Methods and materials for nt-3 gene therapy

Abstract

The present disclosure relates to methods and materials for neurotrophic factor 3 (NT-3) gene therapy. The present disclosure relates to recombinant adeno-associated virus (rAAV) delivery of NT-3 polynucleotides. The present disclosure provides rAAVs and methods of using the rAAVs for NT-3 gene therapy to improve muscle strength, stimulate muscle growth, and treat muscle consumption disorders such as muscular dystrophy and fibula amyotrophic neuropathy.

Description

[0001] This application is a divisional application of the invention application with the application date of October 19, 2018, the Chinese national application number of 201880068238.4, and the invention name of “Methods and Materials for NT-3 Gene Therapy”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Patent Application No. 62 / 574,828 filed on October 20, 2017, U.S. Provisional Patent Application No. 62 / 676687 filed on May 25, 2018, and U.S. Provisional Patent Application No. 62 / 741,335 filed on October 4, 2018, the disclosures of which are hereby incorporated by reference in their entireties.

[0004] U.S. Government Statement of Interest

[0005] This invention was made with government support under Grant Nos. NS105986 and U01-NS066914 from the National Institutes of Health. The government has certain rights in this invention.

[0006] Incorporation by Reference into the Sequence Listing

[0007] This application contains a Sequence Listing in computer readable form as a separate part of the disclosure (file name: 53122A_Seqlisting.txt; 17,133 bytes - ASCII text file; created on October 18, 2018), which is incorporated herein by reference in its entirety. Technical Field

[0008] The present disclosure relates to recombinant adeno-associated virus (rAAV) delivery of neurotrophin 3 (NT-3) polynucleotides. The present disclosure provides rAAV and methods of using rAAV for NT-3 gene therapy to improve muscle strength, stimulate muscle growth, and treat neuropathy and muscle wasting disorders such as Charcot-Marie-Tooth neuropathy. Background Art

[0009] Recent studies have demonstrated that neurotrophin 3 (NT-3) is a multipotent molecule with previously unknown or underappreciated features. In addition to its well-established effects on peripheral nerve regeneration and Schwann cells (SC), NT-3 also has anti-inflammatory and immunomodulatory effects. Yang et al., Mel Titer, 22(2):440-450(2014). NT-3 has recently been shown to be able to alleviate spontaneous autoimmune peripheral polyneuropathy in rodent models of chronic inflammatory demyelinating peripheral nerve disorders that occur in humans. Yalvac et al., Gene therapy, 23(1):95-102(2015).

[0010] Charcot-Marie-Tooth (CMT) neuropathy is the most common inherited neuropathy. CMT1 includes five types of CMT, which are caused when four genes are mutated. This group includes the majority of people with CMT. These genes are associated with SCs and the myelin sheath surrounding axons, but they interact in different ways and are therefore phenotypically heterogeneous. CMT type 1A is the result of a DNA duplication on chromosome 17p11 containing the PMP22 gene, which leads to the development of the classic CMT1 phenotype. Patients present with clinical signs in the first two decades of life, resulting in severe disability requiring mobility assistive devices. In these patients, peripheral nerve regeneration is incomplete due to long-term axonotmesis and denervation occurring as part of the chronic neuropathy. NT-3 is a trophic factor secreted by Schwann cells (SCs) that supports nerve regeneration. The ability of denervated SCs to survive is crucial for nerve regeneration, as SCs provide both growth factors and the basement membrane (i.e., the scaffold that promotes axonal growth). Long-term denervation leads to reduced regenerative capacity, which is associated with decreased expression of regeneration-related SC molecules (neurotrophic factors (NTFs) and their receptors), resulting in denervated SC atrophy, disruption of the band of Bungner, and loss of the SC basement membrane scaffold.

[0011] Previous studies have shown that in the TremblerJ (TrJ) mouse model of Charcot-Marie-Tooth (CMT) neuropathy, NT-3 gene therapy not only improved nerve regeneration with an increase in SC number, myelinated fiber density, and myelin sheath thickness, but also increased muscle fiber diameter, which was demonstrated in the anterior and posterior compartment muscles of the hindlimb. Sahenk et al., Mol Ther [Molecular Therapy], 22(3):511-521 (2014). Previous studies have shown that the known neuropathic phenotype produces multiple changes in skeletal muscle, including a shift from fast type II fibers to slow type I fibers. In TrJ mice, the extensor digitalis longus, which is mainly composed of fast type fibers, has a significantly higher percentage of slow fibers compared to wild type (WT), and the percentage of type I fibers in the soleus muscle increases significantly with age. Nicks et al., J Neuropathol Exp Neurol [Journal of Neuropathology and Experimental Neurology], 72(10):942-954 (2013). Interestingly, aging is also accompanied by similar changes in the proportion of slow fibers in humans and other mammals. Larsson L, Moss R, J Physiol., 472:595–614 (1993); Larsson et al., Am J Physiol., 272:C638–C649 (1997).

[0012] CMT1A is inherited as an autosomal dominant disorder and is the most common type of CMT. Typically, it is caused by a 1.5 Mb duplication at 17p11.2 that includes the peripheral myelin protein 22 (PMP22) gene, which is generated by unequal exchange of homologous chromosomes (1). It is a slowly progressive disease with no known cure. Symptoms most often begin in the first two decades of life. High arches and hammer toes are present. Mobility assistive devices, such as ankle-foot orthoses, are required. Less common severe pediatric cases may be wheelchair- or ventilator-dependent. Typically, 90% of patients have a motor nerve conduction velocity (NCV) in the ulnar nerve between 16 and 35 m / s or less (2). Even though the genetic defect primarily involves Schwann cells (SCs), the clinical electrophysiological presentation is a length-dependent sensorimotor demyelinating neuropathy. The clinical presentation is significantly influenced by axonal degeneration caused by impaired Schwann cell (SC)-axon interactions (3).

[0013] There is currently no treatment for this condition. Ascorbic acid supplementation is highly recommended to help, but multiple studies have failed to show benefit. Neither low-dose (1-2 g / day)(4-6) nor high-dose therapy (3-4 g / day) has been shown to be beneficial(6,7). Initial human trials of NT-3 showed clinical efficacy after 24 weeks of treatment, which was accompanied by an increase in the number of myelinated nerve fibers in post-treatment sural nerve biopsies(8). J In mice, subcutaneous NT-3 treatment improved axonal regeneration and enhanced myelination. However, the short serum half-life of NT-3 proved to be a major obstacle to sustained subcutaneous administration, and the product was discontinued.

[0014] Many musculoskeletal diseases have been shown to cause decreased muscle strength. These include, but are not limited to, hereditary or recessive myopathy (such as muscular dystrophy), muscle wasting diseases (such as cachexia that may be caused by underlying diseases (such as acquired immunodeficiency disease (AIDS), rheumatoid arthritis, cancer, chronic obstructive pulmonary disease (COPD) and cirrhosis), muscle atrophy or weakness (such as sarcopenia that may be caused by aging), long-term disuse (such as paralysis, coma, long-term bed rest and ICU stay), weakness caused by surgery (such as joint replacement surgery), drug-induced myopathy and rhabdomyolysis. The muscle pathology of these diseases and conditions is mediated in part or in whole by a combination of immune, inflammatory and fibrotic reactions. Agents that can block these reactions and / or stimulate the regeneration of damaged tissue will be able to slow down or reverse the disease progression of these obstacles.

[0015] Adeno-associated virus (AAV) is a replication-deficient parvovirus with a single-stranded DNA genome of about 4.7 kb in length, including 145 nucleotide inverted terminal repeats (ITRs). There are a variety of AAV serotypes. The nucleotide sequences of the genomes of AAV serotypes are known. For example, the complete genome of AAV-1 is provided in GenBank Accession No. NC_002077; the complete genome of AAV-2 is provided in GenBank Accession No. NC_001401 and Srivastava et al., J. Virol. [Journal of Virology], 45: 555-564 {1983); the complete genome of AAV-3 is provided in GenBank Accession No. NC_1829; the complete genome of AAV-4 is provided in GenBank Accession No. NC_001829; the AAV-5 genome is provided in GenBank Accession No. AF085716; the complete genome of AAV-6 The AAV-7 and AAV-8 genomes are provided in GenBank Accession No. NC_001862; at least a portion of the AAV-7 and AAV-8 genomes are provided in GenBank Accession Nos. AX753246 and AX753249, respectively; the AAV-9 genome is provided in Gao et 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), encapsidation / packaging, and host cell chromosomal integration are contained in AAV ITR. Three AAV promoters (named p5, p19, and p40 for their relative map positions) drive expression of two AAV internal open reading frames encoding the rep and cap genes. The two rep promoters (p5 and p19) plus differential splicing of a single AAV intron (at nucleotides 2107 and 2227) result in the production of four rep proteins (rep 78, rep68, rep52, and rep40) from the rep gene. The Rep proteins have a variety of enzymatic properties that are ultimately responsible for replication of the viral genome. The cap gene is expressed from the p40 promoter, and it encodes three capsid proteins, VP1, VP2, and VP3. Alternative splicing and non-shared translation start sites are responsible for the production of three related capsid proteins. A single consensus polyadenylation site is located at map position 95 of the AAV genome. The life cycle and genetics of AAV are reviewed in Muzyczka, Current Topics in Microbiology and Immunology, 158:97-129 (1992).

