP muscle lever (Aurora Scientific) together with ASI 610A Dynamic Muscle Control v5.500 software, and a High-Power, Bi-Phase Stimulator and Dual-Mode Lever System for each muscle lever. The data were analyzed using ASI 611A Dynamic Muscle Analysis v5.300 software. Briefly, (1) the mouse was anesthetized; (2) dissection was performed to expose the TA tendon, a string was attached to the tendon, and the TA and knee were exposed; (3) measurements were taken manually every 100 seconds for 15 minutes using a trigger and a tension of 20 mN; (4) the data were analyzed using DMCv5.500. The left TA was harvested for histology: cryosectioned and then immunostained for Pax7 and MyoG and / or laminin / WGA prior to imaging with Zeiss software (Figure 15).

[0161] LX9211 (SAT-3003) treatment resulted in a significant increase in maximum tetanic force (nM, Figure 16), specific force (mN / mm 2 , Figure 17), and twitch force (mN, Figure 18), normalized to the control. A dose-dependent effect was observed; 1 mg / kg of LX9211 SAT-3003 was associated with a greater increase in maximum tetanic force, specific force, and twitch force compared to lower doses.

[0162] These data add support for the use of AAK1 inhibition (e.g., using LX9211) in promoting muscle cell / tissue regeneration for the treatment of muscle tissue injury and muscle diseases (including muscular dystrophy). Further, these data introduce a dose-dependent effect of the AAK1 inhibitor, LX9211, on muscle strength.

[0163] The various embodiments described herein can be combined to provide further embodiments.

[0164] Aspects of the embodiments can be modified if necessary to use concepts from various patents, applications, and publications to provide still further embodiments.

[0165] These and other modifications can be made to the embodiments in light of the above description. In general, in the following claims, the terms used should not be construed as limiting the claims to the specific embodiments disclosed herein and the claims should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Thus, the claims are not limited by the present disclosure.

[0166] All of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications referred to herein are hereby incorporated by reference in their entirety.

Claims

1. A composition for use in increasing asymmetric cell division of skeletal muscle stem cells in vitro or ex vivo, comprising an inhibitor selected from adapter-related kinase 1 (AAK1), cyclin G-related kinase (GAK), or myristoylated and / or palmitoylated serine / threonine kinase 1 (MPSK1), wherein the inhibitor selected from adapter-related kinase 1 (AAK1), cyclin G-related kinase (GAK), or myristoylated and / or palmitoylated serine / threonine kinase 1 (MPSK1) comes into contact with the skeletal muscle stem cells and increases the asymmetric cell division of the skeletal muscle stem cells.

2. A composition comprising an inhibitor of AAK1, GAK, or MPSK1 for use in increasing asymmetric cell division of skeletal muscle stem cells, or for use in increasing skeletal muscle tissue growth or regeneration in a subject.

3. The composition for use according to claim 2, wherein the subject has damaged or injured skeletal muscle tissue, and, if necessary, the skeletal muscle tissue is damaged or injured as a result of physical injury or accident, disease, genetic mutation, infection, overuse, loss of blood circulation, muscle atrophy, muscle wasting, dystrophic muscle, or aging.

4. The composition for use according to claim 2, for use in the treatment of muscular dystrophy.

5. (a) The skeletal muscle stem cells are pathological skeletal muscle stem cells containing mutations associated with muscular dystrophy, or the subject has muscular dystrophy, optionally Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), Emery-Dreyfus muscular dystrophy, Landouzy-Dejerine muscular dystrophy, facioscapulohumeral muscular dystrophy (FSH), limb-girdle muscular dystrophy, von Graef-Fuchs muscular dystrophy, oculopharyngeal muscular dystrophy (OPMD), myotonic dystrophy (Steinert disease), or congenital muscular dystrophy, or (b) The damaged or injured muscle stem cells include mutations in the dystrophin gene, A composition for use according to any one of claims 2 to 4.

6. The composition for use according to claim 2, wherein the skeletal muscle stem cells have reduced asymmetric cell division compared to normal healthy skeletal muscle stem cells.

7. The composition for use according to claim 2, wherein the inhibitor inhibits the expression of AAK1, GAK, or MPSK1 by, as necessary, transcription, translation, post-translational modification, or the stability of the protein component, or by inhibiting the gene encoding the protein component, and, as necessary, the inhibitor binds to a polynucleotide sequence that regulates the expression of AAK1, GAK, or MPSK1, wherein the nucleotide sequence is located within the gene of AAK1, GAK, or MPSK1, and / or, as necessary, the inhibitor binds to a polynucleotide sequence encoding AAK1, GAK, or MPSK1, or a polynucleotide sequence complementary to the polynucleotide sequence encoding AAK1, GAK, or MPSK1, wherein the polynucleotide sequence is located within the gene or mRNA of AAK1, GAK, or MPSK1.

8. The composition for use according to claim 7, wherein the polynucleotide sequence is DNA or RNA, and / or optionally the inhibitor comprises a polynucleotide sequence, optionally the inhibitor comprises a DNA polynucleotide sequence and / or an RNA polynucleotide sequence, and / or optionally the inhibitor comprises shRNA, microRNA, gRNA, siRNA, aptamer, or antisense oligonucleotide, and optionally the inhibitor comprises a guide RNA that targets the AAK1 gene and a polynucleotide sequence encoding the CRISPR-Cas protein.

9. The inhibitor inhibits the activity of AAK1, GAK, or MPSK1, and optionally the inhibitor binds to AAK1, GAK, or MPSK1, and / or optionally the inhibitor comprises a polypeptide, and optionally the inhibitor comprises an antibody or a functional fragment thereof that binds to AAK1, GAK, or MPSK1, and / or optionally the inhibitor The composition for use according to claim 2, wherein the inhibitor is an organic molecule, for example, an organic low molecular weight, and optionally the inhibitor is selected from the group consisting of SGC-AAK1-1, LP-935509, LP-922761, BMT-090605, BMT-124110, LP-927443, and BMS-901715, and / or optionally the inhibitor inhibits AAK1, GAK, or MPSK1 kinase activity or AAK1, GAK, or MPSK1 ATP binding activity.

10. (a) The inhibitor AAK1, GAK, or MPSK1 does not substantially inhibit the proliferation or cell cycle progression of the skeletal muscle stem cells. (b) The cells are mammalian, and / or human, as is appropriate. (c) The inhibitor inhibits AAK1, GAK, and / or MPSK1. The composition for use according to claim 2.

11. (a) the method increases skeletal muscle tissue regeneration in the subject, and / or (b) the inhibitor AAK1, GAK, or MPSK1 is administered to the subject systemically or locally, as necessary, at the site of tissue damage or injury. The composition for use according to claim 2.

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