Use and combination therapy of myostatin inhibitor

Abstract

To provide a method for treating spinal muscular atrophy (SMA) of a subject.SOLUTION: A muscle condition of SMA or the like is treated by using an agent inhibiting myostatin signaling. Furthermore, the invention includes a combination therapy including a myostatin inhibitor and a neuron-modulating factor. In one aspect, provided is a method for treating spinal muscular atrophy (SMA) of a subject including: a step of administering myostatin inhibitor of an effective amount for treating SMA to the subject, in which the subject receives SMN-modulating factor therapy or is expected to receive SMN-modulating factor therapy; and a step of treating spinal muscular atrophy of the subject by the administration.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Application No. 62 / 349,596, filed June 13, 2016, entitled "Methods and Compositions for Treating Spinal Muscular Atrophy," No. 62 / 470,157, filed March 10, 2017, entitled "Uses of Myostatin Inhibitors," No. 62 / 486,934, filed April 18, 2017, entitled "Uses of Myostatin Inhibitors," No. 62 / 511,702, filed May 26, 2017, entitled "Uses of Myostatin Inhibitors," and No. 62 / 512,254, filed May 30, 2017, entitled "Uses of Myostatin Inhibitors," the contents of each of which are incorporated herein by reference in their entirety. [Background technology]

[0002] Myostatin, also known as growth differentiation factor 8 and abbreviated as GDF-8 or GDF8, is a key regulator of muscle homeostasis. Loss of myostatin, as well as mutations that result in pharmacological inhibition of myostatin activity, have been shown to increase muscle growth in multiple species, including humans. Over the past two decades, this identification has prompted numerous groups to develop myostatin pathway antagonists as therapeutic agents for treating muscle conditions such as disuse atrophy, sarcopenia, and cachexia. Summary of the Invention [Problem to be solved by the invention]

[0003] In recent years, at least six myostatin inhibitor drug candidates, including small molecules and biologics, have entered the clinical stage but have failed due to undesirable side effects (e.g., risk of toxicity), lack of meaningful efficacy, or both. Despite the undisputed biological role of myostatin in regulating muscle growth, in many cases, despite satisfactory preclinical results, translation into safe and effective drugs has been unsuccessful. The lack of successful translation of myostatin inhibitors into clinical development in numerous muscle conditions, including muscular dystrophy, cachexia, sarcopenia, sporadic inclusion body myositis (SIBM), and inactivity, presents a challenge in the art. [Means for solving the problem]

[0004] The present invention encompasses the recognition that the in vivo effects of myostatin inhibition depend on the biological context. According to the present invention, factors that confer target muscle responsiveness to myostatin inhibition therapy include i) the anabolic state of the target muscle, ii) the degree of functional innervation by motor neurons, and, optionally, iii) the type of muscle fiber contained in the target muscle. Thus, as described in further detail herein, these factors should be considered to maximize or enhance the benefits of myostatin inhibition.

[0005] The inventors of the present disclosure have identified a set of criteria that can provide useful guidance for determining biological contexts (e.g., clinical conditions) in which myostatin inhibition can have an optimal effect on muscle: i) The muscle being treated to promote hypertrophy retains or regains sufficient anabolic capacity; ii) the muscle being treated to promote hypertrophy and / or prevent atrophy retains or regains at least partial functional innervation of motor neurons, and in some cases, iii) The muscle to be treated has motor functions that depend on fast-twitch (e.g., type II) fibers. That it is necessary.

[0006] Thus, the profile of suitable clinical attributes identified herein can determine the likelihood of responsiveness to myostatin inhibition in muscle, providing guidance for selecting suitable clinical indications and for identifying patient populations likely to benefit from such therapy. In some cases where a patient lacks or is deficient in one or more of the attributes, additional agents (e.g., therapeutic agents) may be co-administered to compensate for the deficiency in order to enhance or optimize efficacy. Thus, combination therapies incorporating such agents in combination with inhibitors of myostatin are encompassed by the present invention.

[0007] Accordingly, the present invention provides various embodiments of methods for treating a muscle condition in a subject, including administering to a subject a therapeutically effective amount of a myostatin inhibitor to enhance muscle / motor function, wherein i) the target muscle retains or regains full anabolic capacity and the intended clinical outcome comprises promoting muscle hypertrophy, ii) the target muscle retains or regains at least partial innervation of functional motor neurons and the intended clinical outcome comprises promoting muscle hypertrophy and / or preventing muscle atrophy, and / or iii) the target muscle affected by the muscle condition comprises or is enriched in fast-twitch muscle fibers that are primarily type II fibers.

[0008] In some embodiments, the subject retains the anabolic capacity (ability to grow) of the target muscle. Such a subject may be a pediatric patient. In other embodiments, the subject has weakened or impaired anabolic capacity of the target muscle but has regained sufficient anabolic capacity. The limited anabolic capacity may be due to age, a medical condition (injury, disease, etc.), side effects of medication, overall health, or any combination thereof. In some embodiments, the subject has amyotrophic lateral sclerosis (ALS), sarcopenia, cachexia, SIBM, immunodeficiency, muscular dystrophy, or any combination thereof. In some embodiments, the subject is being treated with an anabolic stimulator, e.g., an agent that boosts cellular anabolic pathways. In some embodiments, the subject receives a combination therapy including a myostatin inhibitor and an anabolic stimulator.

[0009] In some embodiments, the subject retains at least partial functional innervation of the target muscle by motor neurons, e.g., a functional neuromuscular junction. In some embodiments, the subject has a condition associated with partial denervation of motor neurons. In some embodiments, the subject has a condition associated with impaired neurotransmission. In some embodiments, the impaired neurotransmission comprises neuronal hyperexcitability, impaired synaptic vesicle transport or release, and / or impaired function or availability of mitochondria to support neuromuscular signaling. In some embodiments, the subject has restored or enhanced at least partial functional motor neuron innervation of the target muscle and / or at least partially normal neurotransmission between the motor neuron and the target muscle. In some embodiments, the subject has been treated with an agent that promotes motor neuron function (i.e., a neuronal therapeutic agent). In some embodiments, such an agent at least partially corrects neurotransmission and / or membrane excitability. In some embodiments, the agent is a corrector of a genetic defect. In some embodiments, the subject is treated with an agent that corrects a genetic defect with the goal of promoting motor neuron function so that at least partial innervation or function can be maintained or restored (restored).

[0010] In some embodiments, the subject has spinal muscular atrophy (SMA). In some embodiments, the genetic defect is a mutation in survival motor neuron 1 (SMN1). In some embodiments, the agent (e.g., a corrector agent) is a splice modifier or a gene therapy agent. In some embodiments, the splice modifier is a small molecule agent, and in other embodiments, the splice modifier is a nucleic acid agent, such as an RNA-based agent. In some embodiments, In some embodiments, the corrective factor therapy promotes the function and / or survival of motor neurons. In some embodiments, the corrective factor therapy slows the progression of SMA.

[0011] In some embodiments, the subject has been treated with a corrective agent and / or is likely to be treated with a corrective agent.For example, the subject is treated with a corrective agent within about 6 months of myostatin inhibitor therapy, for example, within about 6 months before or after administration of the myostatin inhibitor.In some embodiments, such a subject has or has been diagnosed with non-ambulatory SMA.In some embodiments, such a subject has type I SMA, type II SMA, or non-ambulatory type III SMA.

[0012] According to the present invention, an effective amount of a myostatin inhibitor for treating a muscular condition is an amount that achieves both clinical efficacy and safety. In some embodiments, the effective amount is an amount that enhances muscle function, such as force production and motor function. In some embodiments, the effective amount is an amount that enhances motor function requiring fast-twitch muscle fibers (e.g., type II fibers). In some embodiments, motor function includes eccentric contraction of the muscle. In some embodiments, an effective amount of myostatin therapy is effective to slow or attenuate the progression of the disease (e.g., muscle atrophy), maintain disease status (e.g., as measured / monitored by suitable motor function tests, plasma protein markers, metabolic markers, etc.), slow the loss of alpha-motor neurons, prevent or delay the appearance of immature muscle markers, prevent, attenuate, or delay intramuscular fat deposition (e.g., fatty replacement of muscle tissue), prevent metabolic dysregulation, prevent or reduce the frequency of bone loss or fractures, increase the Expanded Hammersmith Functional Motor Scale score by ≧1 point compared to controls not receiving the myostatin inhibitor, slow the rate of deterioration, delay regression (e.g., progressive decrease) of the Expanded Hammersmith Functional Motor Scale over 12 months, 24 months, or 36 months, and / or prevent or reduce the incidence of CHOP The amount is sufficient to increase the INTEND score by ≥ 1 point compared to a control not receiving a myostatin inhibitor and / or to increase the MFM-32 score by ≥ 1 point compared to a control not receiving a myostatin inhibitor.

[0013] In some embodiments, the muscle condition treated with a myostatin inhibitor is associated with a neuromuscular disease, including, but not limited to, amyotrophic lateral sclerosis (ALS), congenital myasthenic syndrome, congenital myopathy, spastic fasciculation syndrome, Duchenne muscular dystrophy (DMD), glycogen storage disease type II, hereditary spastic paraplegia, inclusion body myositis (IBM), Isaac syndrome, Kearns-Sayre syndrome, Lambert-Eaton myasthenic syndrome, mitochondrial myopathy, muscular dystrophy, myasthenia gravis, myotonic dystrophy, peripheral neuropathy, spinal-bulbar muscular atrophy, spinal muscular atrophy (SMA), spinal muscular atrophy with respiratory distress type 1, stiff-person syndrome, Troyer syndrome, and Guillain-Barré syndrome.

[0014] In some embodiments, the muscle condition treated with a myostatin inhibitor according to the present invention is spinal muscular atrophy (SMA).

[0015] In embodiments where the neuromuscular disease is SMA, the genetic defect may include a mutation in the SMN1 gene. To promote the function of motor neurons affected by the mutation, the subject may be treated with a corrector agent targeted to correct the genetic defect. In some embodiments, the agent is a splice modifier, which may be a nucleic acid-based (e.g., RNA-based) agent or a small molecule agent. In some embodiments, the subject is treated with the corrector agent within about six months of receiving the myostatin inhibitor, e.g., within about six months before or within about six months after administration of the myostatin inhibitor.

[0016] The present invention further contemplates that suitable inhibitors of myostatin signaling may be used as monotherapy (without SMN modifiers) to treat less severe forms of SMA in which patients retain the ability to walk. Suitable patients for such therapy include ambulatory patients with SMA type III and SMA type IV. In some embodiments, such treatment can slow the progression of SMA so that patients retain the ability to walk for a longer period than controls before the pathology transitions to non-ambulatory forms. [Brief explanation of the drawings]

[0017] [Figure 1] Figure 1 provides an overview of myostatin activation. Each dimeric precursor polypeptide contains a prodomain and a growth factor domain. In an initial proteolytic step by a proprotein convertase, promyostatin is cleaved between the prodomain and the growth factor domain, producing latent myostatin. In this form, the prodomain is still physically associated with the growth factor domain. Latent myostatin is then cleaved by a toroidal protease, after which active mature myostatin is released from the latent complex.

[0018] [Figure 2] Figures 2A and 2B provide an overview of dexamethasone-induced atrophy in mice. Figure 2A shows the experimental model of single-dose SRK-015 treatment in healthy and atrophy-induced mice. Figure 2B provides the changes in gastrocnemius muscle weight measured at the indicated time points.

[0019] [Figure 3]Figures 3A-3E provide five graphs showing that administration of muSRK-015P to healthy animals enhances muscle function: (Figure 3A) Maximum force (normalized to limb length) generated by the gastrocnemius muscle as a function of stimulation frequency in muSRK-015P-treated and control mice, (Figure 3B) Maximum EDL force per muscle length as a function of stimulation frequency in muSRK-015P-treated and control mice, (Figure 3C & 3D) GA and EDL muscle weights in muSRK-015P-treated and control mice, respectively, and (Figure 3E) Mean type IIB fiber area in muSRK-015P-treated and control mice.

[0020] [Figure 4] FIG. 4 provides a graph showing the effect of muSRK-015P on maximal force of the plantar flexor muscles (gastrocnemius, soleus, and plantaris) in Δ7 SMA and wild-type mice treated with SMN correctors.

[0021] [Figure 5] Figures 5A-5C provide three graphs showing the effects of muSRK-015P on (Figure 5A) muscle weight, (Figure 5B) mean myofiber cross-sectional area, and (Figure 5C) myofiber cross-sectional area frequency distribution in Δ7 SMA mice treated with SMN correctors and muSRK-015P or vehicle.

[0022] [Figure 6] Figures 6A-6B provide two graphs showing the effect of muSRK-015P on (Figure 6A) GA muscle mass and (Figure 6B) hindlimb grip strength in an acute contusion spinal cord injury model.

[0023] [Figure 7]Figures 7A-7D show target binding analysis of serum and muscle from SMNΔ7 mice. Figure 7A shows an immunoblot measuring circulating latent myostatin after 4 weeks of treatment with muSRK-015P. Figure 7B shows the immunoblot demonstrating target binding in muscle. Figure 7C shows a TGX unstained gel that allows visualization and quantification of total lane protein content for normalization during UV imaging. Figure 7D shows quantification of latent myostatin signal in muscle from mice treated with muSRK-015P compared to the latent myostatin present in WT mice.

[0024] [Figure 8] Figures 8A-8C provide PK and PD data for SRK-015 in scid mice. Figure 8A shows the PK analysis of SRK-015. Lean mass was measured by qNMR at the indicated time points after dose administration. Figure 8B shows the increase in lean mass compared to the IgG control. Target binding of SRK-015 in serum and muscle was assessed by analyzing the levels of latent myostatin in serum and muscle using Western blots as shown in Figure 8C.

[0025] [Figure 9] Figures 9A-9B provide SRK-015 PK data in cynomolgus monkeys. SRK-015 concentrations were assessed by ELISA. Figure 9A shows serum SRK-015 concentrations during the week after the first antibody dose. Figure 9B shows serum SRK-015 concentrations during the final 5 weeks of the study, following the last of eight weekly antibody doses.

[0026] [Figure 10-1]Figures 10A-10D provide data demonstrating that SRK-015 is pharmacologically effective at multiple doses in cynomolgus monkeys. Muscle weights were determined 5 weeks after the last dose of SRK-015 in cynomolgus monkeys. Figure 10A shows the increase in gastrocnemius muscle mass after SRK-015 treatment. Figure 10B shows the increase in biceps muscle mass after SRK-015 treatment. The time course of target binding in monkey serum was analyzed by semiquantitative Western blot analysis. Figure 10C shows target binding data from monkeys administered 3 mg / kg or 30 mg / kg of SRK-015 once weekly. As shown in Figure 10D, SRK-015 bound to latent myostatin at both doses tested. [Figure 10-2] Figures 10A-10D provide data demonstrating that SRK-015 is pharmacologically effective at multiple doses in cynomolgus monkeys. Muscle weights were determined 5 weeks after the last dose of SRK-015 in cynomolgus monkeys. Figure 10A shows the increase in gastrocnemius muscle mass after SRK-015 treatment. Figure 10B shows the increase in biceps muscle mass after SRK-015 treatment. The time course of target binding in monkey serum was analyzed by semiquantitative Western blot analysis. Figure 10C shows target binding data from monkeys administered 3 mg / kg or 30 mg / kg of SRK-015 once weekly. As shown in Figure 10D, SRK-015 bound to latent myostatin at both doses tested.

[0027] [Figure 11-1] Figures 11A-11B provide data showing muscle performance in SMNΔ7 mice treated from birth with a therapeutically sufficient dose of SMN-C1. After 4 weeks of treatment with muSRK-015P, weight gain (Figure 11A) and gastrocnemius muscle mass (Figure 11B) were measured relative to PBS control animals. Plantar flexor and masseter muscle performance was measured using the 305C muscle lever system (Figure 11B). [Figure 11-2]Figures 11A-11B provide data showing muscle performance in SMNΔ7 mice treated from birth with a therapeutically sufficient dose of SMN-C1. After 4 weeks of treatment with muSRK-015P, weight gain (Figure 11A) and gastrocnemius muscle mass (Figure 11B) were measured relative to PBS control animals. Plantar flexor and masseter muscle performance was measured using the 305C muscle lever system (Figure 11B).

[0028] [Figure 12] FIG. 12 is a schematic diagram showing the selection of certain patient populations likely to benefit from myostatin inhibition, either alone or in combination with additional agents (e.g., anabolic stimulants and / or neuronal enhancers). DETAILED DESCRIPTION OF THE INVENTION

[0029] The present invention is based, at least in part, on the recognition that the effectiveness of myostatin inhibition depends, at least in part, on the status of the target muscle, and that certain conditions must be met to confer a benefit on muscle function. Myostatin inhibition is particularly suitable for treating clinical indications with the following attributes: i) the muscle to be treated retains or regains full / robust anabolic capacity (e.g., younger subjects), ii) the muscle to be treated (i.e., the target muscle) retains or regains at least partial functional innervation by motor neurons (e.g., sufficient neuromuscular signaling between the target muscle and innervating motor neurons), and / or iii) the muscle to be treated is required for motor function that is dependent on type II fibers, e.g., addressing an unmet medical need by boosting fast-twitch (glycolytic) muscle fiber function, with motor outcome assessments driven by fast-twitch fiber activity. Based on these attributes, various embodiments of therapies and combination therapies are disclosed herein.

[0030] In the context of this application, the term "combination therapy" refers to the combined administration of two or more biologically active agents (e.g., drugs) used in conjunction with one another. Combination therapy may include a single formulation or may include multiple formulations. The combined administration may be simultaneous or sequential. The combined administration may be by the same route of administration or by different routes of administration. As long as there is overlap in the effects of the two (or more) therapeutic agents in a subject to achieve a complementary, additive, or synergistic clinical effect, it is considered combination therapy.

[0031] Criterion (i) above indicates that the target muscle is sufficiently active in that it maintains or regains the ability to synthesize cellular components (e.g., constitutive metabolism (i.e., anabolism)) rather than promoting their breakdown. Muscles with anabolic capacity therefore have the capacity to grow ("hypertrophy") rather than to weaken ("atrophy"). Myostatin has long been identified as a negative regulator of muscle mass; indeed, several groups have tested myostatin inhibitors in various muscle conditions but have failed to produce clinically meaningful results; to date, these studies have neglected to take into account the importance of this "anabolic" basal environment within which myostatin inhibition can exert its effect on promoting muscle growth. Indeed, most of the clinical indications in which myostatin inhibitors have been tested to date involve patient populations whose muscles are prone to a catabolic state. The inventors of the present application have taken into consideration the concept that factors controlling muscle synthesis and muscle breakdown are in dynamic equilibrium and have recognized that inhibition of myostatin signaling can produce muscle-building effects as long as the target muscle also retains sufficient anabolic activity to drive protein synthesis.

[0032] Two scenarios can be considered for meeting the above criterion (i). In the first scenario, this criterion may be naturally met in younger individuals (e.g., pediatric patients and young adults) who are growing or whose metabolism is robust and whose cellular anabolic pathways are already robust and active. Therefore, these patient populations have a favorable basal environment in which myostatin inhibition can have a clinical effect and are more likely to respond to myostatin inhibition therapy, which can promote muscle growth. In the second scenario, when patient populations treated with myostatin inhibitors are typically older individuals or those who are otherwise considered to have lost at least some of their anabolic mechanisms or their functions (e.g., those suffering from sarcopenia, cachexia, immunodeficiency, infection, etc.), myostatin inhibition may not provide the desired benefit due to a lack of sufficient anabolic activity. However, such deficiencies can be overcome or compensated for by the co-administration of a second agent aimed at boosting the patient's anabolic capacity, thereby making the patient more responsive in promoting muscle hypertrophy to the co-administration of a myostatin inhibitor. Thus, the present invention includes a combination therapy for treating a muscle condition in a catabolic subject, comprising a myostatin inhibitor (i.e., an agent that inhibits myostatin activation, activity, and / or signaling) and an anabolic stimulator (i.e., an agent that boosts anabolic function or promotes protein synthesis). Such agents promote muscle growth. The compound is administered to a subject in an amount effective to enhance muscle synthesis (e.g., promote muscle synthesis over muscle breakdown) and correspondingly improve motor function.

[0033] As used herein, the term "catabolic state" refers to a state in which the balance of synthesis and degradation (e.g., protein synthesis and protein degradation) in a target tissue / cell is tilted toward the latter, resulting in a net catabolic effect on the target. Similarly, as used herein, the term "anabolic state" refers to a state in which the balance of synthesis and degradation (e.g., protein synthesis and protein degradation) in a target tissue / cell is tilted toward the former, resulting in a net anabolic effect on the target. Thus, for a patient population experiencing a catabolic state, a therapy incorporating both a myostatin inhibitor and an anabolic stimulant can achieve improved clinical benefits of myostatin inhibition compared with monotherapy. Typically, the status of a target tissue (e.g., muscle) is measured by determining the circulating levels of various hormones (e.g., IGF-1, testosterone) and / or by determining the level of muscle protein synthesis. Measurement of hormone levels can be performed using serum, saliva, or urine samples by methods known to those skilled in the art, including competitive immunoassays. Measurement of muscle protein synthesis can be performed by methods known to those skilled in the art, including muscle biopsy.

[0034] The recognition of the importance of criterion (ii) is based on the finding that the functions of muscles and the motor neurons that innervate target muscles (collectively referred to as "motor units") are at least partially interdependent, and that a degree of cross-talk (i.e., bidirectional signaling) between the two components (i.e., the neuronal and muscular components) is necessary for maintaining neuromuscular function. It is contemplated that for myostatin inhibition to have a meaningful effect on target muscle function, the muscle must receive sufficient neural input from the innervating motor neurons (i.e., the presence of functional neuromuscular signaling). This is likely relevant to both clinical situations in which the primary desired outcome is to promote muscle growth and prevent muscle loss. As demonstrated in the Examples below, in multiple muscle injury models, myostatin inhibition can prevent and attenuate injury-induced muscle atrophy and metabolic dysregulation. In these animal models, injured muscles at least partially retained neural input rather than completely severed innervating motor nerves. Previous reports in the literature have shown that myostatin inhibition does not enhance muscle function in complete spinal cord injury models. Therefore, without wishing to be bound by any particular theory, it is contemplated that sufficient neuronal input (e.g., neurotransmission) from innervating motor neurons contributes, at least in part, to the beneficial effects of myostatin inhibition in target muscles. Typically, neurotransmission is measured in intact animals by directly stimulating a nerve (e.g., a nerve innervating a muscle) and measuring the contraction of the innervated muscle group. In such measurements, the absence of muscle contraction indicates the absence of neurotransmission. Similarly, a gradual decline in the response measured in the target muscle after repeated stimulation may indicate "fatigue," which may reflect impairments in membrane excitability, synaptic vesicle transport, mitochondrial function / availability, and / or glucose regulation. In SMA, there is a progressive loss of fully innervated neuromuscular junctions, which can be assessed by immunofluorescence. Neurotransmission (e.g., neuromuscular transmission) can also be measured using other electrophysiological methods known to those skilled in the art.