[0016] AAV has unique characteristics that make it useful as a vector for delivering foreign DNA to cells, for example, in gene therapy. AAV infection of cells in culture is non-cytopathic, and natural infection in humans and other animals is silent and asymptomatic. In addition, AAV infects many mammalian cells, making it possible to target many different tissues in vivo. In addition, AAV transduces slowly dividing and non-dividing cells and can persist essentially throughout the life cycle of these cells as transcriptionally active nuclear episomes (extrachromosomal elements). The AAV proviral genome is infectious as cloned DNA in a plasmid, which makes the construction of recombinant genomes feasible. In addition, since the signals directing AAV replication, genome encapsidation, and integration are contained in the ITRs of the AAV genome, part or all of the internal approximately 4.3 kb of the genome (encoding replication and structural capsid proteins, rep-cap) can be replaced with foreign DNA. The rep and cap proteins can be provided in trans. Another notable feature of AAV is that it is a very stable and robust virus. It easily withstands the conditions used to inactivate adenovirus (56°C to 65°C for several hours), making refrigeration of AAV less important. AAV can even be freeze-dried. Finally, AAV-infected cells are not resistant to repeated infection.

[0017] There is a need to develop treatments for CMT neuropathy and other muscle wasting disorders. The present invention provides a gene therapy approach to deliver NT-3 to treat CMT neuropathy and other muscle wasting disorders. Summary of the invention

[0018] The present disclosure provides a method for stimulating muscle growth in a subject. The method comprises administering to a subject in need thereof a therapeutically effective amount of neurotrophin-3 (NT-3), pre-NT-3 or an effective fragment thereof, or a nucleic acid encoding NT-3, pre-NT-3 or an effective fragment thereof. The present disclosure describes a novel effect of NT-3, namely its ability to directly affect protein synthesis and metabolic remodeling in neurogenic muscle.

[0019] In various embodiments of the disclosure, NT-3, pro-NT-3 or an effective fragment thereof, or a nucleic acid encoding NT-3 or an effective fragment thereof of the disclosure is administered intramuscularly.

[0020] In any method of the present disclosure, a nucleic acid encoding NT-3 or its effective fragment is administered using a viral vector. In certain embodiments, the viral vector is an adeno-associated virus (AAV) vector. In a related embodiment, the nucleic acid encoding NT-3 or its effective fragment of the present disclosure is operably connected to a muscle-specific promoter (such as a triple muscle-specific creatine kinase promoter). In various embodiments, the nucleic acid encoding NT-3 or its effective fragment of the present disclosure comprises SEQ ID NO: 1.

[0021] The present disclosure provides a nucleic acid comprising, in order from 5' to 3': (i) a first AAV2 inverted terminal repeat (ITR); (ii) a muscle creatine kinase promoter / enhancer sequence set forth as 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 that is at least 90% identical to SEQ ID NO:2 or 100% identical to SEQ ID NO:2, or is encoded by a nucleotide sequence that is 90% identical to nucleotides 1077-1850 of SEQ ID NO:11 or 100% identical to nucleotides 1077-1850 of SEQ ID NO:11.

[0022] In some embodiments, the nucleic acid of the present disclosure further comprises a chimeric intron listed at nucleotides 892-1024 of SEQ ID NO: 11 at the 3' end of the promoter / enhancer. In addition, the nucleic acid of the present disclosure may further comprise an SV40 polyadenylation signal listed at nucleotides 1860-2059 of SEQ ID NO: 11 at the 3' end of the nucleotide sequence encoding the human NT-3 polypeptide.

[0023] Any nucleic acid of the present disclosure may comprise one or more inverted terminal repeat (ITR) sequences. For example, the nucleic acid may comprise a first ITR listed as nucleotides 7-112 of SEQ ID NO: 11 and / or a second ITR listed as nucleotides 2121-2248 of SEQ ID NO: 11.

[0024] In some embodiments, the nucleic acids comprise a scAAV1.tMCK.NTF3 genome that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO:11.

[0025] The disclosure also provides recombinant adeno-associated virus particles (rAAV) comprising any nucleic acid of the disclosure, wherein the rAAV is infectious. These rAAV particles can be any rAAV serotype, such as AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, or AAVrh.74. In addition, in any rAAV particles of the present invention, the AAV DNA in the rAAV genome is from AAV-1.

[0026] The present disclosure also provides compositions comprising the rAAV of the present disclosure and a pharmaceutically acceptable carrier. For example, these compositions are formulated to treat muscle wasting disorders or neuropathy in a subject in need thereof, or are formulated to stimulate muscle growth in a subject in need thereof.

[0027] In one embodiment, the disclosure provides a method for treating muscle wasting disorders or neuropathy in a human subject in need thereof, the method comprising administering to the human subject a nucleic acid encoding the NT-3 polypeptide; wherein a) the nucleic acid comprises a nucleotide sequence that is 90% identical to the nucleotide sequence of SEQ ID NO: 1; b) the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 1; c) the nucleic acid comprises a nucleic acid sequence encoding an amino acid sequence that is at least 90% identical to SEQ ID NO: 2 or 100% identical to SEQ ID NO: 2, d) the nucleic acid encoding the NT-3 polypeptide is any nucleic acid of the disclosure, e) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the rAAV is administered at a dose that results in sustained expression of a low concentration of the NT-3 polypeptide, f) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the route of administration is intramuscular, and the dose of the rAAV administered is about 1.5x 1012vg / kg to about 6.5x 1012vg / kg, g) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the route of administration is intramuscular, and the dose of rAAV administered is about 2x 1012vg / kg to about 6x 1012vg / kg, h) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the route of administration is intramuscular, and the dose of rAAV administered is about 2x 1012vg / kg, i) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the route of administration is intramuscular, and the dose of rAAV administered is about 4x 1012vg / kg, j) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the route of administration is intramuscular, and the dose of rAAV administered is about 6x 1012vg / kg, k) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the route of administration is intramuscular injection, the concentration is about 2x 1013vg / ml, and 3 to 6 injections of about 0.5 to 1ml are used per muscle for administration, or l) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the route of administration is intramuscular injection, the concentration is about 2x1013vg / ml 13 vg / ml, administered using multiple injections in a total volume of approximately 5 to 14 ml.

[0028] 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 to the human subject a nucleic acid encoding the NT-3 polypeptide; wherein a) the nucleic acid comprises a nucleotide sequence that is 90% identical to the nucleotide sequence of SEQ ID NO: 1; b) the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 1; c) the nucleic acid comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO: 2 or to SEQ ID NO: 3. NO:2 is a nucleic acid sequence with 100% identical amino acid sequence, d) the nucleic acid encoding the NT-3 polypeptide is any nucleic acid of the present disclosure, e) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the rAAV is administered at a dose that results in sustained expression of a low concentration of the NT-3 polypeptide, f) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the route of administration is intramuscular, and the dose of the rAAV administered is about 1.5x1012vg / kg to about 6.5x1012vg / kg, g) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the route of administration is intramuscular, and the dose of the rAAV administered is about 2x1012vg / kg to about 6x1012vg / kg. 1012vg / kg, h) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the route of administration is intramuscular, and the dose of rAAV administered is about 2x 1012vg / kg, i) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the route of administration is intramuscular, and the dose of rAAV administered is about 4x 1012vg / kg, j) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the route of administration is intramuscular, and the dose of rAAV administered is about 6x 1012vg / kg, k) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the route of administration is intramuscular injection, and the concentration is about 2x 1013vg / ml, administered using 3 to 6 injections / muscle of about 0.5 to 1 ml, or 1) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, administered by intramuscular injection at a concentration of about 2x10 13 vg / ml, administered using multiple injections in a total volume of approximately 5 to 14 ml.

[0029] In any method of the present disclosure, the nucleic acid is administered using a viral vector such as an adeno-associated viral vector. Any method of the present disclosure can be performed with a nucleic acid operably linked to a muscle-specific promoter such as a muscle-specific creatine kinase (MCK) promoter. In addition, any method of the present disclosure can be performed with scAAV1.tMCK.NTF3 comprising the NT-3 gene cassette listed in SEQ ID NO: 11.

[0030] In some embodiments, the present disclosure provides a gene therapy method using the human neurotrophin-3 gene (NTF3) using a scAAV1 vector (self-complementary AAV1 serotype) under the control of the muscle-specific promoter tMCK. Specifically, the present disclosure provides a method for treating a subject diagnosed with a muscle wasting disorder or neuropathy, the method comprising administering an AAV vector expressing NT-3. Specifically, these methods include administering the construct scAAV1.tMCK.NTF3 via intramuscular (IM) injection in the gastrocnemius and tibialis anterior muscles. For example, intramuscular delivery of an AAV vector expressing NT-3, such as scAAV1.tMCK.NTF3, initiates local production of NT-3 and secretes it into the circulation, thereby promoting nerve myelination and fiber regeneration, leading to stabilization of the CMT disease phenotype. More specifically, the present disclosure provides a method of ... 12 vg / kg or about 6 x 10 12 The method of administering scAAV1.tMCK.NTF3 at a dose of vg / kg. The scAAV1.tMCK.NTF3 comprises the NT-3 gene cassette listed in SEQ ID NO:11.

