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 of related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 574,828 filed on 20 October 2017, U.S. Provisional Patent Application No. 62 / 676687 filed on 25 May 2018, and U.S. Provisional Patent Application No. 62 / 741,335 filed on 4 October 2018, the disclosures of which are incorporated herein by reference in their entirety.

[0002] Disclosure of U.S. government interests This invention was made with government support under authorization numbers NS105986 and U01-NS066914 granted by the National Institutes of Health. The U.S. Government has certain rights to this invention.

[0003] Reference to sequence listings This application includes, as a separate part of the present disclosure, a computer-readable sequence listing (filename: 53122A_Seqlisting.txt; a 17,133-byte ASCII text file created on 18 October 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) polynucleotide. This disclosure provides rAAV and methods for using rAAV in NT-3 gene therapy to improve muscle strength, stimulate muscle growth, and treat neuropathies 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 undervalued characteristics. In addition to its well-recognized effects on peripheral nerve regeneration and Schwann cells (SCs), NT-3 possesses anti-inflammatory and immunomodulatory effects. Yang et al., Mel Titer, 22(2):440-450 (2014). It has also recently been demonstrated that NT-3 can mitigate spontaneous autoimmune peripheral polyneuropathy in a rodent model of chronic inflammatory demyelinating peripheral neuropathy occurring 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 encompasses five types of CMT, caused by four genes when mutated. This group includes the majority of people affected by CMT. While these genes are related to the myelin sheath surrounding the sclerotocytes (SCs) and axons, they interact in various ways, and therefore the phenotype is heterogeneous. CMT1A is the result of DNA duplication on chromosome 17p11, which includes the PMP22 gene, leading to the development of the classic CMT1 phenotype. Patients develop clinical signs before the age of 20 that cause significant disability requiring walking aids. In these patients, peripheral nerve regeneration is incomplete due to prolonged axonal transection and denervation that occur as part of chronic neuropathy. NT-3 is a trophic factor secreted by Schwann cells (SCs) that aids in nerve regeneration. The ability of denervated SCs to survive is crucial for nerve regeneration because SCs provide both growth factors and basement membranes, which are scaffolds that promote axonal growth. Prolonged denervation leads to a decrease in regenerative capacity associated with reduced expression of regenerative SC molecules (neurotrophic factors (NTFs) and their receptors), resulting in denervated sclerosing cell atrophy, destruction of the Büngner zone, 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 improves nerve regeneration with increased SC number, myelinated fiber density, and myelin thickness, but also increases muscle fiber diameter, as seen 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 phenotype of neuropathy can result in diverse changes in skeletal muscle, including a shift from fast-twitch type II fibers to slow-twitch type I fibers. In TrJ mice, the extensor digitorum longus, which is mainly composed of fast-twitch type fibers, has been found to have 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 has dramatically increased with age. Nicks et al., J Neuropathol Exp Neurol, 72(10):942-954 (2013). Interestingly, similar changes in slow-twitch muscle fiber production occur in humans and other mammals in parallel with aging. 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, which is produced by unequal crossover of homologous chromosomes (1). It is a disease that progresses slowly without known treatment. Symptoms almost always begin before the age of 20. Cavus pedis and hammertoe are present. Walking aids such as short leg braces are required. Less frequently, but in severe pediatric cases, wheelchair or ventilator dependence may occur. Typically, 90% of patients have a motor nerve conduction velocity (NCV) in the ulnar nerve of 16–35 m / s or less (2). Even if the genetic defect is mainly involved in Schwann cells (SCs), the electrophysiological clinical picture is length-dependent sensorimotor demyelinating neuropathy. This clinical presentation is significantly influenced by axonal degeneration resulting from impaired Schwann cell (SC)-axonal interactions (3).

[0009] Currently, there is no treatment for this condition. Ascorbic acid supplements have been highly touted as potentially helpful, but numerous studies have shown them to be ineffective. Both low-dose (1-2 g / day) (4-6) and high-dose therapy (3-4 g / day) have been found to be unhelpful (6, 7). Initially, a clinical trial of NT-3 demonstrated clinical efficacy 24 weeks after treatment, accompanied by an increase in the number of myelinated nerve fibers in sural nerve biopsies (8). J In mice, subcutaneous NT-3 therapy improved axonal regeneration and promoted myelin formation. However, the short half-life of NT-3 in the blood proved to be a major obstacle to continuous subcutaneous administration, and the 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 myopathy (such as muscular dystrophy), muscle wasting disorders (such as cachexia that may result from underlying conditions such as acquired immunodeficiency syndrome (AIDS), rheumatoid arthritis, cancer, chronic obstructive pulmonary disease (COPD), and cirrhosis), conditions of muscle atrophy or attenuation (such as sarcopenia, which may be a result of aging), weakness induced by prolonged non-use (such as paralysis, coma, prolonged bed rest, and ICU stay), surgery (such as artificial joint replacement), drug-induced myopathy, and rhabdomyolysis. The muscle pathology of these diseases and conditions is mediated, partially or entirely, by a combination of immune, inflammatory, and fibrotic responses. Drugs capable of blocking these responses and / or stimulating the regeneration of damaged tissue may be able to slow or halt the progression of disease in these disorders.

[0011] Adeno-associated virus (AAV) is a replication-deficient parvovirus whose single-stranded DNA genome is approximately 4.7 kb long and contains 145 nucleotide inverted terminal repeats (ITRs). Multiple serotypes of AAV exist. The nucleotide sequences of the AAV serotype genomes are publicly known. For example, the complete genome of AAV-1 is available under GenBank accession number NC_002077; the complete genome of AAV-2 is available under GenBank accession number NC_001401 and under Srivastava et al., J. Virol., 45:555-564 (1983); the complete genome of AAV-3 is available under GenBank accession number NC_1829; the complete genome of AAV-4 is available under GenBank accession number NC_001829; the AAV-5 genome is available under GenBank accession number AF085716; the complete genome of AAV-6 is available under GenBank accession number NC_001862; at least portions of the AAV-7 and AAV-8 genomes are available under GenBank accession numbers AX753246 and AX753249, respectively; and the AAV-9 genome is available under Gao et al. The AAV-10 genome is provided in al., J. Virol., 78:6381-6388 (2004); 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), capsid formation / packaging, and integration into host cell chromosomes are contained within the AAV ITR. Three AAV promoters (named p5, p19, and p40 due to their relative map locations) drive the expression of two AAV internal open reading frames encoding the rep and cap genes. Two rep promoters (p5 and p19) associated with the differential splicing of a single AAV intron (at nucleotides 2107 and 2227) generate four rep proteins (rep 78, rep 68, rep 52, and rep 40) from the rep gene. The rep proteins possess multiple enzymatic properties that ultimately contribute to the replication of the viral genome.The cap gene is expressed from the p40 promoter and encodes three capsid proteins: VP1, VP2, and VP3. Alternative splicing and non-consensus translation initiation sites are involved in the production 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 outlined in Muzyczka, Current Topics in Microbiology and Immunology, 158:97-129 (1992).

[0012] AAV possesses 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 non-cytotoxic, and natural infection in humans and other animals is subclinical and asymptomatic. Furthermore, AAV infects many mammalian cells and can target many different tissues in vivo. Additionally, AAV can gradually transduce dividing and non-dividing cells and, as a transcriptionally active nuclear episome (extrachromosomal factor), can essentially persist for the lifetime of those cells. The AAV proviral genome is infectious as cloned DNA in a plasmid, enabling the construction of recombinant genomes. Furthermore, because signals directing AAV replication, genomic capsid formation, and integration are contained within the ITR of the AAV genome, some or all of the approximately 4.3 kb inside the genome (encoding the replication and structural capsid proteins, rep-cap) can be replaced with foreign DNA. The rep and cap proteins can be supplied trans. Another important characteristic of AAV is that it is an extremely stable and robust virus. Since AAV readily withstands the conditions used to inactivate adenoviruses (56°C to 65°C for several hours), chilling of AAV is not particularly important. AAV can even be freeze-dried. Finally, cells infected with AAV are not resistant to co-infection. [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) [Overview of the project] [Means for solving the problem]

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

[0015] This disclosure provides a method for stimulating muscle growth in a subject. The method involves 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. This disclosure describes the novel effects of NT-3, its ability to directly influence protein synthesis and metabolic remodeling in neurogenic muscle.

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

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

[0018] This 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 described in 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 or 100% identical to SEQ ID NO: 2, or 90% identical to or 100% identical to nucleotides 1077-1850 of SEQ ID NO: 11.

[0019] In one embodiment, the nucleic acid of the Disclosure further comprises a chimeric intron described on the 3' side of the promoter / enhancer, as described on nucleotides 892-1024 of SEQ ID NO: 11. Furthermore, the nucleic acid of the Disclosure may further comprise an SV40 polyadenylation signal described on nucleotides 1860-2059 of SEQ ID NO: 11, as described on the 3' side of the nucleotide sequence encoding the human NT-3 polypeptide.

[0020] Any of the nucleic acids of this disclosure may comprise one or more inverted end repeat (ITR) sequences. For example, a nucleic acid may comprise a first ITR described in nucleotides 7-112 of SEQ ID NO: 11, and / or a second ITR described in nucleotides 2121-2248 of SEQ ID NO: 11.

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

[0022] This disclosure also provides recombinant adeno-associated virus particles (rAAV) that are infectious and contain any of the nucleic acids of this disclosure. The rAAV particles may 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] This disclosure also provides compositions comprising the rAAV and pharmaceutically acceptable carriers thereof. For example, these compositions are formulated to treat muscle wasting diseases or neuropathy in subjects requiring them, or these compositions are formulated to stimulate muscle growth in subjects requiring them.

[0024] In one embodiment, the present disclosure is a method for treating a muscle-wasting disease or neuropathy in a human subject in need thereof, comprising the step of administering a nucleic acid encoding an NT-3 polypeptide to the human subject; wherein a) the nucleic acid comprises a nucleotide sequence 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% or 100% identical to SEQ ID NO: 2; and d) the nucleic acid encoding an NT-3 polypeptide is the present disclosure e) The nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and rAAV is administered in a dose that results in sustained expression of low concentrations of NT-3 polypeptide, f) The nucleic acid encoding the 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 1.5 × 10¹² vg / kg to approximately 6.5 × 10¹² vg / kg, and g) NT-3 polypeptide h) The nucleic acid encoding the recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the route of administration is intramuscular, and the dose of rAAV administered is approximately 2 × 10¹² vg / kg to approximately 6 × 10¹² vg / kg, i) The nucleic acid encoding the 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 2 × 10¹² vg / kg, i) The nucleic acid encoding the 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 4 × 10¹² vg / kg, and (j) the nucleic acid encoding the 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 × 10¹² vg / kg, and (k) the nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV)scAAV1.tMCK.NTF3, and the route of administration is approximately 0.The administration method is intramuscular injection at a concentration of approximately 2 × 10¹³ vg / ml, administered using 3 to 6 injections of 5 to 1 ml per muscle, or (l) the nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, administered via multiple injections of a total volume of approximately 5 to 14 ml, at a rate of approximately 2 × 10¹³ vg / ml. 13 This invention provides a method involving intramuscular injection at a concentration of vg / ml.

[0025] In another embodiment, the present disclosure provides a method for improving muscle strength or stimulating muscle growth in a human subject in need thereof, comprising the steps of administering a nucleic acid encoding an NT-3 polypeptide to the human subject; wherein a) the nucleic acid comprises a nucleotide sequence 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% or 100% identical to SEQ ID NO: 2; and d) the nucleic acid encoding an NT-3 polypeptide is the present disclosure e) The nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and rAAV is administered in a dose that results in sustained expression of low concentrations of NT-3 polypeptide, f) The nucleic acid encoding the 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 1.5 × 10¹² vg / kg to approximately 6.5 × 10¹² vg / kg, and g) NT-3 polypeptide h) The nucleic acid encoding the recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the route of administration is intramuscular, and the dose of rAAV administered is approximately 2 × 10¹² vg / kg to approximately 6 × 10¹² vg / kg, i) The nucleic acid encoding the 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 2 × 10¹² vg / kg, i) The nucleic acid encoding the 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 4 × 10¹² vg / kg, and (j) the nucleic acid encoding the 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 × 10¹² vg / kg, and (k) the nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV)scAAV1.tMCK.NTF3, and the route of administration is approximately 0.The administration method is intramuscular injection at a concentration of approximately 2 × 10¹³ vg / ml, administered using 3 to 6 injections of 5 to 1 ml per muscle, or (l) the nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, administered via multiple injections of a total volume of approximately 5 to 14 ml, at a rate of approximately 2 × 10¹³ vg / ml. 13 This invention provides a method involving intramuscular injection at a concentration of vg / ml.

[0026] In any of the methods of this disclosure, the nucleic acid is administered using a viral vector, such as an adeno-associated virus vector. Any of the methods of this disclosure may be carried out using a nucleic acid operably ligated to a muscle-specific promoter, such as a muscle-specific creatine kinase (MCK) promoter. Furthermore, any of the methods of this disclosure may be carried out using scAAV1.tMCK.NTF3, which contains the NT-3 gene cassette described in SEQ ID NO: 11.

[0027] In one embodiment, the Disclosure provides a gene therapy method using the human neurotrophin-3 gene (NTF3) under the control of a muscle-specific promoter, tMCK, with an scAAV1 vector, an autocomplementary AAV1 serotype. The Disclosure provides a method for treating a subject diagnosed with 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 into the gastrocnemius and tibialis anterior muscles. For example, intramuscular delivery of an AAV vector expressing NT-3, e.g., scAAV1.tMCK.NTF3, initiates local production and secretion into the circulation of NT-3, thereby promoting myelination and fiber regeneration, leading to stabilization of the CMT disease phenotype. More specifically, the Disclosure provides a method for approximately 2 × 10⁻⁶ 12 Dosage of vg / kg or approximately 6 × 10 12 This document provides a method for administering scAAV1.tMCK.NTF3 at a dose of vg / kg. scAAV1.tMCK.NTF3 contains the NT-3 gene cassette described in Sequence ID No. 11.

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

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

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

[0031] In one embodiment, the present disclosure provides a method of treating a muscle wasting disease or neuropathy in a human subject who needs it, the method comprising administering recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3 to the human subject, wherein the route of administration is an intramuscular route and the dose of rAAV administered is from about 1.0×10 12 vg / kg to about 7×10 12 vg / kg, or from about 1.5×10 12 vg / kg to about 6.5×10 12 vg / kg, or from about 2×10 12 vg / kg to about 6×1012 This provides a method for obtaining a value of vg / kg.

[0032] In another embodiment, the present disclosure provides a method for treating a muscle-wasting disease or neuropathy in a human subject in need thereof, comprising the step of 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 approximately 1.0 × 10⁻¹⁴ 12 vg / kg, or approximately 1.5 × 10⁻⁶ 12 vg / kg, or approximately 2 × 10⁻⁶ 12 vg / kg, or approximately 3 × 10⁻⁶ 12 vg / kg, or approximately 4 × 10 12 vg / kg, or approximately 5 × 10 12 vg / kg, or approximately 6 × 10⁻⁶ 12 vg / kg, or approximately 7 × 10⁻⁶ 12 vg / kg, or approximately 8 × 10 12 vg / kg, or approximately 9 × 10 12 vg / kg, or approximately 1 × 10⁻⁶ 13 This provides a method for obtaining a value of vg / kg.

[0033] In another embodiment, the present disclosure provides a method for treating a muscle wasting disease or neuropathy in a human subject in need thereof, comprising the step of administering recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3 to the human subject, wherein the route of administration is approximately 1 × 10⁻⁶ 13 The present invention provides a method of intramuscular injection at a concentration of vg / ml. For example, rAAV is administered using 3 to 6 injections per muscle, for example, with each injection volume being 0.5 to 1 ml, 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.

[0034] In an exemplary embodiment, the present disclosure provides a method for treating a muscle wasting disease or neuropathy in a human subject in need thereof, comprising the step of administering recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3 to the human subject, wherein the route of administration is approximately 2 × 10¹⁶ injections per muscle (each injection volume being 0.5 to 1 ml), each being administered by 3 to 6 injections per muscle (each injection volume being 0.5 to 1 ml). 13 The method provides intramuscular injection at a concentration of vg / ml. 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.

[0035] In one embodiment, a method for improving muscle strength or stimulating muscle growth in a human subject requiring such improvement, comprising the step of 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 approximately 1.0 × 10⁻⁶ 12 vg / kg ~ approx. 7×10 12 vg / kg, or approximately 1.5 × 10⁻⁶ 12 vg / kg ~ approx. 6.5×10 12 vg / kg, or approximately 2 × 10⁻⁶ 12 vg / kg ~ approx. 6×10 12 This provides a method for obtaining a value of vg / kg.

[0036] In another embodiment, the Disclosure provides a method for improving muscle strength or stimulating muscle growth in a human subject in need thereof, comprising the step of 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 approximately 1.0 × 10⁻¹⁴ 12 vg / kg, or approximately 1.5 × 10⁻⁶ 12 vg / kg, or approximately 2 × 10⁻⁶ 12 vg / kg, or approximately 3 × 10⁻⁶ 12 vg / kg, or approximately 4 × 10 12 vg / kg, or approximately 5 × 10 12 vg / kg, or approximately 6 × 10⁻⁶ 12 vg / kg, or approximately 7 × 10⁻⁶ 12 vg / kg, or approximately 8 × 1012 vg / kg, or approximately 9 × 10 12 vg / kg, or approximately 1 × 10⁻⁶ 13 This provides a method for obtaining a value of vg / kg.

[0037] In exemplary embodiments, the present disclosure provides a method for improving muscle strength or stimulating muscle growth in a human subject requiring such improvement, comprising the steps of administering recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3 to the human subject, wherein the route of administration is approximately 1 × 10⁻⁶ 13 The present invention provides a method of intramuscular injection at a concentration of vg / ml. For example, rAAV is administered using 3 to 6 injections per muscle, for example, with each injection volume being 0.5 to 1 ml, 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.

[0038] In exemplary embodiments, the present disclosure provides a method for improving muscle strength or stimulating muscle growth in a human subject requiring such improvement, comprising the step of administering recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3 to the human subject, wherein the route of administration is 3 to 6 injections per muscle (each injection volume being 0.5 to 1 ml), with low doses (2 × 10 per patient). 12 (vg / kg) and high dose (6 x 10 per patient) 12 Approximately 1 × 10⁻¹⁶ doses are administered at vg / kg. 13 The method provides intramuscular injection at a concentration of vg / ml. 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.

[0039] In any of the methods of this 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 invention, administration of scAAV1.tMCK.NTF3 results in an improvement in muscle strength in the upper or lower limb of the subject, which is measured, for example, as a decrease in the composite score on the CMT Pediatric Scale (CMTPeds). Furthermore, in any of the methods of the present invention, administration of scAAV1.tMCK.NTF3 results in a reduction or cessation of disease progression over a period of two years. Disease progression is measured by CMTPeds.

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

[0041] In one aspect of this disclosure, in any of the methods, the subject suffers from hereditary neuropathy, such as Charcot-Marie-Tooth (CMT) neuropathy, such as CMT1A, CMT2K, CMT4A, CMTRIA, and axonal and demyelinating neuropathy caused by autosomal recessive, autosomal dominant, or X-linked genetic mutations. Hereditary neuropathy may be caused by any of the genetic mutations shown in Table 1. Furthermore, hereditary neuropathy may be transthyretin amyloid neuropathy caused by mutations in the transthyretin (TTR) gene, such as Val30Met, Ile107Val, and Ser77Tyr.

[0042] In another aspect of this disclosure, in any of the methods, the subject suffers from an acquired neuropathy involving axonal loss and / or impaired nerve regeneration. Acquired neuropathy is a peripheral neuropathy caused by any disorder or disease known to cause neuropathy. For example, the subject suffers from a peripheral neuropathy caused by diabetes, human immunodeficiency virus (HIV) infection, thyroid disorders such as hypothyroidism, hypoglycemia, uremia, renal failure, hepatic dysfunction, hepatic failure, polycythemia, connective tissue disorders, cancer, Lyme disease, celiac disease, leprosy, porphyria, Sjögren's syndrome, poliovirus infection, acromegaly, lipid / glycolipid metabolism disorders, West Nile disease, amyloidosis, mitochondrial disorders, benign monoclonal gamma globulinemia (MGUS), or abnormal protein disorders such as POEMS syndrome. The subject suffers from a peripheral neuropathy caused by vitamin B 12 They suffer from nutritional / vitamin deficiencies, such as vitamin E deficiency or copper deficiency.

[0043] In a further aspect of this disclosure, in any of the methods described herein, the subject is suffering from autoimmune peripheral polyneuropathy, acute inflammatory demyelinating polyneuropathy (AIDP), chronic inflammatory demyelinating polyneuropathy (CIDP), mononeuropathy due to vasculitis, paraneuropathy, idiopathic ganglionitis, amyotrophic lateral sclerosis, multifocal motor conduction block neuropathy, or lower motor neuron syndrome.