[0035] The requirement for sufficient neuronal signaling means that myostatin inhibitors may not provide optimal benefit if neuromuscular crosstalk is completely lost or disrupted (i.e., functional neuromuscular signaling is absent), either by injury or in certain disease situations. This concept led the inventors to recognize that in neuromuscular diseases involving genetic defects that impair motor neurons, the target muscle itself may remain intact during the early stages of the disease, but its function may gradually decline due to the lack of sufficient neuronal input from motor neurons to the muscle and feedback from the muscle to the motor neuron (i.e., the lack of functional neuromuscular signaling). Therefore, Therefore, it is contemplated herein that an intervention (e.g., a pharmacological intervention) that promotes nerve-muscle signaling that targets and corrects or restores the underlying neuronal deficits should enhance the benefits of myostatin inhibition.

[0036] Thus, the present invention includes combination therapies for treating neuromuscular diseases. Such combination therapies include i) inhibitors of myostatin signaling (e.g., agents that inhibit myostatin activation, activity, and / or signaling), and ii) neuronal therapeutic agents (e.g., neuronal corrective factors, neuronal enhancers, etc.) that include agents targeted at treating motor neurons to correct neuronal defects (e.g., disease-causing genetic mutations). The myostatin inhibitor and the agent for treating motor neurons can be administered in combination with each other as a combination therapy in amounts effective to enhance motor function.

[0037] Suitable patient populations for myostatin inhibitor therapy to treat neuromuscular diseases include those receiving neurotherapeutic agents (e.g., those receiving neuronal modifiers / enhancers). Based on the concept that functional motor units involve bidirectional signaling between target muscles and innervating motor neurons, it is contemplated that enhancing the function of one can positively affect the function of the other, and vice versa. Thus, a subpopulation of patients who have received neurotherapeutic agents but do not respond to them in a clinically meaningful manner can be made more responsive to neurotherapeutic agents in combination with myostatin inhibitor therapy. Similarly, a subpopulation of patients who are responsive to neurotherapeutic agents can achieve additional clinical benefits when receiving myostatin inhibitor therapy.

[0038] In some embodiments, the methods of the present invention are suitable for treating or preventing muscle conditions or disorders and neuromuscular diseases. As used herein, the term "muscle condition" or "muscle disorder" refers to a disease, condition, or disorder in which muscles do not function normally, or a disease, condition, or disorder in which muscles function normally but generate less force due to a reduced amount of muscle capacity. As used herein, the term "neuromuscular disease" refers to any disease caused by or associated with disruption of signal transduction or information transmission between neurons and muscle tissue. In some embodiments, impaired neurological signaling occurs due to damage to neuronal structure, preventing neurons from transmitting signals to their targets. In other embodiments, the neuronal structure remains intact, but there is a functional disruption or defect, e.g., blockage, at the neuromuscular junction, such that the neuron's signaling ability is affected. In some embodiments, the disruption of signal transduction is associated with denervation, e.g., partial loss or impairment of the nerve supply or neuronal input to its target muscle. In some embodiments, denervation is induced by injury. Suitable neuromuscular diseases or conditions that can be treated according to the present invention include, but are not limited to, amyotrophic lateral sclerosis (ALS), congenital myasthenic syndromes, congenital myopathies, spastic fasciculation syndromes, Duchenne muscular dystrophy (DMD), glycogen storage disease type II, hereditary spastic paraplegia, inclusion body myositis (IBM), Isaac syndrome, Kearns-Sayre syndrome, Lambert-Eaton myasthenic syndrome, mitochondrial myopathy, muscular dystrophies, myasthenia gravis, myotonic dystrophy, peripheral neuropathy, spinal-bulbar muscular atrophy, spinal muscular atrophy (SMA), spinal muscular atrophy with respiratory distress type 1, stiff-person syndrome, Troyer syndrome, and Guillain-Barré syndrome.

[0039] In any of the above embodiments, the combined administration of an agent aimed at enhancing / promoting neuronal function or correcting / restoring the underlying neuronal deficit (collectively referred to as a "neuronal therapeutic agent") and a myostatin inhibitor is useful. Additionally, such combined therapy may further include an anabolic stimulant (i.e., an anabolic stimulant) for patients whose target muscles may be in or at risk of becoming catabolic. Such anabolic stimulators, when used in combination, can enhance the benefits of myostatin inhibitors. Accordingly, the present invention includes a method for treating a neuromuscular disorder in a patient, comprising administering to the patient an effective amount of a combination therapy comprising a myostatin inhibitor, a neuronal enhancer / modifier (i.e., a neuronal therapeutic agent), and an anabolic stimulator.

[0040] The above-described recognition of factors that may influence the outcome of myostatin inhibitor therapy is further illustrated in FIG.

[0041] Criterion (iii) incorporates the concept that muscles are differentially affected by myostatin inhibition based, in part, on their fiber type. Evidence provided herein suggests that muscles rich in fast-twitch fibers (e.g., type II fibers), including glycolytic fast-twitch fibers, may be particularly sensitive to myostatin inhibition. Thus, myostatin inhibitor therapy may preferentially benefit muscles rich in fast-twitch fibers (e.g., muscles containing type II fibers) and enhance motor function requiring or dependent on fast-twitch fibers. As shown in Figure 2, the gastrocnemius muscle was found to be responsive to myostatin inhibition therapy. It should be noted that the gastrocnemius muscle is known to contain approximately 75% fast-twitch glycolytic fibers.

[0042] Thus, the present invention is based, at least in part, on the recognition that patients suffering from neurological disorders that impair motor function may benefit from a combination of both agents that target muscle function (such as muscle enhancers) and agents that target neural function, such as splice regulators and gene modifiers (sometimes commonly referred to as "neuronal therapeutics"). The present invention is particularly useful for treating conditions involving impaired signaling between motor neurons and their target muscles (such as neuromuscular disorders). The present invention is particularly useful for treating conditions involving partial, but not complete, loss of neurons that innervate muscles.

[0043] The present invention encompasses the recognition that inhibition of myostatin signaling may be advantageous in the treatment of conditions in which highly metabolic, fast-twitch muscle-rich muscles are particularly susceptible. In particularly useful embodiments, therapeutic regimens for treating such conditions include inhibitors or antagonists of myostatin signaling in combination with agents that treat motor neurons that innervate fast-twitch muscle fibers.

[0044] Such conditions may be associated with genetic mutations that result in impaired axonal transport or its regulation, impaired vesicle transport or its regulation, impaired neurotransmission or its regulation, impaired mitochondrial function or utilization, or any combination thereof. In some embodiments, such common mutations may cause impaired energy production, energy expenditure, glucose utilization, or their regulation.

[0045] In some embodiments, the condition is spinal muscular atrophy (SMA). definition

[0046] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0047] Unless in the working examples or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood to be modified in all instances by the term "about." When used in reference to percentages, the term "about" can mean ±1%. Additionally, the term "about" can mean within ±1% of the value.

[0048] The terms "administer," "administering," or "administration" include any method of delivering a myostatin inhibitor, a neuron modifying factor, e.g., an SMN modifying factor, and / or an anabolic stimulating factor, e.g., a pharmaceutical composition, to a subject's system or to a specific area within or on a subject (systemic administration and local administration, respectively).

[0049] As used herein, the term "responder" refers to a patient who has a positive predicted response to a therapeutic / biological drug. Similarly, as used herein, the term "non-responder patient" refers to a patient who has a negative or absent predicted response to a therapeutic / biological drug. The term "poor responder" as used herein refers to a patient who has a positive predicted response to a therapeutic / biological drug but does not achieve complete treatment of the disease / disorder and would benefit from further therapy and achieve a further and / or improved clinical response.

[0050] The term "predicted response" or the like, as used herein, refers to determining the likelihood that a patient will respond favorably or unfavorably to a given therapy / biological drug. In particular, the term "prediction," as used herein, relates to the individual evaluation of any parameter that may be useful in determining a patient's progress. As those skilled in the art will understand, predicting clinical response to treatment with a biological drug preferably, but not necessarily, 100% accurately diagnoses or evaluates the subject. However, this term requires that a statistically significant portion of subjects can be identified as having a high probability of having a positive response. Those skilled in the art can easily determine whether a subject is statistically significant using various well-known statistical evaluation tools, such as confidence interval determination, p-value determination, Student's t-test, Mann-Whitney test, etc. For details, see Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York, 1983. Preferred confidence intervals are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%. The p-value is preferably 0.2, 0.1, or 0.05.

[0051] The term "clinical response" as used herein refers to the response of a subject suffering from a pathology treatable with a biological drug to said biological drug. Standard criteria may vary from disease to disease and are discussed in more detail herein.

[0052] Patients who would "benefit from muscle growth" include both healthy patients and patients with diseases and / or disorders involving loss of muscle mass and / or strength. In one embodiment, a patient who would benefit from muscle growth is a subject with a muscle disease or disorder, such as SMA.

[0053] As used herein, the terms "comprising" or "comprises" are used in reference to compositions, methods, and their corresponding components that are essential to the invention, but leave open the possibility of including non-specified elements, whether essential or not.

[0054] The term "consisting of" refers to compositions, methods, and their corresponding components described herein, but excludes any element not recited in that description of an embodiment.

[0055] The term "control" or "control sample," as used herein, refers to any clinically or scientifically relevant comparison sample, population, or counterpart, including, for example, a sample from a healthy subject, a sample from a subject having a deficiency that may cause or render the subject susceptible to a particular disease or condition, a subject having a disease or condition of interest, a sample from a subject treated with a pharmaceutical carrier, a sample from a subject prior to treatment, Placebo or buffer treated subjects or samples, as well as untreated subjects or samples, etc. are included.

[0056] The term "control level" refers to an accepted or predetermined level of a biological marker, e.g., the level of the marker obtained before treatment or before the onset of a disease, or before the administration of a drug, e.g., a myostatin inhibitor or SMN modifier. The level of the biological marker present in a subject or population of subjects having one or more particular characteristics, e.g., the presence or absence of a particular disease or condition, e.g., SMA.

[0057] The term "reduction," as used herein, refers to a statistically significant decrease in such levels in the context of a disease symptom. A reduction may be, for example, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% below the level of detection by a detection method. A reduction may also be, for example, about 1-10%, 10-20%, 1-30%, 20-50%, 30-60%, 40-70%, 50-80%, or 60-90% below the level of detection by a detection method. In certain embodiments, a reduction is to a level that is accepted as within the normal range for individuals without such a disorder, which may also be referred to as normalization of levels.

[0058] The term "increase," e.g., in the context of a disease symptom, such as loss of function or mass, e.g., loss of muscle mass associated with a disease, refers to a statistically significant increase in such levels. The increase may be, for example, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or higher than the level of detection of the detection method. The increase may also be, for example, about 1-10%, 10-20%, 1-30%, 20-50%, 30-60%, 40-70%, 50-80%, or 60-90% higher than the level of detection of the detection method. In certain embodiments, the increase is to a level that is accepted as within the normal range for individuals without such disorder, which may also be referred to as normalization of the level. In certain embodiments, the increase is a normalization of the level of a sign or symptom of the disease, an increase in the difference between the subject level of the sign of the disease and the normal level of the sign of the disease.

[0059] As used herein, the term "denervation" refers to the loss or impairment of nerve supply or neuronal input to a target tissue, such as muscle tissue. Thus, "partial denervation" may be associated with a partial impairment of neuromuscular signaling between a target muscle and innervating motor neurons. Causes of denervation include disease (e.g., genetic disorders of motor neurons), chemical toxicity, physical injury, or intentional surgical severing of nerves. Denervation may be partial (also referred to as incomplete denervation) or complete. Partial denervation may be, for example, at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% loss or impairment of nerve supply or neuronal input to the target tissue. In some embodiments, partial denervation includes loss or impairment of about 1-10%, 10-20%, 1-30%, 20-50%, 30-60%, 40-70%, 50-80%, 60-90% of the nerve supply or neuronal input to that target tissue. As described in more detail herein, partial denervation and neuromuscular damage are measured, for example, using compound muscle action potential and motor unit number estimation.

[0060] As used herein, "determining" refers to performing an assay or using a method to ascertain the state of someone or something, e.g., the presence, absence, level, or degree of a particular condition, biomarker, disease state, or physiological condition. It is understood that:

[0061] "Onset" or "progression" of a disease refers to the initial manifestation and / or subsequent progression of the disease. Disease onset is detectable and can be assessed using standard clinical techniques. However, onset also refers to progression, which may be undetectable. For purposes of this disclosure, onset or progression refers to the biological course of symptoms. "Onset" includes occurrence, recurrence, and onset. As used herein, "onset" or "onset" of a myopathy-related disease / disorder includes initial onset and / or recurrence.

[0062] 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. The abbreviation "eg" is derived from the Latin exempli gratia and is used herein to indicate a non-limiting example. Thus, the abbreviation "eg" is synonymous with the term "for example."

[0063] As used herein, the terms "effective amount" and "effective dose" refer to any amount or dose of a compound or composition sufficient to achieve its intended purpose, i.e., a desired biological or pharmacological response in a tissue or subject, at an acceptable benefit / risk ratio. For example, in certain embodiments of the invention, the intended purpose may be to inhibit myostatin activation in vivo to achieve a clinically meaningful outcome associated with myostatin inhibition.

[0064] Measurement of the relevant intended purpose may be objective (i.e., measurable by some assay or marker) or subjective (i.e., an indication or feeling of effect is provided by the subject). In some embodiments, a therapeutically effective amount is an amount that, when administered to a patient population that meets certain clinical criteria for a disease, disorder, or condition (e.g., as determined by manifest symptoms, disease progression / stage, genetic profile, etc.), results in a statistically significant therapeutic response in that population.

[0065] In some embodiments, an effective amount is an amount that, when administered according to a particular regimen, produces a positive clinical outcome where the adverse effects (e.g., toxicity) are at a reasonably acceptable level, such that the adverse effects, if any, are sufficiently tolerable for the patient to continue the treatment regimen and the benefits of the therapy outweigh the risk of toxicity. One of skill in the art will understand that in some embodiments of the invention, a unit dosage can be considered to comprise an effective amount if it contains an amount appropriate for administration in the context of a dosing regimen that correlates with a positive outcome.

[0066] A therapeutically effective amount is generally administered in a dosage regimen that may include multiple unit doses. For any particular pharmaceutical agent, the therapeutically effective amount (and / or the appropriate unit dose within an effective dosage regimen) may vary, for example, depending on the route of administration, the combination with other pharmaceutical agents. In some embodiments, the specific therapeutically effective amount (and / or unit dose) for any particular patient may depend on various factors, including the disorder being treated and the severity of the disorder, the activity of the specific pharmaceutical agent used, the specific composition used, the patient's age, weight, overall health, sex, and diet, the time of administration, the route of administration, and / or the excretion or metabolic rate of the specific pharmaceutical agent used, the duration of treatment, and similar factors well known in the medical field.

[0067] "Treating" or "preventing" a disease or disorder means delaying or preventing the onset of such disease or disorder, reversing, alleviating, ameliorating, inhibiting, slowing, or halting the progression, intensification, or worsening of the conditions associated with such disease or disorder, or the progression or severity of such conditions. Although a complete treatment or prevention of the disease or disorder is not required, in one embodiment, the symptoms of the disease or disorder are alleviated by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%.

[0068] As used herein, the term "neuronal therapeutic agent" refers to an agent that aims to improve (e.g., enhance or restore) neuronal function. Neuronal therapeutic agents are useful for treating conditions involving impaired signaling between motor neurons and their target muscles (e.g., neuromuscular disorders). Specifically, neuronal therapeutic agents are particularly useful for treating conditions involving partial, but not complete, loss of neurons that innervate muscles. In one embodiment, a "neuronal therapeutic agent" may be a gene therapy agent, a small molecule, or an antisense oligonucleotide, as described in more detail herein. In one embodiment, a "neuronal therapeutic agent" is an "SMN corrector," as described in more detail herein. In some embodiments, a neuronal therapeutic agent is an agent capable of fully restoring motor neuron function to a cell (e.g., a cell in a subject). In some embodiments, a neuronal therapeutic agent is an agent capable of partially restoring motor neuron function to a cell (e.g., a cell in a subject). In some embodiments, a neuron therapeutic agent is an agent capable of restoring at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more of motor neuron function to a cell (e.g., a cell in a subject). Those skilled in the art will understand that motor neuron function typically includes membrane excitability, axonal transport, vesicle transport, neurotransmitter release, mitochondrial function, and / or mitochondrial availability, and that such function is measured using assays known to those skilled in the art. Spinal Muscular Atrophy (SMA)

[0069] Spinal muscular atrophy (SMA) is a debilitating and often fatal neuromuscular disease and the most common genetic cause of infant mortality (1). SMA is one of the most common rare diseases, with approximately 1 in 54 individuals being a carrier and approximately 1 in 11,000 children born with SMA. SMA is an autosomal recessive genetic disorder involving mutations or deletions in the survival motor neuron 1 (SMN1) gene. Specifically, SMA is caused by reduced levels of the SMN protein, and sufficient amounts of SMN protein are required to promote the survival of anterior horn cells in the spinal cord. Loss of motor neurons leads to severe muscle atrophy that often leads to death due to respiratory failure (2).

[0070] SMA patients lack a functional SMN1 gene, but the paralogous gene SMN2 produces low levels of functional SMN protein through alternative splicing that shortens the transcript. There is considerable interest in suitable methods and compositions for treating SMA patients that are effective in preventing muscle atrophy and promoting neuronal survival. SMA is clinically heterogeneous, and patients are classified based on the severity of the disease.

[0071] Type 0 is the most severe form of SMA and is diagnosed prenatally due to reduced fetal movement in the womb. Patients require ventilator support at birth. Type 1 SMA is typically diagnosed between the ages of six months and six months, and patients never gain enough strength to sit independently. Without intervention, most Type 1 patients do not survive past two years without respiratory support. People with Types II and III produce greater amounts of SMN protein and have less severe, but still life-altering, forms of SMA. Type II patients are diagnosed between six and eighteen months of age. Type II patients can sit unaided but cannot walk unaided. With Type III SMA, patients are diagnosed after the age of 18 months and can sit and walk unaided, but may become wheelchair dependent later in life. Type IV SMA is adult-onset, has a milder phenotype, and is non- It remains rare (1, 3). Stratification of SMA by type is a useful clinical paradigm, but disease phenotypes exist as more of a continuum than discrete categories (4).

[0072] The clinical heterogeneity of SMA is due, in part, to the genetic complexity of the disease. Mutations in the SMN1 gene lead to SMA (5), but in humans, a nearly identical gene, SMN2, is located very close to SMN1 (6). The main difference between these genes is a C-to-T transition that creates an exon splice silencer and results in the removal of exon 7 from the final mRNA transcript. The truncated SMN protein is unstable and rapidly degraded. Nevertheless, approximately 10% of the mRNA produced from SMN2 is correctly spliced ​​to produce full-length SMN protein, but this amount is insufficient to fully compensate for the loss of SMN1. The copy number of SMN2 varies among individuals; generally, higher numbers (3–4 copies) are associated with milder forms of SMA (1–4).

[0073] The role of SMN in regulating motor neuron survival and function is not fully understood, but its best-characterized functions are snRNP biogenesis and pre-mRNA splicing (2). Although motor neurons appear to be particularly sensitive to reduced SMN protein levels, SMN is ubiquitously expressed, and other organ systems, including the liver, spleen, digestive system, autonomic nervous system, and bone, are also affected in SMA patients (3). As noted above, severe skeletal muscle atrophy is observed in SMA patients, primarily due to loss of motor neuron innervation. However, not all muscles are equally affected, with axial muscles generally exhibiting greater atrophy and denervation than limb muscles (2, 7). In SMA patients, the diaphragm is largely spared due to the preservation of the phrenic nerve (8). Interestingly, fast-twitch type II muscle fibers exhibit significantly greater atrophy than slow-twitch type I fibers (9). The degree of muscle atrophy is directly related to the degree of innervation, and nerve-innervated muscles are less affected by loss of SMN protein and exhibit less atrophy (7, 8, 10). Nevertheless, SMN protein appears to play a direct role in skeletal muscle, as myogenic cells isolated from mouse models of SMA exhibit dysregulated myogenic gene expression, differentiate prematurely, and are associated with poor myotube formation (11, 12). In addition, evidence of muscle pathology has been shown in presymptomatic mice, and muscle-specific deletion of SMN exon 7 is associated with severe muscular dystrophy (13, 14). Treatment Approaches for SMA - SMN Modifiers

[0074] Multiple therapeutic approaches to restore SMN protein levels are under investigation: SMN1 gene replacement therapy, small molecules that modulate SMN2 splicing, and the use of antisense oligonucleotides (ASOs) to block the SMN2 intronic splicing silencer and thus increase inclusion of exon 7.

[0075] SMN1 gene replacement therapy using adeno-associated viral vectors (AAV) has shown benefit in mouse models of SMA, and AVXS-101, an AAV9-SMN1 vector derived from AveXis, is currently in phase I clinical trials (see NCT02122952) (15).

[0076] Other approaches have focused on modulating SMN2 splicing so that exon 7 is retained in a higher percentage of transcripts, leading to increased production of full-length SMN protein. Novartis and PTC Therapeutics / Roche have both developed small molecules that selectively enhance SMN2 exon 7 inclusion, leading to increased full-length SMN protein levels and therapeutic efficacy in mouse models of SMA (16-19). These small molecules from both companies are currently in phase 2 clinical trials (clinical trials NCT02913482, NCT03032172, NCT02908685, NCT022688552). (See references). Oral administration of RG7800, SMN-C2, and SMN-C3 in mild and severe preclinical models of SMA showed that the compounds increased SMN protein levels in both brain and muscle tissue of treated mice compared with vehicle. The molecules also efficiently crossed the blood-brain barrier (BBB). In a severe SMA mouse model, both compounds normalized motor behavior and increased body weight and survival compared with vehicle. However, clinical programs were put on hold due to safety concerns.

