Methods of treatment for muscular disorders using small molecule modulation of alpha7 integrin

Abstract

Disclosed herein are α7 integrin modulatory agents and methods of using such to treat muscular disorders, disease, degeneration, and / or muscular dystrophy. The methods include administering an effective amount of an α7 integrin modulatory agent to the subject with a muscular disorder, disease, degeneration, and / or dystrophy, wherein the α7 integrin modulatory agent increases α7 integrin expression or activity as compared to α7 integrin expression or activity prior to treatment, thereby treating the subject. Also disclosed are methods of enhancing muscle regeneration, repair, or maintenance in a subject and methods of enhancing α7 integrin expression by use of the disclosed α7 integrin modulatory agents. Methods of preventing or reducing muscle injury or damage in a subject are also disclosed.

Description

METHODS OF TREATMENT FOR MUSCULAR DISORDERS USING SMALL MOLECULE MODULATION OF ALPHA7 INTEGRINCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 USC §1 19(e) of US Provisional Application No. 63 / 528,569 filed July 24, 2023, and US Provisional Application No. 63 / 539,416 filed September 20, 2023, which are incorporated herein by reference in their entirety for all purposes.FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with Government support under Agreement No. 9R420D030543-02A, awarded by the National Institute of Neurological Disorders. The United States Government has certain rights in the invention.BACKGROUND

[0003] Muscle disorders and / or degeneration can result in weakness, pain, paralysis, and impaired quality of life. Muscles disorders are caused by ageing, injury, overuse, cancer, inflammation, infections, and / or genetic mutations. Some treatments for muscles disorders are often dependent on the cause of the disorder and do not always contribute to improving quality of life for patients living with a muscle disorder. Muscular dystrophies are muscular disorders caused by genetic mutations. There are over 30 types of muscular dystrophies, including Duchenne muscular dystrophy, the most common form of muscular dystrophy. Symptoms of muscular disorders include weakness, impaired movement, inability to walk, and trouble breathing and / or swallowing. Lifespan is reduced for patients with muscular dystrophy. There is no cure for muscular dystrophies and the effectiveness of treatments varies.

[0004] Duchenne muscular dystrophy is an X-chromosome-linked disease and is caused by a loss of function mutation in the dystrophin gene. Duchenne muscular dystrophy affects 1 in 3500 live male births with patients suffering from chronic muscle degeneration and weakness. Clinical symptoms are first detected between the ages of 2 and 5 years and, by the time the patient is in their teens, the ability for independent ambulation is lost. Death typically occurs in the patient before they are 30 years old due to cardiopulmonary failure.

[0005] Dystrophin is a protein that localizes on skeletal and cardiac muscles cell plasma membranes and connects cell cytoskeletons to extracellular matrix laminin. Without functionaldystrophin, important connections between muscle cells and fibers are compromised, leading to the impaired muscles function observed in Duchenne muscular dystrophy.

[0006] Integrins are transmembrane receptors that, like dystrophin, play a role in the connections between muscle cells and cell-to-extracellular matrix adhesion. Integrins also have a role in signaling pathways that regulate, amongst other things, cell proliferation and intracellular cytoskeleton organization. The a7pi integrin, in particular, is a laminin receptor on skeletal and cardiac muscle cells that provides a transmembrane connection between basal lamina and muscle fibers. The a7|31 integrin is a heterodimeric receptor protein made up of the a7 chain subunit and the [31 chain subunit. The a7 chain is encoded by the integrin a-7 (ITA7) gene and the [31 chain is encoded by the integrin subunit |3 1 (ITGB1 ) gene. Transgenically enhanced expression of a7 integrin in mice was found to increase the overall amount of ct7[31 integrins in muscle tissues. Burkin et al., 2001 , J. of Cell Biol. 152(6): 1207-1218.

[0007] Increasing the amount of a7[31 integrin is known to ameliorate and / or prevent the symptoms of muscle disorders and degeneration including those caused by sarcopenia, muscle injury, muscle disorders, and muscular dystrophy. Haroon et al., 2022, Aging 14(1 ): 28-53; Rozo et al., 2016, Nat Med. 22(8): 889-896; McClure et al., 2016, Acta Biomaterialia 39: 44-54; Boppart et al., 2006, Am J Physiol 290: C1660-C1665. Increasing levels of a7 integrin in a mouse model for muscular dystrophy significantly prolonged lifespan of the mouse subjects. Burkin etal., 2001 , J. of Cell Biol. 152(6): 1207-1218. Further, increased transgenic expression of a7 integrin does not negatively disrupt global gene expression profiles. Liu et al., 2008, Am J Physiol Cell Physiol. 294(2): C627-40. Further still, increasing the amounts of the a7[31 integrin protein generally promotes muscle cell proliferation, adhesion, endurance, regeneration, and resistance to apoptosis. Id. Boppart & Mahmassani, 2019, Am J Physiol 317: C629-C641 ;McClure et al., 2016, supra; Valero et al., 2011 , PLoS ONE 7(1 ): e29760.

[0008] Despite the known beneficial outcomes of increasing a7 integrin levels for muscle health, many nuances and complexities of a7 integrin transcriptional regulation remain unknown.SUMMARY OF THE INVENTION

[0009] This disclosure relates to methods of treating muscle disease or disorder by administering to a subject, compounds that increase expression of a7 integrin in muscle cells.

[0010] In one aspect, the present disclosure provides a method for treating a subject with a muscle disease or disorder, comprising: administering an effective amount of an a7 integrin modulatory agent to the subject with the muscle disease or disorder, wherein the a7 integrinmodulatory agent comprises at least one of the following compounds: TRLILI, TD l-011536, GA- 017, VT02956, T65, and T74.

[0011] In one aspect, the present disclosure provides a method for treating a subject with muscle degeneration or atrophy, comprising: administering an effective amount of an a7 integrin modulatory agent to the subject with the muscle degeneration, wherein the a7 integrin modulatory agent comprises at least one of the following compounds: TRULI, TDI-011536, GA- 017, VT02956, T65, and T74.

[0012] In one aspect, the present disclosure provides a method for treating a subject with a muscular dystrophy, comprising: administering an effective amount of an a7 integrin modulatory agent to the subject with muscular dystrophy, wherein the a7 integrin modulatory agent comprises at least one of the following compounds: TRULI, TDI-01 1536, GA-017, VT02956, T65, and T74.

[0013] In one aspect, the present disclosure provides a method for treating a subject with a muscle disease or disorder, comprising administering to the subject an effective amount of an a7 integrin modulatory agent, wherein the a7 integrin modulatory agent inhibits activity of Latsl and / or Lats2 kinases thereby increasing expression of a7 integrin, and wherein the a7 integrin modulatory agent comprises at least one of the following compounds: TRULI, TDI-011536, GA- 017, VT02956, T65, and T74

[0014] In some embodiments, a method is disclosed for treating a subject with muscle degeneration or atrophy where the muscle degeneration or atrophy is a decrease in muscle strength of at least 20% (e.g., 20% or at least 20%, 25% or at least 25%, 30% or at least 30%, 35% or at least 35%, 40% or at least 40%, 45% or at least 45%, 50% or at least 50%, 55% or at least 55%, 60% or at least 60%, 65% or at least 65%, 70% or at least 70%, 75% or at least 75%, 80% or at least 80%, or 85% or at least 85%) relative to a control population.

[0015] In some embodiments, a method is disclosed for treating a subject with muscle degeneration or atrophy where the muscle degeneration or atrophy is a decrease in muscle mass of at least 20% (e.g., 20% or at least 20%, 25% or at least 25%, 30% or at least 30%, 35% or at least 35%, 40% or at least 40%, 45% or at least 45%, 50% or at least 50%, 55% or at least 55%, 60% or at least 60%, 65% or at least 65%, 70% or at least 70%, 75% or at least 75%, 80% or at least 80%, or 85% or at least 85%), in the subject relative to a measurement of muscle mass in the subject at a prior time point. In various embodiments, the prior time point may be one year, two years, three years, four years, or five years earlier. In some cases, the prior time point may be from 1 month to 10 years earlier, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11months earlier, or 1 , 1 .5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 years earlier.

[0016] In some embodiments, the muscular dystrophy treated is merosin deficient congenital muscular dystrophy Type 1 A (MDC1 A), merosin deficient congenital muscular dystrophy Type 1 D (MDC1 D), limb-girdle muscular dystrophy (LGMD), Duchenne muscular dystrophy (DMD), Fukuyama congenital muscular dystrophy (FCMD), or Facioscapulohumeral muscular dystrophy (FSHD).

[0017] In some embodiments, the muscular dystrophy is Duchenne muscular dystrophy.

[0018] In any of the embodiments discussed above or herein, the a7 integrin modulatory agent may be TRULL

[0019] In any of the embodiments discussed above or herein, the a7 integrin modulatory agent may be TDI-01 1536.

[0020] In any of the embodiments discussed above or herein, the a7 integrin modulatory agent may be GA-017.

[0021] In any of the embodiments discussed above or herein, the a7 integrin modulatory agent may be VT02956.

[0022] In any of the embodiments discussed above or herein, the a7 integrin modulatory agent may be T65.

[0023] In any of the embodiments discussed above or herein, the a7 integrin modulatory agent may be T74.

[0024] In some embodiments, the methods disclosed herein further comprise administering to the subject an additional therapeutic agent.

[0025] In some embodiments, the additional therapeutic agent is a protein, a growth factor satellite cells, stem cells, myocytes, an a7p 1 integrin modulatory agent, or an additional a7 integrin modulatory agent.

[0026] In any of the embodiments discussed above or herein, the subject can be human.

[0027] In various embodiments, any of the features or components of embodiments discussed above or herein may be combined, and such combinations are encompassed within the scope of the present disclosure. Any specific value discussed above or herein may be combined with another related value discussed above or herein to recite a range with the values representing the upper and lower ends of the range, and such ranges and all values falling within such ranges are encompassed within the scope of the present disclosure. Each of the values discussed above or herein may be expressed with a variation of 1%, 5%, 10% or 20%. Other embodiments will become apparent from a review of the ensuing detailed description.BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 A shows two digital images of X-gal staining demonstrating that a7[3gal+ / myoblasts express -galactosidase which increases upon differentiation to myotubes.

[0029] Figure 1 B shows a digital image of a Western analysis of a7|3gal+ / ' myoblasts differentiated from 0-72 hours shows a corresponding increase in both a7 integrin and [3- galactosidase, where a-Tubulin was used as a loading control.

[0030] Figure 2A shows the structure of TRULL

[0031] Figure 2B shows the structure of TDI-01 1536.

[0032] Figure 2C shows the structure of GA-017.

[0033] Figure 2D shows the structure of VT02956.

[0034] Figure 2E shows the structure of T65.

[0035] Figure 2F shows the structure of T74.

[0036] Figure 3A shows the a7 integrin reporter dose dependent response curves for administration of S969, TDI-01 1536, PY-60, and TRULL

[0037] Figure 3B shows the a7 integrin reporter non-linear regression curve-fit for administration of S969, TDI-01 1536, PY-60, and TRULL

[0038] Figure 4 shows the results of an ITGA7+ / LACZassay in myotubes using various small molecule combinations.

[0039] Figure 5 shows the GA-017 dose-response curve relative to other ITGA7 small molecule enhancers depicted in Figure 3A.

[0040] Figure 6 shows the dose response curves for S969, TDI-011536, TRULI, VT02956, and GA-017 in the ITGA7+ / LacZ myotube assay.