[0044] Acquired neuropathy can be toxic neuropathy. For example, toxic neuropathy can be caused by chloramphenicol, chloroquine, colchicine, disulfiram, etanercept, ethambutol, gold, hydroxychloroquine, nitrofurantoin, metronidazole, stabuzin, zalcitabine, infliximab, leflunomide, thalidomide, or chemotherapeutic agents (cisplatin, cytarabine, bortezomib, docetaxel, lenalidomide, misonidazole, oxaliplatin, paclitaxel, procarbazine, suramin, thalidomide, vin). It is the result of the toxic effects of prescribed medications such as blastine or vincristine, anti-alcohol drugs such as disulfiram, anticonvulsants such as phenytoin or dilantine, cardiac or blood pressure medications (statins, amiodarone, hydralazine, procainamide, perhexylline, etc.), antibiotics (fluoroquinolones, isoniazid, Cipro, Levaquin, Flagyl, or metronidazole, etc.), 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 this disclosure, in any of the methods described herein, the subject is a cancer patient suffering from acquired neuropathy. For example, the cancer patient develops neuropathy associated with nutritional deficiencies, chemotherapy side effects, and / or paraneoplastic syndromes.

[0046] In yet another embodiment of this disclosure, in any of the methods, the subjects are surgical patients suffering from acquired neuropathy. For example, the surgical patients develop neuropathy after undergoing bariatric surgery, multiple orthopedic surgeries, or multiple surgeries for “compression nerve.”

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

[0048] In another aspect of this disclosure, in any of the methods, the subject is Duchenne muscular dystrophy, Becker muscular dystrophy, myotonic muscular dystrophy, sarcoglycanopathy, myotonic dystrophy, Emery-Dreyfus muscular dystrophy, congenital muscular dystrophy, merosin-deficient congenital muscular dystrophy, Bethlemmyopathy, Ullrich congenital muscular dystrophy, facioscapulohumeral muscular dystrophy, spinal muscular dystrophy, vertebral tonic 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 2H, 2I, 2J, 2K, 2L, 2M, 2N, 2O, and 2Q; muscle-ocular-brain diseases; Fukuyama-Walker-Warburg syndrome; myasthenic syndrome; congenital myasthenia gravis; inclusion body myopathy; inclusion body myositis; dermatomyositis; central nucleus myopathy; Miyoshi type myopathy; mitochondrial myopathy; nemaline myopathy; Nonaka myopathy; myasthenia gravis; or polymyositis.

[0049] In one embodiment, the disclosure provides the 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 drug for stimulating muscle growth in a subject. For example, the drug is formulated for intramuscular administration.

[0050] In exemplary embodiments, the drug comprises a nucleic acid encoding NT-3, pro-NT-3, or an effective fragment thereof, where the nucleic acid is contained within a viral vector. In relevant embodiments, the viral vector is an adeno-associated viral vector. In various embodiments, the nucleic acid is operably ligated 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 nucleic acids encoding an NT-3 polypeptide for the manufacture of a drug for treating muscle wasting disease or neuropathy in human subjects, wherein a) the nucleic acid comprises a nucleotide sequence 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 or 100% identical to SEQ ID NO: 2; d) the nucleic acid encoding an NT-3 polypeptide is any of the nucleic acids of the present disclosure; e) NT - The nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the dose of rAAV is such that it results in sustained expression of low concentrations of NT-3 polypeptide, f) the nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the drug is formulated for intramuscular administration, and the dose of rAAV is approximately 1.5 × 10¹² vg / kg to approximately 6.5 × 10¹² vg / kg, g) the nucleic acid encoding the NT-3 polypeptide is recombinant A The drug is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the drug is formulated for intramuscular administration, with a dose of rAAV of approximately 2 × 10¹² vg / kg to approximately 6 × 10¹² vg / kg, and the nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the drug is formulated for intramuscular administration, with a dose of rAAV of approximately 2 × 10¹² vg / kg, and the nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) sc j) The nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the drug is formulated for intramuscular administration, with a dose of rAAV of approximately 4 × 10¹² v g / kg; k) The nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the drug is formulated for intramuscular administration, with a dose of rAAV of approximately 6 × 10¹² v g / kg; k) The nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the drug is approximately 0.Formulated for intramuscular injection at a concentration of approximately 2 × 10¹³ vg / ml, administered using 3 to 6 injections of 5 to 1 ml per muscle, or (l) the nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the drug is administered using multiple injections of a total volume of approximately 5 to 14 ml, at a concentration of approximately 2 × 10¹³ vg / ml. 13 It is formulated for intramuscular injection at a concentration of vg / ml and is also available for use.

[0052] This disclosure relates to the use of doses of nucleic acids encoding an NT-3 polypeptide for the manufacture of agents for improving muscle strength or stimulating muscle growth in human subjects, wherein a) the nucleic acid comprises a nucleotide sequence 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 or 100% identical to SEQ ID NO: 2; d) the nucleic acid encoding an NT-3 polypeptide is any of the nucleic acids of this disclosure; e) N f) The nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the dose of rAAV is such that it results in sustained expression of low concentrations of NT-3 polypeptide, and g) the nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the composition is formulated for intramuscular administration, and the dose of rAAV is approximately 1.5 × 10¹² vg / kg to approximately 6.5 × 10¹² vg / kg, and g) the nucleic acid encoding the NT-3 polypeptide is recombinant The adeno-associated virus (rAAV) scAAV1.tMCK.NTF3 is formulated for intramuscular administration, with a dose of rAAV of approximately 2 × 10¹² vg / kg to approximately 6 × 10¹² vg / kg, and the nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the drug is formulated for intramuscular administration, with a dose of rAAV of approximately 2 × 10¹² vg / kg, and the nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) s j) The nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the drug is formulated for intramuscular administration, with a dose of rAAV of approximately 4 × 10¹² v g / kg; k) The nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the drug is formulated for intramuscular administration, with a dose of rAAV of approximately 6 × 10¹² v g / kg; k) The nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the drug is approximately 0.Formulated for intramuscular injection at a concentration of approximately 2 × 10¹³ vg / ml, administered using 3 to 6 injections of 5 to 1 ml per muscle, or (l) the nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the drug is administered using multiple injections of a total volume of approximately 5 to 14 ml, at a concentration of approximately 2 × 10¹³ vg / ml. 13 It is formulated for intramuscular injection at a concentration of vg / ml and is also available for use.

[0053] For example, any of the agents of this disclosure may include a nucleic acid formulated for administration using a viral vector, such as an adeno-associated virus vector. Furthermore, any of the agents of this disclosure may include a nucleic acid operably linked to a muscle-specific promoter, for example, the muscle-specific promoter is a muscle-specific creatine kinase promoter (MCK). In another embodiment, in the agent or in any of the disclosures, scAAV1.tMCK.NTF3 includes the NT-3 gene cassette described in SEQ ID NO: 11. In one embodiment, the disclosure provides the use of recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3 for the preparation of an agent for treating muscle-wasting disease or neuropathy in a human subject requiring such use, wherein the agent results in sustained expression of a low concentration of NT-3 protein.

[0054] In another embodiment, the disclosure provides the use of recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3 for the preparation of a drug for stimulating muscle growth in human subjects requiring it, wherein the dose results in sustained expression of a low concentration of NT-3 protein.

[0055] In one embodiment, the disclosure relates to the use of recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3 for the preparation of a drug for treating muscle wasting disease or neuropathy in a human subject requiring it, wherein the drug is formulated for an intramuscular route of administration, and the drug is approximately 1.0 × 10 12 vg / kg ~ approx. 7×10 12vg / kg, or approximately 1.5 × 10⁻⁶ 12 vg / kg ~ approx. 6.5×10 12 vg / kg, or approximately 2 × 10⁻⁶ 12 vg / kg ~ approx. 6×10 12 This provides a dosage of rAAV in vg / kg, including its use.

[0056] In another embodiment, the Disclosure relates to the use of recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3 for the preparation of a drug for treating muscle wasting disease or neuropathy in human subjects requiring it, wherein the drug is formulated for an intramuscular route of administration, and the drug is approximately 1.0 × 10 12 vg / kg, or approximately 1.5 × 10⁻⁶ 12 vg / kg, or approximately 2 × 10⁻⁶ 12 vg / kg, or approximately 3 × 10⁻⁶ 12 vg / kg, or approximately 4 × 10 12 vg / kg, or approximately 5 × 10 12 vg / kg, or approximately 6 × 10⁻⁶ 12 vg / kg, or approximately 7 × 10⁻⁶ 12 vg / kg, or approximately 8 × 10 12 vg / kg, or approximately 9 × 10 12 vg / kg, or approximately 1 × 10⁻⁶ 13 This provides a dosage of rAAV in vg / kg, including its use.

[0057] In another embodiment, the Disclosure relates to the use of recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3 for the preparation of a drug for treating muscle wasting disease or neuropathy in human subjects requiring it, wherein the drug is formulated for an intramuscular route of administration, and the drug is approximately 2 × 10 13 The invention provides a use including a concentration of rAAV in vg / ml. For example, the drug is administered to the medial and lateral heads of the gastrocnemius and tibialis anterior muscles in each leg by 3 to 6 injections per muscle (e.g., each injection volume is 0.5 to 1 ml), where a total of 5 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 exemplary embodiments, the present disclosure relates to the use of recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3 for the preparation of a drug for treating muscle wasting disease or neuropathy in human subjects requiring it, wherein the drug is formulated for an intramuscular route of administration and administered by 3 to 6 injections per muscle (each injection volume being 0.5 to 1 ml) for approximately 1 × 10 13 The use of rAAV is provided, including doses of vg / ml. 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.

[0059] In one embodiment, the disclosure relates to the use of recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3 for the preparation of a drug for improving muscle strength or stimulating muscle growth in a human subject requiring it, wherein the drug is formulated for an intramuscular route of administration, and the drug is approximately 1.0 × 10 12 vg / kg ~ approx. 7×10 12 vg / kg, or approximately 1.5 × 10⁻⁶ 12 vg / kg ~ approx. 6.5×10 12 vg / kg, or approximately 2 × 10⁻⁶ 12 vg / kg ~ approx. 6×10 12 This provides a dosage of rAAV in vg / kg, including its use.

[0060] In another embodiment, the disclosure relates to the use of recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3 for the preparation of a drug for improving muscle strength or stimulating muscle growth in a human subject requiring it, wherein the drug is formulated for an intramuscular route of administration, and the drug is approximately 1.0 × 10 12 vg / kg, or approximately 1.5 × 10⁻⁶ 12 vg / kg, or approximately 2 × 10⁻⁶ 12 vg / kg, or approximately 3 × 10⁻⁶ 12 vg / kg, or approximately 4 × 10 12 vg / kg, or approximately 5 × 10 12 vg / kg, or approximately 6 × 10⁻⁶ 12 vg / kg, or approximately 7 × 10⁻⁶ 12vg / kg, or about 8×10 12 vg / kg, or about 9×10 12 vg / kg, or about 1×10 13 vg / kg, and provides for use, comprising a dose of rAAV.

[0061] In another embodiment, the disclosure provides for use of recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3 for the preparation of an agent for improving muscle strength or stimulating muscle growth in a human subject in need thereof, wherein the agent is formulated for an intramuscular route of administration, the agent is administered at low dose (2×10 12 vg / kg per patient) and high dose (6×10 12 vg / kg per patient), and comprises a concentration of rAAV of about 2×10 13 vg / ml. In certain embodiments, 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., each injection volume is 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.

[0062] In an exemplary embodiment, the disclosure provides for use of recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3 for the preparation of an agent for improving muscle strength or stimulating muscle growth in a human subject in need thereof, wherein the agent is formulated for an intramuscular route of administration, the agent is administered at low dose (2×10 12 vg / kg per patient) and high dose (6×10 12 vg / kg per patient), using 3 - 6 injections per muscle (each injection volume is 0.5 - 1 ml), and comprises a concentration of rAAV of about 2×10 13 vg / 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.

[0063] In any use of this disclosure, the agent is formulated for bilateral intramuscular injection into the medial and lateral heads of the gastrocnemius and tibialis anterior muscles. Furthermore, in any use of the present invention, the agent results in improved muscle strength in the upper or lower limb of the subject, which is measured, for example, as a decrease in the composite score on the CMT Pediatric Scale (CMTPeds). Furthermore, in any use of the present invention, the agent results in a reduction or cessation of disease progression over a period of two years. Disease progression is measured by CMTPeds.

[0064] In one aspect of this disclosure, in any use of this disclosure, the subject suffers from Charcot-Marie-Tooth (CMT) neuropathy, such as CMT1A, CMT2K, CMT4A, CMTRIA, and hereditary neuropathy such as axonal and demyelinating neuropathy caused by autosomal recessive, autosomal dominant, or X-linked genetic mutations. Hereditary neuropathy may be caused by any of the genetic mutations shown in Table 1. Furthermore, hereditary neuropathy may be transthyretin amyloid neuropathy caused by mutations in the transthyretin (TTR) gene, such as Val30Met, Ile107Val, and Ser77Tyr.

[0065] In another aspect of this disclosure, in any of the methods of the present invention, the subject suffers from an acquired neuropathy with axonal loss and / or impaired nerve regeneration. Acquired neuropathy is a peripheral neuropathy caused by any disorder or disease that causes neuropathy. For example, the subject suffers from a peripheral neuropathy caused by diabetes, human immunodeficiency virus (HIV) infection, thyroid disorders such as hypothyroidism, hypoglycemia, uremia, renal failure, hepatic dysfunction, hepatic failure, polycythemia, connective tissue disorders, cancer, Lyme disease, celiac disease, leprosy, porphyria, Sjögren's syndrome, poliovirus infection, acromegaly, lipid / glycolipid metabolism disorders, West Nile disease, amyloidosis, mitochondrial disorders, benign monoclonal gamma globulinemia (MGUS), or abnormal protein disorders such as POEMS syndrome. The subject suffers from a peripheral neuropathy caused by vitamin B 12 You may have a nutritional / vitamin deficiency, such as a deficiency of vitamin E or copper.

[0066] Furthermore, in any use of this disclosure, the subject is suffering from autoimmune peripheral polyneuropathy, acute inflammatory demyelinating polyneuropathy (AIDP), chronic inflammatory demyelinating polyneuropathy (CIDP), mononeuropathy due to vasculitis, paraneuropathy, idiopathic ganglionitis, amyotrophic lateral sclerosis, multifocal motor conduction block neuropathy, or lower motor neuron syndrome.

[0067] Acquired neuropathy is a type of toxic neuropathy. For example, toxic neuropathy can be caused by chloramphenicol, chloroquine, colchicine, disulfiram, etanercept, ethambutol, gold, hydroxychloroquine, nitrofurantoin, metronidazole, stabuzin, zalcitabine, infliximab, leflunomide, thalidomide, or chemotherapeutic agents (cisplatin, cytarabine, bortezomib, docetaxel, lenalidomide, misonidazole, oxaliplatin, paclitaxel, procarbazine, suramin, thalidomide, vinyl syrup). This is the result of the toxic effects of prescribed medications such as blastine or vincristine, anti-alcohol drugs such as disulfiram, anticonvulsants such as phenytoin or dilantine, cardiac or blood pressure medications (statins, amiodarone, hydralazine, procainamide, perhexylline, etc.), antibiotics (fluoroquinolones, isoniazid, Cipro, Levaquin, Flagyl, or metronidazole, etc.), 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 use of this disclosure, the subject is a cancer patient suffering from acquired neuropathy. For example, the cancer patient develops neuropathy associated with nutritional deficiencies, chemotherapy side effects, and / or paraneoplastic syndromes.

[0069] In yet another embodiment, in any use of this disclosure, the subject is a surgical patient suffering from acquired neuropathy. For example, the surgical patient develops neuropathy after undergoing bariatric surgery, multiple orthopedic surgeries, or multiple surgeries for “compression nerve.”

[0070] In another embodiment, in any use of the Disclosure, the subject suffers from hereditary myopathy, neuromuscular disease, muscle atrophy, drug-induced myopathy, sarcopenia, cachexia, type II muscle fiber atrophy, hereditary muscular dystrophy, age-related muscle atrophy, or acquired autoimmune primary muscle disease.

[0071] In another embodiment, in any use of the present disclosure, the subject matter includes Duchenne muscular dystrophy, Becker muscular dystrophy, myotonic muscular dystrophy, sarcoglycanopathy, myotonic dystrophy, Emery-Dreyfus muscular dystrophy, congenital muscular dystrophy, merosin-deficient congenital muscular dystrophy, Bethlemmyopathy, Ullrich congenital muscular dystrophy, facioscapulohumeral muscular dystrophy, spinal muscular dystrophy, vertebral tonic 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 2H, 2I, 2J, 2K, 2L, 2M, 2N, 2O, and 2Q; muscle-ocular-brain diseases; Fukuyama-Walker-Warburg syndrome; myasthenic syndrome; congenital myasthenia gravis; inclusion body myopathy; inclusion body myositis; dermatomyositis; central nucleus myopathy; Miyoshi type myopathy; mitochondrial myopathy; nemaline myopathy; Nonaka myopathy; myasthenia gravis; or polymyositis.

[0072] In one embodiment, the 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 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 contained in a viral vector, such as an adeno-associated virus vector. In various embodiments, the nucleic acid is operably ligated to a muscle-specific promoter, such as a triple muscle-specific creatine kinase promoter. In various embodiments, the composition comprises a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 1.

[0074] This disclosure relates to a composition comprising a nucleic acid encoding an NT-3 polypeptide for use in treating muscle wasting disease or neuropathy in human subjects, wherein a) the nucleic acid comprises a nucleotide sequence 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% or 100% identical to SEQ ID NO: 2; d) the nucleic acid encoding the NT-3 polypeptide is any of the nucleic acids of this disclosure; and e) the NT-3 polypeptide f) The nucleic acid encoding the peptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and rAAV is administered in a dose that results in sustained expression of low concentrations of NT-3 polypeptide, and g) the nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the composition is formulated for intramuscular administration, and the dose of rAAV is approximately 1.5 × 10¹² vg / kg to approximately 6.5 × 10¹² vg / kg, and g) the nucleic acid encoding the NT-3 polypeptide is recombinant adeno The composition is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the composition is formulated for intramuscular administration, with a dose of rAAV of approximately 2 × 10¹² vg / kg to approximately 6 × 10¹² vg / kg, and the nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the composition is formulated for intramuscular administration, with a dose of rAAV of approximately 2 × 10¹² vg / kg, and the nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAA j) The composition is V1.tMCK.NTF3, and the composition is formulated for intramuscular administration, with a dose of rAAV of approximately 4 × 10¹² vg / kg; k) The nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the composition is formulated for intramuscular administration, with a dose of rAAV administered of approximately 6 × 10¹² vg / kg; k) The nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the composition is approximately 0.Formulated for intramuscular injection at a concentration of approximately 2 × 10¹³ vg / ml, administered using 3 to 6 injections of 5 to 1 ml per muscle, or (l) the nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the composition is administered using multiple injections of a total volume of approximately 5 to 14 ml, at a concentration of approximately 2 × 10¹³ vg / ml. 13 We also provide compositions formulated for intramuscular injection at concentrations of vg / ml.

[0075] In another embodiment, the Disclosure relates to a composition comprising a nucleic acid encoding an NT-3 polypeptide for use in human subjects to improve muscle strength or stimulate muscle growth, wherein a) the nucleic acid comprises a nucleotide sequence 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 or 100% identical to SEQ ID NO: 2; and d) the nucleic acid encoding the NT-3 polypeptide comprises any of the nucleic acids of the Disclosure. e) The nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the dose of rAAV is such that it results in sustained expression of low concentrations of NT-3 polypeptide; f) The nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the composition is formulated for intramuscular administration, with a dose of rAAV of approximately 1.5 × 10¹² vg / kg to approximately 6.5 × 10¹² vg / kg; g) The nucleic acid encoding the NT-3 polypeptide is h) The nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the composition is formulated for intramuscular administration, with a dose of rAAV of approximately 2 × 10¹² vg / kg to approximately 6 × 10¹² vg / kg, and i) the nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the composition is formulated for intramuscular administration, with a dose of rAAV of approximately 2 × 10¹² vg / kg, and i) the nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rA j) The rAAV is scAAV1.tMCK.NTF3, and the composition is formulated for intramuscular administration, with a dose of approximately 4 × 10¹² vg / kg; k) The nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the composition is formulated for intramuscular administration, with a dose of approximately 6 × 10¹² vg / kg; k) The nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the composition is approximately 0.Formulated for intramuscular injection at a concentration of approximately 2 × 10¹³ vg / ml, administered using 3 to 6 injections of 5 to 1 ml per muscle, or (l) the nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the composition is administered using multiple injections of a total volume of approximately 5 to 14 ml, at a concentration of approximately 2 × 10¹³ vg / ml. 13 The present invention provides a composition formulated for intramuscular injection at a concentration of vg / ml.

[0076] For example, any of the compositions of this disclosure may include nucleic acids formulated for administration using a viral vector, such as an adeno-associated virus vector. Furthermore, any of the compositions of this disclosure may include nucleic acids operably linked to a muscle-specific promoter, for example, the muscle-specific promoter is a muscle-specific creatine kinase promoter (MCK). In another embodiment, in a composition or in any of the disclosure, scAAV1.tMCK.NTF3 includes the NT-3 gene cassette described 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 the treatment of muscle-wasting diseases or neuropathy in human subjects requiring the sustained expression of low concentrations of NT-3 protein.

[0078] In another embodiment, the disclosure provides the use of recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3 to stimulate muscle growth in human subjects requiring it, resulting in sustained expression of low concentrations of NT-3 protein.

[0079] In one embodiment, the present disclosure relates to a composition comprising a dose of recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3 for the treatment of muscle wasting disease or neuropathy in a human subject requiring such treatment, wherein the composition is formulated for intramuscular administration and the dose of rAAV administered is approximately 1.0 × 10⁻¹⁴ 12 vg / kg ~ approx. 7×1012 vg / kg, or about 1.5×10 12 vg / kg to about 6.5×10 12 vg / kg, or about 2×10 12 vg / kg to about 6×10 12 vg / kg, and provides a composition.