[0077] Another clinical-stage small molecule SMN2 splice modulator, LMI070, was discontinued after preclinical animal studies showed damage to peripheral nerves and blood vessels in the spinal cord, testes, and kidneys.

[0078] To date, one muscle-directed drug, CK-212107, targets skeletal muscle troponin to alter contractility.

[0079] Additional small molecule-based SMN2 splice correctors are described, for example, in U.S. Patent Application Publication No. US2009 / 0031435, published January 29, 2009, and U.S. Patent No. 8,399,437, published March 19, 2013, the contents of each of which are incorporated herein by reference in their entirety. However, it should be understood that other small molecule splice correctors, including SMN2 splice correctors known in the art, will be apparent to those of skill in the art and are within the scope of the present disclosure.

[0080] A third approach is the use of antisense oligonucleotides (ASOs) to block the SMN2 intron splicing silencer, thus increasing exon 7 inclusion, again rescuing disease, for example, in mouse models of SMA (20-22). Biogen / Ionis has developed nusinersen, an ASO splice modifier that has shown clinical efficacy and was recently approved by the FDA and marketed as Spinraza™ (23-25). However, each dose of nusinersen requires intrathecal delivery under general anesthesia. Additionally, while the antisense modifier nusinersen has proven promising, its clinical efficacy is considered modest, with 60% of patients with childhood-onset SMA (Type 1) reported to be non-responders. 43% of patients treated with nusinersen did not achieve a ≥3-point increase on the Hammersmith Functional Motor Scale (Extended) (HFMSE), and the mean increase in treated patients was less than 6 points compared to placebo. Therefore, improvement achieved with nusinersen treatment is only partial.

[0081] All of these molecules have demonstrated significant efficacy preclinically, and in the case of nusinersen, clinically; however, none provide a complete cure for the disease. In mouse models, both small molecules and ASO splice modifiers significantly reduce disease severity, yet treated animals exhibit deficits in lifespan, weight, muscle mass, and muscle function compared to healthy controls (21, 26). In a double-blind clinical trial of infantile-onset SMA, nusinersen provided clinically meaningful benefit at interim analysis (41% of the treatment group demonstrated improvement in motor milestones using the Hammersmith Infant Neurological Examination, compared with 0% of placebo). The motor milestones achieved were significant in type 1 patients, with 5 of 81 treated patients able to sit unassisted (though milestones are rarely achieved in these patients). Nevertheless, these patients did not achieve a full range of developmental milestones, and the milestones achieved would be considered disappointing in normal individuals (25). In a second placebo-controlled trial in Type 2 SMA, nusinersen again demonstrated clinically meaningful improvements, with a 5.9-point increase in Hammersmith Functional Motor Scale-Extended (HFMSE) score compared to the placebo group. The maximum score on the HFMSE is 66 points, and most type 2 patients score less than 20 (27, 28). Nevertheless, in this trial, 43% of patients failed to achieve at least a 3-point improvement in motor function (25). These results suggest that SMN2 splice regulators may have a significant effect on SMA disease course and patient quality of life, but further gain-of-function is required to further improve disease burden reduction.

[0082] As used herein, the term "SMN modifying factor" refers to any therapeutic agent or compound that can be used to increase or improve SMN gene expression (e.g., SMN1 gene expression and / or SMN2 gene expression), SMN protein production, and / or functional SMN activity. SMN modifying factors include, for example, splice correctors / modifiers that alter the splicing of the SMN2 transcript. It should be noted that systemically delivered SMN splice modifiers may also affect SMN splicing in other (i.e., non-neuronal) tissues in which SMN is expressed.

[0083] An "SMN modifier" may be a central modifier or a systemic modifier. Central modifiers are administered intrathecally directly to the central nervous system (CNS). In contrast, systemic modifiers can be administered by any route, for example orally, and affect not only the CNS but also other tissues throughout the body.

[0084] In some embodiments, a "functional SMN protein" is capable of promoting motor neuron function and / or survival. In some embodiments, a "functional SMN protein" is capable of fully restoring motor neuron function to a cell (e.g., a cell in a subject). In some embodiments, a functional SMN protein is capable of partially restoring motor neuron function to a cell (e.g., a cell in a subject). In some embodiments, a functional SMN protein is capable of restoring at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more of motor neuron function to a cell (e.g., a cell in a subject). In some embodiments, the full-length SMN protein is the result of protein translation (e.g., in a cell) of a correctly spliced ​​SMN mRNA. In some embodiments, the functional SMN protein is encoded from an SMN2 mRNA that includes exon 7.

[0085] In one embodiment, the "SMN modifying factor" may be a gene therapy agent, a small molecule, or an antisense oligonucleotide, as described in more detail herein. In some embodiments, the SMN modifying factor is an oligonucleotide molecule. In some embodiments, the SMN modifying factor is an antisense molecule. In some embodiments, the SMN modifying factor may be an antisense molecule that increases expression of the SMN2 gene. In some embodiments, the SMN modifying factor may be an antisense molecule that increases expression of an SMN2 mRNA that includes exon 7. In some embodiments, the SMN modifying factor may be an antisense molecule that increases expression of a functional SMN protein, e.g., an SMN protein encoded by an SMN2 mRNA that includes exon 7. For example, an antisense oligonucleotide aimed at inhibiting the intron splice silencer site (ISS) in intron 7 of the SMN2 gene may modulate pre-mRNA processing, leading to a greater likelihood of inclusion of exon 7 in the mature mRNA transcript of SMN2, resulting in increased production of functional SMN protein.

[0086] The terms "splice corrector," "splice regulator," and "splice modifier" as used herein are interchangeable and refer to a splice that corrects aberrant splicing of an RNA transcript, such as that encoded by the SMN2 gene, and / or enhances the expression of the SMN protein. "SMN2 splice corrector" refers to an agent that modulates the expression of SMN2. In some embodiments, the SMN2 splice corrector increases the inclusion of exon 7 in the SMN2 pre-mRNA. In some embodiments, increasing the inclusion of exon 7 in the SMN2 pre-mRNA leads to increased expression of functional SMN protein (e.g., from the SMN2 gene) in a cell or subject, such as an SMN protein that can promote neuronal function and / or survival.

[0087] In one embodiment, the SMN corrector may be gene therapy. As used herein, the term "gene therapy" refers to any procedure that uses nucleic acids to heal, cure, or otherwise improve a subject's condition. Gene therapy requires that nucleic acids be delivered into specific cells. Delivery methods include viral or non-viral means, which are known in the art. See, for example, Patil et al., AAPS J., Vol. 7 (I): E6 1-E77 (2005); Gascon et al., Non-Viral Delivery Systems in Gene Therapy (2013); Somiari et al., Molecular Therapy, Vol. 2(3), pp. 178-187 (2000); Herweijer, H. and J. A. Wolff, Gene therapy, Vol. 10(6): pp. 453-458 (2003); and Nayerossadat et al., Advanced biomedical Research, Vol. 1(2):1-11 (2012). Viral means for delivering gene therapy include the use of viral vectors. Viral vectors are genetically engineered viruses that can carry a therapeutic gene payload and are reprogrammed to infect and subsequently transfer said payload to specific tissues without the side effects typically associated with wild-type viral infection. Several viruses, including retroviruses, adenoviruses, herpes simplex viruses, lentiviruses, poxviruses, and Epstein-Barr viruses, can be used as viral vectors. Viral vectors are safer than wild-type viruses, but they may induce an immune response, which may necessitate the use of non-viral delivery methods. In one embodiment, the viral vector is an AAV viral vector. Non-viral delivery methods include, but are not limited to, physical methods such as naked DNA injection, electroporation, gene gun bombardment, and ultrasound, as well as biochemical methods. Magnetofection, another delivery technique, combines physical and biochemical elements.

[0088] In some embodiments, an "effective amount" of an SMN modifying agent is an amount of agent capable of fully restoring motor neuron function to a cell (e.g., a cell in a subject). In some embodiments, an SMN modifying agent is an agent capable of partially restoring motor neuron function to a cell (e.g., a cell in a subject). In some embodiments, an "effective amount" of an SMN modifying agent is an amount of agent capable of restoring at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more of motor neuron function to a cell (e.g., a cell in a subject). Those skilled in the art will understand that motor neuron function typically includes membrane excitability, axonal transport, vesicle transport, neurotransmitter release, mitochondrial function, and / or mitochondrial availability.

[0089] In some embodiments, the SMN correcting factor is an agent, e.g., a small molecule or oligonucleotide (e.g., an antisense oligonucleotide), that increases expression of functional SMN protein, e.g., by promoting inclusion of exon 7 in SMN2 mRNA transcripts. In some embodiments, the cell is a cell within a subject, e.g., a subject to which the SMN correcting factor is administered. In some embodiments, the SMN correcting factor increases the relative amount of SMN2 mRNA that includes exon 7 compared to SMN2 mRNA that does not include exon 7 in a cell, e.g., a cell in a subject. In some embodiments, an "effective amount" of an SMN correcting factor increases the amount of correctly spliced ​​SMN2 mRNA in a cell (e.g., a cell in a subject) by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% of the SMN2 mRNA in the cell. , 90%, 95%, 99%, or more contain exon 7. In some embodiments, an "effective amount" of an SMN modifying factor increases the level of SMN2 mRNA containing exon 7 in a subject by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more. In some embodiments, an "effective amount" of an SMN modifying factor increases the level of functional SMN protein in a subject by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more. Improving muscle function by targeting myostatin - myostatin inhibitors

[0090] One therapeutic approach to improving patient motor function is to directly target skeletal muscle to reduce muscle atrophy and therefore improve muscle strength in subjects with muscle conditions, such as SMA. Inhibition of myostatin (also known as growth differentiation factor 8 or GDF-8) offers a promising approach to increasing muscle mass and function in patients with muscle conditions, such as SMA. Myostatin is a member of the TGFβ superfamily and is a key negative regulator of muscle growth. Genetic loss of myostatin results in a significant increase in muscle mass, which is due to both myocyte hypertrophy and hyperplasia (29). Similarly to myostatin loss-of-function mutations, pharmacological inhibition of myostatin increases muscle mass, but this is mediated by muscle hypertrophy rather than hyperplasia (30). Additionally, evidence from animal models suggests that blocking myostatin signaling prevents muscle atrophy associated with limb immobilization, cancer cachexia, and corticosteroid treatment (31-34). Since its initial description in knockout mice, myostatin mutations and associated muscle hypertrophy have been identified in cattle, dogs, and humans. Loss of myostatin is not thought to cause any adverse effects (35-37).

[0091] The profound effect of myostatin loss on muscle mass, along with the lack of pathology observed with myostatin mutations, suggests that this growth factor is an important therapeutic target for conditions in which muscle wasting is a prominent feature, including sarcopenia, cancer cachexia, muscular dystrophies, and disuse atrophy (38). Several companies are pursuing various approaches to inhibit myostatin and thus increase muscle mass and strength. The most common approaches to myostatin inhibition are (1) antibodies that bind and inhibit the mature growth factor (commonly referred to as "neutralizing" antibodies), (2) antibodies against the myostatin receptor ActRIIB, (3) soluble ligand traps such as ActRIIB-Fc, and (4) viral-mediated expression of myostatin inhibitors such as follistatin (39-43). However, in addition to targeting myostatin, many of these therapies also inhibit related family members such as GDF11 and activin. Because the amino acid sequences of mature myostatin and GDF11 are 90% identical, generating antibodies that specifically bind to myostatin but not GDF11 is extremely challenging. Because myostatin, GDF11, and activin all signal through ActRIIB, antibodies that block ActRIIB or soluble ActRIIB ligand traps will inhibit the activity of all three growth factors (44). Follistatin, an endogenous inhibitor of myostatin, also binds and inhibits GDF11 and activin (45). Some of these molecules also bind, albeit with lower affinity, to less related growth factors such as BMP9 and BMP10. This lack of specificity has the potential for unwanted side effects. While the clear role of GDF11 during development has been elucidated, its postnatal biological role and the effects of GDF11 inhibition remain unclear. GDF11 has been suggested to be both a pro- and anti-aging factor, and to be beneficial and detrimental to muscle growth and regeneration (46-53). Activin A regulates the release of follicle-stimulating hormone (FSH), ovarian follicle development, and ovarian fibrosis. Myostatin is important for multiple reproductive functions, including growth, development, and postmenstrual endometrial repair (54). BMP9 is involved in maintaining vascular epithelial integrity, and inhibition of this ligand is thought to be linked to the telangiectasia and gingival bleeding observed in patients treated with ACE-031, an ActRIIb-Fc fusion (41). Collectively, these observations point to the importance of developing selective inhibitors of myostatin signaling to minimize the risk of adverse effects that may be caused by unintended inhibition of one or more signaling pathways of related growth factors. The inventors of the present disclosure recognized that the specificity of myostatin inhibition is particularly important for minimizing the risk of toxicity of treatment in younger patients who are still growing, as well as in patients who, in some cases, must undergo long-term therapy, including lifelong management of the disease.

[0092] Although various emerging myostatin inhibitors, including those listed above, have all significantly increased muscle mass and strength in rodents, none have succeeded in achieving their primary clinical endpoints in patients (31, 32, 34, 38, 44, 55-57). Pfizer's MYO-029, an anti-myostatin antibody, has been shown to have poor pharmacological properties, which likely contributed to its clinical failure (58). Acceleron's ACE-031 was discontinued due to adverse bleeding events resulting from BMP9 inhibition (41). In other cases, the underlying causes of failure are less clear. For example, several companies are using these molecules in clinical trials in elderly patients (e.g., older, frail, fall-prone individuals with sarcopenia). Patients in these trials have shown modest increases in muscle mass (approximately 2-3%), but without corresponding improvements in muscle strength (38, 40, 59). One possibility for this non-association is that these trials were not long enough to detect improvements in muscle function. It's possible that the central nervous system needs additional time to adapt to larger muscles. Alternatively, the reduced anabolic capacity of older adults (i.e., reduced IGF-1 and testosterone expression, reduced muscle protein synthesis) may have limited the effectiveness of myostatin inhibition in this population (60, 61). Taken together, these results caution that even when some muscle growth is achieved, it does not necessarily translate into improved muscle function (e.g., strength, force production) and therefore motor function, such as the ability to perform a specific motor task.

[0093] As used herein, the term "myostatin inhibitor" refers to any agent capable of blocking or antagonizing myostatin signaling. Such agents can include small molecule antagonists of myostatin and biological antagonists of myostatin (e.g., protein fragments and antibodies). In some embodiments, the myostatin inhibitor can be an antibody (including fragments thereof, such as domain antibodies (dAbs) as described in, for example, U.S. Patent Nos. 6,291,158, 6,582,915, 6,593,081, 6,172,197, and 6,696,245), a small molecule inhibitor, an adnectin, an affibody, a DARPin, an anticalin, an avimer, a versabody, or a gene therapy agent. Myostatin inhibitors or antagonists known in the art to date include, but are not limited to, PF06252616 (Pfizer), Trevogrumab (Regneron), ACE-083 (Acceleron), BMS-986089 (BMS), follistatin (Nationwide), ACE-031 (Acceleron), Myo-029 (Wyeth), LY2495655 (Eli Lilly), Pinta-745 (Atara), Bimagrumab / BYM338 (Novartis), and the anti-latent myostatin antibodies described in PCT / JP2015 / 006323 (Chugai Pharmaceutical) or any derivatives thereof (such as affinity matured derivatives or humanized derivatives or fragments), and the anti-myostatin antibodies described herein, such as SRK-015 or antigen-binding fragments thereof. The use of myostatin inhibitors encompassed by the present invention may also be achieved by administering them as monoclonal antibodies. These include antibody mimetics such as monobodies and single-domain antibodies. Monobodies are synthetic binding proteins that typically use the fibronectin type III domain (FN3) as a molecular scaffold. Monobodies include Adnectin™, which is based on the 10th fibronectin type III domain. One example of an Adnectin is BMS-986089.

[0094] Preferably, the myostatin inhibitor is myostatin-selective. Many agents described in the literature as blocking myostatin activity have been found to be non-selective. Indeed, many such agents also affect other related growth factors, particularly GDF11, which shares high sequence homology (approximately 90% identity) with myostatin. To reduce the potential for adverse effects, agents that specifically block myostatin signaling (without affecting other growth factor signaling) are preferred. This may be particularly important for pediatric populations and young adults who are still growing and anabolically active. For example, GDF11 plays a critical role during early development. Therefore, preserving intact GDF11 signaling during individual development may be important to avoid disruption of normal developmental processes. Similarly, highly selective intervention of myostatin signaling over other biological pathways may be advantageous in situations where long-term therapy, which may in some cases include lifelong treatment, is warranted. In this way, any undesirable side effects and toxicities that accumulate over time and cause long-term adverse effects can be minimized or prevented.

[0095] Suitable inhibitors of myostatin include biologics such as antibodies (e.g., SRK-015). Biologics include: i) classes of antibodies or antigen-binding fragments thereof that inhibit the activation step of myostatin from its precursor; ii) classes of antibodies or antigen-binding fragments thereof that neutralize mature myostatin activity; and iii) classes of antibodies or antigen-binding fragments thereof that block the interaction of myostatin with its receptor. In some embodiments, antibodies in (i) above are preferred. Non-limiting examples of such antibodies are disclosed, for example, in PCT / US2015 / 059468 and PCT / US2016 / 052014, each of which is incorporated herein by reference in its entirety. In some embodiments, antibodies or antigen-binding portions thereof suitable for practicing the present invention comprise one or more CDR sequences, variable heavy and light chain sequences, or heavy and light chain sequences selected from those set forth in Table 1, Table 2, or Table 3 below. [Table 1] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 3-1] [Table 3-2] [Table 3-3]

[0096] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable domain (CDRH3) comprising a complementarity determining region 3 comprising the sequence set forth in any one of SEQ ID NOs: 10-11. In one embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable domain (CDRL3) comprising a complementarity determining region 3 comprising the sequence set forth in any one of SEQ ID NOs: 22-23. In one embodiment, the antibody or antigen-binding fragment thereof comprises six complementarity determining regions (CDRs): CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, where CDRH1 comprises the sequence set forth in any one of SEQ ID NOs: 1-3. , CDRH2 comprises the sequence shown in any one of SEQ ID NOs: 4 to 9, CDRH3 comprises the sequence shown in any one of SEQ ID NOs: 10 to 11, CDRL1 comprises the sequence shown in any one of SEQ ID NOs: 12 to 17, CDRL2 comprises the sequence shown in any one of SEQ ID NOs: 18 to 21, and CDRL3 comprises the sequence shown in any one of SEQ ID NOs: 22 to 23.

[0097] In one embodiment, CDRH1 comprises the sequence set forth in SEQ ID NO: 1 or 2, CDRH2 comprises the sequence set forth in SEQ ID NO: 4 or 5, CDRH3 comprises the sequence set forth in SEQ ID NO: 10, CDRL1 comprises the sequence set forth in SEQ ID NO: 12 or 13, CDRL2 comprises the sequence set forth in SEQ ID NO: 18 or 19, and CDRL3 comprises the sequence set forth in SEQ ID NO: 22.

[0098] In one embodiment, CDRH1 comprises the sequence set forth in SEQ ID NO: 1 or 3, CDRH2 comprises the sequence set forth in SEQ ID NO: 6 or 7, CDRH3 comprises the sequence set forth in SEQ ID NO: 11, CDRL1 comprises the sequence set forth in SEQ ID NO: 14 or 15, CDRL2 comprises the sequence set forth in SEQ ID NO: 20 or 21, and CDRL3 comprises the sequence set forth in SEQ ID NO: 23.

[0099] In one embodiment, CDRH1 comprises the sequence set forth in SEQ ID NO: 1 or 3, CDRH2 comprises the sequence set forth in SEQ ID NO: 8 or 9, CDRH3 comprises the sequence set forth in SEQ ID NO: 11, CDRL1 comprises the sequence set forth in SEQ ID NO: 16 or 17, CDRL2 comprises the sequence set forth in SEQ ID NO: 20 or 21, and CDRL3 comprises the sequence set forth in SEQ ID NO: 23.

[0100] In a preferred embodiment, a suitable monoclonal antibody for use in the present invention is SRK-015. SRK-015 binds to the "arm" region within the prodomain of the pro / latent myostatin complex and inhibits the release of mature growth factors (i.e., GDF-8) from the latent / inactive complex. The myostatin arm region is provided herein as SEQ ID NO: 116 (RELIDQYDVQRDDSSDGSLEDDDYHATTETIITMPTESDFLMQVDGKPKCCFFKFSSKIQYNKVVKAQLWIYLRPVETPTTVFVQILRLIKPMKDGTRYTGIRSLKLDMNPGTGIWQSIDVKTVLQNWLKQPESNLGIEIKALDENGHDLAVTFPGPGEDGLNPFLEVKVTDTPKRSRR). Other domains of myostatin are known to those of skill in the art and are listed, for example, in at least Table 2 of WO 16 / 073879, published May 12, 2016, the entire contents of which are expressly incorporated herein by reference.

[0101] SRK-015 binding is specific to pro- / latent myostatin; therefore, SRK-015 does not bind to mature GDF-8 or GDF-11 (or any other members of the TGFβ superfamily of growth factors), thereby allowing selective targeting of myostatin signaling without affecting other biological pathways. In addition to this binding specificity, SRK-015 has demonstrated favorable pharmacokinetic (PK) and pharmacodynamic (PD) properties in both mice and non-human primates (see Examples 8 and 9). In some embodiments, suitable dosages of SRK-015 for administration to human patients to treat SMA range from 1 to 30 mg / kg, e.g., 1 to 5 mg / kg, 3 to 5 mg / kg, 3 to 10 mg / kg, 5 to 10 mg / kg, 5 to 15 mg / kg, 5 to 20 mg / kg, 10 to 20 mg / kg, etc. In some embodiments, patients are administered SRK-015 once a week, once every two weeks, once every three weeks, once a month, once every six weeks, etc. Combination therapy for treating spinal muscular atrophy

[0102] Surprisingly, we discovered that target muscles can be more responsive to myostatin inhibition in the presence of modifiers designed to promote or improve motor neuron function, based on the recognition that neural input contributes to muscle function, leading to improved motor function. Additionally, the most severe forms of SMA typically present early in very young children, a population with robust anabolic abilities that are generally well-suited for myostatin intervention. Furthermore, SMA patients have difficulty completing simple motor activities and tasks that often involve the function of fast-twitch muscle fibers. In summary, SMA is a clinical condition that would benefit from myostatin inhibition if SMN modifiers are present in the underlying environment.