[0041] Figure 7 shows Western blotting results for C2C12 myotube lysates treated for 48 hour with either DMSO, 2 mM VT02956, 2 mM TDI-011536, 20 mM S969, or 10 mM GA-017 and probed for a7 integrin using a rabbit polyclonal antibody and normalized to Revert Total Protein Stain.

[0042] Figure 8 shows the Western blot results of Figure 7 as quantitated by Image J and graphed.

[0043] Figure 9 shows the Immunofluorescence of C2C12 myotubes treated with DMSO, 20 mM S969, 10 mM TRULI, or 2 mM TDI-011536 and probed for active Yap, phospo-Yap S127, and DAPI. The nuclear localization of active Yap shows inhibition of LATS1 / 2 kinase by the small molecules.DETAILED DESCRIPTION

[0044] Disclosed herein are a7 integrin expression modulatory agents and methods of using such to treat a condition associated with impaired a7 integrin expression, such as muscle diseases, disorders, and degeneration, including muscular dystrophy.

[0045] In one embodiment, a method for treating a subject with a muscular disease or disorder, muscle degeneration, or muscular dystrophy is disclosed. The method includes administering an effective amount of an a7 integrin modulatory agent to the subject, wherein the a7 integrin modulatory agent is TRULI, TDI-011536, GA-017, or VT02956, or any combination thereof. In some embodiments, the a7 integrin modulatory agent is administered with an additional therapeutic agent. Different o7 integrin modulatory agents can be administered in combinations and / or can be administered in suitable pharmaceutical carriers.

[0046] An a7 integrin modulatory agent can be administered by any suitable method, such as topically, parenterally (such as intravenously, subcutaneously, or intraperitoneally), or orally. In a specific example, the a7 integrin modulatory agent is administered systemically, such as through parenteral administration, such as stomach injection or peritoneal injection.

[0047] The disclosed methods also may be used to enhance repair or maintenance, or prevent damage to, other tissues and organs. For example, the methods of the present disclosure can be used to treat symptoms of muscular dystrophy stemming from effects to cells or tissue other than skeletal muscle, such as impaired or altered brain function, smooth muscles, or cardiac muscles.

[0048] The foregoing and other features of the disclosure will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.

[0049] Disclosed herein are a7 integrin expression modulatory agents and methods of using such to treat muscles disorders that can be improved by increased expression of a7 integrin.

[0050] In some embodiments, the muscular disorder is muscular dystrophy. In some embodiments, the muscular dystrophy is merosin deficient congenital muscular dystrophy Type 1 A (MDC1 A), merosin deficient congenital muscular dystrophy Type 1 D (MDC1 D), limb-girdle muscular dystrophy (LGMD), Duchenne muscular dystrophy (DMD), Fukuyama congenital muscular dystrophy (FCMD) or Facioscapulohumeral muscular dystrophy (FSHD).

[0051] In some particular embodiments, the muscular dystrophy is DMD, MDC1 A or FCMD.

[0052] In one particular embodiment, the muscular dystrophy is DMD.

[0053] In some embodiments, the a7 integrin modulatory agent is administered with an additional therapeutic agent.

[0054] In some embodiments, the additional therapeutic agent is a costameric protein, a growth factor, satellite cells, stem cells, myocytes or an additional a7 integrin modulatory agent.

[0055] In some embodiments, the additional a7 integrin modulatory agent is laminin-1 11 , a laminin-1 11 fragment, valproic acid, or a valproic acid analog.

[0056] In some embodiments, the method further includes selecting a subject with muscular dystrophy.

[0057] In some embodiments, the selecting a subject with muscular dystrophy includes diagnosing the subject with muscular dystrophy prior to administering an effective amount of the a7 integrin modulatory agent to the subject.

[0058] In other embodiments, a method of enhancing muscle regeneration, repair, or maintenance in a subject is disclosed.

[0059] In some embodiments, the method includes administering the a7 modulatory agent prior to the subject experiencing muscle damage or disease.

[0060] In some embodiments, the method is a method of enhancing muscle maintenance in a subject.

[0061] In some embodiments, the a7 integrin modulatory agent is administered to the subject prior to the subject exercising.

[0062] In some embodiments, the a7 integrin modulatory agent is administered to a subject at risk of acquiring a muscle disease or damage, such as an elderly subject (e.g., >50 years of age, >60 years of age, or >70 years of age).

[0063] In some embodiments, the method also includes selecting a subject in need of enhancing muscle regeneration, repair, or maintenance.

[0064] In some embodiments, selecting a subject in need of enhancing muscle regeneration, repair, or maintenance includes diagnosing the subject with a condition characterized by impaired muscle regeneration prior to administering an effective amount of the a7 integrin modulatory agent to the subject.

[0065] In some embodiments, selecting a subject in need of enhancing muscle regeneration, repair, or maintenance comprises diagnosing the subject with a condition characterized by impaired production of a component of a7 integrin prior to administering an effective amount of the a7 integrin modulatory agent to the subject.

[0066] In some embodiments, the a7 integrin modulatory agent is administered with an additional therapeutic agent.

[0067] In some embodiments, the additional therapeutic agent is a costameric protein, a growth factor, satellite cells, stem cells, myocytes or an additional a7 integrin modulatory agent.

[0068] In further embodiments, a method of prospectively preventing or reducing muscle injury or damage in a subject is disclosed.

[0069] In some embodiments, the subject is at risk of developing a muscle injury or damage.

[0070] In some embodiments, the cell is a muscle cell.

[0071] In some embodiments, the muscle cell is present in a mammal, and wherein contacting the cell with an agent comprises administering the agent to the mammal.

[0072] In some embodiments, the subject is a human.Terms and Definitions

[0073] The following explanations of terms and methods are provided to better describe the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. The singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. The term “comprises” means “includes.” Thus, “comprising A or B,” means “including A, B, or A and B,” without excluding additional elements.

[0074] It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below.

[0075] Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

[0076] Unless indicated otherwise, the nomenclature of substituents that are not explicitly defined herein are arrived at by naming the terminal portion of the functionality followed by the adjacent functionality toward the point of attachment.

[0077] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. All sequences provided in the disclosed Genbank Accession numbers are incorporated herein by reference as available on July 31 ,2023. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0078] In order to facilitate review of the various embodiments of this disclosure, the following explanations of specific terms are provided:

[0079] Administration: To provide or give a subject one or more agents, such as an agent that increases a7 integrin expression and / or treats one or more symptoms associated with a muscle disease or disorder (e.g., muscular dystrophy), by any effective route. Exemplary routes of administration include, but are not limited to, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), oral, sublingual, rectal, transdermal, intranasal, vaginal and inhalation routes.

[0080] Agent: Any protein, nucleic acid molecule (including chemically modified nucleic acids), compound, antibody, small molecule, organic compound, inorganic compound, or other molecule of interest. Agent can include a therapeutic agent, a diagnostic agent or a pharmaceutical agent. A therapeutic or pharmaceutical agent is one that alone or together with an additional compound induces the desired response (such as inducing a therapeutic or prophylactic effect when administered to a subject, including treating a subject with a muscle disease or disorder (e.g., muscular dystrophy).

[0081] In some examples, an agent can act directly or indirectly to alter the expression and / or activity of o7. In a particular example, a therapeutic agent significantly increases the expression and / or activity of o7 (which is a muscular dystrophy associated molecule) thereby treating one or more signs or symptoms associated with a muscle disease or disorder (e.g., muscular dystrophy). An example of a therapeutic agent is one that can increase the expression and / or activity of the o7 gene or gene product, for example as measured by a clinical response (such as a decrease in one or more signs or symptoms associated with the muscle disease or disorder (e.g., muscular dystrophy), an improvement in muscular health, regeneration, repair or maintenance of a muscle cell or tissue). “Improving muscular health” refers to an improvement in muscular health compared with a preexisting state or compared with a state which would occur in the absence of treatment. For example, improving muscular health may include enhancing muscle regeneration, maintenance, or repair. Improving muscular health may also include prospectively treating a subject to prevent or reduce muscular damage or injury. “Regeneration” refers to the repair of cells or tissue, such as muscle cells or tissue (or organs) which includes muscle cells, following injury or damage to at least partially restore the muscle or tissue to a condition similar to which the cells or tissue existed before the injury or damageoccurred. Regeneration also refers to facilitating repair of cells or tissue in a subject having a disease affecting such cells or tissue to eliminate or ameliorate the effects of the disease. In more specific examples, regeneration places the cells or tissue in the same condition or an improved physiological condition as before the injury or damage occurred or the condition which would exist in the absence of disease. “Maintenance” of cells or tissue, such as muscle cells or tissue (or organs) which includes muscle cells, refers to maintaining the cells or tissue in at least substantially the same physiological condition, such as maintaining such condition even in the presence of stimulus which would normally cause damage, injury, or disease. “Repair” of cells or tissue, such as muscle cells or tissue (or organs) which includes muscle cells, refers to the physiological process of healing damage to the cells or tissue following damage or other trauma.

[0082] A “pharmaceutical agent” is a chemical compound or composition capable of inducing a desired therapeutic or prophylactic effect when administered to a subject, alone or in combination with another therapeutic agent(s) or pharmaceutically acceptable carriers. In a particular example, a pharmaceutical agent significantly increases the expression and / or activity of a7 thereby treating a condition or disease associated with decreased a7 expression / activity, such as muscular dystrophy.

[0083] Analog or Derivative: A compound which is sufficiently homologous to a compound such that it has a similar functional activity for a desired purpose as the original compound. Analogs or derivatives refers to a form of a substance, which has at least one functional group altered, added, or removed, compared with the parent compound.

[0084] Biological activity: The beneficial or adverse effects of an agent on living matter. When the agent is a complex chemical mixture, this activity is exerted by the substance's active ingredient or pharmacophore, but can be modified by the other constituents. Activity is generally dosage-dependent and it is not uncommon to have effects ranging from beneficial to adverse for one substance when going from low to high doses. In one example, the agent significantly increases the biological activity of a7 which reduces one or more signs or symptoms associated with a muscle disease or disorder (e.g., muscular dystrophy).

[0085] Contacting: Placement in direct physical association, including both a solid and liquid form. Contacting an agent with a cell can occur in vitro by adding the agent to isolated cells or in vivo by administering the agent to a subject.

[0086] Control: A sample or standard used for comparison with a test sample, such as a biological sample obtained from a patient (or plurality of patients) without a particular disease or condition, such as a muscle disease or disorder (e.g., muscular dystrophy). In someembodiments, the control is a sample obtained from a healthy patient (or plurality of patients) (also referred to herein as a “normal” control), such as a normal biological sample. In some embodiments, the control is a historical control or standard value (e.g., a previously tested control sample or group of samples that represent baseline or normal values (e.g., expression values), such as baseline or normal values of a particular gene such as a ct7p 1 gene, gene product in a subject without a muscular dystrophy). In some examples, the control is a standard value representing the average value (or average range of values) obtained from a plurality of patient samples (such as an average value or range of values of the gene or gene products, such as the a7[31 gene or gene products, in the subjects without a muscular dystrophy).

[0087] Decrease: To reduce the quality, amount, or strength of something. In one example, a therapy decreases one or more symptoms associated with the muscle disease or disorder (e.g., muscular dystrophy), for example as compared to the response in the absence of the therapy.

[0088] Diagnosis: The process of identifying a disease, such as a muscle disease or disorder (e.g., muscular dystrophy), by its signs, symptoms and results of various tests. The conclusion reached through that process is also called “a diagnosis.” Forms of testing commonly performed include blood tests, medical imaging, urinalysis, and biopsy.