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

[0081] In another embodiment, the present disclosure is a composition comprising a dose of recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3 for treating muscle wasting diseases or neuropathies in a human subject who needs it, the composition is formulated for intramuscular administration, and the dose of rAAV administered is a low dose (2×10 per patient 12 vg / kg) and a high dose (6×10 per patient 12 vg / kg) and is administered at about 2×10 13A composition is provided in vg / ml. In one embodiment, this composition 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 (each injection volume being 0.5 to 1 ml). A total of 5 to 14 mL of vector is administered to the medial and lateral heads of the gastrocnemius and tibialis anterior muscles in each leg.

[0082] In exemplary embodiments, the Disclosure relates to a composition comprising a dose of recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3 for the treatment of muscle wasting disease or neuropathy in human subjects requiring it, wherein the composition is formulated for intramuscular administration, and the dose of rAAV to be administered is a low dose (2 × 10 per patient) using 3 to 6 injections per muscle (each injection volume being 0.5 to 1 ml). 12 (vg / kg) and high dose (6 x 10 per patient) 12 Approximately 2 × 10⁻¹⁶ doses are administered at vg / kg. 13 A composition is provided in vg / ml. A total of 5 mL to 14 mL of the vector is administered to the medial and lateral heads of the gastrocnemius and tibialis anterior muscles in each leg.

[0083] In one embodiment, the Disclosure relates to 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 approximately 1.0 × 10⁻⁶ 12 vg / kg ~ approx. 7×10 12 vg / kg, or approximately 1.5 × 10⁻⁶ 12 vg / kg ~ approx. 6.5×10 12 vg / kg, or approximately 2 × 10⁻⁶ 12 vg / kg ~ approx. 6×10 12 The present invention provides a composition with a concentration of vg / kg.

[0084] In another embodiment, the Disclosure relates to 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 approximately 1.0 × 10⁻¹⁴ 12 vg / kg, or approximately 1.5 × 10⁻⁶ 12 vg / kg, or approximately 2 × 10⁻⁶ 12 vg / kg, or approximately 3 × 10⁻⁶ 12 vg / kg, or approximately 4 × 10 12 vg / kg, or approximately 5 × 10 12 vg / kg, or approximately 6 × 10⁻⁶ 12 vg / kg, or approximately 7 × 10⁻⁶ 12 vg / kg, or approximately 8 × 10 12 vg / kg, or approximately 9 × 10 12 vg / kg, or approximately 1 × 10⁻⁶ 13 The present invention provides a composition with a concentration of vg / kg.

[0085] In another embodiment, the Disclosure relates to 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 requiring it, wherein the composition is formulated for intramuscular administration and the dose of rAAV administered is a low dose (2 × 10 per patient). 12 (vg / kg) and high dose (6 x 10 per patient) 12 Approximately 2 × 10⁻¹⁰ doses are administered at vg / kg. 13 A composition is provided in vg / ml. In one embodiment, this composition 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 (each injection volume being 0.5 to 1 ml). A total of 5 to 14 mL of vector is administered to the medial and lateral heads of the gastrocnemius and tibialis anterior muscles in each leg.

[0086] In exemplary embodiments, the Disclosure relates to 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 requiring it, wherein the composition is formulated for intramuscular administration, and the dose of rAAV administered is a low dose (2 × 10 per patient) using 3 to 6 injections per muscle (each injection being 0.5 to 1 ml). 12 (vg / kg) and high dose (6 x 10 per patient) 12 Approximately 2 × 10⁻¹⁶ doses are administered at vg / kg. 13 A composition is provided in vg / ml. A total of 5 mL to 14 mL of the vector is administered to the medial and lateral heads of the gastrocnemius and tibialis anterior muscles in each leg.

[0087] In any of the compositions of this 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 compositions of this invention, administration of scAAV1.tMCK.NTF3 results in an improvement in muscle strength in the upper or lower limb of the subject, which is measured, for example, as a decrease in the composite score on the CMT Pediatric Scale (CMTPedS). Furthermore, in any of the compositions of this disclosure, administration of this composition results in a reduction or cessation of disease progression over a period of two years. Disease progression is measured by CMTPedS. CMTPedS is an 11-item scale consisting of the Functional Dexterity Test, Nine-Hole Peg Test (9HPT), manual dynamometer tests for handgrip, plantar flexion, and dorsiflexion strength, needle prick and vibration sensation, Bruininks-Oseletzky balance test, gait assessment, long jump, and 6-minute walk test (6MWT). Effectiveness is defined as the cessation of decline in abilities measured by this scale two years after gene transfer.

[0088] In any aspect of the present disclosure, the subject in any of the compositions suffers from hereditary neuropathy, such as Charcot-Marie-Tooth (CMT) neuropathy, such as CMT1A, CMT2K, CMT4A, CMTRIA, and axonal and demyelinating neuropathy caused by autosomal recessive, autosomal dominant, or X-linked genetic mutations. Hereditary neuropathy may be caused by any of the genetic mutations shown in Table 1. Furthermore, hereditary neuropathy may be transthyretin amyloid neuropathy caused by mutations in the transthyretin (TTR) gene, such as Val30Met, Ile107Val, and Ser77Tyr.

[0089] In another aspect of this disclosure, in any of the compositions, the subject suffers from an acquired neuropathy with impaired axonal loss and / or nerve regeneration. Acquired neuropathy is a peripheral neuropathy caused by any disorder or disease that causes neuropathy. For example, the subject suffers from a peripheral neuropathy caused by diabetes, human immunodeficiency virus (HIV) infection, thyroid disorders such as hypothyroidism, hypoglycemia, uremia, renal failure, hepatic dysfunction, hepatic failure, polycythemia, connective tissue disorders, cancer, Lyme disease, celiac disease, leprosy, porphyria, Sjögren's syndrome, poliovirus infection, acromegaly, lipid / glycolipid metabolism disorders, West Nile disease, amyloidosis, mitochondrial disorders, benign monoclonal gamma globulinemia (MGUS), or abnormal protein disorders such as POEMS syndrome. The subject suffers from a peripheral neuropathy caused by vitamin B 12 They suffer from nutritional / vitamin deficiencies, such as vitamin E deficiency or copper deficiency.

[0090] Furthermore, in any of the compositions of this disclosure, the subject suffers from autoimmune peripheral polyneuropathy, acute inflammatory demyelinating polyneuropathy (AIDP), chronic inflammatory demyelinating polyneuropathy (CIDP), mononeuropathy due to vasculitis, paraneuropathy, idiopathic ganglionitis, amyotrophic lateral sclerosis, multifocal motor conduction block neuropathy, or lower motor neuron syndrome.

[0091] In any of the compositions of this disclosure, acquired neuropathy is toxic neuropathy. For example, toxic neuropathy is caused by chloramphenicol, chloroquine, colchicine, disulfiram, etanercept, ethambutol, gold, hydroxychloroquine, nitrofurantoin, metronidazole, stabidine, zalcitabine, infliximab, leflunomide, thalidomide, or chemotherapeutic agents (cisplatin, cytarabine, bortezomib, docetaxel, lenalidomide, misonidazole, oxaliplatin, paclitaxel, procarbazine, suramin, thalidomide, bin It is the result of the toxic effects of prescribed medications such as blastine or vincristine, anti-alcohol drugs such as disulfiram, anticonvulsants such as phenytoin or dilantine, heart or blood pressure medications (statins, amiodarone, hydralazine, procainamide, perhexylline, etc.), antibiotics (fluoroquinolones, isoniazid, Cipro, Levaquin, Flagyl, or metronidazole, etc.), 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 aspect of this disclosure, in any of the compositions, the subject is a cancer patient suffering from acquired neuropathy. For example, the cancer patient develops neuropathy associated with nutritional deficiencies, chemotherapy side effects, and / or paraneoplastic syndromes.

[0093] In yet another aspect of this disclosure, in any of the compositions, the subject is a surgical patient suffering from acquired neuropathy. For example, the surgical patient develops neuropathy after undergoing bariatric surgery, multiple orthopedic surgeries, or multiple surgeries for “compression nerve.”

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

[0095] In another aspect of this disclosure, any of the compositions may include Duchenne muscular dystrophy, Becker muscular dystrophy, myotonic muscular dystrophy, sarcoglycanopathy, myotonic dystrophy, Emery-Dreyfus muscular dystrophy, congenital muscular dystrophy, merosin-deficient congenital muscular dystrophy, Bethlemmyopathy, Ullrich congenital muscular dystrophy, facioscapulohumeral muscular dystrophy, spinal muscular dystrophy, vertebral tonic 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 2H, 2I, 2J, 2K, 2L, 2M, 2N, 2O, and 2Q; muscle-ocular-brain diseases; Fukuyama-Walker-Warburg syndrome; myasthenic syndrome; congenital myasthenia gravis; inclusion body myopathy; inclusion body myositis; dermatomyositis; central nucleus myopathy; Miyoshi type myopathy; mitochondrial myopathy; nemaline myopathy; Nonaka myopathy; myasthenia gravis; or polymyositis.

[0096] In another embodiment of this disclosure, in any of the compositions, the subject is suffering from traumatic nerve injury, such as nerve injury caused by compression, double crush, or transection. 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 described in nucleotides 147-860 of SEQ ID NO: 11; (iii) Nucleotide sequences encoding human NT-3 polypeptides; and (iv) Second AAV2 ITR sequence nucleic acids containing; 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. (Item 2) The nucleic acid according to item 1, further comprising a chimeric intron described in nucleotides 892-1024 of sequence number 11 on the 3' side of the promoter / enhancer. (Item 3) The nucleic acid according to item 1 or 2, further comprising the SV40 polyadenylation signal described in nucleotides 1860-2059 of SEQ ID NO: 11 at the 3' end of the nucleotide sequence encoding a human NT-3 polypeptide. (Item 4) The first ITR is described in nucleotides 7-112 of SEQ ID NO: 11, and / or The second ITR is a nucleic acid as described in any one of items 1 to 3, as listed in nucleotides 2121 to 2248 of sequence number 11. (Item 5) The first ITR is described in nucleotides 7-112 of sequence number 11, The second ITR is a nucleic acid as described in any one of items 1 to 3, as listed in nucleotides 2121 to 2248 of sequence number 11. (Item 6) Nucleic acids containing the scAAV1.tMCK.NTF3 genome, which is at least 90% identical to the nucleotide sequence described in Sequence ID No. 11. (Item 7) Nucleic acids containing the scAAV1.tMCK.NTF3 genome described in Sequence ID No. 11. (Item 8) Recombinant adeno-associated virus particles (rAAV) containing nucleic acids described in any one of items 1-7, which are infectious. (Item 9) The rAAV particle according to item 8, wherein the rAAV is 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) The rAAV particle described in 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 rAAV particles and a pharmaceutically acceptable carrier as described in any one of items 8 to 10. (Item 12) The composition according to item 11, wherein the composition is formulated to treat muscle wasting disease or neuropathy in a subject requiring it. (Item 13) The composition according to item 11, wherein the composition is formulated to stimulate muscle growth in a subject requiring it. (Item 14) A method for treating muscle wasting disease or neuropathy in a human subject requiring such treatment, comprising the step of administering a nucleic acid encoding an NT-3 polypeptide to the human subject; wherein, a) The nucleic acid contains 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 the nucleic acid described in any one of items 1 to 7, e) The nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the rAAV is administered in a dose that results in sustained expression of a low concentration of NT-3 polypeptide. f) The nucleic acid encoding the 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 1.5 × 10¹² vg / kg to approximately 6.5 × 10¹² vg / kg. g) The nucleic acid encoding the 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 2 × 10¹² vg / kg to approximately 6 × 10¹² vg / kg. h) The nucleic acid encoding the 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 2 × 10¹² vg / kg. i) The nucleic acid encoding the 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 4 × 10¹² vg / kg. j) The nucleic acid encoding the 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 × 10¹² vg / kg. k) The nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the route of administration is intramuscular injection at a concentration of approximately 2 × 10¹³ vg / ml, administered in 3 to 6 injections of approximately 0.5 to 1 ml per muscle, or l) The nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the route of administration is approximately 2 × 10 by multiple injections with a total volume of approximately 5 to 14 ml. 13 The method involves intramuscular injection at a concentration of vg / ml. (Item 15) A method for improving muscle strength or stimulating muscle growth in a human subject requiring such improvement, comprising the step of administering a nucleic acid encoding an NT-3 polypeptide to the human subject; wherein, a) The nucleic acid contains 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 the nucleic acid described in any one of items 1 to 7, e) The nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the rAAV is administered in a dose that results in sustained expression of a low concentration of NT-3 polypeptide. f) The nucleic acid encoding the 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 1.5 × 10¹² vg / kg to approximately 6.5 × 10¹² vg / kg. g) The nucleic acid encoding the 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 2 × 10¹² vg / kg to approximately 6 × 10¹² vg / kg. h) The nucleic acid encoding the 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 2 × 10¹² vg / kg. i) The nucleic acid encoding the 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 4 × 10¹² vg / kg. j) The nucleic acid encoding the 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 × 10¹² vg / kg. k) The nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the route of administration is intramuscular injection at a concentration of approximately 2 × 10¹³ vg / ml, administered in 3 to 6 injections of approximately 0.5 to 1 ml per muscle, or l) The nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the route of administration is approximately 2 × 10 by multiple injections with a total volume of approximately 5 to 14 ml. 13 The method involves intramuscular injection at a concentration of vg / ml. (Item 16) The method according to item 14 or 15, wherein the nucleic acid is administered using a viral vector. (Item 17) The method according to item 16, wherein the viral vector is an adeno-associated virus vector. (Item 18) The method according to any one of items 14 to 17, wherein the nucleic acid is operably linked to a muscle-specific promoter. (Item 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) The method according to any one of items 14 to 19, wherein the scAAV1.tMCK.NTF3 comprises the NT-3 gene cassette described in Sequence ID No. 11. (Item 21) The method according to any one of items 14 to 20, wherein the route of administration is intramuscular injection. (Item 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) The method according to any one of items 15 to 22, wherein the muscle strength to be improved in the subject is located in the upper or lower limb. (Item 24) The method according to any one of items 15 to 22, wherein the improvement in muscle strength is measured as a decrease in the composite score on the CMT Pediatric Scale (CMTPeds) or as a decrease in disease progression over a two-year period. (Item 25) A composition comprising a nucleic acid encoding an NT-3 polypeptide for use in treating muscle wasting disease or neuropathy in human subjects, wherein: a) The nucleic acid contains 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 the nucleic acid described in any one of items 1 to 7, e) The nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the rAAV is administered in a dose that results in sustained expression of a low concentration 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 an intramuscular route of administration, and the dose of the rAAV is about 1.5 × 10⁻¹⁴ 12 vg / kg ~ approx. 6.5×10 12 It is 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 an intramuscular route of administration, and the dose of the rAAV is approximately 2 × 10⁻¹⁴ 12 vg / kg ~ approx. 6×10 12 It is 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 an intramuscular route of administration, and the dose of the rAAV is approximately 2 × 10⁻¹⁴ 12 It is 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 an intramuscular route of administration, and the dose of the rAAV is approximately 4 × 10⁻¹⁴ 12 It is 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 an intramuscular route of administration, and the dose of rAAV administered is approximately 6 × 10⁻¹⁴ 12 It is vg / kg. k) The nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the composition is administered by approximately 2 × 10⁻¹⁶ injections of approximately 0.5–1 ml per muscle in 3–6 doses. 13 Formulated for intramuscular injection at a concentration of vg / ml, or l) The nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the composition is administered by multiple injections in a total volume of approximately 5-14 ml, resulting in approximately 2 × 10 13 A composition formulated for intramuscular injection at a concentration of vg / ml. (Item 26) A composition comprising a nucleic acid encoding an NT-3 polypeptide for use in improving muscle strength or stimulating muscle growth in human subjects, wherein a) The nucleic acid contains 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 the nucleic acid described in any one of items 1 to 7, e) The nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the rAAV is in a dose that results in sustained expression of a low concentration 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 an intramuscular route of administration, and the dose of the rAAV is about 1.5 × 10⁻¹⁴ 12 vg / kg ~ approx. 6.5×10 12 It is 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 an intramuscular route of administration, and the dose of the rAAV is approximately 2 × 10⁻¹⁴ 12 vg / kg ~ approx. 6×10 12 It is 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 an intramuscular route of administration, and the dose of the rAAV is approximately 2 × 10⁻¹⁴ 12 It is 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 an intramuscular route of administration, and the dose of the rAAV is approximately 4 × 10⁻¹⁴ 12 It is 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 an intramuscular route of administration, and the dose of the rAAV is approximately 6 × 10⁻¹⁴ 12 It is vg / kg. k) The nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the composition is administered by approximately 2 × 10⁻¹⁶ injections of approximately 0.5–1 ml per muscle in 3–6 doses. 13 Formulated for intramuscular injection at a concentration of vg / ml, or l) The nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the composition is administered by multiple injections in a total volume of approximately 5-14 ml, resulting in approximately 2 × 10 13 A composition formulated for intramuscular injection at a concentration of vg / ml. (Item 27) The composition according to item 25 or 26, wherein the nucleic acid is formulated for administration using a viral vector. (Item 28) The composition according to item 27, wherein the viral vector is an adeno-associated virus vector. (Item 29) The composition according to any one of items 25 to 28, wherein the nucleic acid is operably linked to a muscle-specific promoter. (Item 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) The composition according to any one of items 25 to 29, wherein the scAAV1.tMCK.NTF3 comprises the NT-3 gene cassette described in Sequence ID No. 11. (Item 32) The composition according to any one of items 25 to 31, wherein the composition is formulated for intramuscular injection. (Item 33) The composition according to any one of items 25 to 32, wherein the composition is formulated for bilateral intramuscular injection into the medial and lateral heads of the gastrocnemius and tibialis anterior muscles. (Item 34) The composition according to any one of items 26 to 33, wherein the improved muscle strength is located within the upper or lower limb of the subject. (Item 35) The composition according to any one of items 26 to 33, wherein the improvement in muscle strength is measured as a decrease in the composite score on the CMT Pediatric Scale (CMTPeds) or as a decrease in disease progression over a period of two years. (Item 36) Use of nucleic acids encoding NT-3 polypeptide for the manufacture of agents for the treatment of muscle wasting diseases or neuropathy in human subjects, wherein: a) The nucleic acid contains 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 the nucleic acid described in any one of items 1 to 7, e) The nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the rAAV is in a dose that results in sustained expression of a low concentration of NT-3 polypeptide. f) The nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the drug is formulated for intramuscular administration, and the dose of rAAV is approximately 1.5 × 10⁻¹⁴ 12 vg / kg ~ approx. 6.5×10 12 It is vg / kg. g) The nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the drug is formulated for intramuscular administration, and the dose of rAAV is approximately 2 × 10⁻¹⁴ 12 vg / kg ~ approx. 6×10 12 It is vg / kg. h) The nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the drug is formulated for intramuscular administration, and the dose of rAAV is approximately 2 × 10⁻¹⁴ 12 It is vg / kg. i) The nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the drug is formulated for intramuscular administration, and the dose of rAAV is approximately 4 × 10⁻¹⁴ 12 It is vg / kg. j) The nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the drug is formulated for an intramuscular route of administration, and the dose of rAAV administered is approximately 6 × 10⁻¹⁴ 12 It is vg / kg. k) The nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the drug is formulated for intramuscular injection at a concentration of approximately 2 × 10¹³ vg / ml, administered in 3 to 6 injections of approximately 0.5 to 1 ml per muscle, or l) The nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the drug is administered by multiple injections in a total volume of approximately 5-14 ml, resulting in approximately 2 × 10 13 Formulated for intramuscular injection at a concentration of vg / ml. (Item 37) The use of a dose of nucleic acid encoding an NT-3 polypeptide for the manufacture of a drug for improving muscle strength or stimulating muscle growth in human subjects, wherein: a) The nucleic acid contains 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 the nucleic acid described in any one of items 1 to 7, e) The nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the rAAV is in a dose that results in sustained expression of a low concentration 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 an intramuscular route of administration, and the dose of the rAAV is about 1.5 × 10⁻¹⁴ 12 vg / kg ~ approx. 6.5×10 12 It is vg / kg. g) The nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the drug is formulated for intramuscular administration, and the dose of rAAV is approximately 2 × 10⁻¹⁴ 12 vg / kg ~ approx. 6×10 12 It is vg / kg. h) The nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the drug is formulated for intramuscular administration, and the dose of rAAV is approximately 2 × 10⁻¹⁴ 12 It is vg / kg. i) The nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the drug is formulated for intramuscular administration, and the dose of rAAV is approximately 4 × 10⁻¹⁴ 12 It is vg / kg. j) The nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the drug is formulated for an intramuscular route of administration, and the dose of rAAV administered is approximately 6 × 10⁻¹⁴ 12 It is vg / kg. k) The nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the drug is administered using approximately 0.5 to 1 ml per muscle in 3 to 6 injections, resulting in approximately 2 × 10 13 Formulated for intramuscular injection at a concentration of vg / ml, or l) The nucleic acid encoding the NT-3 polypeptide is recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the drug is administered by multiple injections in a total volume of approximately 5-14 ml, resulting in approximately 2 × 10 13 Formulated for intramuscular injection at a concentration of vg / ml. (Item 38) The use described in item 36 or 37, wherein the nucleic acid is formulated for administration using a viral vector. (Item 39) The use described in any one of items 36 to 38, wherein the viral vector is an adeno-associated virus vector. (Item 40) The use according to any one of items 36 to 39, wherein the nucleic acid is operably linked to a muscle-specific promoter. (Item 41) The use described in any one of items 36 to 40, wherein the muscle-specific promoter is a muscle-specific creatine kinase promoter. (Item 42) The use of scAAV1.tMCK.NTF3 as described in any one of items 36 to 41, comprising the NT-3 gene cassette described in Sequence ID No. 11. (Item 43) The drug is formulated for intramuscular injection, as described in any one of items 36-42. (Item 44) The drug is formulated for bilateral intramuscular injection into the medial and lateral heads of the gastrocnemius and tibialis anterior muscles, as described in any one of items 36-43. (Item 45) The use described in any one of items 37-44, wherein the muscle strength to be improved in the subject is located in the upper or lower limb. (Item 46) The use described in any one of items 37-45, where the improvement in muscle strength is measured as a decrease in the composite score on the CMT Pediatric Scale (CMTPeds) or as a decrease in disease progression over a two-year period. (Item 47) The method, composition, or use described in any one of items 14 to 46, for which the subject is at risk of developing muscle atrophy. (Item 48) The method, composition, or use described in any one of items 14 to 46, wherein the subject is suffering from muscle atrophy. (Item 49) The method, composition, or use described in any one of items 14 to 46, wherein the subject is suffering from muscular dystrophy. (Item 50) The method, composition, or use described in any one of items 14 to 46, wherein the subject is suffering from neuropathy. (Item 51) The method, composition, or use described in any one of items 14 to 46, wherein the subject is suffering from Charcot-Marie-Tooth (CMT) neuropathy. (Item 52) The method, composition, or use described in item 51, wherein the subject has one of the genetic mutations shown in Table 1. (Item 52) The method, composition, or use described in any one of items 14 to 46, wherein the subject is suffering from transthyretin amyloid neuropathy. (Item 53) The method, composition, or use described in item 52, wherein the subject has one of the genetic mutations Val30Met, Ile107Val, and Ser77Tyr. (Item 54) The aforementioned conditions include cancer, diabetes, human immunodeficiency virus (HIV) infection, thyroid disorders, hypothyroidism, hypoglycemia, uremia, renal failure, hepatic dysfunction, hepatic failure, polycythemia, connective tissue disorders, Lyme disease, celiac disease, leprosy, porphyria, Sjögren's syndrome, poliovirus infection, acromegaly, lipid / glycolipid metabolism disorders, West Nile disease, amyloidosis, mitochondrial disorders, abnormal protein disorders, benign monoclonal gamma globulinemia (MGUS), POEMS syndrome, nutritional / vitamin deficiencies, and vitamin B 12 The method, composition, or use described in any one of items 14-46 for a patient suffering from acquired neuropathy caused by a deficiency, vitamin E deficiency, or copper deficiency. (Item 55) The method, composition, or use described in any one of items 14 to 46, wherein the subject suffers from hereditary myopathy, peripheral neuropathy, toxic neuropathy, autoimmune peripheral polyneuropathy, acute inflammatory demyelinating polyneuropathy (AIDP), chronic inflammatory demyelinating polyneuropathy (CIDP), mononeuropathy due to vasculitis, 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 can be more easily understood by referring to the following figures. [Brief explanation of the drawing]