[0103] Thus, the present invention provides combination therapies for treating neuromuscular disorders, such as SMA, that achieve improved clinical benefits for patients compared to monotherapy with each agent alone. Specifically, targeting affected muscles with a specific inhibitor of myostatin signaling to enhance muscle function, combined with a corrective agent aimed at improving the patient's motor neuron function, results in a more beneficial clinical outcome compared to either agent alone. Such effects may be complementary, additive, or synergistic compared to monotherapy. A combination therapy that provides a complementary effect means that the total clinical benefit provided by the combination is greater than the clinical benefit of a single therapy (e.g., either neuron therapy alone or myostatin inhibitor therapy alone). A combination therapy that provides an additive effect means that the clinical benefit of the combined agents reflects the sum of the individual therapies. A combination therapy that provides a synergistic effect means that the overall benefit achieved by the combination is greater than the additive effect of each agent alone. Furthermore, in some embodiments, the additive or synergistic effect of the combination therapy may allow for less frequent dosing of one or more of the therapeutic agents and / or the use of lower doses of one or more of the therapeutic agents compared to administration of the monotherapeutic agents alone. In other embodiments, lower dosages and / or less frequent dosing of one or more of the therapeutic agents of the combination therapy may result in reduced toxicity due to fewer side effects from one or more of the therapeutic agents. Thus, in some embodiments, the effective amount of an agent (such as an SMN modifier) ​​used as a component of a combination therapy to treat a disease of interest (such as SMA) is less than the effective amount of the same agent used as a monotherapy.

[0104] Thus, one aspect of the present invention provides the use of a myostatin inhibitor to treat SMA, particularly in patients who are also receiving therapy to address motor neuron loss, such as an SMN-modifying factor. Accordingly, the present invention encompasses a method for treating spinal muscular atrophy (SMA) in a subject who is being treated with an SMN-modifying factor, the method comprising administering a myostatin inhibitor.

[0105] In some embodiments, the myostatin inhibitor comprises an antibody or antigen-binding fragment thereof that binds to mature myostatin, a pro-form of myostatin (e.g., pro- and / or latent myostatin), or a myostatin receptor in an amount effective to treat SMA. In some embodiments, a suitable antibody binds to mature myostatin but also binds to GDF11. In some embodiments, a suitable antibody selectively binds to mature myostatin but does not bind to GDF11. In some embodiments, a suitable antibody binds to mature and latent myostatin but does not bind to pro-myostatin. In some embodiments, a suitable antibody binds to pro- and latent myostatin but does not bind to mature myostatin. In some embodiments, such antibodies inhibit a step in myostatin activation by stabilizing the pro-myostatin complex. In some embodiments, such antibodies inhibit a step in myostatin activation by interfering with one or more steps of proteolysis. For example, in some embodiments, such antibodies inhibit protease-dependent cleavage of the myostatin prodomain. In some embodiments, the protease is a furin or furin-like protease, or a tolloid or tolloid-like protease. In ... The antibody may be pH sensitive in that it binds to the antigen and dissociates at acidic pH.

[0106] According to the present invention, myostatin inhibitors can be administered to SMA patients who are either responsive, hyporesponsive, or non-responsive to SMN-modifying factor therapy. In the case of hyporesponsive or non-responsive individuals, simultaneous inhibition of myostatin signaling can improve neuromuscular signaling, in part by enhancing muscle function, thereby making the innervating motor neurons of non-responsive individuals more responsive to SMN-modifying factors. Without wishing to be bound by any particular theory, it is contemplated that enhancement of the muscle component can affect the neuronal component through positive feedback, and vice versa, due to the bidirectional nature of neuromuscular signaling.

[0107] Although SMN modifying factor low responders and / or non-responders may benefit from myostatin inhibition, a more preferred patient population nevertheless includes those who are SMN modifying factor responders. It is contemplated that myostatin inhibition therapy used in combination with SMN modifying factor therapy may further improve motor function in such individuals.

[0108] "Combination therapy" in this invention is intended to mean that the pharmacological effects of one drug (such as an SMN modifier) ​​overlap in vivo with the pharmacological effects of another drug (such as a myostatin inhibitor). Thus, the two drugs need not be administered as a single formulation, nor at the same time, nor by the same route. For example, depending on the PK / PD of each drug, it is contemplated that patients will generally receive an SMN modifier and a myostatin inhibitor within six months of each other for best results.

[0109] As discussed above, suitable "SMN correctors" include splice modifiers, SMN gene replacement or gene therapy agents, SMN transcriptional enhancers, SMN protein translation enhancers, and SMN protein stabilizers. In some embodiments, such SMN correctors may be small molecule agents, biologics, or nucleic acids. In some embodiments, the SMN corrector is a small molecule splice modifier of Smn2. In some embodiments, the SMN corrector is an antisense RNA splice modifier of Smn2. In some embodiments, gene therapy involves introducing one or more transgenes into a patient. In some embodiments, gene transfer is achieved by using a suitable vector, such as a viral vector and a lipid-based carrier. In the case of viral vector-mediated gene delivery, gene therapy may involve using a particular serotype for initial treatment and then a different serotype for subsequent treatments to minimize adverse immune responses in the subject. In some embodiments, gene therapy involves targeted genome editing, such as CRISPR / Cas9 technology or modifications thereof. Non-limiting examples of SMN modifiers used in combination with myostatin inhibitors according to the present disclosure include, but are not limited to, the following: nusinersen (Biogen), AVXS-101 (AveXis), RG7916 (Roche / PTC / SMAF), RG7800 (Roche / PTC), olesoxime (Roche / Trophos), VY-SMN101 (Voyager / Genzyme), LMI070 (Novartis), SMN gene therapy (Genzyme / Sanofi), and antisense oligonucleotides (RaNA). Patient population

[0110] The combination therapies described herein may be suitable for treating any type of SMA in a subject, including SMA Types I, II, III, and IV. "Subject," "individual," or "patient" are used interchangeably herein and refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, mice, rats, monkeys, humans, farm animals, sport animals, and pets. In embodiments, the subject is a human. It is subject to approval.

[0111] Patient populations that can benefit from the therapies described herein include those with non-ambulatory SMA and those with ambulatory SMA. These include patients with SMA type I, SMA type II, SMA type III, or SMA type IV. In some embodiments, the subject has type II SMA. In some embodiments, the subject has non-ambulatory type III SMA. In some embodiments, the subject has type I SMA. In some embodiments, the subject has ambulatory type III SMA.

[0112] In some embodiments of the present invention, combination therapy including a myostatin inhibitor therapy (e.g., a myostatin inhibitor) and a neuronal therapeutic agent (e.g., an SMN modifying agent) is considered for non-ambulatory forms of SMA, such as Type I, Type II, and non-ambulatory Type III SMA. In other embodiments, myostatin inhibitor monotherapy is considered for ambulatory forms of SMA, such as ambulatory Type III and Type IV SMA. In addition, myostatin inhibitor monotherapy may be suitable for treating subjects identified by genetic screening as carriers of SMN gene mutations. Such genetic screening can be performed on newborn / infant subjects as well as in utero (e.g., fetuses). Because disease severity is highly dependent on the copy number and expression of the Smn2 gene, genotyping alone may be insufficient to distinguish very young patients between those who will eventually develop severe forms of SMA and those who will develop milder forms of SMA. For this and other reasons, the decision to initiate a neuronal therapeutic agent, such as an SMN modifying agent, may be considered premature. Nevertheless, identifying mutations in Smn1 may justify early pharmacological intervention, including myostatin inhibition, which may provide clinical benefit in the meantime.

[0113] In some embodiments, the subject with SMA is between 0 and 6 months of age. The subject with SMA may be between 6 and 15 months of age. In other embodiments, the subject with SMA may be <3 years of age. In other embodiments, the subject with SMA may be >3 years of age.

[0114] In one embodiment, the subject has been identified as a carrier of an SMN mutation in a manner known to those of skill in the art, including genetic screening, e.g., has a mutation in the SMN gene associated with SMA, hi one embodiment, the subject has been identified as a carrier of an SMN mutation, e.g., by genetic screening, either in utero or as an infant.

[0115] As used herein, a subject may suffer from partial damage to neuromuscular function.Neuromuscular function and / or neuromuscular damage are measured using methods generally known to those skilled in the art and described in more detail herein.For example, damage to neuromuscular function may be measured using compound muscle action potential (CMAP), which measures the success of muscle contraction in response to nerve stimulation.In addition, damage to neuromuscular function may be measured using motor unit number estimation (MUNE), which determines the number of motor units that form a given nerve.

[0116] The methods described herein may further comprise selecting a subject. In some embodiments, the subject has or is at risk of developing a muscle condition or disorder, such as SMA. In some embodiments, the subject has or is at risk of developing a disease or disorder associated with impaired neurological signal transduction. In one embodiment, the subject can be selected based on genetic screening, for example, identifying a gene mutation associated with a disease, such as SMA, for example, a gene mutation in SMN. In one embodiment, the subject can be selected based on genetic screening within 24 hours of birth. In another embodiment, the subject can be selected based on genetic screening in utero. It is possible. Dosage and Administration

[0117] To practice the methods disclosed herein, an effective amount of the pharmaceutical composition described above can be administered to a subject (e.g., a human) in need of treatment by a suitable route, such as intravenous administration, e.g., as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, inhalation, or topical routes.

[0118] The terms "administer," "administering," or "administration" include any method for delivering an antibody or antigen-binding fragment thereof, e.g., a pharmaceutical composition comprising such an antibody or antigen-binding fragment or agent, to a subject's system or to a specific area in or on a subject (systemic administration and local administration, respectively).

[0119] In some embodiments, a subject is administered a myostatin inhibitor and / or SMN modifier approximately weekly, biweekly, monthly, etc. Typically, suitable dosages of myostatin inhibitors include between about 0.1 and 30 mg / kg. Empirical considerations, such as half-life, will generally contribute to determining the dosage. Such myostatin inhibitors can be administered by intravenous injection / infusion. In some embodiments, such myostatin inhibitors can be administered subcutaneously, e.g., under the skin. In other embodiments, myostatin inhibitors can be administered intrathecally, e.g., intraspinal. Similarly, SMN modifiers, e.g., splice modifiers, can be administered orally, e.g., by mouth.

[0120] In one embodiment, the subject is receiving an SMN modifying factor prior to administration of the myostatin inhibitor. In another embodiment, the subject is receiving an SMN modifying factor simultaneously with administration of the myostatin inhibitor. In another embodiment, the subject will receive an SMN modifying factor after administration of the myostatin inhibitor.

[0121] In one embodiment, the subject receives the SMN modifying factor within 6 months of receiving the myostatin inhibitor. In one embodiment, the subject receives the SMN modifying factor within 3 months of receiving the myostatin inhibitor. In one embodiment, the subject receives the SMN modifying factor within 6 months, 5 months, 4 months, 3 months, 2 months, or 1 month of receiving the myostatin inhibitor. In one embodiment, the subject receives the SMN modifying factor within 4 weeks, 3 weeks, 2 weeks, or 1 week of receiving the myostatin inhibitor. In one embodiment, the subject receives the SMN modifying factor on the same day as receiving the myostatin inhibitor.

[0122] In one embodiment, the subject will receive the SMN modifying factor within 6 months of receiving the myostatin inhibitor. In one embodiment, the subject will receive the SMN modifying factor within 3 months of receiving the myostatin inhibitor. In one embodiment, the subject will receive the SMN modifying factor within 6 months, 5 months, 4 months, 3 months, 2 months, or 1 month of receiving the myostatin inhibitor. In one embodiment, the subject will receive the SMN modifying factor within 4 weeks, 3 weeks, 2 weeks, or 1 week of receiving the myostatin inhibitor.

[0123] In one embodiment, the SMN modifying factor component of combination therapy is antisense nucleotide, and is administered to the central nervous system of the target by intrathecal injection.In one embodiment, antisense nucleotide is administered to the target every few months, for example, once a month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months or every 12 months.In another embodiment, initial treatment can comprise more frequent dose administration, and then less frequent maintenance dose administration thereafter.

[0124] In another embodiment, the SMN modifier component of the combination therapy is a small molecule and is orally administered to the subject. In one embodiment, the small molecule is administered to the subject once daily. In another embodiment, the small molecule is administered to the subject once weekly, once every two weeks, or once monthly.

[0125] In one embodiment, the SMN corrector component of the combination therapy is a gene therapy and is administered by intravenous injection. In one embodiment, the SMN corrector is a gene therapy and is administered by intrathecal injection. In one embodiment, the initial treatment may involve more frequent doses followed by less frequent maintenance doses. Less frequent maintenance doses may be preferred to avoid an inappropriate immune response to the gene therapy.

[0126] In one embodiment, the myostatin inhibitor component of the combination therapy is administered to the subject by intravenous administration. In one embodiment, the myostatin inhibitor component of the combination therapy is administered to the subject by oral administration. In one embodiment, the myostatin inhibitor component of the combination therapy is administered to the subject by subcutaneous injection. In one embodiment, the myostatin inhibitor is administered to the subject once daily, once weekly, once every two weeks, or once monthly. In one embodiment, initial treatment may involve more frequent dose administration, followed by less frequent maintenance doses.

[0127] An "effective amount" for treating SMA, as used herein, may be an amount that achieves clinical efficacy, including, but not limited to, delaying or attenuating muscle atrophy, delaying the loss of alpha-motor neurons, preventing or reducing the expression of muscle markers, preventing, attenuating, or delaying intramuscular fat deposition (fatty replacement of muscle tissue), preventing or delaying the use of a ventilator / respirator, delaying the time until a patient becomes wheelchair-bound, increasing the Expanded Hammersmith Motor Scale score by ≥ 1 point compared to an untreated control group or ≥ 1 point from a baseline measured before modifying factor treatment, delaying the progressive decline of the Expanded Hammersmith Motor Scale over 12, 24, or 36 months, CHOP Increase in INTEND score by ≥ 3 points compared to untreated controls, increase in MFM-32 score by at least 1 point compared to untreated controls, delay in transition from ambulatory to non-ambulatory SMA, reduction in hospitalizations, etc. Each of these measures is described in more detail below.

[0128] As used herein, the term "treating" refers to the application or administration of a composition containing one or more active agents to a subject having a disease / disorder, symptom of a disease / disorder, or predisposition to a disease / disorder associated with myopathy, for the purpose of curing, relieving, alleviating, altering, remedying, ameliorating, improving, or affecting the disorder, symptom of the disease, or predisposition to the disease / disorder.

[0129] Although combination therapy comprising myostatin inhibitors and neuronal modifying factors is generally preferred, in some cases, monotherapy of myostatin inhibitors may be considered.Suitable patient populations for which such monotherapy is considered include those with milder forms of SMA, such as ambulatory type III SMA and type IV (e.g., adult-onset) SMA.Based on the requirements of criterion (ii) discussed in detail above, for example, patients with ambulatory ability retain sufficient neuromuscular function.Therefore, myostatin inhibition can provide the benefit of enhancing the muscle function of such patients even in the absence of simultaneous neuronal modifying factor therapy.Therefore, monotherapy comprising myostatin inhibitors for treating milder forms of SMA (e.g., ambulatory SMA) is encompassed by the present invention.

[0130] In some embodiments, myostatin inhibitor monotherapy for treating patients with ambulatory Type III SMA can help slow disease progression to non-ambulatory SMA. In some embodiments, patients receiving myostatin inhibitor monotherapy can be compared with a control group (similar In some cases, motor function can be preserved or even improved compared to patients (diagnosed with SMA but not receiving monotherapy). However, it should be noted that such patients respond to myostatin inhibitors in the absence of a neurotherapeutic agent that can boost the benefits of myostatin inhibition. In some embodiments, myostatin inhibitor monotherapy for treating ambulatory patients with SMA comprises administering to the subject an amount of a myostatin inhibitor effective to produce a clinically meaningful outcome. In such embodiments, a clinically meaningful clinical outcome corresponds to an improved expanded Hammersmith Functional Motor Scale score that is at least 1 point (≧1) higher than an untreated control group or at least 1 point (≧1) higher than a baseline measured before myostatin inhibitor treatment. In some embodiments, a meaningful clinical outcome may correspond to an improved expanded Hammersmith Functional Motor Scale score that is at least 1 point (≧1) higher than a baseline or modified value measured before receiving SMA modifying factor therapy or myostatin inhibitor therapy, respectively. In other embodiments, the expanded Hammersmith Functional Motor Scale score is at least 2 points (≧2), at least 3 points (≧3), at least 4 points (≧4), at least 5 points (≧5), at least 6 points (≧6), at least 7 points (≧7), at least 8 points (≧8), at least 9 points (≧9), at least 10 points (≧10), at least 12 points (≧12), at least 15 points (≧15), at least 20 points (≧20), at least 25 points (≧25), at least 30 points (≧30), at least 35 points (≧35), at least 40 points (≧40), at least 45 points (≧45), at least 50 points (≧50), or at least 60 points (≧60) higher than the baseline or modified value measured before receiving SMN modifying factor therapy or myostatin inhibitor therapy, respectively. In some embodiments, a meaningful clinical outcome may correspond to an improved expanded Hammersmith Functional Motor Scale score that is at least 1 point (≧1) higher than an untreated control group.In some embodiments, a meaningful clinical outcome may correspond to an improved Expanded Hammersmith Functional Motor Scale score that is at least 2 points (≧2), at least 3 points (≧3), at least 4 points (≧4), at least 5 points (≧5), at least 6 points (≧6), at least 7 points (≧7), at least 8 points (≧8), at least 9 points (≧9), or at least 10 points (≧10), at least 12 points (≧12), at least 15 points (≧15), at least 20 points (≧20), at least 25 points (≧25), at least 30 points (≧30), at least 35 points (≧35), at least 40 points (≧40), at least 45 points (≧45), at least 50 points (≧50), or at least 60 points (≧60) higher than an untreated control group.

[0131] In some embodiments, the myostatin inhibitor therapy provided herein can help maintain disease status in a patient population receiving a myostatin inhibitor compared to a control group not receiving a myostatin inhibitor. Maintaining disease status refers to preventing further deterioration of affected muscles or delaying or slowing the rate of disease progression, as assessed, for example, by changes in the patient's motor function over time. Thus, even if no improvement in motor function test scores is demonstrated, myostatin inhibitor therapy can provide clinical benefit by countering disease progression. Thus, such clinical benefit may manifest over a longer period of observation, until a patient population treated with a myostatin inhibitor maintains their previous test scores or shows a slower rate of score reduction over time, compared to a control group. Biological effects of myostatin inhibitors and SMN modifiers

[0132] The clinical effects of myostatin inhibitors, alone or in combination with SMN modifiers described herein, can be monitored and / or efficacy assessed by various means. Exemplary such biologically beneficial effects are provided herein. Beneficial biological effects in elephants can be achieved by administering a myostatin inhibitor in combination with an SMN-modifying factor. In some embodiments, the myostatin inhibitor and / or SMN-modifying factor are administered in an amount effective to produce one or more of the biological effects described below.

[0133] The ability to assess functional scales that can be reliably measured in SMA patients is important for tracking the disease progression of patients and the effect of therapy over time.Muscle function can be assessed by physiological measurements such as muscle strength and force generation, but motor function scales monitor disease progression in a way that conveys more meaning and relevance to patients' functionality in daily life than measures that quantify strength itself.Not intended to be limiting, the list of some known motor function assessment tests that can be used to evaluate SMA patients is provided below.Other tests include, but are not limited to, gross motor function scale (GMFM), 6-minute walk test, 10-meter walk / run test, floor rise time test, timed up and go test (TUG), and stair climbing test, and these methods are well known to those skilled in the art. Expanded Hammersmith Functional Motor Scale

[0134] The disease severity of patients with SMA, both before, during, and after treatment with the myostatin inhibitors described herein, can be classified using a number of tests and assays known to those skilled in the art. The Hammersmith Functional Motor Scale, Extended (HFMSE) is a validated endpoint for SMA type II and non-ambulatory type III, and is well known to those skilled in the art. The test system includes 33 items (e.g., motor tasks or activities) that assess motor function. Items that are short-duration motor activities primarily driven by muscle strength and require type II fast-twitch muscle fibers include: sitting unsupported for 3 seconds, lying down from a seated position, rotating from supine to prone, performing a 3-second push-up position, rising from a kneeling position, climbing up and down four steps, and jumping forward 12 inches.

[0135] In some embodiments, the subject has a baseline Expanded Hammersmith Functional Rating Scale score of <66, e.g., <65, <60, <55, <50, <40, <35, <30, <25, <20, etc., prior to receiving the modifier or myostatin inhibitor therapy ("baseline"). In one embodiment, the subject has a baseline Expanded Hammersmith Functional Rating Scale score of <50 prior to receiving the modifier or myostatin inhibitor. In one embodiment, the subject has a baseline Expanded Hammersmith Functional Rating Scale score of <40 prior to receiving the modifier or myostatin inhibitor. In one embodiment, the subject has a baseline Expanded Hammersmith Functional Rating Scale score of <35 prior to receiving the modifier or myostatin inhibitor. In one embodiment, the subject has a baseline Expanded Hammersmith Functional Rating Scale score of <30 prior to receiving the modifier or myostatin inhibitor. In one embodiment, the subject has a baseline expanded Hammersmith Functional Motor Scale score of ≦25 before receiving the modifier or myostatin inhibitor. In one embodiment, the subject has a baseline expanded Hammersmith Functional Motor Scale score of ≦20 before receiving the modifier or myostatin inhibitor.

[0136] In some embodiments, the subject has an increased (or "modified") Expanded Hammersmith Functional Motor Scale score after SMN modifying factor therapy. In some embodiments, the subject has an increased (or "modified") score after receiving SMN modifying factors that is at least 3 points, at least 4 points, at least 5 points, at least 6 points, at least 7 points, at least 8 points, at least 9 points, at least 10 points, or less than baseline. In some embodiments, the subject has an increased Expanded Hammersmith Functional Motor Scale score after SMN modifying factor therapy. In some embodiments, the subject improves their score by at least 3 points, at least 4 points, at least 5 points, at least 6 points, at least 7 points, at least 8 points, at least 9 points, at least 10 points, at least 11 points, at least 12 points, at least 13 points, at least 14 points, or at least 15 points. In some embodiments, upon further treatment with a myostatin inhibitor (i.e., combination therapy), the subject further improves their score by at least 3 points, at least 4 points, at least 5 points, at least 6 points, at least 7 points, at least 8 points, at least 9 points, or at least 10 points, either from a baseline measured before receiving SMN modifying factor therapy or compared to an untreated control group.

