Methods and materials for NT-3 gene therapy

Abstract

To provide recombinant adeno-associated virus (rAAV) delivery of a neurotrophin 3 (NT-3) polynucleotide.SOLUTION: The disclosure provides rAAV and methods of using the rAAV for NT-3 gene therapy to improve muscle strength, stimulate muscle growth and treat muscle wasting disorders, such as muscular dystrophy and Charcot-Marie-Tooth neuropathy. The disclosure provides methods of stimulating muscle growth in a subject. The method comprises administering a therapeutically effective amount of neurotrophin-3 (NT-3), pro-NT-3 or an effective fragment thereof, or a nucleic acid encoding NT-3, pro-NT-3 or an effective fragment thereof, to a subject in need thereof. The disclosure describes novel effect of NT-3, its ability to directly influence the protein synthesis and metabolic remodeling in neurogenic muscle.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 574,828, filed October 20, 2017, U.S. Provisional Patent Application No. 62 / 676,687, filed May 25, 2018, and U.S. Provisional Patent Application No. 62 / 741,335, filed October 4, 2018, the disclosures of which are incorporated herein by reference in their entireties.

[0002] Statement of U.S. Government Interest This invention was made with government support under Grant Nos. NS105986 and U01-NS066914 awarded by the National Institutes of Health. The United States Government has certain rights in this invention.

[0003] INCORPORATION-BY-REFERENCE TO SEQUENCE LISTING This application contains as a separate part of this disclosure a Sequence Listing in computer readable form (Filename: 53122A_Seqlisting.txt; 17,133 byte ASCII text file created on October 18, 2018), which is incorporated herein by reference in its entirety.

[0004] This disclosure relates to recombinant adeno-associated virus (rAAV) delivery of neurotrophin 3 (NT-3) polynucleotides. This disclosure provides rAAV and methods of using rAAV for NT-3 gene therapy to improve muscle strength, stimulate muscle growth, and treat neuropathic and muscle-wasting diseases, such as Charcot-Marie-Tooth neuropathy. [Background technology]

[0005] Recent studies have demonstrated that neurotrophin 3 (NT-3) is a versatile molecule with previously unknown or underappreciated features. In addition to its well-recognized effects on peripheral nerve regeneration and Schwann cells (SCs), NT-3 has anti-inflammatory and immunomodulatory effects. Yang et al., Mel Titer, 22(2):440-450 (2014). It has recently been demonstrated that NT-3 can alleviate spontaneous autoimmune peripheral polyneuropathy in a rodent model of chronic inflammatory demyelinating peripheral neuropathy that occurs in humans. Yalvac et al., Gene therapy, 23(1):95-102 (2015).

[0006] Charcot-Marie-Tooth (CMT) neuropathy is the most common hereditary neuropathy. CMT1 comprises five types of CMT caused by four genes when mutated. This group comprises the majority of individuals with CMT. These genes are related to SCs and the myelin sheath surrounding axons, but they interact in various ways, resulting in heterogeneous phenotypes. CMT1A is the result of a DNA duplication on chromosome 17p11, which contains the PMP22 gene, leading to the classic CMT1 phenotype. Patients develop clinical signs before the age of 20, causing significant disability requiring walking aids. In these patients, peripheral nerve regeneration is incomplete due to the prolonged axotomy and denervation that occur as part of the chronic neuropathy. NT-3 is a trophic factor secreted by Schwann cells (SCs) that supports nerve regeneration. The ability of denervated SCs to survive is important for nerve regeneration because they provide both growth factors and a basement membrane, a scaffold that promotes axonal growth. Long-term denervation leads to a decreased regenerative capacity associated with a decreased expression of regeneration-related SC molecules (neurotrophic factors (NTFs) and their receptors), resulting in atrophy of denervated SCs, disruption of Büngner's zones, and loss of the SC basement membrane scaffold.

[0007] Previous studies have shown that NT-3 gene therapy in the TremblerJ (TrJ) mouse model of Charcot-Marie-Tooth (CMT) neuropathy not only improved nerve regeneration with increased SC number, myelinated fiber density, and myelin thickness, but also increased muscle fiber diameter in the anterior and posterior muscles of the hindlimb (Sahenk et al., Mol Ther, 22(3):511-521 (2014)). Previous studies have shown that the neuropathy phenotype results in diverse changes in skeletal muscle, including a switch from fast-twitch type II fibers to slow-twitch type I fibers. In TrJ mice, the extensor digitorum longus (extensor digitalis longus), which is primarily composed of fast-twitch type II fibers, has a significantly higher percentage of slow-twitch fibers compared to wild-type (WT) mice, and the percentage of type I fibers in the soleus muscle dramatically increased with age. Nicks et al., J Neuropathol Exp Neurol, 72(10):942-954 (2013). Interestingly, similar changes in slow muscle fiber production occur in parallel with aging in humans and other mammals. Larsson L, Moss R, J Physiol., 472:595-614 (1993); Larsson et al., Am J Physiol., 272:C638-C649 (1997).

[0008] CMT1A, inherited as an autosomal dominant disorder, is the most common type of CMT. In most cases, it is caused by a 1.5 Mb duplication at 17p11.2 containing the peripheral myelin protein 22 (PMP22) gene, generated by unequal crossover of homologous chromosomes (1). It is a slowly progressive disease without known treatment. Symptoms most often begin before the age of 20. Pes cavus and hammertoe are present. Gait aids, such as ankle-foot orthoses, are required. Although rare, severe pediatric cases can lead to wheelchair or ventilator dependence. Typically, 90% of patients have a motor nerve conduction velocity (NCV) in the ulnar nerve of 16–35 m / s or less (2). Even though the genetic defect primarily involves Schwann cells (SCs), the electrophysiological clinical picture is that of a length-dependent sensorimotor demyelinating neuropathy. This clinical picture is significantly influenced by axonal degeneration due to impaired Schwann cell (SC)-axon interactions ( 3 ).

[0009] Currently, there is no cure for this condition. Ascorbic acid supplements have been much touted as helpful, but many studies have shown no benefit. Both low-dose (1-2 g / day) (4-6) and high-dose therapy (3-4 g / day) have proven to be of no benefit (6, 7). Initial clinical trials of NT-3 demonstrated clinical efficacy after 24 weeks of treatment, with an increase in the number of myelinated nerve fibers in post-treatment sural nerve biopsies (8). Tr J Subcutaneous NT-3 treatment improved axonal regeneration and promoted myelination in mice. However, the short serum half-life of NT-3 proved to be a major obstacle to sustained subcutaneous administration, and this product was discontinued.

[0010] Numerous musculoskeletal disorders have been shown to result in muscle weakness. These include, but are not limited to, hereditary or recessive myopathies (e.g., muscular dystrophies), muscle-wasting disorders (e.g., cachexia, which may result from underlying conditions such as acquired immunodeficiency syndrome (AIDS), rheumatoid arthritis, cancer, chronic obstructive pulmonary disease (COPD), and liver cirrhosis), muscle atrophy or muscle wasting conditions (e.g., sarcopenia, which may result from aging), prolonged disuse (e.g., paralysis, coma, prolonged bed rest, and ICU stay), weakness induced by surgery (e.g., total joint replacement), drug-induced myopathy, and rhabdomyolysis. The muscle pathology of these diseases and conditions is mediated, in part or in whole, by a combination of immune, inflammatory, and fibrotic responses. Agents capable of blocking these responses and / or stimulating regeneration of damaged tissue could slow or halt disease progression in these disorders.

[0011] Adeno-associated virus (AAV) is a replication-deficient parvovirus whose single-stranded DNA genome is approximately 4.7 kb in length, containing 145 nucleotide inverted terminal repeats (ITRs). Multiple serotypes of AAV exist. The nucleotide sequences of the genomes of AAV serotypes are known. For example, the complete genome of AAV-1 is provided under GenBank accession number NC_002077; the complete genome of AAV-2 is provided under GenBank accession number NC_001401 and Srivastava et al., J. Virol., 45:555-564 {1983); the complete genome of AAV-3 is provided under GenBank accession number NC_1829; the complete genome of AAV-4 is provided under GenBank accession number NC_001829; the AAV-5 genome is provided under GenBank accession number AF085716; the complete genome of AAV-6 is provided under GenBank accession number NC_001862; at least portions of the AAV-7 and AAV-8 genomes are provided under GenBank accession numbers AX753246 and AX753249, respectively; and the AAV-9 genome is provided by Gao et al. The AAV-10 genome is provided in Mol. Ther., 13(1):67-76 (2006); and the AAV-11 genome is provided in Virology, 330(2):375-383 (2004). Cis-acting sequences that direct viral DNA replication (rep), encapsidation / packaging, and host cell chromosomal integration are contained within the AAV ITRs. Three AAV promoters (designated p5, p19, and p40 for their relative map positions) drive expression of two AAV internal open reading frames encoding the rep and cap genes. Two rep promoters (p5 and p19) associated with differential splicing of a single AAV intron (at nucleotides 2107 and 2227) result in the generation of four rep proteins (rep 78, rep 68, rep 52, and rep 40) from the rep gene. The Rep proteins possess multiple enzymatic properties that are ultimately responsible for replicating the viral genome.The cap gene is expressed from the p40 promoter and encodes the three capsid proteins VP1, VP2, and VP3. Alternative splicing and non-consensus translation initiation sites are responsible for the generation of the three related capsid proteins. A single consensus polyadenylation site is located at map position 95 of the AAV genome. The life cycle and genetics of AAV are reviewed in Muzyczka, Current Topics in Microbiology and Immunology, 158:97-129 (1992).

[0012] AAV has unique characteristics that make it attractive as a vector for delivering foreign DNA to cells, for example, in gene therapy. AAV infection of cells in culture is noncytopathic, and natural infection in humans and other animals is silent and asymptomatic. Furthermore, AAV can infect many mammalian cell types and potentially target many different tissues in vivo. Furthermore, AAV can gradually transduce dividing and nondividing cells and persist as transcriptionally active nuclear episomes (extrachromosomal elements) essentially for the lifespan of those cells. The AAV proviral genome is infectious as cloned DNA in a plasmid, making the construction of recombinant genomes feasible. Furthermore, because signals directing AAV replication, genome encapsidation, and integration are contained within the ITRs of the AAV genome, part or all of the internal approximately 4.3 kb region of the genome (encoding the replication and structural capsid protein, rep-cap) can be replaced with foreign DNA. The rep and cap proteins can be provided in trans. Another important feature of AAV is that it is an extremely stable and robust virus. Cryopreservation of AAV is less critical because AAV readily withstands the conditions used to inactivate adenovirus (56-65°C for several hours). AAV can even be lyophilized. Finally, cells infected with AAV are not resistant to superinfection. [Prior art documents] [Non-patent literature]

[0013] [Non-Patent Document 1] Yang et al.,Mel Titer,22(2):440-450(2014) [Non-patent document 2] Yalvac et al.,Gene therapy,23(1):95-102(2015) [Non-patent document 3] Sahenk et al.,Mol Ther,22(3):511-521(2014) [Non-patent document 4] Nicks et al.,J Neuropathol Exp Neurol,72(10):942-954(2013) [Non-Patent Document 5] Larsson L, Moss R, J Physiol.,472:595-614(1993) [Non-patent document 6] Larsson et al.,Am J Physiol.,272:C638-C649(1997) [Non-Patent Document 7] Gao et al., J. Virol., 78:6381-6388 (2004) [Non-patent document 8] Mol. Ther., 13(1):67-76(2006) [Non-Patent Document 9] Virology, 330(2):375-383(2004) [Non-Patent Document 10] Muzyczka,Current Topics in Microbiology and Immunology,158:97-129(1992) Summary of the Invention [Means for solving the problem]

[0014] There is a need to develop therapies for CMT neuropathy and other muscle wasting diseases. The present invention provides a gene therapy method for delivering NT-3 for the treatment of CMT neuropathy and other muscle wasting diseases.

[0015] The present disclosure provides a method for stimulating muscle growth in a subject. The method comprises administering to a subject in need thereof a therapeutically effective amount of neurotrophin-3 (NT-3), pro-NT-3, or an effective fragment thereof, or a nucleic acid encoding NT-3, pro-NT-3, or an effective fragment thereof. The present disclosure describes a novel effect of NT-3: its ability to directly affect protein synthesis and metabolic remodeling in neurogenic muscle.

[0016] In various embodiments of the present disclosure, NT-3, pro-NT-3, or an effective fragment thereof, or a nucleic acid encoding NT-3 or an effective fragment thereof of the present disclosure is administered intramuscularly.

[0017] In any of the methods of the present disclosure, the nucleic acid encoding NT-3 or an effective fragment thereof is administered using a viral vector. For certain embodiments, the viral vector is an adeno-associated virus (AAV) vector. In related embodiments, the nucleic acid encoding NT-3 or an effective fragment thereof of the present disclosure is operably linked to a muscle-specific promoter, such as a muscle-specific creatine kinase triplet promoter. In various embodiments, the nucleic acid encoding NT-3 or an effective fragment thereof of the present disclosure comprises SEQ ID NO: 1.

[0018] The present disclosure provides a nucleic acid comprising, in 5' to 3' order: (i) a first AAV2 inverted terminal repeat (ITR); (ii) a muscle creatine kinase promoter / enhancer sequence set forth at nucleotides 147-860 of SEQ ID NO:11; (iii) a nucleotide sequence encoding a human NT-3 polypeptide; and (iv) a second AAV2 ITR sequence, wherein the human NT-3 polypeptide has an amino acid sequence encoded by a nucleotide sequence that is at least 90% identical to SEQ ID NO:2 or 100% identical to SEQ ID NO:2, or 90% identical to nucleotides 1077-1850 of SEQ ID NO:11 or 100% identical to nucleotides 1077-1850 of SEQ ID NO:11.

[0019] In certain embodiments, a nucleic acid of the present disclosure further comprises a chimeric intron 3' to the promoter / enhancer, as set forth at nucleotides 892 to 1024 of SEQ ID NO: 11. Additionally, a nucleic acid of the present disclosure may further comprise an SV40 polyadenylation signal 3' to the nucleotide sequence encoding the human NT-3 polypeptide, as set forth at nucleotides 1860 to 2059 of SEQ ID NO: 11.

[0020] Any of the nucleic acids of the present disclosure can include one or more inverted terminal repeat (ITR) sequences. For example, a nucleic acid can include a first ITR set forth in nucleotides 7-112 of SEQ ID NO:11 and / or a second ITR set forth in nucleotides 2121-2248 of SEQ ID NO:11.

[0021] In one embodiment, the nucleic acid comprises a scAAV1.tMCK.NTF3 genome that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO:11.

[0022] The present disclosure also provides an infectious recombinant adeno-associated virus particle (rAAV) comprising any of the nucleic acids of the present disclosure. The rAAV particle can be any rAAV serotype, such as AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, or AAVrh.74. Furthermore, in any of the rAAV particles of the present invention, the AAV DNA in the rAAV genome is derived from AAV-1.

[0023] The present disclosure also provides compositions comprising an rAAV of the present disclosure and a pharmaceutically acceptable carrier. For example, the compositions are formulated to treat a muscle-wasting disease or neuropathy in a subject in need thereof, or the compositions are formulated to stimulate muscle growth in a subject in need thereof.

[0024] In one embodiment, the disclosure provides a method of treating a muscle wasting disease or neuropathy in a human subject in need thereof, comprising administering to the human subject a nucleic acid encoding an NT-3 polypeptide; wherein a) the nucleic acid comprises a nucleotide sequence that is 90% identical to the nucleotide sequence of SEQ ID NO:1, b) the nucleic acid comprises the nucleotide sequence of SEQ ID NO:1; c) the nucleic acid comprises a nucleic acid sequence that encodes an amino acid sequence that is at least 90% identical to SEQ ID NO:2 or 100% identical to SEQ ID NO:2, and d) the nucleic acid encoding the NT-3 polypeptide is a nucleic acid sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO:2 or 100% identical to SEQ ID NO:2, e) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the rAAV is administered at a dose that results in sustained expression of a low concentration of the NT-3 polypeptide; f) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the route of administration is intramuscular, and the dose of the rAAV administered is about 1.5 x 10 vg / kg to about 6.5 x 10 vg / kg; g) the NT-3 polypeptide is the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the route of administration is intramuscular, and the dose of the rAAV administered is about 2×10 vg / kg to about 6×10 vg / kg; h) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the route of administration is intramuscular, and the dose of the rAAV administered is about 2×10 vg / kg to about 6×10 vg / kg; i) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV)scAAV1.tMCK.NTF3, the route of administration is intramuscular, and the dose of rAAV administered is approximately 4 x 10 vg / kg; j) the nucleic acid encoding an NT-3 polypeptide is recombinant adeno-associated virus (rAAV)scAAV1.tMCK.NTF3, the route of administration is intramuscular, and the dose of rAAV administered is approximately 6 x 10 vg / kg; k) the nucleic acid encoding an NT-3 polypeptide is recombinant adeno-associated virus (rAAV)scAAV1.tMCK.NTF3, and the route of administration is approximately 0.or l) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the route of administration is intramuscular injection at a concentration of about 2 x 10 administered using multiple injections in a total volume of about 5 to 14 ml. 13 The method is an intramuscular injection at a concentration of 0.15 mg / ml.

[0025] In another embodiment, the disclosure provides a method of improving muscle strength or stimulating muscle growth in a human subject in need thereof, comprising administering to the human subject a nucleic acid encoding an NT-3 polypeptide; wherein a) the nucleic acid comprises a nucleotide sequence that is 90% identical to the nucleotide sequence of SEQ ID NO:1, b) the nucleic acid comprises the nucleotide sequence of SEQ ID NO:1; c) the nucleic acid comprises a nucleic acid sequence that encodes an amino acid sequence that is at least 90% identical to SEQ ID NO:2 or 100% identical to SEQ ID NO:2, and d) the nucleic acid encoding the NT-3 polypeptide is a nucleic acid sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO:2, e) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the rAAV is administered at a dose that results in sustained expression of a low concentration of the NT-3 polypeptide; f) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the route of administration is intramuscular, and the dose of the rAAV administered is about 1.5 x 10 vg / kg to about 6.5 x 10 vg / kg; g) the NT-3 polypeptide is the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the route of administration is intramuscular, and the dose of the rAAV administered is about 2×10 vg / kg to about 6×10 vg / kg; h) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the route of administration is intramuscular, and the dose of the rAAV administered is about 2×10 vg / kg to about 6×10 vg / kg; i) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV)scAAV1.tMCK.NTF3, the route of administration is intramuscular, and the dose of rAAV administered is approximately 4 x 10 vg / kg; j) the nucleic acid encoding an NT-3 polypeptide is recombinant adeno-associated virus (rAAV)scAAV1.tMCK.NTF3, the route of administration is intramuscular, and the dose of rAAV administered is approximately 6 x 10 vg / kg; k) the nucleic acid encoding an NT-3 polypeptide is recombinant adeno-associated virus (rAAV)scAAV1.tMCK.NTF3, and the route of administration is approximately 0.or l) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the route of administration is intramuscular injection at a concentration of about 2 x 10 administered using multiple injections in a total volume of about 5 to 14 ml. 13 The method is an intramuscular injection at a concentration of 0.15 mg / ml.

[0026] In any of the methods of the present disclosure, the nucleic acid is administered using a viral vector, such as an adeno-associated viral vector. Any of the methods of the present disclosure can be performed using a nucleic acid operably linked to a muscle-specific promoter, such as the muscle-specific creatine kinase (MCK) promoter. Additionally, any of the methods of the present disclosure can be performed using scAAV1.tMCK.NTF3, which contains the NT-3 gene cassette set forth in SEQ ID NO:11.

[0027] In one embodiment, the present disclosure provides a gene therapy method using a scAAV1 vector, a self-complementary AAV1 serotype, and the human neurotrophin-3 gene (NTF3) under the control of a muscle-specific promoter, tMCK. The present disclosure provides a method for treating a subject diagnosed with a muscle wasting disease or neuropathy, comprising administering an AAV vector expressing NT-3. In particular, the method comprises administering the construct scAAV1.tMCK.NTF3 by intramuscular (IM) injection in the gastrocnemius and tibialis anterior muscles. For example, intramuscular delivery of an AAV vector expressing NT-3, such as scAAV1.tMCK.NTF3, initiates local production and secretion of NT-3 into the circulation, thereby promoting nerve myelination and fiber regeneration and resulting in stabilization of the CMT disease phenotype. More specifically, the present disclosure provides a method for treating a subject diagnosed with a muscle wasting disease or neuropathy, comprising administering an AAV vector expressing NT-3 by intramuscular (IM) injection in the gastrocnemius and tibialis anterior muscles. 12 vg / kg dose or approximately 6 x 10 12 The present invention provides a method for administering scAAV1.tMCK.NTF3 at a dose of 1000 mg / kg, which contains the NT-3 gene cassette set forth in SEQ ID NO:11.

[0028] The present disclosure also provides a method for administering an AAV vector expressing NT-3 as an alternative gene therapy for treating muscle wasting diseases or neuropathy. NT-3 has a short half-life, and the disclosed method involves administering an AAV vector for sustained release of NT-3 protein, even if the subject expresses endogenous NT-3 protein. As an alternative gene therapy, administration of an AAV vector provides sustained delivery of NT-3 protein through continuous secretion by muscle cells. This continuous, sustained, low blood concentration of NT-3 protein provides therapeutic benefit with minimal risk of toxicity. Systemic production of NT-3 through gene therapy is also a more convenient and cost-effective treatment option compared to repeated injections of purified NT-3 peptide.

[0029] The present disclosure provides a method for treating a muscle wasting disease or neuropathy in a human subject in need thereof, comprising administering to the human subject a dose of recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3 that results in sustained expression of low levels of NT-3 protein.

[0030] The present disclosure also provides a method of stimulating muscle growth in a human subject in need thereof, comprising administering to the human subject a dose of recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3 that results in sustained expression of low levels of NT-3 protein.

[0031] In one embodiment, the disclosure provides a method of treating a muscle wasting disease or neuropathy in a human subject in need thereof, comprising administering recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3 to the human subject, wherein the route of administration is intramuscular and the dose of rAAV administered is about 1.0 x 10 12 vg / kg ~ approx. 7×10 12 vg / kg, or approximately 1.5 × 10 12 vg / kg ~ approx. 6.5×10 12 vg / kg, or approximately 2 × 10 12 vg / kg ~ approx. 6×1012 vg / kg.

[0032] In another embodiment, the disclosure provides a method of treating a muscle wasting disease or neuropathy in a human subject in need thereof, comprising administering recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3 to the human subject, wherein the route of administration is intramuscular and the dose of rAAV administered is about 1.0 x 10 12 vg / kg, or approximately 1.5 × 10 12 vg / kg, or approximately 2 × 10 12 vg / kg, or approximately 3 × 10 12 vg / kg, or approximately 4 × 10 12 vg / kg, or approximately 5 × 10 12 vg / kg, or approximately 6 × 10 12 vg / kg, or approximately 7 × 10 12 vg / kg, or approximately 8 × 10 12 vg / kg, or approximately 9 × 10 12 vg / kg, or approximately 1 × 10 13 vg / kg.

[0033] In another embodiment, the disclosure provides a method of treating a muscle wasting disease or neuropathy in a human subject in need thereof, comprising administering to the human subject recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, wherein the route of administration is 13 In one embodiment, the rAAV is administered intramuscularly at a concentration of 0.5 to 100 mg / ml. For example, the rAAV is administered using 3 to 6 injections per muscle, each injection volume being 0.5 to 1 ml, with a total of 5 ml to 14 ml of vector administered into the medial and lateral heads of the gastrocnemius and tibialis anterior muscles in each leg.

[0034] In an exemplary embodiment, the disclosure provides a method of treating a muscle wasting disease or neuropathy in a human subject in need thereof, comprising administering to the human subject recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, wherein the route of administration is approximately 2 x 10 administered using 3-6 injections per muscle (each injection volume being 0.5-1 ml). 13 The method provides for intramuscular injection of vector at a concentration of 1000 mg / ml into the medial and lateral heads of the gastrocnemius and tibialis anterior muscles in each leg, with a total of 5 mL to 14 mL of vector administered.

[0035] In one embodiment, a method of improving muscle strength or stimulating muscle growth in a human subject in need thereof comprises administering recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3 to the human subject, wherein the route of administration is intramuscular and the dose of rAAV administered is about 1.0 x 10 12 vg / kg ~ approx. 7×10 12 vg / kg, or approximately 1.5 × 10 12 vg / kg ~ approx. 6.5×10 12 vg / kg, or approximately 2 × 10 12 vg / kg ~ approx. 6×10 12 vg / kg.

[0036] In another embodiment, the disclosure provides a method of improving muscle strength or stimulating muscle growth in a human subject in need thereof, comprising administering recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3 to the human subject, wherein the route of administration is intramuscular and the dose of rAAV administered is about 1.0 x 10 12 vg / kg, or approximately 1.5 × 10 12 vg / kg, or approximately 2 × 10 12 vg / kg, or approximately 3 × 10 12 vg / kg, or approximately 4 × 10 12 vg / kg, or approximately 5 × 10 12 vg / kg, or approximately 6 × 10 12 vg / kg, or approximately 7 × 10 12 vg / kg, or approximately 8 × 1012 vg / kg, or approximately 9 × 10 12 vg / kg, or approximately 1 × 10 13 vg / kg.

[0037] In an exemplary embodiment, the disclosure provides a method of improving muscle strength or stimulating muscle growth in a human subject in need thereof, comprising administering to the human subject recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, wherein the route of administration is 13 In one embodiment, the rAAV is administered intramuscularly at a concentration of 0.5 to 100 mg / ml. For example, the rAAV is administered using 3 to 6 injections per muscle, each injection volume being 0.5 to 1 ml, with a total of 5 ml to 14 ml of vector administered into the medial and lateral heads of the gastrocnemius and tibialis anterior muscles in each leg.

[0038] In an exemplary embodiment, the disclosure provides a method of improving muscle strength or stimulating muscle growth in a human subject in need thereof, comprising administering to the human subject recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, wherein the route of administration is a low dose (2 x 10 per patient) using 3-6 injections per muscle (each injection volume being 0.5-1 ml). 12 vg / kg) and high dose (6 × 10 per patient 12 1 × 10 vg / kg 13 The method provides for intramuscular injection of vector at a concentration of 1000 mg / ml into the medial and lateral heads of the gastrocnemius and tibialis anterior muscles in each leg, with a total of 5 mL to 14 mL of vector administered.

[0039] In any of the methods of the present disclosure, the route of administration of scAAV1.tMCK.NTF3 is intramuscular bilateral injection into the medial and lateral heads of the gastrocnemius and tibialis anterior muscles. Furthermore, in any of the methods of the present disclosure, administration of scAAV1.tMCK.NTF3 results in improved muscle strength in the subject's upper or lower limbs, for example, as measured by a reduction in the composite score on the CMT Pediatric scale (CMTPeds). Furthermore, in any of the methods of the present disclosure, administration of scAAV1.tMCK.NTF3 results in a reduction or halt of disease progression over a two-year period. Disease progression is measured by the CMTPeds.

[0040] Muscle strength can also be measured using electromyography, hand-held myometry, fixed-system dynamometers, manual muscle testing, and / or functional / activity tests such as the Jebsen test, as well as the 6-minute walk test, timed rise from supine position, and other tests. It is measured using timed tests that assess how long it takes a subject to perform a specific task, such as timed rise from floor, 10 meter walk / run, timed climb 4 steps, and timed descent 4 steps.

[0041] In one embodiment of the present disclosure, in any of the methods, the subject suffers from a hereditary neuropathy, such as a Charcot-Marie-Tooth (CMT) neuropathy, such as CMT1A, CMT2K, CMT4A, and CMTRIA, as well as axonal and demyelinating neuropathies caused by autosomal recessive, autosomal dominant, or X-linked genetic mutations. The hereditary neuropathy can be caused by any of the genetic mutations listed in Table 1. Additionally, the hereditary neuropathy can be a transthyretin amyloid neuropathy caused by the following genetic mutations: a mutation in the transthyretin (TTR) gene, such as Val30Met, Ile107Val, and Ser77Tyr.