[0098] [Figure 1-1] Graphs and images illustrating AAV1.NT-3-induced fiber type remodeling in TrJ muscle are shown. Representative images of SDH-stained tissue sections of AAV1.tMCK.NT-3-treated Trembler J (TrJ) (Figure 1A) and untreated (TrJ-PBS) gastrocnemius muscle (Figure 1B) 16 weeks after injection. Slow-twitch oxidative (STO, arrows), fast-twitch oxidative (FTO, arrowheads), and fast-twitch glycolytic (FTG, asterisks) fibers are shown (Figure 1B). Aerobic fibers (oxidative fibers) are reduced in a. In TrJ-PBS muscle (Figure 1B), an increased number of small STO fibers and angular fibers of all fiber types are present along smaller populations of fibers that conform to the neuronal changes. Scale bars for a and b = 30 gm. Switching of fiber type from STO to FTO / FTG fibers in TrJ muscle by NT-3 gene therapy (Figure 1C). The mean percentage of STOs 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 in the normalization of fiber type distribution by NT-3 in TrJ neurogenic muscle. [Figure 1-2]Graphs and images illustrating AAV1.NT-3-induced fiber type remodeling in TrJ muscle are shown. Representative images of SDH-stained tissue sections of AAV1.tMCK.NT-3-treated Trembler J (TrJ) (Figure 1A) and untreated (TrJ-PBS) gastrocnemius muscle (Figure 1B) 16 weeks after injection. Slow-twitch oxidative (STO, arrows), fast-twitch oxidative (FTO, arrowheads), and fast-twitch glycolytic (FTG, asterisks) fibers are shown (Figure 1B). Aerobic fibers (oxidative fibers) are reduced in a. In TrJ-PBS muscle (Figure 1B), an increased number of small STO fibers and angular fibers of all fiber types are present along smaller populations of fibers that conform to the neuronal changes. Scale bars for a and b = 30 gm. Switching of fiber type from STO to FTO / FTG fibers in TrJ muscle by NT-3 gene therapy (Figure 1C). The mean percentage of STOs 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 in the 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 analyses of mTOR targets, Phospho(P)-4EBP1 (Thr37 / 46), and P-S6 (Ser235 / 236) in TrJ (Figure 2A) and wild-type (WT) (Figure 2B) gastrocnemius muscle 16 weeks after injection. Graphs show the expression levels of phosphorylated protein forms normalized to GAPDH. Coomassie blue stained membranes represent equivalent gel loading. Error bars are ±SEM; n=5-6 in each group, *P<0.05, unpaired t-test. (Figure 2C) Relative expression of glycolysis (1-1K1 and PK1) and aerobic system 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 test 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 analyses of mTOR targets, Phospho(P)-4EBP1 (Thr37 / 46), and P-S6 (Ser235 / 236) in TrJ (Figure 2A) and wild-type (WT) (Figure 2B) gastrocnemius muscle 16 weeks after injection. Graphs show the expression levels of phosphorylated protein forms normalized to GAPDH. Coomassie blue stained membranes represent equivalent gel loading. Error bars are ±SEM; n=5-6 in each group, *P<0.05, unpaired t-test. (Figure 2C) Relative expression of glycolysis (1-1K1 and PK1) and aerobic system 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 test followed by Tukey's multiple comparison test. [Figure 3-1]Graphs and images showing the direct effects of NT-3 on myotubes are shown. (Figure 3A) Representative Western blot images and analyses 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 minutes. The density values ​​of the phosphorylated protein bands were normalized to GAPDH and shown as a percentage in the control group. Coomassie blue stained membranes represent equivalent gel loading. Myotubes were incubated with NT-3 (100 ng / ml) for 48 hours, and then the relative mRNA expression of metabolic markers (PGC1a, HK1, PK1) was detected by qPCR (Figure 3B), and glucose consumption for lactate production in cell culture medium was detected by ELISA (Figure 3C). (Figure 3D) Relative expression levels of myogenin, NT-3 receptor, P75NTR, and TrkC in myoblasts relative to myotubes after 48 hours of NT-3 (100 ng / ml) treatment. GAPDH was used as a housekeeping gene in the analysis. Results shown are 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 effects of NT-3 on myotubes are shown. (Figure 3A) Representative Western blot images and analyses 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 minutes. The density values ​​of the phosphorylated protein bands were normalized to GAPDH and shown as a percentage in the control group. Coomassie blue stained membranes represent equivalent gel loading. Myotubes were incubated with NT-3 (100 ng / ml) for 48 hours, and then the relative mRNA expression of metabolic markers (PGC1a, HK1, PK1) was detected by qPCR (Figure 3B), and glucose consumption for lactate production in cell culture medium was detected by ELISA (Figure 3C). (Figure 3D) Relative expression levels of myogenin, NT-3 receptor, P75NTR, and TrkC in myoblasts relative to myotubes after 48 hours of NT-3 (100 ng / ml) treatment. GAPDH was used as a housekeeping gene in the analysis. Results shown are mean ± SEM from at least three independent experiments (*P<0.05, Student's paired t-test). [Figure 4] The graph shows the blood concentrations 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] This study shows the relative mRNA expression of P75NTR and TrkC in TrJ and WT gastrocnemius muscles. GAPDH was used as a housekeeping gene in the analysis. The results shown are 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] The constraint map and ORF analysis pAAV.tMCK.NTF3 are shown. [Figure 8] This 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. [Modes for carrying out the invention]

[0099] We investigated the increase in fiber size induced by AAV.NT-3 treatment in TrJ muscle to determine whether this increase is simply a result of nerve regeneration, or whether NT-3 can directly affect muscle protein synthesis unrelated to nerve regeneration, thereby increasing muscle fiber size.

[0100] The novel effects of NT-3, including its ability to directly influence protein synthesis and metabolic remodeling in neurogenic muscle, are disclosed herein.

[0101] The studies described herein first evaluated the effect of AAV.NT-3 gene therapy on the oxidative state of TrJ muscle 16 weeks after gene injection and found that increased muscle fiber size was associated with changes in the oxidative state of muscle fibers relative to the normalization of fiber type ratios observed in the wild. Treatment resulted in a decrease in the percentage of slow-twitch (STO) fibers, while increasing the intermediate and fast-twitch (FTO and FTG) fiber populations, reflecting a reversal of the pattern observed in untreated TrJ muscle. The increase in fiber size induced by NT-3 was most pronounced in the FTG fiber population. Next, we investigated whether mammalian targets of rapamycin complex 1 (mTORC1) activation played a role in NT-3 induced muscle protein synthesis, particularly with an emphasis on the preferential hypertrophic growth of glycolytic fibers. mTORC1 regulates translation and ribosome biosynthesis through the 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 associated with the activation of cellular glycolysis, and the activation of cellular glycolysis is involved in the increased translation of glycolytic enzymes or their transcription 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) as evidence of mTORC1 activation. In parallel, the expression levels of mitochondrial biosynthesis regulators (peroxisome proliferator-activated receptor y 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 can directly induce Akt / mTOR pathway activation in TrkC-expressing myotubes, but not in myoblasts. Furthermore, myogenin expression levels were significantly higher in myotubes, while p75NTR expression was downregulated compared to myoblasts, indicating that NT-3-induced myoblast differentiation is related to mTORC1 activation.

[0102] The findings described herein offer many suggestions for the potential use of NT-3 not only for the treatment of neuropathy affecting both nerves and muscles, but also for muscle wasting diseases including aging, cancer cachexia or type II muscle fiber atrophy, as well as for hereditary or acquired autoimmune primary muscle diseases associated with impaired hypertrophic growth phase of regeneration, which may involve disruption of mTORC1 signaling and defective mitochondrial biosynthesis.

[0103] This disclosure relates to a method for stimulating muscle growth in subjects. The method includes 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. Subjects in need of muscle growth stimulation 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 the subject using autocomplementary adeno-associated virus (scAAV) type 1 under the control of a muscle-specific tMCK promoter. In another embodiment, the present invention provides a gene therapy method for increasing muscle strength in a subject in need, for example, a subject diagnosed with or suffering from a muscle wasting disease such as CMT.

[0105] Preclinical trials were conducted using trembler J mouse 9 (Tr), a natural mouse model for CMT1. J The delivery of the construct AAV1.tMCK.NTF3 to the gastrocnemius muscle demonstrated improvements in nerve regeneration, myelin formation, myelinated fiber density, sciatic nerve complex muscle action potential amplitude, and functional performance and hind limb grip strength in the rotorod test (see Example 3).

[0106] As used herein, terms such as “treatment” and “treating” refer to obtaining a desired pharmacological or physiological effect. This effect may be therapeutic in that it results in the partial or complete cure of a disease or adverse effects caused by a disease. As used herein, “treatment” encompasses any treatment of a disease in mammals, particularly humans, and may include suppressing a disease or condition, i.e., stopping its onset; and reducing a disease, i.e., causing its regression.

[0107] In this specification, prevention refers to any effect that provides benefit to subjects at risk of developing conditions or diseases such as 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 subjects for the methods described herein, without excessive adverse side effects, taking into consideration 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 drug that achieves the goal of improving the severity and incidence of the disease. Therapeutic effectiveness may be measured by evaluating the reduction of symptoms in subjects in response to NT-3 administration.

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

[0111] As used herein, the term “diagnosis” may encompass determining the likelihood of a subject developing a disease, or the presence or nature of a disease in a subject. As used herein, the term “diagnosis” may also encompass determining the severity of the disease and the likelihood of its outcome or the onset or recovery from the disease (generally referred to as prognosis). “Diagnosis” may also encompass a diagnosis in relation to reasonable treatment, where the diagnosis guides treatment, including the initial selection of treatment and modifications to treatment (e.g., adjustment of dosage or medication plan).

[0112] As used herein, “subject” can be any animal and may also be called a patient. Preferably, the subject is a vertebrate, and more preferably, the subject is a mammal such as livestock (e.g., cattle, horses, pigs) or pets (e.g., dogs, cats). 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 nucleotides in length. This term includes DNA molecules (e.g., eDNA or genomic or synthetic DNA) and RNA molecules (e.g., mRNA or synthetic RNA), as well as DNA or RNA analogs containing unnatural nucleotide analogs, unnatural nucleoside bonds, or both. Nucleic acids can have any topological structure. For example, nucleic acids can be single-stranded, double-stranded, triple-stranded, quadruple-stranded (quadruplexed), partially double-stranded, branched, hairpin, circular, or padlocked conformation.

[0114] As used herein, the term “gene” refers to a nucleotide sequence that directs the synthesis of an enzyme or other polypeptide molecule (e.g., may include coding sequences, e.g., consecutive open reading frames (ORFs) that code for a polypeptide) or that may be functional in itself in an organism. Genes in an organism can be clustered in an operon as defined herein, where an operon is separated from other genes and / or operons by intergenetic DNA. Individual genes contained within an operon can overlap without intergenetic DNA between them.

[0115] As used herein, the term "AAV" is a standard abbreviation for adeno-associated virus. Adeno-associated viruses are single-stranded DNA parvoviruses that grow only within cells, with specific functions provided by co-infecting helper viruses. There are currently 13 characterized serotypes of AAV. General information and an overview 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, since it is well known that various serotypes are quite closely related structurally and functionally, even at the genetic level, these same principles can be applied to further AAV serotypes. (See, for example, Blacklowe, 1988, pp. 165-174 of Parvoviruses and Human Disease, JR Pattison, ed.; and Rose, Comprehensive Virology 3:1-61 (1974)). For example, all AAV serotypes appear to exhibit very similar replication characteristics mediated by homologous rep genes; all possess three related capsid proteins, such as those expressed in AAV2. The similarity is further suggested by extensive cross-hybridization between serotypes along genome length; and heteroduplex analysis revealing the presence of similar self-annealing regions at the ends corresponding to “inverted terminal repeats” (ITRs). Similar infectivity patterns also suggest that replication function in each serotype is under similar regulatory control.

[0116] The terms “vector” or “expression vector” refer to any type of genetic construct containing nucleic acid that encodes RNA that can be transcribed. Expression vectors may contain various regulatory sequences, structural genes (e.g., the gene of interest), and nucleic acid sequences that perform other functions as well.

[0117] As used herein, the term "vector" generally refers to a DNA molecule derived from a plasmid or bacteriophage, into which a DNA fragment can be inserted or cloned. A recombinant vector contains one or more unique restriction sites and can be self-replicating in a defined host or intermediate organism such that the cloned sequence is reproducible. A vector contains a promoter operably linked to a gene or coding region such that when the vector is transfected into a recipient cell, RNA is expressed.

[0118] As used herein, an "AAV vector" refers to a vector containing one or more polynucleotides (or transgenes) of interest flanked by AAV inverted terminal repeats (ITRs). Such AAV vectors can be replicated and packaged into infectious virus 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 virus particle" or "AAV vector particle" refers to a virus particle composed of at least one AAV capsid protein and a capsid-formed polynucleotide AAV vector. If the particle contains a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome such as a transgene to be delivered to a mammalian cell), it is typically referred to as an "AAV vector particle" or simply an "AAV vector". Thus, the production of AAV vector particles necessarily includes the production of AAV vectors because such vectors are contained within the AAV vector particles.

[0120] As used herein, the term "about" refers to a deviation of + / - 10% from a 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 muscular atrophy 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 plasmid. The advantage of herpes simplex virus vectors is their natural tropism for sensory neurons. However, viral vectors related to adenovirus are the most popular because of their low risk of insertional mutagenesis and immunogenicity, the absence of their endogenous viral genes, and their ability to be produced at high titers. While Kantor et al. have reviewed various methods of gene delivery into the central nervous system, Goins et al. have described methods of gene therapy for the treatment of chronic pain in the peripheral nervous system. 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, which are resident glial cells of the peripheral nerves, has been reported using various viral vectors. Mason et al., Curr. Gene Ther. 11, 75-89 (2011). When the vector is within a viral vector and the vector is packaged, the virion can be used to infect cells. When naked DNA is used, transfection or transformation procedures such as those suitable for a particular host cell can be used. Formulations of naked DNA using polymers, liposomes, or nanospheres can be used for gene delivery. The nucleic acid can be administered in any desired format that provides a sufficiently efficient delivery level, including complexation to viral particles, liposomes, nanoparticles, and polymers.

[0124] Nucleic acids (e.g., cDNA or transgenes) that encode genes whose expression alleviates peripheral neuropathy can be cloned into expression cassettes having regulatory elements such as (constitutive or regulated) promoters that drive transgene expression and the downstream polyadenylation sequences of the nucleic acid. For example, regulatory elements that are 1) specific to a tissue or region of the body; 2) constitutive; and / or 3) inducible / regulated may be used.

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

[0126] AAV is the preferred vector for use in treating muscle atrophy by gene therapy. Gene delivery mediated by AAV 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 of neurological disorders, and no adverse effects associated with the use of this vector have been reported from clinical trials to date: Adeno-associated viruses are nonpathogenic dependent viruses of the Parvoviridae family that require helper functions from other viruses, such as adenoviruses or herpes simplex viruses, to complete their life cycle. Wild-type (WT) AAV is characterized by a single-stranded DNA (ssDNA) genome with approximately 5kb inverted terminal repeat sequences (ITRs) at both ends surrounded by a capsid.

[0127] Adenovirus vectors used to deliver transgenes to cells for applications such as in vivo gene therapy, in vitro testing, and / or production of transgene products are generally obtained from adenoviruses by deleting the initial region 1 (El) gene (Berkner, KL, Curr. Top. Micro. Immunol. 158 L39-66 1992). The deletion of the El gene results in defects in the replication of such adenovirus vectors, significantly reducing the expression of any remaining viral genes present within the vector. Recombinant adenovirus vectors have several advantages for use as gene delivery vehicles, including tropism to both dividing and non-dividing cells, potential for minimal pathogenicity, the ability to replicate to high titers for vector strain preparation, and the ability to carry large inserts. However, the presence of remaining viral genes in adenovirus vectors is considered potentially detrimental.

[0128] Therefore, in one embodiment, an adenovirus vector having deletions of various adenovirus gene sequences. In particular, a pseudoadenoviral vector (PAV), also known as a "gutless adenovirus" or miniadenovirus vector, is an adenovirus vector derived from an adenovirus genome that contains the minimum cis-acting nucleotide sequence required for the replication and packaging of the vector genome and may contain one or more transgenes (see U.S. Patent No. 5,882,877, incorporated herein by reference, which covers pseudoadenovirus vectors (PAVs) and methods for producing PAVs). Such PAVs, capable of accommodating up to approximately 36 kb of foreign nucleic acid, are advantageous because they optimize the vector's carrying capacity while reducing the possibility of a host immune response to the vector or the generation of a virus capable of replication. A PAV vector contains a 5' inverted terminal repeat (ITR) and a 3' ITR nucleotide sequence that contain the origin of replication, as well as a cis-acting nucleotide sequence required for packaging the PAV genome, and can accommodate one or more transgenes along with appropriate regulatory elements, such as promoters and enhancers.

[0129] AAV The recombinant AAV genome of the present invention comprises the nucleic acid molecule of the present invention and one or more AAV ITRs adjacent to the nucleic acid molecule. The AAV DNA in the rAAV genome may be derived from any AAV serotype from which the recombinant virus may originate, 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 (e.g., Gao et al., PNAS, 99:11854-11859 (2002); and Viral Vectors for Gene Therapy: Methods and Protocols, ed. Machida, Humana). See Press, 2003). Furthermore, pseudotype AAV vectors may also be used in the methods described herein. Pseudotype AAV vectors contain the genome of one AAV serotype in the capsid of a second AAV serotype; for example, an AAV vector containing an AAV2 capsid and an AAV1 genome, or an AAV vector containing an AAV5 capsid and an AAV2 genome. (Auricchio et al., (2001) Hum. Mol. Genet., 10(26):3075-81). The production of pseudotype rAAV is disclosed, for example, in International Publication No. 01 / 83692. Other types of rAAV variants, such as rAAV with capsid mutations, are also conceivable. For example, Marsic et al., Molecular See Therapy, 22(11):1900-1909 (2014). As shown in the background section above, nucleotide sequences of various AAV serotype genomes are known in the art. AAV1, AAV6, AAV8, or AAVrh.74 may be used to promote skeletal muscle-specific expression.