[0089] Effective amount: An amount of agent that is sufficient to generate a desired response, such as reducing or inhibiting one or more signs or symptoms associated with a condition or disease. When administered to a subject, a dosage will generally be used that will achieve target tissue / cell concentrations. In some examples, an “effective amount” is one that treats one or more symptoms and / or underlying causes of any of a disorder or disease. In some examples, an “effective amount” is a therapeutically effective amount in which the agent alone with an additional therapeutic agent(s) (for example anti-pathogenic agents), induces the desired response such as treatment of a muscle disease or disorder (e.g., muscular dystrophy, such as DMD, FCMD or MDC1A).

[0090] In particular examples, it is an amount of an agent capable of increasing a7 gene expression or activity by least 5%, 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 98%, or even at least 100% (elimination of the disease to a point beyond detection).

[0091] In some examples, an effective amount is an amount of a pharmaceutical preparation that alone, or together with a pharmaceutically acceptable carrier or one or more additional therapeutic agents, induces the desired response.

[0092] In one example, a desired response is to increase the subject’s survival time by slowing the progression of the disease, such as slowing the progression of muscular dystrophy. The disease does not need to be completely inhibited for the pharmaceutical preparation to be effective. For example, a pharmaceutical preparation can decrease the progression of the disease by a desired amount, for example by at least 5%, at least 10%, at least 15%, at least20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 98%, or even at least 100%, as compared to the progression typical in the absence of the pharmaceutical preparation.

[0093] In another or additional example, it is an amount sufficient to partially or completely alleviate symptoms of the muscle disease or disorder (e.g., muscular dystrophy) within the subject. Treatment can involve only slowing the progression of the disease temporarily, but can also include halting or reversing the progression of the disease permanently.

[0094] Effective amounts of the agents described herein can be determined in many different ways, such as assaying for a reduction in of one or more signs or symptoms associated with the muscle disease or disorder e.g., muscular dystrophy) in the subject or measuring the expression level of one or more molecules known to be associated with the muscle disease or disorder (e.g., muscular dystrophy). Effective amounts also can be determined through various in vitro, in vivo or in situ assays, including the assays described herein.

[0095] The disclosed therapeutic agents can be administered in a single dose, or in several doses, for example daily, during a course of treatment. However, the effective amount can be dependent on the source applied (for example a nucleic acid molecule isolated from a cellular extract versus a chemically synthesized and purified nucleic acid), the subject being treated, the severity and type of the condition being treated, and the manner of administration.

[0096] Expression: The process by which the coded information of a gene is converted into an operational, non-operational, or structural part of a cell, such as the synthesis of a protein. Gene expression can be influenced by external signals. For instance, exposure of a cell to a hormone may stimulate expression of a hormone-induced gene. Different types of cells can respond differently to an identical signal. Expression of a gene also can be regulated anywhere in the pathway from DNA to RNA to protein. Regulation can include controls on transcription, translation, RNA transport and processing, degradation of intermediary molecules such as mRNA, or through activation, inactivation, compartmentalization or degradation of specific protein molecules after they are produced. In an example, expression, such as expression ofa7 integrin, can be regulated to treat one or more signs or symptoms associated with muscular dystrophy.

[0097] The expression of a nucleic acid molecule can be altered relative to a normal (wild type) nucleic acid molecule. Alterations in gene expression, such as differential expression, include but are not limited to: (1 ) overexpression; (2) underexpression; or (3) suppression of expression. Alternations in the expression of a nucleic acid molecule can be associated with, and in fact cause, a change in expression of the corresponding protein.

[0098] Protein expression can also be altered in some manner to be different from the expression of the protein in a normal (wild type) situation. This includes but is not necessarily limited to: (1 ) a mutation in the protein such that one or more of the amino acid residues is different; (2) a short deletion or addition of one or a few (such as no more than 10-20) amino acid residues to the sequence of the protein; (3) a longer deletion or addition of amino acid residues (such as at least 20 residues), such that an entire protein domain or sub-domain is removed or added; (4) expression of an increased amount of the protein compared to a control or standard amount; (5) expression of a decreased amount of the protein compared to a control or standard amount; (6) alteration of the subcellular localization or targeting of the protein; (7) alteration of the temporally regulated expression of the protein (such that the protein is expressed when it normally would not be, or alternatively is not expressed when it normally would be); (8) alteration in stability of a protein through increased longevity in the time that the protein remains localized in a cell; and (9) alteration of the localized (such as organ or tissue specific or subcellular localization) expression of the protein (such that the protein is not expressed where it would normally be expressed or is expressed where it normally would not be expressed), each compared to a control or standard. Controls or standards for comparison to a sample, for the determination of differential expression, include samples believed to be normal (in that they are not altered for the desired characteristic, for example a sample from a subject who does not have muscular dystrophy, such as DMD, FCMD or MDC1 A) as well as laboratory values (e.g., range of values), even though possibly arbitrarily set, keeping in mind that such values can vary from laboratory to laboratory.

[0099] Laboratory standards and values can be set based on a known or determined population value and can be supplied in the format of a graph or table that permits comparison of measured, experimentally determined values.

[0100] Extracellular matrix: An extracellular structure of a tissue or a layer thereof, including the arrangement, composition, and forms of one or more matrix components, such as proteins, including structural proteins such as collagen and elastin, proteins such as fibronectin andlaminins, and proteoglycans. The matrix may comprise fibrillic collagen, having a network of fibers. In some examples, the extracellular matrix is connected to cells through the costameric protein network.

[0101] Increase: To enhance the quality, amount, or strength of something. In one example, an agent increases the activity or expression of a7pi , for example relative to an absence of the agent. In a particular example, an agent increases the activity or expression of a7[31 by at least 5%, 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 98%, or even at least 100%, including between 10% to 95%, 20% to 80%, 30% to 70%, 40% to 50%, such as 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 100%. Such increases can be measured using the methods disclosed herein.

[0102] In a particular example, a therapy increases (also known as up-regulates) the expression of a7, such as an increase of at least 5%, 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 98%, or even at least 100% in a7 expression, thereby treating / alleviating one or more signs or symptoms associated with muscular dystrophy. In some examples, an increase in expression refers to an increase in a a7 gene product. An a7 gene product can be RNA (such as mRNA, rRNA, tRNA, and structural RNA) or protein.

[0103] Gene upregulation includes any detectable increase in the production of an a7 gene product. In certain examples, production of a a7 gene product increases by at least 2-fold, for example at least 3-fold or at least 4-fold, as compared to a control (such an amount of gene expression in a normal cell). In one example, a control is a relative amount of a7 gene expression or protein expression in a biological sample taken from a subject who does not have a muscle disease or disorder (e.g., muscular dystrophy, such as DMD, FCMD or MDC1 A).Such increases can be measured using the methods disclosed herein. For example, “detecting or measuring expression of a7” includes quantifying the amount of the gene, gene product or modulator thereof present in a sample. Quantification can be either numerical or relative. Detecting expression of the gene, gene product or modulators thereof can be achieved using any method known in the art or described herein, such as by measuring nucleic acids by PCR (such as RT-PCR) and proteins by ELISA. In primary embodiments, the change detected is an increase or decrease in expression as compared to a control, such as a reference value or a healthy control subject. In some examples, the detected increase or decrease is an increase ordecrease of at least two-fold compared with the control or standard. Controls or standards for comparison to a sample, for the determination of differential expression, include samples believed to be normal (in that they are not altered for the desired characteristic, for example a sample from a subject who does not have a muscle disease or disorder (e.g., muscular dystrophy, such as DMD, FCMD or MDC1 A)) as well as laboratory values (e.g., range of values), even though possibly arbitrarily set, keeping in mind that such values can vary from laboratory to laboratory.

[0104] Laboratory standards and values can be set based on a known or determined population value and can be supplied in the format of a graph or table that permits comparison of measured, experimentally determined values.

[0105] In other embodiments of the methods, the increase or decrease is of a diagnostically significant amount, which refers to a change of a sufficient magnitude to provide a statistical probability of the diagnosis.

[0106] The level of expression in either a qualitative or quantitative manner can detect nucleic acid or protein. Exemplary methods include microarray analysis, RT-PCR, Northern blot, Western blot, and mass spectrometry.

[0107] Inhibiting a disease or condition: A phrase referring to reducing the development of a disease or condition, for example, in a subject who is at risk for a disease or who has a particular disease. Particular methods of the present disclosure provide methods for inhibiting muscular dystrophy and / or muscle disorders.

[0108] Integrin: A cell surface transmembrane glycoprotein receptor. Integrins are involved in many biological processes such as wound healing, blood clot formation, gene regulation, and immune responses. Integrins can regulate tissue specific cell adhesion molecules. Integrins are heterodimeric non-covalently associated glycoproteins composed of two subunits. The subunits, which are designated a and [3, have approximate molecular weights of 150-180 kilodaltons and 90-110 kilodaltons, respectively.

[0109] The a7[31 integrin is a major laminin receptor expressed in skeletal muscle. The ct7[31 integrin plays a role in the development of neuromuscular and myotendinous junctions. In the adult, the a7pi integrin is concentrated at junctional sites and found in extrajunctional regions where it mediates the adhesion of the muscle fibers to the extracellular matrix.

[0110] The o7|31 integrin is important for muscle regeneration. For example, during embryonic development, the a7[31 integrin regulates myoblast migration to regions of myofiber formation. It has been found that MyoD (myogenic determination protein) transactivates a7 integrin gene expression in vitro, which would increase a7 integrin levels in activated satellitecells. Human, mouse and rat myoblast cell lines derived from satellite cells express high levels of a7 integrin. Elevated o7 integrin mRNA and protein are detected in the skeletal muscle of 5 week old mdx mice, which correlates with the period of maximum muscle degeneration and regeneration. In addition, the a7B1 integrin associates with muscle specific pi -integrin binding protein (MIBP), which regulates laminin deposition in C2C12 myoblasts. Laminin provides an environment that supports myoblast migration and proliferation. Finally, enhanced expression of the a7 integrin in dystrophic skeletal muscle results in increased numbers of satellite cells.

[0111] The sequences for a7|31 integrin subunits are publicly available on GenBank, see, for example Gene Accession No. NM 001 144116 (human) and NM 008398.2 (mouse) for a7 integrin, and Gene Accession No. NM 002211 for [31 integrin (also known as CD29), each of which is herein incorporated by reference as available on July 31 , 2023.

[0112] Laminin: Any of the family of glycoproteins that are typically involved in the formation and maintenance of extracellular matrices. Laminin is a heterotrimers formed from an a chain, a |3 chain, and a y chain. The various chains of a particular laminin can affect the properties of the molecule. In some aspects of the present disclosure, fragments, derivatives, or analogs of various laminins can be used, such as laminins having at least a portion at least substantially homologous to the laminin a1 chain. A “fragment of laminin,” as used herein, refers to a portion of a substance, such as laminin. A fragment may be, in some examples, a particular domain or chain of a protein. For example, particular embodiments of the present disclosure involve administering a fragment of laminin-1 corresponding to at least a portion of (or all of) the laminin ct 1 chain. Fragments may be synthetic or may be derived from larger parent substances.

[0113] In some aspects, laminins may be administered as a mixture of laminins, including fragments, analogs, and derivatives thereof. Suitable methods for preparing analogs of laminin domains are disclosed in U.S. Patent No. 6,933,280, incorporated by reference herein to the extent not inconsistent with this disclosure.

[0114] The laminin materials or compositions of the present disclosure may be delivered as discrete molecules or may be complexed with, or conjugated to, another substance. For example, the laminin may be combined with a carrier, such as to aid in delivery of the laminin to a site of interest or to increase physiological uptake or incorporation of the laminin.