[0031] The present disclosure also provides methods for administering an AAV vector expressing NT-3 as an alternative gene therapy to treat muscle wasting disorders or neuropathy. NT-3 has a short half-life, and the methods of the present disclosure include administering an AAV vector for sustained release of NT-3 protein, even if the subject expresses endogenous NT-3 protein. As an alternative gene therapy, administration of the AAV vector provides sustained delivery of NT-3 protein through sustained secretion of muscle cells. This continuous, sustained low circulating NT-3 protein level provides a therapeutic effect with minimal risk of toxicity. Systemic production of NT-3 by gene therapy is also a more convenient and cost-effective treatment option when compared to repeated injections of purified NT-3 peptide.

[0032] The present disclosure provides methods for treating muscle wasting disorders or neuropathy in a human subject in need thereof, comprising the step of administering to the human subject a dose of recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3 that results in sustained expression of low concentrations of NT-3 protein.

[0033] The present disclosure also provides methods for stimulating muscle growth in a human subject in need thereof, comprising the step of administering to the human subject a dose of recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3 that results in sustained expression of low concentrations of NT-3 protein.

[0034] In one embodiment, the present disclosure provides methods for treating muscle wasting disorders or neuropathy in a human subject in need thereof, the methods comprising the step of administering to the human subject a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, wherein the administration route is intramuscular, and the dose of the rAAV administered is about 1.0 x 10 12 vg / kg to about 7x 10 12 vg / kg, or about 1.5 x 10 12 vg / kg to about 6.5 x 10 12 vg / kg, or about 2 x 10 12 vg / kg to about 6x 10 12 vg / kg.

[0035] In another embodiment,

[0036] The present disclosure provides a method for treating muscle wasting disorders or neuropathy in a human subject in need thereof, comprising the step of administering to the human subject a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, wherein the administration route is intramuscular, and the dose of the rAAV administered is about 1.0 x 10 12 vg / kg, or about 1.5 x 10 12 vg / kg, or about 2 x 10 12 vg / kg, or about 3x 10 12 vg / kg, or about 4 x 10 12 vg / kg, or about 5x 10 12 vg / kg, or about 6x10 12 vg / kg, or about 7 x 10 12 vg / kg, or about 8 x 10 12 vg / kg, or about 9 x 10 12 vg / kg, or about 1x 10 13 vg / kg.

[0037] In another embodiment, the present disclosure provides a method for treating muscle wasting disorders or neuropathy in a human subject in need thereof, comprising the step of administering to the human subject a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, wherein the administration route is about 1 x 10 13 For example, rAAV is administered using 3 to 6 injections per muscle, e.g., each injection volume will be 0.5 to 1 ml, with a total of 5 to 14 mL of vector administered to the medial and lateral heads of the gastrocnemius and tibialis anterior muscles in each leg.

[0038] In an exemplary embodiment, the present disclosure provides a method for treating muscle wasting disorders or neuropathy in a human subject in need thereof, comprising the step of administering to the human subject a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, wherein the administration route is intramuscular injection at a concentration of about 2 x 10 13 vg / ml, respectively, using 3 to 6 injections / muscle (each injection volume will be 0.5 to 1 ml). A total of 5 mL to 14 mL of vehicle is administered to the medial and lateral heads of the gastrocnemius and tibialis anterior muscles in each leg.

[0039] In one embodiment, the present disclosure provides a method for improving muscle strength or stimulating muscle growth in a human subject in need thereof, the method comprising the step of administering to the human subject a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, wherein the administration route is an intramuscular route, and the dose of the rAAV administered is about 1.0x10 12 vg / kg to about 7x 10 12 vg / kg, or about 1.5 x 10 12 vg / kg to about 6.5 x 10 12 vg / kg, or about 2 x 10 12 vg / kg to about 6x 10 12 vg / kg.

[0040] In another embodiment, the present disclosure provides a method for improving muscle strength or stimulating muscle growth in a human subject in need thereof, the method comprising the step of administering to the human subject a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, wherein the administration route is an intramuscular route, and the dose of the rAAV administered is about 1.0 x 10 12 vg / kg, or about 1.5 x 10 12 vg / kg, or about 2 x 10 12 vg / kg, or about 3x 1012 vg / kg, or about 4 x 10 12 vg / kg, or about 5 x 10 12 vg / kg, or about 6 x 10 12 vg / kg, or about 7 x 10 12 vg / kg, or about 8 x 10 12 vg / kg, or about 9x10 12 vg / kg, or about 1x 10 13 vg / kg.

[0041] In an exemplary embodiment, the present disclosure provides a method for improving muscle strength or stimulating muscle growth in a human subject in need thereof, the method comprising the step of administering to the human subject a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, wherein the administration route is about 1 x 10 13 For example, rAAV is administered using 3 to 6 injections per muscle, e.g., each injection volume will be 0.5 to 1 ml, with a total of 5 to 14 mL of vector administered to the medial and lateral heads of the gastrocnemius and tibialis anterior muscles in each leg.

[0042] In an exemplary embodiment, the present disclosure provides a method for improving muscle strength or stimulating muscle growth in a human subject in need thereof, the method comprising the step of administering to the human subject a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, wherein the administration route is intramuscular injection at a concentration of about 1 x 10 13 vg / ml, using 3 to 6 injections / muscle at low doses (2 x 10 12 vg / kg) and high dose (6x 10 12 vg / kg) (each injection volume will be 0.5 to 1 ml). A total of 5 mL to 14 mL of vehicle will be administered to the medial and lateral heads of the gastrocnemius and tibialis anterior muscles in each leg.

[0043] In any of the methods of the present disclosure, wherein the route of administration of scAAV1.tMCK.NTF3 is bilateral intramuscular injection into the medial and lateral heads of the gastrocnemius and tibialis anterior muscles. Additionally, in any of the methods of the present invention, administration of scAAV1.tMCK.NTF3 results in improved muscle strength of the upper or lower extremities in the subject, and, for example, the improvement in muscle strength is measured as a decrease in the composite score on the CMT Pediatric Rating Scale (CMTPeds). Additionally, 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 two-year period. Disease progression is measured by CMTPeedS.

[0044] Muscle strength is also measured using electromyography, handheld myometry, fixed system myometry, manual muscle testing, and / or functional / mobility tests such as the Jebsen test, and timed tests that assess how long it takes a subject to complete a specific task, such as the 6-minute walk test, timed ascent from ground level, 10-meter walk / run, timed ascent of 4 steps, and timed descent of 4 steps.

[0045] In one aspect of the disclosure, in any method, the subject suffers from a hereditary neuropathy such as Charcot-Marie-Tooth (CMT) neuropathy, e.g., CMT1A, CMT2K, CMT4A, CMTRIA, and axonal and demyelinating neuropathy caused by autosomal recessive gene variants or autosomal dominant gene variants or X-linked gene variants. The hereditary neuropathy may be caused by any of the gene variants provided in Table 1. In addition, the hereditary neuropathy may be a transthyretin amyloid neuropathy caused by a transthyretin (TTR) gene mutation, such as the following gene variants: Val30Met, Ile107Val, and Ser77Tyr.

[0046] In another aspect of the disclosure, in any method, the subject suffers from an acquired neuropathy with axonal loss and / or impaired nerve regeneration. The acquired neuropathy is a peripheral neuropathy caused by any disorder or disease known to cause neuropathy. For example, the subject suffers from peripheral neuropathy caused by: diabetes, human immunodeficiency virus (HIV) infection, thyroid disorders (such as hypothyroidism), hypoglycemia, uremia, renal insufficiency, liver dysfunction, liver failure, polycythemia, connective tissue disorders, cancer, Lyme disease, celiac disease, leprosy, porphyria, Sjögren's syndrome, poliomyelitis, acromegaly, lipid / glycolipid metabolism disorders, West Nile syndrome, amyloidosis, mitochondrial disorders, dysproteinemic disorders such as monoclonal gammapathy of unknown significance (MGUS) or POEMS syndrome. The subject suffers from nutritional / vitamin deficiencies such as vitamin B 12 deficiency, vitamin E deficiency, or copper deficiency.

[0047] In another aspect of the disclosure, in any of the methods described herein, the subject has autoimmune peripheral polyneuropathy, acute inflammatory demyelinating polyradiculoneuropathy (AIDP), chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), vasculitic mononeuritis multiplex, paraneuropathy, idiopathic ganglionitis, amyotrophic lateral sclerosis, multifocal motor block neuropathy, or lower motor neuron syndrome.