[0137] For example, many non-ambulatory SMA patients have baseline Hammersmith scores ranging from 15 to 30 points out of a total of 66 points. As a result of SMN modifying factor therapy, such patients can improve their scores by an average of 4 to 10 points over their corresponding baseline. With combination therapy including a myostatin inhibitor, such patients can further improve their scores. In some embodiments, such patients improve their expanded Hammersmith Functional Rating Scale scores by 1 to 20 points over their corresponding baseline. In some embodiments, such patients improve their expanded Hammersmith Functional Rating Scale scores by at least 1 point over their already-corrected scores measured after SMN modifying factor therapy.

[0138] It should be noted that in SMA patients, differences of as little as one point between various motor test scoring systems can be clinically significant. To appreciate this, an illustrative example based on the standard HFMSE system test items in a non-ambulatory SMA patient is provided below.

[0139] Tasks 1 and 2 of the HFMSE exam involve sitting upright (without back support) for 3+ seconds. Two points are awarded if the patient can sit for a count of three or more without hand support, one point if they can maintain balance for a count of three using one hand for support, and no points if they need both hands for balance. In real-life settings, the difference between being able to sit without using the hands for support (2 points) and needing to use one hand even for balance (1 point) is significant because in the former case, the patient can use both hands to perform activities (e.g., holding objects) while sitting upright. Task 3 of the exam assesses whether the patient can raise one hand to touch the head above ear level while sitting. The ability to perform this seemingly simple task can mean the difference between being able to comb one's hair or put on a hat without assistance. Motor Function Scale (MFM)

[0140] The Motor Function Scale (MFM) test provides a genetic scale for assessing various parameters of motor function in SMA patients with varying degrees of disease severity, including ambulatory and non-ambulatory children and adults between approximately 6 and 62 years of age. Multiple revisions of the MFM exist for use in various patient populations. For example, the MFM32 is suitable for children older than 6 years of age, and a modified version, the MFM20, has been validated in children younger than 6 years of age. The MFM has been successfully used in clinical trials to monitor or detect changes in patient motor function that reflect deterioration over time (see, e.g., clinicaltrials.gov NCT02628743).

[0141] In one embodiment, a subject with SMA who is being administered a therapy or combination therapy described herein exhibits at least a two-fold, at least a three-fold, at least a four-fold, or at least a five-fold increase in MFM score following administration of the therapy or combination therapy. Upper Limb Module (ULM)

[0142] The Upper Limb Module (ULM) test was specifically designed as an add-on module to provide an assessment of arm function. The ULM is intended to capture performance in activities of daily living that are not typically included in measures of gross motor function. The assessment includes nine activities that children can reliably perform and take approximately 10 minutes to complete. The ULM has been used in multicenter settings and clinical trials.

[0143] In one embodiment, a subject with SMA who is being administered a therapy or combination therapy described herein exhibits at least a 2-fold, at least a 3-fold, at least a 4-fold, or at least a 5-fold increase in ULM score following administration of the therapy or combination therapy. Revised Upper Limb Module

[0144] The Revised Upper Limb Module (RULM) allows for the assessment of arm function in individuals with SMA and has demonstrated good validity and reliability, making it suitable for use in clinical studies. The RULM included 20 activities that children as young as 30 months of age could successfully complete. These included tasks such as reaching from the knee to a table, picking up small objects, pressing a button, tearing paper, opening a Ziploc container, raising the hand above the shoulder, and lifting objects of various weights to various heights. Outcome measures are tests used by researchers to evaluate whether a particular treatment in a clinical trial is showing any effect on patients. Using the correct outcome measures is important to ensure that a clinical trial can demonstrate treatment success. According to SMA News Today, the RULM effectively captured progressive muscle loss on the weaker end of the disease spectrum. Six-minute walk test

[0145] The 6-minute walk test (6MWT) has been reported to be a reliable and valid functional assessment of patients with SMA and can capture the fatigue component of the disease. For example, fatigue observed in SMA test patients was reflected in a 17% decrease in walking speed from the first minute to the last minute of the 6MWT.

[0146] In one embodiment, a subject with SMA who is being administered a therapy or combination therapy described herein exhibits at least a two-fold, at least a three-fold, at least a four-fold, or at least a five-fold increase in RULM score following administration of the therapy or combination therapy. CHOP INTEND score

[0147] The CHOP INTEND is a clinician-rated questionnaire developed to assess motor skills in spinal muscular atrophy type 1. Sixteen items are scored from 0 to 4. Global scores range from 0 to 64, with higher scores indicating better motor skills. (Glanzman AM, Mazzone E, Main M, Pelliccioni M, Wood J, Swoboda KJ, Scott C, Pane M, Messina S, Bertini E, Mercuri E, Finkel RS. The Children's Hospital of Philadelphia Infant Test of Neuromuscular Disorders (CHOP) (See CHOPS INTEND): test development and reliability., Neuromuscul Disord., March 2010;20(3):155-61.) The CHOPS INTEND has been validated and shown to be reliable in SMA type 1 subjects. The CHOPS INTEND is derived in part from the TIMP (Test of Infant Motor Function) and is designed to measure motor function in frail infants with neuromuscular disease. This test involves active movement (voluntary movement). This includes assessment of reflex movements (e.g., goal-directed) and evoked reflexes, but does not include assessment of breathing or suckling.

[0148] In one embodiment, a subject with SMA who is being administered a therapy or combination therapy described herein exhibits at least a 2-fold, at least a 3-fold, at least a 4-fold, or at least a 5-fold increase in at least one of the 16 CHOP criteria following administration of the therapy or combination therapy. CMAP test

[0149] Neuromuscular damage can be assessed using compound muscle action potential (CMAP) testing, which provides electrical stimulation of a nerve and records compound muscle action potentials from surface electrodes overlying the muscles supplied by that nerve. This test may involve stimulating the wrist, elbow, and, less frequently, the axillary and brachial plexus.

[0150] The CMAP measures the summed voltage response of individual muscle fiber action potentials. Typically, a CMAP is obtained by placing electrodes on the target muscle and administering supramaximal stimulation (i.e., stimulation with an intensity significantly greater than that required to activate all nerve or muscle fibers in contact with the electrode) repeated every 30–60 seconds for 2–3 minutes until a stable baseline amplitude is achieved. The subject then contracted the target muscle for 2–5 minutes, with brief (3–4 second) pauses every 15 seconds to prevent muscle ischemia. CMAP recordings were made every minute during muscle exercise and every 1–2 minutes after 30 minutes of exercise or until no further decrease in CMAP amplitude was observed. CMAP amplitude is typically measured in millivolts (mV). The percentage amplitude decrease was calculated by subtracting the minimum post-exercise amplitude from the maximum post-exercise amplitude and dividing it by the maximum post-exercise amplitude. In CMAP studies performed in a group of people without muscle disease, CMAP amplitude decreases ranged from 5.4% to 28.8% (average 15%). A decrease in CMAP amplitude of greater than 40% was considered diagnostic of muscle disease.

[0151] In some embodiments, the subject's CMAP amplitude is reduced by at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 90%, or more. In some embodiments, the negative CMAP peak amplitude of a subject with a muscle disease (e.g., SMA) is substantially lower than the corresponding negative CMAP peak amplitude of a subject without a muscle disease (a control subject). In one embodiment, the negative CMAP peak amplitude of a subject with a muscle disease is at least 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 90%, or more lower than the corresponding negative CMAP peak amplitude of a control subject. CMAP testing, as described herein, can be used to determine the effectiveness of therapy by comparing CMAP decline before and after treatment.

[0152] In one embodiment, a subject having SMA and being administered a therapeutic agent or combination therapy described herein exhibits at least a two-fold, at least a three-fold, at least a four-fold, or at least a five-fold increase in CMAP following administration of the therapeutic agent or combination therapy. MUNE exam

[0153] The motor unit number estimation (MUNE) is a test that can be used to determine the approximate number of motor neurons in a muscle or muscle group. The MUNE test provides a calculated value that represents the estimated number of motor neurons or axons (motor control inputs) that supply the muscle or muscle group being tested. In addition, the MUNE test provides a means to measure motor unit size, allowing for tracking of motor neuron loss. The MUNE test is typically most frequently used in neuromuscular disorders such as amyotrophic lateral sclerosis and spinal muscular atrophy.

[0154] Typically, in the MUNE test, bipolar electrodes on the skin surface are used to measure the movement of the electrodes at the site of placement. The nerve was stimulated with enough intensity to activate all of its motor axons, corresponding to activating all of the motor units (i.e., motor neurons or axons) and constituent muscle fibers, resulting in complete depolarization and muscle contraction. The electrical impulses generated by this muscle activity were recorded with electrodes placed on the muscle at the skin's surface. In healthy muscles, all motor units and all of their muscle fibers are activated simultaneously during this test, generating a maximal motor response, the compound motor action potential (CMAP). The amplitude of the CMAP corresponds to the total number of motor units and muscle fibers activated. The amplitudes of the three responses for each site were summed and then divided by 9 to obtain the average single motor unit action potential (SMUP) amplitude. This amplitude was then divided by the maximum compound motor unit action potential (CMAP) amplitude to obtain the MUNE.

[0155] The mean MUNE for normal healthy subjects was 225 (±87), compared with 41.9 (±39) for subjects with, for example, a muscle disease (e.g., ALS or SMA) at baseline. Subjects with a muscle condition or disorder show a clear gradual decline over time, with a mean decline rate as high as approximately 9% per month. In one embodiment, the mean monthly decline rate of MUNE values ​​for subjects with a muscle disease or disorder is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, or greater compared to the corresponding MUNE values ​​for control subjects without muscle disease. It is also conceivable that subjects with muscle disease may have normal CMAP amplitude measurements but MUNE values ​​that are less than 50% of those of control subjects. In one embodiment, subjects with normal CMAP amplitude values ​​have MUNE values ​​that are at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60% or more lower than the corresponding MUNE values ​​of control subjects without muscle disease.

[0156] In one embodiment, a subject with SMA who is administered a therapeutic agent or combination therapy described herein exhibits an increase in MUNE values ​​of at least 2-fold, at least 3-fold, at least 4-fold, or at least 5-fold after administration of the therapeutic agent or combination therapy. Effects on muscle mass and / or function in human subjects

[0157] Administration of a myostatin inhibitor and / or muscle tissue function in a human subject. In some embodiments, the muscle tissue is selected from the group consisting of smooth muscle tissue, skeletal muscle tissue, and cardiac muscle tissue. Smooth muscle tissue is composed of long, tapered cells, is generally unmyelinated, has a much higher actin / myosin ratio, lacks prominent sarcomeres, and differs from striated muscle in that it can contract much shorter than its resting length. Smooth muscle cells are found around the intestine and uterus, particularly in the walls of blood vessels. Cardiac muscle tissue is striated or unmyelinated tissue responsible for the pumping action of the vertebrate heart. Individual cardiac muscle cells do not fuse with each other to form multinucleated structures like striated muscle tissue. Skeletal muscle tissue is under voluntary control. Muscle fibers are syncytial and contain myofibrils and longitudinally aligned sarcomeres. Skeletal muscle fibers exist in two basic types depending on their expression of specific myosin heavy chain (MHC) isoforms: slow-twitch (e.g., type I fibers) and fast-twitch (e.g., type II fibers). Slow-twitch fibers are typically configured to work better aerobically and help enable long-term endurance activities such as long-distance running, while fast-twitch fibers typically fatigue more quickly but are configured to work better anaerobically and are used for powerful explosive movements such as sprinting. The distinction between slow-twitch and fast-twitch fibers is based on histochemical staining for myosin adenosine triphosphatase (ATPase) and myosin heavy chain type. Slow-twitch fibers (primarily type I fibers) are MHC isoform I, and the three fast-twitch isoforms (primarily type II fibers) are MHC isoform IIa, MHC isoform IId, and MHC isoform IIb (S. Schiaffino, J. Muscle Res. Cell. Motil., 10 (1989)). In some embodiments, the mass and / or function of fast-twitch muscle tissue in a human subject is increased. In other embodiments, the mass and / or function of slow-twitch muscle tissue in a human subject is increased.

[0158] In some embodiments, administration of an effective amount of a myostatin inhibitor, such as an antibody or antigen-binding fragment thereof described herein, to a subject can result in an increase in muscle mass. Preferably, such an increase in muscle mass is clinically meaningful to the benefit or otherwise improve the health status of the subject. For example, a clinically meaningful change in muscle mass can improve a patient's mobility, self-care, metabolism, etc. In some embodiments, the increase in muscle mass is an increase in one or more lean muscles. In some embodiments, such an increase in muscle mass is a systemic effect, such that muscles throughout the body or substantially throughout the body show a measurable effect. In other embodiments, the effect is localized to a specific group / type of muscle.

[0159] In some embodiments, muscle tissue, e.g., lean muscle tissue, mass is increased by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100%. In other embodiments, muscle tissue, e.g., lean muscle tissue, mass is increased by at least about 1-5%, 5-10%, 10-20%, 1-30%, 1-40%, 1-50%, 10-50%, 20-30%, 20-60%, 30-80%, 40-90%, or 50-100%. Such increases in muscle mass can be estimated or measured by any suitable known method, including measuring cross-sectional area (e.g., forearm cross-section) by MRI, circumference, diaphragm width (e.g., by ultrasound), etc.

[0160] In some embodiments, administering an effective amount of an antibody or antigen-binding fragment thereof described herein to a subject can result in enhanced muscle function. Muscle function can be assessed by various measures, including, but not limited to, force generation, grip strength (e.g., maximal grip strength), endurance, muscle oxidative capacity, dynamic grip endurance, etc. In some embodiments, serum creatinine levels are used as a biomarker that has been validated to indicate muscle mass, albeit with limited sensitivity.

[0161] In some embodiments, administration of a myostatin inhibitor increases locomotor function in a human subject. In some embodiments, locomotor function in a human subject is increased by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100%. In other embodiments, locomotor function in a human subject is increased by at least about 1-5%, 5-10%, 10-20%, 1-30%, 1-40%, 1-50%, 10-50%, 20-30%, 20-60%, 30-80%, 40-90%, or 50-100%.

[0162] In another embodiment, administration of a myostatin inhibitor increases muscle strength in a human subject. In some embodiments, muscle strength in a human subject is increased by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100%. In other embodiments, muscle strength in a human subject is increased by at least about 1-5%, 5-10%, 10-20%, 1-30%, 1-40%, 1-50%, 10-50%, 20-30%, 20-60%, 30-80%, 40-90%, or 50-100%.

[0163] In some embodiments, administration of a myostatin inhibitor and / or SMN modifying agent can produce clinically meaningful changes in muscle function corresponding to functional enhancement in the patient. In some embodiments, functional enhancement includes improvements in the patient's mobility, self-care, metabolism, etc. . Effect on the level of intramuscular fat deposits

[0164] The administration of myostatin inhibitor and / or SMN modifying factor affects the level of intramuscular fat deposition in human subjects.In one embodiment, the fat replacement of muscle tissue is prevented, alleviated or delayed.As used herein, the term " adipose tissue " refers to the fat that contains the connective tissue that stores fat.Adipose tissue is derived from preadipocytes.

[0165] The mass of adipose tissue can be determined by any method known to those skilled in the art. For example, adipose tissue can be measured by dual-energy X-ray absorptiometry (DXA). Quantification of intramuscular fat deposition can also be determined using magnetic resonance imaging (MRI). For example, MR dual-echo dual-flip angle spoiled gradient recalled (SPGR) MRI technique or three-point Dixon MRI technique can be used to assess the level of intramuscular fat deposition in a subject. The aforementioned MRI techniques and protocols for quantifying intramuscular fat deposition are described, for example, in Leroy-Willig et al., Magnetic Resonance Imaging, Vol. 15, No. 7, pp. 737-744, 1997, and Gaeta et al., Skeletal Radiol, DOI 10.1007 / s00256-011-1301-5. and the contents of these documents are incorporated herein by reference in their entireties. However, it should be recognized that other methods of determining and quantifying intramuscular fat deposits are known in the art and would be apparent to one of ordinary skill in the art.

[0166] In some embodiments, the replacement level of intramuscular fat deposits is reduced by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100% following administration of the therapeutic agent to the subject. Effects on quality of life in human subjects

[0167] Assessment of the quality of life of patients with severe or chronic conditions, such as those with SMA, may include an integrated approach to assessing various aspects of physical, mental, social, and other parameters. Generally, a higher degree of quality of life is associated with factors such as: accessibility to assistive technology, community re-establishment, lower limb functionality and walking and wheelchair mobility, mental health, severity of neurological impairment and autonomic dysfunction, pain management, functional independence and self-care, upper limb strength, and spasticity control. Administration of the myostatin inhibitors described herein increases the quality of life of human subjects, achieving clinically meaningful improvements as measured by standardized quality of life tests / systems.

[0168] Several suitable tests for assessing a patient's quality of life are known in the art, including, but not limited to, the Spinal Cord Independence Measure (SCIM), Functional Independence Measure (FIM), and Incontinence Quality of Life Questionnaire (I-QOL). Questionnaire), Life Satisfaction Questionnaire (LISAT-9, LISAT-11), Quality of Life Index (QLI), Quality of Life Profile for Adults with Physical Disabilities (QOLP-PD, Quality of Life Profile for Adults with Physical Disabilities), Quality of Well-Being (QWB, Quality of Well-Being) and Quality of Well-Being-Self-Administered (QWB-SA, Quality of Well-Being-Self-Administered), Qualiveen, Satisfaction with Life Scale (SWLS, Deiner Scale), Short Form 36 (SF-36), Disease Impact Profile 68 ( SIP68), and the World Health Organization Quality of Life-BREF (WHOQOL-BREF).

[0169] In some embodiments, quality of life is assessed according to the SF-36 quality of life scoring system, a validated scoring system in which an 8-point change is considered clinically meaningful. In some embodiments, administration of an effective amount of a myostatin inhibitor results in a clinically meaningful improvement in a standardized quality of life test score.

[0170] As used herein, the term "clinically meaningful improvement" refers to a significant improvement over standard levels. In some embodiments, a patient's SF-36 Quality of Life score increases by at least 8 points after treatment with an effective amount of an antibody or antigen-binding fragment thereof described herein, compared to the patient's score before treatment. In some embodiments, the patient achieves a higher score as assessed by the SF-36 Quality of Life test, e.g., an increase of at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, or 50 points in the SF-36 Quality of Life scoring system. In other embodiments, the score on the SF-36 Quality of Life scoring system increases by at least about 8-10, 10-15, 15-20, 20-30, 30-40, 40-50, 8-20, 8-30, 8-40, or 8-50 points.

[0171] In some embodiments, one or more quality of life scales are used to assess a patient's quality of life before or after treatment with an inhibitor of myostatin signaling disclosed herein. Advantages of this test include: i) it is easy to administer, ii) it assesses both physical function and mental health, and iii) it is highly validated across several clinical indications. Preventing muscle loss or atrophy

[0172] Administration of an effective amount of a myostatin inhibitor and / or SMN modifier prevents, delays, or alleviates muscle loss or atrophy in a human subject at risk of developing muscle loss and / or atrophy. In some embodiments, muscle loss or atrophy is reduced or prevented by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100%. In other embodiments, muscle loss or atrophy is reduced or prevented by at least about 1-5%, 5-10%, 10-20%, 1-30%, 1-40%, 1-50%, 10-50%, 20-30%, 20-60%, 30-80%, 40-90%, or 50-100% compared to a control group not receiving the myostatin inhibitor and / or SMN modifier.

[0173] In a particular embodiment, the above-referenced control group has only been treated with an SMN modifying factor. In a different embodiment, the above-referenced control group has not been treated with an SMN modifying factor.

[0174] Administration of an effective amount of a myostatin inhibitor and / or SMN modifying factor can prevent further deterioration of affected muscles or delay or slow the rate of disease progression in such patients. In some embodiments, administration of an effective amount of a myostatin inhibitor and / or SMN modifying factor can delay muscle loss or atrophy in human subjects at risk of developing muscle loss and / or atrophy compared to a control group not receiving the myostatin inhibitor and / or SMN modifying factor. In some embodiments, muscle loss or atrophy is delayed for at least 1 month, 2 months, or more in human subjects receiving the myostatin inhibitor and / or SMN modifying factor compared to a control group not receiving the myostatin inhibitor and / or SMN modifying factor. The treatment is delayed for 1 month, 3 months, 6 months, 8 months, 12 months, 2 years, 3 years, 5 years, or 10 years. In a particular embodiment, the above-referenced control group has only been treated with an SMN modifying factor. In a different embodiment, the above-referenced control group has not been treated with an SMN modifying factor.

[0175] Prevention of further deterioration of affected muscles refers to maintaining disease status including, for example, maintaining motor function test scores for a longer period of time compared to controls, slower disease progression as measured / monitored by motor function tests, fewer hospitalizations, fewer injuries (e.g., fractures), longer time to needing mechanical ventilation, longer time to becoming wheelchair bound, etc.

[0176] Prevention of muscle loss or atrophy by using the myostatin inhibitors and / or SMN modifiers described herein can be readily monitored or assessed by any suitable method for assessing motor function for the affected muscles. Effects on bone homeostasis

[0177] The administration of an effective amount of myostatin inhibitor can provide clinically significant protection to patient's bones.Such effects include but are not limited to the increase in patient's bone density, increase in bone mass, increase in bone mineral density, increase in bone strength, prevent bone loss, and reduce fracture frequency.Those skilled in the art are familiar with the suitable techniques that can be used to measure various parameters of bone homeostasis.Such techniques include imaging techniques such as micro-CT scanning and central dual-energy x-ray absorptiometry (central DXA) test. Effects on metabolic control

[0178] Administration of an effective amount of a myostatin inhibitor can provide a clinically meaningful effect on metabolic control, including, but not limited to, preventing a patient from developing metabolic dysregulation and alleviating a patient's metabolic dysregulation. i) Effect on insulin sensitivity in human subjects

[0179] Methods for measuring insulin sensitivity are known in the art, such as the glucose tolerance test and the fasting insulin or glucose test. During a glucose tolerance test, a fasting patient ingests a 75-gram oral dose of glucose, and then blood glucose levels are measured over the next two hours. A blood glucose level below 7.8 mmol / L (140 mg / dL) is considered normal, a blood glucose level between 7.8 and 11.0 mmol / L (140 to 197 mg / dL) is considered impaired glucose tolerance (IGT), and a blood glucose level greater than or equal to 11.1 mmol / L (200 mg / dL) is considered diabetes. For the fasting insulin test, a fasting serum insulin level greater than 25 mIU / L or 174 pmol / L is considered insulin resistant. In some embodiments, the metabolic rate is increased by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100%. In other embodiments, the metabolic rate is increased by at least about 1-5%, 5-10%, 10-20%, 1-30%, 1-40%, 1-50%, 10-50%, 20-30%, 20-60%, 30-80%, 40-90%, or 50-100%. ii) Effects on adipose tissue levels in human subjects

[0180] Administration of a myostatin inhibitor, such as an antibody or antigen-binding fragment thereof that specifically binds to pro / latent myostatin, affects levels in adipose tissue in a human subject. As used herein, the term "adipose tissue" refers to fat, including the connective tissue that stores fat. Adipose tissue is derived from preadipocytes. Its primary role is to store energy in the form of lipids, but it also serves as a cushion or insulator for the body. There are two types of adipose tissue: white adipose tissue (WAT), which stores energy, and brown adipose tissue (BAT), which generates body heat.