[0115] In specific examples, the laminin administered includes or consists of laminin-1 (LAM- 1 11 ), which includes the chains a1 [31 y1 . In further examples, the laminin administered includes or consists of laminin-2, which includes the chains a2p 1 y 1 . In yet further examples, the laminin administered includes or consists of laminin-4, which includes the chains a2p2y1 .

[0116] Laminins may be obtained from any suitable source. For example, laminin-1 may be obtained from placental tissue or from Engelbreth-Holm-Swarm murine sarcoma. Suitable methods of isolating various laminins are disclosed in U.S. Patent No. 5,444,158, incorporated by reference herein to the extent not inconsistent with the present disclosure.

[0117] Modulatory Agent: Any molecule or compound that affects the expression of a gene or affects the amount of protein in a cell, tissue, or organ. For example, an a7 integrin modulatory agent can increase the expression of a7 and / or increase the amount of a7 integrin chain and ct7f31 integrin protein.

[0118] Muscle: Any myoblast, myocyte, myofiber, myotube or other structure composed of muscle cells. Muscles or myocytes can be skeletal, smooth, or cardiac. Muscle may also refer to, in particular implementations of the present disclosure, cells or other materials capable of forming myocytes, such as stem cells and satellite cells.

[0119] Muscle disorder: A problem with muscle cells or tissue including those caused by a muscular dystrophy. Muscle disorders also include muscle injury, weakness, atrophy, ageing (sarcopenia), impaired regeneration, impaired motor neuron activity or function, impaired cardiac muscle function under hypoxia, or any other condition leading to impaired muscle ability or integrity.

[0120] Muscle degeneration (or muscle atrophy): A decrease in size, mass, and / or strength of muscle tissue that can result in progressive weakness. Muscle degeneration can be caused by disease, poor nutrition, and / or a lack of physical activity due to injury, illness, and / or age. Muscle degeneration (whether size, mass or strength) can be determined relative to a control population (e.g., based on age and gender), or determined relative to a prior measurement made in the individual subject (e.g., from 1 month to 10 years earlier).

[0121] Muscular dystrophy: A term used to refer to a group of genetic disorders that lead to progressive muscle weakness. Muscular dystrophy can result in skeletal muscle weakness and defects in skeletal muscle proteins, leading to a variety of impaired physiological functions. No satisfactory treatment of muscular dystrophy exists. Existing treatments typically focus on ameliorating the effects of the disease and improving the patient’s quality of life, such as through physical therapy or through the provision of orthopedic devices.

[0122] Mutated genes associated with muscular dystrophy are responsible for encoding a number of proteins associated with the costameric protein network. Such proteins include laminin-2, collagen, dystroglycan, integrins, caveolin-3, ankyrin, dystrophin, a-dystrobrevin, vinculin, plectin, BPAGIb, muscle LIM protein, desmin, actinin-associated LIM protein, a-actin, titin, telethonin, cypher, myotilin, and the sarcoglycan / sarcospan complex. 1

[0123] The most common form of muscular dystrophy is DMD, affecting 1 in 3,500 live male births. DMD is an X-linked recessive disorder characterized by a mutation in the gene that codes for dystrophin. Dystrophin is a cytoskeletal protein about 430 kDa in size. This protein works to connect the cell’s cytoskeleton and extracellular matrix. The loss of dystrophin in DMD patients leads to a loss of muscle fiber attachment at the extracellular matrix during contraction, which ultimately leads to progressive fiber damage, membrane leakage and a loss of muscle function. Most patients die before they reach the age of 30 due to respiratory or cardiac failure.

[0124] Beckers muscular dystrophy (also known as Benign pseudohypertrophic muscular dystrophy) is related to DMD in that both result from a mutation in the dystrophin gene, but in DMD no functional dystrophin is produced making DMD much more severe than BMD. BMD is an X-linked recessive inherited disorder characterized by slowly progressive muscle weakness of the legs and pelvis. BMD is a type of dystrophinopathy, which includes a spectrum of muscle diseases in which there is insufficient dystrophin produced in the muscle cells, results in instability in the structure of muscle cell membrane. This is caused by mutations in the dystrophin gene, which encodes the protein dystrophin. The pattern of symptom development of BMD is similar to DMD, but with a later, and much slower rate of progression.

[0125] Congenital muscular dystrophies are caused by gene mutations. FCMD and MDC1 A are examples of congenital muscular dystrophies. MDC1 A is a congenital muscular dystrophy due to a genetic mutation in the LAMA2 gene which results in lack of or complete loss of Iaminin-a2 protein. This loss of Iaminin-a2 leads to an absence of laminins-211 / 221 . Laminins- 211 / 221 are major components of the extracellular matrix and play a key role in muscle cell development. During muscle cell differentiation laminin binds to the a7pi integrin. Without Iaminin-a2, muscle fibers are unable to adhere to the basement membrane and myotubes undergo apoptosis. Muscle regeneration also fails, leading to a loss of muscle repair and an increase in muscle fibrosis and inflammation. This chronic tissue injury is a major cause of morbidity and mortality in MDC1 A.

[0126] Congenital Muscular Dystrophies (CMD) and Limb-Girdle muscular dystrophy (LGMD) are common forms of highly heterogeneous muscular dystrophies which can be distinguished by their age at onset. In CMD, onset of symptoms is at birth or within the first 6 months of life; in LGMD onset of symptoms is in late childhood, adolescence or even adult life. Inheritance in LGMD can be autosomal dominant (LGMD type 1 ) or autosomal recessive (LGMD type 2), CMD is recessively inherited. CMD and LGMD can overlap both clinically and genetically

[0127] MDC1 A is a progressive muscle wasting disease that results in children being confined to a wheelchair, requiring ventilator assistance to breathe and premature death. Symptoms aredetected at birth with poor muscle tone and “floppy” baby syndrome. DMD, BMD and LGMD are progressive muscle degenerative diseases usually diagnosed at 3-5 years of age when children show developmental delay including ability to walk and climb stairs. The disease is progressive and children are usually confined to a wheelchair in their teens and require ventilator assistance.

[0128] Fukuyama congenital muscular dystrophy (FCMD) is an inherited condition that predominantly affects the muscles, brain, and eyes. Congenital muscular dystrophies are a group of genetic conditions that cause muscle weakness and wasting (atrophy) beginning very early in life. Fukuyama congenital muscular dystrophy affects the skeletal muscles, which are muscles the body uses for movement. The first signs of the disorder appear in early infancy and include a weak cry, poor feeding, and weak muscle tone (hypotonia). Weakness of the facial muscles often leads to a distinctive facial appearance including droopy eyelids (ptosis) and an open mouth. In childhood, muscle weakness and joint deformities (contractures) restrict movement and interfere with the development of motor skills such as sitting, standing, and walking. Fukuyama congenital muscular dystrophy also impairs brain development. People with this condition have a brain abnormality called cobblestone lissencephaly, in which the surface of the brain develops a bumpy, irregular appearance (like that of cobblestones). These changes in the structure of the brain lead to significantly delayed development of speech and motor skills and moderate to severe intellectual disability. Social skills are less severely impaired. Most children with Fukuyama congenital muscular dystrophy are never able to stand or walk, although some can sit without support and slide across the floor in a seated position. More than half of all affected children also experience seizures. Other signs and symptoms of Fukuyama congenital muscular dystrophy include impaired vision, other eye abnormalities, and slowly progressive heart problems after age 10. As the disease progresses, affected people may develop swallowing difficulties that can lead to a bacterial lung infection called aspiration pneumonia. Because of the serious medical problems associated with Fukuyama congenital muscular dystrophy, most people with the disorder live only into late childhood or adolescence.

[0129] Fukuyama congenital muscular dystrophy is seen almost exclusively in Japan, where it is the second most common form of childhood muscular dystrophy (after Duchenne muscular dystrophy). Fukuyama congenital muscular dystrophy has an estimated incidence of 2 to 4 per 100,000 Japanese infants.

[0130] Fukuyama congenital muscular dystrophy is caused by mutations in the FKTN gene which encodes fukutin. The most common mutation in the FKTN gene reduces the amount of fukutin produced within cells. A shortage of fukutin likely prevents the normal modification of a-dystroglycan, which disrupts that protein's normal function. Without functional a-dystroglycan to stabilize muscle cells, muscle fibers become damaged as they repeatedly contract and relax with use. The damaged fibers weaken and die over time, leading to progressive weakness and atrophy of the skeletal muscles.

[0131] Defective a-dystroglycan also affects the migration of neurons during the early development of the brain. Instead of stopping when they reach their intended destinations, some neurons migrate past the surface of the brain into the fluid-filled space that surrounds it. Because Fukuyama congenital muscular dystrophy involves a malfunction of a-dystroglycan, this condition is described as a dystroglycanopathy.

[0132] Facioscapulohumeral muscular dystrophy (FSHD) is a form of muscular dystrophy associated with progressive muscle weakness and loss of muscle tissue. Unlike DMD and BMD which mainly affect the lower body, FSHD affects the upper body mainly the face, shoulder and upper arm muscles. However, it can affect muscles around the pelvis, hips, and lower leg.Symptoms for FSHD often do not appear until age 10 - 26, but it is not uncommon for symptoms to appear much later. In some cases, symptoms never develop. Symptoms are usually mild and very slowly become worse. Facial muscle weakness is common, and may include eyelid drooping, inability to whistle, decreased facial expression, depressed or angry facial expression, difficulty pronouncing words, shoulder muscle weakness (leading to deformities such as pronounced shoulder blades (scapular winging) and sloping shoulders), weakness of the lower, hearing loss and possible heart conditions.

[0133] Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers (vehicles) useful in this disclosure are conventional. Remington’s Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 19th Edition (1995), describes compositions and formulations suitable for pharmaceutical delivery of one or more agents, such as one or more a7 modulatory agents.

[0134] In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations can include injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. In addition to biologically-neutral carriers, pharmaceutical agents to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate, sodium lactate, potassium chloride, calcium chloride, and triethanolamine oleate.

[0135] Sample (or Biological Sample): A biological specimen containing genomic DNA, RNA (including mRNA), protein, or combinations thereof, obtained from a subject. Examples include, but are not limited to, peripheral blood, urine, saliva, tissue biopsy, surgical specimen, and autopsy material. In one example, a sample includes muscle biopsy, such as from a subject with DMD, FCMD, or MDCI A.

[0136] Signs or symptoms: Any subjective evidence of disease or of a subject's condition, e.g., such evidence as perceived by the subject; a noticeable change in a subject's condition indicative of some bodily or mental state. A “sign” is any abnormality indicative of disease, discoverable on examination or assessment of a subject. A sign is generally an objective indication of disease. Signs include, but are not limited to any measurable parameters such as tests for detecting muscular dystrophy, including measuring creatine kinase levels, electromyography (to determine if weakness is caused by destruction of muscle tissue rather than by damage to nerves) or immunohistochemistry / immunoblotting / immunoassay (e.g., ELISA) to measure muscular dystrophy-associated molecules, such as a7 integrin. In one example, reducing or inhibiting one or more symptoms or signs associated with muscular dystrophy, includes increasing the activity or expression of a7 integrin by a desired amount, for example by at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100%, as compared to the activity and / or expression in the absence of the treatment. Symptoms of muscular dystrophy include, but are not limited to, muscle weakness and loss, difficulty running, difficulty hopping, difficulty jumping, difficulty walking, difficulty breathing, fatigue, skeletal deformities, muscle deformities (contractions of heels; pseudohypertrophy of calf muscles), heart disease (such as dilated cardiomyopathy), elevated creatine phosphokinase (CK) levels in blood or combinations thereof.