[0048] The acquired neuropathy may be a toxic neuropathy. For example, toxic neuropathy is the result of the toxic effects of prescription drugs such as chloramphenicol, cholinequinol, colchicine, disulfiram, etanercept, ethambutol, gold, hydroxychloroquine, nitrofurantoin, metronidazole, stavudine, zalcitabine, infliximab, leflunomide, thalidomide, or chemotherapy agents such as cisplatin, cytarabine, bortezomib, docetaxel, lenalidomide, misondiazole, oxaliplatin, paclitaxel, procarbazine, suramin, thalidomide, vinblastine, or vincristine, or anti-alcohol drugs such as disulfiram, or anticonvulsants such as phenytoin or dilantin, or heart or blood pressure medications such as statins, amiodarone, hydralazine, procainamide, perhexiline, or antibiotics such as fluoroquinolones, isoniazid, ciprofloxacin, levofloxacin, metronidazole, or metronidazole, or skin disease treatments such as dapoxetine. This toxic neuropathy may also be caused by chronic alcohol abuse or vitamin B6 toxicity.

[0049] In another aspect of the 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.

[0050] In yet another aspect of the disclosure, in any of the methods, the subject is a surgical patient with acquired neuropathy. For example, the surgical patient developed neuropathy after undergoing bariatric surgery, multiple plastic surgeries, or multiple "entrapped nerve" surgeries.

[0051] In another aspect of the disclosure, in any of the methods, the subject suffers from a genetic myopathy, a neuromuscular disease, muscular atrophy, a drug-induced myopathy, sarcopenia, cachexia, type II myofiber dystrophy, a genetically determined muscular dystrophy, age-related muscular atrophy, or an acquired autoimmune primary muscle disorder.

[0052] In another aspect of the disclosure, in any of the methods, the subject has Duchenne muscular dystrophy, Becker muscular dystrophy, myotonic dystrophy, sarcoglycanopathy, myotonic dystrophy, Emery-Dreifuss muscular dystrophy, congenital muscular dystrophy, fascin deficiency congenital muscular dystrophy, Bethlem myopathy, Ullrich congenital muscular dystrophy, muscular dystrophy, facioscapulohumeral muscular dystrophy, spinal muscular dystrophy, rigid spinal 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, eye, and brain disease; Fukuyama-Walker-Warburg syndrome; myasthenic syndrome; congenital muscular weakness; inclusion body myopathy; inclusion body myositis; dermatomyositis; centronuclear myopathy; Myoshi myopathy; mitochondrial myopathy; nemaline myopathy; Nonaka myopathy; myasthenia gravis; or polymyositis.

[0053] In one embodiment, the disclosure provides a therapeutically effective amount of neurotrophin-3 (NT-3), pre-NT-3 or an effective fragment thereof, or a nucleic acid encoding NT-3, pre-NT-3 or an effective fragment thereof for the manufacture of a medicament for stimulating muscle growth in a subject. For example, the medicament is formulated for intramuscular administration.

[0054] In an exemplary embodiment, the drug comprises a nucleic acid encoding NT-3, pre-NT-3 or an effective fragment thereof, wherein the nucleic acid is in a viral vector. In a related embodiment, the viral vector is an adeno-associated viral vector. In various embodiments, the nucleic acid is operably linked to a muscle-specific promoter such as a triple muscle-specific creatine kinase promoter. In various embodiments, the nucleic acid comprises SEQ ID NO: 1.

[0055] The present invention also provides the use of a nucleic acid encoding the NT-3 polypeptide for the manufacture of a medicament for treating muscle wasting disorders or neuropathy in a human subject, wherein: a) the nucleic acid comprises a nucleotide sequence that is 90% identical to the nucleotide sequence of SEQ ID NO: 1; b) the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 1; c) the nucleic acid comprises a nucleic acid sequence that encodes an amino acid sequence that is at least 90% identical to SEQ ID NO: 2 or 100% identical to SEQ ID NO: 2, d) the nucleic acid encoding the NT-3 polypeptide is any nucleic acid of the present disclosure, e) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the rAAV is at a dose that results in sustained expression of a low concentration of the NT-3 polypeptide, f) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the medicament is formulated for intramuscular administration, and the dose of the rAAV is about 1.5x 1012 vg / kg to about 6.5x 1012vg / kg, g) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the drug is formulated for intramuscular administration, and the dose of the rAAV is about 2x 1012vg / kg to about 6x 1012vg / kg, h) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the drug is formulated for intramuscular administration, and the dose of the rAAV is about 2x 1012vg / kg, i) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the drug is formulated for intramuscular administration, and the dose of the rAAV is about 4x 1012vg / kg. 1012vg / kg, j) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the drug is formulated for intramuscular administration, and the dose of rAAV administered is about 6x 1012vg / kg, k) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the drug is formulated for intramuscular injection, the concentration is about 2x 1013vg / ml, and 3 to 6 injections of about 0.5 to 1ml are administered per muscle, or l) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the drug is formulated for intramuscular injection, the concentration is about 2x 1013vg / ml, and 3 to 6 injections of about 0.5 to 1ml are administered per muscle. 13 vg / ml, administered using multiple injections in a total volume of approximately 5 to 14 ml.

[0056] The present disclosure also provides a use of a certain dose of a nucleic acid encoding the NT-3 polypeptide for the manufacture of a medicament for improving muscle strength or stimulating muscle growth in a human subject, wherein: a) the nucleic acid comprises a nucleotide sequence that is 90% identical to the nucleotide sequence of SEQ ID NO: 1; b) the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 1; c) the nucleic acid comprises a nucleic acid sequence that encodes an amino acid sequence that is at least 90% identical to SEQ ID NO: 2 or 100% identical to SEQ ID NO: 2, d) the nucleic acid encoding the NT-3 polypeptide is any nucleic acid of the present disclosure, e) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the rAAV is at a dose that results in sustained expression of a low concentration of the NT-3 polypeptide, f) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the composition is formulated for intramuscular administration, and the dose of the rAAV is about 1.5 x 1012 vg / kg to about 6.5 x 1012vg / kg, g) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the drug is formulated for intramuscular administration, and the dose of the rAAV is about 2x 1012vg / kg to about 6x 1012vg / kg, h) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the drug is formulated for intramuscular administration, and the dose of the rAAV is about 2x 1012vg / kg, i) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the drug is formulated for intramuscular administration, and the dose of the rAAV is about 4x 1012vg / kg. 1012vg / kg, j) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the drug is formulated for intramuscular administration, and the dose of rAAV administered is about 6x 1012vg / kg, k) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the drug is formulated for intramuscular injection, the concentration is about 2x1013vg / ml, and 3 to 6 injections of about 0.5 to 1ml are administered per muscle, or l) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the drug is formulated for intramuscular injection, the concentration is about 2x1013vg / ml, and 3 to 6 injections of about 0.5 to 1ml are administered per muscle. 13 vg / ml, administered using multiple injections in a total volume of approximately 5 to 14 ml.

[0057] For example, any drug of the present disclosure may include a nucleic acid formulated for administration using a viral vector such as an adeno-associated viral vector. In addition, any drug of the present 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 any drug or the present disclosure, scAAV1.tMCK.NTF3 includes the NT-3 gene cassette listed in SEQ ID NO: 11. In one embodiment, the present disclosure provides the use of a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3 for the preparation of a drug for treating muscle wasting disorders or neuropathy in a human subject in need thereof, wherein the drug results in sustained expression of low concentrations of NT-3 protein.

[0058] In another embodiment, the present disclosure provides the use of recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3 for the preparation of a medicament for stimulating muscle growth in a human subject in need thereof, wherein the dose results in sustained expression of low concentrations of NT-3 protein.

[0059] In one embodiment, the disclosure provides a use of a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3 for the preparation of a medicament for treating a muscle wasting disorder or a neuropathy in a human subject in need thereof, wherein the medicament is formulated for an intramuscular route of administration, and wherein the medicament comprises about 1.0 x 10 12 vg / kg to about 7x 10 12 vg / kg, or about 1.5 x 10 12 vg / kg to about 6.5 x 10 12 vg / kg, or about 2 x 10 12 vg / kg to about 6x 10 12 vg / kg rAAV dose.

[0060] In another embodiment, the disclosure provides a use of a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3 for the preparation of a medicament for treating a muscle wasting disorder or a neuropathy in a human subject in need thereof, wherein the medicament is formulated for an intramuscular route of administration, and wherein the medicament comprises about 1.0 x 10 12 vg / kg, or about 1.5 x 10 12 vg / kg, or about 2 x 10 12 vg / kg, or about 3x 10 12 vg / kg, or about 4 x 10 12 vg / kg, or about 5x 10 12 vg / kg, or about 6x1012 vg / kg, or about 7x10 12 vg / kg, or about 8 x 10 12 vg / kg, or about 9 x 10 12 vg / kg, or about 1x 10 13 vg / kg rAAV dose.

[0061] In another embodiment, the disclosure provides a use of a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3 for the preparation of a medicament for treating a muscle wasting disorder or a neuropathy in a human subject in need thereof, wherein the medicament is formulated for an intramuscular route of administration, and wherein the medicament comprises about 2 x 10 13 For example, the drug 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, for example, each injection volume will be 0.5 to 1 ml, wherein 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.

[0062] In an exemplary embodiment, the disclosure provides a use of a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3 for the preparation of a medicament for treating a muscle wasting disorder or a neuropathy in a human subject in need thereof, wherein the medicament is formulated for an intramuscular route of administration, and wherein the medicament comprises about 1 x 10 13 vg / ml of rAAV dose, respectively, will be administered using 3 to 6 injections / muscle (each injection volume will be 0.5 to 1 ml). A total of 5 mL to 14 mL of vector will be administered to the medial and lateral heads of the gastrocnemius and tibialis anterior muscles in each leg.