[0042] In another embodiment of the present disclosure, in any of the methods, the subject suffers from acquired neuropathy accompanied by axonal loss and / or impaired nerve regeneration. Acquired neuropathy is peripheral neuropathy caused by any disorder or disease known to cause neuropathy. For example, the subject suffers from peripheral neuropathy caused by diabetes, human immunodeficiency virus (HIV) infection, thyroid disorders such as hypothyroidism, hypoglycemia, uremia, renal failure, liver dysfunction, liver failure, polycythemia, connective tissue disorders, cancer, Lyme disease, celiac disease, leprosy, porphyria, Sjogren's syndrome, poliovirus infection, acromegaly, disorders of lipid / glycolipid metabolism, West Nile disease, amyloidosis, mitochondrial disorders, benign monoclonal gammopathy (MGUS) or paraprotein disorders such as POEMS syndrome. The subject may also receive vitamin B 12 Suffering from a nutritional / vitamin deficiency, such as iron deficiency, vitamin E deficiency, or copper deficiency.

[0043] In a further aspect of the disclosure, in any of the methods described herein, the subject is afflicted with autoimmune peripheral polyneuropathy, acute inflammatory demyelinating polyneuropathy (AIDP), chronic inflammatory demyelinating polyneuropathy (CIDP), vasculitic mononeuritis multiplex, paraneuropathy, idiopathic ganglionitis, amyotrophic lateral sclerosis, multifocal motor conduction block neuropathy, or lower motor neuron syndrome.

[0044] The acquired neuropathy can be a toxic neuropathy. For example, the toxic neuropathy can be caused by chloramphenicol, chloroquine, colchicine, disulfiram, etanercept, ethambutol, gold, hydroxychloroquine, nitrofurantoin, metronidazole, stavudine, zalcitabine, infliximab, leflunomide, thalidomide, or chemotherapeutic agents (cisplatin, cytarabine, bortezomib, docetaxel, lenalidomide, misonidazole, oxaliplatin, paclitaxel, procarbazine, suramin, thalidomide, vincristine ... Toxic neuropathy is the result of the toxic effects of prescribed medications, such as anti-alcohol drugs such as benzodiazepines (e.g., thiazolidine, benzocaine, or vincristine), or anti-alcohol drugs such as disulfiram, or anticonvulsants such as phenytoin or dilantin, or heart or blood pressure medications (e.g., statins, amiodarone, hydralazine, procainamide, perhexiline), or antibiotics (e.g., fluoroquinolones, isoniazid, Cipro, Levaquin, Flagyl, or metronidazole), or skin condition medications such as dapsone. Toxic neuropathy can also be caused by long-term alcohol abuse or vitamin B6 toxicity.

[0045] In another aspect of the present disclosure, in any of the methods described herein, the subject is a cancer patient suffering from an acquired neuropathy, for example, the cancer patient has neuropathy associated with nutritional deficiency, chemotherapy side effects, and / or paraneoplastic syndromes.

[0046] In yet another aspect of the present disclosure, in any of the methods, the subject is a surgical patient suffering from an acquired neuropathy, for example, a surgical patient who has developed neuropathy after bariatric surgery, multiple orthopedic surgeries, or multiple surgeries for "entrapped nerves."

[0047] In another aspect of the disclosure, in any of the methods, the subject is afflicted with a hereditary myopathy, a neuromuscular disease, muscle atrophy, drug-induced myopathy, sarcopenia, cachexia, type II muscle fiber atrophy, a genetic muscular dystrophy, age-related muscle atrophy, or an acquired autoimmune primary muscle disorder.

[0048] In another embodiment of the disclosure, in any of the methods, the subject is diagnosed with Duchenne muscular dystrophy, Becker muscular dystrophy, myotonic muscular dystrophy, sarcoglycanopathy, myotonic dystrophy, Emery-Dreifuss muscular dystrophy, congenital muscular dystrophy, merosin-deficient congenital muscular dystrophy, Bethlem myopathy, Ullrich congenital muscular dystrophy, facioscapulohumeral muscular dystrophy, spinal muscular dystrophy, spondylotonic muscular dystrophy, distal muscular dystrophy, oculopharyngeal muscular dystrophy, congenital muscular dystrophy (MDC) 1A, 1B, 1C, and 1D; limb-girdle muscular dystrophy (LGMD) 1A, 1B, 1C, 1D, 1E, 1F, 1G, 1H, 2A, 2B, 2C, 2D, 2E, 2F, 2G If you have 2H, 2I, 2J, 2K, 2L, 2M, 2N, 2O, or 2Q; muscle-eye-encephalopathy; Fukuyama-Walker-Warburg syndrome; myasthenic syndrome; congenital myasthenia; inclusion body myopathy; inclusion body myositis; dermatomyositis; centronuclear myopathy; Miyoshi myopathy; mitochondrial myopathy; nemaline myopathy; Nonaka myopathy; myasthenia gravis; or polymyositis.

[0049] In one embodiment, the present disclosure provides use of a therapeutically effective amount of neurotrophin-3 (NT-3), pro-NT-3, or an effective fragment thereof, or a nucleic acid encoding NT-3, pro-NT-3, or an effective fragment thereof, for the manufacture of a medicament for stimulating muscle growth in a subject. For example, the medicament is formulated for intramuscular administration.

[0050] In exemplary embodiments, the agent comprises a nucleic acid encoding NT-3, pro-NT-3, or an effective fragment thereof, wherein the nucleic acid is in a viral vector. In related embodiments, the viral vector is an adeno-associated viral vector. In various embodiments, the nucleic acid is operably linked to a muscle-specific promoter, such as a triplet muscle-specific creatine kinase promoter. In various embodiments, the nucleic acid comprises SEQ ID NO:1.

[0051] The present invention relates to the use of a nucleic acid encoding an NT-3 polypeptide for the manufacture of a medicament for treating a muscle wasting disease or neuropathy in a human subject, wherein a) the nucleic acid comprises a nucleotide sequence that is 90% identical to the nucleotide sequence of SEQ ID NO: 1; b) the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 1; c) the nucleic acid comprises a nucleic acid sequence that encodes an amino acid sequence that is at least 90% identical to SEQ ID NO: 2 or 100% identical to SEQ ID NO: 2; d) the nucleic acid encoding the NT-3 polypeptide is any of the nucleic acids disclosed herein; and e) the nucleic acid encoding an NT-3 polypeptide is any of the nucleic acids disclosed herein. f) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the rAAV is at a dose that results in sustained expression of a low concentration of the NT-3 polypeptide; f) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the agent is formulated for intramuscular administration, and the dose of the rAAV is about 1.5 x 10 vg / kg to about 6.5 x 10 vg / kg; g) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the agent is formulated for intramuscular administration, and the dose of the rAAV is about 1.5 x 10 vg / kg to about 6.5 x 10 vg / kg; a) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the medicament is formulated for intramuscular administration, and the dose of the rAAV is about 2×10 vg / kg to about 6×10 vg / kg; b) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the medicament is formulated for intramuscular administration, and the dose of the rAAV is about 2×10 vg / kg; c) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the medicament is formulated for intramuscular administration, and the dose of the rAAV is about 2×10 vg / kg; AAV1.tMCK.NTF3, the agent is formulated for intramuscular administration, and the dose of the rAAV is about 4 x 10 vg / kg; j) the nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the agent is formulated for intramuscular administration, and the dose of the rAAV administered is about 6 x 10 vg / kg; k) the nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the agent is formulated for intramuscular administration, and the dose of the rAAV administered is about 0.or l) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the agent is formulated for intramuscular injection at a concentration of about 2 x 10 vg / ml administered using 3 to 6 injections per muscle in a total volume of about 5 to 14 ml. 13 The use is also provided, wherein the compound is formulated for intramuscular injection at a concentration of 0.15 mg / ml.

[0052] The present disclosure relates to the use of a dose of a nucleic acid encoding an NT-3 polypeptide for the manufacture of a medicament for improving muscle strength or stimulating muscle growth in a human subject, wherein a) the nucleic acid comprises a nucleotide sequence that is 90% identical to the nucleotide sequence of SEQ ID NO:1, b) the nucleic acid comprises the nucleotide sequence of SEQ ID NO:1; c) the nucleic acid comprises a nucleic acid sequence that encodes an amino acid sequence that is at least 90% identical to SEQ ID NO:2 or 100% identical to SEQ ID NO:2, d) the nucleic acid encoding the NT-3 polypeptide is any of the nucleic acids of the present disclosure, and e) the N f) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the rAAV is at a dose that results in sustained expression of a low concentration of the NT-3 polypeptide; f) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the composition is formulated for an intramuscular route of administration, and the dose of the rAAV is from about 1.5 x 10 vg / kg to about 6.5 x 10 vg / kg; g) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the composition is formulated for an intramuscular route of administration, and the dose of the rAAV is from about 1.5 x 10 vg / kg to about 6.5 x 10 vg / kg; e) the nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the medicament is formulated for intramuscular administration, and the dose of the rAAV is about 2 x 10 vg / kg to about 6 x 10 vg / kg; h) the nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the medicament is formulated for intramuscular administration, and the dose of the rAAV is about 2 x 10 vg / kg; i) the nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the medicament is formulated for intramuscular administration, and the dose of the rAAV is about 2 x 10 vg / kg. cAAV1.tMCK.NTF3, the agent is formulated for intramuscular administration, and the dose of the rAAV is about 4 x 10 vg / kg; j) the nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the agent is formulated for intramuscular administration, and the dose of the rAAV administered is about 6 x 10 vg / kg; k) the nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the agent is formulated for intramuscular administration, and the dose of the rAAV administered is about 0.or l) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the agent is formulated for intramuscular injection at a concentration of about 2 x 10 vg / ml administered using 3 to 6 injections per muscle in a total volume of about 5 to 14 ml. 13 The use is also provided, wherein the compound is formulated for intramuscular injection at a concentration of 0.15 mg / ml.

[0053] For example, any of the medicaments of the present disclosure may comprise a nucleic acid formulated for administration using a viral vector, such as an adeno-associated viral vector. Furthermore, any of the medicaments of the present disclosure may comprise a nucleic acid operably linked to a muscle-specific promoter, for example, the muscle-specific promoter is the muscle-specific creatine kinase promoter (MCK). In another embodiment, in any of the medicaments or disclosed herein, scAAV1.tMCK.NTF3 comprises the NT-3 gene cassette set forth in SEQ ID NO: 11. In one embodiment, the present disclosure provides use of recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3 for the preparation of a medicament for treating muscle wasting disease or neuropathy in a human subject in need thereof, wherein the medicament results in sustained expression of low levels of NT-3 protein.

[0054] In another embodiment, the present disclosure provides use of recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3 for the preparation of a medicament for stimulating muscle growth in a human subject in need thereof, wherein the dose results in sustained expression of low levels of NT-3 protein.

[0055] In one embodiment, the disclosure provides a use of recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3 for the preparation of a medicament for treating a muscle wasting disease or neuropathy in a human subject in need thereof, wherein the medicament is formulated for an intramuscular route of administration, and the medicament is administered in an amount of about 1.0 x 10 12 vg / kg ~ approx. 7×10 12vg / kg, or approximately 1.5 × 10 12 vg / kg ~ approx. 6.5×10 12 vg / kg, or approximately 2 × 10 12 vg / kg ~ approx. 6×10 12 The use includes a dose of rAAV that is 0.05 mg / kg.

[0056] In another embodiment, the disclosure provides a use of recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3 for the preparation of a medicament for treating a muscle wasting disease or neuropathy in a human subject in need thereof, wherein the medicament is formulated for an intramuscular route of administration, and the medicament is administered in an amount of about 1.0 x 10 12 vg / kg, or approximately 1.5 × 10 12 vg / kg, or approximately 2 × 10 12 vg / kg, or approximately 3 × 10 12 vg / kg, or approximately 4 × 10 12 vg / kg, or approximately 5 × 10 12 vg / kg, or approximately 6 × 10 12 vg / kg, or approximately 7 × 10 12 vg / kg, or approximately 8 × 10 12 vg / kg, or approximately 9 × 10 12 vg / kg, or approximately 1 × 10 13 The use includes a dose of rAAV that is 0.05 mg / kg.

[0057] In another embodiment, the disclosure provides a use of recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3 for the preparation of a medicament for treating a muscle wasting disease or neuropathy in a human subject in need thereof, wherein the medicament is formulated for an intramuscular route of administration, and the medicament is administered in an amount of about 2 x 10 13 For example, the agent is administered to the medial and lateral heads of the gastrocnemius and tibialis anterior muscles in each leg using 3 to 6 injections per muscle (e.g., each injection volume is 0.5 to 1 ml), respectively, where a total of 5 ml to 14 ml of vector is administered to the medial and lateral heads of the gastrocnemius and tibialis anterior muscles in each leg.

[0058] In an exemplary embodiment, the disclosure provides a use of recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3 for the preparation of a medicament for treating a muscle wasting disease or neuropathy in a human subject in need thereof, wherein the medicament is formulated for intramuscular administration, and the medicament is administered using 3 to 6 injections per muscle (each injection volume being 0.5 to 1 ml), each of which is administered to approximately 1 x 10 13 The present invention provides for use in a method comprising administering a dose of rAAV in a dose range of 1000-1400 mg / mL, wherein a total of 5 mL to 14 mL of vector is administered into the medial and lateral heads of the gastrocnemius and tibialis anterior muscles in each leg.

[0059] In one embodiment, the disclosure provides a use of recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3 for the preparation of a medicament for improving muscle strength or stimulating muscle growth in a human subject in need thereof, wherein the medicament is formulated for an intramuscular route of administration, and the medicament is administered in an amount of about 1.0 x 10 12 vg / kg ~ approx. 7×10 12 vg / kg, or approximately 1.5 × 10 12 vg / kg ~ approx. 6.5×10 12 vg / kg, or approximately 2 × 10 12 vg / kg ~ approx. 6×10 12 The use includes a dose of rAAV that is 0.05 mg / kg.

[0060] In another embodiment, the disclosure provides a use of recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3 for the preparation of a medicament for improving muscle strength or stimulating muscle growth in a human subject in need thereof, wherein the medicament is formulated for an intramuscular route of administration, and the medicament is administered in an amount of about 1.0 x 10 12 vg / kg, or approximately 1.5 × 10 12 vg / kg, or approximately 2 × 10 12 vg / kg, or approximately 3 × 10 12 vg / kg, or approximately 4 × 10 12 vg / kg, or approximately 5 × 10 12 vg / kg, or approximately 6 × 10 12 vg / kg, or approximately 7 × 10 12vg / kg, or approximately 8 × 10 12 vg / kg, or approximately 9 × 10 12 vg / kg, or approximately 1 × 10 13 The use includes a dose of rAAV that is 0.05 mg / kg.

[0061] In another embodiment, the disclosure provides a use of recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3 for the preparation of a medicament for improving muscle strength or stimulating muscle growth in a human subject in need thereof, wherein the medicament is formulated for an intramuscular route of administration and the medicament is administered at a low dose (2 x 10 per patient). 12 vg / kg) and high dose (6 × 10 per patient 12 Approximately 2 × 10 13 The present invention provides a method for administering rAAV to a patient, comprising administering the rAAV to a patient in a concentration of 0.5-1.0 vg / ml. In one embodiment, the agent is administered to the medial and lateral heads of the gastrocnemius and tibialis anterior muscles in each leg using 3-6 injections per muscle (e.g., 0.5-1 ml each). A total of 5-14 ml of vector is administered to the medial and lateral heads of the gastrocnemius and tibialis anterior muscles in each leg.

[0062] In an exemplary embodiment, the disclosure provides a use of recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3 for the preparation of a medicament for improving muscle strength or stimulating muscle growth in a human subject in need thereof, wherein the medicament is formulated for intramuscular administration route and the medicament is administered at a low dose (2 x 10 per patient) using 3-6 injections per muscle (each injection volume being 0.5-1 ml). 12 vg / kg) and high dose (6 × 10 per patient 12 Approximately 2 × 10 13 The present invention provides for the use of rAAV containing a concentration of rAAV in 100 mg / ml of rAAV. A total of 5 mL to 14 mL of vector is administered into the medial and lateral heads of the gastrocnemius and tibialis anterior muscles in each leg.

[0063] In any of the uses of the present disclosure, the agent is formulated for intramuscular bilateral injection into the medial and lateral heads of the gastrocnemius and tibialis anterior muscles. Further, in any of the uses of the present invention, the agent results in an improvement in muscle strength in the subject's upper or lower limbs, for example, the improvement in muscle strength is measured as a reduction in the composite score on the CMT Pediatric scale (CMTPeds). Further, in any of the uses of the present invention, the agent results in a reduction or halt of disease progression over a two-year period. Disease progression is measured by the CMTPeds.

[0064] In one embodiment of the present disclosure, in any of the uses of the present disclosure, the subject suffers from a hereditary neuropathy, such as Charcot-Marie-Tooth (CMT) neuropathy, e.g., CMT1A, CMT2K, CMT4A, CMTRIA, and axonal and demyelinating neuropathies caused by autosomal recessive, autosomal dominant, or X-linked genetic mutations. The hereditary neuropathy may be caused by any of the genetic mutations listed in Table 1. Additionally, the hereditary neuropathy may be a transthyretin amyloid neuropathy caused by a mutation in the transthyretin (TTR) gene, such as the following genetic mutations: Val30Met, Ile107Val, and Ser77Tyr.

[0065] In another embodiment of the present disclosure, in any of the methods of the present invention, the subject suffers from acquired neuropathy accompanied by axonal loss and / or impaired nerve regeneration. Acquired neuropathy is peripheral neuropathy caused by any disorder or disease that causes neuropathy. For example, the subject suffers from peripheral neuropathy caused by diabetes, human immunodeficiency virus (HIV) infection, thyroid disorders such as hypothyroidism, hypoglycemia, uremia, renal failure, liver dysfunction, liver failure, polycythemia, connective tissue disorders, cancer, Lyme disease, celiac disease, leprosy, porphyria, Sjogren's syndrome, poliovirus infection, acromegaly, disorders of lipid / glycolipid metabolism, West Nile disease, amyloidosis, mitochondrial disorders, benign monoclonal gammopathy (MGUS) or paraprotein disorders such as POEMS syndrome. The subject may also receive vitamin B 12 The patient may be suffering from a nutritional / vitamin deficiency, such as a vitamin D deficiency, vitamin E deficiency or copper deficiency.

[0066] Additionally, in any of the uses of the present disclosure, the subject is suffering from autoimmune peripheral polyneuropathy, acute inflammatory demyelinating polyneuropathy (AIDP), chronic inflammatory demyelinating polyneuropathy (CIDP), vasculitic mononeuritis multiplex, paraneuropathy, idiopathic ganglionitis, amyotrophic lateral sclerosis, multifocal motor conduction block neuropathy, or lower motor neuron syndrome.

[0067] The acquired neuropathy is a toxic neuropathy. For example, the toxic neuropathy may be caused by chloramphenicol, chloroquine, colchicine, disulfiram, etanercept, ethambutol, gold, hydroxychloroquine, nitrofurantoin, metronidazole, stavudine, zalcitabine, infliximab, leflunomide, thalidomide, or chemotherapeutic agents (cisplatin, cytarabine, bortezomib, docetaxel, lenalidomide, misonidazole, oxaliplatin, paclitaxel, procarbazine, suramin, thalidomide, vincristine ... It is the result of the toxic effects of prescribed medications, such as anti-alcohol drugs (such as cyclosporine or vincristine), or anti-alcohol drugs such as disulfiram, or anticonvulsants such as phenytoin or dilantin, or heart or blood pressure medications (such as statins, amiodarone, hydralazine, procainamide, perhexiline), or antibiotics (such as fluoroquinolones, isoniazid, Cipro, Levaquin, Flagyl, or metronidazole), or skin condition medications such as dapsone. Toxic neuropathy can be caused by long-term alcohol abuse or vitamin B6 toxicity.

[0068] In another embodiment, in any of the uses of the present disclosure, the subject is a cancer patient suffering from an acquired neuropathy, for example, the cancer patient has developed a neuropathy associated with nutritional deficiency, chemotherapy side effects, and / or paraneoplastic syndromes.

[0069] In yet another aspect, in any of the uses of the present disclosure, the subject is a surgical patient suffering from an acquired neuropathy, for example, a surgical patient who has developed neuropathy after bariatric surgery, multiple orthopedic surgeries, or multiple surgeries for "entrapped nerves."

[0070] In another aspect, in any of the uses of the present disclosure, the subject is suffering from a hereditary myopathy, a neuromuscular disease, muscle atrophy, drug-induced myopathy, sarcopenia, cachexia, type II muscle fiber atrophy, a genetic muscular dystrophy, age-related muscle atrophy, or an acquired autoimmune primary muscle disease.

[0071] In another aspect, in any of the uses of the present disclosure, the subject is a patient with Duchenne muscular dystrophy, Becker muscular dystrophy, myotonic muscular dystrophy, sarcoglycanopathy, myotonic dystrophy, Emery-Dreifuss muscular dystrophy, congenital muscular dystrophy, merosin-deficient congenital muscular dystrophy, Bethlem myopathy, Ullrich congenital muscular dystrophy, facioscapulohumeral muscular dystrophy, spinal muscular dystrophy, spondylotonic muscular dystrophy, distal muscular dystrophy, oculopharyngeal muscular dystrophy, congenital muscular dystrophy (MDC) 1A, 1B, 1C, and 1D; limb-girdle muscular dystrophy (LGMD) 1A, 1B, 1C, 1D, 1E, 1F, 1G, 1H, 2A, 2B, 2C, 2D, 2E, 2F, 2G If you have 2H, 2I, 2J, 2K, 2L, 2M, 2N, 2O, or 2Q; muscle-eye-encephalopathy; Fukuyama-Walker-Warburg syndrome; myasthenic syndrome; congenital myasthenia; inclusion body myopathy; inclusion body myositis; dermatomyositis; centronuclear myopathy; Miyoshi myopathy; mitochondrial myopathy; nemaline myopathy; Nonaka myopathy; myasthenia gravis; or polymyositis.

[0072] In one embodiment, the present disclosure provides a composition comprising a therapeutically effective amount of neurotrophin-3 (NT-3), pro-NT-3, or an effective fragment thereof, or a nucleic acid encoding NT-3, pro-NT-3, or an effective fragment thereof, for use in stimulating muscle growth in a subject. For example, the composition of the present disclosure is formulated for intramuscular administration.

[0073] In exemplary embodiments, the composition comprises a nucleic acid encoding NT-3, pro-NT-3, or an effective fragment thereof. In related embodiments, the nucleic acid is in a viral vector, such as an adeno-associated viral vector. In various embodiments, the nucleic acid is operably linked to a muscle-specific promoter, such as a muscle-specific creatine kinase promoter triplet. In various embodiments, the composition comprises a nucleic acid comprising the nucleotide sequence of SEQ ID NO:1.

[0074] The present disclosure provides a composition comprising a nucleic acid encoding an NT-3 polypeptide for use in treating a muscle wasting disease or neuropathy in a human subject, wherein a) the nucleic acid comprises a nucleotide sequence that is 90% identical to the nucleotide sequence of SEQ ID NO:1; b) the nucleic acid comprises the nucleotide sequence of SEQ ID NO:1; c) the nucleic acid comprises a nucleic acid sequence that encodes an amino acid sequence that is at least 90% identical to SEQ ID NO:2 or 100% identical to SEQ ID NO:2; d) the nucleic acid encoding the NT-3 polypeptide is any of the nucleic acids of the present disclosure; and e) the NT-3 polypeptide is an NT-3 polypeptide. f) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the rAAV is administered at a dose that results in sustained expression of a low concentration of the NT-3 polypeptide; f) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the composition is formulated for an intramuscular route of administration, and the dose of the rAAV is about 1.5 x 10 vg / kg to about 6.5 x 10 vg / kg; g) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the composition is formulated for an intramuscular route of administration, and the dose of the rAAV is about 1.5 x 10 vg / kg to about 6.5 x 10 vg / kg; a) the nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the composition is formulated for intramuscular administration, and the dose of the rAAV is about 2 x 10 vg / kg to about 6 x 10 vg / kg; b) the nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the composition is formulated for intramuscular administration, and the dose of the rAAV is about 2 x 10 vg / kg to about 6 x 10 vg / kg; c) the nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the composition is formulated for intramuscular administration, and the dose of the rAAV is about 2 x 10 vg / kg; j) the nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the composition is formulated for intramuscular administration and the dose of rAAV administered is about 6 x 10 vg / kg; k) the nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the composition is formulated for intramuscular administration and the dose of rAAV administered is about 0.or l) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the composition is formulated for intramuscular injection at a concentration of about 2 x 10 vg / ml administered using 3-6 injections per muscle in a total volume of about 5-14 ml. 13 Also provided is a composition formulated for intramuscular injection at a concentration of 1000 mg / ml.

[0075] In another embodiment, the disclosure provides a composition comprising a nucleic acid encoding an NT-3 polypeptide for use in improving muscle strength or stimulating muscle growth in a human subject, wherein a) the nucleic acid comprises a nucleotide sequence that is 90% identical to the nucleotide sequence of SEQ ID NO:1; b) the nucleic acid comprises the nucleotide sequence of SEQ ID NO:1; c) the nucleic acid comprises a nucleic acid sequence that encodes an amino acid sequence that is at least 90% identical to SEQ ID NO:2 or 100% identical to SEQ ID NO:2; and d) the nucleic acid encoding the NT-3 polypeptide is any of the nucleic acids of the disclosure. e) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, wherein the rAAV is at a dose that results in sustained expression of a low concentration of the NT-3 polypeptide; f) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, wherein the composition is formulated for intramuscular administration and the dose of the rAAV is from about 1.5 x 10 vg / kg to about 6.5 x 10 vg / kg; g) the nucleic acid encoding the NT-3 polypeptide is , a) the nucleic acid encoding an NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the composition is formulated for intramuscular administration, the dose of the rAAV is about 2 x 10 vg / kg to about 6 x 10 vg / kg; b) the nucleic acid encoding an NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the composition is formulated for intramuscular administration, the dose of the rAAV is about 2 x 10 vg / kg to about 6 x 10 vg / kg; c) the nucleic acid encoding an NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the composition is formulated for intramuscular administration, the dose of the rAAV is about 2 x 10 vg / kg. j) the nucleic acid encoding an NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the composition is formulated for intramuscular administration, the dose of the rAAV is about 4 x 10 vg / kg; k) the nucleic acid encoding an NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the composition is formulated for intramuscular administration, the dose of the rAAV is about 6 x 10 vg / kg;or l) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the composition is formulated for intramuscular injection at a concentration of about 2 x 10 vg / ml administered using 3-6 injections per muscle in a total volume of about 5-14 ml. 13 The composition is formulated for intramuscular injection at a concentration of 1000 mg / ml.

[0076] For example, any of the compositions of the present disclosure can include a nucleic acid formulated for administration using a viral vector, such as an adeno-associated viral vector. Additionally, any of the compositions of the present disclosure can include a nucleic acid operably linked to a muscle-specific promoter, for example, the muscle-specific promoter is the muscle-specific creatine kinase promoter (MCK). In another embodiment, in any of the compositions or disclosed herein, scAAV1.tMCK.NTF3 includes the NT-3 gene cassette set forth in SEQ ID NO:11.

[0077] In one embodiment, the disclosure provides a composition comprising a dose of recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3 for treating a muscle wasting disease or neuropathy in a human subject in need thereof, which results in sustained expression of low levels of NT-3 protein.

[0078] In another embodiment, the present disclosure provides the use of recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3 to stimulate muscle growth in a human subject in need thereof, resulting in sustained expression of low levels of NT-3 protein.

[0079] In one embodiment, the disclosure provides a composition comprising a dose of recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3 for treating a muscle wasting disease or neuropathy in a human subject in need thereof, wherein the composition is formulated for intramuscular administration and the dose of rAAV administered is about 1.0 x 10 12 vg / kg ~ approx. 7×1012 vg / kg, or approximately 1.5 × 10 12 vg / kg ~ approx. 6.5×10 12 vg / kg, or approximately 2 × 10 12 vg / kg ~ approx. 6×10 12 vg / kg of the composition.

[0080] In another embodiment, the disclosure provides a composition comprising a dose of recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3 for treating a muscle wasting disease or neuropathy in a human subject in need thereof, wherein the composition is formulated for intramuscular administration and the dose of rAAV administered is about 1.0 x 10 12 vg / kg, or approximately 1.5 × 10 12 vg / kg, or approximately 2 × 10 12 vg / kg, or approximately 3 × 10 12 vg / kg, or approximately 4 × 10 12 vg / kg, or approximately 5 × 10 12 vg / kg, or approximately 6 × 10 12 vg / kg, or approximately 7 × 10 12 vg / kg, or approximately 8 × 10 12 vg / kg, or approximately 9 × 10 12 vg / kg, or approximately 1 × 10 13 vg / kg of the composition.