[0130] The DNA plasmid of the present invention comprises the rAAV genome of the present invention. The DNA plasmid is transferred to a cell tolerant of infection by an AAV helper virus (e.g., adenovirus, E1 deletion adenovirus, or herpesvirus) for assembly of the rAAV genome into infectious viral particles. Techniques for producing rAAV particles, in which the AAV genome, rep and cap genes, and helper virus function are provided to the cell to be packaged, are standard in the art. The production of rAAV requires that the following components be present in a single cell (indicated herein as the packaging cell): the rAAV genome, the AAV rep and cap genes isolated from the rAAV genome (i.e., not in the rAAV genome), and the helper virus function. The AAV rep and cap genes may be derived from any AAV serotype from which the recombinant virus may originate, and may be derived from an AAV serotype different from the rAAV genome ITR, 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 International Publication No. 01 / 83692, which is incorporated herein by reference in its entirety.

[0131] The 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 an rAAV genome lacking the AAV rep and cap genes, the AAV rep and cap genes isolated from the rAAV genome, and selectable markers such as the neomycin resistance gene, is incorporated into the cell 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 a synthetic linker 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). Next, the packaging cell line is infected with a helper virus such as adenovirus. The advantages of this method are that the cells are selectable and it is suitable for large-scale production of rAAV. Another example of a suitable method is to use adenovirus or baculovirus instead of plasmid to introduce the rAAV genome and / or rep and cap genes into packaging cells.

[0132] The general principles of rAAV production are outlined, 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 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); US Patent No. 5,173,414; International Publication No. 95 / 13365 and corresponding US Patent No. 5,658,776; International Publication No. 95 / 13392; International Publication No. 96 / 17947; PCT / US98 / 18600; International Publication No. 97 / 09441 (PCT / US96 / 14423); International Publication No. 97 / 08298 (PCT / US96 / 13872); International Publication No. 97 / 21825 (PCT / US96 / 20777); International Publication No. 97 / 06243 (PCT / FR96 / 01064); International Publication No. 99 / 11764; Perrin et al. This information is found in: 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 No. 5,786,211; U.S. Patent No. 5,871,982; and U.S. Patent No. 6,258,595. The above literature is incorporated herein by reference in whole, with particular emphasis on the literature relating to rAAV production.

[0133] Therefore, the present invention provides packaging cells that produce infectious rAAV. In one embodiment, the packaging cells may be stably transformed cancer cells such as HeLa cells, 293 cells, and PerC.6 cells (same 293 cell lines). In another embodiment, the packaging cells may be non-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 monkey fetal lung cells).

[0134] The recombinant AAV of the present invention (i.e., infectious capsidized rAAV particles) comprises an rAAV genome. In exemplary embodiments, 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 may be configured to contain the nucleic acid molecules of the present invention are described in International Patent Application No. PCT / US2012 / 047999 (International Publication No. 2013 / 016352), which is incorporated herein by reference in whole.

[0135] rAAV can be purified by methods standard 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. Patent No. 6,566,118 and International Publication No. 98 / 09657.

[0136] In another embodiment, the present invention envisions a composition comprising the rAAV of the present invention. The composition of the present invention comprises rAAV and a pharmaceutically acceptable carrier. The composition may also include other components such as diluents and auxiliary agents. Acceptable carriers, diluents and auxiliary agents are harmless to the recipient and preferably inactive at the dose and concentration used, and include buffers such as phosphates, citrates, 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 nonionic surfactants such as Tween, Pluronics®, or polyethylene glycol (PEG).

[0137] The titer of rAAV administered in the method of the present invention varies depending, for example, on the specific rAAV, the method of administration, the therapeutic target, the individual, and the targeted cell type, and can be determined by methods standard in the art. The titer of rAAV is approximately 1 × 10⁶ 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 Or it may be in the range of DNase-resistant particles (DRPs) or higher. The dose may also be expressed in units of viral genome (vg).

[0138] The present invention envisions a method for transducing target cells with rAAV in vitro or in vitro. The in vivo method comprises administering an effective dose, or multiple effective doses, of a composition comprising rAAV of the present invention to an animal (including humans) 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 a dose that alleviates (eliminates or reduces) at least one symptom associated with the disorder / disease condition being treated, slows or prevents the progression of the disorder / disease condition, reduces the severity of the disease, results in disease remission (partial or complete), and / or prolongs survival.

[0139] In particular, the actual administration of rAAV according to the present invention may be carried out by using any physical method to deliver the rAAV recombinant vector to the target tissue of an animal. Administration according to the present invention includes, but is not limited to, injection into muscle, 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 carriers or other components that may be co-administered with rAAV (however, compositions that degrade DNA should be avoided in conventional methods using rAAV). The capsid protein of rAAV may be modified to target specific target tissues, such as muscle, which rAAV is intended for. See, for example, International Publication No. 02 / 053703, which is incorporated herein by reference in whole. Pharmaceutical compositions may be prepared as injectable formulations or as topical formulations delivered to muscle by transdermal transport. Many formulations for both intramuscular injection and transdermal transport have been previously developed and may be used in the implementation of the present invention. 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 may be 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, and muscle creatine kinase sequence elements [Johnson et al.]. The present invention provides a method for transduction into muscle cells and muscle tissue induced by muscle-specific regulatory elements, including but not limited to: regulatory elements derived from mouse creatine kinase enhancer (mCK) elements, skeletal fast-twitch muscle troponin C gene, slow-twitch muscle cardiac troponin C gene, and slow-twitch muscle troponin I gene; promoters including hypoxia-inducible nuclear factors (Semenza et al., Proc Natl Acad Sci USA, 88:5680-5684 (1991)), steroid-inducible elements, and glucocorticoid response elements (GRE) (see 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. This invention envisions the sustained expression of miRNAs from transduced muscle fibers.

[0142] "Muscle cells" or "muscle tissue" means cells or groups of cells derived from any type of muscle (e.g., skeletal muscle and smooth muscle from the digestive tract, bladder, blood vessels, or cardiac tissue). Such muscle cells, such as myoblasts, muscle cells, myotubes, cardiomyocytes, and cardiomyocytes, may 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-defective rAAV of the present invention, which results in the 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 have developed an AAV expression cassette carrying the human NT-3 cDNA coding sequence under the control of either a CMV promoter or a triple muscle-specific creatine kinase (tMCK) promoter. The inventors have previously shown that improvement in the motor function, histopathology, and electrophysiology of the peripheral nerves can be achieved using a recombinant AAV1 vector and that it can increase neurotrophin-3 expression in tremble (Try) mice, a model of Charcot-Marie-Tooth disease variant CMT1A. See Sahenk et al., Mol Ther. 22(3):511-21(2014), the disclosure of which is incorporated herein by reference.

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

[0146] Dosage and Route of Administration The present invention provides for local and systemic administration of an effective dose of rAAV and the compositions of the present invention, including the combination therapies of the present invention. For example, systemic administration is administration to the circulatory system such that the whole body is affected. Systemic administration includes enteral administration such as absorption through the gastrointestinal tract, and parenteral administration via injection, infusion, or transplantation.

[0147] Therefore, the 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 may vary depending, for example, on the specific rAAV, administration method, therapeutic goal, individual, and targeted cell type, and may be determined by methods standard in the art. Two or more doses, for example, one, two, three, or more doses may be administered. The potency of rAAV in the dose is approximately 1 × 10⁶ 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 x 10 13 , about 1×10 14 , or ~approximately 1 x 10 15 Or it may be in the range of DNase-resistant particles (DRPs) or higher. The dose may also be expressed in units of viral genome (vg) (i.e., 1 × 10⁻⁶ each). 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 15A method for titrating AAV is described in Clark et al., Hum. Gene Ther., 10:1031-1039 (1999).

[0149] In one embodiment of the above method, where the administration route is the intramuscular (IM) route, the dose of rAAV administered is approximately 1.5 × 10⁻⁶. 12 ~At least approximately 6.5 × 10 12 The values ​​are in vg / kg. (All ranges in this specification are intended to represent the individual values ​​within that range, as well as the individual upper and lower limits within each range.) In one embodiment of the above method, where the route of administration is intramuscular (IM), the dose of rAAV administered is 2 × 10⁻¹⁶. 12 The value is vg / kg. In one embodiment of the above method, where the route of administration is intramuscular (IM), the dose of rAAV administered is 4 × 10⁻¹⁴. 12 The value is vg / kg. In one embodiment of the above method, where the route of administration is intramuscular (IM), the dose of rAAV administered is 6 × 10⁻¹⁴. 12 It is vg / kg.

[0150] Human patients are the subjects envisioned herein for treatment. Human patients are the subjects envisioned herein for treatment by intramuscular (IM) delivery. Such patients include, for example: i) adult subjects (over 18 years of age) diagnosed with CMT1A, ii) showing a 1.5 Mb duplication in 17p11.2 including the peripheral myelin protein 22 (PMP22) gene, iii) males and females of any ethnic or racial group, iv) showing decreased ankle dorsiflexion strength (should have full range of motion against gravity but be unable to maintain full dorsiflexion against gravity or be unable to stand on tiptoe for more than 3 seconds (Northstar criteria)), iv) abnormal nerve conduction velocity, v) ability to cooperate with clinical evaluation and repetitive nerve conduction studies, and vi) sexually active subjects, willingness to use reliable contraception during the study. Suitable patients should not include, for example, those having: i) Active viral infection based on clinical observation or serological evidence of HIV or hepatitis A, B, or C infection; ii) Ongoing immunosuppressive therapy or immunosuppressive therapy within 6 months of the start of the trial (e.g., corticosteroids, cyclosporine, tacrolimus, methotrexate, cyclophosphamide, intravenous immunoglobulin); iii) Persistent leukopenia or leukocytosis (WBC ≤ 3.5 K / μL or ≥ 20.0 K / μL) or 1.5 K / μL iv) absolute neutrophil count less than L, AAV1-binding antibody titer greater than 1:50 as determined by ELISA immunoassay, v) need for comorbidity or long-term drug therapy due to the unnecessary risk of gene transfer, in the opinion of the principal investigator (PI), vi) ankle contracture or surgery that interferes with appropriate muscle strength testing, vii) pregnancy, lactation, or planning for pregnancy, viiii) other causes of neuropathy, and / or ix) limb surgery within the past six months. In an exemplary clinical protocol, CMT1A patients are administered a total amount of vector scAAV1.tMCK.NTF3 divided into 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 are administered one of the following: i) 2 × 10 12 ii) Low-dose vector in vg / kg (total amount) or 6 × 1012 High-dose vectors in vg / kg (total amount).

[0151] In one embodiment, the vector is administered by intramuscular (IM) injection without a diluent. In an alternative embodiment, the composition for intramuscular injection may include an adjuvant such as sesame oil or peanut oil, or an aqueous solution of propylene glycol and a sterile aqueous solution may be used. Such aqueous solutions may be buffered as needed, and the liquid diluent may first be isotonic with physiological saline or glucose. Solutions of rAAV as a free acid (DNA containing acidic phosphate groups) or a pharmacokinetically acceptable salt may be suitably prepared by mixing with a surfactant such as hydroxypropyl cellulose in water. Dispersions of rAAV may also be prepared in glycerol, liquid polyethylene glycol and mixtures thereof, and in oil. Under normal storage and use conditions, these preparations contain preservatives to prevent microbial growth. In this regard, all sterile aqueous media used can be readily obtained by standard techniques well known to those skilled in the art.

[0152] Suitable pharmaceutical carriers, diluents, or excipients for injection applications include sterile aqueous solutions or dispersions, and sterile powders for the immediate preparation of sterile injection solutions or dispersions. In all cases, the form must be sterile and fluid enough to allow for easy injection. It must be stable under manufacturing and storage conditions and protected from contamination by microorganisms such as bacteria and fungi. Carriers may be solvents or dispersion media, including, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Adequate fluidity can be maintained, for example, by the use of coatings such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action can be achieved by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosar, etc. In many cases, it is preferable to include isotonic agents, such as sugars or sodium chloride. Long-term absorption of injectable compositions can be achieved by using absorption-delaying agents, such as aluminum monostearate and gelatin.

[0153] Sterile injectable solutions are prepared by incorporating the required amount of rAAV, along with various other components listed above as needed, into a suitable solvent, followed by filtration sterilization. Generally, dispersions are prepared by incorporating a sterile active ingredient into a sterile vehicle containing a basic dispersion medium and other necessary components listed above. For sterile powders for the preparation of sterile injectable solutions, preferred preparation methods are vacuum drying and freeze-drying techniques, which produce powders of the active ingredient and any additional desired components from its pre-sterile filtered solution.

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

[0155] In another embodiment, an rAAV genome is provided herein. The rAAV genome to be administered contains NT-3 polynucleotides under the control of transcriptional regulatory sequences. The rAAV genome lacks AAV rep and cap DNA. The AAV DNA in the rAAV genome may be derived from any AAV serotype from which the recombinant virus may originate, 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 shown in the background section above, the nucleotide sequences of the genomes of these AAV serotypes are known in the art.

[0156] In one embodiment, 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 the myosin heavy chain (Salva et al. These are muscle-specific regulatory elements, including, but not limited to, the desmin promoter, regulatory elements derived from the skeletal fast-twitch muscle troponin C gene, the slow-twitch muscle cardiac troponin C gene, and the slow-twitch muscle troponin I gene: hypoxia-inducible nuclear factor (Semenza et al., Proc. Natl. Acad. Sci. USA, 88:5680-5684 (1991)), promoters including steroid-inducible elements and glucocorticoid response elements (GRE) (see Mader and White, Proc. Natl. Acad. Sci. USA, 90:5603-5607 (1993)), and other regulatory elements. In some embodiments, the transcriptional regulatory element is the MCK promoter. In some embodiments, the transcriptional regulatory element is the MHCK7 promoter.

[0157] In one embodiment, the NT-3 polynucleotide in the rAAV genome is the NT-3 cDNA described in SEQ ID NO: 1 (corresponding to nucleotides 1077-1850 of SEQ ID NO: 11). In another embodiment, the NT-3 polynucleotide in the rAAV genome is either the NT-3 cDNA described in Genbank accession number NM_001102654 or the NT-3 cDNA sequence described in SEQ ID NO: 1, or a mutant 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 another embodiment, the mutant 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 described as SEQ ID NO: 1 or provided as Genbank accession number NM_001102654 is described as SEQ ID NO: 2. In one embodiment, 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 described as SEQ ID NO: 1 or provided as Genbank accession number NM_001102654 (SEQ ID NO: 2). The amino acid sequence change may be, for example, a substitution, deletion, or insertion of one or more amino acids, preferably a conserved substitution. The mutant NT-3 polypeptide may have any combination of amino acid substitutions, deletions, or insertions, provided that polypeptide activity is preserved. In one embodiment, a mutant NT-3 polypeptide may 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 (Sequence ID 2) encoded by the NT-3 cDNA described as Sequence ID 1 or provided as Genbank accession number NM_001102654.

[0158] In one embodiment, the rAAV genome is the AAV.tMCK.NTF3 genome, and the sequence of its NT-3 gene cassette is described in Sequence ID No. 11 and annotated in Table 4 (see Example 3).

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

[0160] Isolated nucleic acids are also provided, comprising, in 5' to 3' order: (i) a first AAV2 inverted terminal repeat sequence (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 1077-1850 of SEQ ID NO: 11.

[0161] Recombinant AAVs containing the above nucleic acids, as well as rAAVs containing nucleotide sequences that are at least 90% identical to the nucleotide sequence shown in Sequence ID No. 1, are envisioned.

[0162] A DNA plasmid containing the rAAV genome of this disclosure is provided. The DNA plasmid contains the rAAV genome as envisioned herein. The DNA plasmid is transferred to a cell tolerant of infection by an AAV helper virus (e.g., adenovirus, E1 deletion adenovirus, or herpesvirus) for assembly of the rAAV genome into infectious viral particles. Techniques for producing rAAV particles, in which the AAV genome, rep and cap genes, and helper virus function are provided to the cell to be packaged, are standard in the art. The production of rAAV requires that the following components be present in a single cell (indicated herein as the packaging cell): the rAAV genome, the AAV rep and cap genes isolated from the rAAV genome (i.e., not in the rAAV genome), and the helper virus function. The AAV rep and cap genes may originate from any AAV serotype from which the recombinant virus may originate, and may originate from an AAV serotype different from the rAAV genome ITR, 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 International Publication No. 01 / 83692. Other types of rAAV variants, such as rAAV with capsid mutations, are also conceivable. See, for example, Marsic et al., Molecular Therapy, 22(11):1900-1909 (2014).

[0163] The 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 an rAAV genome lacking the AAV rep and cap genes, the AAV rep and cap genes isolated from the rAAV genome, and selectable markers such as the neomycin resistance gene, is incorporated into the cell 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 a synthetic linker 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). Next, the packaging cell line is infected with a helper virus such as adenovirus. The advantages of this method are that the cells are selectable and it is suitable for large-scale production of rAAV. Other examples of suitable methods include using adenoviruses or baculoviruses instead of plasmids to introduce the rAAV genome and / or rep and cap genes into packaging cells. Methods for producing rAAV using self-complementary genomes are also known in the art.

[0164] The general principles of rAAV production are outlined, 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 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); US Patent No. 5,173,414; International Publication No. 95 / 13365 and corresponding US Patent No. 5,658,776; International Publication No. 95 / 13392; International Publication No. 96 / 17947; PCT / US98 / 18600; International Publication No. 97 / 09441 (PCT / US96 / 14423); International Publication No. 97 / 08298 (PCT / US96 / 13872); International Publication No. 97 / 21825 (PCT / US96 / 20777); International Publication No. 97 / 06243 (PCT / FR96 / 01064); International Publication No. 99 / 11764; Perrin et al. This information is found in: 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 No. 5,786,211; U.S. Patent No. 5,871,982; and U.S. Patent No. 6,258,595. The above literature is incorporated herein by reference in whole, with particular emphasis on the literature relating to rAAV production.

[0165] Accordingly, in a further embodiment, the disclosure provides packaging cells that produce infectious rAAV. In one embodiment, the packaging cells may be stably transformed cancer cells such as HeLa cells, 293 cells and PerC.6 cells (same 293 cell lines). In another embodiment, the packaging cells may be non-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 macaque fetal lung cells).

[0166] rAAV can be purified by methods standard 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. Patent No. 6,566,118 and International Publication No. 98 / 09657.

[0167] Therefore, in another embodiment, the disclosure assumes an rAAV comprising an NT-3 polynucleotide. In one embodiment, the rAAV comprises an AAV rh74 capsid and an NT-3 polynucleotide. In one embodiment, the rAAV genome lacks AAV rep and cap DNA. In one embodiment of the method, the rAAV is rAAVrh7.4.tMCK.NTF3. In one embodiment, the rAAV is a self-complementary genome.

[0168] In another embodiment, this disclosure envisions a composition comprising rAAV as described herein. The composition of this disclosure comprises rAAV in a pharmaceutically acceptable carrier. This composition may also include other components such as diluents. Acceptable carriers and diluents are harmless to the recipient and preferably inactive at the dose and concentration used, and include buffers such as phosphates, citrates, 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 nonionic surfactants such as Tween, Pluronics®, or polyethylene glycol (PEG). In one embodiment, the rAAV is Tris, MgCl 2、 It is 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 MgCl2, and 0.001% Pluronic® F68.

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

[0170] Sterile injectable solutions are prepared by incorporating the required amount of rAAV, along with various other components listed above as needed, into a suitable solvent, followed by filtration sterilization. Generally, dispersions are prepared by incorporating a sterile active ingredient into a sterile vehicle containing a basic dispersion medium and other necessary components listed above. For sterile powders for the preparation of sterile injectable solutions, preferred preparation methods are vacuum drying and freeze-drying techniques, which produce powders of the active ingredient and any additional desired components from its pre-sterile filtered solution.

[0171] Stimulation of muscle growth One aspect of the present invention provides a method for stimulating muscle growth in a subject, comprising 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.

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

[0173] Muscles can be classified into three types: skeletal muscle, cardiac muscle, and smooth muscle. Skeletal muscle is muscle tissue capable of generating force and transmitting that force to the skeleton, enabling breathing, movement, and maintaining posture. Cardiac muscle is the muscle of the heart. Smooth muscle is the muscle tissue of the arterial walls and intestinal walls. The methods and compositions of the present invention are mainly applied to skeletal muscle, but may also have a beneficial effect on smooth muscle. "Skeletal muscle" is defined as muscle that interacts with bones, tendons, and joints.

[0174] In one embodiment, the present invention provides a method for treating diseases, disorders, disabilities, and conditions that cause muscle weakness (also referred to herein as musculoskeletal diseases, and muscle dysfunction and muscle wasting diseases). The main categories of musculoskeletal diseases are muscular dystrophy 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 diseases or disorders (including hereditary myopathy, neuromuscular disease, muscle atrophy, drug-induced myopathy), or muscle weakness. The present invention also provides a method for treating neuropathy, such as hereditary CMT and CMT1A, and axonal and demyelinating polyneuropathy, such as chronic inflammatory demyelinating polyneuropathy. The method of treatment comprises administering to a patient in need 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 suffers from a muscle disease 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 one embodiment, NT-3 may be used to treat muscle atrophy. Muscle atrophy is a general term used to describe a condition characterized by the depletion or reduction of muscle tissue resulting from a variety of diseases, disorders, other pathological conditions, or events. Muscle atrophy may, but is not limited to, recovery from severe burns, major artificial joint replacement, neuropathic pain, peripheral neuropathy, necrotizing vasculitis, weightlessness (e.g., astronauts and cosmonauts), prolonged hospitalization, degenerative diseases (e.g., amyotrophic lateral sclerosis) and organ transplantation, as well as spinal cord injury, prolonged hemodialysis, and prolonged immobilization resulting from stroke.