[0137] Subject: Living multi-cellular vertebrate organisms, a category that includes human and non-human mammals.

[0138] Tissue: An aggregate of cells, usually of a particular kind, together with their intercellular substance that form one of the structural materials of an animal and that in animals include connective tissue, epithelium, muscle tissue, and nerve tissue.

[0139] Treating a disease: A therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition related to a muscle disease or disorder (e.g., muscular dystrophy), such as a sign or symptom of a muscle disease or disorder (e.g., muscular dystrophy). Treatment can induce remission or cure of a condition or slow progression, for example, in some instances can include inhibiting the full development of a disease, forexample preventing development of a muscular dystrophy. Prevention of a disease does not require a total absence of disease. For example, a decrease of at least 50% can be sufficient.

[0140] Treating a disease can be a reduction in severity of some or all clinical symptoms of the disease or condition, a reduction in the number of relapses of the disease or condition, an improvement in the overall health or well-being of the subject, by other parameters well known in the art that are specific to the particular disease or condition, and combinations of such factors.

[0141] Under conditions sufficient for: A phrase that is used to describe any environment that permits the desired activity. In one example, includes administering a disclosed agent to a subject sufficient to allow the desired activity. In particular examples, the desired activity is increasing the expression or amount of a7 integrin.OverviewMolecular Pathway and Mechanism

[0142] The Hippo Pathway is a highly conserved developmental signaling pathway which regulates cell proliferation and apoptosis. Via phosphorylation cascades, the pathway functions by causing transcriptional activation or repression of target genes depending on cell signals. The pathway utilizes two pairs of core groups of kinases: Mst1 and Mst2 as one pair, and serine / threonine-protein kinases Latsl and Lats2 as the second pair. When the pathway is active, Mst1 and Mst2 become phosphorylated and subsequently phosphorylate Latsl and Lats2. Phosphorylated Latsl and Lats2 then phosphorylate the transcriptional activators Yap and Taz. When phosphorylated, Yap and Taz hinder cell division and proliferation. However, unphosphorylated Yap and Taz favor the activation of genes associated with cell proliferation and division.

[0143] Presently disclosed is the novel discovery that inhibition of Latsl and Lats2 promotes increased expression of a7 integrin. Methods of using Latsl and Lats2 inhibition to treat muscle diseases and disorders are additionally disclosed herein. By targeting the Latsl / 2 kinases to induce heightened expression of a7 integrin, muscle diseases, disorders, and dystrophies can be treated.

[0144] The novel methods disclosed herein can ameliorate the symptoms of and treat muscle aging (sarcopenia), non-use muscle atrophy (e.g., due to injury or otherwise), and can improve muscle regeneration, muscle mass, cardiac muscle function under hypoxia, motor neuron function and regeneration, and neuromuscular junction maintenance.Compounds for Treating Muscle Diseases or Disorders, including Muscular Dystrophy

[0145] Disclosed herein are compounds that may be used as an a7 integrin modulatory agents in methods disclosed herein. In particular disclosed embodiments, the compound is effective in treating muscular diseases or disorders, including muscular dystrophies. The compound is a small-molecule therapeutic that promotes increased expression of a7 integrin.

[0146] TRULI (Figure 2A):

[0149] VT02956 (Figure 2D):

[0151] T74 (Figure 2F):Methods of UseI. Methods of Treating Muscle Diseases or Disorders and Enhancing Muscle Regeneration

[0152] Disclosed herein are methods of enhancing muscle regeneration, repair or maintenance in a subject. In some examples, the method includes administering an effective amount of an a7 integrin modulatory agent to a subject in need of muscle regeneration, repair or maintenance, wherein the a7 integrin modulatory agent includes TRULI, TDI-011536, GA-017, VT02956, T65, T74, or a combination thereof, wherein the a7 integrin modulatory agent increases a7 integrin expression or activity as compared to a7 integrin expression or activity prior to treatment, thereby enhancing muscle regeneration, repair or maintenance in a subject.

[0153] The disclosed a7 integrin modulatory agents can increase the expression of a7 nucleic acid sequences and amount of a7 integrin protein by activating the a7 promoter. An increase in the expression or activity does not need to be 100% for the agent to be effective. For example, an agent can increase the expression or biological activity by a desired amount, for example by at least 5%, 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 98%, or even at least 100%, including about 15% to about 98%, about 30% to about 95%, about 40% to about 80%, about 50% to about 70%, including about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98% or about 100%, as compared to activity or expression in a control. Methods of assessing a7 integrin expression and activity are known to those of ordinary skill in the art, including those described in the Examples below (e.g., Western blot and ELISA assay with commercially available antibodies).

[0154] Muscle regeneration may benefit, for example, geriatric or other patient populations with reduced muscle repair capability, or simply speed the muscle repair process for otherwise physiologically unimpaired patients. In particular implementations, administration of a a7 integrin modulatory agent can aid muscle repair, or reduction of muscle damage, in athletes or others having activity-induced muscle injury or damage. In yet further implementations, muscle repair in patients suffering from muscle damage, such as through accident or injury, can be augmented by administration of a a7 integrin modulatory agent.

[0155] In some examples, a7 integrin modulatory agent is administered prior to the subject experiencing muscle damage or disease. In some examples, the a7 integrin modulatory agent is administered to the subject prior to the subject exercising.

[0156] In some examples, the method further includes selecting a subject in need of enhancing muscle regeneration, repair, or maintenance. For example, in some instances, selecting a subject in need of enhancing muscle regeneration, repair, or maintenance comprises diagnosing the subject with a condition characterized by impaired muscle regeneration prior to administering an effective amount of the a7 integrin modulatory agent to the subject. Methods for diagnosing and selecting a subject in need of muscle regeneration, repair or maintenance are known to those of ordinary skill in the art and include those provided described herein (including those in the Methods of Treatment of Muscular Dystrophy). As stated above, subjects may be selected based upon their life style (e.g., engaged in moderate to intense exercise or physical activities), age (e.g., elderly population at more risk of experiencing muscle degeneration or injury) or pre-disposition to muscle degeneration or injury (e.g., genetics or previous muscle injury).II. Methods of Treating Muscular Dystrophy

[0157] The a7 integrin has been shown to be a major modifier of disease progression in patients with muscular dystrophy. Increased expression of the a7 integrin in muscle can alleviate muscle disease in mouse models of muscular dystrophy. By use of a muscle cellbased assay (described in Example 1 below), the inventors identified the following molecules that up-regulate a7 integrin expression in muscle: TRULI, TDI-011536, GA-017, and VT02956 (see Figures 2A-2D, 3 and 4). Based on these observations, methods of treatment of muscular dystrophy by increasing the expression or activity of a7 integrin are disclosed.

[0158] In particular, methods are disclosed herein for treating muscular dystrophy, such as DMD, FCMD, LGMD, FSHD, BMD, MDC1 A or MDC1 D. In one example, the method includes administering an effective amount of an a7 integrin modulatory agent to a subject with muscular dystrophy or suspected of having or developing muscular dystrophy, in which the agent increases the biological activity or expression of a7 integrin and thereby, treating the muscular dystrophy in the subject. In some example, the method of treatment inhibits or reduces one or more signs or symptoms associated with muscular dystrophy in the subject.

[0159] The disclosed a7 integrin modulatory agents can increase the expression of a7 integrin nucleic acid sequences and amount of a7pi integrin protein by activating the a7 promoter. An increase in the expression or activity does not need to be 100% for the agent to be effective. For example, an agent can increase the expression or biological activity by a desired amount, for example by at least 5%, 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 least95%, at least 98%, or even at least 100%, including about 15% to about 98%, about 30% to about 95%, about 40% to about 80%, about 50% to about 70%, including about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98% or about 100%, as compared to activity or expression in a control. Methods of assessing a7 integrin expression and activity are known to those of ordinary skill in the art, including those described in the Examples below (e.g., Western blot and ELISA assay with commercially available antibodies).

[0160] In a particular example, the subject is a human.

[0161] In additional aspects, the method involves selecting a subject with muscular dystrophy. In some example, a subject is selected for treatment following diagnosing the subject with muscular dystrophy. For example, the method can include diagnosing the subject as suffering from muscular dystrophy, such as DMD, MDC1A, MDC1 D, LGMD, DMD, FCMD or FSHD.

[0162] Methods of diagnosing a subject with muscular dystrophy are known to those of skill in the art and include, but are not limited to, muscle biopsies and measuring serum creatine kinase levels. Additionally, alterations in biomarker known to be associated with muscular dystrophy may be detected by measuring such levels in serum or urine sample.

[0163] In a further implementation, the method involves diagnosing the subject as suffering from a disease, disorder, or condition characterized by a mutation in the gene encoding a7 integrin. In another implementation, the method involves diagnosing the subject as suffering from a disease, disorder, or condition characterized by a decreased level of a7 integrin expression.

[0164] Alterations in the expression can be measured at the nucleic acid level (such as by real time quantitative polymerase chain reaction or microarray analysis) or at the protein level (such as by Western blot analysis or ELISA). These methods are known to those of skill in the art.

[0165] In some examples, following the measurement of the expression levels of a7 integrin expression or serum creatine kinase levels, the assay results, findings, diagnoses, predictions and / or treatment recommendations are recorded and communicated to technicians, physicians and / or patients, for example. In certain embodiments, computers are used to communicate such information to interested parties, such as, patients and / or the attending physicians. The therapy selected for administered is then based upon these results.

[0166] In one embodiment, the results and / or related information is communicated to the subject by the subject's treating physician. Alternatively, the results may be communicated directly to a test subject by any means of communication, including writing, such as by providingT1a written report, electronic forms of communication, such as email, or telephone. Communication may be facilitated by use of a computer, such as in case of email communications. In certain embodiments, the communication containing results of a diagnostic test and / or conclusions drawn from and / or treatment recommendations based on the test, may be generated and delivered automatically to the subject using a combination of computer hardware and software which will be familiar to artisans skilled in telecommunications. One example of a healthcare-oriented communications system is described in U.S. Pat. No.6,283,761 ; however, the present disclosure is not limited to methods which utilize this particular communications system. In certain embodiments of the methods of the disclosure, all or some of the method steps, including the assaying of samples, diagnosing of diseases, and communicating of assay results or diagnoses, may be carried out in diverse e.g., foreign) jurisdictions.

[0167] In several embodiments, identification of a subject as having muscular dystrophy, such as DMD, LGMD, FSHD, BMD, FCMD, MDC1 D or MDC1 A, results in the physician treating the subject, such as prescribing one or more disclosed o7 integrin modulatory agents for inhibiting or delaying one or more signs and symptoms associated with muscular dystrophy. In additional embodiments, the dose or dosing regimen is modified based on the information obtained using the methods disclosed herein.Administration of an Effective Amount of an a7 Integrin Modulatory Agent

[0168] For any of the disclosed methods, an effective amount of a7 integrin modulatory agent is one when administered by a particular route and concentration induces the desired response (e.g., treatment of muscular dystrophy, enhancing muscle regeneration, repair or maintenance, preventing or reducing muscle injury or damage, or enhancing a7 integrin expression).I. Administration Routes, Formulations and Concentrations

[0169] Methods of administration of the disclosed a7 integrin modulatory agents are routine, and can be determined by a skilled clinician. The disclosed a7 integrin modulatory agents or other therapeutic substance are in general administered topically, nasally, intravenously, orally, intracranially, intramuscularly, parenterally or as implants, but even rectal or vaginal use is possible in principle. The disclosed a7 integrin modulatory agents also may be administered to a subject using a combination of these techniques.