[0081] In another embodiment, the disclosure provides a composition comprising a dose of recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3 for treating a muscle wasting disease or neuropathy in a human subject in need thereof, wherein the composition is formulated for intramuscular administration and the dose of rAAV administered is a low dose (2 x 10 per patient). 12 vg / kg) and high dose (6 × 10 per patient 12 Approximately 2 × 10 13The composition is provided in a volume of 0.5-1.5 vg / ml. In one embodiment, the composition is administered to the medial and lateral heads of the gastrocnemius and tibialis anterior muscles in each leg using 3-6 injections per muscle, each injection volume being 0.5-1 ml. A total of 5 ml-14 ml of vector is administered to the medial and lateral heads of the gastrocnemius and tibialis anterior muscles in each leg.

[0082] In an exemplary embodiment, the disclosure provides a composition comprising a dose of recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3 for treating muscle wasting disease or neuropathy in a human subject in need thereof, wherein the composition is formulated for intramuscular administration and the dose of rAAV administered is a low dose (2 x 10 per patient) using 3-6 injections per muscle (each injection volume being 0.5-1 ml). 12 vg / kg) and high dose (6 × 10 per patient 12 Approximately 2 × 10 administered at vg / kg 13 A total of 5 mL to 14 mL of vector is administered into the medial and lateral heads of the gastrocnemius and tibialis anterior muscles in each leg.

[0083] In one embodiment, the disclosure provides a composition comprising a dose of recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3 for improving muscle strength in a human subject, wherein the composition is formulated for intramuscular administration and the dose of rAAV administered is about 1.0 x 10 12 vg / kg ~ approx. 7×10 12 vg / kg, or approximately 1.5 × 10 12 vg / kg ~ approx. 6.5×10 12 vg / kg, or approximately 2 × 10 12 vg / kg ~ approx. 6×10 12 vg / kg of the composition.

[0084] In another embodiment, the disclosure provides a composition comprising a dose of recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3 for improving muscle strength in a human subject, wherein the composition is formulated for intramuscular administration and the dose of rAAV administered is about 1.0 x 10 12 vg / kg, or approximately 1.5 × 10 12 vg / kg, or approximately 2 × 10 12 vg / kg, or approximately 3 × 10 12 vg / kg, or approximately 4 × 10 12 vg / kg, or approximately 5 × 10 12 vg / kg, or approximately 6 × 10 12 vg / kg, or approximately 7 × 10 12 vg / kg, or approximately 8 × 10 12 vg / kg, or approximately 9 × 10 12 vg / kg, or approximately 1 × 10 13 vg / kg of the composition.

[0085] In another embodiment, the disclosure provides a composition comprising a dose of recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3 for improving muscle strength or stimulating muscle growth in a human subject in need thereof, wherein the composition is formulated for intramuscular administration and the dose of rAAV administered is a low dose (2 x 10 per patient). 12 vg / kg) and high dose (6 × 10 per patient 12 Approximately 2 × 10 13 The composition is provided in a volume of 0.5-1.5 vg / ml. In one embodiment, the composition is administered to the medial and lateral heads of the gastrocnemius and tibialis anterior muscles in each leg using 3-6 injections per muscle, each injection volume being 0.5-1 ml. A total of 5 ml-14 ml of vector is administered to the medial and lateral heads of the gastrocnemius and tibialis anterior muscles in each leg.

[0086] In an exemplary embodiment, the disclosure provides a composition comprising a dose of recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3 for improving muscle strength or stimulating muscle growth in a human subject in need thereof, wherein the composition is formulated for intramuscular administration and the dose of rAAV administered is a low dose (2 x 10 per patient) using 3-6 injections per muscle (each injection volume being 0.5-1 ml). 12 vg / kg) and high dose (6 × 10 per patient 12 Approximately 2 × 10 administered at vg / kg 13 A total of 5 mL to 14 mL of vector is administered into the medial and lateral heads of the gastrocnemius and tibialis anterior muscles in each leg.

[0087] In any of the compositions of the present disclosure, the route of administration of scAAV1.tMCK.NTF3 is intramuscular bilateral injection into the medial and lateral heads of the gastrocnemius and tibialis anterior muscles. Further, in any of the compositions of the present disclosure, administration of scAAV1.tMCK.NTF3 results in improved muscle strength in the subject's upper or lower limbs, for example, improved muscle strength as measured by the CMT Pediatric Scale (CMT). The efficacy of any of the compositions disclosed herein is measured as a reduction in the composite score on the Clinical Performance Test for Pediatric Pediatrics (CMTPedS). Furthermore, in any of the compositions disclosed herein, administration of the composition results in a reduction or halt of disease progression over a two-year period. Disease progression is measured by the CMTPedS. The CMTPedS is an 11-item scale consisting of the Functional Dexterity Test, the Nine-Hole Peg Test (9HPT), handgrip, plantar flexion, and dorsiflexion strength using a manual dynamometer, pinprick and vibration sensation, the Bruyninks-Oserecki test - balance assessment, gait assessment, long jump, and the 6-minute walk test (6MWT). Efficacy is defined as a halt in decline of ability as measured by this scale two years after gene transfer.

[0088] In embodiments of the present disclosure, in any of the compositions, the subject suffers from a hereditary neuropathy, such as Charcot-Marie-Tooth (CMT) neuropathies, such as CMT1A, CMT2K, CMT4A, and CMTRIA, as well as axonal and demyelinating neuropathies caused by autosomal recessive, autosomal dominant, or X-linked genetic mutations. The hereditary neuropathies can be caused by any of the genetic mutations listed in Table 1. Additionally, the hereditary neuropathies can be transthyretin amyloid neuropathy caused by mutations in the transthyretin (TTR) gene, such as the following genetic mutations: Val30Met, Ile107Val, and Ser77Tyr.

[0089] In another embodiment of the present disclosure, in any of the compositions, the subject suffers from acquired neuropathy accompanied by axonal loss and / or impaired nerve regeneration. Acquired neuropathy is peripheral neuropathy caused by any disorder or disease that causes neuropathy. For example, the subject suffers from peripheral neuropathy caused by diabetes, human immunodeficiency virus (HIV) infection, thyroid disorders such as hypothyroidism, hypoglycemia, uremia, renal failure, liver dysfunction, liver failure, polycythemia, connective tissue disorders, cancer, Lyme disease, celiac disease, leprosy, porphyria, Sjogren's syndrome, poliovirus infection, acromegaly, disorders of lipid / glycolipid metabolism, West Nile disease, amyloidosis, mitochondrial disorders, benign monoclonal gammopathy (MGUS) or paraprotein disorders such as POEMS syndrome. The subject may also receive vitamin B 12 Suffering from a nutritional / vitamin deficiency, such as iron deficiency, vitamin E deficiency, or copper deficiency.

[0090] Additionally, in any of the compositions of the present disclosure, the subject is afflicted with autoimmune peripheral polyneuropathy, acute inflammatory demyelinating polyneuropathy (AIDP), chronic inflammatory demyelinating polyneuropathy (CIDP), vasculitic mononeuritis multiplex, paraneuropathy, idiopathic ganglionitis, amyotrophic lateral sclerosis, multifocal motor conduction block neuropathy, or lower motor neuron syndrome.

[0091] In any of the compositions of the present disclosure, the acquired neuropathy is a toxic neuropathy. For example, the toxic neuropathy may be caused by chloramphenicol, chloroquine, colchicine, disulfiram, etanercept, ethambutol, gold, hydroxychloroquine, nitrofurantoin, metronidazole, stavudine, zalcitabine, infliximab, leflunomide, thalidomide, or a chemotherapeutic agent (cisplatin, cytarabine, bortezomib, docetaxel, lenalidomide, misonidazole, oxaliplatin, paclitaxel, procarbazine, suramin, thalidomide, vincristine ... It is the result of the toxic effects of prescribed medications, such as anti-alcohol drugs (such as cyclosporine or vincristine), or anti-alcohol drugs such as disulfiram, or anticonvulsants such as phenytoin or dilantin, or heart or blood pressure medications (such as statins, amiodarone, hydralazine, procainamide, perhexiline), or antibiotics (such as fluoroquinolones, isoniazid, Cipro, Levaquin, Flagyl, or metronidazole) and skin condition medications such as dapsone. Toxic neuropathy can also be caused by long-term alcohol abuse or vitamin B6 toxicity.

[0092] In another embodiment of the present disclosure, in any of the compositions, the subject is a cancer patient suffering from an acquired neuropathy, for example, the cancer patient has developed a neuropathy associated with nutritional deficiency, chemotherapy side effects, and / or paraneoplastic syndromes.

[0093] In yet another aspect of the present disclosure, in any of the compositions, the subject is a surgical patient suffering from an acquired neuropathy, for example, the surgical patient develops neuropathy after bariatric surgery, multiple orthopedic surgeries, or multiple surgeries for "entrapped nerves."

[0094] In another aspect of the disclosure, in any of the above, the subject is suffering from a hereditary myopathy, a neuromuscular disease, muscle atrophy, drug-induced myopathy, sarcopenia, cachexia, type II muscle fiber atrophy, a genetic muscular dystrophy, age-related muscle atrophy, or an acquired autoimmune primary muscle disease.

[0095] In another embodiment of the disclosure, in any of the compositions, the subject is a patient with Duchenne muscular dystrophy, Becker muscular dystrophy, myotonic muscular dystrophy, sarcoglycanopathy, myotonic dystrophy, Emery-Dreifuss muscular dystrophy, congenital muscular dystrophy, merosin-deficient congenital muscular dystrophy, Bethlem myopathy, Ullrich congenital muscular dystrophy, facioscapulohumeral muscular dystrophy, spinal muscular dystrophy, spondylotonic muscular dystrophy, distal muscular dystrophy, oculopharyngeal muscular dystrophy, congenital muscular dystrophy (MDC) 1A, 1B, 1C, and 1D; limb-girdle muscular dystrophy (LGMD) 1A, 1B, 1C, 1D, 1E, 1F, 1G, 1H, 2A, 2B, 2C, 2D, 2E, 2F, 2G If you have 2H, 2I, 2J, 2K, 2L, 2M, 2N, 2O, or 2Q; muscle-eye-encephalopathy; Fukuyama-Walker-Warburg syndrome; myasthenic syndrome; congenital myasthenia; inclusion body myopathy; inclusion body myositis; dermatomyositis; centronuclear myopathy; Miyoshi myopathy; mitochondrial myopathy; nemaline myopathy; Nonaka myopathy; myasthenia gravis; or polymyositis.

[0096] In another aspect of the disclosure, in any of the compositions, the subject is suffering from traumatic nerve injury, such as nerve injury caused by compression, double crush, or amputation. The present invention provides, for example, the following items. (Item 1) From 5' to 3': (i) the first AAV2 inverted terminal repeat (ITR); (ii) the muscle creatine kinase promoter / enhancer sequence set forth in nucleotides 147 to 860 of SEQ ID NO:11; (iii) a nucleotide sequence encoding a human NT-3 polypeptide; and (iv) a second AAV2 ITR sequence A nucleic acid comprising: A nucleic acid having an amino acid sequence encoded by a nucleotide sequence that is at least 90% identical to SEQ ID NO:2 or 100% identical to SEQ ID NO:2, or that is 90% identical to nucleotides 1077 to 1850 of SEQ ID NO:11 or 100% identical to nucleotides 1077 to 1850 of SEQ ID NO:11 (Item 2). 2. The nucleic acid according to item 1, further comprising a chimeric intron set forth in nucleotides 892 to 1024 of SEQ ID NO: 11 on the 3' side of the promoter / enhancer. (Item 3) 3. The nucleic acid according to item 1 or 2, further comprising an SV40 polyadenylation signal sequence represented by nucleotides 1860 to 2059 of SEQ ID NO: 11 on the 3' side of the nucleotide sequence encoding the human NT-3 polypeptide. (Item 4) the first ITR is set forth in nucleotides 7 to 112 of SEQ ID NO: 11, and / or 4. The nucleic acid according to any one of items 1 to 3, wherein the second ITR is set forth in nucleotides 2121 to 2248 of SEQ ID NO: 11. (Item 5) the first ITR is set forth in nucleotides 7 to 112 of SEQ ID NO: 11; 4. The nucleic acid according to any one of items 1 to 3, wherein the second ITR is set forth in nucleotides 2121 to 2248 of SEQ ID NO: 11. (Item 6) A nucleic acid comprising the scAAV1.tMCK.NTF3 genome that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO:11. (Item 7) A nucleic acid comprising the scAAV1.tMCK.NTF3 genome set forth in SEQ ID NO:11. (Item 8) 8. A recombinant adeno-associated virus particle (rAAV) comprising the nucleic acid of any one of items 1 to 7, which is infectious. (Item 9) 9. The rAAV particle of item 8, wherein the rAAV is of the serotype AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, or AAVrh.74. (Item 10) 11. The rAAV particle of any one of items 8 to 10, wherein the AAV DNA in the rAAV genome is derived from AAV-1. (Item 11) A composition comprising the rAAV particles of any one of items 8 to 10 and a pharmaceutically acceptable carrier. (Item 12) 12. The composition of claim 11, wherein the composition is formulated for treating a muscle wasting disease or neuropathy in a subject in need thereof. (Item 13) 12. The composition of claim 11, wherein the composition is formulated to stimulate muscle growth in a subject in need thereof. (Item 14) 1. A method of treating a muscle wasting disease or neuropathy in a human subject in need thereof, comprising administering to said human subject a nucleic acid encoding an NT-3 polypeptide; wherein: a) the nucleic acid comprises a nucleotide sequence that is 90% identical to the nucleotide sequence of SEQ ID NO: 1; b) the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 1; c) the nucleic acid comprises a nucleic acid sequence encoding an amino acid sequence that is at least 90% identical to SEQ ID NO:2 or 100% identical to SEQ ID NO:2; d) the nucleic acid encoding the NT-3 polypeptide is the nucleic acid according to any one of items 1 to 7; e) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the rAAV is administered at a dose that results in sustained expression of low levels of NT-3 polypeptide; f) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the route of administration is intramuscular, and the dose of the rAAV administered is about 1.5 x 10 vg / kg to about 6.5 x 10 vg / kg; g) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the route of administration is intramuscular, and the dose of the rAAV administered is about 2 x 10 vg / kg to about 6 x 10 vg / kg; h) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the route of administration is intramuscular, and the dose of the rAAV administered is about 2 x 10 vg / kg; i) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the route of administration is intramuscular, and the dose of the rAAV administered is about 4 x 10 vg / kg; j) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the route of administration is intramuscular, and the dose of the rAAV administered is about 6 x 10 vg / kg; k) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the route of administration is intramuscular injection at a concentration of about 2 x 10 vg / ml administered using 3-6 injections per muscle of about 0.5-1 ml; or l) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the route of administration is about 2 x 10 administered using multiple injections in a total volume of about 5-14 ml. 13 The method is an intramuscular injection at a concentration of 0.05 mg / ml. (Item 15) 1. A method of improving muscle strength or stimulating muscle growth in a human subject in need thereof, comprising administering to said human subject a nucleic acid encoding an NT-3 polypeptide; wherein: a) the nucleic acid comprises a nucleotide sequence that is 90% identical to the nucleotide sequence of SEQ ID NO: 1; b) the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 1; c) the nucleic acid comprises a nucleic acid sequence encoding an amino acid sequence that is at least 90% identical to SEQ ID NO:2 or 100% identical to SEQ ID NO:2; d) the nucleic acid encoding the NT-3 polypeptide is the nucleic acid according to any one of items 1 to 7; e) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the rAAV is administered at a dose that results in sustained expression of low levels of NT-3 polypeptide; f) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the route of administration is intramuscular, and the dose of the rAAV administered is about 1.5 x 10 vg / kg to about 6.5 x 10 vg / kg; g) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the route of administration is intramuscular, and the dose of the rAAV administered is about 2 x 10 vg / kg to about 6 x 10 vg / kg; h) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the route of administration is intramuscular, and the dose of the rAAV administered is about 2 x 10 vg / kg; i) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the route of administration is intramuscular, and the dose of the rAAV administered is about 4 x 10 vg / kg; j) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the route of administration is intramuscular, and the dose of the rAAV administered is about 6 x 10 vg / kg; k) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the route of administration is intramuscular injection at a concentration of about 2 x 10 vg / ml administered using 3-6 injections per muscle of about 0.5-1 ml; or l) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the route of administration is about 2 x 10 administered using multiple injections in a total volume of about 5-14 ml. 13 The method is an intramuscular injection at a concentration of 0.05 mg / ml. (Item 16) Item 17. The method according to Item 14 or 15, wherein the nucleic acid is administered using a viral vector. Item 17. The method of item 16, wherein the viral vector is an adeno-associated viral vector. (Item 18) 18. The method of any one of items 14 to 17, wherein the nucleic acid is operably linked to a muscle-specific promoter. (Item 19) 19. The method according to any one of items 14 to 18, wherein the muscle-specific promoter is a muscle-specific creatine kinase promoter. (Item 20) 20. The method of any one of items 14 to 19, wherein the scAAV1.tMCK.NTF3 comprises the NT-3 gene cassette set forth in SEQ ID NO: 11. (Item 21) 21. The method according to any one of items 14 to 20, wherein the route of administration is intramuscular injection. (Item 22) 22. The method according to any one of items 14 to 21, wherein the route of administration is intramuscular bilateral injection into the medial and lateral heads of the gastrocnemius and tibialis anterior muscles. (Item 23) 23. The method of any one of items 15 to 22, wherein the muscle strength to be improved in the subject is in the upper or lower limbs. (Item 24) 23. The method of any one of items 15 to 22, wherein the improvement in muscle strength is measured as a reduction in the composite score on the CMT Pediatric Scale (CMTPeds) or as a reduction in disease progression over a two-year period. (Item 25) 1. A composition comprising a nucleic acid encoding an NT-3 polypeptide for use in treating a muscle wasting disease or neuropathy in a human subject, wherein: a) the nucleic acid comprises a nucleotide sequence that is 90% identical to the nucleotide sequence of SEQ ID NO: 1; b) the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 1; c) the nucleic acid comprises a nucleic acid sequence encoding an amino acid sequence that is at least 90% identical to SEQ ID NO:2 or 100% identical to SEQ ID NO:2; d) the nucleic acid encoding the NT-3 polypeptide is the nucleic acid according to any one of items 1 to 7; e) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the rAAV is administered at a dose that results in sustained expression of low levels of NT-3 polypeptide; f) the nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the composition is formulated for intramuscular administration, and the dose of the rAAV is about 1.5 x 10 12 vg / kg ~ approx. 6.5×10 12 vg / kg, g) the nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the composition is formulated for intramuscular administration, and the dose of the rAAV is about 2×10 12 vg / kg ~ approx. 6×10 12 vg / kg, h) the nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the composition is formulated for intramuscular administration, and the dose of the rAAV is about 2×10 12 vg / kg, i) the nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the composition is formulated for intramuscular administration, and the dose of the rAAV is about 4×10 12 vg / kg, j) the nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the composition is formulated for intramuscular administration, and the dose of rAAV administered is about 6×10 12 vg / kg, k) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the composition is administered using 3-6 injections per muscle of about 0.5-1 ml. 13 formulated for intramuscular injection at a concentration of 0.05 mg / ml, or l) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the composition is administered using multiple injections in a total volume of about 5-14 ml. 13 The composition is formulated for intramuscular injection at a concentration of 1000 mg / ml. (Item 26) 1. A composition comprising a nucleic acid encoding an NT-3 polypeptide for use in improving muscle strength or stimulating muscle growth in a human subject, wherein: a) the nucleic acid comprises a nucleotide sequence that is 90% identical to the nucleotide sequence of SEQ ID NO: 1; b) the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 1; c) the nucleic acid comprises a nucleic acid sequence encoding an amino acid sequence that is at least 90% identical to SEQ ID NO:2 or 100% identical to SEQ ID NO:2; d) the nucleic acid encoding the NT-3 polypeptide is the nucleic acid according to any one of items 1 to 7; e) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the rAAV is at a dose that results in sustained expression of a low level of NT-3 polypeptide; f) the nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the composition is formulated for intramuscular administration, and the dose of the rAAV is about 1.5 x 10 12 vg / kg ~ approx. 6.5×10 12 vg / kg, g) the nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the composition is formulated for intramuscular administration, and the dose of the rAAV is about 2×10 12 vg / kg ~ approx. 6×10 12 vg / kg, h) the nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the composition is formulated for intramuscular administration, and the dose of the rAAV is about 2×10 12 vg / kg, i) the nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the composition is formulated for intramuscular administration, and the dose of the rAAV is about 4×10 12 vg / kg, j) the nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the composition is formulated for intramuscular administration, and the dose of the rAAV is about 6×10 12 vg / kg, k) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the composition is administered using 3-6 injections per muscle of about 0.5-1 ml. 13 formulated for intramuscular injection at a concentration of 0.05 mg / ml, or l) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the composition is administered using multiple injections in a total volume of about 5-14 ml. 13 The composition is formulated for intramuscular injection at a concentration of 1000 mg / ml. (Item 27) 27. The composition of claim 25 or 26, wherein the nucleic acid is formulated for administration using a viral vector. (Item 28) 28. The composition of item 27, wherein the viral vector is an adeno-associated viral vector. (Item 29) 29. The composition of any one of items 25 to 28, wherein the nucleic acid is operably linked to a muscle-specific promoter. (Item 30) 30. The composition according to any one of items 25 to 29, wherein the muscle-specific promoter is a muscle-specific creatine kinase promoter. (Item 31) 30. The composition of any one of items 25 to 29, wherein the scAAV1.tMCK.NTF3 comprises the NT-3 gene cassette set forth in SEQ ID NO: 11. (Item 32) 32. The composition of any one of items 25 to 31, wherein the composition is formulated for intramuscular injection. (Item 33) 33. The composition of any one of items 25 to 32, wherein the composition is formulated for intramuscular bilateral injection into the medial and lateral heads of the gastrocnemius and tibialis anterior muscles. (Item 34) 34. The composition of any one of items 26 to 33, wherein the improved muscle strength is in the upper or lower limbs of the subject. (Item 35) 34. The composition of any one of items 26 to 33, wherein the improvement in muscle strength is measured as a reduction in the composite score on the CMT Pediatric Scale (CMTPeds) or as a reduction in disease progression over a two-year period. (Item 36) 1. Use of a nucleic acid encoding an NT-3 polypeptide for the manufacture of a medicament for treating a muscle wasting disease or neuropathy in a human subject, comprising: a) the nucleic acid comprises a nucleotide sequence that is 90% identical to the nucleotide sequence of SEQ ID NO: 1; b) the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 1; c) the nucleic acid comprises a nucleic acid sequence encoding an amino acid sequence that is at least 90% identical to SEQ ID NO:2 or 100% identical to SEQ ID NO:2; d) the nucleic acid encoding the NT-3 polypeptide is the nucleic acid according to any one of items 1 to 7; e) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the rAAV is at a dose that results in sustained expression of a low level of NT-3 polypeptide; f) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the agent is formulated for intramuscular administration, and the dose of the rAAV is about 1.5 x 10 12 vg / kg ~ approx. 6.5×10 12 vg / kg, g) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the agent is formulated for intramuscular administration, and the dose of the rAAV is about 2×10 12 vg / kg ~ approx. 6×10 12 vg / kg, h) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the agent is formulated for intramuscular administration, and the dose of the rAAV is about 2×10 12 vg / kg, i) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the agent is formulated for intramuscular administration, and the dose of the rAAV is about 4×10 12 vg / kg, j) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the agent is formulated for intramuscular administration, and the dose of the rAAV administered is about 6×10 12 vg / kg, k) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the agent is formulated for intramuscular injection at a concentration of about 2 x 10 vg / ml administered using 3 to 6 injections per muscle of about 0.5 to 1 ml; or l) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the agent is administered using multiple injections in a total volume of about 5-14 ml, or about 2 x 10 13 Formulated for intramuscular injection at a concentration of vg / ml, use. (Item 37) 1. Use of a dose of a nucleic acid encoding an NT-3 polypeptide for the manufacture of a medicament for improving muscle strength or stimulating muscle growth in a human subject, comprising: a) the nucleic acid comprises a nucleotide sequence that is 90% identical to the nucleotide sequence of SEQ ID NO: 1; b) the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 1; c) the nucleic acid comprises a nucleic acid sequence encoding an amino acid sequence that is at least 90% identical to SEQ ID NO:2 or 100% identical to SEQ ID NO:2; d) the nucleic acid encoding the NT-3 polypeptide is the nucleic acid according to any one of items 1 to 7; e) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the rAAV is at a dose that results in sustained expression of a low level of NT-3 polypeptide; f) the nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the composition is formulated for intramuscular administration, and the dose of the rAAV is about 1.5 x 10 12 vg / kg ~ approx. 6.5×10 12 vg / kg, g) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the agent is formulated for intramuscular administration, and the dose of the rAAV is about 2×10 12 vg / kg ~ approx. 6×10 12 vg / kg, h) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the agent is formulated for intramuscular administration, and the dose of the rAAV is about 2×10 12 vg / kg, i) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the agent is formulated for intramuscular administration, and the dose of the rAAV is about 4×10 12 vg / kg, j) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the agent is formulated for intramuscular administration, and the dose of the rAAV administered is about 6×10 12 vg / kg, k) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the agent is administered using 3-6 injections per muscle of about 0.5-1 ml, or 13 formulated for intramuscular injection at a concentration of 0.05 mg / ml, or l) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the agent is administered using multiple injections in a total volume of about 5-14 ml, or about 2 x 10 13 Formulated for intramuscular injection at a concentration of vg / ml, use. (Item 38) 38. The use according to item 36 or 37, wherein the nucleic acid is formulated for administration using a viral vector. (Item 39) 39. The use according to any one of items 36 to 38, wherein the viral vector is an adeno-associated viral vector. (Item 40) 40. The use of any one of items 36 to 39, wherein the nucleic acid is operably linked to a muscle-specific promoter. (Item 41) 41. The use according to any one of items 36 to 40, wherein the muscle-specific promoter is a muscle-specific creatine kinase promoter. (Item 42) 42. The use of any one of items 36 to 41, wherein the scAAV1.tMCK.NTF3 comprises the NT-3 gene cassette set forth in SEQ ID NO: 11. (Item 43) 43. The use according to any one of items 36 to 42, wherein the medicament is formulated for intramuscular injection. (Item 44) 44. The use according to any one of items 36 to 43, wherein the medicament is formulated for intramuscular bilateral injection into the medial and lateral heads of the gastrocnemius and tibialis anterior muscles. (Item 45) 45. The use according to any one of items 37 to 44, wherein the muscle strength to be improved in the subject is in the upper or lower limbs. (Item 46) 46. ​​The use of any one of items 37 to 45, wherein the improvement in muscle strength is measured as a reduction in the composite score on the CMT Pediatric Scale (CMTPeds) or as a reduction in disease progression over a two-year period. (Item 47) 47. The method, composition or use according to any one of items 14 to 46, wherein the subject is at risk of developing muscle wasting. (Item 48) 47. The method, composition or use according to any one of items 14 to 46, wherein the subject is suffering from muscle wasting. (Item 49) 47. The method, composition or use according to any one of items 14 to 46, wherein the subject suffers from muscular dystrophy. (Item 50) 47. The method, composition or use according to any one of items 14 to 46, wherein the subject is suffering from a neuropathy. (Item 51) 47. The method, composition or use according to any one of items 14 to 46, wherein the subject is suffering from Charcot-Marie-Tooth (CMT) neuropathy. (Item 52) 52. The method, composition or use according to item 51, wherein the subject has one of the genetic variations shown in Table 1. (Item 52) 47. The method, composition or use according to any one of items 14 to 46, wherein the subject is suffering from transthyretin amyloid neuropathy. (Item 53) 53. The method, composition or use according to item 52, wherein the subject has one of the genetic variations Val30Met, Ile107Val, and Ser77Tyr. (Item 54) The subject is diagnosed with cancer, diabetes, human immunodeficiency virus (HIV) infection, thyroid disorders, hypothyroidism, hypoglycemia, uremia, renal failure, liver dysfunction, hepatic failure, polycythemia, connective tissue disorders, Lyme disease, celiac disease, leprosy, porphyria, Sjogren's syndrome, poliovirus infection, acromegaly, disorders of lipid / glycolipid metabolism, West Nile disease, amyloidosis, mitochondrial disorders, paraprotein diseases, benign monoclonal gammopathy (MGUS), POEMS syndrome, nutritional / vitamin deficiencies, vitamin B 12 47. The method, composition or use according to any one of items 14 to 46, wherein the patient is suffering from an acquired neuropathy caused by a deficiency of vitamin E or a copper deficiency. (Item 55) 47. The method, composition, or use according to any one of items 14 to 46, wherein the subject is suffering from hereditary myopathy, peripheral neuropathy, toxic neuropathy, autoimmune peripheral polyneuropathy, acute inflammatory demyelinating polyneuropathy (AIDP), chronic inflammatory demyelinating polyneuropathy (CIDP), vasculitic mononeuritis multiplex, paraneuropathy, idiopathic ganglionitis, amyotrophic lateral sclerosis, multifocal motor conduction block neuropathy, or lower motor neuron syndrome, neuromuscular disease, muscle atrophy, drug-induced myopathy, sarcopenia, cachexia, type II muscle fiber atrophy, age-related muscle atrophy, or acquired autoimmune primary muscle disease.