[0063] In one embodiment, the disclosure provides a use of a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3 for the preparation of a medicament for improving muscle strength or stimulating muscle growth in a human subject in need thereof, wherein the medicament is formulated for intramuscular administration, and wherein the medicament comprises about 1.0 x 10 12 vg / kg to about 7x 10 12 vg / kg, or about 1.5 x 10 12 vg / kg to about 6.5 x 10 12 vg / kg, or about 2 x 10 12 vg / kg to about 6x 10 12 vg / kg rAAV dose.

[0064] In another embodiment, the disclosure provides a use of a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3 for the preparation of a medicament for improving muscle strength or stimulating muscle growth in a human subject in need thereof, wherein the medicament is formulated for intramuscular administration, and wherein the medicament comprises about 1.0 x 10 12 vg / kg, or about 1.5 x 10 12 vg / kg, or about 2 x 10 12 vg / kg, or about 3x 10 12 vg / kg, or about 4 x 10 12 vg / kg, or about 5x 10 12 vg / kg, or about 6x10 12 vg / kg, or about 7x10 12 vg / kg, or about 8 x 10 12 vg / kg, or about 9 x 10 12 vg / kg, or about 1x 10 13 vg / kg rAAV dose.

[0065] In another embodiment, the disclosure provides a use of a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3 for the preparation of a medicament for improving muscle strength or stimulating muscle growth in a human subject in need thereof, wherein the medicament is formulated for intramuscular administration, and wherein the medicament comprises about 2 x 10 13 vg / ml rAAV concentration, with low doses (2 x 10 12 vg / kg) and high dose (6x 10 12 vg / kg). In some embodiments, the drug is administered to the medial and lateral heads of the gastrocnemius and tibialis anterior muscles in each leg using 3 to 6 injections / muscle, for example, each injection volume is 0.5 to 1 ml. A total of 5 mL to 14 mL of the vehicle will be administered to the medial and lateral heads of the gastrocnemius and tibialis anterior muscles in each leg.

[0066] In an exemplary embodiment, the present disclosure provides a use of a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3 for the preparation of a medicament for improving muscle strength or stimulating muscle growth in a human subject in need thereof, wherein the medicament is formulated for an intramuscular route of administration, and wherein the medicament comprises about 2 x 10 13 vg / ml rAAV concentration, using 3 to 6 injections / muscle at low doses (2 x 10 12 vg / kg) and high dose (6x 10 12vg / kg) (each injection volume will be 0.5 to 1 ml). A total of 5 mL to 14 mL of vehicle will be administered to the medial and lateral heads of the gastrocnemius and tibialis anterior muscles in each leg.

[0067] In any purposes of the present disclosure, the medicine is formulated for intramuscular bilateral injection to the medial head and lateral head of the gastrocnemius and tibialis anterior muscles. In addition, in any purposes of the present invention, the medicine causes the muscle strength of the upper limbs or lower limbs in the subject to be improved, and for example the improvement of muscle strength is measured as the reduction of the comprehensive score on the CMT pediatric scale (CMTPedS). In addition, in any purposes of the present invention, the medicine causes a reduction or cessation of disease progression in a two-year period. Disease progression is measured by CMTPedic.

[0068] In one aspect of the disclosure, in any use of the disclosure, the subject suffers from a hereditary neuropathy (e.g., Charcot-Marie-Tooth (CMT) neuropathy, e.g., CMT1A, CMT2K, CMT4A, CMTRIA), and axonal and demyelinating neuropathy caused by autosomal recessive gene variants or autosomal dominant gene variants or X-linked gene variants. The hereditary neuropathy may be caused by any of the gene variants provided in Table 1. In addition, the hereditary neuropathy may be a transthyretin amyloid neuropathy caused by a transthyretin (TTR) gene mutation, such as the following gene variants: Val30Met, Ile107Val, and Ser77Tyr.

[0069] In another aspect of the disclosure, in any method of the invention, the subject suffers from an acquired neuropathy with axonal loss and / or impaired nerve regeneration. The acquired neuropathy is a peripheral neuropathy caused by any disorder or disease that causes neuropathy. For example, the subject suffers from peripheral neuropathy caused by: diabetes, human immunodeficiency virus (HIV) infection, thyroid disorders (such as hypothyroidism), hypoglycemia, uremia, renal insufficiency, liver dysfunction, liver failure, polycythemia, connective tissue disorders, cancer, Lyme disease, celiac disease, leprosy, porphyria, Sjögren's syndrome, poliomyelitis, acromegaly, lipid / glycolipid metabolism disorders, West Nile syndrome, amyloidosis, mitochondrial disorders, dysproteinemia disorders such as monoclonal gammopathy of undetermined significance (MGUS) or POEMS syndrome. The subject may suffer from nutritional / vitamin deficiencies, such as vitamin B 12 deficiency, vitamin E deficiency, or copper deficiency.

[0070] Additionally, in any use of the present disclosure, the subject suffers from autoimmune peripheral polyneuropathy, acute inflammatory demyelinating polyradiculoneuropathy (AIDP), chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), vasculitic mononeuritis multiplex, paraneuropathy, idiopathic ganglionitis, amyotrophic lateral sclerosis, multifocal motor block neuropathy, or lower motor neuron syndrome.

[0071] This acquired neuropathy is a toxic neuropathy. For example, toxic neuropathy is the result of the toxic effects of prescription drugs such as chloramphenicol, cholinequinol, colchicine, disulfiram, etanercept, ethambutol, gold, hydroxychloroquine, nitrofurantoin, metronidazole, stavudine, zalcitabine, infliximab, leflunomide, thalidomide, or chemotherapy agents such as cisplatin, cytarabine, bortezomib, docetaxel, lenalidomide, misondiazole, oxaliplatin, paclitaxel, procarbazine, suramin, thalidomide, vinblastine, or vincristine, or anti-alcohol drugs such as disulfiram, or anticonvulsants such as phenytoin or dilantin, or heart or blood pressure medications such as statins, amiodarone, hydralazine, procainamide, perhexiline, or antibiotics such as fluoroquinolones, isoniazid, ciprofloxacin, levofloxacin, metronidazole, or metronidazole, or skin disease treatments such as dapoxetine. This toxic neuropathy may be caused by chronic alcohol abuse or vitamin B6 toxicity.

[0072] In another aspect, in any use of the present 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.

[0073] In yet another aspect, in any use of the present disclosure, the subject is a surgical patient with acquired neuropathy. For example, the surgical patient develops neuropathy after undergoing bariatric surgery, multiple plastic surgeries, or multiple "entrapped nerve" surgeries.

[0074] In another aspect, in any use of the present disclosure, the subject suffers from a hereditary myopathy, a neuromuscular disease, muscular atrophy, a drug-induced myopathy, sarcopenia, cachexia, type II myofiber dystrophy, a genetically determined muscular dystrophy, age-related muscular atrophy, or an acquired autoimmune primary muscle disorder.

[0075] In another aspect, in any use of the present disclosure, the subject has Duchenne muscular dystrophy, Becker muscular dystrophy, myotonic dystrophy, sarcoglycanopathy, myotonic dystrophy, Emery-Dreifuss muscular dystrophy, congenital muscular dystrophy, fascin deficiency congenital muscular dystrophy, Bethlem myopathy, Ullrich congenital muscular dystrophy, muscular dystrophy, facioscapulohumeral muscular dystrophy, spinal muscular dystrophy, rigid spinal 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, eye, and brain disease; Fukuyama-Walker-Warburg syndrome; myasthenic syndrome; congenital muscular weakness; inclusion body myopathy; inclusion body myositis; dermatomyositis; centronuclear myopathy; Myoshi myopathy; mitochondrial myopathy; nemaline myopathy; Nonaka myopathy; myasthenia gravis; or polymyositis.

[0076] In one embodiment, the present disclosure provides a composition comprising a therapeutically effective amount of neurotrophin-3 (NT-3), pre-NT-3 or an effective fragment thereof, or a nucleic acid encoding NT-3, pre-NT-3 or an effective fragment thereof, for stimulating muscle growth in a subject. For example, the composition of the present disclosure is formulated for intramuscular administration.

[0077] In an exemplary embodiment, the composition comprises a nucleic acid encoding NT-3, pre-NT-3 or an effective fragment thereof. In a related embodiment, the nucleic acid is in a viral vector such as an adeno-associated viral vector. In various embodiments, the nucleic acid is operably linked to a muscle-specific promoter such as a triple muscle-specific creatine kinase promoter. In various embodiments, the composition comprises a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 1.