[0170] Suitable solid or liquid pharmaceutical preparation forms are, for example, aerosols, (micro)capsules, creams, drops, drops or injectable solution in ampoule form, emulsions, granules, powders, suppositories, suspensions, syrups, tablets, coated tablets, and alsopreparations with protracted release of active compounds, in whose preparation excipients and additives and / or auxiliaries such as binders, coating agents, disintegrants, flavorings, lubricants, solubilizers, sweeteners, or swelling agents are customarily used as described above. The pharmaceutical agents are suitable for use in a variety of drug delivery systems. For a brief review of various methods for drug delivery, see Langer, “New Methods of Drug Delivery,” Science 249:1527-1533 (1990), incorporated by reference herein to the extent not inconsistent with the present disclosure.

[0171] The disclosed a7 integrin modulatory agents or other therapeutic agents of the present disclosure can be formulated into therapeutically-active pharmaceutical agents that can be administered to a subject parenterally or orally. Parenteral administration routes include, but are not limited to epidermal, intraarterial, intramuscular (IM and depot IM), intraperitoneal (IP), intravenous (IV), intrasternal injection or infusion techniques, intranasal (inhalation), intrathecal, injection into the stomach, subcutaneous injections (subcutaneous (SQ and depot SQ), transdermal, topical, and ophthalmic.

[0172] The disclosed a7 integrin modulatory agents or other therapeutic agents can be mixed or combined with a suitable pharmaceutically acceptable excipients to prepare pharmaceutical agents. Pharmaceutically acceptable excipients include, but are not limited to, alumina, aluminum stearate, buffers (such as phosphates), glycine, ion exchangers (such as to help control release of charged substances), lecithin, partial glyceride mixtures of saturated vegetable fatty acids, potassium sorbate, serum proteins (such as human serum albumin), sorbic acid, water, salts or electrolytes such as cellulose-based substances, colloidal silica, disodium hydrogen phosphate, magnesium trisilicate, polyacrylates, polyalkylene glycols, such as polyethylene glycol, polyethylene-polyoxypropylene-block polymers, polyvinyl pyrrolidone, potassium hydrogen phosphate, protamine sulfate, group 1 halide salts such as sodium chloride, sodium carboxymethylcellulose, waxes, wool fat, and zinc salts, for example. Liposomal suspensions may also be suitable as pharmaceutically acceptable carriers.

[0173] Upon mixing or addition of one or more disclosed a7 integrin modulatory agents and / or other therapeutic agents, the resulting mixture may be a solid, solution, suspension, emulsion, or the like. These may be prepared according to methods known to those of ordinary skill in the art. The form of the resulting mixture depends upon a number of factors, including the intended mode of administration and the solubility of the agent in the selected carrier.

[0174] Pharmaceutical carriers suitable for administration of the disclosed a7 integrin modulatory agents or other therapeutic agents include any such carriers known to be suitable for the particular mode of administration. In addition, the disclosed a7 integrin modulatoryagents or other therapeutic substance can also be mixed with other inactive or active materials that do not impair the desired action, or with materials that supplement the desired action, or have another action.

[0175] Methods for solubilizing may be used where the agents exhibit insufficient solubility in a carrier. Such methods are known and include, but are not limited to, dissolution in aqueous sodium bicarbonate, using cosolvents such as dimethylsulfoxide (DMSO), and using surfactants such as TWEEN® (ICI Americas, Inc., Wilmington, DE).

[0176] The disclosed a7 integrin modulatory agents or other therapeutic agents can be prepared with carriers that protect them against rapid elimination from the body, such as coatings or time-release formulations. Such carriers include controlled release formulations, such as, but not limited to, microencapsulated delivery systems. A disclosed a7 integrin modulatory agents or other therapeutic agent is included in the pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect, typically in an amount to avoid undesired side effects, on the treated subject. The therapeutically effective concentration may be determined empirically by testing the compounds in known in vitro and in vivo model systems for the treated condition. For example, mouse models of muscular dystrophy may be used to determine effective amounts or concentrations that can then be translated to other subjects, such as humans, as known in the art.

[0177] Injectable solutions or suspensions can be formulated, using suitable non-toxic, parenterally-acceptable diluents or solvents, such as 1 ,3-butanediol, isotonic sodium chloride solution, mannitol, Ringer’s solution, saline solution, or water; or suitable dispersing or wetting and suspending agents, such as sterile, bland, fixed oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid; a naturally occurring vegetable oil such as coconut oil, cottonseed oil, peanut oil, sesame oil, and the like; glycerine; polyethylene glycol; propylene glycol; or other synthetic solvent; antimicrobial agents such as benzyl alcohol and methyl parabens; antioxidants such as ascorbic acid and sodium bisulfite; buffers such as acetates, citrates, and phosphates; chelating agents such as ethylenediaminetetraacetic acid (EDTA); agents for the adjustment of tonicity such as sodium chloride and dextrose; and combinations thereof. Parenteral preparations can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass, plastic, or other suitable material. Buffers, preservatives, antioxidants, and the like can be incorporated as required. Where administered intravenously, suitable carriers include physiological saline, phosphate-buffered saline (PBS), and solutions containing thickening and solubilizing agents such as glucose, polyethyleneglycol, polypropyleneglycol, and mixtures thereof. Liposomal suspensions, including tissue- targeted liposomes, may also be suitable as pharmaceutically acceptable carriers.

[0178] For topical application, one or more disclosed a7 integrin modulatory agents, or other therapeutic agent may be made up into a cream, lotion, ointment, solution, or suspension in a suitable aqueous or non-aqueous carrier. Topical application can also be accomplished by transdermal patches or bandages which include the therapeutic substance. Additives can also be included, e.g., buffers such as sodium metabisulphite or disodium edetate; preservatives such as bactericidal and fungicidal agents, including phenyl mercuric acetate or nitrate, benzalkonium chloride, or chlorhexidine; and thickening agents, such as hypromellose.

[0179] If the disclosed a7 integrin modulatory agent, or other therapeutic agent is administered orally as a suspension, the pharmaceutical agents can be prepared according to techniques well known in the art of pharmaceutical formulation and may contain a suspending agent, such as alginic acid or sodium alginate, bulking agent, such as microcrystalline cellulose, a viscosity enhancer, such as methylcellulose, and sweeteners / flavoring agents. Oral liquid preparations can contain conventional additives such as suspending agents, e.g., gelatin, glucose syrup, hydrogenated edible fats, methyl cellulose, sorbitol, and syrup; emulsifying agents, e.g., acacia, lecithin, or sorbitan monooleate; non-aqueous carriers (including edible oils), e.g., almond oil, fractionated coconut oil, oily esters such as glycerine, propylene glycol, or ethyl alcohol; preservatives such as methyl or propyl p-hydroxybenzoate or sorbic acid; and, if desired, conventional flavoring or coloring agents. When formulated as immediate release tablets, these agents can contain dicalcium phosphate, lactose, magnesium stearate, microcrystalline cellulose, and starch and / or other binders, diluents, disintegrants, excipients, extenders, and lubricants.

[0180] If oral administration is desired, one or more disclosed o7 integrin modulatory agents, or other therapeutic substances can be provided in a composition that protects it from the acidic environment of the stomach. For example, he disclosed a7 integrin modulatory agents or other therapeutic agents can be formulated with an enteric coating that maintains its integrity in the stomach and releases the active compound in the intestine. The disclosed a7 integrin modulatory agents, or other therapeutic agent can also be formulated in combination with an antacid or other such ingredient.

[0181] Oral compositions generally include an inert diluent or an edible carrier and can be compressed into tablets or enclosed in gelatin capsules. For the purpose of oral therapeutic administration, one or more of the disclosed a7 integrin modulatory agents, or other therapeutic substances can be incorporated with excipients and used in the form of capsules, tablets, ortroches. Pharmaceutically compatible adjuvant materials or binding agents can be included as part of the composition.

[0182] The capsules, pills, tablets, troches, and the like can contain any of the following ingredients or compounds of a similar nature: a binder such as, but not limited to, acacia, corn starch, gelatin, gum tragacanth, polyvinylpyrrolidone, or sorbitol; a filler such as calcium phosphate, glycine, lactose, microcrystalline cellulose, or starch; a disintegrating agent such as, but not limited to, alginic acid and corn starch; a lubricant such as, but not limited to, magnesium stearate, polyethylene glycol, silica, or talc; a gildant, such as, but not limited to, colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; disintegrants such as potato starch; dispersing or wetting agents such as sodium lauryl sulfate; and a flavoring agent such as peppermint, methyl salicylate, or fruit flavoring.

[0183] When the dosage unit form is a capsule, it can contain, in addition to material of the above type, a liquid carrier, such as a fatty oil. In addition, dosage unit forms can contain various other materials that modify the physical form of the dosage unit, for example, coatings of sugar and other enteric agents. One or more of the disclosed a7 integrin modulatory agents, or other therapeutic agent can also be administered as a component of an elixir, suspension, syrup, wafer, tea, chewing gum, or the like. A syrup may contain, in addition to the active compounds, sucrose or glycerin as a sweetening agent and certain preservatives, dyes and colorings, and flavors.

[0184] When administered orally, the compounds can be administered in usual dosage forms for oral administration. These dosage forms include the usual solid unit dosage forms of tablets and capsules as well as liquid dosage forms such as solutions, suspensions, and elixirs. When the solid dosage forms are used, they can be of the sustained release type so that the compounds need to be administered less frequently.

[0185] In some examples, one or more of the disclosed a7 integrin modulatory agents and / or a therapeutic agent is injected into the stomach of a subject is incorporated systemically in the subject, such as in diverse muscle groups. Examples of methods and compositions for administering therapeutic substances which include proteins include those discussed in Banga, Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems 2ed.(2005); Mahato, Biomaterials for Delivery and Targeting of Proteins and Nucleic Acids (2004); McNally, Protein Formulation and Delivery, 2ed. (2007); and Kumar et aL, “Novel Delivery Technologies for Protein and Peptide Therapeutics,” Current Pharm. Biotech., 7:261 -276 (2006); each of which is incorporated by reference herein to the extent not inconsistent with the present disclosure.

[0186] In some implementations, the effective amount of one or more of the disclosed a7 integrin modulatory agents is administered as a single dose per time period, such as every three or four months, month, week, or day, or it can be divided into at least two unit dosages for administration over a period. Treatment may be continued as long as necessary to achieve the desired results. For instance, treatment may continue for about 3 or 4 weeks up to about 12-24 months or longer, including ongoing treatment. The compound can also be administered in several doses intermittently, such as every few days (for example, at least about every two, three, four, five, or ten days) or every few weeks (for example at least about every two, three, four, five, or ten weeks).

[0187] Particular dosage regimens can be tailored to a particular subject, condition to be treated, or desired result. For example, when the methods of the present disclosure are used to treat muscular dystrophy or similar conditions, an initial treatment regimen can be applied to arrest the condition. Such initial treatment regimen may include administering a higher dosage of one or more of the disclosed a7 integrin modulatory agents, or administering such material more frequently, such as daily. After a desired therapeutic result has been obtained, such as a desired level of muscle regeneration, a second treatment regimen may be applied, such as administering a lower dosage of one or more of the disclosed a7 integrin modulatory agents or administering such material less frequently, such as monthly, bi-monthly, quarterly, or semiannually. In such cases, the second regimen may serve as a “booster” to restore or maintain a desired level of muscle regeneration. Similar treatment regimens may be used for other subjects with reduced or impaired muscle regeneration capabilities, such as geriatric subjects.