[0097] The present invention may be more readily understood by reference to the following figures. [Brief explanation of the drawings]

[0098] [Figure 1-1] Graphs and images are shown demonstrating AAV1.NT-3-induced fiber type remodeling in TrJ muscle. Representative images of SDH-stained tissue sections from AAV1.tMCK.NT-3-treated Trembler J (TrJ) (Figure 1A) and untreated (TrJ-PBS) gastrocnemius muscles (Figure 1B) at 16 weeks post-injection. Slow-twitch oxidative (STO, arrow), fast-twitch oxidative (FTO, arrowhead), and fast-twitch glycolytic (FTG, asterisk) fibers are indicated (Figure 1B). Oxidative fibers are reduced in a. In TrJ-PBS muscle (Figure 1B), increased numbers of small STO fibers and angular fibers of all fiber types are present, along with a small population of types consistent with neural alterations. Scale bar for a, b = 30 gm. Fiber type switching from STO to FTO / FTG fibers in TrJ muscle by NT-3 gene therapy (Figure 1C). The mean percentage of STO in both treatment groups (obtained from n = 3-5 mice in each group) was not significantly different from wild-type (WT) muscle, indicating a change to normalization of fiber type distribution by NT-3 in TrJ neurogenic muscle. [Figure 1-2]Graphs and images are shown demonstrating AAV1.NT-3-induced fiber type remodeling in TrJ muscle. Representative images of SDH-stained tissue sections from AAV1.tMCK.NT-3-treated Trembler J (TrJ) (Figure 1A) and untreated (TrJ-PBS) gastrocnemius muscles (Figure 1B) at 16 weeks post-injection. Slow-twitch oxidative (STO, arrow), fast-twitch oxidative (FTO, arrowhead), and fast-twitch glycolytic (FTG, asterisk) fibers are indicated (Figure 1B). Oxidative fibers are reduced in a. In TrJ-PBS muscle (Figure 1B), increased numbers of small STO fibers and angular fibers of all fiber types are present, along with a small population of types consistent with neural alterations. Scale bar for a, b = 30 gm. Fiber type switching from STO to FTO / FTG fibers in TrJ muscle by NT-3 gene therapy (Figure 1C). The mean percentage of STO in both treatment groups (obtained from n = 3-5 mice in each group) was not significantly different from wild-type (WT) muscle, indicating a change to normalization of fiber type distribution by NT-3 in TrJ neurogenic muscle. [Figure 2-1] Graphs and images showing the effects of AAV 1.NT3 treatment on mTOR signaling and metabolic markers are shown. Representative Western blot images and analysis of mTOR targets, Phospho(P)-4EBP1 (Thr37 / 46), and P-S6 (Ser235 / 236), in TrJ (Figure 2A) and wild-type (WT) (Figure 2B) gastrocnemius muscles at 16 weeks post-injection. Graphs show expression levels of phosphorylated forms of proteins normalized to GAPDH. Coomassie blue-stained membranes represent equivalent gel loading. Error bars are ± SEM; n = 5–6 per group; *P < 0.05, unpaired t-test. (Figure 2C) Relative expression of glycolytic (1-1K1 and PK1) and aerobic regulators (PGC1α) by qPCR; GAPDH was used as a housekeeping gene. Error bars are ±SEM; n=5-6 in each group, *P<0.05, one-way anova followed by Tukey's multiple comparison test. [Figure 2-2] Graphs and images showing the effects of AAV 1.NT3 treatment on mTOR signaling and metabolic markers are shown. Representative Western blot images and analysis of mTOR targets, Phospho(P)-4EBP1 (Thr37 / 46), and P-S6 (Ser235 / 236), in TrJ (Figure 2A) and wild-type (WT) (Figure 2B) gastrocnemius muscles at 16 weeks post-injection. Graphs show expression levels of phosphorylated forms of proteins normalized to GAPDH. Coomassie blue-stained membranes represent equivalent gel loading. Error bars are ± SEM; n = 5–6 per group; *P < 0.05, unpaired t-test. (Figure 2C) Relative expression of glycolytic (1-1K1 and PK1) and aerobic regulators (PGC1α) by qPCR; GAPDH was used as a housekeeping gene. Error bars are ±SEM; n=5-6 in each group, *P<0.05, one-way anova followed by Tukey's multiple comparison test. [Figure 3-1]Graphs and images showing the direct effect of NT-3 on myotubes are shown. (Figure 3A) Representative Western blot images and analysis of the Akt / mTOR pathway, Phospho(P)-Akt (Ser473), P-4EBP1 (Thr37 / 46), and P-S6 (Ser235 / 236) in myotubes incubated with recombinant human NT-3 (100 ng / ml) or PBS (control) for 30 min. Density values ​​of phosphorylated protein bands were normalized to GAPDH and expressed as a percentage of the control group. Coomassie blue-stained membranes represent equivalent gel loading. Myotubes were incubated with NT-3 (100 ng / ml) for 48 h, and then the relative mRNA expression of metabolic markers (PGC1a, HK1, PK1) was detected by qPCR (Figure 3B), and glucose consumption relative to lactate production in the cell culture medium was detected by ELISA (Figure 3C). (Figure 3D) Relative expression levels of myogenin and NT-3 receptors, p75NTR and TrkC, in myoblasts versus myotubes after 48 hours of NT-3 (100 ng / ml) treatment. GAPDH was used as a housekeeping gene in the analysis. Results shown are the mean ± SEM from at least three independent experiments (*P < 0.05, Student's paired t-test). [Figure 3-2]Graphs and images showing the direct effect of NT-3 on myotubes are shown. (Figure 3A) Representative Western blot images and analysis of the Akt / mTOR pathway, Phospho(P)-Akt (Ser473), P-4EBP1 (Thr37 / 46), and P-S6 (Ser235 / 236) in myotubes incubated with recombinant human NT-3 (100 ng / ml) or PBS (control) for 30 min. Density values ​​of phosphorylated protein bands were normalized to GAPDH and expressed as a percentage of the control group. Coomassie blue-stained membranes represent equivalent gel loading. Myotubes were incubated with NT-3 (100 ng / ml) for 48 h, and then the relative mRNA expression of metabolic markers (PGC1a, HK1, PK1) was detected by qPCR (Figure 3B), and glucose consumption relative to lactate production in the cell culture medium was detected by ELISA (Figure 3C). (Figure 3D) Relative expression levels of myogenin and NT-3 receptors, p75NTR and TrkC, in myoblasts versus myotubes after 48 hours of NT-3 (100 ng / ml) treatment. GAPDH was used as a housekeeping gene in the analysis. Results shown are the mean ± SEM from at least three independent experiments (*P < 0.05, Student's paired t-test). [Figure 4] 1 shows a graph depicting the blood levels of NT-3 in treated and untreated mice.At the endpoint, serum was collected from each mouse and circulating NT-3 levels were detected by ELISA. [Figure 5] Relative mRNA expression of p75NTR and TrkC in TrJ and WT gastrocnemius muscles is shown. GAPDH was used as a housekeeping gene in the analysis. Results shown are the mean ± SEM from at least three independent experiments (*P<0.05, Student's t-test). [Figure 6]A schematic diagram of the construct AAV.tMCK.NTF3 (SEQ ID NO:11) is shown. The vector contains a muscle-specific tMCK promoter (SEQ ID NO:3), a chimeric intron (SEQ ID NO:5), a consensus Kozak sequence (SEQ ID NO:6), NTF3 cDNA (SEQ ID NO:1), and a polyadenylation signal (SEQ ID NO:7). [Figure 7] Restriction map and ORF analysis of pAAV.tMCK.NTF3 are shown. [Figure 8] 1 shows the location of intramuscular (IM) injection of AAV.tMCK.NTF3 in human subjects. [Figure 9-1] The nucleotide sequence of AAV.tMCK.NTF3 (SEQ ID NO: 11) is shown. [Figure 9-2] The nucleotide sequence of AAV.tMCK.NTF3 (SEQ ID NO: 11) is shown. DETAILED DESCRIPTION OF THE INVENTION

[0099] The increase in fiber size induced by AAV.NT-3 treatment in TrJ muscle was examined to determine whether this increase was simply a result of nerve regeneration or whether NT-3 could directly affect muscle protein synthesis independent of nerve regeneration, thereby increasing muscle fiber size.

[0100] Disclosed herein is a novel effect of NT-3: its ability to directly affect protein synthesis and metabolic remodeling in neurogenic muscle.

[0101] The study described herein first evaluated the effects of AAV.NT-3 gene therapy on the oxidative status of TrJ muscles 16 weeks after gene injection and found that the increase in myofiber size was associated with changes in myofiber oxidative status relative to the normalization of fiber type ratios seen in WT. Treatment resulted in a decrease in the percentage of slow-twitch (STO) fibers, while the intermediate-twitch and fast-twitch (FTO and FTG) fiber populations increased, reflecting a reversal of the pattern seen in untreated TrJ muscles. The NT-3-induced increase in fiber size was most pronounced for the FTG fiber population. Next, we investigated whether mammalian target of rapamycin complex 1 (mTORC1) activation played a role in NT-3-induced muscle protein synthesis, with a particular emphasis on preferential hypertrophic growth of glycolytic fibers. mTORC1 regulates translation and ribosome biogenesis through phosphorylation of the translation regulators eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) and S6 kinase 1 (S6K1). Laplante M, Sabatini D., Cell, 149(2):274-293(2012). Furthermore, mTORC1 is involved in the activation of cellular glycolysis, which involves increased translation of glycolytic enzymes or their transcriptional regulators. Duvel et al. al., Molecular Cell, 39(2):171-183 (2010). Histochemical changes in TrJ muscle were accompanied by increased phosphorylation levels of 4E-BP1 and S6 protein (S6P), evidence of mTORC1 activation. In parallel, the expression levels of mitochondrial biogenesis regulators (peroxisome proliferator-activated receptor γ coactivator 1a, PGC1α) and glycolysis markers (hexokinase-1, HK1, and pyruvate kinase 1, PK1) increased in TrJ muscle. These changes were not significant in AAV.NT-3-treated WT muscle. Furthermore, in vitro studies showed that recombinant NT-3 could directly induce Akt / mTOR pathway activation in TrkC-expressing myotubes but not in myoblasts. Furthermore, myogenin expression levels were significantly higher in myotubes, whereas p75NTR expression was downregulated compared with myoblasts, indicating that NT-3-induced myoblast differentiation is associated with mTORC1 activation.

[0102] The findings described herein have many implications for the potential use of NT-3 not only for the treatment of neuropathies with benefits to both nerve and muscle, but also for muscle wasting diseases including aging, cancer cachexia, or type II muscle fiber atrophy, as well as inherited or acquired autoimmune primary muscle diseases associated with disturbances in mTORC1 signaling and impaired hypertrophic growth phase of regeneration, which may involve defective mitochondrial biogenesis.

[0103] The present disclosure relates to a method for stimulating muscle growth in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of neurotrophin-3 (NT-3), pro-NT-3, or an effective fragment thereof, or a nucleic acid encoding NT-3, pro-NT-3, or an effective fragment thereof. Subjects in need of stimulated muscle growth include those suffering from muscular dystrophy or muscle atrophy.

[0104] The present invention provides a method for inhibiting muscle wasting, comprising administering an AAV vector to deliver the NTF3 gene encoding neurotrophin-3 (NT-3). In one embodiment, the present invention provides a gene therapy method for treating Charcot-Marie-Tooth disease type 1A (CMT1A), in which the NTF3 gene encoding NT-3 is delivered to a subject using a self-complementary adeno-associated virus (scAAV) type 1 under the control of the muscle-specific tMCK promoter. In another embodiment, the present invention provides a gene therapy method for increasing muscle strength in a subject in need thereof, for example, a subject diagnosed with or suffering from a muscle-wasting disease such as CMT.

[0105] Preclinical studies have been conducted in trembler J mice, a natural mouse model for CMT1 (Tr J demonstrated that delivery of the construct AAV1.tMCK.NTF3 to the gastrocnemius muscle of rats improved nerve regeneration, myelination, myelinated fiber density, sciatic nerve compound muscle action potential amplitude, and functional performance in the rotarod test and hindlimb grip strength (see Example 3).

[0106] As used herein, the terms "treatment," "treating," and the like refer to obtaining a desired pharmacological or physiological effect. This effect may be therapeutic in that it is a partial or complete cure of a disease or adverse effects resulting from a disease. "Treatment," as used herein, encompasses any treatment of a disease in a mammal, particularly a human, and may include inhibiting the disease or condition, i.e., halting its development; and relieving the disease, i.e., causing regression of the disease.

[0107] Prevention as used herein refers to any action that benefits a subject at risk of suffering from a condition or disease, such as a neuropathy, demyelinating polyneuropathy, muscle wasting disease or atrophy.

[0108] As used herein, "pharmaceutically acceptable" means that the compound or composition is suitable for administration to a subject for the methods described herein without undue adverse side effects having regard to the severity of the disease and the need for treatment.

[0109] The terms "therapeutically effective" and "pharmacologically effective" are intended to qualify the amount of agent that achieves the goal of ameliorating disease severity and frequency of occurrence. The effectiveness of treatment can be measured by assessing the alleviation of symptoms in a subject in response to administration of NT-3.

[0110] The term "effective fragment" refers to a portion of a polynucleotide sequence that encodes a functional fragment of an NT-3 polypeptide. The term "effective fragment" also refers to a portion of an NT-3 polypeptide amino acid sequence that retains NT-3 growth factor activity. Exemplary NT-3 growth factor activities include supporting the survival and differentiation of existing neurons, and inducing and supporting the growth and differentiation of new neurons and synapses. Additionally, NT-3 activity includes stimulating muscle growth and muscle function.

[0111] As used herein, the term "diagnosis" can include determining the likelihood that a subject will develop a disease, or the presence or nature of a disease in a subject. As used herein, the term diagnosis also includes determining the severity and likely outcome of a disease or the likelihood of development or recovery from a disease (commonly referred to as a prognosis). "Diagnosis" can also include diagnosis in the context of rational treatment, where diagnosis guides treatment, including initial selection of treatment, modification of treatment (e.g., adjustment of dosage or dosing regimen), etc.

[0112] As used herein, a "subject" can be any animal and can also be referred to as a patient. Preferably, the subject is a vertebrate, and more preferably, the subject is a mammal, such as a livestock animal (e.g., cow, horse, pig) or a pet (e.g., dog, cat). In some embodiments, the subject is a human.

[0113] The term "polynucleotide" or "nucleic acid molecule" refers to a polymeric form of nucleotides at least 10 bases in length. The term includes DNA molecules (e.g., eDNA or genomic or synthetic DNA) and RNA molecules (e.g., mRNA or synthetic RNA), as well as analogs of DNA or RNA that contain non-natural nucleotide analogs, non-natural internucleoside linkages, or both. Nucleic acids can be in any topological structure. For example, nucleic acids can be single-stranded, double-stranded, triple-stranded, quadruplexed, partially double-stranded, branched, hairpinned, circular, or in a padlocked conformation.

[0114] The term "gene," as used herein, refers to a nucleotide sequence that directs the synthesis of an enzyme or other polypeptide molecule (e.g., may include a coding sequence, e.g., a contiguous open reading frame (ORF) that encodes a polypeptide), or that may itself be functional in an organism. Genes in an organism can be clustered in operons, as defined herein, where the operon is separated from other genes and / or operons by intergenic DNA. Individual genes contained in an operon can overlap without intergenic DNA between individual genes.

[0115] As used herein, the term "AAV" is the standard abbreviation for adeno-associated virus. Adeno-associated virus is a single-stranded DNA parvovirus that grows only in cells in which certain functions are provided by a coinfecting helper virus. Currently, there are 13 characterized serotypes of AAV. General information and reviews of AAV can be found, for example, in Carter, 1989, Handbook of Parvoviruses, Vol. 1, pp. 169-228, and Berns, 1990, Virology, pp. 1743-1764, Raven Press, New York. However, these same principles can be applied to additional AAV serotypes, as it is well known that the various serotypes are structurally and functionally quite closely related, even at the genetic level. (See, e.g., Blacklowe, 1988, pp. 165-174 of Parvoviruses and Human Disease, J.R.P.Tattison, ed.; and Rose, Comprehensive Virology 3:1-61 (1974)). For example, all AAV serotypes appear to exhibit very similar replication properties mediated by homologous rep genes; all possess three related capsid proteins, such as those expressed in AAV2. The degree of similarity is further suggested by extensive cross-hybridization between serotypes along the length of the genome; and heteroduplex analysis, which reveals the presence of similar self-annealing segments at the ends corresponding to the "inverted terminal repeats" (ITRs). The similar infectivity patterns also suggest that the replication functions in each serotype are under similar regulatory control.

[0116] The term "vector" or "expression vector" refers to any type of genetic construct containing a nucleic acid encoding an RNA capable of being transcribed. Expression vectors can contain various regulatory sequences, structural genes (e.g., genes of interest), and nucleic acid sequences that serve other functions as well.

[0117] "Vector" refers to a DNA molecule, usually derived from a plasmid or bacteriophage, into which a fragment of DNA can be inserted or cloned. Recombinant vectors contain one or more unique restriction sites and may be capable of autonomous replication in a defined host or vector so that the cloned sequence can be reproduced. A vector contains a promoter operably linked to a gene or coding region such that RNA is expressed upon transfection into a recipient cell.

[0118] As used herein, "AAV vector" refers to a vector containing one or more polynucleotides of interest (or transgenes) flanked by AAV interterminal repeats (ITRs). Such AAV vectors can be replicated and packaged into infectious viral particles when present in a host cell transfected with a vector encoding and expressing the rep and cap gene products.

[0119] An "AAV virion" or "AAV viral particle" or "AAV vector particle" refers to a viral particle composed of at least one AAV capsid protein and an encapsidated polynucleotide AAV vector. When a particle contains a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, such as a transgene delivered to a mammalian cell), it is typically referred to as an "AAV vector particle" or simply an "AAV vector." Thus, production of an AAV vector particle necessarily includes production of an AAV vector, since such a vector is contained within the AAV vector particle.

[0120] As used herein, the term "about" refers to a + / -10% deviation from the base value.

[0121] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0122] Gene therapy for peripheral neuropathy In one aspect, the present invention provides a method of treating a subject suffering from muscle wasting using gene therapy.

[0123] Vectors that can be used to deliver therapeutic nucleic acids include viral and non-viral vectors. Suitable vectors that can be used include adenovirus, adeno-associated virus, retrovirus, lentivirus, HSV (herpes simplex virus), and plasmids. The advantage of herpes simplex virus vectors is their natural tropism for sensory neurons. However, adenovirus-related viral vectors are the most popular due to their low risk of insertional mutagenesis and immunogenicity, their lack of endogenous viral genes, and their ability to be produced at high titers. Kantor et al. outline various methods for gene transfer into the central nervous system, while Goins et al. describe a method of gene therapy for the treatment of chronic peripheral nervous system pain. See Kantor et al., Adv Genet. 87, 125-197 (2014), and Goins et al., Neurobiol. Dis. 48(2), 255-270 (2012), the disclosures of which are incorporated herein by reference. In particular, successful gene delivery to Schwann cells, resident glial cells of peripheral nerves, has been reported using various viral vectors. Mason et al., Curr. Gene Ther. 11, 75-89 (2011). If the vector is within a viral vector and the vector is packaged, virions can be used to infect cells. If naked DNA is used, transfection or transformation procedures appropriate for the particular host cell can be used. Formulations of naked DNA using polymers, liposomes, or nanospheres can be used for gene delivery. Nucleic acids can be administered in any desired format that results in sufficiently efficient delivery levels, including complexation to viral particles, liposomes, nanoparticles, and polymers.

[0124] A nucleic acid (e.g., a cDNA or transgene) encoding a gene whose expression alleviates peripheral neuropathy can be cloned into an expression cassette with regulatory elements, such as a promoter (constitutive or regulatable) that drives transgene expression and a polyadenylation sequence downstream of the nucleic acid. For example, regulatory elements can be used that are 1) specific to a tissue or region of the body; 2) constitutive; and / or 3) inducible / regulatable.

[0125] In some embodiments, muscle-specific regulatory elements are used. Examples of muscle-specific regulatory elements include the mammalian muscle creatine kinase (MCK) promoter, mammalian desmin promoter, mammalian troponin I (TNNI2) promoter, or mammalian skeletal alpha-actin (ASKA) promoter. Muscle-specific enhancers useful in the present invention are selected from the group consisting of the mammalian MCK enhancer, mammalian DES enhancer, and vertebrate troponin I IRE (TNI IRE, hereafter referred to as FIRE) enhancer. One or more of these muscle-specific enhancer elements can be used in combination with the muscle-specific promoters of the present invention to provide tissue-specific regulatory elements.

[0126] AAV is a preferred vector for use in treating muscle atrophy through gene therapy. AAV-mediated gene delivery has emerged as an effective and safe means for both preclinical and clinical trials of neurological disorders. (Ojala et al., Neuroscientist., 21(1):84-98 (2015)) Currently, AAV is the most widely used vector in clinical trials for neurological disorders, and no adverse effects associated with the use of this vector have been reported in clinical trials to date. Adeno-associated virus is a nonpathogenic, dependent virus of the Parvoviridae family that requires helper functions from other viruses, such as adenovirus or herpes simplex virus, to achieve its life cycle. Wild-type (WT) AAV is characterized by a single-stranded DNA (ssDNA) genome with approximately 5 kb of inverted terminal repeats (ITRs) at both ends, enclosed by a capsid.

[0127] Adenoviral vectors used to deliver transgenes to cells for applications such as in vivo gene therapy and in vitro testing and / or production of transgene products are generally derived from adenoviruses by deletion of the early region 1 (E1) gene (Berkner, KL, Curr. Top. Micro. Immunol. 158 L39-66 1992). Deletion of the E1 gene renders such adenoviral vectors replication defective and significantly reduces expression of any remaining viral genes present in the vector. Recombinant adenoviral vectors have several advantages for use as gene delivery vehicles, including tropism for both dividing and non-dividing cells, minimal pathogenic potential, the ability to replicate to high titers for preparation of vector stocks, and the ability to carry large inserts. However, it is believed that the presence of remaining viral genes in adenoviral vectors may be harmful.

[0128] Thus, in certain embodiments, adenoviral vectors have deletions of various adenoviral gene sequences. In particular, pseudoadenoviral vectors (PAVs), also known as "gutless adenoviruses" or miniadenoviral vectors, are adenoviral vectors derived from the genome of adenoviruses that contain minimal cis-acting nucleotide sequences required for replication and packaging of the vector genome and may contain one or more transgenes (see U.S. Pat. No. 5,882,877, which is incorporated herein by reference and covers pseudoadenoviral vectors (PAVs) and methods for producing PAVs). Such PAVs, which can accommodate up to approximately 36 kb of foreign nucleic acid, are advantageous because they optimize the carrying capacity of the vector while reducing the possibility of a host immune response against the vector or the generation of replication-competent virus. PAV vectors contain 5' inverted terminal repeat (ITR) and 3' ITR nucleotide sequences containing origins of replication, as well as cis-acting nucleotide sequences required for packaging of the PAV genome, and can accommodate one or more transgenes along with appropriate regulatory elements, such as promoters, enhancers, etc.

[0129] AAV A recombinant AAV genome of the invention comprises a nucleic acid molecule of the invention and one or more AAV ITRs flanking the nucleic acid molecule. The AAV DNA in the rAAV genome can be derived from any AAV serotype from which a recombinant virus can be derived, including, but not limited to, AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, and AAV-13 (see, e.g., Gao et al., PNAS, 99:11854-11859 (2002); and Viral Vectors for Gene Therapy: Methods and Protocols, ed. Machida, Humana Press, 2003). Furthermore, pseudotyped AAV vectors can also be used in the methods described herein. Pseudotyped AAV vectors contain the genome of one AAV serotype within the capsid of a second AAV serotype; for example, an AAV vector containing an AAV2 capsid and an AAV1 genome, or an AAV5 capsid and an AAV2 genome. (Auricchio et al., (2001) Hum. Mol. Genet., 10(26):3075-81). The production of pseudotyped rAAV is disclosed, for example, in WO 01 / 83692. Other types of rAAV variants, such as rAAVs with capsid mutations, are also contemplated. See, for example, Marsic et al., Molecular See, Therapy, 22(11):1900-1909 (2014). As noted in the Background section above, the nucleotide sequences of the genomes of various AAV serotypes are known in the art. To promote skeletal muscle-specific expression, AAV1, AAV6, AAV8, or AAVrh.74 can be used.

[0130] The DNA plasmids of the invention comprise the rAAV genome of the invention. The DNA plasmids are transferred to cells permissive for infection with an AAV helper virus (e.g., adenovirus, E1-deleted adenovirus, or herpesvirus) for assembly of the rAAV genome into infectious viral particles. Techniques for generating rAAV particles, in which the packaged AAV genome, rep and cap genes, and helper virus functions are provided in the cell, are standard in the art. rAAV production requires the presence of the following components in a single cell (referred to herein as a packaging cell): the rAAV genome, AAV rep and cap genes separated from (i.e., not in) the rAAV genome, and helper virus functions. The AAV rep and cap genes may be derived from any AAV serotype from which a recombinant virus can be derived, and may be derived from an AAV serotype different from the rAAV genome ITRs, including, but not limited to, AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAVrh.74, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, and AAV-13. The production of pseudotyped rAAV is disclosed, for example, in WO 01 / 83692, which is incorporated herein by reference in its entirety.

[0131] A method for generating packaging cells involves creating a cell line that stably expresses all the necessary components for AAV particle production. For example, a plasmid (or multiple plasmids) containing a rAAV genome lacking the AAV rep and cap genes, AAV rep and cap genes isolated from the rAAV genome, and a selectable marker such as a neomycin resistance gene is integrated into the cell's genome. The AAV genome has been introduced into bacterial plasmids by procedures such as GC tailing (Samulski et al., 1982, Proc. Natl. Acad. S6. USA, 79:2077-2081), addition of synthetic linkers containing restriction endonuclease cleavage sites (Laughlin et al., 1983, Gene, 23:65-73), or direct blunt-end ligation (Senapathy & Carter, 1984, J. Biol. Chem., 259:4661-4666). The packaging cell line is then infected with a helper virus such as adenovirus. The advantages of this method are that the cells are selectable and are suitable for large-scale production of rAAV. Another example of a suitable method uses adenovirus or baculovirus, rather than a plasmid, to introduce the rAAV genome and / or the rep and cap genes into the packaging cells.

[0132] The general principles of rAAV production are reviewed in, for example, Carter, 1992, Current Opinions in Biotechnology, 1533-539; and Muzyczka, 1992, Curr. Topics in Microbial. and Immunol., 158:97-129). Various techniques have been proposed by Ratschin et al.,Mol.Cell.Biol.4:2072(1984);Hermonat et al.,Proc.Natl.Acad.Sci.USA,81:6466(1984);Tratschin et al.,Mo1.Cell.Biol.5:3251(1985);McLaughlin et al. al., J. Virol., 62:1963 (1988); and Lebkowski et al., 1988 Mol. Cell. Biol., 7: 349 (1988). Samulski et al. al. (1989, J. Virol., 63:3822-3828); U.S. Pat. No. 5,173,414; WO 95 / 13365 and corresponding U.S. Pat. No. 5,658,776; WO 95 / 13392; WO 96 / 17947; PCT / US98 / 18600; WO 97 / 09441 (PCT / US96 / 14423); WO 97 / 08298 (PCT / US96 / 13872); WO 97 / 21825 (PCT / US96 / 20777); WO 97 / 06243 (PCT / FR96 / 01064); WO 99 / 11764; Perrin et al. al. (1995) Vaccine 13:1244-1250; Paul et al. (1993) Human Gene Therapy 4:609-615; Clark et al. (1996) Gene Therapy 3:1124-1132; U.S. Patent Nos. 5,786,211; 5,871,982; and 6,258,595. The above references are incorporated herein by reference in their entireties, with particular emphasis on the sections of the literature relevant to rAAV production.