[0181] Brown adipose tissue (BAT) is known to be involved in the dissipation of chemical energy in response to cold or excessive food intake, and also has the ability to regulate energy balance. Activation of brown adipose tissue has been shown to improve glucose homeostasis and insulin sensitivity in humans, suggesting that anyone with insulin dysfunction can benefit from BAT activation (Stanford et al., J Clin Invest., 2013, 123(1):215-223).

[0182] Beige adipose tissue is generated as a result of the browning of WAT, also known as beiging. This occurs when adipocytes within WAT depots develop characteristics of BAT. Beige adipocytes take on a multivesicular appearance (containing several lipid droplets) and increase expression of uncoupling protein 1 (UCP1). In this way, these normally energy-storing white adipocytes become energy-releasing adipocytes (Harms et al., Nature Medicine. 2013, 19(10):1252-63).

[0183] Visceral or abdominal fat (also known as organ fat or intra-abdominal fat) is located inside the abdominal cavity and packed between organs (such as the stomach, liver, intestines, and kidneys). Visceral fat differs from subcutaneous fat, which is located under the skin, and intramuscular fat, which is scattered among skeletal muscles. While fat in the lower body, such as the thighs and buttocks, is subcutaneous and not a uniformly spaced tissue, abdominal fat is mostly visceral and semi-liquid. Excess visceral fat is known as central obesity or "belly fat," and is characterized by excessive abdominal protrusion. Newly developed tests, such as the body volume index (BVI), are specifically designed to measure abdominal volume and abdominal fat. Excess visceral fat has also been linked to type 2 diabetes, insulin resistance, inflammatory diseases, and other obesity-related disorders (Mokdad et al., JAMA: The Journal of the American Medical Association, 2001, 289(1):76-9).

[0184] Adipose tissue mass can be determined by any method known to those skilled in the art, for example, adipose tissue can be measured by dual energy X-ray absorptiometry (DXA).

[0185] Administration of a myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof that specifically binds to pro / latent myostatin, increases the level of brown adipose tissue and / or beige adipose tissue in a human subject, while administration of a myostatin inhibitor, e.g., an anti-pro / latent myostatin antibody or antigen-binding portion thereof, decreases the level of white adipose tissue and visceral adipose tissue in a human subject.

[0186] In some embodiments, the level of brown or beige adipose tissue is increased by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100%. In other embodiments, the level of brown or beige adipose tissue is increased by at least about 1-5%, 5-10%, 10-20%, 1-30%, 1-40%, 1-50%, 10-50%, 20-30%, 20-60%, 30-80%, 40-90%, or 50-100%.

[0187] In some embodiments, the level of white or visceral adipose tissue is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, The reduction is 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100%. In other embodiments, the level of white or visceral adipose tissue is reduced by at least about 1-5%, 5-10%, 10-20%, 1-30%, 1-40%, 1-50%, 10-50%, 20-30%, 20-60%, 30-80%, 40-90%, or 50-100%. iii) Effect on fat to muscle tissue ratio in human subjects

[0188] Administration of a myostatin inhibitor, such as an antibody or antigen-binding fragment thereof that specifically binds to pro / latent myostatin, reduces the ratio of fat to muscle tissue in a human subject. In some embodiments, the ratio of fat to muscle tissue is reduced by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100%. In other embodiments, the ratio of fat to muscle tissue is reduced by at least about 1-5%, 5-10%, 10-20%, 1-30%, 1-40%, 1-50%, 10-50%, 20-30%, 20-60%, 30-80%, 40-90%, or 50-100%. iv) Effect on glucose uptake in human subjects

[0189] Administration of a myostatin inhibitor, such as an antibody or antigen-binding fragment thereof that specifically binds to pro- or latent myostatin, affects glucose uptake by tissues in a human subject. In some embodiments, glucose uptake by muscle tissue is increased. For example, glucose uptake by muscle tissue is increased by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100%. In some embodiments, glucose uptake by muscle tissue is increased by at least about 1-5%, 5-10%, 10-20%, 1-30%, 1-40%, 1-50%, 10-50%, 20-30%, 20-60%, 30-80%, 40-90%, or 50-100%.

[0190] In other embodiments, glucose uptake by white adipose tissue, liver tissue, and vascular tissue is reduced. In some embodiments, glucose uptake by white adipose tissue, liver tissue, and vascular tissue is reduced by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100%. In other embodiments, glucose uptake by white adipose tissue, liver tissue, and vascular tissue is reduced by at least about 1-5%, 5-10%, 10-20%, 1-30%, 1-40%, 1-50%, 10-50%, 20-30%, 20-60%, 30-80%, 40-90%, or 50-100%. v) Effect on intramuscular fat infiltration in human subjects

[0191] Administration of a myostatin inhibitor, e.g., an antibody or antigen-binding fragment thereof that specifically binds to pro / latent myostatin, reduces intramuscular fat infiltration in a human subject. In some embodiments, intramuscular fat infiltration is reduced by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, or 100%. In other embodiments, intramuscular fat infiltration is reduced by at least about 1-5%, 5-10%, 10-20%, 1-30%, 1-40%, 1-50%, 10-50%, 20-30%, 20-60%, 30-80%, 40-90%, or 50-100%. Effect of delayed α-motor neuron loss

[0192] Administration of an effective amount of a myostatin inhibitor can slow the loss of α-motor neurons. Those skilled in the art are familiar with suitable techniques that can be used to measure various parameters indicative of α-motor neuron loss. Such techniques include homogenous Immunofluorescence methods that use imaging techniques such as high-throughput time-resolved fluorescence (HTRF, Cisbio Bioassays), micro-CT scanning, and light and electron microscopy. Biomarker expression

[0193] Changes in the levels of certain biomarkers (e.g., plasma biomarkers) of SMA can be measured to monitor the progression of pathology and the patient's response to treatment. Suitable methods and assays for measuring changes in the levels of suitable biomarkers from patient samples are known in the art. Thus, the detection methods of the present invention can be used to detect mRNA, protein, cDNA, or genomic DNA, for example, in biological samples in vitro and in vivo. For example, in vitro techniques for detecting mRNA include Northern hybridization and in situ hybridization. In vitro techniques for detecting marker proteins include enzyme-linked immunosorbent assay (ELISA), Western blot, immunoprecipitation, and immunofluorescence. In vitro techniques for detecting genomic DNA include Southern hybridization. In vivo techniques for detecting mRNA include polymerase chain reaction (PCR), Northern hybridization, and in situ hybridization. Furthermore, in vivo techniques for detecting marker proteins include introducing a labeled antibody against the protein or a fragment thereof into a subject. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.

[0194] More particularly, monitoring the effect of an agent (e.g., a myostatin inhibitor and / or an SMN modifier) ​​on the expression levels of a marker can be applied not only to basic drug screening, but also to clinical trials, as well as to assessing disease maintenance and progression, and assessing a patient's responsiveness to a particular therapy. For example, the effectiveness of an agent that affects marker expression can be monitored in biological samples collected from a subject being treated for SMA, e.g., before, during, and / or after treatment, by measuring changes in the levels of the marker and / or the expression levels of the marker over time. In a preferred embodiment, the present invention provides a method for monitoring the effectiveness of a subject's treatment with an agent, comprising the steps of: (i) obtaining a pre-administration sample from the subject before administering the agent; (ii) detecting the expression level of one or more selected markers of the present invention in the pre-administration sample; (iii) obtaining one or more post-administration samples from the subject; (iv) detecting the expression level of the marker in the post-administration sample; (v) comparing the expression level of the marker in the pre-administration sample with the expression level of the marker in one or more post-administration samples; and (vi) evaluating the effectiveness of the treatment regimen and, if necessary, modifying or adjusting the treatment accordingly. For example, increased expression of a marker gene during the course of treatment may indicate that the dosage is ineffective and that an increase in dosage is desirable. Conversely, decreased expression of a marker gene may indicate that the treatment is effective and that there is no need to modify the dosage.

[0195] Suitable biomarkers include the plasma proteins disclosed in Kobayashi et al. (2013) PLOS ONE, Vol. 8, No. 4, page e60113. Due to ease of sample collection, one or more biomarkers present in a serum sample are preferred, although in certain cases muscle biomarkers collected, for example, by tissue biopsy, may be used.

[0196] In some embodiments, the SMA plasma protein marker is selected from the list of: CILP2 (cartilage intermediate layer protein 2), TNXB (tenascin XB), CLEC3B (C-type lectin domain family 3, member B (tetranectin)), TNXB (tetranectin Nascin XB), ADAMTSL4 (ADAMTS-like 4), THBS4 (Thrombospondin 4), COMP (Cartilage Oligomeric Matrix Protein), CRTAC1 (Cartilage Acidic Protein 1), F13B (Coagulation Factor XIII, B Polypeptide), PEPD (Peptidase D), LUM (Lumican), CD93 (Complement Component 1, q Subcomponent, Receptor 1), Complement C2 / B Mix, APCS (Amyloid P Component, Serum), VTN (Vitronectin), DPP4 (Dipeptidyl Peptidase 4 (CD26, Adenosine Deaminase Complex Protein 2)), CRP (C-Reactive Protein, Pentraxin B) associated), HBB (hemoglobin beta), GSN (gelsolin), NCAM1 (neural cell adhesion molecule 1), CFI factor I (complement), APOA4 (apolipoprotein AIV), VTN (vitronectin), F13A1 (coagulation factor XIII, A1 polypeptide), INHBC (inhibin, beta C), RPS27A (ubiquitin and ribosomal protein S27a precursor), CDH13 (cadherin 13, H-cadherin (heart)), complement C2 / B mixture, C2 complement component 2, CP (ceruloplasmin (ferroxidase)), HBA (hemoglobin subunit alpha), QSOX1 (quiescin Q6), LRG1 (Leucine-rich alpha 2-glycoprotein 1), C9 (Complement component 9), SERPINA10 (Serpin peptidase inhibitor, Clade A (alpha 1 antiproteinase, antitrypsin), member 10), ALP (Alkaline phosphatase, Liver / Bone / Kidney), fc-gamma receptor III-A / B mixture, PROC (Protein C (Inactivator of coagulation factors Va and VIIIa)), VCAM1 (Vascular cell adhesion molecule 1), GAPDH (Glyceraldehyde-3-phosphate dehydrogenase), OMD (Osteomodulin), IGKVD41 (Immunoglobulin kappa variable 41), IGFBP6 (Insulin-like growth factor binding protein 6), PTPRG (Tyrosine phosphatase protein, receptor type G), S100A9 (S100 calcium-binding protein A9 (calgranulin B)), VNN1 (Vanin1), SERPIND (serpin peptidase inhibitor, clade D (heparin cofactor), member 1), CA1 (carbonic anhydrase I), CTSD (cathepsin D (lysosomal aspartyl peptidase)), HP (haptoglobin), SELENBP1 (selenium-binding protein 1), ORM2 (orosomucoid 2), PRDX2 (peroxiredoxin 2), AOC3 (amine oxidase, copper-containing 3 (vascular adhesion protein 1)), COL6A3 (collagen, type VI, alpha 3), PZP (pregnancy plasma protein), COL 6A1 (collagen, type VI, alpha 1), PARK7 (Parkinson's disease (autosomal recessive, early-onset) 7), THBS1 (thrombospondin 1), CAT (catalase), LCP1 (lymphocyte cytosolic protein 1 (L-plastin)), AFM (afamin), HPR (haptoglobin-related protein), SELL1 (selectin L (lymphocyte adhesion molecule 1)), ENG (endoglin), PFN1 (profilin 1), PI16 (peptidase inhibitor 16), SERPINA6 (serpin peptidase inhibitor, clade A (afamin)), F9 (Coagulation Factor IX), PROCR (Protein C Receptor, Endothelial), ORM1 (Orosomucoid 1), NEO1 (Neogenin Homologue 1), MMRN2 (Multimerin 2), LGB (Beta-Lactoglobulin), CNTN4 (Contactin 4), SHBG (Sex Hormone Binding Globulin), CA2 (Carbonic Anhydrase II), IGFBP5 (Insulin-Like Growth Factor Binding Protein 5), PLTP (Phospholipid Transfer Protein), FGA (Fibrinogen Alpha Chain), TPM4 (tropomyosin 4), MB (myoglobin), SPP1 (osteopontin), AXL (AXL receptor tyrosine kinase), APSC (amyloid P component, serum), CRP (C-reactive protein, pentraxin-related), CCL22 (chemokine (CC motif) ligand 22 (macrophage-derived chemokine)), THBD (thrombomodulin), CALCA (calcitonin), LEP (leptin), NPPB (brain natriuretic peptide b), MMP2 (matrix metalloproteinase 2), CK (creatine kinaseMuscle / bone), ACE (angiotensin converting enzyme), FAPB3 (fatty acid binding protein (heart)), CD40 (CD40 ligand), MIF (macrophage migration inhibitory factor), ANGPT2 (angiopoietin 2), AHSG (alpha-2-H S-glycoprotein (fetuin A), CFH (complement factor H), IL8 (interleukin 8), C3 (complement component 3), PPY (pancreatic polypeptide), VEGFA (vascular endothelial growth factor), TF (transferrin), PGF (placental growth factor), EGF (epidermal growth factor), GSTA1 (glutathione S-transferase alpha), SOD1 (superoxide dismutase 1), VCAM1 (vascular cell adhesion molecule 1), PAI1 (plasminogen activator inhibitor 1), CSF1 (macrophage colony-stimulating factor 1), S100A12 (S100 protein A12), VTN (vitronectin), FASLG (Fas ligand), A1M (alpha-1-microglobulin), AST (aspartate transaminase), ACCT (alpha-1-antichymotrypsin), CCL3 (chemokine (CC motif) ligand 3 (macrophage inflammatory protein 1 beta)), SORT1 (sortilin), TBG (thyroxine-binding globulin), APOA1 (apolipoprotein A1), MPO myeloperoxidase, B2M (beta-2 microglobulin), EPO (erythropoietin), MMP10 (matrix metalloproteinase 10), PROS1 (vitamin K-dependent protein S), MMP7 (matrix metalloproteinase 7), AGER (receptor for advanced glycation end products), IL18 (interleukin-18), CCL11 (chemokine CC motif ligand 11), IGA (immunoglobulin A), C-peptide (proinsulin C-peptide), A2M (alpha-2-macroglobulin), PDGF BB (Platelet-derived growth factor), CCL16 (Chemokine CC motif ligand 16), IL1A (Interleukin-1 alpha), APOA4 (Apolipoprotein A4), MMP9 (Matrix metalloproteinase 9), SPP1 (Osteopontin), CLEC3B (C-type lectin domain family 3, member B (Tetranectin)), IGFBP6 (Insulin-like growth factor binding protein 6), FABP4 (Fatty acid binding protein (adipocyte)), CHI3L1 (Chitinase 3-like 1 (YKL-40)), LEP (Leptin), CTSD (Cathepsin D), MST1 (Macrophage-stimulating 1 (Hepatocyte growth factor-like)), MIF (Macrophage migration inhibitory factor), S100A4 (S100 calcium ion transporter) um-binding protein A4), GLO1 (glyoxalase 1 (lactoylglutathione lyase)), ENG (endoglin), FTL1 (Fms-related tyrosine kinase 1 (vascular endothelial growth factor receptor)), ERBB2 (human epidermal growth factor receptor 2 (HER2)), NDKB (nucleoside phosphatase kinase isoform B), PRDX-4 (peroxiredoxin 4), PLAUR (plasminogen activator, urokinase receptor), IL6R (interleukin-6 receptor), CCL24 (chemokine (C-C motif) ligand 24 (eotaxin 2)), GSN (gelsolin), PSAT1 (phosphoserine aminotransferase 1), and TGFB1 (transforming growth factor beta 1).

[0197] In some embodiments, the biomarkers are selected from the following list representing the top 13 SMA motor function regressors in two SMA populations: COMP, AXL, CD93 PEPD, THBS4, LUM, MB, DPP4, SPP1, CHI3L1, CDH13, APCS, and LEP.

[0198] Additionally or alternatively, any suitable physiological measurement known in the art can be performed to assess muscle function, including electrical impedance myography (EIM), quantitative muscle magnetic resonance imaging (qMRI), dual-energy x-ray absorptiometry (DEXA), and the like. Therapies to promote muscle hypertrophy

[0199] In another aspect, the present invention provides the use of a myostatin inhibitor to promote muscle hypertrophy and improve muscle function in a subject in which the anabolic capacity of the muscle is preserved or restored, and the anabolic pathways (cellular machinery for protein production) are well preserved (i.e., functional and active).

[0200] While this is typically true for young / growing subjects (e.g., pediatric populations), in older subjects, muscles may lose robust anabolic capacity; in other words, the anabolic-catabolic balance tends to tilt toward the latter. Because myostatin inhibition provides optimal benefits in such subjects, it is contemplated that agents that stimulate anabolic pathways be administered in combination with myostatin inhibitors. By simultaneously boosting the anabolic arm of the cellular pathway, the target muscle can be made more responsive to the effects of myostatin inhibition.

[0201] Typically, muscle function in subjects with preserved or restored anabolic capacity is assessed by administering an anabolic hormone, such as testosterone, to the subject and measuring its effect on muscle growth and strength. The response of such subjects to anabolic hormones can be measured by methods known to those skilled in the art.

[0202] In some embodiments, a subject who would benefit from increased muscle mass is administered a myostatin inhibitor, such as those described herein. If the myostatin inhibitor treatment does not provide a significant benefit to the subject, further administration of an anabolic stimulator can be considered as a combination therapy to boost the effects of the myostatin inhibitor in accordance with the present disclosure.

[0203] Subjects who can benefit from enhanced muscle growth may or may not exhibit clinical symptoms of myopathy. Thus, such use can provide health benefits that may include increased muscle mass, enhanced ability to perform certain motor functions (tasks), etc. to generally "healthy" individuals who can nonetheless benefit from improved muscle function.

[0204] The myostatin inhibitors described herein, whether as monotherapy or combination therapy, are suitable for treating patients who exhibit one or more clinical symptoms of myopathy.As used herein, the term "myopathy" refers to a muscle condition characterized by impaired muscle structure or function, which typically results in muscle weakness.In addition, "myopathy" may include muscle conditions characterized by normal muscle structure but abnormal or dysfunctional neuronal input, which then affects muscle function.In addition, "myopathy" may include inflammatory myopathy and / or autoimmune myopathy, such as myasthenia gravis.

[0205] Myopathy includes muscle conditions that are neuromuscular or musculoskeletal in nature. In some embodiments, the myopathy is a hereditary myopathy. Hereditary myopathy includes, but is not limited to, dystrophies, myotonia, congenital myopathies (e.g., nemaline myopathy, multi / minicore myopathy, and centronuclear myopathy), mitochondrial myopathy, familial periodic myopathy, inflammatory myopathy, and metabolic myopathy (e.g., glycogen storage disease and lipid storage disorder). In some embodiments, the myopathy is an acquired myopathy. Acquired myopathies include, but are not limited to, exogenous substance-induced myopathies (e.g., drug-induced myopathies and glucocorticoid myopathies, alcoholic myopathies, and myopathies due to other toxic agents), myositis (e.g., dermatomyositis, polymositis, and inclusion body myositis), myositis ossificans, rhabdomyolysis, and Myopathy includes myoglobinuria, urinary tract infections, and disuse atrophy. In some embodiments, the myopathy is disuse atrophy caused by prolonged muscle disuse, which can lead to a deterioration in normal muscle function. Disuse atrophy may be the result of hospitalization, a fracture (e.g., a hip fracture), or nerve injury. In some embodiments, the myopathy is associated with a disease or disorder such as amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), renal failure, AIDS, a heart condition, and / or cachexia syndrome due to cancer. In some embodiments, the myopathy is associated with aging. In some embodiments, the myopathy is associated with a condition called myoglobinuria. The myopathy is associated with sarcopenia. In some embodiments, the myopathy is associated with paraspinal muscular atrophy (PMA).

[0206] In some embodiments, the myopathy is a primary myopathy. In one embodiment, the primary myopathy comprises disuse atrophy. In some embodiments, the disuse atrophy is associated with hip fracture, joint replacement, critical care myopathy, spinal cord injury, or stroke. In some embodiments, the myopathy is genetic muscle weakness associated with, for example, muscular dystrophy.

[0207] In some embodiments, the myopathy is a secondary myopathy in which muscle loss or dysfunction is secondary to disease pathology. In some embodiments, the secondary myopathy includes denervation or cachexia. In some embodiments, the secondary myopathy is caused by denervation associated with monitor neuron dysfunction. In some embodiments, the motor neuron dysfunction is due to a genetic mutation affecting motor neurons. Diseases known to involve motor neuron mutations include, but are not limited to, amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA). In some embodiments, the secondary myopathy is cachexia associated with renal failure, AIDS, a cardiac condition, cancer, or aging. In some embodiments, the secondary myopathy is caused by nerve injury, including unwanted nerve injury incurred during a medical procedure such as surgery. The adverse effects of such injury on the function of target tissues (e.g., target muscles) can be effectively treated by administration of a myostatin inhibitor described herein. For example, such administration can prevent and / or alleviate myopathy and / or promote recovery.

[0208] If the intended clinical outcome of myopathy treatment is primarily to promote muscle growth (hypertrophy), patients may initially be administered a myostatin inhibitor, such as those described herein, as monotherapy. Response to therapy should be monitored for clinical efficacy. If meaningful benefit is not obtained from monotherapy within a reasonable time frame, e.g., within 1 to 6 months of initiating myostatin inhibitor therapy, supplementation with an anabolic stimulator as a combination therapy may be considered.