[0078] The present disclosure also provides a composition comprising a nucleic acid encoding a NT-3 polypeptide for use in treating a muscle wasting disorder or a neuropathy in a human subject, wherein: a) the nucleic acid comprises a nucleotide sequence that is 90% identical to the nucleotide sequence of SEQ ID NO: 1; b) the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 1; c) the nucleic acid comprises a nucleic acid sequence that encodes an amino acid sequence that is at least 90% identical to SEQ ID NO: 2 or 100% identical to SEQ ID NO: 2, d) the nucleic acid encoding the NT-3 polypeptide is any nucleic acid of the present disclosure, e) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the rAAV is administered at a dose that results in sustained expression of a low concentration of the NT-3 polypeptide, f) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the composition is formulated for intramuscular administration, and the dose of the rAAV is about 1.5 x 1012 vg / kg to about 6.5 x 1012vg / kg, g) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the composition is formulated for intramuscular administration, and the dose of the rAAV is about 2x 1012vg / kg to about 6x 1012vg / kg, h) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the composition is formulated for intramuscular administration, and the dose of the rAAV is about 2x 1012vg / kg to about 6x 1012vg / kg 1012vg / kg, i) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the composition is formulated for intramuscular administration, and the dose of the rAAV is about 4x1012vg / kg, j) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the composition is formulated for intramuscular administration, and the dose of the administered rAAV is about 6x 1012vg / kg, k) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the composition is formulated for intramuscular injection, and the concentration is about 2x 1013 vg / ml, administered using 3 to 6 injections / muscle of about 0.5 to 1 ml, or 1) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the composition is formulated for intramuscular injection at a concentration of about 2 x 10 13 vg / ml, administered using multiple injections in a total volume of approximately 5 to 14 ml.

[0079] In another embodiment, the present disclosure provides a composition comprising a nucleic acid encoding the NT-3 polypeptide, the composition is used to improve muscle strength or stimulate muscle growth in a human subject, wherein a) the nucleic acid comprises a nucleotide sequence that is 90% identical to the nucleotide sequence of SEQ ID NO: 1; b) the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 1; c) the nucleic acid comprises a nucleic acid sequence that encodes an amino acid sequence that is at least 90% identical to SEQ ID NO: 2 or 100% identical to SEQ ID NO: 2, d) the nucleic acid encoding the NT-3 polypeptide is any nucleic acid of the present disclosure, e) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, and the rAAV is at a dose that results in sustained expression of a low concentration of the NT-3 polypeptide, f) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the composition is formulated for intramuscular administration, and the dose of the rAAV is about 1.5 x 1012 vg / kg to about 6.5 x 1012vg / kg, g) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the composition is formulated for intramuscular administration, and the dose of the rAAV is about 2x 1012vg / kg to about 6x 1012vg / kg, h) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the composition is formulated for intramuscular administration, and the dose of the rAAV is about 2x 1012vg / kg, i) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the composition is formulated for intramuscular administration, and the dose of the rAAV is about 4x 1012vg / kg. 1012vg / kg, j) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the composition is formulated for intramuscular administration, and the dose of the rAAV is about 6x 1012vg / kg, k) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the composition is formulated for intramuscular injection, the concentration is about 2x 1013vg / ml, and 3 to 6 injections of about 0.5 to 1ml are administered per muscle, or l) the nucleic acid encoding the NT-3 polypeptide is a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3, the composition is formulated for intramuscular injection, the concentration is about 2x 1013vg / ml, and 3 to 6 injections of about 0.5 to 1ml are administered per muscle. 13 vg / ml, administered using multiple injections in a total volume of approximately 5 to 14 ml.

[0080] For example, any composition of the disclosure may include a nucleic acid formulated for administration using a viral vector such as an adeno-associated viral vector. In addition, any composition of the disclosure may include a nucleic acid operably linked to a muscle-specific promoter, such as a muscle-specific creatine kinase promoter (MCK). In another embodiment, in any composition or disclosure, scAAV1.tMCK.NTF3 includes the NT-3 gene cassette listed in SEQ ID NO: 11.

[0081] In one embodiment, the present disclosure provides a composition comprising recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3 for treating muscle wasting disorders or neuropathy in a human subject in need thereof, wherein the composition results in sustained expression of low concentrations of NT-3 protein.

[0082] In another embodiment, the present disclosure provides the use of a recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3 for stimulating muscle growth in a human subject in need thereof, wherein the rAAV results in sustained expression of low concentrations of NT-3 protein.

[0083] In one embodiment, the present disclosure provides a composition comprising a dose of recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3 for treating a muscle wasting disorder or a neuropathy in a human subject in need thereof, wherein the composition is formulated for intramuscular administration, and the dose of rAAV administered is about 1.0 x 10 12 vg / kg to about 7x 10 12 vg / kg, or about 1.5 x 10 12 vg / kg to about 6.5 x 10 12 vg / kg, or about 2 x 10 1 2vg / kg to about 6x10 12 vg / kg.

[0084] In another embodiment, the present disclosure provides a composition comprising a dose of recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3 for treating a muscle wasting disorder or a neuropathy in a human subject in need thereof, wherein the composition is formulated for intramuscular administration, and the dose of rAAV administered is about 1.0 x 10 12 vg / kg, or about 1.5 x 10 12 vg / kg, or about 2 x 10 12 vg / kg, or about 3x 10 12 vg / kg, or about 4 x 10 12vg / kg, or about 5x10 12 vg / kg, or about 6x10 12 vg / kg, or about 7 x 10 12 vg / kg, or about 8 x 10 12 vg / kg, or about 9 x 10 12 vg / kg, or about 1x 10 13 vg / kg.

[0085] In another embodiment, the disclosure provides a composition comprising a dose of recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3 for treating a muscle wasting disorder or a neuropathy in a human subject in need thereof, wherein the composition is formulated for intramuscular administration and the dose of rAAV administered is about 2 x 10 13 vg / ml, at low doses (2x 10 12 vg / kg) and high dose (6x 10 12 vg / kg). In some embodiments, the composition is administered using 3 to 6 injections / muscle (each injection volume will be 0.5 to 1 ml) to the medial and lateral heads of the gastrocnemius and tibialis anterior muscles in each leg, respectively. A total of 5 mL to 14 mL of the vehicle will be administered to the medial and lateral heads of the gastrocnemius and tibialis anterior muscles in each leg.

[0086] In an exemplary embodiment, the present disclosure provides a composition comprising a dose of recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3 for treating a muscle wasting disorder or a neuropathy in a human subject in need thereof, wherein the composition is formulated for intramuscular administration, and the dose of the rAAV administered is about 2 x 10 13 vg / ml, using 3 to 6 injections / muscle at low doses (2 x 10 12 vg / kg) and high dose (6x10 12 vg / kg) (each injection volume will be 0.5 to 1 ml). A total of 5 mL to 14 mL of vehicle will be administered to the medial and lateral heads of the gastrocnemius and tibialis anterior muscles in each leg.

[0087] In one embodiment, the present disclosure provides a composition comprising a dose of recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3 for improving muscle strength in a human subject, wherein the composition is formulated for intramuscular administration, and the dose of rAAV administered is about 1.0 x 10 12 vg / kg to about 7x 10 12vg / kg, or about 1.5 x 10 12 vg / kg to about 6.5 x 10 12 vg / kg, or about 2 x 10 12 vg / kg to about 6x 10 12 vg / kg.

[0088] In another embodiment, the present disclosure provides a composition comprising a dose of recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3 for improving muscle strength in a human subject, wherein the composition is formulated for intramuscular administration, and the dose of rAAV administered is about 1.0 x 10 12 vg / kg, or about 1.5 x 10 12 vg / kg, or about 2 x 10 12 vg / kg, or about 3x 10 12 vg / kg, or about 4 x 10 12 vg / kg, or about 5 x 10 12 vg / kg, or about 6x10 12 vg / kg, or about 7 x 10 12 vg / kg, or about 8 x 10 12 vg / kg, or about 9 x 10 12 vg / kg, or about 1x 10 13 vg / kg.

[0089] In another embodiment, the present disclosure provides a composition comprising a dose of recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3 for improving muscle strength or stimulating muscle growth in a human subject in need thereof, wherein the composition is formulated for intramuscular administration, and the dose of rAAV administered is about 2 x 10 13 vg / ml, at low doses (2x 10 12 vg / kg) and high dose (6x 10 12 vg / kg). In some embodiments, the composition is administered using 3 to 6 injections / muscle (each injection volume will be 0.5 to 1 ml) to the medial and lateral heads of the gastrocnemius and tibialis anterior muscles in each leg, respectively. A total of 5 mL to 14 mL of the vehicle will be administered to the medial and lateral heads of the gastrocnemius and tibialis anterior muscles in each leg.

[0090] In an exemplary embodiment, the present disclosure provides a composition comprising a dose of recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3 for improving muscle strength or stimulating muscle growth in a human subject in need thereof, wherein the composition is formulated for intramuscular administration, and the dose of rAAV administered is about 2 x 10 13 vg / ml, using 3 to 6 injections / muscle at low doses (2 x 10 12 vg / kg) and high dose (6x10 12 vg / kg) (each injection volume will be 0.5 to 1 ml. A total of 5 mL to 14 mL of vehicle will be administered to the medial and lateral heads of the gastrocnemius and tibialis anterior muscles in each leg.