[0133] Thus, the present invention provides packaging cells that produce infectious rAAV. In one embodiment, the packaging cells can be stably transformed cancer cells such as HeLa cells, 293 cells, and PerC.6 cells (an allogeneic 293 cell line). In another embodiment, the packaging cells are cells that are not transformed cancer cells, such as low-passage 293 cells (human fetal kidney cells transformed with adenovirus E1), MRC-5 cells (human fetal fibroblasts), WI-38 cells (human fetal fibroblasts), Vero cells (monkey kidney cells), and FRhL-2 cells (rhesus fetal lung cells).

[0134] The recombinant AAV of the invention (i.e., infectious, encapsidated rAAV particles) comprise a rAAV genome. In an exemplary embodiment, the genomes of both rAAVs lack AAV rep and cap DNA, i.e., there is no AAV rep or cap DNA between the ITRs of the genome. Examples of rAAVs that can be constructed to comprise nucleic acid molecules of the invention are described in International Patent Application No. PCT / US2012 / 047999 (WO 2013 / 016352), the entire contents of which are incorporated herein by reference.

[0135] rAAV can be purified by standard methods in the art, such as column chromatography or cesium chloride gradients. Methods for purifying rAAV vectors from helper viruses are known in the art, including, for example, those disclosed in Clark et al., Hum. Gene Ther., 10(6):1031-1039 (1999); Schenpp and Clark, Methods Mol. Med., 69 427-443 (2002); U.S. Pat. No. 6,566,118 and WO 98 / 09657.

[0136] In another embodiment, the present invention contemplates a composition comprising the rAAV of the present invention. The composition of the present invention comprises an rAAV and a pharmaceutically acceptable carrier. The composition may also contain other ingredients, such as diluents and auxiliary agents. Acceptable carriers, diluents, and auxiliary agents are non-toxic to recipients and are preferably inert at the dosages and concentrations used, and include buffers such as phosphate, citrate, or other organic acids; antioxidants such as ascorbic acid; low molecular weight polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or non-ionic surfactants such as Tween, Pluronics, or polyethylene glycol (PEG).

[0137] The titer of the rAAV administered in the methods of the invention will vary depending, for example, on the particular rAAV, the method of administration, the therapeutic goal, the individual, and the cell type being targeted, and can be determined by standard methods in the art. The titer of the rAAV is approximately 1 x 10 per ml. 6 , about 1×10 7 , about 1×10 8 , about 1×10 9 , about 1×10 10 , about 1×10 11 , about 1×10 12 , about 1×10 13 ~Approx. 1×10 14 Doses may also be expressed in units of viral genomes (vg).

[0138] Methods of transducing target cells with rAAV in vitro or in vivo are contemplated by the present invention. In vivo methods involve administering an effective dose, or effective doses, of a composition comprising an rAAV of the present invention to an animal (including a human) in need thereof. If the dose is administered before the onset of the disorder / disease, the administration is prophylactic. If the dose is administered after the onset of the disorder / disease, the administration is therapeutic. In embodiments of the present invention, an effective dose is one that alleviates (eliminates or reduces) at least one symptom associated with the disorder / disease state being treated, slows or prevents the progression of the disorder / disease state, reduces the extent of the disease, results in remission (partial or complete) of the disease, and / or prolongs survival.

[0139] In particular, the actual administration of the rAAV of the present invention can be carried out by using any physical method that delivers the rAAV recombinant vector to the target tissue of an animal. Administration in accordance with the present invention includes, but is not limited to, injection into muscle, the bloodstream, and / or direct injection into the liver. Simply resuspending rAAV in phosphate-buffered saline has been demonstrated to be sufficient to provide a vehicle useful for muscle tissue expression, and there are no known limitations on the carriers or other components that can be co-administered with rAAV (although compositions that degrade DNA should be avoided in typical rAAV-based methods). The capsid protein of rAAV can be modified to target the rAAV to a specific target tissue of interest, such as muscle. See, for example, International Publication No. WO 02 / 053703, which is incorporated herein by reference in its entirety. Pharmaceutical compositions can be prepared as injectable formulations or as topical formulations delivered to muscle via transdermal delivery. Numerous formulations for both intramuscular injection and transdermal delivery have been previously developed and can be used in the practice of the present invention. The rAAV may be used with any pharmaceutically acceptable carrier to facilitate administration and handling.

[0140] Transduction can be performed using gene cassettes containing tissue-specific regulatory elements. For example, one embodiment of the present invention includes gene cassettes containing regulatory elements from the actin and myosin gene families, such as the myoD gene family [see Weintraub et al., Science, 251:761-766 (1991)], the muscle cell-specific enhancer-binding factor MEF-2 [Cserjesi and Olson, Mol Cell Biol 11:4854-4862 (1991)], the human skeletal actin gene [Muscat et al., Mol Cell Biol, 7:4089-4099 (1987)], regulatory elements from the cardiac actin gene, muscle creatine kinase sequence elements [Johnson et al. al., Mol Cell Biol, 9:3393-3399 (1989)] and mouse creatine kinase enhancer (mCK) element, regulatory elements derived from the fast skeletal troponin C gene, the slow cardiac troponin C gene, and the slow troponin I gene: hypoxia-inducible nuclear factor (Semenza et al., Proc Natl Acad Sci USA, 88:5680-5684 (1991)), promoters containing steroid-inducible elements and glucocorticoid response elements (GRE) (Mader and White, Proc. Natl. Acad. Sci. USA 90:5603-5607 (1993)), and other regulatory elements.

[0141] Muscle tissue is an attractive target for in vivo DNA delivery because it is not a vital organ and is easily accessible. The present invention envisions sustained expression of miRNAs from transduced muscle fibers.

[0142] "Muscle cell" or "muscle tissue" refers to a cell or group of cells derived from any type of muscle (e.g., skeletal and smooth muscle, e.g., from the gastrointestinal tract, bladder, blood vessels, or heart tissue). Such muscle cells, such as myoblasts, myocytes, myotubes, cardiomyocytes, and cardiomyoblasts, can be differentiated or undifferentiated.

[0143] The term "transduction" is used to refer to the administration / delivery of the coding region of NT-3 to recipient cells either in vivo or in vitro via the replication-deficient rAAV of the present invention, resulting in expression of NT-3 by the recipient cells.

[0144] In one embodiment, the gene therapy is NT-3 gene therapy via recombinant adeno-associated virus (AAV) delivery. The inventors developed an AAV expression cassette carrying the human NT-3 cDNA coding sequence under the control of either a CMV promoter or a triplet muscle-specific creatine kinase (tMCK) promoter. The inventors previously demonstrated that improvements in peripheral nerve motor function, histopathology, and electrophysiology can be achieved using a recombinant AAV1 vector to increase neurotrophin-3 expression in tremble (Try) mice, a model of Charcot-Marie-Tooth variant CMT1A. See Sahenk et al., Mol Ther. 22(3):511-21 (2014), the disclosure of which is incorporated herein by reference.

[0145] Thus, the present invention provides a method of administering an effective dose (or doses administered essentially simultaneously or at intervals) of an rAAV encoding NT-3 to a patient in need thereof.

[0146] Dose and route of administration The present invention provides for local and systemic administration of effective doses of rAAV and compositions of the present invention, including combination therapies of the present invention. For example, systemic administration is administration into the circulatory system so that the entire body is affected. Systemic administration includes enteral administration, such as absorption through the digestive tract, and parenteral administration via injection, infusion, or implantation.

[0147] Thus, routes of administration of rAAV contemplated in the above methods include, but are not limited to, intraperitoneal (IP), intramuscular (IM), and intravascular (including, for example, intra-arterial limb perfusion (ILP) and intravenous (IV) routes.

[0148] The dose of rAAV administered in the methods disclosed herein will vary depending, for example, on the particular rAAV, the method of administration, the therapeutic goal, the individual, and the targeted cell type, and can be determined by standard methods in the art. Two or more doses can be administered, for example, 1, 2, 3, or more doses. The titer of rAAV in a dose can be about 1 x 10 per ml. 6 , about 1×10 7 , about 1×10 8 , about 1×10 9 , about 1×10 10 , about 1×1 11 , about 1×10 12 , about 1.5×10 12 , about 1×10 12 , about 3×10 12 , about 4×10 12 , about 5×10 12 , about 6×10 12 , about 6.5×10 12 , about 7×10 12 , 1×10 13 , about 1×10 14 , or ~approximately 1 × 10 15 Doses may also be expressed in units of viral genomes (vg) (i.e., 1 x 10 or 1 x 10, respectively). 7 vg, 1×10 8 vg, 1×10 9 vg, 1×10 10 vg, 1×10 11 vg, 1×10 12 vg, approx. 1.5×10 12 vg, approx. 1×10 12 vg, approx. 3×10 12 vg, approx. 4×10 12 vg, approx. 5×10 12 vg, approx. 6×10 12 vg, approx. 6.5×10 12 vg, approx. 7×10 12 vg, 1×10 13 vg, 1×10 14 vg, 1×10 15). A method for titrating AAV is described in Clark et al., Hum. Gene Ther., 10:1031-1039 (1999).

[0149] In certain embodiments of the above methods, where the route of administration is intramuscular (IM), the dose of rAAV administered is about 1.5×10 12 ~ at least about 6.5 × 10 12 (All ranges herein are intended to represent each individual value within that range, as well as the individual upper and lower limits of each range.) In certain embodiments of the above methods, where the route of administration is intramuscular (IM), the dose of rAAV administered is 2 x 10 12 In certain embodiments of the above methods, where the route of administration is intramuscular (IM), the dose of rAAV administered is 4×10 12 In certain embodiments of the above methods, where the route of administration is intramuscular (IM), the dose of rAAV administered is 6×10 12 vg / kg.

[0150] Human patients are contemplated herein for treatment. Human patients are contemplated herein for treatment by intramuscular (IM) delivery. Such patients include, for example: i) adult subjects (over 18 years of age) diagnosed with CMT1A, ii) exhibiting a 1.5 Mb duplication at 17p11.2 containing the peripheral myelin protein 22 (PMP22) gene, iii) males and females of any ethnic or racial group, iv) exhibiting ankle dorsiflexion weakness (should have full ROM against gravity, but unable to maintain full dorsiflexion against gravity or unable to stand on tiptoes for more than 3 seconds (Northstar criteria)), iv) abnormal nerve conduction velocity, v) ability to cooperate with clinical evaluation and repeated nerve conduction studies, and vi) for sexually active subjects, willingness to practice reliable contraception during the study. Suitable patients should not, for example, have: i) active viral infection based on clinical observation or serologic evidence of HIV, or hepatitis A, B, or C infection; ii) ongoing immunosuppressive therapy or immunosuppressive therapy within 6 months of study initiation (e.g., corticosteroids, cyclosporine, tacrolimus, methotrexate, cyclophosphamide, intravenous immunoglobulin); iii) persistent leukopenia or leukocytosis (WBC ≤ 3.5K / μL or ≥ 20.0K / μL) or ≥ 1.5K / μL iv) an absolute neutrophil count of less than 1 L; iv) an AAV1-binding antibody titer of 1:50 or greater as determined by ELISA immunoassay; v) concurrent illness or need for prolonged drug therapy, which, in the opinion of the laboratory principal investigator (PI), would pose an unnecessary risk for gene transfer; vi) ankle contracture or surgery that would prevent adequate muscle strength testing; vii) pregnancy, breastfeeding, or planned pregnancy; viii) other causes of neuropathy; and / or ix) limb surgery within the past 6 months. In an exemplary clinical protocol, CMT1A patients receive a total dose of the vector scAAV1.tMCK.NTF3 divided between the medial and lateral heads of the gastrocnemius and tibialis anterior (TA) muscles of the leg, which preferentially cause ankle weakness and instability in CMT. Subjects receive one of the following: i) 2 x 10 12 vg / kg (total) of low dose vector or ii) 6 × 1012 vg / kg (total) high dose vector.

[0151] In one embodiment, the vector is administered by intramuscular (IM) injection without a diluent. In alternative embodiments, compositions for intramuscular injection contain an adjuvant such as sesame oil or peanut oil, or aqueous propylene glycol solutions, as well as sterile aqueous solutions, may be used. Such aqueous solutions may be buffered, if necessary, and the liquid diluent is first rendered isotonic with saline or glucose. Solutions of rAAV as a free acid (DNA contains acidic phosphate groups) or a pharmacologically acceptable salt may be prepared in water, suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions of rAAV may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, as well as in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. In this regard, all sterile aqueous media employed are readily obtainable by standard techniques well known to those skilled in the art.

[0152] Pharmaceutical carriers, diluents, or excipients suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0153] Sterile injectable solution is prepared by incorporating the required amount of rAAV into a suitable solvent with various other ingredients as listed above, as needed, followed by filtration sterilization.Generally, dispersion is prepared by incorporating sterilized active ingredient into a sterile vehicle containing a basic dispersion medium and the other necessary ingredients listed above.For the preparation of sterile powder for sterile injectable solution, the preferred preparation method is vacuum drying and freeze-drying technology, which produces a powder of active ingredient and any additional desired ingredients from its previously sterile-filtered solution.

[0154] Transduction with rAAV can also be performed in vitro. In one embodiment, the desired target muscle cells are removed from a subject, transduced with rAAV, and reintroduced into the subject. Alternatively, syngeneic or xenogeneic muscle cells can be used if they do not elicit an inappropriate immune response in the subject.

[0155] In another aspect, a rAAV genome is provided herein. The genome of the administered rAAV comprises an NT-3 polynucleotide under the control of a transcriptional regulatory sequence. The rAAV genome lacks AAV rep and cap DNA. The AAV DNA in the rAAV genome can be derived from any AAV serotype from which a recombinant virus can be derived, including, but not limited to, AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, and AAVrh.74. As noted in the background section above, the nucleotide sequences of the genomes of these AAV serotypes are known in the art.

[0156] In certain embodiments, the transcriptional regulatory sequences of the rAAV genome include those derived from the actin and myosin gene families, e.g., the myoD gene family [see Weintraub et al., Science, 251:761-766 (1991)], the muscle cell-specific enhancer-binding factor MEF-2 [Cserjesi and Olson, Mol. Cell. Biol., 11:4854-4862 (1991)], the human skeletal actin gene [Muscat et al., Mol. Cell. Biol., 7:4089-4099 (1987)], regulatory elements derived from the cardiac actin gene, the muscle creatine kinase (MCK) promoter [Johnson et al., Mol. Cell. Biol., 9:3393-3399 (1989)] and the MCK enhancer, the MHCK7 promoter (a modified form of the MCK promoter incorporating an enhancer from myosin heavy chain (Salva et al.) Muscle-specific regulatory elements include, but are not limited to, regulatory elements derived from the desmin promoter, the fast skeletal troponin C gene, the slow cardiac troponin C gene, and the slow troponin I gene; hypoxia-inducible nuclear factor (Semenza et al., Proc. Natl. Acad. Sci. USA, 88:5680-5684 (1991)); promoters containing steroid-inducible elements and glucocorticoid response elements (GREs) (see Mader and White, Proc. Natl. Acad. Sci. USA, 90:5603-5607 (1993)); and other regulatory elements. In one embodiment, the transcriptional regulatory element includes the MCK promoter. In one embodiment, the transcriptional regulatory element includes the MHCK7 promoter.

[0157] In some embodiments, the NT-3 polynucleotide in the rAAV genome is the NT-3 cDNA set forth in SEQ ID NO: 1 (corresponding to nucleotides 1077-1850 of SEQ ID NO: 11). In some embodiments, the NT-3 polynucleotide in the rAAV genome is the NT-3 cDNA set forth in Genbank Accession No. NM_001102654 or the NT-3 cDNA sequence set forth in SEQ ID NO: 1, or a variant polynucleotide having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the NT-3 cDNA. In some embodiments, the variant NT-3 polynucleotide encodes the same NT-3 polypeptide as the polypeptide encoded by the NT-3 cDNA of SEQ ID NO: 1. The amino acid sequence of the NT-3 polypeptide encoded by the NT-3 cDNA set forth as SEQ ID NO: 1 or provided under GenBank Accession No. NM_001102654 is set forth as SEQ ID NO: 2. In certain embodiments, a mutant NT-3 polynucleotide encodes a mutant NT-3 polypeptide having at least one amino acid sequence change compared to the amino acid sequence of the polypeptide encoded by the NT-3 cDNA set forth as SEQ ID NO: 1 or provided under GenBank Accession No. NM_001102654 (SEQ ID NO: 2). The amino acid sequence change can be, for example, a substitution, deletion, or insertion of one or more amino acids, preferably a conservative substitution. The mutant NT-3 polypeptide can have any combination of amino acid substitutions, deletions, or insertions, provided that the activity of the polypeptide is maintained. In one embodiment, a variant NT-3 polypeptide can have several amino acid changes such that its amino acid sequence shares at least 60, 70, 80, 85, 90, 95, 97, 98, 99, or 99.5% identity with the amino acid sequence encoded by the NT-3 cDNA set forth as SEQ ID NO: 1 or provided as Genbank Accession No. NM_001102654 (SEQ ID NO: 2).

[0158] In one embodiment, the rAAV genome is the AAV.tMCK.NTF3 genome, the sequence of whose NT-3 gene cassette is set forth in SEQ ID NO: 11 and annotated in Table 4 (see Example 3).

[0159] In yet another aspect, there is provided an isolated nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO: 11. In one embodiment, the isolated nucleic acid consists of the nucleotide sequence set forth in SEQ ID NO:11.

[0160] Also provided is an isolated nucleic acid comprising, in 5' to 3' order: (i) a first AAV2 inverted terminal repeat (ITR) (SEQ ID NO:4); (ii) a muscle creatine kinase promoter sequence (SEQ ID NO:3); (iii) a nucleotide sequence encoding a human NT-3 polypeptide (SEQ ID NO:1); and (iv) a second AAV2 ITR sequence (SEQ ID NO:8), wherein the human NT-3 polypeptide is at least 90% identical to SEQ ID NO:2, 100% identical to SEQ ID NO:2, or has an amino acid sequence encoded by nucleotides 1077 to 1850 of SEQ ID NO:11.

[0161] Recombinant AAVs comprising the above nucleic acids are contemplated, as well as rAAVs comprising a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO:1.

[0162] DNA plasmids containing the rAAV genome of the present disclosure are provided. The DNA plasmids contain the rAAV genomes envisioned herein. The DNA plasmids are transferred to cells permissive for infection with an AAV helper virus (e.g., adenovirus, E1-deleted adenovirus, or herpesvirus) for assembly of the rAAV genome into infectious viral particles. Techniques for generating rAAV particles, in which the packaged AAV genome, rep and cap genes, and helper virus functions are provided in the cell, are standard in the art. rAAV production requires the following components to be present in a single cell (referred to herein as a packaging cell): the rAAV genome, AAV rep and cap genes separated from the rAAV genome (i.e., not in the rAAV genome), and helper virus functions. The AAV rep and cap genes may be derived from any AAV serotype from which a recombinant virus can be derived, and may be derived from an AAV serotype different from the rAAV genome ITRs, including, but not limited to, AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, and AAV rh74. The production of pseudotyped rAAV is disclosed, for example, in WO 01 / 83692. Other types of rAAV variants, such as rAAVs with capsid mutations, are also contemplated. See, for example, Marsic et al., Molecular Therapy, 22(11):1900-1909 (2014).

[0163] A method for generating packaging cells involves creating a cell line that stably expresses all the necessary components for AAV particle production. For example, a plasmid (or multiple plasmids) containing a rAAV genome lacking the AAV rep and cap genes, AAV rep and cap genes isolated from the rAAV genome, and a selectable marker such as a neomycin resistance gene is integrated into the cell's genome. The AAV genome has been introduced into a bacterial plasmid by procedures such as GC tailing (Samulski et al., 1982, Proc. Natl. Acad. S6. USA, 79:2077-2081), addition of a synthetic linker containing a restriction endonuclease cleavage site (Laughlin et al., 1983, Gene, 23:65-73), or direct blunt-end ligation (Senapathy & Carter, 1984, J. Biol. Chem., 259:4661-4666). The packaging cell line is then infected with a helper virus such as adenovirus. The advantages of this method are that the cells are selectable and suitable for large-scale production of rAAV. Another example of a suitable method uses adenovirus or baculovirus, rather than a plasmid, to introduce the rAAV genome and / or the rep and cap genes into the packaging cells. Methods for producing rAAV using self-complementary genomes are also known in the art.

[0164] The general principles of rAAV production are reviewed, for example, in Carter, 1992, Current Opinions in Biotechnology, 1533-539; and Muzyczka, 1992, Curr. Topics in Microbial and Immunol., 158:97-129. Various techniques are described in Ratschin et al., Mol. Cell. Biol. 4:2072 (1984); Hermonat et al., Proc. Natl. Acad. Sci. USA, 81:6466 (1984); Tratschin et al., Mol. Cell. Biol. 5:3251 (1985); McLaughlin et al., J. Virol., 62:1963 (1988); and Lebkowski et al., 1988. Mol.Cell.Biol.,7:349(1988).Samulski et al. (1989, J. Virol., 63:3822-3828); U.S. Pat. No. 5,173,414; WO 95 / 13365 and corresponding U.S. Pat. No. 5,658,776; WO 95 / 13392; WO 96 / 17947; PCT / US98 / 18600; WO 97 / 09441 (PCT / US96 / 14423); WO 97 / 08298 (PCT / US96 / 13872); WO 97 / 21825 (PCT / US96 / 20777); WO 97 / 06243 (PCT / FR96 / 01064); WO 99 / 11764; Perrin et al. al. (1995) Vaccine 13:1244-1250; Paul et al. (1993) Human Gene Therapy 4:609-615; Clark et al. (1996) Gene Therapy 3:1124-1132; U.S. Patent Nos. 5,786,211; 5,871,982; and 6,258,595. The above references are incorporated herein by reference in their entireties, with particular emphasis on the sections of the literature relevant to rAAV production.

[0165] Thus, in a further aspect, the present disclosure provides packaging cells that produce infectious rAAV. In one embodiment, the packaging cells can be stably transformed cancer cells such as HeLa cells, 293 cells, and PerC.6 cells (an allogeneic 293 cell line). In another embodiment, the packaging cells are cells that are not transformed cancer cells, such as low-passage 293 cells (human fetal kidney cells transformed with adenovirus E1), MRC-5 cells (human fetal fibroblasts), WI-38 cells (human fetal fibroblasts), Vero cells (monkey kidney cells), and FRhL-2 cells (rhesus fetal lung cells).

[0166] rAAV can be purified by standard methods in the art, such as column chromatography or cesium chloride gradients. Methods for purifying rAAV vectors from helper viruses are known in the art, including, for example, those disclosed in Clark et al., Hum. Gene Ther., 10(6):1031-1039 (1999); Schenpp and Clark, Methods Mol. Med., 69 427-443 (2002); U.S. Pat. No. 6,566,118 and WO 98 / 09657.

[0167] Thus, in another aspect, the present disclosure contemplates an rAAV comprising an NT-3 polynucleotide. In some embodiments, the rAAV comprises an AAV rh74 capsid and an NT-3 polynucleotide. In some embodiments, the genome of the rAAV lacks AAV rep and cap DNA. In some embodiments of this method, the rAAV is rAAVrh7.4.tMCK.NTF3. In some embodiments, the rAAV is a self-complementary genome.

[0168] In another aspect, the present disclosure contemplates a composition comprising the rAAV described herein. The composition of the present disclosure comprises rAAV in a pharmaceutically acceptable carrier. The composition may also contain other ingredients, such as a diluent. Acceptable carriers and diluents are non-toxic to recipients and preferably inert at the dosages and concentrations used, and include buffers such as phosphate, citrate, or other organic acids; antioxidants such as ascorbic acid; low molecular weight polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or non-ionic surfactants such as Tween, Pluronics, or polyethylene glycol (PEG). In one embodiment, the rAAV is administered in Tris, MgCl , or a mixture thereof. 2、 Formulated in NaCl and pluronic F68. In one embodiment, rAAV is formulated in 200 mM NaCl containing 20 mM Tris (pH 8.0), 1 mM MgCl, and 0.001% pluronic F68.

[0169] Combination therapies are also contemplated herein. As used herein, combination includes simultaneous or sequential treatment. Combinations of the methods of the present disclosure with standard medical treatments (e.g., corticosteroids and / or immunosuppressants) are specifically contemplated, as are combinations with novel therapies. In various embodiments, the subject is treated with a corticosteroid before, during, or after the subject is treated according to the methods contemplated herein (or with any permutation of a combination of two or more of the three possibilities). For example, a combination includes administering a corticosteroid, such as prednisolone, before, during, and / or after administration of the rAAV vector.

[0170] Sterile injectable solution is prepared by incorporating the required amount of rAAV into a suitable solvent with various other ingredients as listed above, as needed, followed by filtration sterilization.Generally, dispersion is prepared by incorporating sterilized active ingredient into a sterile vehicle containing a basic dispersion medium and the other necessary ingredients listed above.For the preparation of sterile powder for sterile injectable solution, the preferred preparation method is vacuum drying and freeze-drying technology, which produces a powder of active ingredient and any additional desired ingredients from its previously sterile-filtered solution.

[0171] Stimulating muscle growth One aspect of the present invention provides a method for stimulating muscle growth in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of neurotrophin-3 (NT-3), pro-NT-3, or an effective fragment thereof, or a nucleic acid encoding NT-3, pro-NT-3, or an effective fragment thereof.

[0172] In certain embodiments, the methods of the present invention may be used to increase muscle strength, muscle mass, or muscle endurance, and reduce muscle fatigue in a subject.

[0173] Muscles can be classified into three types: skeletal, cardiac, and smooth. Skeletal muscle is muscle tissue that can generate force and transmit that force to the skeleton, allowing for breathing, movement, and maintaining posture. Cardiac muscle is the muscle of the heart. Smooth muscle is the muscle tissue of the arterial and intestinal walls. The methods and compositions of the present invention are primarily applicable to skeletal muscle, but may also have a beneficial effect on smooth muscle. "Skeletal muscle" and "skeletal muscle" are defined as muscles that have interactions with bones, tendons, and joints.

[0174] In certain embodiments, the present invention provides methods for the treatment of diseases, illnesses, disorders, and conditions that cause muscle weakness (also referred to herein as musculoskeletal disorders and muscle dysfunction and muscle wasting disorders). The main categories of musculoskeletal disorders are muscular dystrophies and muscle atrophy.

[0175] In one embodiment, the present invention provides a method for treating musculoskeletal disorders, including diseases, disorders, or conditions that cause muscle dysfunction and muscle wasting (including hereditary myopathies, neuromuscular diseases, muscle atrophy, and drug-induced myopathies), or conditions that cause muscle weakness. The present invention also provides a method for treating neuropathies, such as hereditary CMT and CMT1A, and axonal and demyelinating polyneuropathies, such as chronic inflammatory demyelinating polyneuropathy. The method of treatment comprises administering to a patient in need thereof a therapeutically effective amount of neurotrophin-3 (NT-3), pro-NT-3, or an effective fragment thereof, or a nucleic acid encoding NT-3, pro-NT-3, or an effective fragment thereof. In one embodiment, the subject is suffering from a muscle disorder selected from the group consisting of sarcopenia, cachexia, type II muscle fiber atrophy, and acquired autoimmune primary muscle diseases associated with impaired hypertrophic growth phase of regeneration.

[0176] In some embodiments, NT-3 can be used to treat muscle atrophy. Muscle atrophy is a general term used to describe a condition characterized by the wasting or loss of muscle tissue due to various diseases, disorders, other conditions, or events. Muscle atrophy can be the result of, but is not limited to, recovery from severe burns, major joint replacement surgery, neuropathic pain, peripheral neuropathy, necrotizing vasculitis, weightlessness (e.g., astronauts and cosmonauts), long-term hospitalization, degenerative diseases (e.g., amyotrophic lateral sclerosis) and organ transplants, as well as long-term immobilization due to spinal cord injury, long-term hemodialysis, and stroke.

[0177] In some embodiments, NT-3 can be used to treat disuse muscle atrophy. Disuse muscle atrophy is a condition characterized by the wasting or loss of muscle tissue due to prolonged inactivity. Disuse muscle atrophy can be the result of, but is not limited to, recovery from severe burns, major joint replacement surgery, neuropathic pain, weightlessness (e.g., astronauts and cosmonauts), long-term hospitalization, anorexia, and organ transplantation, as well as long-term immobilization due to spinal cord injury, long-term hemodialysis, and stroke.

[0178] In certain embodiments, NT-3 can be used to treat age-related muscle atrophy, a condition characterized by the wasting or loss of muscle tissue and its replacement by fibrotic tissue as a subject ages.