[0188] When particular methods of the present disclosure are used to prevent or mitigate muscle damage, such as damage caused by exertion or injury, the subject is typically treated a sufficient period of time before the exertion or injury in order to provide therapeutic effect. For example, the subject may be treated at least about 24 hours before the expected activity or potential injury, such as at least about 48 hours, about 72 hours, about 1 week, about 2 weeks, about three weeks, or about 4 weeks or more prior.

[0189] When embodiments of the method of the present disclosure are used to prevent or treat a muscle injury, one or more of the disclosed a7 integrin modulatory agents or other therapeutic substance can be applied directly to, or proximately to, the area to be treated. For example, the substance can be injected into or near the area. In further examples, the substance can be applied topically to the area to be treated. Treatment is typically initiated prior to the injury to several weeks following the injury. In more specific implementations, the treatment is initiated between about 12 and about 72 hours following injury, such as betweenabout 24 and about 48 hours following injury. In some cases, a single administration of the substance is effective to provide the desired therapeutic effect. In further examples, additional administrations are provided in order to achieve the desired therapeutic effect.

[0190] Amounts effective for various therapeutic treatments of the present disclosure may, of course, depend on the severity of the disease and the weight and general state of the subject, as well as the absorption, inactivation, and excretion rates of the therapeutically-active compound or component, the dosage schedule, and amount administered, as well as other factors known to those of ordinary skill in the art. It also should be apparent to one of ordinary skill in the art that the exact dosage and frequency of administration will depend on the particular a7 integrin modulatory agent, or other therapeutic substance being administered, the particular condition being treated, the severity of the condition being treated, the age, weight, general physical condition of the particular subject, and other medication the subject may be taking. Typically, dosages used in vitro may provide useful guidance in the amounts useful for in vivo administration of the pharmaceutical composition, and animal models may be used to determine effective dosages for treatment of particular disorders. For example, mouse models of muscular dystrophy may be used to determine effective dosages that can then be translated to dosage amount for other subjects, such as humans, as known in the art. Various considerations in dosage determination are described, e.g., in Gilman et al., eds., Goodman And Gilman's: The Pharmacological Bases of Therapeutics, 8th ed., Pergamon Press (1990); and Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Co., Easton, Pa. (1990), each of which is herein incorporated by reference to the extent not inconsistent with the present disclosure.

[0191] In specific examples, the one or more disclosed a7 integrin modulatory agents is administered to a subject in an amount sufficient to provide a dose of the agent of between about 10 fmol / g and about 500 nmol / g, such as between about 2 nmol / g and about 20 nmol / g or between about 2 nmol / g and about 10 nmol / g. In additional examples, the a7 integrin modulatory agent is administered to a subject in an amount sufficient to provide a dose of between about 0.01 pg / kg and about 1000 mg / kg or between about 0.1 mg / kg and about 1000 mg / kg, in particular examples this amount is provided per day or per week. In another example, the disclosed a7 integrin modulatory agent is administered to a subject in an amount sufficient to provide a dose of agent of between about 0.2 mg / kg and about 2 mg / kg. In further examples, the a7 integrin modulatory agent is administered to a subject in an amount sufficient to provide a concentration of a7 integrin modulatory agent in the administrated material of between about 5 nM and about 500 nM, such as between about 50 nM and about 200 nm, or about 100 nM. Inother examples, the a7 integrin modulatory agent is administered to a subject between about 500 pg / ml and about 1 pg / ml, such as about 300 pg / ml and about 3 pg / ml, about 200 pg / ml and about 20 pg / ml, including 500 pg / ml, 400 pg / ml, 300 pg / ml, 250 pg / ml, 200 pg / ml, 150 pg / ml, 100 pg / ml, 50 pg / ml, 25 pg / ml, 12.5 pg / ml, 6.25 pg / ml, 3.125 pg / ml, 2.5 pg / ml and 1.25 pg / ml.II. Desired Response

[0192] One or more disclosed a7 integrin modulatory agents and / or additional therapeutic agents are administered by a specific route and / or concentration to generate a desired response. In some examples, a desired response refers to an amount effective for lessening, ameliorating, eliminating, preventing, or inhibiting at least one symptom of a disease, disorder, or condition treated and may be empirically determined. In various embodiments of the present disclosure, a desired response is muscle regeneration, reductions or prevention of muscle degeneration, promotion of muscle maintenance, reduction or prevention of muscle injury or damage, reduction or prevention in one more signs or symptoms associated with muscular dystrophy.

[0193] In particular, indicators of muscular health, such as muscle cell regeneration, maintenance, or repair, can be assessed through various means, including monitoring markers of muscle regeneration, such as transcription factors such as Pax7, Pax3, MyoD, MRF4, and myogenin. For example, increased expression of such markers can indicate that muscle regeneration is occurring or has recently occurred. Markers of muscle regeneration, such as expression of embryonic myosin heavy chain (eMyHC), can also be used to gauge the extent of muscle regeneration, maintenance, or repair. For example, the presence of eMyHC can indicate that muscle regeneration has recently occurred in a subject.

[0194] Muscle cell regeneration, maintenance, or repair can also be monitored by determining the girth, or mean cross sectional area, of muscle cells or density of muscle fibers. Additional indicators of muscle condition include muscle weight and muscle protein content. Mitotic index (such as by measuring BrdU incorporation) and myogenesis can also be used to evaluate the extent of muscle regeneration.

[0195] In particular examples, the improvement in muscle condition, such as regeneration, compared with a control is at least about 10%, such as at least about 30%, or at least about 50% or more, including an at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, including a 10% to 90% decrease, 20% to 80% increase, 30% to 70% increase or a 40% to 60% increase (e.g., a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 200% or more increase).III. Additional Treatments or Therapeutic Agents

[0196] In particular examples, prior to, during, or following administration of an effective amount of an agent that reduces or inhibits one or more signs or symptoms associated with muscular dystrophy, the subject can receive one or more other therapies. In one example, the subject receives one or more treatments prior to administration of a disclosed a7 modulatory agent. Examples of such therapies include, but are not limited to, laminin-1 11 protein therapy, which works to stabilize the sarcolemma and reduce muscle degeneration. In some examples, a source of muscle cells can be added to aid in muscle regeneration and repair. In some aspects of the present disclosure, satellite cells are administered to a subject in combination with laminin therapy. U.S. Patent Publication 2006 / 0014287, incorporated by reference herein to the extent not inconsistent with the present disclosure, provides methods of enriching a collection of cells in myogenic cells and administering those cells to a subject. In further aspects, stem cells, such as adipose-derived stem cells, are administered to the subject. Suitable methods of preparing and administering adipose-derived stem cells are disclosed in U.S. Patent Publication 2007 / 0025972, incorporated by reference herein to the extent not inconsistent with the present disclosure. Additional cellular materials, such as fibroblasts, can also be administered, in some examples.

[0197] Additional therapeutic agents include agents which enhance the effect of the disclosed a7[31 modulatory agents, such as a component of the extracellular matrix, such as an integrin, dystrophin, dystroglycan, utrophin, or a growth factor. In some examples, the additional therapeutic agent reduces or enhances expression of a substance that enhances the formation or maintenance of the extracellular matrix. In some examples, the additional substance can include aggrecan, angiostatin, cadherins, collagens (including collagen I, collagen III, or collagen IV), decorin, elastin, enactin, endostatin, fibrin, fibronectin, osteopontin, tenascin, thrombospondin, vitronectin, and combinations thereof. Biglycans, glycosaminoglycans (such as heparin), glycoproteins (such as dystroglycan), proteoglycans (such as heparan sulfate), and combinations thereof can also be administered.

[0198] In some examples, growth stimulants such as cytokines, polypeptides, and growth factors such as brain-derived neurotrophic factor (BDNF), CNF (ciliary neurotrophic factor), EGF (epidermal growth factor), FGF (fibroblast growth factor), glial growth factor (GGF), glial maturation factor (GMF) glial-derived neurotrophic factor (GDNF), hepatocyte growth factor (HGF), insulin, insulin-like growth factors, kerotinocyte growth factor (KGF), nerve growth factor (NGF), neurotropin-3 and -4, PDGF (platelet-derived growth factor), vascular endothelial growthfactor (VEGF), and combinations thereof may be administered with one of the disclosed methods.Clinical Trials

[0199] To obtain regulatory approval for the use of one or more of the disclosed a7 modulatory agents to treat a muscular disorder, clinical trials are performed. As is known in the art, clinical trials progress through phases of testing, which are identified as Phases I, II, III, and IV.

[0200] Initially, the disclosed a7 modulatory agents are evaluated in a Phase I trial. Typically Phase I trials are used to determine the best mode of administration (for example, by pill or by injection), the frequency of administration, and the toxicity for the compounds. Phase I studies frequently include laboratory tests, such as blood tests and biopsies, to evaluate the effects of the potential therapeutic in the body of the patient. For a Phase I trial, a small group of patients with a muscular disorder are treated with a specific dose of a disclosed a7|31 modulatory agent. During the trial, the dose is typically increased group by group in order to determine the maximum tolerated dose (MTD) and the dose-limiting toxicities (DLT) associated with the compound. This process determines an appropriate dose to use in a subsequent Phase II trial.

[0201] A Phase II trial can be conducted to further evaluate the effectiveness and safety of the disclosed a7 modulatory agents. In Phase II trials, a disclosed a7 modulatory agent is administered to groups of patients with a muscular disorder using the dosage found to be effective in Phase I trials.

[0202] Phase III trials focus on determining how a disclosed a7 modulatory agent compares to the standard, or most widely accepted, treatment. In Phase III trials, patients are randomly assigned to one of two or more “arms”. In a trial with two arms, for example, one arm will receive the standard treatment (control group) and the other arm will receive a disclosed a7 modulatory agent treatment (investigational group).

[0203] Phase IV trials are used to further evaluate the long-term safety and effectiveness of a disclosed a7|31 modulatory agent. Phase IV trials are less common than Phase I, II and III trials and take place after a disclosed a7 modulatory agent has been approved for standard use.I. Eligibility of Patients for Clinical Trials

[0204] Participant eligibility criteria can range from general (for example, age, sex, type of disease) to specific (for example, type and number of prior treatments, disease characteristics, blood cell counts, organ function). In one embodiment, eligible patients have been diagnosed with a muscular disorder. Eligibility criteria may also vary with trial phase. Patients eligible forclinical trials can also be chosen based on objective measurement of a muscular disorder and failure to respond to other muscular disorder treatments. For example, in Phase I and II trials, the criteria often exclude patients who may be at risk from the investigational treatment because of abnormal organ function or other factors. In Phase II and III trials additional criteria are often included regarding disease type and stage, and number and type of prior treatments.

[0205] Phase I trials usually include 15 to 30 participants for whom other treatment options have not been effective. Phase II trials typically include up to 100 participants who have already received drug therapy, but for whom the treatment has not been effective.

[0206] Participation in Phase III trials is often restricted based on the previous treatment received. Phase III trials usually include hundreds to thousands of participants. This large number of participants is necessary in order to determine whether there are true differences between the effectiveness of a disclosed a7 modulatory agent and the standard treatment. Phase III can include patients ranging from those newly diagnosed with a muscular disorder to those with re-occurring signs and / or symptoms associated with a muscular disorder or a muscular disorder that did not respond to prior treatment.