[0091] In any composition of the present disclosure, the administration route of scAAV1.tMCK.NTF3 is intramuscular bilateral injection into the medial and lateral heads of the gastrocnemius and tibialis anterior muscles. In addition, in any composition of the present invention, the administration of scAAV1.tMCK.NTF3 results in improved muscle strength of the upper or lower limbs in the subject, and, for example, the improvement in muscle strength is measured as a reduction in the composite score on the CMT Pediatric Scale (CMTPedS). In addition, in any composition of the present disclosure, the administration of the composition results in a reduction or cessation of disease progression over a two-year period. Disease progression is measured by CMTPeedS. CMTPeedS is an 11-item scale consisting of: functional agility test, nine-hole column test (9HPT), hand grip strength, plantar flexion and dorsiflexion strength (using a handheld myometer), acupuncture and vibration sensation, Bruininks Oseretsky test-balance assessment, gait assessment, long jump and six-minute walk test (6MWT). Efficacy was defined as cessation of decline in performance as measured by this scale at 2 years after gene transfer.

[0092] In aspects of the present disclosure, in any of the compositions, the subject suffers from a hereditary neuropathy such as Charcot-Marie-Tooth (CMT) neuropathy, e.g., CMT1A, CMT2K, CMT4A, CMTRIA, and axonal and demyelinating neuropathy caused by autosomal recessive gene variants or autosomal dominant gene variants or X-linked gene variants. The hereditary neuropathy may be caused by any of the gene variants provided in Table 1. In addition, the hereditary neuropathy may be a transthyretin amyloid neuropathy caused by a transthyretin (TTR) gene mutation, such as the following gene variants: Val30Met, Ile107Val, and Ser77Tyr.

[0093] In another aspect of the disclosure, in any composition, the subject suffers from an acquired neuropathy with axonal loss and / or impaired nerve regeneration. The acquired neuropathy is a peripheral neuropathy caused by any disorder or disease that causes neuropathy. For example, the subject suffers from peripheral neuropathy caused by: diabetes, human immunodeficiency virus (HIV) infection, thyroid disorders (such as hypothyroidism), hypoglycemia, uremia, renal insufficiency, liver dysfunction, liver failure, polycythemia, connective tissue disorders, cancer, Lyme disease, celiac disease, leprosy, porphyria, Sjögren's syndrome, poliomyelitis, acromegaly, lipid / glycolipid metabolism disorders, West Nile syndrome, amyloidosis, mitochondrial disorders, dysproteinemia disorders such as monoclonal gammopathy of undetermined significance (MGUS) or POEMS syndrome. The subject suffers from a nutritional / vitamin deficiency, such as vitamin B 12 deficiency, vitamin E deficiency, or copper deficiency.

[0094] Additionally, in any of the compositions of the present disclosure, the subject suffers from autoimmune peripheral polyneuropathy, acute inflammatory demyelinating polyradiculoneuropathy (AIDP), chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), vasculitic mononeuritis multiplex, paraneuropathy, idiopathic ganglionitis, amyotrophic lateral sclerosis, multifocal motor block neuropathy, or lower motor neuron syndrome.

[0095] In any of the compositions of the present disclosure, the acquired neuropathy is a toxic neuropathy. For example, toxic neuropathy is the result of the toxic effects of prescription drugs such as chloramphenicol, cholinequinol, colchicine, disulfiram, etanercept, ethambutol, gold, hydroxychloroquine, nitrofurantoin, metronidazole, stavudine, zalcitabine, infliximab, leflunomide, thalidomide, or chemotherapy agents such as cisplatin, cytarabine, bortezomib, docetaxel, lenalidomide, misondiazole, oxaliplatin, paclitaxel, procarbazine, suramin, thalidomide, vinblastine or vincristine, or anti-alcohol drugs such as disulfiram, or anticonvulsants such as phenytoin or dilantin, or heart or blood pressure medications such as statins, amiodarone, hydralazine, procainamide, perhexiline, or antibiotics such as fluoroquinolones, isoniazid, ciprofloxacin, levofloxacin, metronidazole or metronidazole, and skin disease treatments such as dapoxetine. This toxic neuropathy is also caused by long-term alcohol abuse or vitamin B6 toxicity.

[0096] In another aspect of the 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.

[0097] In yet another aspect of the disclosure, in any of the compositions, the subject is a surgical patient with acquired neuropathy. For example, the surgical patient develops neuropathy after undergoing bariatric surgery, multiple plastic surgeries, or multiple "entrapped nerve" surgeries.

[0098] In another aspect of the disclosure, in any, the subject suffers from a hereditary myopathy, a neuromuscular disease, muscular atrophy, a drug-induced myopathy, sarcopenia, cachexia, type II myofiber dystrophy, a genetically determined muscular dystrophy, age-related muscular atrophy, or an acquired autoimmune primary muscle disorder.

[0099] In another aspect of the disclosure, in any of the compositions, the subject has Duchenne muscular dystrophy, Becker muscular dystrophy, myotonic dystrophy, sarcoglycanopathy, myotonic dystrophy, Emery-Dreifuss muscular dystrophy, congenital muscular dystrophy, fascin deficiency congenital muscular dystrophy, Bethlem myopathy, Ullrich congenital muscular dystrophy, trophy, facioscapulohumeral muscular dystrophy, spinal muscular dystrophy, rigid spinal 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, eye, and brain disease; Fukuyama-Walker-Warburg Syndrome; myasthenic syndrome; congenital muscular weakness; inclusion body myopathy; inclusion body myositis; dermatomyositis; centronuclear myopathy; Myoshi myopathy; mitochondrial myopathy; nemaline myopathy; Nonaka myopathy; myasthenia gravis; or polymyositis.

[0100] In another aspect of the disclosure, in any of the compositions, the subject suffers from a traumatic nerve injury, such as a nerve injury caused by compression, entrapment, or transection. BRIEF DESCRIPTION OF THE DRAWINGS

[0101] The present invention may be more readily understood with reference to the following drawings, in which:

[0102] Figure 1 A to Figure 1 C provides a graph and images showing AAV1.NT-3-induced fiber type remodeling in TrJ muscle. At 16 weeks after injection, AAV1.tMCK.NT-3-treated TremblerJ (TrJ) ( Figure 1 Gastrocnemius muscle (A) and untreated (TrJ-PBS) Figure 1 B) Representative images of SDH-stained tissue sections. Slow oxidation (STO, arrow), fast oxidation (FTO, arrow head), and fast glycolysis (FTG, asterisk) are shown ( Figure 1 B). The oxidative fibers in a were reduced. In TrJ-PBS muscle ( Figure 1 In B), all fiber types showed an increased number of small STO fibers and angular fibers, as well as a small type group compatible with neurogenic changes. For a, b, scale bar = 30 gm. TrJ muscle ( Figure 1 C), fiber type shifts from STO to FTO / FTG fibers. The mean STO percentages in both treatment groups (derived from n=3-5 mice in each group) were not significantly different from wild-type (WT) muscle, suggesting a shift toward normalization of fiber type distribution in TrJ neurogenic muscles with NT-3.

[0103] Figure 2 A to Figure 2 C provides graphs and images showing the effects of AAV 1.NT3 treatment on mTOR signaling and metabolic markers. At 16 weeks post-injection, Figure 2 A) and wild type (WT) ( Figure 2 B) Representative Western blot images and analysis of mTOR targets in gastrocnemius muscle, namely phospho (P)-4EBP1 (Thr37 / 46) and P-S6 (Ser235 / 236). These graphs show the expression levels of phosphorylated forms of the proteins normalized to GAPDH. Coomassie blue-stained membranes represent equal gel loadings. Error bars are ± SEM; n = 5-6 in each group, *P < 0.05, unpaired t-test. ( Figure 2 C) Relative expression of glycolytic (1-1K1 and PK1) and oxidative regulators (PGC1α) by qPCR; GAPDH was used as a housekeeping gene. Error bars are ± SEM; n = 5-6 in each group, *P < 0.05, one-way ANOVA followed by Tukey's multiple comparison test.

[0104] Figure 3 A to Figure 3 D provides diagrams and images showing the direct effects of NT-3 on myotubes. Figure 3A) Representative Western blot images and analysis of the Akt / mTOR pathway, phospho (P)-Akt (Ser473), P-4EBP1 (Thr37 / 46), and P-S6 (Ser235 / 236) in myotubes incubated with recombinant human NT-3 (100 ng / ml) or PBS (control) for 30 min. Density values ​​of phosphorylated protein bands were normalized to GAPDH and shown as a percentage of the control group. Coomassie blue-stained membranes represent equal gel loading. Myotubes were incubated with NT-3 (100 ng / ml) for 48 h, and then the relative mRNA expression of metabolic markers (PGC1a, HK1, PK1) was detected by qPCR ( Figure 3 B), and the relationship between glucose consumption and lactate production in cell culture medium was detected by ELISA ( Figure 3 C). Figure 3 D) Relative expression levels of myogenin and NT-3 receptors p75NTR and TrkC in myoblasts and myotubes after NT-3 (100 ng / ml) treatment for 48 h. 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.

[0105] Figure 4 A graph showing serum levels of NT-3 in treated and untreated mice is provided. At the endpoint, serum was collected from each mouse and circulating NT-3 levels were measured by ELISA.