[0209] Alternatively, myopathy patients with a catabolic basal environment are likely to require a concomitant anabolic boost to realize the full effects of myostatin inhibition and achieve meaningful muscle growth, and are therefore considered candidates for combination therapy. Such patient populations include, but are not limited to, those who are elderly (e.g., 65 years of age or older), those who exhibit clinical symptoms of sarcopenia, those who exhibit clinical symptoms of cachexia, those who exhibit symptoms of osteoporosis, those suffering from frequent or chronic infections, those with conditions that cause systemic immunodeficiency, and those with severe injuries or diseases that cause prolonged immobility.

[0210] Thus, the present invention encompasses combination therapies comprising a myostatin inhibitor and an anabolic stimulator, which may be advantageous for treating any condition in which a patient would benefit from improved exercise and / or metabolic function but whose anabolic capacity is impaired.

[0211] A treatment regimen aimed at achieving both myostatin inhibition and anabolic stimulation may be particularly advantageous for the elderly population. Thus, the present disclosure encompasses combination therapies incorporating both an inhibitor of myostatin signaling and an agent that boosts or promotes anabolic pathways, such as an anabolic stimulator. For example, such combination therapies may be useful for treating age-related muscle conditions, such as sarcopenia. This is because anabolic activity is gradually lost as part of the normal aging process, as evidenced by decreased protein synthesis, slower metabolism, etc., and in this context, myostatin inhibition may be beneficial for its muscle-building effects. This is based on the observation that myostatin inhibition may be less effective in exerting its effects. Similarly, such combination therapy may be useful in treating conditions such as cachexia, sporadic inclusion body myositis (SIBM), and disuse-related muscle wasting. It is contemplated that the muscle-building effects of myostatin inhibition can be fully achieved in the presence of an agent that restores or boosts the activity of cellular anabolic mechanisms. This may at least partially explain why so many clinical myostatin inhibitors to date have shown limited success in achieving clinically meaningful outcomes. Such studies have typically been performed in older patients whose anabolic capacity may have been weakened by age or other conditions that preferentially drive the balance toward a catabolic rather than an anabolic state. This recognition sheds light on the selection of appropriate patient populations likely to respond to myostatin inhibitor therapy, and the present invention encompasses such recognition.

[0212] As used herein, an agent that boosts cellular anabolic pathways includes any agent that stimulates or promotes protein synthesis over protein degradation, and may be collectively referred to herein as an "anabolic stimulator." Typically, anabolic stimulation in a tissue / organ can lead to hypertrophy as a result of greater net positive protein synthesis than protein degradation in the tissue / organ. In contrast, when catabolic pathways dominate over anabolic pathways, the net result may include tissue / organ atrophy due to promotion of protein degradation over protein synthesis. Therefore, the net outcome is likely to be a balance between these opposing functional branches of signaling in vivo.

[0213] Many anabolic stimulants are known in the art.Anabolic stimulants include, but are not limited to, IGF1 agonist, anabolic hormones, testosterone, steroids (such as androgens, oxymetholone, estrogens, progestogens, etc.), GH / somatotropin, parathyroid hormone (PTH), prostaglandins, leptin, statins, and any derivatives thereof.Any agent that promotes or stimulates protein synthesis and / or generally increases metabolic rate can function as anabolic stimulant.

[0214] According to the present invention, anabolic stimulators can be administered to patients who are either responsive, hyporesponsive, or non-responsive to myostatin inhibitor therapy. In hyporesponsive or non-responsive individuals, simultaneous stimulation of anabolic pathways can improve the subject's anabolic capacity, thereby allowing the myostatin inhibitor to benefit from improved exercise and / or metabolic function.

[0215] Nevertheless, while myostatin inhibitor low-responders and / or non-responders may benefit from anabolic stimulation, alternative patient populations include those who are myostatin inhibitor responders. It is contemplated that myostatin inhibitor therapy used in combination with anabolic stimulator therapy may further improve motor function in such individuals. Therapies to prevent muscle wasting

[0216] In some embodiments, the methods of the present invention are suitable for preventing muscle loss (atrophy). Prevention of muscle atrophy may be desirable across a wide range of patient populations, including those in generally good health. Thus, the present invention is useful in any situation in which the intended clinical outcome involves preventing muscle loss. This is based, at least in part, on the concept that myostatin plays a broader role as a "metabolic switch" that senses the body's energy expenditure (such as glucose levels) and controls muscle homeostasis by promoting muscle breakdown and / or fat synthesis. The myostatin inhibitory therapy described herein can be used to counter this effect.

[0217] Therefore, patients suitable for myostatin inhibition therapy include those suffering from a variety of genetic disorders, muscle conditions, metabolic disorders, injuries, etc. Injuries can include, but are not limited to, injuries or damage to muscles, bones, tendons, and nerves. Patients with severe injuries or illnesses that result in prolonged immobility can also benefit from myostatin inhibition therapy.

[0218] In some embodiments, the methods of the present invention are suitable for preventing muscle atrophy. Muscle atrophy is a highly regulated catabolic process that occurs during periods of disuse (e.g., disuse atrophy) and / or in response to injury or increased systemic inflammation (e.g., cachexia). Muscle atrophy can encompass a wide range of clinical conditions, including systemic conditions such as spinal cord injury and more localized conditions such as vocal cord paresis / vocal cord paralysis. As used herein, the term "vocal cord paresis / vocal cord paralysis" refers to a condition resulting from abnormal nerve input to the laryngeal muscles (larynx muscles). Paralysis can involve complete blockage of nerve impulses, resulting in no movement, while paresis can involve partial blockage of nerve impulses, resulting in weak or abnormal laryngeal muscle movement. In some embodiments, the anti-myostatin antibody or antigen-binding fragment thereof is administered locally, for example, by direct local injection into the affected vocal cord muscles.

[0219] In some embodiments, the methods of the present invention are suitable for treating or preventing muscle conditions and disorders, including paraspinal muscular atrophy (PMA). In one embodiment, the antibodies or antigen-binding fragments thereof described herein are used in a method for treating postoperative paraspinal muscular atrophy, i.e., paraspinal muscular atrophy that is atrophy of the paraspinal muscles following surgery. In one embodiment, the method of treatment comprises treating nerve injury-dependent muscle atrophy. In one embodiment, the method of treatment described herein comprises treating postoperative nerve injury-dependent muscle atrophy. In one embodiment, the method of treatment comprises treating postoperative muscle atrophy, and the surgery is spinal surgery. In one embodiment, the method of treatment comprises treating postoperative muscle atrophy, and the spinal surgery is a lumbar or lumbar procedure, such as a lumbar fusion, a lumbar nonfusion procedure, a posterior lumbar fusion, an anterior lumbar fusion, a minimally invasive (MIS) posterior lumbar decompression, a minimally invasive (MIS) posterior lumbar fusion, or a similar non-MIS procedure. In one embodiment, the method of treatment includes treating paraspinal muscle atrophy after lumbar fusion surgery. In one embodiment, the method of treatment includes treating paraspinal muscle atrophy after posterior lumbar fusion surgery. In one embodiment, the method of treatment includes treating paraspinal muscle atrophy after non-MIS lumbar fusion surgery. In some embodiments, administration of an effective amount promotes or accelerates recovery from conditions such as injury, surgery, and other medical procedures. Suitable such conditions may include conditions associated with nerve damage (whether resulting from injury or surgical or other clinical procedures).

[0220] Another aspect of the present disclosure includes a method of treating a subject having a disease or condition associated with a congenital myopathy. Exemplary congenital myopathies include, but are not limited to, X-linked myotubular myopathy, autosomal dominant centronuclear myopathy, autosomal recessive centronuclear myopathy, nemaline myopathy, and congenital fiber disparity myopathy.

[0221] Another aspect of the present disclosure includes a method of treating a subject having a muscle disease or condition associated with muscular dystrophies, including, but not limited to, Duchenne, Becker, facioscapulohumeral (FSH), and limb-girdle muscular dystrophies.

[0222] Another aspect of the present disclosure includes a method of treating a subject having a urogynecologic-related disease or condition, glottic disorder (stenosis), extraocular myopathy, carpel tunnel, Guillain-Barre, or osteosarcoma. Non-limiting embodiments for carrying out the present invention include the following.

[0223] In one embodiment, the present invention provides a myostatin inhibitor for use in treating spinal muscular atrophy (SMA) in a subject undergoing or expected to undergo SMN modifying factor therapy.

[0224] In some embodiments of the present invention referred to above, the subject has non-ambulatory SMA. In certain embodiments, the non-ambulatory SMA is SMA type I, type II, or type III. In one embodiment, the subject has non-ambulatory SMA type III.

[0225] In any of these embodiments described herein, the subject may have a baseline Expanded Hammersmith Functional Rating Scale score of ≦65. In certain embodiments, the subject has a baseline Expanded Hammersmith Functional Rating Scale score of ≦60, ≦55, ≦50, ≦45, ≦40, ≦35, ≦30, ≦25, or ≦20. In certain embodiments, the invention provides a myostatin inhibitor for use in treating spinal muscular atrophy (SMA) in a subject receiving or expected to receive SMN modifying factor therapy, wherein the subject has a modified Expanded Hammersmith Functional Rating Scale score of ≦60, ≦55, ≦50, ≦45, ≦40, ≦35, ≦30, ≦25, or ≦20 after receiving SMN modifying factor therapy. In certain embodiments of the invention referenced above, the subject has a modified Expanded Hammersmith Functional Rating Scale score of at least 1 point improvement over their baseline score after receiving SMN modifying factor therapy. In one embodiment, the baseline Expanded Hammersmith Functional Rating Scale score is determined before administering a myostatin inhibitor to the subject. In one embodiment, the baseline Expanded Hammersmith Functional Rating Scale score is determined before administering an SMN modifying factor to the subject. In one embodiment, the baseline Expanded Hammersmith Functional Rating Scale score is determined before administering a myostatin inhibitor and an SMN modifying factor to the subject. In certain other embodiments of the invention referenced above, the subject does not show improvement in the modified Expanded Hammersmith Functional Rating Scale score after receiving SMN modifying factor therapy.

[0226] In any of these embodiments described herein, the SMN modifying factor therapy comprises a) a splice modifier, b) an SMN gene replacement or gene therapy, c) an SMN transcriptional enhancer, d) an SMN protein translation enhancer, or e) an SMN protein stabilizer.

[0227] In certain embodiments, the SMN modifying factor is a central modifying factor or a systemic modifying factor. The term "central modifying factor," as used herein, refers to an SMN modifying factor administered directly to the CNS via an intrathecal route. The term "central modifying factor," as used herein, refers to an SMN modifying factor that is administered systemically (e.g., orally) and affects not only the CNS but other tissues as well. In different embodiments, the (a) splice modifier is an RNA-based splice modifier. In certain embodiments described herein, the (a) splice modifier is administered to the subject intrathecally (i.e., into the spinal canal or intrathecally so as to reach the cerebrospinal fluid (CSF)). In different embodiments, the SMN modifying factor therapy includes (b) SMN gene replacement or gene therapy delivered by a vector, which vector is optionally a viral vector. The terms "vector" and "viral vector" shall have their ordinary meanings and will be apparent to those skilled in the art of biochemistry.

[0228] In one embodiment, the splice modifier of (a) above is a small molecule splice modifier. In certain embodiments, the small molecule splice modifier is administered orally (i.e., the substance is taken by mouth). In certain embodiments, the small molecule splice modifier is , twice daily, once daily, every other day, twice weekly, or once weekly.

[0229] In one embodiment, the present invention provides a myostatin inhibitor for use in treating SMA in a subject who has ambulatory SMA and is not receiving SMN-modifying factor therapy. In certain embodiments of the invention described above, the ambulatory SMA is ambulatory Type III or Type IV. In further embodiments of the invention described above, the subject has a baseline Expanded Hammersmith Functional Rating Scale score of ≧40, ≧45, ≧50, or ≧55. In different embodiments of the invention described above, the subject has a baseline Expanded Hammersmith Functional Rating Scale score in the range of ≧48 to ≦58.

[0230] In one embodiment, the invention provides a myostatin inhibitor for use in treating SMA in a subject who has been identified as a carrier of an SMN mutation and who has not received SMN-modifying factor therapy. The terms "carrier of an SMN mutation" shall have their ordinary meaning, and in this context shall refer to an individual or other organism who has inherited an SMN recessive (i.e., non-dominant) allele but does not display the trait or symptoms of the disease caused by the SMN dominant allele. In further embodiments of the invention described above, the subject is identified as a carrier by in utero (i.e., inside the womb) or infant genetic screening.

[0231] In one embodiment, the present invention provides a myostatin inhibitor for use in increasing muscle mass in a subject, wherein i) the target muscle is in an anabolic state and / or ii) the subject is treated with an anabolic stimulating factor.

[0232] In further embodiments of the invention as described above, the subject suffers from sarcopenia, cachexia, infection, prolonged immobility or the subject is > 65 years old.

[0233] In one embodiment, the present invention provides a myostatin inhibitor for use in preventing muscle loss in a subject, wherein the subject is suffering from a condition involving partial impairment of neuromuscular function, and the subject is receiving neuronal therapy to treat or enhance motor neurons. In a further embodiment of the invention described above, the condition is a genetic disorder or injury that affects neuronal function.

[0234] In any of these embodiments described herein, the myostatin inhibitor comprises a) a small molecule antagonist of myostatin signaling, or b) an antibody or antigen-binding portion thereof that binds to i) pro / latent myostatin, ii) mature myostatin, or iii) the myostatin receptor. In further embodiments of the invention described above, the antibody or antigen-binding portion thereof binds to pro / latent myostatin but not mature myostatin or GDF11. In a different embodiment of the invention described above, the antibody or antigen-binding portion thereof binds to mature myostatin but not mature GDF11. In another embodiment of the invention described above, the antibody or antigen-binding portion thereof is administered to a subject at a dosage ranging from 1 mg / kg to 30 mg / kg. In a different embodiment of the invention described above, the antibody or antigen-binding portion thereof is administered to a subject twice weekly, once weekly, once every two weeks, or once monthly. In any of the above embodiments describing myostatin inhibitors, the antibody or antigen-binding portion thereof is administered to a subject intravenously (i.e., injected into a vein) or subcutaneously (i.e., injected into the skin).

[0235] In one embodiment, the present invention provides the use of a myostatin inhibitor for use in the treatment of SMA, wherein the myostatin inhibitor is administered as a monotherapy or a combination therapy. In one embodiment, the present invention provides the use of a myostatin inhibitor for use in the treatment of SMA, wherein the target muscle is in an anabolic state and / or undergoes anabolic stimulation. The present invention provides the use of a myostatin inhibitor to promote muscle growth in a subject treated with a stimulatory factor.

[0236] In one embodiment, the present invention provides the use of a myostatin inhibitor to prevent muscle atrophy in a subject suffering from a condition involving partial impairment of neuromuscular function and being treated with neuronal therapy. In certain embodiments of the present invention described herein, the condition is ALS. In one embodiment, the present invention provides the use of a myostatin inhibitor in the manufacture of a medicament for treating SMA. In a different embodiment, the present invention provides a pharmaceutical composition comprising the myostatin inhibitor of embodiment C1 and an excipient. In certain embodiments, the pharmaceutical composition described above is formulated for intravenous or subcutaneous administration.

[0237] In one embodiment, provided herein is a method for promoting muscle hypertrophy, the method comprising administering to a subject who may benefit from muscle growth and who is a poor responder to a myostatin inhibitor an effective amount of an anabolic stimulator such that the effect of the myostatin inhibitor is boosted. In one embodiment, the anabolic stimulator and the myostatin inhibitor are administered as a combination therapy.

[0238] In one embodiment, the subject suffers from a condition selected from the group consisting of sarcopenia, cachexia, chronic SCI, chronic or frequent infections, osteoporosis, and frequent falls / fractures. In one embodiment, the subject is 65 years of age or older.

[0239] In one embodiment, the subject is further treated with a neuronal therapy. In one embodiment, the subject has amyotrophic lateral sclerosis (ALS) or SMA.

[0240] In one embodiment, the myostatin inhibitor is a small molecule antagonist of myostatin or a biological antagonist of myostatin, hi another embodiment, the biological antagonist of myostatin is an antibody or antigen-binding portion thereof.

[0241] In one embodiment, disclosed herein is a method for treating a muscular condition in a human subject, the method comprising: selecting a human subject having a target muscle with full anabolic capacity and in which functional neuromuscular signaling between the target muscle and innervating motor neurons is partially impaired; and administering to the human subject a myostatin inhibitor in an amount effective to enhance function of the target muscle, thereby treating the muscular condition in the human subject. In one embodiment, the target muscle retains or restores at least partial innervation of motor neurons. In one embodiment, the target muscle comprises fast-twitch type II fibers. In one embodiment, the human subject is a pediatric subject.

[0242] In one embodiment, the method further comprises administering to the subject an anabolic stimulator. In one embodiment, the anabolic stimulator is administered to the subject prior to, concurrently with, or after administration of the myostatin inhibitor. In one embodiment, the muscle condition is associated with a defect of motor neurons. In one embodiment, the defect is a genetic defect. In one embodiment, the genetic defect is a mutation of Smn1.

[0243] In one embodiment, the method further comprises administering to the human subject an agent that corrects the genetic defect. In one embodiment, the agent is a splice regulator. In one embodiment, the agent is a small molecule agent or a nucleic acid agent. In one embodiment, the agent increases motor neuron function. In one embodiment, the motor neuron function includes membrane excitability, axonal transport, vesicle transport, neurotransmitter release, mitochondrial function, and / or mitochondrial availability. In one embodiment, the agent is an SMN corrector.

[0244] In one embodiment, the muscle condition is associated with a neuromuscular disease. In one embodiment, the neuromuscular disease is spinal muscular atrophy (SMA). In one embodiment, the SMA is SMA type I, SMA type II, or SMA type III. In one embodiment, SMA type III is ambulatory SMA or non-ambulatory SMA.

[0245] In accordance with the present invention, any of the myostatin inhibitors described herein may be formulated into pharmaceutical compositions comprising the myostatin inhibitor and, optionally, excipients. Such pharmaceutical compositions are used to treat a disease or condition in a human subject, either as a monotherapy or combination therapy, in accordance with the present disclosure.

[0246] The present invention encompasses the use of any of the myostatin inhibitors described herein for the manufacture of a medicament suitable for administration to a human subject in accordance with the present disclosure.

[0247] This invention is further illustrated by the following examples which should not be construed as limiting. [Example]

[0248] Example 1 SRK-015: specific inhibition of myostatin activation Like other TGF-β family members, myostatin is secreted as an inactive precursor termed promyostatin, in which the presence of a prodomain blocks access of the growth factor to its receptor. Myostatin activation results from two distinct proteolytic cleavage steps (Figure 1). Promyostatin is first cleaved by proprotein convertases such as furin, which recognize the RXXR site between the prodomain and the mature growth factor (30, 62). After cleavage, the growth factor and prodomain remain together and form a latent complex (latent myostatin) that cannot bind to its receptor. The active growth factor is released after a second cleavage by members of the BMP / tolloid family (e.g., TLL-2, tolloid-like protein 2) (63). After activation, myostatin binds to a receptor complex consisting of a type I receptor (Alk4 / 5) and a type II receptor (ActRIIA / B), leading to the phosphorylation and activation of Smad2 / 3. Ultimately, activation of this signaling pathway leads to reduced protein synthesis and increased protein degradation (64). Myostatin has been detected in two compartments in vivo: in the circulation and in muscle. The latter is predominantly in a latent form, while the latter is predominantly in muscle, where it is bound to extracellular matrix proteins such as perlecan (65-68).

[0249] Using a novel approach, we have discovered and developed a myostatin inhibitor with a unique mechanism of action and significantly improved specificity over most other inhibitors. As noted above, the mature forms of myostatin and GDF11 are 90% identical, making it difficult to identify antibodies specific to myostatin. However, the prodomains of these growth factors are only 43% identical. Therefore, we have generated antibodies that target and specifically bind to the precursor form of myostatin, inhibiting the activation of mature myostatin from its latent form.