[0179] In certain embodiments, NT-3 can be used to treat sarcopenia, a condition characterized by the wasting or loss of muscle tissue and its replacement by fibrotic tissue as a subject ages.

[0180] In one embodiment, NT-3 can be used to treat muscle wasting in cachexia. Cachexia is weight loss, muscle atrophy, fatigue, weakness, and significant loss of appetite as a result of chronic illness in people who are not actively trying to lose weight. The muscle wasting component of cachexia can be the result of, but is not limited to, cancer, multiple sclerosis, tuberculosis, acquired immunodeficiency syndrome, human immunodeficiency virus, malnutrition, Parkinson's disease, emphysema, heart failure, motor neuron disease, cystic fibrosis, dementia, sarcopenia, chronic obstructive pulmonary disease, kidney disease, and kidney failure.

[0181] In certain embodiments, NT-3 can be used to treat muscle wasting resulting from viral infections (e.g., HIV, Epstein-Barr virus), bacterial infections (e.g., mycobacteria and rickettsia), post-polio syndrome, and parasitic infections (e.g., trypanosome and schistosome) in which subjects are at risk for developing muscle wasting.

[0182] Neurotrophin-3 In one embodiment, a therapeutically effective amount of NT-3, pro-NT-3, or an NT-3 analog thereof is administered to a subject to stimulate muscle growth. Neurotrophin 3 (NT-3) is a neurotrophic factor in the NGF (nerve growth factor) family of neurotrophins. NT-3 is a protein growth factor with activity on specific neurons in the peripheral and central nervous system; it is best known for its role in supporting the survival and differentiation of existing neurons and promoting the growth and differentiation of new neurons and synapses.

[0183] The present disclosure includes blocking peptides substantially similar to at least a portion of the amino acid sequence of the extracellular region of Cx26. As used herein, the term "portion" refers to an amino acid sequence within the extracellular region of Cx26 that comprises at least four amino acids. In further embodiments, a portion refers to an amino acid sequence that is at least six amino acids long, at least eight amino acids long, or at least ten amino acids long. Thus, a blocking peptide consists of at least four, six, eight, or ten amino acids. Similarly, the blocking peptides described herein can have a maximum size. The maximum size of a blocking peptide relates to the overall size of the peptide, including any additional sequence linked to the peptide, such as a protein transduction domain. In certain embodiments, a blocking peptide has a maximum size of less than about 200 amino acids, while in other embodiments, a blocking peptide has a maximum size of less than about 100 amino acids. In other embodiments, a blocking peptide has a maximum size of 75 amino acids or less, 50 amino acids or less, 40 amino acids or less, 30 amino acids or less, or 20 amino acids or less.

[0184] As used herein, the term "polypeptide" refers to an oligopeptide, peptide, or protein sequence, or a fragment, portion, or subunit thereof, and to natural or synthetic molecules. The term "polypeptide" also includes amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and can contain any type of modified amino acid. The term "polypeptide" also includes peptides and polypeptide fragments, motifs, etc., glycosylated polypeptides, all "mimetic" and "peptidomimetic" polypeptide forms, and retro-inversion peptides (also called all-D-retro or mtro-enantio peptides).

[0185] "Substantially similar" means that a given amino acid (or nucleic acid) sequence shares at least 85%, more preferably at least 90%, and even more preferably at least 95% identity with a reference sequence. Identity or homology with such sequences is defined herein as the percentage of amino acid residues in a candidate sequence that are identical with a known peptide, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology, not counting conservative substitutions as part of the sequence identity. N-terminal, C-terminal, or internal extensions, deletions, or insertions into the peptide sequence should not be construed as affecting homology.

[0186] Substantially similar peptides include those that differ by one or more amino acid changes, where the changes, e.g., substitution, addition, or deletion of amino acid residues, do not abolish the properties of the related peptide, such as its ability to associate with FAK or NANOG. Furthermore, only sequences describing or encoding proteins in which only conservative substitutions have been made in conserved regions are substantially similar overall. Preferably, substantially similar sequences also retain the unique activity of the polypeptide.

[0187] Examples of conservative substitutions include the substitution of a non-polar (hydrophobic) residue such as isoleucine, leucine, or methionine for another. Similarly, the present invention contemplates the substitution of one polar (hydrophilic) residue between arginine and lysine, between glutamine and asparagine, and between glycine and serine. Furthermore, the substitution of a basic residue such as lysine, arginine, or histidine for another, or the substitution of an acidic residue such as aspartic acid or glutamic acid for another, is also contemplated. Examples of non-conservative substitutions include the substitution of a non-polar (hydrophobic) residue such as isoleucine, valine, leucine, alanine, or methionine for a polar (hydrophilic) residue such as cysteine, glutamine, glutamic acid, or lysine, and / or the substitution of a polar residue for a non-polar residue.

[0188] The phrase "conservative substitution" also includes the use of chemically derivatized residues in place of underivatized residues, so long as the peptide retains the required ability to bind to NT-3. Substantially similar peptides also include the presence of additional amino acids or the deletion of one or more amino acids that do not affect the required ability to bind to NT-3; for example, substantially similar peptides can contain an N- or C-terminal cysteine, whereby, if necessary, the peptide can be covalently bound to a carrier protein, such as albumin. Such binding can reduce the removal of the peptide from the blood and also reduce the rate of proteolysis of the peptide. Furthermore, for the purposes of the present invention, peptides containing D-amino acids in place of L-amino acids are also included in the term "conservative substitution." The presence of such D-isomers can help minimize proteolytic activity and removal of the peptide.

[0189] In one embodiment, pro-neurotrophin-3 protein (pro-NT-3) is administered to a subject. The pro-form of neurotrophin-3 is an approximately 30 kDa precursor form of NT-3 that is converted to mature NT by enzymatic cleavage and removal of the approximately 15 kDa N-terminal prodomain. See Tauris et al., Eur. J Neurosci, 33(4), 622-631 (2011).

[0190] Treatment of muscle atrophy The present invention provides methods for treating subjects with peripheral muscle atrophy. Pyruvate compounds can be used to provide prophylactic and / or therapeutic treatment. For example, pyruvate compounds can be administered prophylactically to a subject prior to the onset of peripheral neuropathy. Prophylactic (i.e., preventive) administration is effective in reducing the likelihood of subsequent development of peripheral neuropathy in a subject or reducing the severity of subsequent peripheral neuropathy. Preventive treatment can be provided to subjects at high risk for developing peripheral neuropathy, such as those with a family history of peripheral neuropathy. Expression of myelin protein 22 (PMP22) mutations accounts for 70-80% of all cases of Charcot-Marie-Tooth neuropathy, and therefore, their presence can be useful as a criterion for selecting patients for treatment with the pyruvate compounds described herein.

[0191] Alternatively, the compounds of the present invention can be administered therapeutically to a subject already suffering from a peripheral neuropathy. In one embodiment of therapeutic administration, administration of the compound is effective in eliminating the peripheral neuropathy; in another embodiment, administration of the pyruvate compound is effective in reducing the severity of the peripheral neuropathy or extending the lifespan of a subject suffering from it. In one embodiment, the method of treatment comprises administering to a subject a therapeutically effective amount of a pyruvate compound in a pharmaceutically acceptable formulation over a significant period of time.

[0192] CMT mutants Charcot-Marie-Tooth (CMT) hereditary neuropathies (CMT) refer to a group of disorders characterized by chronic motor and sensory polyneuropathy, also known as hereditary motor and sensory neuropathies. Autosomal dominant CMT neuropathy types include demyelinating (also known as CMT1), axonal non-demyelinating (also known as CMT2), and dominant intermediate CMT (DI-CMT). Other neuropathies equivalent to CMT include distal hereditary motor neuropathy (dHMN), distal spinal muscular atrophy (DSMA), and Dejerine-Sottas syndrome (DSS). A description and classification of CMT neuropathies is provided in Bird, GeneReviews, University of Seattle, Washington. Provided by Washington Seattle PIM 20301532, Updated 2018 Jun 28.

[0193] Currently, there are over 70 known genetic mutations in CMT-associated genes. These genetic mutations are shown in Table 1 below using the classification system of Magy et al. (Neurology 90:e870-6, 2018). The inheritance mode for each CMT-associated genetic mutation is autosomal dominant (AD), autosomal recessive (AR), or X-linked (XL). The neuropathy for each CMT-associated genetic mutation is axonal (Ax), demyelinating (De), or intermediate (In). The "Other designations" shown in Table 1 are those used in other classification systems, including dominant intermediate CMT (DI-CMT), distal spinal muscular dystrophy (DSMA), hereditary sensory and autonomic neuropathy (HSAN), and distal hereditary motor neuropathy (dHMN).

[0194] [Table 1-1]

[0195] [Table 1-2]

[0196] [Table 1-3]

[0197] [Table 1-4]

[0198] [Table 1-5]

[0199] Administration and Formulation The vectors or peptides used with certain embodiments of the present invention can be incorporated into pharmaceutical compositions suitable for administration to a subject. In certain embodiments, the pharmaceutical composition comprises a vector of the present invention and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate-buffered saline, dextrose, glycerol, ethanol, and the like, as well as combinations thereof. In many cases, it may be preferable to include an isotonic agent in the composition, for example, a sugar, a polyalcohol such as mannitol or sorbitol, or sodium chloride. Pharmaceutically acceptable carriers may further contain minor amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives, or buffers, which enhance the shelf life or effectiveness of the vector or pharmaceutical composition.

[0200] The vector or peptide may be administered acutely (i.e., at the onset or shortly after an event leading to muscle wasting), or prophylactically (e.g., before scheduled surgery or before signs or symptoms appear), or during the course of muscle wasting to reduce or ameliorate the progression of symptoms that would otherwise occur. The timing and intervals of administration vary depending on the subject's symptoms and may be administered at intervals of a few hours to a few days, over a period of several hours, days, weeks, or longer, as can be determined by one of skill in the art.

[0201] A composition containing a vector or peptide is generally administered intravenously. When administered intravenously, the composition may be combined with other ingredients such as carriers and / or adjuvants. The peptide may also be covalently bound to a protein carrier such as albumin to minimize removal of the peptide. There are no limitations on the nature of other ingredients, except that such ingredients must be pharmaceutically acceptable, effective for their intended administration, and not reduce the activity of the active ingredient of the composition.

[0202] Pharmaceutical forms suitable for injection include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the final solution form must be sterile and fluid. Typical carriers include, for example, aqueous solutions buffered with water (i.e., biocompatible buffers), solvents or dispersion media containing ethanol, polyols (glycerol, propylene glycol, polyethylene glycol, suitable mixtures thereof, etc.), surfactants, or vegetable oils. Sterilization can be carried out by any art-recognized technique, including, but not limited to, filtration or the addition of antibacterial or antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, or thimerosal. Additionally, isotonic agents, such as sugars or sodium chloride, can be incorporated into the compositions of the present invention.

[0203] Sterile injectable solutions containing the peptides of the present invention can be prepared by incorporating the required amount of the compound in an appropriate solvent with the various ingredients listed above, as needed, followed by sterilization, preferably filtered sterilization. To obtain a sterile powder, the solution can be vacuum-dried or freeze-dried, as needed.

[0204] When the peptides of the present invention are administered orally, such pharmaceutical compositions containing an effective dose of the peptide may also contain an inert diluent, such as an assimilable edible carrier, may be in hard or soft shell gelatin capsules, may be compressed into tablets, or may be in an elixir, suspension, syrup, etc. Thus, the peptides of the present invention are formulated for convenient and effective administration in a pharmaceutically effective amount with a therapeutically effective amount of a suitable pharmaceutically acceptable carrier.

[0205] As used herein, the phrase "effective amount" or "therapeutically effective amount" refers to an amount of an agent sufficient to stimulate muscle growth or reduce or prevent muscle atrophy. The exact amount required will vary from subject to subject, depending on the subject's species, age, and general condition, the specific therapeutic agent, its method and / or route of administration, and the like. However, it will be understood that the total daily usage of the compounds and compositions of the present invention can be determined by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject or organism will depend on various factors, including the disorder being treated and the severity of the disorder; the activity of the specific compound used; the specific composition used; the subject's age, weight, overall health, sex, and dietary habits; the timing, route of administration, and excretion rate of the specific composition used; the duration of treatment; drugs used in combination with or simultaneously with the specific composition used; and similar factors well known in the medical arts.

[0206] The vector or peptide may be administered in a manner compatible with the dosage form and in an amount that is therapeutically effective. The systemic dosage will depend on the age, weight, and condition of the patient, as well as the route of administration. For example, a suitable dose of peptide for administration to an adult ranges from about 0.001 to about 20.0 mg per kilogram of body weight. The peptide should preferably be administered in an amount of at least about 50 mg per administration, more preferably in an amount of up to about 500 mg to about 1 gram per administration. Because the peptide compositions of the present invention are eventually cleared from the bloodstream, readministration of the composition is indicated and preferred. [Example]

[0207] Aspects and embodiments of the present invention will now be illustrated by the following examples, however, there are various other embodiments that fall within the scope of the present invention, which should not be limited to the specific examples set forth herein.

[0208] Example 1 AAV1.NT-3 gene therapy increases muscle fiber diameter by activating the MTOR pathway and metabolic remodeling in a mouse model of CMT NT-3 has well-recognized effects on peripheral nerves and Schwann cells, promoting axon regeneration and associated myelination. We evaluated the effects of AAV.NT-3 gene therapy on the oxidative status of neurogenic muscle from Trembled (TrJ) mice 16 weeks after gene injection and found that the increase in muscle fiber size was associated with changes in muscle fiber oxidative status, normalizing the fiber type ratio seen in wild-type mice. The NT-3-induced increase in fiber size was most pronounced for the fast-twitch fiber population. These changes in TrJ muscle were accompanied by increased phosphorylation levels of 4E-BP1 and S6 protein, evidence of mTORC1 activation. In parallel, the expression levels of the mitochondrial biogenesis regulator PGC-1α and markers of glycolysis (HK1 and PK1) increased in TrJ muscle. In vitro studies demonstrated that recombinant NT-3 could directly induce Akt / mTOR pathway activation in TrkC-expressing myotubes, but not in myoblasts. Concomitantly, myogenin expression levels were significantly higher in myotubes, whereas p75NTR expression was downregulated compared with myoblasts, indicating that NT-3-induced myoblast differentiation is associated with mTORC1 activation. These studies demonstrate for the first time that NT-3 increases myofiber diameter in neurogenic muscles through direct activation of the mTOR pathway, and that the increase in fiber size is more pronounced for fast-twitch fibers.

[0209] method Animals, treatment protocols, and histopathology: TrJ mice (B6.D2-Pmp22Tr-J / J) and C57BL / 6 wild-type mice were obtained from the Jackson Laboratory (Barrington, MA, USA). The left gastrocnemius muscles of 9- to 12-week-old TrJ mice were injected with PBS (n=6) or 3 × 10 10 A separate cohort of TrJ mice was injected with either 1 × 10 11 vg of single-stranded AAV1.CMV.NT-3 to induce high NT-3 expression levels for comparison (n=6). WT mice were injected with PBS (n=6) or 3x10 10Mice were administered either the scAAV1.tMCK.NT-3 vector (vg) or the scAAV1.tMCK.NT-3 vector (n=4). Groups of mice were euthanized, and their muscles were harvested 16 weeks after gene injection and processed for cryostat sectioning. Succinate dehydrogenase (SDH) enzyme histochemistry was used to assess metabolic fiber type differentiation using a standard protocol established in-house. Fiber type-specific diameter measurements were obtained from 12-µm-thick SDH-stained cross sections. Three images (per animal per section per section) representing three distinct regions of the gastrocnemius muscle along the midline axis (the deep region primarily composed of STO, the intermediate region showing a checkerboard appearance of STO and FTO or FTG fibers, and the superficial region primarily composed of FTG fibers) were taken at 20x magnification using an Olympus BX41 microscope and SPOT camera. This technique was chosen to capture changes in the oxidative status of fibers within each region in response to treatment-induced metabolic changes. The diameters of dark (STO), intermediate (FTO) and light (FTG) fibers were determined using the Zeiss Measurements were made using Axiovision LE4 software by measuring the shortest distance across muscle fibers and expressed as a percentage of the total. Average fiber diameters (mean ± SEM) were obtained from combining all three fiber types in each cohort. An average of 1250 fibers (960–1486) were measured per group.

[0210] AAV NT-3 Vector Production and Efficacy: The design of self-complementary AAV viral vectors with serotype 1 NT-3 under the tMCK or CMV promoter was previously described and generated at the Viral Vector Core at Nationwide Children's Hospital (Columbus, OH). (Sahenk et al., Mol Ther, 22(3):511-521 (2014)). Aliquots of virus were kept at -80°C until use. Blood samples were collected from treated and untreated mice by intraocular bleeding under anesthesia at 6 and 16 weeks postinjection, and serum was assayed for NT-3 levels using a capture ELISA.

[0211] C2C12 myoblast culture and myotube formation: C2C12 myoblasts were cultured in growth medium (GM) consisting of DMEM Medium (Gibco-Invitrogen, #10569010, Carlsbad, CA) supplemented with 10% FBS (Fisher Scientific, #26-140-079, Carlsbad, CA) and 1% penicillin / streptomycin solution (Gibco-Invitrogen, #15640055, Carlsbad, CA) at 37°C and 5% CO in a humidified chamber. Myotube formation was induced in confluent cultures by replacing GM with differentiation medium [DMEM (Gibco-Invitrogen, #11965092, Carlsbad, CA) supplemented with 5% horse serum (Gibco-Invitrogen, #26050088, Carlsbad, CA) and 1% penicillin / streptomycin solution (Invitrogen, #15640055, Carlsbad, CA)]. All subsequent assays for myotubes, including QPCR, Western blot, and ELISA, were initiated 3 days after induction of myotube formation. Both myoblasts and myotubes were exposed to recombinant human NT-3 (Pepprotech, Rocky Hill, NJ) at a concentration of 100 ng / ml in 6-well plates. Culture media was collected for glucose consumption and lactate formation by using glucose and lactate assay kits (Eton Bioscience, San Diego, CA) according to the manufacturer's instructions.

[0212] QPCR experiments: Total RNA was isolated from the gastrocnemius muscles of NT-3-treated and untreated control mice at the endpoint. Total RNA was isolated from myoblasts and myotubes before and 48 hours after NT-3 treatment. cDNA was synthesized using the mirVana RNA Isolation Kit (Life Technologies, #AM1560, TX, USA) followed by the Trascriptor First Strand cDNA Synthesis Kit (Roche, #04379012001, Roche, USA) according to the manufacturer's instructions. Other qPCR experiments were performed using iTaq™ universal SYBR® Green supermix (Biorad, #1725122, Hercules, CA, USA). Primer sequences for PGC 1α (Cunningham et al., Nature, 450(7170):736-740(2007)) and GAPDH (Toscano et al., Mol Ther, 18(5):1035-1045(2010)) (housekeeping genes) can be found in the literature. Other primer sequences can be found in Primer Band. Wang et al. al., Nucleic acids research, 40 (Database issue): D1144-1149 (2012). All qPCR experiments were performed using an ABI 7500 Real Time PCR machine, and the results were analyzed using Data Assist Software (ABI).

[0213] Protein extraction and Western blot experiments: Frozen gastrocnemius muscle blocks were cut into 20 μm-thick sections and placed in small 2 ml plastic tubes (15–20 sections per block). They were homogenized in lysis buffer (RIPA lysis buffer (Thermo Fisher, #89900, USA) with 1x Halt protease inhibitor (Thermo Fisher, #78429, USA) and 1x phosphatase inhibitor (Sigma, #P0044, USA) for 20 seconds three times using an automatic pellet mixer and disposable pestle. For in vitro signal transduction assays, myoblasts and myotubes were harvested in small 2 ml tubes after 30 minutes of incubation with NT-3 (100 ng / ml) and lysed as described above. The lysates were centrifuged at 13,000 rpm for 10 minutes at 4°C, and the supernatant was carefully collected. Protein concentrations were measured using a BCA Protein Assay Kit (Thermo Fisher, #23252, Waltham, MA, USA). Protein samples (10–40 μg) were electrophoresed in 4–12% Bolt® Bis-Tris Plus precast 10- or 15-well polyacrylamide gels (Thermo Fisher, #NW04120BOX) and transferred to PDVF membranes (GE Healthcare, #10600021, Pittsburgh, USA). The membranes were blocked with 5% bovine serum albumin (BSA, Bedford, MA, USA) in TBS buffer containing 0.05% Tween-20 (TBS-T, Amresco, OH, USA) for 2 h at room temperature and then incubated with the appropriate primary antibody in TBS-TS buffer containing 5% BSA overnight at 4°C in a cold room. The primary antibodies used in this study were as follows: anti-phospho S6 protein Ser235 / 236 (#4858), anti-S6 protein (#2217), anti-Phospho Akt Ser473 (#4060), anti-Akt (#9272), anti-phospho 4E-BP1 thr37 / 46 (#2855), anti-4E-BP1 (#9644), anti-GAPDH (Santa cruz, #sc365062).After washing five times for 5 min on an orbital shaker with TBS-T, the membrane was incubated with secondary antibodies [HRP-conjugated anti-rabbit (#HAF008), HRP-conjugated anti-mouse (HAF007)] from R&D Systems (Minneapolis, MN, USA) in 5% nonfat dry milk in TBS-T buffer for 1 h. The membrane was washed again with TBS-T as above and then incubated with ECL Prime Western detection reagent (Amersham, #RPN2232) for 1–3 min. After incubation with PBS (NJ, USA), the membranes were exposed to X-ray film (Denville, #E3018, MA, USA) using multiple exposure times. Protein bands on the film were photographed using a camera (Sony A600, Japan), and band intensities were quantified using Quantity-One software (BioRad, v.4.6.9). The relative content of the analyzed protein in each sample was determined by normalizing the band intensity to the content of GAPDH in the same sample. The membranes were stained with 0.1% Coomassie Brilliant Blue R stain (Thermo Fisher, USA), rinsed, and photographed to confirm equal protein loading in each lane.

[0214] Statistics: For muscle fiber size comparison between treated and untreated groups, statistical analysis was performed using one-way analysis of variance (Anova) in Graphpad Prism 6 software. Student's t-test or one-way Anova was performed when applicable for other statistical analyses. The significance level was set at P<0.05. In all experiments, results were presented as mean ± SEM.

[0215] result AAV1.NT-3-induced fiber type remodeling in TrJ muscles Previously, a switch from fast-twitch to slow-twitch fibers in the TrJ muscle was observed as part of a neuropathic phenotype (Nicks et al., J Neuropathol Exp Neurol, 72(10):942-954 (2013)). In this study, metabolic fiber type differentiation was assessed in the TrJ and age-matched WT gastrocnemius muscles using SDH staining by collecting samples from the deep, intermediate, and superficial regions of the muscle as described. At 16 weeks after gene injection, there was a significant decrease in STO fibers, particularly FTO and FTG fiber size, along with an increase in their size, compared with the untreated (PBS) group, which showed neural alterations, small, angular fibers, and a population of types (Figure 1A and B). Quantitative analysis showed that both the number and percentage of STO fibers per unit area in the TrJ-PBS muscle were significantly higher than in WT muscle, consistent with previous studies. 1 × 10 cells containing both promoters, resulting in either low (tMCK) or high (CMV) NT-3 expression in the treatment groups (Figure 4). 11 A dose of 100 mg of AAV1.NT-3 demonstrated a fiber type switch from STO to FTO / FTG fibers. The mean density or percentage of STO in both treatment groups (obtained from n = 3–5 mice in each group) was not significantly different from WT muscle, indicating a normalization of fiber type distribution by NT-3 (Figure 1C). Furthermore, WT muscles treated with AAV1.NT-3 did not exhibit a significant change in fiber type distribution profile.

[0216] NT-3 treatment in TrJ mice showed differential effects on increasing muscle fiber size (Table 2). When NT-3 was expressed under the control of the tMCK promoter, a significant diameter increase was observed only in FTG fibers. In a second treatment cohort, in which high NT-3 expression was achieved with the CMV promoter, there was a significant diameter increase in all fiber types. Interestingly, this dose-dependent NT-3 effect was seen only in neurogenic TrJ muscles; at the same time point, i.e., 16 weeks after gene injection, no significant diameter change was observed in any of the fiber types in WT muscles with NT-3 gene therapy.

[0217] Table 2 shows that the increase in fiber size in neurogenic TrJ muscles after NT-3 gene therapy was more pronounced for fast-twitch fibers.

[0218] [Table 2]

[0219] AAV-NT-3 improved mTOR signaling and metabolic markers in TrJ muscle The histological findings described above in TrJ muscles in response to AAV1.NT-3 treatment prompted us to investigate whether mTORC1 activation plays a role in NT-3-induced myofiber hypertrophy. mTORC1 activity was assessed by the phosphorylation levels of its downstream substrates, 4EBP-1 and ribosomal S6P, in muscle samples from the groups. In TrJ muscles treated with AAV1.NT-3, the levels of phosphorylated 4EBP-1 and S6P were significantly increased compared with untreated counterparts from the TrJ-PBS control (Figure 2A). In contrast, we found that NT-3 treatment did not significantly affect the phosphorylation levels of 4EBP-1 and S6P in WT muscles (Figure 2B).

[0220] Through 4E-BP, mTORC1 regulates the synthesis of nuclear-encoded mitochondrial proteins, controlling mitochondrial activity and biogenesis, and thus regulating energy expenditure and production

[17] . Accordingly, PGC 1α, a master regulator of mitochondrial biogenesis, was upregulated in NT-3-treated TrJ muscles, indicating that NT-3 can counteract the defective PGC 1α expression levels seen in neurogenic muscle (Figure 2C). Consistent with the lack of mTORC1 activation, no change in PGC 1α expression levels was observed in WT muscles treated with NT-3 (Figure 2C). The increase in fiber size in TrJ muscles induced by NT-3 gene therapy primarily occurred in FTO and FTG fibers, which have higher glycolytic activity than slow-twitch fibers

[18] . Consistent with this, we also found that the expression of the rate-limiting enzymes of glycolysis, HK1 and PK1, was upregulated in treated TrJ muscles, suggesting an increase in glycolytic flux. These changes were not significant in WT muscles treated with AAV.NT-3.

[0221] NT-3 activates the Akt / mTORC1 pathway via the TrkC receptor in C2C12 myotubes The in vivo studies described herein demonstrated that the increased fiber size and fiber type remodeling induced by AAV1.NT-3 treatment in TrJ muscle are associated with mTORC1 activation. However, the question of whether this change is simply a result of nerve regeneration or whether NT-3 can directly alter muscle protein synthesis and cellular metabolism independently of nerve regeneration remains to be resolved. As a next step, the direct effect of NT-3 on the mTOR pathway in an in vitro system was investigated by exposing C2C12 myoblasts and myotubes to recombinant NT-3 without nerve influence. The results showed that NT-3 could induce Akt / mTOR pathway activation in myotubes (Figure 3A), but not in myoblasts. Treatment of myotubes with 100 ng of recombinant NT-3 for 30 min resulted in significantly higher phosphorylation of Akt, 4EBP1, and S6P compared to the control group (Figure 3A). In another set of experiments, we found that NT-3 significantly enhanced the expression of the mitochondrial biogenesis marker PGC 1α and the glycolysis marker PK1 in myotubes after 48 h of incubation (Figure 3B). Accordingly, analysis of the supernatant at this time point showed increased glucose consumption and lactate production in NT-3-treated myotubes compared with controls (Figure 3C). No effect of NT-3 on HK1 expression levels was observed under the conditions under which these experiments were performed.

[0222] The expression of p75NTR and TrkC receptors, and myogenin, markers of myoblast entry into the differentiation pathway, was analyzed in myoblast and myotube cultures. NT-3 exerts its biological effects through binding to its preferred receptor, TrkC, or the low-affinity neurotrophin receptor p75NTR. Schecterson LC, Bothwell M, Neuron, 9(3):449-463 (1992). p75NTR is expressed in C2C12 myoblasts and is downregulated during muscle differentiation. Seidl et al., Journal of Cellular Physiology, 176(1):10-21 (1998). It has been shown that the neurotrophic factor NGF influences muscle differentiation and cell growth via p75NTR, and downregulation of p75NTR is thought to be essential for muscle differentiation. Similar to the effects of NGF, NT-3 was also found to promote myogenin expression in myotubes, and as expected, this was associated with significantly higher expression of p75NTR in myoblasts compared to myotubes, whereas TrkC expression levels did not differ in both groups (Figure 3D). NT-3 did not differentially affect the expression levels of these receptors in myoblasts or myotubes.