[0207] One skilled in the art will appreciate that clinical trials should be designed to be as inclusive as possible without making the study population too diverse to determine whether the treatment might be as effective on a more narrowly defined population. The more diverse the population included in the trial, the more applicable the results could be to the general population, particularly in Phase III trials. Selection of appropriate participants in each phase of clinical trial is considered to be within the ordinary skills of a worker in the art.II. Assessment of Patients Prior to Treatment

[0208] Prior to commencement of the study, several measures known in the art can be used to first classify the patients. Patients can first be assessed, for example by determining serum creatine kinase (CK) levels or other indicators of a muscle disorder, such as increased levels of muscle inflammation, apoptosis, muscle loss, myotube hypertrophy, and / or decreased myofibers stability and cell survival.III. Administration of a Disclosed a7 Modulatory Agent in Clinical Trials

[0209] A disclosed a7 modulatory agent is typically administered to the trial participants orally. A range of doses of the agent can be tested. Provided with information from preclinical testing, a skilled practitioner can readily determine appropriate dosages of agent for use in clinical trials. In one embodiment, a dose range is from about 100 pg / kg and about 5000 mg / kg of the subject’s weight, such as1 mg / kg and about 2000 mg / kg of the subject’s weight, about 100 mg / kg and about 1500 mg / kg of the subject’s weight, about 100 pg / kg and about 2000 mg / kg ofthe subject’s weight, about 200 mg / kg and about 1000 mg / kg of the subject’s weight, about 200 mg / kg and about 750 mg / kg of the subject’s weight, about 250 mg / kg and about 500 mg / kg of the subject’s weight, about 100 pm and about 500 mM. In some embodiments, subjects are given a disclosed a7 modulatory agent orally at 10 to 60 mg / kg of body weight per day. For example, 10-15 mg / kg of a disclosed a7 modulatory agent is administered for two weeks and if well tolerated the dose is increased by 5-10 mg / kg / week to achieve optimal clinical response. In some examples, the daily dose does not exceed 60 mg / kg of body weight and is given for a minimum of 6 months with liver function monitored every two weeks to monthly.IV. Pharmacokinetic Monitoring

[0210] To fulfill Phase I criteria, distribution of the disclosed a7 modulatory agent is monitored, for example, by chemical analysis of samples, such as blood, collected at regular intervals. For example, samples can be taken at regular intervals up until about 72 hours after the start of treatment.

[0211] If analysis is not conducted immediately, the samples can be placed on dry ice after collection and subsequently transported to a freezer to be stored at -70 °C until analysis can be conducted. Samples can be prepared for analysis using standard techniques known in the art and the amount of the disclosed a7 modulatory agent present can be determined, for example, by high-performance liquid chromatography (HPLC). Pharmacokinetic data can be generated and analyzed in collaboration with an expert clinical pharmacologist and used to determine, for example, clearance, half-life and maximum plasma concentration.V. Monitoring of Patient Outcome

[0212] The endpoint of a clinical trial is a measurable outcome that indicates the effectiveness of a compound under evaluation. The endpoint is established prior to the commencement of the trial and will vary depending on the type and phase of the clinical trial. Examples of endpoints include, for example, decline in serum CK levels, inflammation, apoptosis, and muscle loss. For example, at least a 10% reduction in serum CK levels indicates the patient is responsive to the treatment.

[0213] The following examples are provided to illustrate certain particular features and / or embodiments. These examples should not be construed to limit the invention to the particular features or embodiments described.EXAMPLESExample 1LacZ reporter gene in a7Pgal+Amuscle cells reports the transcriptional activity of the a7 integrin promoter

[0214] Objective: This Example shows that the LacZ reporter gene in a7Pgal+Amuscle cells faithfully reports the transcriptional activity of the a7 integrin promoter and can be used screen for a7 modulatory agents.

[0215] Methods: An a7 integrin null mouse was produced in which exon 1 of the gene encoding the a7 integrin was replaced by the LacZ reporter. In these mice, all the transcriptional regulatory elements of the a7 integrin promoter were retained, allowing p - galactosidase to report expression of 7 integrin. A primary myoblast cell line (designated a7pgal+A) isolated from 10 day old a7+Apups were analyzed for the ability of p -galactosidase to report o7 integrin expression. o7Pgal+Amyoblasts were differentiated and subjected to X-gal staining and western analysis (Figures. 1 A and 1 B).

[0216] Results: These results demonstrate that p-galactosidase expression in a7Pgal+Amuscle cells increased upon myogenic differentiation consistent with the expression pattern of a7 integrin in myoblasts and myotubes. These results confirm that the LacZ reporter gene in a7Pgal+Amuscle cells faithfully reports the transcriptional activity of the a7 integrin promoter. The activity of the a7 integrin promoter was measured by p-galactosidase cleavage of the non- fluorescent compound fluorescein di- p-D-galactopyranoside (FDG) to fluorescein. This assay was used to screen compound libraries to identify molecules that up-regulate a7 integrin expression in muscle.Example 2Activity of TRULI, TDI-011536, TDI-011536, S969, SU9516 and PY-60 in enhancing expression of a7 integrin in muscle cells

[0217] Objective: The ability of disclosed compounds to increase alpha integrin expression was assayed using the muscle-cell based Itga7 screen.

[0218] ITGA7+ / LACZ Screening Assay: ITGA7+ / LACZ cells were differentiated into myotubes in black walled Olympus TC-treated 96-well plates using standard procedures. The compounds TRULI, TDI-011536, S-969, SU9516 and PY-60 were added either individually or in combination to the differentiation media at doses ranging from 1 nM to 100 pM with negative control normalization wells receiving an equal volume of DMSO. The structure of S-969 can be found in US20170088837A1 . PY-60 is a robust activator of Yap transcriptional activity bybinding to and inhibiting function of annexin A2. Uninhibited, annexin 2 represses Yap activity. At 48 hours post-treatment, the media / compounds were aspirated, and cells were lysed by incubation in 50 pL MPER (ThermoFisher) reagent for 3 minutes at room temperature. The levels of p-Galactosidase, produced from the LACZ gene insertion into ITGA7, was assayed by adding 50 pL of the reaction solution (Na-Phosphate Buffer, MgCI2, FDG) and incubating at 37°C for 5 minutes. Following incubation, 100 pL of 2X TE was added to stop the reaction prior to determination of released fluorescein signal. The compound data was then normalized to DMSO treated wells to produce a fold-difference to DMSO and graphed (Figures 3A, 3B, and 4).

[0219] Results: Presently disclosed a7 integrin modulatory agents more potently increased transcription and translation of a7 integrin than comparators. Administration of S969 increased levels of the a7 reporter in a similar manner previously observed. Administration of TRULI alone or TDI-01 1526 alone increased the a7 reporter by 3-fold to 4-fold compared to vehicle control (Figures 3A, 3B and 5). Administration of GA-017 increased a7 reporter by approximately 2-fold compared to vehicle control (Figure 5). Combining S-969 with the other compounds did not result in a further increase in reporter activity (Figure 4). Interestingly, PY- 60, which blocks Annexin A2 to activate Yap, did not increase a7 integrin reporter levels. This suggests that the increased a7 integrin levels are caused by Lats1 / 2 inhibition and not Yap activation.

[0220] Conclusion: These results confirm that the disclosed compounds potently enhance a7 integrin expression in muscle. Thus, the presently disclosed compounds modulate and enhance a7 integrin in muscle cells and can be useful for treatment of muscle disorders and muscular dystrophy.Example 3 Alpha? Integrin Expression and Protein Levels are Increased by LATS1 / 2 Inhibitors

[0221] Methods: The ITGA7+ / LacZ myotube assay as discussed herein was used to assess expression levels and proteins levels of alpha? in response to treatment with S969, TDI-011536, TRULI, VT02956, and GA-017 modulatory agents. For western blotting, C2C12 myotube lysates were treated for 48 hour with either DMSO, 2 mM VT02956, 2 mM TDI-011536, 20 mM S969, or 10 mM GA-017 and probed for alpha? integrin using a rabbit polyclonal antibody (B2- 76) and normalized to Revert Total Protein Stain. In an immunofluorescence assay, C2C12 myotubes were treated with DMSO, 20 mM S969, 10 mM TRULI, or 2 mM TDI-011536 and probed for active Yap, phospo-Yap S127, and DAPL

[0222] Results: S969, TDI-011536, TRULI, VT02956, and GA-017 each increased expression of alpha? integrin as shown in Figure 6. In Figure 7, western blotting results show increased protein levels of alpha? integrin after treatment with the modulatory agents. Western blotting results are shown as quantitated in Figure 8. As shown in Figure 9, the nuclear localization of active Yap shows inhibition of LATS1 / 2 kinase by the modulatory agents.

Claims

What is claimed is:1 . A method for treating a subject with a muscle disease or disorder, comprising: administering an effective amount of an a7 integrin modulatory agent to the subject with the muscle disease or disorder, wherein the a7 integrin modulatory agent comprises at least one of the following compounds: TRULI, TDI-011536, GA-017, VT02956, T65, and T74.

2. The method of claim 1 , wherein the muscle disease or disorder is muscle degeneration, comprising: administering an effective amount of an a7 integrin modulatory agent to the subject with the muscle degeneration, wherein the a7 integrin modulatory agent comprises at least one of the following compounds: TRULI, TDI-01 1536, GA-017, VT02956, T65, and T74.

3. The method of claim 1 , wherein the muscle disease or disorder is muscular dystrophy, comprising: administering an effective amount of an a7 integrin modulatory agent to the subject with muscular dystrophy, wherein the a7 integrin modulatory agent comprises at least one of the following compounds: TRULI, TDI-01 1536, GA-017, VT02956, T65, and T74.

4. A method for treating a subject with a muscle disease or disorder, comprising: administering to the subject an effective amount of an a7 integrin modulatory agent, wherein the a7 integrin modulatory agent increases expression of a7 integrin, and wherein the a7 integrin modulatory agent comprises at least one of the following compounds: TRULI, TDI-011536, GA- 017, VT02956, T65, and T74.

5. The method of claim 2, wherein the muscle degeneration is a decrease in muscle strength of at least 20% relative to a control population.

6. The method of claim 2, wherein the muscle degeneration is a decrease in muscle mass of at least 20% in the subject relative to a measurement of muscle mass in the subject at a prior time point.

7. The method of claim 6, wherein the prior time point is one year, two years, three years, four years, or five years earlier.

8. The method of claim 3, wherein the muscular dystrophy is selected from the group consisting of merosin deficient congenital muscular dystrophy Type 1A (MDC1 A), merosin deficient congenital muscular dystrophy Type 1 D (MDC1 D), limb-girdle muscular dystrophy (LGMD), Duchenne muscular dystrophy (DMD), Fukuyama congenital muscular dystrophy (FCMD), and Facioscapulohumeral muscular dystrophy (FSHD).

9. The method of claim 3, wherein the muscular dystrophy is Duchenne muscular dystrophy.

10. The method of any one of claims 1 to 9, wherein the a7 integrin modulatory agent is TRULL11 . The method of any one of claims 1 to 9, wherein the a7 integrin modulatory agent is TDI-011536.

12. The method of any one of claims 1 to 9, wherein the a7 integrin modulatory agent is GA-017.

13. The method of any one of claims 1 to 9, wherein the a7 integrin modulatory agent is VT02956.

14. The method of any one of claims 1 to 9, wherein the a7 integrin modulatory agent is T65.

15. The method of any one of claims 1 to 9, wherein the a7 integrin modulatory agent is T74.

16. The method of any one of claims 1 to 15, further comprising administering to the subject an additional therapeutic agent.

17. The method of claim 16, wherein the additional therapeutic agent is a protein, a growth factor satellite cells, stem cells, myocytes, an a7|31 integrin modulatory agent, or an additional a7 integrin modulatory agent.

18. The method of any one of claims 1 -17, wherein the subject is a human.

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