[0250] Antibody SRK-015P (the "P" denotes the parent molecule) was optimized to generate SRK-015, a fully human monoclonal antibody. SRK-015 differs from its parent clone by five residues in the variable domain outside of the complementarity-determining regions. Both antibodies bind pro- and latent myostatin with high affinity (single-digit nanomolar range, e.g., 2-9 nM) but show no detectable binding to any form of GDF11 or activin A, or to mature BMP9, BMP10, or TGFβ1. When incubated with human myostatin (expressed and purified from a mammalian expression system in vitro), furin, and mTTL-2 proteases, SRK-015 inhibited the release of mature myostatin, as measured by activation of luciferase expression in a Smad2 / 3 reporter cell line. SRK-015 was ineffective in a similar protease activity assay using pro-GDF11 as a substrate, again demonstrating its specificity for pro- and latent myostatin. SRK-015 did not inhibit the signaling ability of mature myostatin in this assay. Both SRK-015 and SRK-015P exhibited similar functional activity against human and mouse promyostatin in reporter assays, and the EC50 values ​​of the two antibodies in promyostatin activation assays using human and mouse promyostatin were similar. Analysis of protein fragments produced after cleavage of latent myostatin by mTLL-2 demonstrated that SRK-015 inhibited the second (tolloid-mediated) proteolytic step required for myostatin activation. SRK-015 was shown to be capable of binding to the myostatin prodomain and inhibiting tolloid cleavage. Purified recombinant latent myostatin was incubated with TLL-2 tolloid protease in the presence of increasing concentrations of SRK-015. Samples were resolved by reducing SDS-PAGE and probed by Western blot using an antibody that recognizes the myostatin prodomain. Protease-dependent production of cleavage fragments was inhibited with increasing amounts of the antibody, indicating that binding of SRK-015 to latent myostatin inhibited the second tolloid-mediated cleavage step. Example 2 In situ localization of myostatin proform

[0251] Because SRK-015 binds to pro- and latent forms of myostatin, we investigated the localization of these pro-forms in mouse skeletal muscle to confirm that these forms of myostatin are present in the extracellular space of muscle and can be bound and inhibited by the SRK-015 antibody. Cryosections of tibialis anterior muscle from healthy mice were immunostained with the antibody GDF8_068, which binds to pro- and latent forms of myostatin but not to mature growth factors. The tissue was also immunostained with an antibody against laminin, a component of the muscle extracellular matrix. Results indicated that the majority of myostatin precursor detected in muscle was present in the extracellular space, with little signal detected intracellularly. Significant co-staining occurred in the interstitial extracellular space and interstitial perinuclear areas. To confirm the specificity of the antibody staining, samples were immunostained with GDF8-068 preincubated with a 100-fold molar excess of purified promyostatin or pro-GDF11. Preincubation with promyostatin completely abolished the signal, whereas preincubation with pro-GDF11 had no effect on staining. These data indicate that pro- and latent myostatin are present in the extracellular space of skeletal muscle and therefore can be bound and inhibited by SRK-015. Example 3 SRK-015 prevents dexamethasone-induced muscle atrophy

[0252] SRK-015 increased muscle mass in healthy animals and reduced muscle loss in a dexamethasone-induced atrophy model. As shown in Figure 2, administration of a single 20 mg / kg dose of SRK-015 to healthy male mice (vehicle group) resulted in a significant increase in muscle mass after 15 days (17.5% vs. IgG control). A similar increase in muscle mass was observed in female mice (data not shown). SRK-015 was also effective in preventing muscle loss during dexamethasone treatment. Chronic administration of dexamethasone resulted in a significant loss of muscle mass by day 8 (16% decrease, IgG vehicle vs. IgG Dex). A single 20 mg / kg dose of SRK-015 to dexamethasone-treated mice prevented muscle atrophy throughout the course of the study, with no significant differences observed between the treatment group and the IgG vehicle control group (Figure 2). Example 4 SRK-015P enhances muscle mass and function in healthy animals

[0253] Treatment with SRK-015 resulted in gains in muscle function in addition to increases in muscle mass. To evaluate SRK-015-mediated functional effects, we used a murine version of SRK-015P (muSRK-015P), in which the human IgG4 constant region was replaced with that of mouse IgG1, limiting the immune response to the antibody over the 4-week study. All three antibodies, muSRK-015, muSRK-015P, and SRK-015, bind mouse promyostatin with similar affinities (Kd of 2.57 nM, 2.88 nM, and 8.35 nM, respectively). Healthy 10-week-old C57BL / 6 mice were treated weekly with vehicle (PBS) or 20 mg / kg muSRK-015P for 4 weeks. After treatment, neurally evoked function of the plantarflexor muscle groups (gastrocnemius, soleus, and plantaris) was investigated in vivo across a physiologically relevant activation range. Treated animals demonstrated a 19% increase in isometric torque production at frequencies higher than 60 Hz (P = 0.003) (Figure 3A). A 31% increase in maximal contraction rate was observed, but the force-frequency response was unchanged (data not shown). To confirm the direct effect of myostatin inhibition on muscle, independent of neural function and blood supply, in vitro force measurements were performed on isolated EDL muscles. In vitro assessment of EDL force production demonstrated similar increases in function: a 24% increase at 80 Hz (P = 0.024), a 28% increase at 100 Hz (P = 0.010), and a 27% increase at 150 Hz (P = 0.011) (Figure 3B). No changes in EDL contraction and relaxation velocity or the force-frequency relationship were observed after treatment (data not shown). As expected, 4 weeks of treatment with muSRK-015P also affected muscle mass, increasing gastrocnemius muscle by 22% (P = 0.009, Figure 3C) and EDL muscle by 34% (P = 0.007, Figure 3D). Normalization of force to muscle weight revealed no differences between vehicle and either the gastrocnemius or EDL treatment groups, indicating that the hypertrophy induced by muSRK-015P treatment did not negatively affect muscle quality or excitability (data not shown).Histological analysis of the plantar flexors revealed a 27% increase in total cross-sectional area (P = 0.019, data not shown) and a 29% increase in the cross-sectional area of ​​type IIB muscle fibers (P = 0.009, Figure 3E). There was no change in the cross-sectional area of ​​type I, IIA, or IIX fibers, nor was there any change in fiber type distribution after treatment (data not shown). Example 5 Myostatin inhibition improves muscle function in SMA mice treated with splice correctors

[0254] To evaluate the ability of SRK-015 to improve muscle function in SMA, we used a pharmacological model of mutant SMA. In this model, administration of varying doses of the small molecule SMN2 splice regulator SMN-C1 can attenuate disease severity (17, 26). The basis for this model is the Δ7 mouse model of severe SMA. These mice lack the only endogenous mouse SMN gene and express two copies of human SMN2 and two copies of SMN lacking exon 7 (Smn- / -, hSMN2, SMNΔ7). Due to the severity of disease in this model, the median survival of these mice is 13 days, which is insufficient time to evaluate the efficacy of potential therapeutic agents (70). Administration of a low dose (0.1 mg / kg / day) of SMN-C1 to Δ7 mice from birth extended survival, with 70% of treated mice surviving to postnatal day (PND) 52, although disease severity remained high. Treatment with a high dose of 3 mg / kg / day of SMN-C1 results in a milder form of SMA, with mice appearing largely healthy and exhibiting only minor deficits in weight and function. An intermediate model of severity can be achieved by administering 0.1 mg / kg / day of SMN-C1 to Δ7 mice for the first 24 days of life, followed by a transition to 3 mg / kg / day (low-high treatment). The phenotype of these low-high treated mice is roughly intermediate between the low-dose and high-dose phenotypes in terms of weight and muscle function (17, 26, 71). The low-high SMN-C1 paradigm also allows for the development of splice-like proteins after diagnosis. This is useful for evaluating the therapeutic potential of combination therapy with SMN2 splice regulators, as it mimics patients with severe SMA who begin treatment with a corrector (i.e., nusinersen) months or years after birth. Initiating treatment with a second therapeutic agent concurrently with high-dose SMN-C1 allows for the determination of the efficacy of the combination therapy.

[0255] To evaluate the efficacy of myostatin activation inhibition in combination with SMN2 splice correction, Δ7 mice were subjected to a low-high SMN-C1 regimen and administered vehicle or a weekly dose of 20 mg / kg muSRK-015P. On PND 24, a switch to high-dose SMN-C1 was initiated simultaneously. Mice were treated with 3 mg / kg / day SMN-C1 and muSRK-015P or vehicle for an additional 4 weeks. Untreated wild-type mice served as controls. At the end of the study, muscle weight, fiber cross-sectional area, and neural evoked function of the plantar flexor and masseter muscles were assessed. In Δ7 mice, the masseter muscle was the most severely affected muscle, while the gastrocnemius muscle was relatively spared (7).

[0256] Although muSRK-015P did not improve masseter function, muSRK-015P treatment resulted in a significant 60% increase in maximal torque generation by the plantar flexors (Figure 4A, Figure 4B). Interestingly, no significant increase in gastrocnemius mass or mean cross-sectional area of ​​the plantar flexors was observed (Figure 5A, Figure 5B). However, the frequency distribution of muscle fiber cross-sectional area showed that animals treated with muSRK-015P had more fiber cross-sectional area (1000–2000 μm). 2 The vehicle-treated mice had muscle fibers between 1000 μm and 1000 μm. 2A change was observed in that the myostatin-inhibited mice had smaller muscle fibers than the control mice (Figure 5C). In this model, treatment significantly increased force production but not muscle mass. The reason for this is unclear. To date, all animal studies performed with this molecule have resulted in significant increases in muscle mass, and there have been no published reports that myostatin inhibition improves muscle function in the absence of mass gain. One possibility is that treatment with muSRK-015P can improve muscle health and / or quality, resulting in increased neurotrophic factor expression and improved motor neuron survival. Muscle expression of multiple neurotrophic factors (i.e., HGF, neurotrophin-4, and GDNF) has been shown to support motor neuron survival and growth (73-75). Example 6 The benefits of myostatin inhibition require sufficient muscle innervation

[0257] The difference in efficacy of muSRK-015P on the gastrocnemius (which constitutes the majority of the plantar flexor muscles) and masseter may be due to two aspects of SMA disease pathology. First, myostatin inhibition is known to preferentially result in hypertrophy of fast-twitch glycolytic muscle fibers (type IIB in mice), as shown here ((76, 77) and Figure 3E). Mouse gastrocnemius muscles are primarily composed of type IIB fibers (approximately 75%), whereas masseter muscles are significantly less abundant, between 10 and 25% (78, 79). Second, as it is a more severely affected muscle, denervation of the masseter muscle is more extensive than denervation of the gastrocnemius (7). To be effective, myostatin inhibition relies on sufficient muscle innervation, and treatment with myostatin inhibitors is ineffective on muscles below the level of injury in a model of complete spinal cord injury (80). In contrast, muSRK-015P significantly preserved muscle mass and function below the level of injury in a contusion model of spinal cord injury, with only partial denervation (Figure 6). In this study, 8-week-old female C57BL / 6 mice (n = 6–8) underwent laminectomy at thoracic level 9 (T9) followed by a severe (65 kDyne) spinal cord contusion injury using an Infinite Horizon Compactor device. Placebo control animals underwent laminectomy only. Immediately after injury, mice were administered vehicle (PBS), IgG control, or 40 mg / kg muSRK-015P. PBS and IgG control have been shown to be equivalent in multiple models (data not shown). Weekly treatments were administered, and the study was terminated on day 14. (Figure 6A) Mice that underwent partial severe SCI showed significant atrophy of hindlimb muscles, whereas animals treated with muSRK-015P were protected from this atrophy. P<0.05. (Figure 6B) Muscle function testing was performed 7 and 14 days after SCI. Hindlimb grip strength was assessed using a digital force gauge. Mice treated with vehicle and IgG showed significant loss of grip strength, whereas mice treated with muSRK-015P retained limb grip strength significantly below the lesion level. P<0.001. Data are presented as mean ± SEM and analyzed by one-way ANOVA followed by Tukey's post-hoc comparison.

[0258] Importantly, SMA patients treated with nusinersen showed improvement in compound muscle action potential (CMAP) amplitude, a measure that should normally only be reduced in these patients, indicating that SMN2 splice regulation maintains sufficient muscle innervation in at least some muscles and that myostatin inhibition is effective ( 25 ).

[0259] These preclinical results, particularly the significant effect of muSRK-015P on improving muscle function in two models of partial denervation (SMA and incomplete spinal cord injury), provided support for the potential of SRK-015P to significantly restore muscle function in SMA patients.Treatment with SRK-015 may be in combination with a splice corrector or as monotherapy in patients with milder forms of the disease (i.e., Type III SMA) where sufficient innervation remains. Example 7 Target binding in SMNΔ7 mice

[0260] SMNΔ7 mice were treated with 0.1 mg / kg / day of SMN-C1, an SMN splice regulator, from birth until postnatal day (PND) 24. On PND 24, mice were switched to a high dose of SMN-C1 (3 mg / kg / day) and treatment with vehicle or 20 mg / kg / week of muSRK-015P was initiated. Mice received weekly antibody treatment for 4 weeks. At the end of the study, serum and TA muscle were collected and analyzed for target binding by fluorescent Western blot. Samples from 3–5 animals per group were used for these analyses.

[0261] Target binding was observed in SMNΔ7 mice treated with muSRK-015P. Because the bound target is expected to have the half-life of an antibody and accumulate in the circulation and target tissues, binding with muSRK-015P leads to the accumulation of latent myostatin. We developed a Western blot assay using an antibody against the myostatin prodomain to assess latent myostatin levels in serum and muscle after antibody administration. We previously determined the specificity of the antibody using samples from myostatin knockout animals. As indicated, antibody dosing began on postnatal day 24, while the SMN-C1 corrector was switched from a low dose to a high dose. After 4 weeks of dosing, serum and tibialis anterior muscle were collected and target binding was assessed. As shown in Figure 7, the results demonstrate binding of latent myostatin in the circulation (as indicated by target accumulation upon antibody binding) after 4 weeks of treatment with muSRK-015P (see immunoblot in Figure 7A). Lane 1 is recombinant purified promyostatin containing a small amount of latent myostatin. Similarly, Figure 7B shows target binding in muscle. After 4 weeks of treatment, accumulation of latent myostatin was observed in the muscle of mice treated with muSRK-015P, as indicated by antibody binding. Lane 1 is recombinant pro / latent myostatin. To allow for lane normalization, samples were run on a TGX unstained gel, allowing visualization and quantification of total lane protein content upon UV imaging (Figure 7C). Quantification of latent myostatin signal in muscle showed a threefold increase in target accumulation in the muSRK-015P group, indicating target binding. The latent myostatin signal in each lane was normalized to the total protein content of that lane and compared to the latent myostatin present in WT mice (Figure 7D). Example 8 Pharmacokinetics and pharmacodynamics of SRK-015 in scid mice

[0262] Male scid mice were administered a single 5 mg / kg IV dose of SRK-015. Separate cohorts of mice were sacrificed 4 hours, 2, 8, 15, 22, 29, and 56 days after dose administration, and serum and TA muscle were collected for PK and target binding assays. N = 4 or 8 per group. Figure 8A shows the PK analysis of SRK-015. SRK-015 levels were determined from serum samples collected at the indicated times after dose administration. Analysis was performed using an anti-human IgG ELISA. The half-life of 5 mg / kg SRK-015 in scid mice was approximately 20.3 days.

[0263] Similarly, as shown in Figure 8B, administration of a single 5 mg / kg dose to these animals resulted in a significant increase in lean mass compared to the IgG control. Lean mass was measured using qNMR at the indicated time points after dose administration. In addition, SRK-015 demonstrates long-term target binding in serum and muscle after a single 5 mg / kg dose (see Figure 8C). Target binding was no longer observed 56 days after dose administration, as most SRK-015 had been cleared at this time point (data not shown). Target binding was assessed by analyzing the levels of latent myostatin in serum and muscle using Western blot. Because the bound target is expected to have the half-life of an antibody and accumulate in the circulation and target tissues, binding with SRK-015 results in the accumulation of latent myostatin. After determining the specificity of the antibody using samples from myostatin knockout animals, an antibody against the myostatin prodomain was used in this assay. Significant target binding occurs in muscle within 2 days and in the circulation within 4 hours and is maintained up to 29 days after dose administration. Samples from 3 mice from each cohort were analyzed. Example 9 Pharmacokinetics and pharmacodynamics of SRK-015 in cynomolgus monkeys

[0264] The PK characteristics of SRK-015 were also determined in cynomolgus monkeys. Male animals aged 2-3 years (mean age 34 months) were administered weekly doses of 3 mg / kg or 30 mg / kg of SRK-015 for 8 weeks. After the final dose, animals were followed for an additional 5 weeks in the absence of drug administration. Samples from this post-dose period were used to calculate PK parameters. Figure 9A shows SRK-015 concentrations during the week after the first antibody dose. Antibody levels were assessed by ELISA. Figure 9B shows SRK-015 concentrations during the final 5 weeks of the study, following the last of the 8 weekly antibody doses, indicating saturation at higher doses.

[0265] The pharmacokinetic data obtained for SRK-015 in cynomolgus monkeys are summarized below in Table 4. [Table 4]

[0266] At the end of the study, animals were sacrificed 5 weeks after the last dose, as described above, and muscle weights were determined. The gastrocnemius and biceps brachii muscles (Figures 10A and 10B, respectively) were significantly higher in SRK than in rhesus mammary gland. Mass gain after -015 treatment: Gastrocnemius muscle weight increased by 21% in the 3 mg / kg group and 23% in the 30 mg / kg group compared to vehicle controls. * indicates a statistically significant difference versus vehicle by one-way ANOVA, P<0.007. Biceps brachii weight increased by 18% in the 3 mg / kg group and 25% in the 30 mg / kg group compared to vehicle controls. *indicates a statistically significant difference from vehicle by one-way ANOVA, P<0.002. As shown in Figures 10C and 10D, SRK-015 bound to latent myostatin at both doses tested. Figure 10C shows the time course of target binding in the serum of monkeys administered 3 mg / kg or 30 mg / kg weekly. Serum samples were collected on the indicated study days and analyzed by semiquantitative Western blot analysis. Accumulation of latent myostatin, indicative of SRK-015 binding, is evident by day 8. Target continues to accumulate with administration of additional doses. Latent myostatin levels were quantified as described in Example 7 above. The effect of SRK-015 on muscle weight indicates that even at the lower end of the dosage range (e.g., 3 mg / kg / week), SRK-015 is at or near the target binding levels required for maximal efficacy. Example 10 Muscle performance in SMNΔ7 mice treated from birth with therapeutically sufficient doses of SMN-C1

[0267] SMNΔ7 mice were administered the SMN splice regulator SMN-C1 at 3 mg / kg / day starting on postnatal day (PND) 1. Treatment with this dose of SMN-C1 resulted in significant disease correction and was intended to mimic milder forms of SMA, such as ambulatory type III or type IV SMA. On PND 24, mice were treated with muSRK-015P (20 mg / kg / week). Control groups received PBS. On PND 52, mouse muscle function was assessed in the plantar flexor muscle groups (gastrocnemius, soleus, and plantaris) and masseter muscle. After euthanasia, individual muscles were isolated and weighed (Figures 11A and 11B). Untreated wild-type mice of the same genetic background served as controls.

[0268] Muscle performance was measured using the 305C muscle lever system (Aurora Scientific Inc., Aurora, CAN) as described above.

[0269] After 4 weeks of treatment with muSRK-015P, SMNΔ7 mice showed a 17.7% increase in body weight compared to PBS control animals (P = 0.0021) (Figure 11A). Treatment with muSRK-015P also resulted in an increase in mass of multiple hindlimb muscles, including the gastrocnemius (26.5% increase, P = 0.0071) (Figure 11A). This increase in mass reflected functional gain. Mice treated with muSRK-015P showed a 22–37% improvement in plantar flexor force generation across a physiologically relevant frequency range (40–80 Hz) (Figure 11B). No effect of antibody treatment was observed in the masseter muscle, a muscle more severely affected than the gastrocnemius in this model. References [ka] [ka] [ka] [ka] [ka] [ka]

[0270] Various features and embodiments of the invention referred to in individual sections above also apply mutatis mutandis to other sections where appropriate. Consequently, features specified in one section may be combined with features specified in other sections where appropriate.

[0271] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the following claims.

Claims

1. 1. A method for treating spinal muscular atrophy (SMA) in a subject, comprising: administering to the subject a myostatin inhibitor in an amount effective to treat SMA, wherein the subject is undergoing or is expected to undergo SMN modifying factor therapy; thereby treating spinal muscular atrophy in said subject. A method comprising:

2. 2. The method of claim 1, wherein the subject is expected to receive the SMN modifying factor therapy within 6 months of administration of the myostatin inhibitor.

3. The myostatin inhibitor is an antibody or antigen-binding portion thereof that inhibits activation of myostatin by binding to the pro-form of myostatin; an antibody or antigen-binding portion thereof that neutralizes myostatin activity by binding to mature myostatin; an antibody or antigen-binding portion thereof that binds to a myostatin receptor; a prodomain or fragment thereof that binds to mature myostatin; a soluble myostatin receptor fragment that binds mature myostatin, and Small molecule antagonists of myostatin The method according to any one of claims 1 to 2, wherein the compound is selected from the group consisting of:

4. The method of claim 3, wherein the antibody or its antigen-binding portion binds to the pro domain of the pro-type of myostatin.

5. The method of claim 3 or 4, wherein the antibody or its antigen-binding portion binds to pro / latent myostatin.

6. The method of claim 5, wherein the antibody or its antigen-binding portion does not bind to mature myostatin that is not related to the pro-form of myostatin.

7. 10. The method of any one of the preceding claims, wherein the subject is an SMN corrector responder.

8. The method of any one of claims 1 to 7, wherein the subject is a non-responder or a poor responder to an SMN corrector.

9. The SMN modifying factor is a) splice modifiers, b) SMN gene replacement or gene therapy agents; c) SMN transcription enhancer; d) an SMN protein translation enhancer, or e) SMN protein stabilizer 10. The method of any one of the preceding claims, comprising:

10. 10. The method of any one of the preceding claims, wherein the SMN corrector is an antisense RNA or a small molecule.

11. Any of the preceding claims, wherein the subject has a non-ambulatory SMA or an ambulatory SMA. The method according to any one of claims 1 to 5.

12. 10. The method of any one of the preceding claims, wherein the subject has been diagnosed with SMA Type I, SMA Type II, or SMA Type III.

13. A method for treating ambulatory SMA in a subject, comprising administering to a subject with ambulatory SMA an effective amount of a myostatin inhibitor, wherein the subject is not receiving SMN-modifying factor therapy.

14. A method for treating SMA in a subject, comprising administering to a subject genetically identified as a carrier of an Smn mutation an amount of a myostatin inhibitor effective to prevent muscle atrophy, wherein the subject is not receiving Smn modifying factor therapy.

15. The method of any one of the preceding claims, wherein the subject has a baseline expanded Hammersmith Functional Motor Rating Scale score of ≦65 prior to administration of the myostatin inhibitor.

16. An effective amount for treating SMA is a) slowing or reducing muscle atrophy; b) delaying the loss of α-motor neurons; c) preventing or delaying the onset of immature muscle markers; d) preventing, reducing, or delaying intramuscular fat deposition characterized by fatty replacement of muscle tissue; e) An increase in the Expanded Hammersmith Functional Rating Scale score of ≥ 1 point compared to an untreated control group or ≥ 1 point from baseline measured before treatment; f) delaying the progressive decline of the Expanded Hammersmith Motor Scale over 12, 24, or 36 months; g) an increase in CHOP INTEND score of ≥ 1 point compared to untreated controls; and / or h) Increase in MFM-32 score by at least 1 point compared to untreated controls 10. The method of claim 1, wherein the amount is effective to

17. The method of any one of claims 1 to 16, wherein the antibody or antigen-binding fragment thereof is administered by intravenous injection or infusion.

18. The method of any one of claims 1 to 16, wherein the antibody or antigen-binding fragment thereof is administered by subcutaneous injection.

19. 1. A method for promoting muscle hypertrophy, comprising: administering to a subject who may benefit from muscle growth and who is a poor responder to a myostatin inhibitor an effective amount of an anabolic stimulant such that the effect of said myostatin inhibitor is boosted. A method comprising:

20. 20. The method of claim 19, wherein the anabolic stimulator and the myostatin inhibitor are administered as a combination therapy.

21. 21. The method of claim 19 or 20, wherein the subject is suffering from a condition selected from the group consisting of sarcopenia, cachexia, chronic SCI, chronic or frequent infections, osteoporosis, and frequent falls / fractures.

22. 22. The method of any one of claims 19 to 21, wherein the subject is 65 years of age or older.

23. The method of any one of claims 19 to 22, wherein the subject is further treated with a neuronal therapy.

24. 24. The method of claim 23, wherein the subject has amyotrophic lateral sclerosis (ALS) or SMA.

25. The method of any one of claims 19 to 24, wherein the myostatin inhibitor is a small molecule antagonist of myostatin or a biological antagonist of myostatin.

26. 26. The method of claim 25, wherein the biological antagonist of myostatin is an antibody or an antigen-binding portion thereof.

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