[0223] explanation Evidence is presented herein that NT-3 may have a direct effect on neurogenic muscle metabolism, resulting in increased fiber size and fiber type remodeling relative to normalization via mTORC1 activation. The increase in fiber size was most pronounced for type II fibers, particularly the FTO subtype. Furthermore, it is shown that NT-3 may induce Akt / mTOR pathway activation in myotubes, but not directly in myoblasts, as an important contributor to its in vivo effects in neurogenic muscle. Interestingly, NT-3 gene therapy in WT muscle at the same dose did not affect these characteristics, although the effects of NT-3 on denervated WT muscle using a gene therapy paradigm were not tested. The differential effect of NT-3 on type II muscle fiber subtypes was previously demonstrated in rat gastrocnemius muscle 8 months after nerve repair with or without local delivery of NT-3 to the nerve crush site, where both the proportion and size of type IIb fibers were found to return to normal. Sterne et al., J Cell Biol, 139(3):709-715 (1997). However, this effect could be interpreted as NT-3-promoted axonal regeneration with beneficial consequences in motor target organs, and the potential for NT-3 to specifically affect a subset of motor neurons that specify the type IIb muscle fiber phenotype. It could be argued that the findings from our in vivo studies could represent a combined effect of NT-3 on both nerve and muscle. However, our in vitro data highlight the evidence that NT-3 exerts a direct effect on muscle metabolism through activation of Akt / mTORC1, and that this direct effect is likely important in neurogenic muscle, resulting in a preferential size increase in FTG, i.e., type Erb fibers.

[0224] In conditional transgenic mice, expressing a constitutively active form of Akt resulted in muscle hypertrophy due to the growth of type III muscle fibers. Izumiya et al., Cell metabolism, 7(2):159-172 (2008). This was associated with upregulation of transcripts involved in glycolysis, increased glucose consumption and lactate production (which was associated with lower insulin levels), increased blood glucagon levels, and resistance to high-fat diet-induced obesity. Conversely, mTOR inactivation was associated with a decrease in glycolytic enzymes, PK1 and HK1. Risson et al., J Cell Biol, 187(6):859-874 (2009). In our study, AAV.NT-3 treatment in TrJ muscle increased both FTG fiber size and the expression of glycolytic enzymes PK-1 and HK-1. Furthermore, in vitro studies showed that NT-3 increased glucose uptake and lactate formation in myotubes, along with upregulation of PK-1. These results suggest that NT-3 may be involved in controlling whole-body metabolism by regulating fast-twitch / glycolytic fibers, although further studies are needed to characterize its role in detail. Furthermore, AAV.NT-3 treatment was able to prevent the defective expression of PGC1α seen in TrJ neurogenic muscle, along with the elevated levels of activated 4E-BP1. It has previously been suggested that mTOR regulates mitochondrial biogenesis and metabolism through 4E-BP1 / PGC1α in skeletal muscle. Tsai et al., J Clin Invest, 125(8):2952-2964(2015). NT-3 may also play a role in promoting oxidative phosphorylation through activation of 4EBP1 and PGC lα in muscle.

[0225] NT-3 is first found at high levels in the central nervous system (CNS) during fetal development and is reduced in the adult brain, suggesting that NT-3 plays an important role during early neural development [28, 29]. NT-3 is also important in peripheral nerves and has a positive effect on many stages of neuromuscular development. In Xenopus nerve-muscle cocultures, muscle-derived NT-3 significantly promotes the maturation of synaptic transmission at the neuromuscular junction [30-33]. Furthermore, NT-3 enhances the survival of SCs, a key component of the neuromuscular system

[34] . NT-3 is expressed in SCs to promote nerve regeneration and is a critical component of the autocrine survival loop, ensuring SC survival and differentiation in adult nerves [35-39]. Studies in the CMT1A mouse model have observed several important biological effects of NT-3: (i) an increase in SC numbers, (ii) an increase in the number of myelinated fibers, and (iii) normalization of the axonal neurofilament cytoskeleton [5, 40]. Another NT-3 effect of particular interest herein is the increase in myelin thickness, which was recognized as morphological evidence that NT-3 can affect myelin protein production.

[0226] Previous studies have provided considerable evidence that mTORC1 plays a role in regulating myelination in the CNS. Transgenic overexpression of constitutively active Akt kinase is sufficient to promote myelin membrane growth in the CNS through mTOR signaling [41, 42], and IGF-1-stimulated protein synthesis in oligodendrocyte precursor cells requires the PI3K, Jakt, and MER / ERK pathways

[43] . The ability of NT-3 to target the translational machinery to stimulate myelin protein synthesis was first demonstrated in primary cultures of oligodendrocytes

[44] . NT-3 was found to upregulate 4EBP1 phosphorylation in oligodendrocytes via the PI3K / mTOR pathway. Eliminating mTOR function, particularly in SCs, using a gene inactivation approach affected their ability to myelinate normally

[45] . Indeed, in mutants, myelin sheaths were found to be thinner, internode lengths were shorter, and axon hypertrophy growth was reduced. Concomitantly, downstream targets of mTOR, S6 and 4E-BP1, were less phosphorylated

[45] .

[0227] Given these previous studies, it was possible that the effect of NT-3 on increasing muscle fiber diameter was mediated by the same mechanism (direct effect via activation of mTORC1). It is important to note that NT-3 gene therapy in WT muscle at the same dose used in TrJ did not induce significant changes in fiber type size or type distribution. These observations in WT muscle suggest that the effects of NT-3 are not directed at highly differentiated or normally functioning cells, but rather act on remodeled cellular metabolism that may result from pathological processes. One supporting piece of evidence is that NT-3 does not alter functional recovery after crush injury in WT animals, resulting in only slightly more axons than control or NGF-treated animals

[46] . Consistent with these observations, our toxicity studies evaluating scAAV1.tMCK.NT-3 demonstrated a dose of 1 × 10, 10-fold higher than the highest dose proposed for clinical trials for the treatment of CMT1A. 13vg / kg and showed no treatment-related toxicity or histopathological abnormalities in organ tissues in C57BL / 6 or TrJ mice.

[0228] During muscle development, p75NTR is transiently expressed in myoblasts, which will form myotubes / myofibers or differentiate into satellite cells

[23] . The receptor's temporal expression pattern indicates that p75NTR mediates myoblast survival before differentiation, and that the activity of this receptor during myogenesis is important for muscle development

[23] . Similar to the effects of NGF

[21] , NT-3 was found to promote myogenin expression in myotubes. As expected, this was associated with significantly higher expression of p75NTR in myoblasts, which was significantly downregulated in myotubes. Examination of p75NTR and TrkC expression in TrJ and WT muscle samples revealed significantly higher expression levels of both in neural TrJ muscle compared to WT, and these levels were reduced in response to NT-3 (Figure 5). Although intriguing, this observation does not allow for conclusions about which cell types express these receptors, or whether they are expressed in satellite cells or SCs, in all muscle fibers, or in only one subtype of muscle fiber. Limited data are available about the expression of NT-3 and other neurotrophins and their receptors in human muscle diseases. However, recent studies combining histological examination of muscle biopsies with molecular and cellular analysis of primary muscle progenitor cells have shown that p75NTR is expressed by most satellite cells in vivo and is a marker of regenerating fibers in inflammatory and dystrophic muscle [47, 48]. Our findings in neurogenic muscle are particularly intriguing, and more comprehensive studies of the mechanisms underlying various disease processes are warranted.

[0229] As a conserved Ser / Thr kinase, mTOR is a central regulator of cell growth by integrating signals from nutrients, growth factors, energy status, and environmental stress. The crucial role of mTOR in cell biology and pathobiology, particularly in muscle, which possesses remarkable metabolic and morphological adaptability, is currently attracting significant attention. mTOR binds to raptor to form mTORC1, and muscle-specific inactivation of raptor has been shown to result in muscle atrophy, reduced oxidative capacity, and increased glycogen storage, resulting in dystrophic features that are most pronounced in aerobic muscles

[49] . Meanwhile, reduced mTOR activity exacerbates myopathic features in both slow- and fast-twitch muscles, exhibiting metabolic changes similar to those observed in raptor-deficient muscles, including reduced oxidative metabolism, altered mitochondrial regulation, and glycogen accumulation

[26] . Using a cardiotoxicity-induced muscle necrosis / regeneration cycle paradigm, we recently demonstrated impaired regeneration in a mouse model of limb-girdle muscular dystrophy type 2A, demonstrating that calpain-3-deficient muscle is associated with disrupted mTORC1 signaling and defective mitochondrial biogenesis (under investigation). Taken together, the findings described herein have many implications for the potential use of NT-3 not only for the treatment of neuropathies with benefits for both nerve and muscle, but also for muscle-wasting disorders, including aging, cancer cachexia, or type II muscle fiber atrophy, as well as inherited or acquired autoimmune primary muscle diseases associated with impaired hypertrophic growth phase of regeneration [50-52]. Understanding the role of disrupted mTOR signaling in these disorders should enable the development of novel combination therapies in which NT-3 may play an important role.

[0230] Example 2 NT-3 delivery using the scAAV1.tMCK.NFT3 vector The composition administered is a non-replicating recombinant adeno-associated virus designated scAAV1.tMCK.NTF3, shown in Figure 3. The vector contains the human NT-3 gene under the control of the tMCK muscle-specific promoter. The in vivo biological effect was assessed by transfection of the vector (1 x 10) into the gastrocnemius muscle of C57B16 mice. 11 vg) followed by quantification of circulating NT-3 in serum by ELISA at 4–6 weeks after gene injection.

[0231] First, it was demonstrated that ssAAV1.CMV.NTF3 delivered to the gastrocnemius muscle resulted in sustained and therapeutic NT-3 blood concentrations sufficient to provide functional, electrophysiological, and histopathological improvement of the TrJ nerve. Next, it was investigated whether it was possible to generate the required vector dose and achieve the same level of expression by packaging the expression cassette with scAAV1. A dose-response study was performed in C57BL / 6 mice, using three doses (3 × 10 9 vg, 1×10 10 vg and 3×10 10 Serum NT-3 ELISA data were compared after intramuscular injection of scAAV1.tMCK.NTF3 and scAAV1.CMV.NTF3 at 1 × 10 11 Administration of 100 mg of the sc.rAAV1.CMV.NTF3 vector resulted in significantly higher NT-3 levels than the same dose of single-stranded vector, consistent with the higher efficacy using the self-complementary vector. A half-log lower dose (3 x 10 10 vg), both CMV and tMCK vectors produced biological responses, 1 × 10 11The NT-3 serum concentrations were comparable to those obtained from mice receiving a vg dose of ss.AAV1.CMV.NTF3. NT-3 levels (mean ± SEM) from TrJ mice at 24 weeks post-injection. There were significant differences in NT-3 levels among all seven groups (p < 0.0001). Significant differences in NT-3 levels were observed for the highest and mid-dose vectors for both the promoter and control. However, analysis failed to find significant differences for the lower doses for both vectors. Serum NT-3 levels were compared among all groups (PBS, CMV 3E+09 / 1E+10 / 3E+10, and tMCK 3E+09 / 1E+10 / 3E+10) using the Kruskal-Wallis test. NT-3 levels were compared between each group and the PBS (control) group using the Mann-Whitney U test, adjusting for multiple comparisons using the Bonferroni correction. See Sahenk et al., Mol. Ther. 22(3):511-521, 2014, which is incorporated herein by reference in its entirety.

[0232] Increased muscle diameter at 40 weeks of treatment: The effect of NT-3 gene therapy was demonstrated with ssAAV1.CMV.NTF3 (1 × 10) compared to PBS. 11 In a subset of animals injected with TrJ mice (vg), muscle fiber size was assessed 40 weeks after injection. Neurogenic changes characterized by atrophic angular fibers and group atrophy were evident in muscles from untreated mice, while evidence of nerve regeneration as an increase in fiber type populations and overall fiber size was discernible as a treatment effect. Muscle fiber size histograms generated from the lateral anterior and posterior muscles of the left lower leg (tibialis anterior and gastrocnemius) showed an increase in fiber diameter.

[0233] Further studies in our laboratory demonstrated that NT-3 stimulates the Akt / mTOR pathway in SC cells, resulting in improved axonal myelination and hypertrophic growth in nerves. NT-3 also has a direct stimulatory effect on myotubes via the Trk-C receptor, indicating its role in increasing fiber diameter in the muscles of TrJ mice. Figure 3A shows that NT-3 increased the phosphorylation of Akt (P-Akt) and mTOR targets, 4EBP-1 (P-4EBP1) and PS6K (P-S6K), in SC and myotube cultures. These studies provide evidence to justify the selection of anterior and posterior muscles of the lower leg for vector delivery in this clinical trial.

[0234] Studies with self-complementary (sc) AAV1 and use of the muscle-specific truncated creatine kinase (tMCK) promoter scAAV allows for lower doses resulting in improved safety and dosage levels that meet production specifications. The use of the tMCK promoter is a worthwhile objective that also provides greater safety by avoiding off-target effects. In the following set of experiments, the efficacy of scAAV1.NTF3 under the control of the CMV promoter was evaluated at three doses (3 x 10) within a half-log range. 9 vg, 1×10 10 vg and 3×10 10 The efficacy of AAV1.NTF3 gene transfer in peripheral nerves of TrJ mice was assessed by electrophysiological (Table 3) and morphological examinations 24 weeks after gene transfer. Evidence of transgene expression was assessed by measuring serum NT-3 levels using ELISA.

[0235] [Table 3]

[0236] Investigators during electrodiagnostic testing were blinded to the treatment groups. There was no statistical difference between AAV1.NTF3.CMV (high dose, HD) and AAV1.NTF3.tMCK (high dose, HD) for CMAP, favoring the use of the muscle-specific tMCK promoter. This was further supported by NT-3 levels in an ELISA assay, where significant differences in NT-3 levels were observed for the highest and mid-dose vectors for both the promoter and the control.

[0237] The complete disclosures of all patents, patent applications, and publications, as well as electronically available materials cited herein, are incorporated by reference. The above detailed description and examples are given for clarity of understanding only. No unnecessary limitations should be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the scope of the invention as defined by the claims.

[0238] Example 3 Construction of NT-3-expressing AAV construct The design of a serotype 1 self-complementary AAV viral vector containing NT-3 cDNA under the tMCK or CMV promoter has been previously described in Sahenk et al., Mol Ther, 22(3):511-521 (2014), which is incorporated herein by reference in its entirety. Aliquots of virus were kept at -80°C until use. Blood samples were collected from treated and untreated mice by intraocular bleeding under anesthesia at 6 and 16 weeks after injection, and serum was assayed for NT-3 levels using a capture ELISA. This construct is referred to herein as scAAV1.tMCK.NTF3.

[0239] The tMCK promoter / enhancer sequence was used to drive muscle-specific gene expression and consisted of the muscle creatine kinase promoter with an additional enhancer element (enh358MCK, 584-bp) fused to it. A triple tandem of MCK enhancers (206-bp) was ligated to the 87-bp basal promoter in the tMCK promoter / enhancer.

[0240] The scAAV1.tMCK.NTF3 drug product was produced by three-plasmid DNA transfection of human HEK293 Master Cell Bank cells with (i) the pAAV.tMCK.NTF3-vector plasmid (see Figure 7), (ii) an AAV1 helper plasmid called R88 / C1 containing the AAV rep2 and Cap1 wild-type genes, and (iii) a helper adenovirus plasmid.

[0241] A schematic diagram of the plasmid with its molecular structure and open reading frame is shown in Figure 7. The AAV vector genome derived from the pAAV.tMCK.NTF3 plasmid is a self-complementary DNA genome containing a human NTF3 cDNA expression cassette flanked by AAV2 inverted terminal repeats (ITRs). It is this sequence that is packaged into AAV1 virions. Plasmid pAAV.tMCK.NTF3 was constructed by inserting the tMCK expression cassette and placing the NTF3 gene sequence into the AAV cloning vector psub201. The human NTF3 gene is expressed from the mouse triple tandem MCK promoter, which is a modification of the previously described CK6 promoter and contains a triplicate E box sequence. The SV40 polyadenylation signal is used for efficient transcription termination. The cassette also contains a chimeric intron for increased gene expression, consisting of a 5' donor site from the first intron of the human β-globin gene, a branch point, and a 3' splice acceptor site from the intron between the leader and body of the immunoglobulin gene heavy chain variable region. The NTF3 expression cassette has a consensus Kozak sequence immediately preceding the ATG start and a 200-bp SV40 polyA signal for efficient mRNA termination. The NTF3 cDNA is included in its entirety (NCBI Reference Sequence: NM_001102654). The only viral sequence included in this vector is the AAV2 inverted terminal repeat, which is required for both viral DNA replication and packaging. The AAV ITRs are nearly identical in both sequences but in opposite orientation. The "left" (mutated) ITR has a terminal resolution site deleted to allow genomic hairpin formation. The identity of all DNA plasmid elements is confirmed by DNA plasmid sequencing of the plasmid source stock.

[0242] Shown in Table 4 are the base pair positions of the relevant molecular structures within the AAV vector DNA plasmid of SEQ ID NO:11.

[0243] [Table 4]

[0244] Example 4 Phase I intramuscular study In the initial Phase I intramuscular (IM) safety study, sustained NT-3 transgene expression is the proposed outcome measure in this first stage, which is important for several reasons. True assessment of toxicity depends on demonstration of gene expression. If muscle is not transduced, toxicity (adverse effects) studies will be more difficult to interpret (lack of transduction versus reduced gene expression). The study provides an opportunity to clearly recognize transgene expression and establish methods to distinguish it from endogenous gene expression.

[0245] This clinical trial is an open-label, single-injection, ascending-dose study in which scAAV1.tMCK.NTF3 will be administered by intramuscular injection into the gastrocnemius and tibialis anterior muscles of both legs of CMT1A subjects with a PMP22 gene duplication. Nine adult CMT1A patients aged 18 years or older will participate in one of two cohorts of this study. The first three subjects will receive the lowest effective dose (2.0 x 10) bilaterally distributed between both legs in Cohort 1. 12 vg / kg. An additional 6 subjects will be enrolled in Cohort 2 at a 3-fold dose escalation (6.0 x 10 12Patients will participate at a dose of 100 mg / kg (vg / kg). Post-gene transfer monitoring will include follow-up visits at 7, 14, 30, 60, 90, 120 days, and at 3-month intervals for the remainder of the two-year study. Safety is the primary endpoint of this clinical gene transfer study. Stopping criteria are based on the occurrence of unacceptable toxicity, defined as any Grade II ocular or systemic toxicity that has not resolved after two weeks or any Grade III or higher toxicity. A secondary endpoint is efficacy, defined as the cessation of decline in performance as measured by the CMT Pediatric Scale (CMTPedS) at two years after gene transfer. The CMTPedS is an 11-item scale consisting of the Functional Dexterity Test, the Nine-Hole Peg Test (9HPT), handgrip, plantarflexion, and dorsiflexion strength using a manual myometer, pinprick and vibration sensation, the Bruyninks-Oserecky balance test, gait assessment, the long jump, and the 6-minute walk test (6MWT). Outcome measures include the 100-meter test (100M), peroneal and ulnar CMAP amplitude and sensory and motor conduction velocities, a sensory test modified to increase pinprick sensitivity, touch and vibration assessment, visual analog scales for pain and fatigue, the Short Form Health Survey (SF-36) as a quality of life measure, and circulating NT-3 levels.

[0246] Patients participating in the study will be of any racial, ethnic, or gender background. Criteria for this disease will be defined and will follow the guidelines previously established by Shy et al. (Neurology 64:1209-1214, 2001 and Neurology 70:378-383, 2008).

[0247] The study inclusion criteria were as follows: Adults (over 18 years of age) diagnosed with CMT1A · Must demonstrate a 1.5 Mb duplication at 17p11.2 containing the peripheral myelin protein 22 (PMP22) gene. Men and women of any ethnic or racial group Must demonstrate ankle dorsiflexion muscle weakness (should have full ROM against gravity, but cannot maintain full dorsiflexion against gravity or be unable to stand on tiptoes for more than 3 seconds (Northstar criteria)) Abnormal nerve conduction velocity Ability to cooperate with clinical evaluation and repeated nerve conduction studies Sexually active subjects must be willing to use reliable contraception for the duration of the study.

[0248] The study exclusion criteria were as follows: Active viral infection based on clinical observation or serological evidence of HIV, or hepatitis A, B, or C infection Ongoing immunosuppressive therapy or immunosuppressive therapy within 6 months of study initiation (e.g., corticosteroids, cyclosporine, tacrolimus, methotrexate, cyclophosphamide, intravenous immunoglobulin) Persistent leukopenia or leukocytosis (WBC ≤ 3.5K / μL or ≥ 20.0K / μL) or absolute neutrophil count < 1.5K / μL Subjects with an AAV1-binding antibody titer of 1:50 or greater as determined by ELISA immunoassay In the opinion of the laboratory principal investigator (PI), unnecessary risks of gene transfer may arise, and the risk of concurrent illness or the need for long-term drug therapy may be reduced. Ankle contractures or surgery that prevent proper muscle testing - Pregnant, breastfeeding, or planning to become pregnant Other causes of neuropathy Limb surgery within the past 6 months

[0249] [Table 5]

[0250] Pre-injection baseline measurements (days -30 to -1) After obtaining written informed consent and completing the hospital registration procedure, the following baseline medical procedures and measurements will be performed. Medical history Concomitant medication intake Physical examination ·Chest X-ray Echocardiogram EKG Hematology blood labs (CBC) Coagulation parameters: platelets, PT / INR, PTT Clinical Chemistry Blood Laboratory Tests: Bilirubin, Blood Urea Nitrogen (BUN), GGT, Alkaline phosphatase, alpha-fetoprotein (AFP), creatinine, amylase, serum protein electrophoresis, electrolytes, glucose, creatine kinase Urine tests ·Virus screening (hepatitis and HIV) Pregnancy test (women of childbearing age only) Hematology: Neutralizing antibodies (AAV1) and ELISpot (NT-3 and AAV1) Serum for ELISA (circulating NT-3) Effectiveness measurement Survey and measurement ·Photos of the injection site

[0251] Prednisolone administration before injection Prior to gene transfer, each patient will receive oral prednisone 1 mg / kg / day, up to a maximum of 60 mg / day, followed by a second dose on the day of gene transfer. Subjects will receive prednisone 24 and 48 hours after gene transfer for a total of four doses.

[0252] Protocol for gene transfer Self-complementary AAV1 carrying the human NTF3 gene under the control of the tMCK promoter (scAAV1.tMCK.NTF3) is administered in a single bilateral intramuscular injection into the medial and lateral heads of the gastrocnemius and tibialis anterior (TA) muscles.

[0253] Gene transfer procedure The gene transfer procedure is as follows: The gene transfer injection procedure is performed under sterile conditions. The vector is delivered without diluent, approximately 10 mL to 28 mL total per patient (divided into three muscles, 5 to 14 mL per limb). The vector will be delivered to the procedure room in a pre-labeled syringe sealed in a double leak-proof bag, which will be transported in a designated, labeled cooler. A total of 5-14 mL of vector is administered into each head and lateral head of the gastrocnemius and TA muscles in a total of 3-6 injections per muscle (each injection volume is 0.5-1.0 mL). See schematic diagrams of injection sites in Figures 8A and 8B. · The injection is made at least 0.25 cm below the fascia and the injection is made along the longitudinal axis of the muscle guided by ultrasound. · Each injection should be spaced approximately 1.5cm apart.

[0254] Hospitalized patient monitoring After the gene transfer injection, the subject will return to their assigned inpatient bed while continuing to closely monitor their vital signs and respiratory function. Vital signs will be monitored approximately every hour for the first four hours, then every four hours until discharge. Safety will be assessed by physical examination and laboratory evaluation. The subject will receive a third dose of oral prednisone (day 1), and a fourth and final dose of prednisone will be given the next day (48 hours after injection, day 2). Patients will be discharged approximately 48 hours after gene transfer (if no side effects are observed).

[0255] Outpatient monitoring After discharge, patients will return for follow-up visits at 7, 14, 30, 60, 90, 120 days, and at 3-month intervals for the remainder of the study, spanning two years after gene transfer. Blood samples obtained at all visits will be evaluated for NT-3 protein expression, as demonstrated using anti-NT-3 antibodies in a serum ELISA. Additionally, patients will be tested for efficacy and research measures at visits 7, 9, 11, 13, and 14. Serum ELISA is a direct measure of functional gene expression, with the secondary outcome measure demonstrating efficacy of the circulating transgene.

[0256] Primary endpoint: Safety is the primary endpoint of this clinical gene transfer study, which will be assessed based on the occurrence of unacceptable toxicity, defined as the occurrence of any single grade III or higher unexpected treatment-related toxicity.

[0257] Safety measurements Safety will be measured in each study by collecting height and weight, vital signs, physical examination and systems review, and a series of hematological laboratory tests. Laboratory tests include CBC, platelets, blood urea nitrogen (BUN), GGT, bilirubin, alkaline phosphatase, alpha-fetoprotein, creatinine, amylase, serum protein electrophoresis, electrolytes, glucose, PT / INR and PTT, CK, and urinalysis. Immunology will consist of ELISA for the detection of neutralizing antibodies to AAV1 and NT-3, and ELISpot for the detection of T-cell responses to AAV1 and NT-3. All adverse events will be recorded and evaluated for relevance to gene transfer.

[0258] Secondary endpoints A secondary endpoint of the clinical gene transfer trial is efficacy, defined as cessation of decline in capacity as measured by the CMT Pediatric Scale (CMTPedS) at 2 years after gene transfer.

[0259] Upper and lower limb muscle strength testing is performed using a hand-held dynamometer for distal movement of the upper limbs (handgrip) and legs (foot dorsiflexion). The test limbs are immobilized for testing, which significantly improves the reliability of isometric foot contraction measurements (29). Upper limb function is measured by the 9-hole peg test (9HPT). Lower limb function is measured by the timed 10-meter sprint / walk (T10MW) test.

[0260] Test result measurement Test outcomes included the 100-meter measurement test (100M), peroneal and ulnar CMAP amplitude and sensory and motor conduction velocity, sensory testing modified to increase sensitivity to pinprick, touch testing and vibration assessment, visual analog scales for pain and fatigue, the Short Form Health Survey (SF-36) as a measure of quality of life, and circulating NT-3 levels.

[0261] Electrophysiological testing includes measurements of ulnar sensory nerve amplitude and compound muscle action potential (CMAP) amplitude of the ulnar nerve (recorded from the abductor digiti minimi muscle) and peroneal nerve (recorded from the tibialis anterior muscle), as well as sensory and motor conduction velocities. Peroneal CMAP amplitude from the tibialis anterior muscle has been shown to be a useful outcome measure for clinical trials in patients with CMT1A (30); for upper limb motor symptoms, ulnar CMAP amplitude was found to be the most informative parameter (31). Foot and hand temperatures are maintained at 32–34°C during these testing procedures. Visual analog scales are used to measure pain and fatigue (32). Short-Term Health Survey (Short Term Health Survey) is also used. The SF-36 (FaceFace Health Survey) will be used as a quality of life record to monitor and compare disease burden before and after treatment.

[0262] Patients are rewarded for an optional fascicular sural nerve biopsy. If selected, biopsies are obtained before the start of treatment (from the left sural region) and at the end of the study (from the right sural region). Biopsies are performed as outpatient visits under local anesthesia. Tissue is processed and examined to assess the effect of NT-3 on myelinated fiber regeneration. The proximal end of the incision (2.5 cm long) is placed exactly 10 cm above the Achilles tendon to match the pre- and post-treatment material levels to the entire length of the nerve (8).

[0263] statistical analysis Safety is the primary endpoint of this clinical gene transfer study. It is assessed based on the occurrence of unacceptable toxicity, defined as the occurrence of any one grade III or higher unexpected treatment-related toxicity. Safety is measured in each study by collecting height and weight, vital signs, physical examination and systems review, and a series of hematological laboratory tests. Laboratory tests include CBC, platelets, blood urea nitrogen (BUN), GGT, bilirubin, alkaline phosphatase, alpha-fetoprotein, creatinine, amylase, serum protein electrophoresis, electrolytes, B12, glucose, PT / INR and PTT, CK, and urinalysis. Immunology tests consist of ELISA for the detection of neutralizing antibodies against AAV1 and NT-3, and ELISpot for the detection of T-cell responses to AAV1 and NT-3. All adverse events are recorded and evaluated for their relevance to gene transfer.