[0177] In one embodiment, NT-3 may be used to treat disuse muscle atrophy. Disuse muscle atrophy is a condition characterized by the depletion or reduction of muscle tissue resulting from prolonged lack of exercise. Disuse muscle atrophy can result from, but is not limited to, recovery from severe burns, major artificial joint replacement, neuropathic pain, weightlessness (e.g., astronauts and cosmonauts), prolonged hospitalization, anorexia nervosa, organ transplantation and spinal cord injury, prolonged hemodialysis, and prolonged immobilization resulting from stroke.

[0178] In one embodiment, NT-3 may be used to treat age-related muscle atrophy. Age-related muscle atrophy is a condition characterized by the depletion or reduction of muscle tissue and its replacement by fibrous tissue associated with aging.

[0179] In one embodiment, NT-3 may be used to treat sarcopenia, a condition characterized by the depletion or reduction of muscle tissue and its replacement by fibrous tissue due to aging.

[0180] In one embodiment, NT-3 may be used to treat muscle wasting in cachexia. Cachexia is a condition characterized by weight loss, muscle atrophy, fatigue, weakness, and significant loss of appetite as a result of a chronic disease in a person who is not actively trying to lose weight. Components of muscle wasting in cachexia may, but are 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 renal failure.

[0181] In one embodiment, NT-3 may 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., trypanosomes and schistosomes) (where the subject is at risk of developing muscle atrophy).

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

[0183] This 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 “a 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 may have a maximum size. The maximum size of a blocking peptide relates to the overall size of the peptide and includes any further sequences linked to the peptide, such as a protein transduction domain. In some 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 oligopeptides, peptides, or protein sequences, or fragments, parts, or subunits thereof, and natural or synthetic molecules. The term “polypeptide” also includes amino acids linked to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and may contain any type of modified amino acids. The term “polypeptide” also includes peptides and polypeptide fragments, motifs, glycosylated polypeptides, all “mimicking” and “peptide-mimicking” 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 related to such sequences is defined herein as the percentage of amino acid residues in a candidate sequence that is identical to a known peptide, after, if necessary, the sequences have been aligned and gaps introduced to achieve maximum percent homology, and conservative substitutions are not considered part of the sequence identity. N-terminal, C-terminal, or internal extensions, deletions, or insertions into the peptide sequence should not be interpreted as affecting homology.

[0186] Substantially similar peptides include those that differ by only one or more amino acid changes, where the change, e.g., substitution, addition, or deletion of an amino acid residue, does not invalidate the properties of the relevant peptide, such as their ability to function as FAK or NANOG. Furthermore, only sequences describing or encoding proteins in which only conserved substitutions are 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 nonpolar (hydrophobic) residue such as isoleucine, leucine, or methionine with another. Similarly, the present invention envisions the substitution of a single polar (hydrophilic) residue between arginine and lysine, glutamine and asparagine, and glycine and serine. Furthermore, substitutions of basic residues such as lysine, arginine, or histidine with another, or substitutions of acidic residues such as aspartic acid or glutamic acid with another are also envisioned. Examples of non-conservative substitutions include the substitution of a nonpolar (hydrophobic) residue such as isoleucine, valine, leucine, alanine, or methionine with a polar (hydrophilic) residue such as cysteine, glutamine, glutamic acid, or lysine, and / or the substitution of a polar residue with a nonpolar residue.

[0188] The term "conservative substitution" also includes the use of chemically derivatized residues instead of non-derivatized residues, as long as the peptide retains the necessary 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 necessary ability to bind to NT-3. For example, substantially similar peptides may contain an N-terminal or C-terminal cysteine, thereby allowing the peptide to be covalently bound to a carrier protein, such as albumin, as needed. Such binding can reduce the removal of the peptide from the blood and also decrease the rate of peptide proteolysis. Furthermore, in the spirit of the present invention, peptides containing D-amino acids instead of L-amino acids are also included in the term "conservative substitution." The presence of such D-isomers can help minimize proteolytic activity and peptide removal.

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

[0190] Treatment for muscle atrophy The present invention provides a method for treating subjects having peripheral muscle atrophy. Pirvate compounds may be used to provide prophylactic and / or therapeutic treatment. Pirvate compounds may be administered prophylactically to subjects, for example, before the onset of peripheral neuropathy. Prophylactic (i.e., preventive) administration is effective in reducing the likelihood of the subsequent onset of peripheral neuropathy in subjects or reducing the severity of peripheral neuropathy that subsequently develops. Prophylactic treatment may be offered to subjects at high risk of developing peripheral neuropathy, for example, subjects with a family history of peripheral neuropathy. The expression of myelin protein 22 (PMP22) mutations accounts for 70-80% of all cases of Charcot-Marie-tooth neuropathy, and therefore their presence may be useful as a criterion for selecting patients to receive treatment with the pirvate compounds described herein.

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

[0192] CMT variant Charcot-Marie-Tooth (CMT) hereditary neuropathy refers to a group of disorders characterized by chronic motor and sensory polyneuropathy, also known as hereditary motor and sensory neuropathy. Autosomal dominant CMT neuropathy types include demyelinating (also called CMT1), non-axonal demyelinating (also called CMT2), and dominant intermediate CMT (DI-CMT). Other neuropathy equivalent to CMT include distal hereditary motor neuropathy (9dHMN), distal spinal muscular atrophy (DSMA), and Degerin-Sottas syndrome (DSS). A description and classification of CMT neuropathy is provided in Bird, GeneReviews, Seattle, Washington: University of Washington Seattle PIM 20301532, Updated 2018 Jun 28.

[0193] Currently, there are over 70 known genetic variations for CMT-related genes. These genetic variations are shown in Table 1 below, using the classification system of Magy et al. (Neurology 90:e870-6, 2018). The mode of inheritance for each CMT-related genetic variation is autosomal dominant (AD), autosomal recessive (AR), or X-linked (XL). The neuropathy for each CMT-related genetic variation is axonal (Ax), demyelinating (De), or intermediate (In). The "Other Notation" shown in Table 1 is a notation used in other classification systems, including dominant-intermediate CMT (DI-CMT), distal spinal muscular dystrophy (DSMA), hereditary sensory-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 A vector or peptide used in conjunction with certain embodiments of the present invention may be incorporated into a pharmaceutical composition suitable for administration to a subject. In certain embodiments, the pharmaceutical composition comprises the vector of the present invention and a pharmaceutically acceptable carrier. As used herein, a “pharmaceutically acceptable carrier” includes any physiologically compatible solvent, dispersion medium, coating, antimicrobial and antifungal agent, isotonic agent and absorption retarder. Examples of pharmaceutically acceptable carriers include water, physiological saline, phosphate-buffered saline, dextrose, glycerol, ethanol, and one or more combinations thereof. Often, it is preferable to include an isotonic agent in the composition, such as sugar, polyhydric alcohols such as mannitol and sorbitol, or sodium chloride. The pharmaceutically acceptable carrier may further contain small amounts of auxiliary substances, such as wetting agents or emulsifiers, preservatives, or buffers, which improve the shelf life or efficacy of the vector or pharmaceutical composition.

[0200] Vectors or peptides may be administered acutely (i.e., at the onset of the condition or immediately after an event causing muscle atrophy), prophylactically (e.g., before a scheduled surgery or before signs or symptoms appear), or during the course of muscle atrophy to reduce or improve the progression of symptoms that would otherwise occur. The timing and interval of administration may vary depending on the condition being treated and may be determined by those skilled in the art, and may be administered over periods of several hours, several days, several weeks, or longer, at intervals of several hours to several days.

[0201] Compositions containing vectors or peptides are generally administered intravenously. When administered intravenously, the composition may be combined with other components such as carriers and / or adjuvants. Peptides may also be covalently bound to protein carriers such as albumin to minimize peptide removal. There are no restrictions on the properties of other components, except that such components must be pharmaceutically acceptable, effective for their intended administration, and must not reduce the activity of the active ingredient of the composition.

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

[0203] The preparation of a sterile injection solution containing the peptides of the present invention is carried out by incorporating the required amount of these compounds, along with the various components listed above as needed, into a suitable solvent, followed by sterilization, preferably by filtration sterilization. To obtain a sterile powder, the above solution is vacuum-dried or freeze-dried as needed.

[0204] When the peptides of the present invention are administered orally, the pharmaceutical compositions containing an effective dose of the peptides may also contain an inert diluent, such as an absorbable food carrier, and may be in hard or soft-shell gelatin capsules, compressed into tablets, or in elixirs, suspensions, syrups, etc. Therefore, the peptides of the present invention are formulated in a pharmaceutically effective amount with a suitable pharmaceutically acceptable carrier in a therapeutically effective amount for convenient and effective administration.

[0205] As used herein, the terms “effective dose” or “therapeutically effective dose” refer to an amount of drug sufficient to stimulate muscle growth or to reduce or prevent muscle atrophy. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the specific therapeutic agent, the method and / or route of administration, etc. However, it will be understood that the total daily dose of the compounds and compositions of the present invention can be determined by the attending physician within reasonable medical judgment. The specific therapeutically effective dose level for any particular subject or organism will depend on a variety of factors, including the disorder being treated and its severity; 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 rate of excretion of the specific composition used; the duration of treatment; drugs used in combination with or concurrently with the specific composition used; and similar factors well known in the art of medicine.

[0206] The vector or peptide may be administered in a manner compatible with the dosage form and in a therapeutically effective amount. The systemic dose is determined according to the patient's age, weight and condition, as well as the route of administration. For example, a suitable dose of the peptide for administration to an adult is in the range of 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 dose, and more preferably in an amount of up to about 500 mg to about 1 gram per dose. Since the peptide composition of the present invention is eventually removed from the bloodstream, re-administration of the composition is indicated and preferred. [Examples]

[0207] Herein, aspects and embodiments of the present invention are illustrated by the following examples. However, there are various other embodiments that fall within the scope of the present invention, and this should not be limited to the specific examples shown herein.

[0208] Example 1 AAV1.NT-3 gene therapy increases muscle fiber diameter by activating the MTOR pathway and metabolic remodeling in a CMT mouse model. NT-3 has a well-recognized effect on peripheral nerves and Schwann cells, promoting axonal regeneration and associated myelin formation. Evaluation of the effect of AAV.NT-3 gene therapy on the oxidative state of neurogenic muscle from Trembled (TrJ) mice 16 weeks after gene injection revealed that increased muscle fiber size was associated with changes in the oxidative state of muscle fibers relative to the normalization of fiber type ratios seen in wild-type mice. The NT-3-induced increase in fiber size was most pronounced in fast-twitch muscle fiber populations. These changes in TrJ muscle were accompanied by increased phosphorylation levels of 4E-BP1 and S6 proteins, evidence of mTORC1 activation. In parallel, the expression levels of the mitochondrial biosynthesis regulator PGC 1α and glycolysis markers (HK1 and PK1) increased in TrJ muscle. In vitro studies showed that recombinant NT-3 can directly induce Akt / mTOR pathway activation in TrkC-expressing myotubes, but not in myoblasts. Along with this, myogenin expression levels were significantly higher in myotubes, while p75NTR expression was downregulated compared to myoblasts, indicating that NT-3-induced myoblast differentiation is related to mTORC1 activation. These studies are the first to show that NT-3 increases muscle fiber diameter in neurogenic muscle by directly activating the mTOR pathway, and that this increase in fiber size is more pronounced in fast-twitch muscle fibers.

[0209] method Animals, treatment protocols, and histopathology: TrJ mice (B6.D2-Pmp22Tr-J / J) and C57BL / 6 wild-type mice were obtained from Jackson Laboratory (Bar Harbor, ME). The left calf muscle of 9-12 week old TrJ mice was treated with PBS (n=6) or 3 × 10⁶ 10 One of the vg autocomplementary (sc)AAV1.tMCK.NT-3 vectors (n=6) was injected. TrJ mice from a different cohort received 1 × 10⁶ injections. 11 High NT-3 expression levels were induced in WT mice by injection of vg single-stranded AAV1.CMV.NT-3 for comparison (n=6). WT mice were given either PBS (n=6) or 3 × 10⁶ mice. 10Each mouse group was administered one of the vg scAAV1.tMCK.NT-3 vectors (n=4). The mice were euthanized, and their muscles were harvested 16 weeks after gene injection and processed for cryostat cleavage. Metabolic fiber type differentiation was evaluated using succinate dehydrogenase (SDH) enzyme histochemistry with a standard protocol established in-house. Muscle fiber type-specific diameter measurements were obtained from 12 μm thick SDH-stained sections. Three images representing three different regions of the gastrocnemius muscle (deep region mainly composed of STO, intermediate region showing a checkered appearance of STO and FTO or FTG, and surface region mainly composed of FTG fibers) along the midline axis were acquired at 20x magnification using an Olympus BX41 microscope and SPOT camera (per animal, per region, per section). This method was chosen to capture changes in the oxidative state of fibers within each region in response to metabolic changes induced by treatment. The diameters of dark (STO), intermediate (FTO), and light (FTG) muscle fibers were measured using Zeiss Axiovision LE4 software by measuring the shortest distance across the muscle fiber and expressed as a percentage of the total. The mean fiber diameter (mean ± SEM) was obtained by 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 potency: The design of an autocomplementary AAV viral vector having serotype 1 containing NT-3 under the tMCK or CMV promoter has been previously described and produced at the Viral Vector Core of Nationwide Children's Hospital (Columbus). Sahenk et al., Mol Ther, 22(3):511-521 (2014). Aliquot viruses were kept at -80°C until use. Blood samples were collected from anesthetized mice treated and untreated by intraocular hemorrhage at 6 and 16 weeks after injection, and serum was assayed for NT-3 levels using capture ELISA.

[0211] C2C12 myoblast culture and myotube formation: C2C12 myoblasts were cultured in a humidified chamber at 37°C and 5% CO2 in a 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). Myotubogenesis was induced in confluent cultured cells 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 blotting, and ELISA, were initiated 3 days after induction of myotubogenesis. 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 were collected for glucose consumption and lactate formation using a glucose and lactate assay kit (Eton Bioscience, San Diego, CA) in accordance with the manufacturer's instructions.

[0212] QPCR experiments: Total RNA was isolated from the gastrocnemius muscle 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. This information is found in 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 blotting: Frozen calf muscle blocks were cut into 20 μm thick sections and placed in small 2 ml plastic tubes (15-20 sections per block). These sections were homogenized in RIPA lysis buffer (Thermo Fisher, #89900, USA) with lx Halt protease inhibitor (Thermo Fisher, #78429, USA) and lx phosphatase inhibitor (Sigma, #P0044, USA) in three 20-second passes using an automated pellet mixer and disposable pestle. In the in vitro signal transduction assay, myoblasts and myotubes were collected in small 2 ml tubes 30 minutes after 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 concentration was measured using the BCA Protein Assay Kit (Thermo Fisher, #23252, Waltham, MA, USA). Protein samples (10-40 tg) were electrophoresed in 4-12% Bolt® Bis-Tris Plus precast 10 or 15-well polyacrylamide gel (Thermo Fisher, #NW04120BOX) and transferred to a PDVF membrane (GE Healthcare, #10600021, Pittsburgh, USA). The membrane was 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 hours at room temperature, and incubated overnight in a cool room at 4°C in TBS-TS buffer containing 5% BSA with an appropriate primary antibody. The primary antibodies used in this test 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), and anti-GAPDH (Santa cruz, #sc365062).The membrane was washed five times for 5 minutes in an orbital shaker containing TBS-T, and then incubated for 1 hour with secondary antibodies [HRP-conjugated anti-rabbit (#HAF008), HRP-conjugated anti-mouse (HAF007)] from R&D Systems (Minneapolis, MN, USA) in 5% skim milk powder in TBS-T buffer. The membrane was washed again with TBS-T in the same manner as above, and then incubated for 1–3 minutes with ECL Prime Western detection reagent (Amersham, #RPN2232 NJ, USA), followed by exposure to X-ray film (Denville, #E3018, MA, USA) using multiple exposure times. Protein bands in the film were imaged using a camera (Sony A600, Japan), and band intensity was quantified using Quantity-One software, BioRad, v.4.6.9. The relative content of the proteins analyzed in each sample was determined by normalizing the band intensity relative to the GAPDH content in the same sample. The membranes were stained with 0.1% Coomassie Brilliant Blue R (Thermo Fisher, USA), rinsed, and photographed to confirm equivalent protein packing in each lane.

[0214] Statistics: Statistical analysis was performed using GraphPad Prism 6 software with one-way analysis of variance (ANOVA) to compare muscle fiber size between the treatment and untreated groups. Student's t-test or one-way ANOVA was performed where applicable to other statistical analyses. The significance level was set at P<0.05. Results for all experiments are presented as mean ± SEM.

[0215] result AAV1.NT-3-induced fiber type remodeling in TrJ muscle Previously, a shift from fast-twitch to slow-twitch fibers was observed in TrJ muscle 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 in TrJ and same-age WT gastrocnemius was evaluated using SDH staining by sampling from deep, intermediate, and surface regions of the muscle as described. At 16 weeks after gene injection, there was a significant decrease in STO fibers, particularly FTO and FTG type fiber size, compared to the untreated (PBS) group, which showed neuronal changes, smaller, more angular fibers, and a population of different types (Figure 1A and B). Quantitative tests showed that both the number and percentage of STO fibers per unit area were significantly higher in TrJ-PBS muscle than in WT muscle, consistent with previous studies. In the treatment group, 1 × 10⁶ cells containing both promoters resulted in either low (tMCK) or high (CMV)NT-3 expression (Figure 4). 11 The vg dose of AAV1.NT-3 indicated a switching of fiber type from STO to FTO / FTG fibers. The mean density or percentage of STOs in both treatment groups (obtained from n=3-5 mice in each group) did not differ significantly from WT muscle, indicating a change in normalization of fiber type distribution by NT-3 (Figure 1C). Furthermore, WT muscle treated with AAV1.NT-3 did not show a significant change in the fiber type distribution profile.

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

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

[0218] [Table 2]

[0219] AAV-NT-3 improved mTOR signaling and metabolic markers in TrJ muscle. The histological findings in TrJ muscle in response to AAV1.NT-3 treatment, as described above, prompted an investigation into whether mTORC1 activation plays a role in NT-3-induced muscle fiber hypertrophy. mTORC1 activity was evaluated by the phosphorylation levels of its downstream substrates, 4EBP-1 and ribosomal S6P, in muscle samples from the group. In TrJ muscle treated with AAV1.NT-3, the levels of phosphorylated 4EBP-1 and S6P were significantly increased compared to the untreated equivalent obtained from TrJ-PBS controls (Figure 2A). In contrast, NT-3 treatment did not significantly affect the phosphorylation levels of 4EBP-1 and S6P in WT muscle (Figure 2B).

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

[17] . Therefore, the master regulator of mitochondrial biosynthesis, PGC 1α, was upregulated in NT-3-treated TrJ muscle, indicating that NT-3 can block the deficiency in PGC 1α expression levels observed in neurogenic muscle (Figure 2C). In response to the lack of mTORC1 activation, no change in PGC 1α expression levels was observed in NT-3-treated WT muscle (Figure 2C). The increase in fiber size in TrJ muscle following NT-3 gene therapy mainly occurs in FTO and FTG fibers, which have higher glycolytic activity than slow-twitch fibers

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

[0221] NT-3 activates the Akt / mTORC1 pathway via TrkC receptors in C2C12 myotubes. The in vivo studies described herein have shown that the increase in fiber size and fiber type remodeling induced by AAV1.NT-3 treatment in TrJ muscle are associated with mTORC1 activation. However, the question remains 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. As a next step, the direct effect of NT-3 on the mTOR pathway in an in vitro system was investigated without nerve influence by exposing C2C12 myoblasts and myotubes to recombinant NT-3. The results showed that NT-3 can 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 minutes resulted in significantly higher phosphorylation of Akt, 4EBP1, and S6P compared to the control group (Figure 3A). In another group of experiments, NT-3 was found to significantly increase the expression of the mitochondrial biosynthesis marker PGC 1α and the glycolysis marker PK1 in myotubes after 48 hours of incubation (Figure 3B). Therefore, analysis of the supernatant at this point showed increased glucose consumption and lactate production in myotubes treated with NT-3 compared to the control (Figure 3C). Under the conditions in which these experiments were conducted, no effect of NT-3 on HK1 expression levels was observed.

[0222] The expression of p75NTR and TrkC receptor, as well as myogenin, which are markers of myoblast migration into the differentiation pathway in myoblast and myotube cultures, was analyzed. NT-3 exerts its biological influence 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 neurotrophic factor NGF influences muscle differentiation and cell growth via p75NTR, and that downregulation of p75NTR is essential for muscle differentiation. Similar to the effects of NGF, NT-3 was found to promote myogenin expression in myotubes, and as predicted, this was associated with significantly higher p75NTR expression in myoblasts compared to myotubes, while TrkC expression levels did not differ in either group (Figure 3D). NT-3 did not have a different effect on the expression levels of these receptors in myoblasts or myotubes.