[0264] The main secondary outcome measure is defined as a lack of reduction in disease severity on the CMT Disease Pediatric Scale score (CMTPedS). The CMTPedS computerized scoring system uses z-scores from a reference sample to determine individual scores based on the number of standard deviations by which a patient's performance differs from that of a healthy population. The CMTPedS was scored at a fixed age of 20 years for all patients throughout the study, which is necessary for two reasons. First, the original CMTPedS scale relies on normative values ​​for individual scale items in calculating final scores, but normative values ​​do not exist for individuals older than 20 years. Second, comparison of children's scores with healthy counterparts is essential for children who are still developing, but it has the limitation of imposing functional decline as children reach their birthdays. Even though children's raw scores on the test do not change, the scoring criteria are designed to become more stringent as they age, reflecting the expected improvement in motor skills typically seen in healthy children. However, in clinical trials of degenerative diseases, actual cessation of progression allows for the continuation of a successful trial. Keeping baseline control data consistent throughout the trial allows for the use of a validated scoring system, which is incorporated into CMTPedS. It also allows for the observation of actual changes in assessments such that cessation of progression can be detected.

[0265] The proportion of patients whose CMTPedS scores improved or remained the same over a 2-year period was estimated using a binomial test with 95% confidence intervals. For study purposes, the proportion of patients with no decline in each of the 11 CMTPedS items was estimated separately, as well as for the 100-meter test (100M), CMAP amplitude, visual analog scale (VAS) for pain intensity, scores on the Short Form Health Survey (SF-36), and circulating NT-3 levels. Furthermore, Pearson or Spearman correlation coefficients were used at each measurement time point to assess the association between circulating NT-3 levels and each outcome measure. Because this study is in its preliminary stages, p values ​​are not adjusted for multiple comparisons. However, a sensitivity analysis was performed to show which associations remained statistically significant after adjustment, based on the Bonferroni-Holm step-down procedure.

[0266] Based on longitudinal natural history data (31, 32), a successful secondary outcome measure was defined as cessation of the rate of decline in standardized composite scores of handgrip and ankle dorsiflexion strength, time to complete the 9-hole peg test, and time to walk / run 10 meters.

[0267] Example 5 Toxicological Testing The purpose of this study was to evaluate the effect of wild-type (C57BL / 6) and Trembler (Tr) mice on the gastrocnemius muscle after a single intramuscular injection. J The objective of this study was to evaluate the safety of a self-complementary adeno-associated virus (scAAV) vector expressing human neurotrophin factor 3 (NTF3) cDNA under the control of the muscle-specific tMCK promoter (scAAV1.tMCK.NTF3) in trembler mice. The results of this report demonstrate that a single intramuscular injection in wild-type and trembler mice is safe and well-tolerated up to 48 weeks after injection.

[0268] Test animals Wild-type C57BL / 6 (negative control) and Tr J(Test article) Mice were given either vehicle (0.9% sterile saline) or 1 x 10 13 The mice were intramuscularly injected with either scAAV1.tMCK.NTF3 at 1000µg / kg. Carrier mice of this strain are available from Jackson Laboratories (stock no. 000664) or Charles River (stock no. 027). J Mice are available from Jackson Laboratories (stock number 002504).

[0269] Trembler mice were 8-10 weeks old at the time of injection. Due to animal availability and known breeding issues of the animal strain, a subset of animals was treated at 12 weeks of age.

[0270] A total of 88 animals (44 males / 44 females) were included in the study. 44 animals (22 C57BL / 6, 22 TrJ) were treated with saline control and 44 animals (22 C57BL / 6, 22 TrJ) were treated with the scAAV1.tMCK.NTF3 test article. Of these numbers, half of the animals from each cohort were euthanized after 24 weeks of treatment, and the remaining animals were euthanized after 48 weeks of treatment. Animals were uniquely identified by ear tags.

[0271] Test Design To evaluate the safety of the scAAV1.tMCK.NTF3 test article, 4-6 week old wild-type C57BL / 6 and 8-12 week old Tr mice were used. J Mice were administered either vehicle (0.9% sterile saline) or 1 x 10 mAb in a total volume of 50 μl by a single intramuscular (IM) injection in the left gastrocnemius muscle. 13 Animals were injected with either scAAV1.tMCK.NTF3 at 1000µg / kg or 100µg / kg of scAAV1.tMCK.NTF3. Animals were observed daily throughout the course of the study for overall health and morbidity. Mortality checks were performed twice daily. Body weights were measured every two weeks. Functional testing for hypersensitivity (hot plate and acoustic startle tests) was performed every 8 weeks. Behavioral testing (rotarod and wire hanging) was performed every two weeks.

[0272] Thermal sensitivity was tested using the rodent hot plate test, starting 2 weeks prior to injection, followed by day 0 and every 8 weeks. To perform the test, animals were placed on a plate surface maintained at a temperature of 55°C, and movement was limited to 100 cm using a 15 cm high Plexiglas wall surrounding the plate surface. 2 The area of ​​the heated surface was restricted to 100°C. A timer was started when the animal was placed on the heated surface, and the reaction latency was recorded to 0.1 seconds by a stopwatch. The following activities were considered as responses to the thermal stimulus: licking or flicking / fluttering of the hind paws, as well as jumping and attempting to escape, were also acceptable responses. If this response was observed, the mouse was immediately removed.

[0273] Acoustic sensitivity was tested 2 weeks before injection, followed by day 0 (baseline), at peak serum NT-3 levels (weeks 8-12), and every 8 weeks thereafter. All animals were tested for acoustic hypersensitivity using the acoustic startle test (AST). Startle reactivity was measured in a single startle chamber (startle chamber) as previously described in Beigneux et al., Behavioral Brain Res. 171:295-302, 2006. Measurements were performed using a SR-Lab (San Diego Instruments, San Diego, CA) ion exchange chamber.

[0274] The chamber consisted of a transparent, non-restrictive Plexiglas cylinder resting on a platform inside a ventilated chamber. A high-frequency loudspeaker inside the chamber generated both a continuous background noise of 65 dB and various sound stimuli. Vibrations of the Plexiglas cylinder caused by the animal's whole-body startle response were converted into an analog signal by a piezoelectric unit attached to the platform. The signal was then digitized and stored by a computer. 65 readings were taken at 1 ms intervals starting from stimulus onset, and the mean amplitude (V avgAcoustic startle responses were determined using a 65 dB background noise level. A 65 dB background noise level was presented for a 5-minute adaptation period and continued throughout the test session. All prepulse inhibition (PPI) test sessions consisted of startle trials (pulse alone), prepulse trials (prepulse + pulse), and no-stimulus trials (no-stimulus). Pulse-alone trials consisted of a 40-ms 120 dB pulse of broadband noise. PPI was measured by prepulse + pulse trials, which consisted of a 20-ms noise prepulse, a 100-ms delay, and then a 40-ms 120 dB startle pulse. Acoustic prepulse intensities were 69, 73, and 81 dB. No-stimulus trials consisted of background noise only. Test sessions began and ended with five presentations of pulse-alone trials; during which each trial type was presented 10 times in a pseudorandom order. The average time between trials was 15 seconds (range 12-30 seconds).

[0275] In the rotarod analysis, at least one week of training was required for all subjects to learn the task equally well. In the accelerating rotation protocol, animals were placed on the rod, which accelerated to 5 rpm and then ascended at 5 rpm / sec. Animals were given three trials per session, which were averaged.

[0276] In the wire hanging test, animals were placed by all four paws on a 2 mm diameter metal wire held horizontally 35 cm above a thick layer of soft bedding. The length of time it took for the mouse to fall off the wire was recorded, and after each fall, the mouse was allowed a 1-minute recovery period. Each test session consisted of three trials, from which scores were averaged.

[0277] At 22 and 46 weeks post-injection (2 weeks before euthanasia), blood was collected from the retro-orbital sinus for hematology tests (red blood cell count, hematocrit, hemoglobin, white blood cell count (total and differential), mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, mean corpuscular volume, mean platelet volume, platelet count, reticulocyte count). Clinical chemistry was analyzed for the following parameters: alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase, bilirubin (total and direct), blood urea nitrogen, creatinine, creatine kinase, glucose, and total protein.

[0278] At 24 and 48 weeks after injection, blood was collected by cardiac puncture, and serum was used for immune testing. Serum samples were collected for anti-AAV1 and anti-NT-3 antibody titers for all animals in each cohort (regardless of treatment or sex). Serum samples were further used for ELISA assays of circulating NT-3 levels.

[0279] At 24 and 48 weeks after injection, Tr J The following tissues from the animals were collected for histopathological analysis: gonads, brain, spleen, kidney, jejunum, colon, pancreas, heart, lung, stomach, liver, inguinal lymph nodes, spinal cord, (right and left) gastrocnemius muscles, and gross lesions (if present). Tissues were fixed in 10% neutral buffered formalin, sectioned, and stained with hematoxylin and eosin. Histological analysis was performed by SNBL USA.

[0280] At 24 and 48 weeks after injection, tissues for in-house histopathological examination were collected from the Tr JSix animals per cohort (3 males, 3 females) were collected, excluding the control cohort (n = 5, 3 males, 2 females). Mice were sacrificed and transcardially perfused with 4% paraformaldehyde in phosphate buffer (0.1 M, pH 7.4). The lumbar spinal cord, dorsal root ganglia (DRG), and entire sciatic nerve segments (from the sciatic notch to the popliteal fossa) were dissected. The proximal half of the sciatic nerve segments and one lumbar DRG were transferred to glutaraldehyde fixative along their in situ length and processed for plastic embedding and 1 μm-thick sectioning. The remaining parts of the sciatic nerve tissue and lumbar DRG were cryoprotected in 30% sucrose, frozen in isopentane cooled in liquid nitrogen, and cut into 12 µm sections for immunohistochemistry for the detection of CGRP positivity in DRG neurons and axons (spinal cord). The brains from TrJ mice were placed in 10% neutral buffered formalin.

[0281] [Table 6]

[0282] Animal administration, observation and analysis Indicated animals were administered a single intramuscular (IM) injection of 50 μL into the left gastrocnemius muscle on day 0. Animals were anesthetized with isoflurane inhalation for a minimum of 15 minutes to ensure accurate dosing. Doses were administered by direct injection into the left gastrocnemius muscle. Care was taken to ensure the entire vector dose was accurately placed into the muscle. After dosing, animals were observed until ambulatory and then returned to their cages. Each animal was observed daily for the entire duration of the study. Body weights were measured every two weeks. Mortality checks were performed twice daily.

[0283] At the appropriate age, mice were overdosed with a ketamine / xylazine mixture (200 mg / kg / 20 mg / kg). Blood was collected by cardiac puncture. Tissues were then harvested and sent for analysis.

[0284] Body weight, mouse hematology, and clinical chemistry are plotted for each cohort at each time point. ELISA assays for anti-AAV1 circulating antibodies, anti-NT-3 circulating antibodies, and circulating NT-3 levels were performed for all endpoints. Hypersensitivity functional tests using a rodent hot plate and an acoustic startle apparatus were performed every 8 weeks for all animals. Behavioral tests were performed every 2 weeks for all animals using the rotarod and wire hanging tests. Morphological histopathology of organs was performed for all animals. In-house histopathology of the lumbar spinal cord and DRG was performed on Tr1 mice containing 5 animals (3 males, 2 females). J Excluding cohorts, six mice (three males, three females) were included per cohort. Histopathological evaluation included immunocytochemical distribution of CGRP positivity (in lumbar DRG neurons and spinal cord) and plastic embedding of sciatic nerves and DRG neurons for analysis of pathological changes.

[0285] result Morbidity and mortality All mice survived the injection procedure and the initial observation period passed without any signs of distress. J Animals died due to failure of the water system. No test article-related mortality occurred.

[0286] body weight The weights of all mice in each group were measured every two weeks throughout the course of the study. All treatment groups maintained consistent weight gain throughout the study.

[0287] In C57BL / 6 male animals, there were no significant differences between test article-injected animals and controls. In female animals, body weight was slightly higher in control animals compared to test article-injected animals. J In male animals, the saline-treated cohort was slightly heavier than the NT-3-treated cohort. J For mice, there was no significant difference in body weight.

[0288] Hematology and Clinical Chemistry There were no test article-related changes in hematology parameters at 22 and 46 weeks or in serum chemistry at 24 and 48 weeks.

[0289] ELISA assay ELISA assays for anti-AAV1 antibodies, anti-NT-3 antibodies, and circulating NT-3 were performed using serum samples collected during necropsies at 24 and 48 weeks.

[0290] To measure circulating anti-AAV1 antibodies, serum samples were collected 24 and 48 weeks after vector administration, and antibody titers were determined by enzyme-linked immunosorbent assay (ELISA) (see Table 7 below). Animals treated with scAAV1.tMCK.NTF3 had increased circulating antibodies to the AAV1 capsid. Antibody titers were similar in both males and females at both time points. No positive titers (>1:50) were detected in animals treated with the saline control.

[0291] [Table 7]

[0292] To measure circulating anti-NT-3 antibodies, serum samples were collected 24 and 48 weeks after vector administration, and antibody titers to NT-3 were determined by enzyme-linked immunosorbent assay (ELISA). All mice had antibody titers of less than 1:50 (considered negative) at 24 and 48 weeks after vector administration. See Table 8.

[0293] [Table 8]

[0294] Circulating NT-3 levels were measured by a standard binding ELISA assay in TrPs collected at 24 and 48 weeks after vector administration. JIntramuscular injection of the scAAV1.tMCK.NTF3 vector resulted in Tr1 at 24 and 48 weeks after vector administration. J This resulted in robust expression and secretion of NT-3 in mice. Table 9 shows the Tr expression and secretion of NT-3 from the saline and vector-injected groups. J Figure 1 shows the mean and standard deviation of serum NT-3 levels at 24 and 48 weeks after injection in rats. Gender did not affect circulating NT-3 levels.

[0295] [Table 9]

[0296] Hypersensitivity Testing Thermal sensitivity testing was performed at baseline (before treatment with any of the test articles in control saline) and every 8 weeks up to 48 weeks after injection in C57BL / 6 and TrJ mice. There were no differences between control and vector-injected mice in the C57BL / 6 and TrJ cohorts. There were no significant differences in withdrawal latency between male and female mice in each cohort.

[0297] The results of hearing sensitivity measurements performed in the acoustic startle test demonstrated that AST was clearly inhibited by prepulses at all intensities (69, 73, and 81 dB), and the level of inhibition was dependent on the prepulse intensity in both wild-type and TrJ mice. Treatment with the test substance did not alter PPI responses in C57BL / 6 wild-type mice. %-PPI was detectably lower in TrJ animals treated with the control substance and improved toward normalization in TrJ animals treated with the test substance (NT-3). TrJ animals treated with NT-3 showed significantly lower %-PPI than controls. J This improvement in animals was seen primarily in male animals.

[0298] Behavioral testing In the rotarod assessment, animals in the C57BL / 6 cohort showed no differences in rotarod functional testing regardless of treatment. Animals in the TrJ NT-3-treated cohort showed significant improvements in rotarod performance compared to control TrJ animals starting at 16 weeks of treatment, which persisted until endpoint.

[0299] In the wire-hanging assessment, animals in the C57BL / 6 cohort showed no differences in wire-hanging functional testing regardless of treatment. Animals in the TrJ NT-3-treated cohort showed significant improvements in wire-hanging ability compared to control TrJ animals starting at 28 weeks of treatment, which persisted until endpoint.

[0300] histopathology Morpho-histopathology was performed. A list of organs and tissues analyzed is shown below in Table 10.

[0301] [Table 10]

[0302] After euthanasia, the lumbar spinal cord, dorsal root ganglia (DRG), and entire sciatic nerve (from the sciatic notch to the popliteal fossa) were removed and processed for histopathological evaluation. A list of individual animals included for in-house histopathology is shown in Table 11.

[0303] [Table 11]

[0304] Immunocytochemical analysis of CGRP distribution For immunocytochemical analysis of CGRP distribution, sections from the lumbar spinal cord were examined at 24 and 48 weeks after injection. This analysis showed that CGRP distribution was significantly higher in the Tr rats than in the control rats, regardless of the treatment. J and did not show increased CGRP reactivity with NT-3 in C57BL / 6 animals.

[0305] Plastic-embedded sections were analyzed for pathological changes. Plastic-embedded left sciatic nerve and left lumbar DRG neurons examined at 24 and 48 weeks after injection showed no pathological changes in C57BL / 6 animals. Age-related changes, such as myelin corrugation / infolding and outfolding, suggestive of axonal atrophy, were seen in both treatment cohorts of C57BL / 6 animals at 48 weeks after treatment. Saline-treated Tr J In animals, there was a marked loss of myelinated fibers and many hypomyelinated or naked axons. J The animals exhibit small myelinated fibers, a visible increase in myelin thickness, and a decrease in naked axons. There were no adverse effects of NT-3 treatment in TrJ mice, as evidenced by the lack of axonal arborization within the DRG.

[0306] Generally, C57BL / 6 and Tr J The absence of treatment-related adverse findings throughout the study in both animals indicates that the treatment was well tolerated up to 48 weeks after injection.

[0307] explanation To evaluate the safety of scAAV1.tMCK.NTF3 delivered by a single intramuscular injection, a toxicity study was designed involving a total of 88 animals (44 males / 44 females). Forty-four animals (22 C57BL / 6, 22 TrJ) were treated with 0.9% sterile saline control and 44 animals (22 C57BL / 6, 22 TrJ) were treated with 1 × 10 13 Animals were treated with the scAAV1.tMCK.NTF3 test article at a concentration of 0.05 mg / kg. Half of the animals from each cohort were euthanized after 24 weeks of treatment, and the remaining animals were euthanized after 48 weeks of treatment.

[0308] The data in this report demonstrate that treatment of animals with doses 10-fold greater than the proposed clinical dose did not result in the development of test-article-related adverse safety effects. Throughout the study, animals were observed daily for general health and morbidity, and mortality checks were performed twice daily. Animals were weighed every two weeks. Functional testing for hypersensitivity (thermal and acoustic) was performed every eight weeks. Behavioral testing was performed every two weeks. All animals survived the injection procedure, and the initial observation period passed without signs of distress. There were four deaths of TrJ animals unrelated to test article administration due to a water system failure. No other deaths were recorded in this study.

[0309] After necropsy, hematology and clinical chemistry were measured and showed no test article-related differences. Anti-AAV1 serum ELISA was performed and showed the expected increase in circulating antibodies in test article-treated animals, which was not sex-specific. Anti-NT-3 serum ELISA was performed on all animals and showed negative antibody titers at all time points. Circulating NT-3 levels were also measured for all treatment groups and were increased only in test article-treated animals. There was no sex-specific effect on circulating NT-3 levels. Thermal hypersensitivity testing was performed on C57BL / 6 or Tr mice. J Hearing sensitivity measurements showed no test article-related changes in C57BL / 6 animals, but no significant changes in the Tr mice treated with the test article. J With improvements to normalization in animals treated with control substances, J This improvement was primarily due to the J Rotarod assessment of motor control in C57BL / 6 animals showed no differences associated with treatment with the test article. Test article-treated TrJ animals were compared with control TrJ animals starting at 16 weeks of treatment. J Similarly, wire hanging assessment showed no differences in the C57BL / 6 cohort related to the test article. JAnimals showed significant improvement in wire hanging time starting at 28 weeks of treatment, which was sustained until endpoint.

[0310] Tissues and organs harvested from all cohorts were evaluated at 24 and 48 weeks of treatment. In summary, minimal infiltration of mononuclear cells was observed in the test article-injected left gastrocnemius muscles of 3 / 4 Group 1 males and 4 / 5 Group 1 females from the 24-week necropsy. However, similar infiltration was also observed in the uninjected gastrocnemius muscles of the remaining Group 1 female (animal 3942). This change was not observed in either the saline-injected left gastrocnemius or the uninjected right gastrocnemius muscles of any Group 2 (control) animals from the 24-week necropsy. At the 48-week necropsy, minimal infiltration of mononuclear cells was again observed in the test article-injected left gastrocnemius muscles of 2 / 5 Group 1 males and 1 / 6 Group 1 females. All other microscopic findings were considered randomly distributed across control and treated animals, background findings for the species, or incident to test article administration.

[0311] In-house histology was performed on lumbar spinal cord sections, dorsal root ganglia, and sciatic nerves from a subset of animals (3 males, 3 females per group). Examination of cross sections from lumbar spinal cord sections showed no increase in CGRP activity with NT-3 in animals, regardless of treatment. Pathological evaluation of plastic-embedded sciatic nerve and lumbar DRG neurons showed no changes beyond age-related differences in C57BL / 6 animals. J In animals, treatment with the test article resulted in amelioration of pathology as evidenced by an increase in small myelinated fibers, increased myelin thickness, and a decrease in naked axons. The lack of axonal branching in the DRG further supports the safety of the test article.

[0312] Generally, the collected data presented in this study is 1 x 10 13The test article scAAV1.tMCK.NTF3 injected directly into the gastrocnemius muscle by a single intramuscular injection at a dose of 1000 mg / kg significantly inhibited the growth of male and female C57BL / 6 wild-type and Tr mice, as evidenced by the multiplex assays shown above. J Both treatments were well tolerated in mice up to 48 weeks after injection.

[0313] References 1.Lupski JR, de Oca-Luna RM, Slaugenhaupt S, et al.DNA duplication associated with Charcot-Marie-Tooth disease type 1A.Cell 1991;66:219-232. 2. Saporta AS, Sottile SL, Miller LJ, Feely SM, Siskind CE, Shy ME. Charcot-Marie-Tooth disease subtypes and genetic testing strategies.Ann Neurol 2011;69:22-33. 3.Sahenk Z.Abnormal Schwann cell-axon interactions in CMT neuropathies.The effects of mutant Schwann cells on the axonal cytoskeleton and regeneration-associated myelination. Ann NY Acad Sci 1999;883:415-426. 4. Verhamme C, de Haan RJ, Vermeulen M, Baas F,de Visser M,van Schaik IN.Oral high dose ascorbic acid treatment for one year in young CMT1A patients:a randomized,do uble-blind,placebo-controlled phase II trial.BMC medicine 2009;7:70. 5.Micallef J,Attarian S,Dubourg O,et al.Effect of ascorbic acid in patients with Charcot-Marie-Tooth disease type 1A:a m ulticentre,randomised,double-blind,placebo-controlled trial.Lancet Neurol 2009;8:1103-1110. 6.Pareyson D,Reilly MM,Schenone A,et al.Ascorbic acid in Charcot-Marie-Tooth disease type 1A (CMT-TRIAAL and CMT-TRAUK): a double-blind randomised trial.Lancet Neurol 2011;10:320-328. 7.Lewis RA,McDermott MP,Herrmann DN,et al.High-dosage ascorbic acid treatment in Charcot-Marie-Tooth disease type 1A:res ults of a randomized,double-masked,controlled trial.JAMA neurology 2013;70:981-987. 8.Sahenk Z,Nagaraja HN,McCracken BS,et al.NT-3 promotes nerve regeneration and sensory improvement in CMT1A mouse models and in patients.Neurology 2005;65:681-689. 9.de Waegh SM,Brady ST.Local control of axonal properties by Schwann cells:neuro filaments and axonal transport in homologous and heterologous nerve grafts.J Neurosci Res 1991;30:201-212. 10.Yin X,Crawford TO,Griffin JW,et al.Myelin-associated glycoprotein is a myelin signal that modulates the caliber of myelinated axons.J Neurosci 1998;18:1953-1962. 11.de Waegh S,Brady ST.Altered slow axonal transport and regeneration in a myelin-deficient mutant mouse:the trembler as an in vivo model for Schwann cell-axon interactions.J Neurosci 1990;10:1855-1865. 12.de Waegh SM,Lee VM,Brady ST.Local modulation of neurofilament phosphorylation,axonal caliber,and slow axonal transport by myelinating Schwann cells.Cell 1992;68:451-463. 13.Adlkofer K,Martini R,Aguzzi A,Zielasek J,Toyka KV,Suter U.Hypermyelination and demyelinating peripheral neuropathy in Pmp22-deficient mice.Nat Genet 1995;11:274-280. 14.Adlkofer K,Frei R,Neuberg DH,Zielasek J,Toyka KV,Suter U.Heterozygous peripheral myelin protein 22-deficient mice are affected by a progressive demyelinating tomaculous neuropathy.J Neurosci 1997;17:4662-4671. 15.Anzini P,Neuberg DH,Schachner M,et al.Structural abnormalities and deficient maintenance of peripheral nerve myelin in mice lacking the gap junction protein connexin 32.J Neurosci 1997;17:4545-4551. 16.Bosio A,Bussow H,Adam J,Stoffel W.Galactosphingolipids and axono-glial interaction in myelin of the central nervous system.Cell Tissue Res 1998;292:199-210. 17.Martini R,Zielasek J,Toyka KV,Giese KP,Schachner M.Protein zero (P0)-deficient mice show myelin degeneration in peripheral nerves characteristic of inherited human neuropathies.Nat Genet 1995;11:281-286. 18.Sahenk Z,Chen L.Abnormalities in the axonal cytoskeleton induced by a connexin32 mutation in nerve xenografts.J Neurosci Res 1998;51:174-184. 19.Sahenk Z,Chen L,Freimer M.A novel PMP22 point mutation causing HNPP phenotype:studies on nerve xenografts.Neurology 1 998;51:702-707. 20.Sahenk Z,Chen L,Mendell JR.Effects of PMP22 duplication and deletions on the axonal cytoskeleton.Ann Neurol 1999;45:16-24. 21.Sanchez I,Hassinger L,Paskevich PA,Shine HD,Nixon RA.Oligodendroglia regulate the regional expansion of axon caliber and local accumulation of neurofilaments during development independently of myelin formation.J Neurosci 1996;16:5095-5105. 22.Sahenk Z,Serrano-Munuera C,Chen L,Kakabadze I,Najagara HN.Evidence for impaired axonal regeneration in PMP22 duplication:studies in nerve xenografts.J Periph er Nerv Syst 2003;8:116-127. 23.Ramer MS,Bishop T,Dockery P,et al.Neurotrophin-3-mediated regeneration and recovery of proprioception following dorsal rhizotomy.Molecular and cellular neurosciences 2002;19:239-249. 24.Helgren ME,Cliffer KD,Torrento K,et al.Neurotrophin-3 administration attenuates deficits of pyridoxine-induced large-fiber sensory neuropathy.J Neurosci 1997;17:372-382. 25.Mizisin AP,Calcutt NA,Tomlinson DR,Gallagher A,Fernyhough P.Neurotrophin-3 reverses nerve conduction velocity deficits in streptozotocin-diabetic rats.J Peripher Nerv Syst 1999;4:211-221. 26.Mizisin AP,Kalichman MW,Bache M,Dines KC,DiStefano PS.NT-3 attenuates functional and structural disorders in sensory nerves of galactose-fed rats.J Neuropathol Exp Neurol 1998;57:803-813. 27.Shy ME,Blake J,Krajewski K,et al.Reliability and validity of the CMT neuropathy score as a measure of disability.Neurology 2005;64:1209-1214. 28.Shy ME,Chen L,Swan ER,et al.Neuropathy progression in Charcot-Marie-Tooth disease type 1A.Neurology 2008;70:378-383. 29.Solari A,Laura M,Salsano E,Radice D,Pareyson D.Reliability of clinical outcome measures in Charcot-Marie-Tooth disease.Neuromuscul Disord 2008;18:19-26. 30.Komyathy K,Neal S,Feely S,et al.Anterior tibialis CMAP amplitude correlations with impairment in CMT1A.Muscle & nerve 2013;47:493-496. 31.Mannil M,Solari A,Leha A,et al.Selected items from the Charcot-Marie-Tooth (CMT) Neuropathy Score and secondary clinical outcome measures serve as sensitive clinical markers of disease severity in CMT1A patients.Neuromuscul Disord 2014;24:1003-1017. 32.Pareyson D,Schenone A,Fabrizi GM,et al.A multicenter,randomized,double-blind,placebo-controlled trial of long-term ascorbic acid treatment in Charcot-Marie-Tooth disease type 1A (CMT-TRIAAL):the st udy protocol [EudraCT no.:2006-000032-27 ].Pharmacological research 2006;54:436-441. 33.Piscosquito G,Reilly MM,Schenone A,et al.Responsiveness of clinical outcome measures in Charcot-Marie-Tooth disease.European journal of neurology :the offici al journal of the European Federation of Neurological Societies 2015;22:1556-1563.

[0314] While the present invention has been described with respect to particular embodiments, it is understood that variations and modifications will occur to those skilled in the art. Therefore, only such limitations should be placed on the invention as appear in the appended claims.

Claims

[Claim 1] The invention as described in the drawings.

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