[0223] explanation Evidence is presented herein that NT-3 may directly influence neurogenic muscle metabolism, resulting in increased fiber size and fiber type remodeling relative to normalization by mTORC1 activation. The increase in fiber size was most pronounced in type II fibers, particularly the FTO subtype. Furthermore, it is shown that NT-3 can induce Akt / mTOR pathway activation in myotubes, a key contributing factor to its in vivo effect in neurogenic muscle, but does not directly induce it in myoblasts. Interestingly, NT-3 gene therapy in WT muscle at the same dose did not affect these properties, but the effect of NT-3 on denervated WT muscle using the gene therapy paradigm was not tested. Different effects of NT-3 on type II muscle fiber subtypes have been previously shown in rat gastrocnemius muscle 8 months after nerve repair with or without local delivery of NT-3 to the nerve crush site, and 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 is interpreted as NT-3-promoted axonal regeneration with beneficial outcomes in motor target organs, and the potential of NT-3 may particularly influence a subset of motor neurons that determine the type IIb muscle fiber phenotype. Findings from our in vivo studies may suggest a combined effect of NT-3 on both nerve and muscle. However, our in vitro data highlight evidence that NT-3 directly influences muscle metabolism through Akt / mTORC1 activation, and that this direct effect is likely important in neurogenic muscle, resulting in FTG, i.e., preferential size increase in 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 the upregulation of transcripts involved in glycolysis, increasing glucose consumption and lactate production (which was associated with lower insulin levels), and increasing blood glucagon levels and tolerance 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 studies, 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 regulating systemic metabolism by modulating fast-twitch / glycolytic fibers, but further studies are needed to characterize its role in detail. In addition, AAV.NT-3 treatment was able to block the deficiency in PGC1α expression levels observed in TrJ neurogenic muscle, along with elevated levels of activated 4E-BP1. In skeletal muscle, it has been previously suggested that mTOR regulates mitochondrial biosynthesis and metabolism via 4E-BP1 / PGC1α. Tsai et al., J Clin Invest, 125(8):2952-2964 (2015). NT-3 may also play a role in promoting oxidative phosphorylation through the activation of 4EBP1 and PGC1α in muscle.

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

[34] . NT-3 is expressed in SCs to promote neurogenesis and is a crucial component of the autocrine survival loop, ensuring SC survival and differentiation in adult neurons [35-39]. In studies using the CMT1A mouse model, several important biological effects of NT-3 were observed, namely (i) an increase in SC number, (ii) an increase in the number of myelinated fibers, and (iii) normalization of the axonal filament cytoskeleton [5, 40]. Another NT-3 effect of particular interest in this specification is the increase in myelin thickness, which was recognized as morphological evidence that NT-3 may influence myelin protein production.

[0226] Previous studies have provided considerable evidence that mTORC1 plays a role in regulating myelin formation in the CNS. Transgenic overexpression of constitutively active Akt kinase was sufficient to promote myelin membrane growth in the CNS via mTOR signaling [41, 42], and IGF-1-stimulated protein synthesis in oligodendrocyte progenitor cells requires the PI3KJ Akt and MER / ERK pathways

[43] . The ability of NT-3, which targets the translational mechanism to stimulate myelin protein synthesis, was first demonstrated in primary oligodendrocyte cultures

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

[45] . Indeed, in mutants, myelin was found to be thin, internodal length was shortened, and axonal hypertrophic growth was reduced. In addition, the downstream targets of mTOR, S6 and 4E-BP1, were not phosphorylated much.

[45]

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

[46] . Our toxicity tests evaluating scAAV1.tMCK.NT-3 were consistent with these observations, showing 1 × 10⁶ doses, which is 10 times higher than the highest dose proposed for clinical trials for the treatment of CMT1A. 13When administered at vg / kg, it does not exhibit organ-tissue-related toxicity or histopathological abnormalities in C57BL / 6 or TrJ mice.

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

[23] . The transient expression pattern of the receptor suggests 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, and as predicted, this was associated with significantly high expression of p75NTR in myoblasts, which was significantly downregulated in myotubes. When p75NTR and TrkC expression were examined in TrJ and WT muscle samples, significantly higher expression levels of both were observed in neuronal TrJ muscle compared to WT, and these levels decreased in response to NT-3 (Figure 5). Interesting, however, this observation does not allow for conclusions as to whether the cell type expresses these receptors, or whether they are expressed in all muscle fibers or only in one subtype of muscle fiber, in satellite cells or SCs. Available data on the expression of NT-3 and other neurotrophins and their receptors in human muscle diseases are limited. However, recent studies combining histological investigations of muscle biopsies with molecular and cellular analyses of major muscle progenitor cells have shown that p75NTR is expressed in vivo by most satellite cells and is a marker of regenerating fibers in inflammatory and dystrophic muscle [47, 48]. Our findings in neurogenic muscle are particularly interesting and require more comprehensive research into the mechanisms of various disease courses.

[0229] As a conserved Ser / Thr kinase, mTOR is a central regulator of cell growth by integrating signals from nutrients, growth factors, energy state, and environmental stress. The important role of mTOR in cell biology and pathobiology, particularly in muscles with superior metabolic and morphological adaptability, is currently of great interest. mTOR binds to raptor to form mTORC1, and muscle-specific inactivation of raptor has been shown to result in muscle atrophy, decreased oxidative capacity, and increased glycogen storage, leading to dystrophic features most pronounced in aerobic muscles

[49] . On the other hand, decreased mTOR activity exacerbates myopathic features in both slow-twitch and fast-twitch muscle fibers, exhibiting metabolic changes similar to those observed in raptor-deficient muscles, including decreased oxidative metabolism, altered mitochondrial regulation, and glycogen storage

[26] . In studies using the paradigm of cardiotoxicity-induced cycles of muscle necrosis / regeneration, we recently demonstrated reduced regeneration in a mouse model of limb-girdle muscular dystrophy type 2A, showing that calpain-3-deficient muscle is associated with disrupted mTORC1 signaling and defective mitochondrial biosynthesis (under investigation). Collectively, the findings described herein offer many suggestions for the potential use of NT-3 not only for the treatment of neuropathy affecting both nerves and muscles, but also for muscle wasting diseases including aging, cancer cachexia, or type II muscle fiber atrophy, as well as hereditary 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 a significant role.

[0230] Example 2 NT-3 delivery using scAAV1.tMCK.NFT3 vector The administered composition is a non-replicating recombinant adeno-associated virus called scAAV1.tMCK.NTF3, as 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 observed in the gastrocnemius muscle of C57B16 mice (1 × 10⁻¹⁶). 11 The test is performed after intramuscular injection of (vg), followed by quantification of circulating NT-3 in serum by ELISA 4–6 weeks after gene infusion.

[0231] First, it was demonstrated that ssAAV1.CMV.NTF3 delivered to the gastrocnemius muscle resulted in sufficiently sustained and therapeutic NT-3 blood concentrations to provide functional, electrophysiological, and histopathological improvements to TrJ neurons. Next, it was investigated whether the same level of expression could be achieved by generating the required vector dose and packaging the expression cassette with scAAV1. Dose-response studies were performed in C57BL / 6 mice using three doses (3 × 10⁶). 9 vg, 1×10 10 VG and 3×10 10 We compared serum NT-3 ELISA data after intramuscular injection of scAAV1.tMCK.NTF3 and scAAV1.CMV.NTF3 (vg). 1×10 11 Administration of vg sc.rAAV1.CMV.NTF3 vector resulted in significantly higher NT-3 levels compared to the same dose of single-stranded vector, consistent with the higher efficacy achieved using autocomplementary vectors. (Half-log low dose (3 × 10)) 10 In the vg) both CMV and tMCK vectors elicited a biological response, 1 × 10 11The NT-3 blood concentrations obtained were equivalent to those obtained from mice administered with vg doses of ss.AAV1.CMV.NTF3. NT-3 levels (mean ± SEM) from TrJ mice at 24 weeks after injection. Significant differences in NT-3 levels exist among all seven groups (p < 0.0001). Significant differences in NT-3 levels were observed for the highest and intermediate doses of the vector for both promoters and controls. However, the analysis did not find significant differences for lower doses for both vectors. Serum NT-3 was 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 was compared among each group and the PBS (control) group using the Mann-Whitney U test, and Bonferroni correction was used to adjust for multiple comparisons. See Sahenk et al., Mol.Ther.22(3):511-521, 2014, which is incorporated herein by reference in its entirety.

[0232] Increase in muscle diameter 40 weeks after treatment: Efficacy of NT-3 gene therapy compared to PBS (ssAAV1.CMV.NTF3(1×10)) 11 In a subset of animals injected with vg), muscle fiber size was evaluated in TrJ mice 40 weeks after injection. Neuronal changes characterized by angular fibers and group atrophy of atrophy were evident in the muscles of untreated mice, while evidence of nerve regeneration as an increase in fiber type population and overall fiber size was recognizable as a therapeutic effect. Muscle fiber size histograms created from the lateral anterior and posterior muscles of the left lower limb (tibialis anterior and gastrocnemius) showed an increase in fiber diameter.

[0233] Further laboratory tests by the inventors showed 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 stimulating effect on myotubes via the Trk-C receptor, which demonstrates its role in increasing fiber diameter in the muscles of TrJ mice. Figure 3A shows that NT-3 increased phosphorylation of Akt (P-Akt) and mTOR targets, 4EBP-1 (P-4EBP1), and PS6K (P-S6K) in SC and myotube cultures. These tests provide evidence justifying the selection of anterior and posterior lower leg muscles for vector delivery in this clinical trial.

[0234] Tests using self-complementary (sc)AAV1 and muscle-specific cleavage-type creatine kinase (tMCK) promoters scAAV enables lower doses and dose levels that meet production standards, resulting in improved safety. The use of the tMCK promoter is a worthwhile objective, as it 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 demonstrated at three doses (3 × 10) within the half-log range. 9 vg, 1×10 10 VG and 3×10 10 The results were compared with muscle-specific tMCK promoters administered in both the vg) model. The efficacy of AAV1.NTF3 gene transfection in TrJ mouse peripheral nerves was evaluated 24 weeks after gene transfection by electrophysiological (Table 3) and morphological tests. Evidence of transgene expression was assessed by measuring serum NT-3 levels using ELISA.

[0235] [Table 3]

[0236] The researchers conducting the electrodiagnostic tests were not informed of the treatment group. There was no statistically significant difference in CMAP between AAV1.NTF3.CMV (high dose, HD) and AAV1.NTF3.tMCK (high dose, HD), and the use of the muscle-specific tMCK promoter was preferred. This was further supported by NT-3 levels in the ELISA assay, where significant differences in NT-3 levels were observed for both the promoter and control vectors at the highest and intermediate doses.

[0237] All patents, patent applications, and publications, as well as the complete disclosures of electronically available materials referenced herein, are incorporated by reference. The above detailed descriptions and examples are provided for clarity of understanding only. No unnecessary limitations should be inferred from them. The present invention is not limited to the exact details shown and described, and variations that are obvious to those skilled in the art fall within the scope of the invention as defined by the claims.

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

[0239] The tMCK promoter / enhancer sequence is used to promote muscle-specific gene expression and consists of a muscle creatine kinase promoter to which an enhancer element (enh358MCK, 584-bp) is added and fused. A triple tandem of the MCK enhancer (206-bp) was ligated to an 87-bp basic promoter in the tMCK promoter / enhancer.

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

[0241] A schematic diagram of the plasmid, including its molecular structure and open reading frame, is shown in 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 an AAV2 inverted terminal repeat (ITR). This sequence is inscribed in the AAV1 virion. Plasmid pAAV.tMCK.NTF3 was constructed by inserting the tMCK expression cassette and integrating the NTF3 gene sequence into the AAV cloning vector psub201. The human NTF3 gene is expressed from a mouse triple-tandem MCK promoter, which is a modification of the previously described CK6 promoter and contains a triple E-box sequence. The SV40 polyadenylation signal is used for efficient transcription termination. The cassette also contains a chimeric intron for increased gene expression and consists of a 5' donor site from the first intron of the human β-globin gene, a branching point, and a 3' splice acceptance site from the intron between the leader and body of the immunoglobulin gene heavy chain variable region. The NTF3 expression cassette has an ATG start and a consensus scozac immediately preceding the 200bp SV40 polyA signal for efficient mRNA termination. The NTF3 cDNA is included in its entirety (NCBI reference sequence: NM_001102654). Only the viral sequence contained in this vector is the inverted terminal repeat sequence of AAV2, which is required for both viral DNA replication and packaging. The AAV ITR is a sequence that is nearly identical in both but in opposite orientation. The "left" (mutant) ITR has a deleted terminal resolution site to allow for genomic hairpin formation. The identity of all DNA plasmid elements is confirmed by DNA plasmid sequencing in plasmid source stocks.

[0242] Table 4 shows the base pair positions of the relevant molecular structures within the AAV vector DNA plasmid of Sequence ID No. 11.

[0243] [Table 4]

[0244] Example 4 Phase I intramuscular study In the initial Phase I intramuscular (IM) safety trial, sustained NT-3 transgene expression is the proposed outcome measure in this first stage, for several reasons. A true assessment of toxicity depends on demonstrating gene expression. If the muscle is not transduced, toxicity (adverse effects) tests become more difficult to interpret (lack of transduction versus reduced gene expression). The trial 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 escalation study in which scAAV1.tMCK.NTF3 is administered by intramuscular injection into the gastrocnemius and tibialis anterior muscles of both legs in CMT1A subjects with PMP22 gene duplication. Nine adult CMT1A patients aged 18 years or older will participate in one of the two cohorts of this trial. The first three subjects will receive the minimum effective dose (2.0 × 10⁶) distributed bilaterally between both arms and legs in Cohort 1. 12 Participants will take the dose escalation (vg / kg). The additional 6 participants will take a 3-fold dose escalation (6.0 × 10) in Cohort 2. 12Participants will take part in the study at a dose of vg / kg. Post-transduction monitoring will include follow-up visits at 7, 14, 30, 60, 90, 120 days, and 3-month intervals for the remainder of the 2-year study. Safety is the primary endpoint of this clinical transduction study. The stopping criterion is based on the occurrence of unacceptable toxicity, defined as the occurrence of any grade II ocular or systemic toxicity or any grade III or higher toxicity that has not resolved after 2 weeks. The secondary endpoint is efficacy, defined as the cessation of decline in ability as measured by the CMT Pediatric Scale (CMTPedS) at 2 years after transduction. CMTPedS is an 11-item scale consisting of the Functional Dexterity Test, the Nine-Hole Peg Test (9HPT), handgrip, plantar flexion, and dorsiflexion strength tests using a manual dynamometer, needle stick and vibration sensitivity, Bruininks-Oseletzky balance test, gait assessment, long jump, and 6-minute walk test (6MWT). Measurement of test results includes the 100-meter measurement test (100M), fibular and ulnar CMAP amplitude and sensory and motor conduction velocity, a sensory test modified to increase sensitivity to needle sticks, touch 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 circulatory NT-3 levels.

[0246] Patients participating in the study will include any racial, ethnic, or gender background. The criteria for this disorder are defined and will follow the guidelines already established by Shy et al. (Neurology 64:1209-1214, 2001 and Neurology 70:378-383, 2008).

[0247] The selection criteria for the exam are as follows: • For adults (over 18 years old) diagnosed with CMT1A. • A 1.5 Mb duplication at 17p11.2, including the peripheral myelin protein 22 (PMP22) gene, must be shown. • Men and women of any ethnic or racial group • Must show weakness in the muscles of ankle dorsiflexion (should have full range of motion against gravity, but unable to maintain full dorsiflexion against gravity, or unable to stand on tiptoe for more than 3 seconds (Northstar criteria)). • Abnormal nerve conduction velocity • Ability to cooperate in clinical evaluation and repetitive nerve conduction studies • Sexually active subjects must be willing to use reliable contraception during the study.

[0248] The exclusion criteria for the exam are as follows: • Active viral infection based on clinical observation or serological evidence of HIV, or hepatitis A, B, or C infection. • Immunosuppressive therapy currently underway or immunosuppressive therapy within 6 months of the start of the trial (e.g., corticosteroids, cyclosporine, tacrolimus, methotrexate, cyclophosphamide, intravenous immunoglobulin) • Persistent leukopenia or leukocytosis (WBC ≤ 3.5 K / μL or ≥ 20.0 K / μL) or absolute neutrophil count less than 1.5 K / μL • Subjects with an AAV1-binding antibody titer of 1:50 or higher as determined by ELISA immunoassay. According to the principal investigator (PI), gene transfer may create unnecessary risks, potentially leading to complications or the need for long-term drug therapy. • Ankle contracture or surgery that interferes with proper muscle strength testing. • Pregnant, breastfeeding, or planning to become pregnant Other causes of neuropathy • Limb surgery within the past 6 months

[0249] [Table 5]

[0250] Baseline measurement before injection (-30 days to -1 day) After obtaining written informed consent and completing the hospital registration procedure, the following baseline medical procedures and measurements will be performed. Medical history • Taking concomitant medications • Physical examination ·Chest X-ray • Echocardiogram EKG Hematological blood laboratory tests (blood labs) (CBC) • Coagulation parameters: platelets, PT / INR, PTT • Clinical chemistry and hematology laboratory tests: bilirubin, blood urea nitrogen (BUN), GGT, Alkaline phosphatase, alpha-fetoprotein (AFP), creatinine, amylase, serum protein electrophoresis, electrolytes, glucose, creatine kinase • Urine test • Viral screening (hepatitis and HIV) • Pregnancy test (for women of childbearing age only) • Hematological immunology: Neutralizing antibodies (AAV1) and ELISpot (NT-3 and AAV1) • Serum for ELISA (circulating NT-3) • Effectiveness measurement • Survey and measurement • Photo of the injection site

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

[0252] Protocol for gene transfer Autocomplementary AAV1, possessing the human NTF3 gene under the control of the tMCK promoter (scAAV1.tMCK.NTF3), is administered as a single bilateral intramuscular injection to 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 and injection procedure is performed under sterile conditions. The vector is delivered without diluents in a total of approximately 10 mL to 28 mL per patient (divided into three muscle groups, 5 to 14 mL per limb). The vector is delivered to the treatment room in a pre-labeled syringe sealed in a double leak-proof bag, which is transported in a condenser with a designated label. A total of 5 mL to 14 mL of vector is administered to the heads and lateral heads of the gastrocnemius and TA muscles in a total of 3 to 6 injections per muscle (each injection volume being 0.5 to 1.0 mL). Refer to the schematic diagrams of the injection sites in Figures 8A and 8B. The injection should be administered at least 0.25 cm below the fascia, and the injection should be performed along the longitudinal axis of the muscle, guided by ultrasound. Each injection should be administered approximately 1.5 cm apart.

[0254] Monitoring of hospitalized patients Following the gene transfer injection, the subject will return to their designated inpatient bed while maintaining close monitoring of vital signs and respiratory function. Vital signs will be monitored approximately every hour for the first four hours, and 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 the fourth and final doses of prednisone will be administered the following day (48 hours after injection, Day 2). The patient will be discharged approximately 48 hours after gene transfer (if no adverse events are observed).

[0255] Outpatient monitoring After discharge, patients will visit the hospital for follow-up appointments at 7, 14, 30, 60, 90, 120 days, and 3-month intervals throughout the remainder of the 2-year trial period following gene transfer. Blood samples obtained at all visits will be evaluated for NT-3 protein expression as demonstrated by serum ELISA using anti-NT-3 antibody. Furthermore, patients will be tested for efficacy and survey measurements at the 7th, 9th, 11th, 13th, and 14th visits. Serum ELISA is a direct measurement of functional gene expression using a secondary outcome measure to demonstrate the efficacy of circulating transgenes.

[0256] Primary endpoints: Safety is the primary endpoint of this clinical gene transduction study. It will be assessed based on the occurrence of unacceptable toxicity, defined as the occurrence of any one Grade III or higher unexpected treatment-related toxicity.

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

[0258] Secondary endpoint The secondary endpoint of clinical gene transfer trials is efficacy, defined as the cessation of decline in ability, as measured by the CMT Pediatric Scale (CMTPedS), two years after gene transfer.

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

[0260] Test result measurement The test results include a 100-meter measurement test (100M), fibular and ulnar CMAP amplitude and sensory and motor conduction velocity, a sensory test modified to increase sensitivity to needle sticks, a touch test and vibration assessment, a visual analog scale for pain and fatigue, a Short Form Health Survey (SF-36) as a measure of quality of life, and circulating NT-3 levels.

[0261] Electrophysiological testing includes ulnar sensory nerve amplitude and ulnar compound muscle action potential (CMAP) amplitude (recorded from the abductor digiti minimi muscle) as well as peroneal nerve (recorded from the tibialis anterior muscle) and measurement of sensory and motor conduction velocity. Fibular 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 has been found to be the most useful parameter (31). Foot and hand temperatures are maintained at 32-34°C during these test procedures. Visual analog scales are used to measure pain and fatigue (32). Short Health Questionnaire (Short The Form Health Survey (SF-36) is used as a quality of life record to monitor and compare disease burden before and after treatment.

[0262] A reward will be given to patients who undergo optional fascicular sural nerve biopsy. If selected, the biopsy will be taken before the start of the procedure (from the left calf) and at the end of the procedure (from the right calf). The biopsy will be performed as an outpatient examination under local anesthesia. The tissue will be processed and examined to evaluate the effect of NT-3 on myelinated fiber regeneration. To match the levels of the material before and after processing to the length of the entire nerve, the proximal end of the incision (2.5 cm in length) will be positioned exactly 10 cm above the Achilles tendon (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 height and weight, vital signs, physical examination and system review, and collection of a series of hematological laboratory tests. Laboratory tests include CBC, platelets, blood urea nitrogen (BUN), GGT, bilirubin, alkaline phosphatase, α-fetoprotein, creatinine, amylase, serum protein electrophoresis, electrolytes, B12, glucose, PT / INR and PTT, CK, and urinalysis. Immunology consists of ELISA for the detection of neutralizing antibodies against AAV1 and antibodies against 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 association with gene transfer.

[0264] The primary secondary outcome measure is defined as the absence of a decrease in disease severity on the CMT Disease Pediatric Scale (CMTPedS) score. The computerized scoring system for CMTPedS uses z-scores from a reference sample to determine individual scores based on the number of standard deviations that differ between a patient's performance and that of the healthy population. Scoring of CMTPedS is performed at a constant age of 20 for all patients throughout the study, which is necessary for two reasons. The first reason is that the original CMTPedS scale relies on reference values ​​for individual scale items when calculating the final score, but reference values ​​do not exist for individuals over 2030 years of age. The second reason is that while comparing scores between children and healthy equivalents is essential for children who are still developing, it has the limitation of imposing a decline in function when the child reaches their birthday. Even though the raw scores of children for the study do not change, the scoring criteria are designed to become stricter with their age, which reflects the expected improvement in motor skills that is typically seen in healthy children. However, in clinical trials for degenerative diseases, successful trials can be continued due to actual halting of disease progression. Maintaining consistent age for baseline control data throughout the trial enables the use of an effective scoring system incorporated into CMTPedS. It also allows for the observation of actual changes in assessments that would detect halting of disease progression.

[0265] The proportion of patients whose CMTPedS score improved or remained the same over a two-year period is estimated using a 95% confidence interval with a binomial test. For the purpose of the study, the proportion of patients without a decline in each of the 11 items of CMTPedS is estimated separately, as well as in the 100-meter measurement test (100M), CMAP amplitude, visual analog scale of pain intensity (VAS), Short Form Health Survey (SF-36) score, and circulating NT-3 level. In addition, Pearson or Spearman correlation coefficients are used at each measurement time point to assess the association between circulating NT-3 level and each outcome scale. Because this study is in a preliminary stage, p-values ​​for multiple comparisons are not adjusted. However, a sensitivity analysis is performed to show which associations are statistically significant after adjustment, based on the Bonferroni-Holm step-down procedure.

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

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

[0268] Test animals Wild-type C57BL / 6 (negative control) and Tr J(Test substance) A mouse was given either a vehicle (0.9% sterile saline) or 50 μl of 1 × 10⁶ of total volume. 13 Intramuscular injection of either scAAV1, tMCK, or NTF3 at vg / kg was administered. Carrier mice of this strain are available from Jackson Laboratories (stock number 000664) or Charles River (stock number 027). J The 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 with the animal strain, a subset of the animals were 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 and 22 TrJ) were treated with a saline control, and 44 animals (22 C57BL / 6 and 22 TrJ) were treated with the test substance scAAV1.tMCK.NTF3. Of these, half of the animals from each cohort were euthanized 24 weeks after treatment, and the remaining animals were euthanized 48 weeks after treatment. Animals were uniquely identified by ear tags.

[0271] Test design To evaluate the safety of the scAAV1.tMCK.NTF3 test substance, 4-6 week old wild-type C57BL / 6 and 8-12 week old Tr J In mice, a vehicle (0.9% sterile saline) or a single intramuscular (IM) injection into the left gastrocnemius muscle was administered, resulting in a total volume of 50 μl, 1 × 10⁻¹⁶. 13 The animals were injected with either scAAV1, tMCK, or NTF3 at a dose of vg / kg. Overall health and morbidity were monitored daily throughout the study. Mortality was checked twice daily. Body weight was measured every two weeks. Hypersensitivity tests (hot plate and auditory startle tests) were performed every eight weeks. Behavioral tests (rotarod and wire hanging) were performed every two weeks.

[0272] Temperature sensitivity was tested using a rodent hot plate test, starting two weeks before injection, followed by test on day 0 and every eight weeks. To perform the test, animals were placed on a plate surface maintained at 55°C and moved 100 cm using a 15 cm high Plexiglas wall surrounding the plate surface. 2 The area was limited. A timer was started when the animal was placed on the heating surface, and the response latency was recorded to the nearest 0.1 seconds using a stopwatch. The following activities were considered responses to the heat stimulus: licking or quickly moving the hind legs (flick) / fluttering, and jumping to escape were also acceptable responses. If this response was observed, the mouse was immediately removed.

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

[0274] The chamber consisted of a transparent, unrestrictive Plexiglas cylinder mounted 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 auditory stimuli. The vibrations of the Plexiglas cylinder, triggered by the whole-body startle response of the animals, were converted into analog signals by a piezoelectric unit mounted on the platform. The signals were then digitized and stored by a computer. 65 readings were taken at 1 ms intervals starting from the start of the stimulus, with the average amplitude (V) measured. avgThe auditory startle response was determined using the following method. A background noise level of 65 dB was presented over a 5-minute adaptation period and continued throughout the test session. All prepulse suppression (PPI) test sessions consisted of a startle test (pulse alone), a prepulse test (prepulse + pulse), and a no-stimulus test (no-stimulus). The pulse alone test consisted of broadband noise with a 40 ms 120 dB pulse. The PPI was measured by the prepulse + pulse test, which consisted of a 20 ms noise prepulse, a 100 ms delay, and then a 40 ms 120 dB startle pulse. The acoustic prepulse intensities were 69, 73, and 81 dB. The no-stimulus test consisted of background noise only. The test session began and ended with five presentations of the pulse alone test; in between, each test type was presented 10 times in a pseudo-random order. The average time between tests was 15 seconds (ranging from 12 to 30 seconds).

[0275] In the rotarod analysis, all subjects required at least one week of training to learn the task to a similar degree. In the accelerated rotation protocol, animals were placed on a rod, which was accelerated to 5 rpm and then ascended at 5 rpm / sec7. Animals underwent three tests per session, and the results were averaged.

[0276] In the wire hanging analysis, animals were placed by their four feet on a 2mm diameter metal wire, which was maintained horizontally on a 35cm thick layer of soft bedding. The length of time until the mouse fell from the wire was recorded, and after each fall, the mouse was given a 1-minute recovery period. Each test session consisted of three tests, and the scores from these tests were averaged.

[0277] At 22 and 46 weeks after injection (two weeks before euthanasia), blood was collected from the retro-orbital sinus for hematological testing (red blood cell count, hematocrit, hemoglobin, white blood cell count (total and fractional), 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, (total and direct) bilirubin, serum urea nitrogen, creatinine, creatine kinase, glucose, and total protein.

[0278] Blood was collected by cardiac puncture 24 and 48 weeks after injection, and serum was used for immunoassay. Serum samples were collected for measurement of 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 the injection, Tr J The following tissues were collected from the animals for histopathological analysis: gonads, brain, spleen, kidneys, jejunum, colon, pancreas, heart, lungs, stomach, liver, inguinal lymph nodes, spinal cord, (right and left) calf muscles, and macroscopic lesions (if present). The tissues were fixed in 10% neutral buffered formalin, sectioned, and stained with hematoxylin and eosin. Histological analysis was performed by SNBL USA.

[0280] Tissue samples for in-house histopathological investigation were collected at 24 and 48 weeks after the injection. JExcluding the control cohort (n=5, 3 males, 2 females), six animals (3 males, 3 females) were collected per cohort. Mice were euthanized and perfused transcardially with 4% paraformaldehyde in phosphate buffer (0.1 M, pH 7.4). The lumbar spinal cord, dorsal root ganglia (DRG), and the entire sciatic nerve (from the ischial notch to the popliteal fossa) were incised. The proximal half of the sciatic nerve and one lumbar DRG were transferred to glutaraldehyde fixative at in situ length and prepared for plastic embedding and sectioning to a thickness of 1 μm. The sciatic nerve tissue and the remainder of the lumbar DRG were cryoprotected in 30% sucrose and frozen in isopentane cooled in liquid nitrogen. The DRG neurons and axons (cut into 12 μm sections for immunohistochemistry for detection of CGRP positivity in the spinal cord) were placed in 10% neutral buffered formalin. The brains from TrJ mice were then placed in 10% neutral buffered formalin.

[0281] [Table 6]

[0282] Administration to animals, observation, and analysis The animals shown were administered a single 50 μL volume via intramuscular (IM) injection into the left gastrocnemius muscle on day 0. To ensure accurate administration, the animals were anesthetized by isoflurane inhalation for a minimum of 15 minutes. The dose was administered by direct injection into the left gastrocnemius muscle. Care was taken to ensure that the entire vector dose was delivered precisely into the muscle. After administration, the animals were observed until they could walk and then returned to their cages. Each animal was observed daily throughout the entire duration of the study. Body weight was measured every two weeks. Mortality checks were performed twice daily.

[0283] Mice of appropriate age were overdosed with a ketamine / xylazine mixture (200 mg / kg / 20 mg / kg). Blood was collected by cardiac puncture. Tissue samples were then taken and sent for analysis.

[0284] Body weight, mouse hematology, and clinical chemistry were plotted for each cohort at each time point. ELISA assays for circulating anti-AAV1 antibody, circulating anti-NT-3 antibody, and circulating NT-3 levels were performed for all endpoints. Hypersensitivity tests using rodent hot plates and auditory startlers were performed every 8 weeks for all animals. Behavioral tests, including rotarod and wire hanging tests, were performed every 2 weeks for all animals. Morphological histopathology of organs was performed for all animals. In-house histopathology of the lumbar spinal cord and DRG was performed on 5 animals (3 males, 2 females) including Tr J Excluding the cohort, the study was performed on 6 mice per cohort (3 males and 3 females). Histopathological evaluation included CGRP-positive immunocytochemical distribution (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 rates All mice survived the injection procedure and the initial observation period passed without any signs of distress. 4 Tr J The animals died due to a malfunction in the water supply system. There were no deaths related to the test substance.

[0286] body weight The body weight of all mice in each group was measured every two weeks throughout the entire study. All treatment groups maintained a constant weight gain throughout the study.

[0287] In C57BL / 6 male animals, there was no significant difference between animals injected with the test substance and the control group. In female animals, the control group had a slightly higher body weight compared to animals injected with the test substance. J In male animals, the saline-treated cohort was slightly heavier than the NT-3-treated cohort. J There was no significant difference in body weight among the mice.

[0288] Hematology and Clinical Chemistry No changes related to the test substance were observed in hematological parameters at weeks 22 and 46, or in serological chemistry at weeks 24 and 48.

[0289] ELISA assay ELISA assays for anti-AAV1 antibodies, anti-NT-3 antibodies, and circulating NT-3 were performed using serum samples collected during autopsies 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 against the AAV1 capsid. Antibody titers were similar in both males and females at both time points. Positive titers (>1:50) were not detected in animals treated with physiological 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 against NT-3 were determined by enzyme-linked immunosorbent assay (ELISA). All mice had antibody titers 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 at 24 and 48 weeks after vector administration. JThis was determined in mouse serum samples. Intramuscular injection of the scAAV1.tMCK.NTF3 vector was used at Tr 24 and 48 weeks after vector administration. J This resulted in steady expression and secretion of NT-3 in mice. Table 9 shows the results from the saline and vector injection groups. J The mean and standard deviation of serum NT-3 levels at 24 and 48 weeks after injection are shown. Sex did not affect circulating NT-3 levels.

[0295] [Table 9]

[0296] Hypersensitivity testing Temperature sensitivity tests were performed at baseline (before treatment of control saline with any of the test substances) and every 8 weeks until 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 retreat response latency between male and female mice in each cohort.

[0297] Auditory sensitivity measurements performed in the auditory startle test demonstrated that AST was clearly inhibited by the prepulse 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 the PPI response 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). J This improvement in animals was observed primarily in male animals.

[0298] Behavioral Test In rotarod evaluation, animals in the C57BL / 6 cohort showed no difference in rotarod function tests regardless of treatment. Animals in the TrJ NT-3 treated cohort showed a significant improvement in rotarod performance compared to control TrJ animals, starting at 16 weeks after treatment, and this improvement persisted to the endpoint.

[0299] In wire-hang assessment, animals in the C57BL / 6 cohort showed no difference in wire-hanging function regardless of treatment. Animals in the TrJ NT-3 treated cohort showed a significant improvement in wire-hanging ability compared to control TrJ animals, initiated 28 weeks after treatment, and this improvement persisted to the endpoint.

[0300] histopathology Morphological histopathology was performed. The 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 the entire sciatic nerve (from the ischial 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, cross-sections from the lumbar spinal cord were examined at 24 and 48 weeks after injection. This analysis was performed regardless of the treated substance. J Furthermore, no increase in CGRP reactivity with NT-3 was observed in C57BL / 6 animals.

[0305] Pathological changes in plastic-embedded sections were analyzed. 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 suggestive of axonal atrophy, such as myelin corrugation / infolding and outfolding, were observed in both treatment cohorts of C57BL / 6 animals at 48 weeks after treatment. J In animals, there was marked loss of myelinated fibers and many hypomyelination or bare axons. Tr treated with the test substance J The animals exhibit small myelinated fibers, a visible increase in myelin thickness, and a decrease in bare axons. NT-3 treatment had no adverse effects in TrJ mice, as evidenced by the absence of axonal branching within the DRG.

[0306] Generally speaking, C57BL / 6 and Tr J The absence of adverse findings related to the treatment throughout the entire study in both animal populations 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). 44 animals (22 C57BL / 6, 22 TrJ) were treated with a 0.9% sterile saline control, and the 44 animals (22 C57BL / 6, 22 TrJ) were subjected to a 1 × 10⁻¹⁶ dose. 13 The animals were treated with the test substance scAAV1.tMCK.NTF3 at a concentration of vg / kg. Of these animals, half from each cohort were euthanized 24 weeks after treatment, and the remaining animals were euthanized 48 weeks after treatment.

[0308] The data in this report demonstrate that treating animals with a dose 10 times higher than the proposed clinical dose did not result in the development of any adverse safety effects related to the test substance. Throughout the study, animals were observed daily for overall health and morbidity, and mortality checks were performed twice daily. Animal body weight was measured every two weeks. Functional tests for hypersensitivity (temperature and acoustics) were performed every eight weeks. Behavioral tests were performed every two weeks. All animals survived the injection procedure and the initial observation period proceeded without signs of distress. Four TrJ animals died due to a water system failure, unrelated to the administration of the test substance. No other deaths were recorded in this study.

[0309] Following dissection, hematological and clinical chemistry measurements showed no differences related to the test substance. Anti-AAV1 serum ELISA showed a predicted increase in circulating antibodies in animals treated with the test substance, and this was not sex-specific. Anti-NT-3 serum ELISA was performed on all animals, and negative antibody titers were observed at all time points. Circulating NT-3 levels were also measured in all treatment groups and increased only in animals treated with the test substance. There was no sex-specific effect on circulating NT-3 levels. Temperature sensitivity testing was performed at C57BL / 6 or Tr J No treatment- or sex-specific changes were observed in the animals. Auditory sensitivity measurements showed no changes related to the test substance in C57BL / 6 animals, however, Tr treated with the test substance J Along with improvements in normalization in animals, Tr treated with the control substance J The impairment in animals was demonstrated. This improvement was mainly due to male Tr J Observed in animals. Rotarod assessment of motor control in C57BL / 6 animals showed no difference associated with treatment with the test substance. TrJ animals treated with the test substance were compared with control Tr at 16 weeks after treatment. J It showed a significant improvement compared to animals. Similarly, wire hanging assessment did not show any difference in the C57BL / 6 cohort related to the test substance. Tr treated with the test substance JThe animals showed a significant improvement in wire-hanging time, starting at 28 weeks after treatment, and this improvement persisted until the endpoint.

[0310] Tissues and organs collected from all cohorts were evaluated at 24 and 48 weeks after treatment. In summary, minimal mononuclear cell infiltration was observed in the left gastrocnemius muscle injected with the test substance in 3 / 4 of Group 1 males and 4 / 5 of Group 1 females from dissection at 24 weeks. However, similar infiltration was observed in the uninjected gastrocnemius muscle in the remaining Group 1 females (animals 3942). This change was not observed in either the left gastrocnemius muscle injected with saline or the uninjected right gastrocnemius muscle in any of the Group 2 (control) animals from dissection at 24 weeks. At 48 weeks, minimal mononuclear cell infiltration was again observed in the left gastrocnemius muscle injected with the test substance in 2 / 5 of Group 1 males and 1 / 6 of Group 1 females. All other microscopic findings were considered to be either randomly distributed across control and treated animals, background findings for the species, or incidental to test substance administration.

[0311] In-house histology was performed on lumbar spinal sections, dorsal root ganglia, and sciatic nerves from a subset of animals (3 males and 3 females per group). Examination of sections from the lumbar spinal region did not show an increase in CGRP activity induced by NT-3 in the animals, regardless of treatment. Pathological evaluation of plastic-embedded sciatic nerves and lumbar DRG neurons did not show any changes beyond age-related differences in the C57BL / 6 animals. J In animals, treatment with the test substance resulted in pathological improvement, as evidenced by an increase in small myelinated fibers, increased myelin thickness, and a decrease in bare axons. The absence of axonal branching in DRGs further supports the safety of the test substance.

[0312] In general, the data collected in this study are 1 × 10⁻⁶. 13The test substance scAAV1.tMCK.NTF3, injected directly into the gastrocnemius muscle by a single intramuscular injection at a dose of vg / kg, was found to affect male and female C57BL / 6 wild-type and Tr, as is evident from the multiple measurements shown above. J Both mice showed good tolerability 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, double-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 multicentre,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:results 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:neurofilaments 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 1998;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 Peripher 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. 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[0314] Although the present invention has been described in terms of specific embodiments, it will be understood that variations and modifications will be conceivable to those skilled in the art. Therefore, only the limitations found in the claims should be made to the present invention.

Claims

1. Adeno-associated virus (AAV) vector, wherein the AAV vector is (i) 5'ITR containing sequence number 4; (ii) MCK promoter containing Sequence ID 3; (iii) Intron containing sequence number 5; (iv) Kossack sequence containing sequence number 6; (v) Nucleotide sequences encoding human NT-3 polypeptide; (vi) SV40 polyadenylation signal; and (vii) 3' ITR containing Sequence ID 8 AAV vectors, including...

2. The AAV vector according to claim 1, wherein the SV40 polyadenylation signal includes sequence number 7.

3. The AAV vector according to claim 1, comprising a sequence that is at least 99% identical to nucleotides 7 to 2248 of sequence number 11.

4. The AAV vector according to claim 3, comprising a sequence that is 100% identical to nucleotides 7 to 2248 of sequence number 11.

5. The AAV vector according to claim 1, wherein the vector has an AAV serotype selected from any one of 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, or their variants.

6. The AAV vector according to claim 5, wherein the AAV serotype is AAVrh.

74.

7. A composition comprising the AAV vector and a pharmaceutically acceptable carrier as described in claim 1.

8. The composition according to claim 7, wherein the route of administration is intramuscular injection.

9. (a) to treat muscle wasting diseases or neuropathy in human subjects; and (b) To improve muscle strength or stimulate muscle growth in human subjects. A composition comprising the AAV vector according to claim 1 for use in one or both of the following:

10. The composition is (a) For use in treating muscle wasting disease or neuropathy in human subjects, wherein the composition is formulated to result in sustained expression of NT-3 polypeptide; or (b) For use in improving muscle strength or stimulating muscle growth in human subjects, wherein the AAV vector has a recombinant adeno-associated virus (rAAV) genome, and the composition is administered at a dose of the rAAV that results in sustained expression of the NT-3 polypeptide. The composition according to claim 9.

11. The composition according to claim 9, wherein the composition is for use in treating a muscle wasting disease or neuropathy in a human subject, the subject suffering from Charcot-Marie-Tooth (CMT) neuropathy.

12. The composition according to claim 11, wherein the composition is for use in treating muscle wasting disease or neuropathy in a human subject, the subject having the genetic mutation Val30Met, Ile107Val, or Ser77Tyr.

13. The composition is intended for use in treating muscle wasting diseases or neuropathy in human subjects, the subjects being transthyretin amyloid neuropathy; cancer, diabetes, human immunodeficiency virus (HIV) infection, thyroid disorders, hypothyroidism, hypoglycemia, uremia, renal failure, hepatic dysfunction, hepatic failure, polycythemia, connective tissue disorders, Lyme disease, celiac disease, leprosy, porphyria, Sjögren's syndrome, poliovirus infection, acromegaly, lipid / glycolipid metabolism disorders, West Nile disease, amyloidosis, mitochondrial disorders, abnormal protein disorders, benign monoclonal gamma globulinemia (MGUS), POEMS syndrome, nutritional / vitamin deficiencies, vitamin B １２ The composition according to claim 9, which is used to describe a patient suffering from: deficiency, vitamin E deficiency, copper deficiency-induced acquired neuropathy; hereditary myopathy, peripheral neuropathy, toxic neuropathy, autoimmune peripheral polyneuropathy, acute inflammatory demyelinating polyneuropathy (AIDP), chronic inflammatory demyelinating polyneuropathy (CIDP), mononeuropathy due to vasculitis, paraneuropathy, idiopathic ganglionitis, amyotrophic lateral sclerosis, multifocal motor conduction block neuropathy, 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.

14. The composition according to claim 9, wherein the composition is for use in improving muscle strength or stimulating muscle growth in human subjects, and the route of administration of the composition is intramuscular injection.

15. The composition according to claim 9, wherein the composition is for use in improving muscle strength or stimulating muscle growth in a human subject, and the improvement in muscle strength is measured as a decrease in the composite score on the CMT Pediatric Scale (CMTPedS) or as a reduction in disease progression over a period of two years.

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