[0106] Figure 5 P75 in TrJ and WT gastrocnemius muscle is shown NTR Relative mRNA expression of TrkC and GAPDH was used as a housekeeping gene in the analysis. The results shown are the mean ± SEM from at least three independent experiments, *P < 0.05, Student's t-test.

[0107] Figure 6 A schematic diagram of the construct AAV.tMCK.NTF3 (SEQ ID NO: 11) is provided. The vector contains the 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).

[0108] Figure 7 Restriction map and ORF analysis of pAAV.tMCK.NTF3 are provided.

[0109] FIG. 8A to FIG. 8BThe locations of IM injection of AAV.tMCK.NTF3 in human subjects are indicated.

[0110] Fig. 9 The nucleotide sequence of AAV.tMCK.NTF3 is provided (SEQ ID NO: 11). DETAILED DESCRIPTION

[0111] The increase in fiber size induced by AAV.NT-3 treatment in TrJ muscles was studied to determine whether this increase was solely a result of reinnervation or whether NT-3 had a direct effect on muscle protein synthesis, independent of nerve regeneration, thereby increasing muscle fiber size.

[0112] Disclosed herein is a novel role for NT-3; namely, its ability to directly influence protein synthesis and metabolic remodeling in neurogenic muscle.

[0113] The work described herein first evaluated the effects of AAV.NT-3 gene therapy on the oxidative status of TrJ muscle at 16 weeks after gene injection and found that the increase in muscle fiber size was associated with a change in the oxidative status of muscle fibers toward the normalization of the fiber type ratio seen in WT. The treatment resulted in a decrease in the percentage of slow-twitch oxidative (STO) fibers, while the population of fast-twitch oxidative and glycolytic (FTO and FTG) fibers increased, reflecting the reversal of the pattern seen in untreated TrJ muscles. The NT-3-induced increase in fiber size was most prominent for the FTG fiber population. It was then investigated whether mammalian target of rapamycin complex 1 (mTORC1) activation plays a role in NT-3-induced muscle protein synthesis, with a particular emphasis on the preferential radial growth of glycolytic fibers. mTORC1 regulates translation and ribosome biogenesis through phosphorylation of the translation regulators, eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1) and S6 kinase 1 (S6K1). Laplante M, Sabatini D., Cell, 149(2): 274-293 (2012). In addition, mTORC1 is associated with the activation of cellular glycolysis, which involves increased translation of glycolytic enzymes or their transcriptional regulators. Duvel et al., Molecular cell, 39(2): 171-183 (2010). Histochemical changes in TrJ muscle were found to be accompanied by increased levels of 4E-BP1 and S6 protein (S6P) phosphorylation, which served as evidence of mTORC1 activation. At the same time, the expression levels of mitochondrial biogenesis regulators (peroxisome proliferator-activated receptor y coactivator 1a, PGC1α) and markers of glycolysis (hexokinase-1, HK1, and pyruvate kinase 1, PK1) increased in TrJ muscle. These changes were not significant in AAV.NT-3 treated WT muscles. In addition, in vitro studies showed that recombinant NT-3 can directly induce Akt / mTOR pathway activation in TrkC-expressing myotubes rather than in myoblasts. In addition, myogenin expression levels were significantly higher in myotubes compared with myoblasts, while p75NTR expression was downregulated, indicating that NT-3-induced myoblast differentiation is associated with mTORC1 activation.

[0114] The findings described herein have many implications for the potential use of NT-3, not only for the treatment of neuropathies, with benefits for both nerves and muscles, but also for muscle wasting conditions, including aging, cancer cachexia or type II myofiber atrophy, and inherited or acquired autoimmune primary muscle disorders associated with an impaired regenerative radial growth phase, in which perturbations in mTORC1 signaling and defective mitochondrial biogenesis may be involved.

[0115] The present disclosure relates to a method for stimulating muscle growth in a subject. The method comprises administering to a subject in need thereof a therapeutically effective amount of neurotrophin-3 (NT-3), pre-NT-3 or an effective fragment thereof, or a nucleic acid encoding NT-3, pre-NT-3 or an effective fragment thereof. Subjects in need of stimulating muscle growth include subjects suffering from muscular dystrophy or muscular atrophy.

[0116] 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 muscular atrophy type 1A (CMT1A), wherein the NTF3 gene encoding NT-3 is delivered to the subject using a self-complementary 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 thereof, such as a subject diagnosed with or suffering from a muscle wasting disorder such as CMT.

[0117] Preclinical studies have demonstrated that delivery of the construct AAV1.tMCK.NTF3 to Trembler J mice9 (Tr J ) (i.e., a naturally occurring mouse model of CMT1) in the gastrocnemius muscle improved nerve regeneration, myelination, myelinated fiber density, sciatic nerve compound muscle action potential amplitude and functional performance in the rotarod test, and hindlimb grip strength (see Example 3).

[0118] As used herein, the terms "treatment", "treating" and the like refer to obtaining a desired pharmacological or physiological effect. The effect may be therapeutic in the sense of partially or completely curing a disease or an adverse effect attributable to the disease. As used herein, "treatment" encompasses any treatment of a disease in a mammal, particularly a human, and may include inhibiting a disease or condition, i.e., preventing its development; and relieving a disease, i.e., causing regression of the disease.

[0119] As used herein, prevention refers to any action that provides a benefit to a subject at risk for a condition or disease, such as a neuropathy, demyelinating polyneuropathy, muscle wasting disorder, or atrophy.

[0120] As used herein, "pharmaceutically acceptable" means that the compound or composition is suitable for administration to a subject in the methods described herein without undue deleterious side effects, depending on the severity of the disease and the necessity of the treatment.

[0121] The terms "therapeutically effective" and "pharmacologically effective" are intended to qualify the amount of an agent that will achieve the goal of improving disease severity and frequency. The effectiveness of treatment can be measured by evaluating the reduction of symptoms in a subject in response to administration of NT-3.

[0122] The term "effective fragment" refers to a portion of a polynucleotide sequence encoding a functional fragment of a NT-3 polypeptide. The term "effective fragment" also refers to a portion of a NT-3 polypeptide amino acid sequence that retains NT-3 growth factor activity. Exemplary NT-3 growth factor activities include supporting the survival and differentiation of existing neurons, and inducing and supporting the growth and differentiation of new neurons and synapses. In addition, NT-3 activities include stimulating muscle growth and muscle function.

[0123] As used herein, the term "diagnosis" can encompass determining the likelihood that a subject will develop a disease, or the presence or nature of a disease in a subject. As used herein, the term diagnosis also encompasses determining the severity of a disease or disease episode and the likely outcome or prospects for recovery (which is often referred to as prognosis). "Diagnosis" can also encompass diagnosis in the context of rational therapy, where the diagnosis guides therapy, including the initial selection of therapy, modification of therapy (e.g., adjustment of dosage or dosage regimen), etc.

[0124] As used herein, a "subject" can be any animal, and may also be referred to as a patient. Preferably, the subject is a vertebrate, and more preferably, the subject is a mammal, such as a domesticated livestock (e.g., cattle, horses, pigs) or a pet (e.g., dogs, cats). In some embodiments, the subject is a human.

[0125] The term "polynucleotide" or "nucleic acid molecule" refers to a polymeric form of nucleotides of at least 10 bases in length. The term includes DNA molecules (e.g., cDNA or genomic or synthetic DNA) and RNA molecules (e.g., mRNA or synthetic RNA), as well as DNA or RNA analogs containing non-natural nucleotide analogs, non-natural internucleoside bonds, or both. Nucleic acids can be in any topological conformation. For example, nucleic acids can be single-stranded, double-stranded, triple-stranded, quadruple-stranded, partially double-stranded, branched, hairpin-shaped, circular, or in a padlocked conformation.

[0126] As used herein, the term "gene" refers to a nucleotide sequence that can direct the synthesis of an enzyme or other polypeptide molecule (e.g., can contain a coding sequence, e.g., a continuous open reading frame (ORF) encoding a polypeptide) or can itself function in an organism. As defined herein, genes in an organism can be clustered in an operon, wherein the operon is separated from other genes and / or operons by intergenic DNA. Individual genes contained in an operon can overlap, and there is no intergenic DNA between individual genes.

[0127] As used herein, the term "AAV" is a standard abbreviation for adeno-associated virus. Adeno-associated virus is a single-stranded DNA small virus that grows only in cells where certain functions are provided by a co-infection helper virus. Thirteen AAV serotypes have been characterized. General information and reviews of AAV can be found in, for example, Carter, 1989, Handbook of Parvoviruses [Small Virus Handbook], Vol. 1, pp. 169-228, and Berns, 1990, Virology [Virology], pp. 1743-1764, Raven Press, (New York). However, it is fully expected that these same principles will apply to other AAV serotypes, because it is well known that the various serotypes are quite closely related in structure and function, even at the genetic level. (See, e.g., Blacklowe, 1988, Parvoviruses and Human Disease, pp. 165-174, JR Pattison, ed.; and Rose, Comprehensive Virology 3: 1-61 (1974)). For example, all AAV serotypes apparently exhibit very similar replication properties mediated by homologous rep genes; and all carry three related capsid proteins, such as those expressed in AAV2. The extent of the relatedness is further indicated by heteroduplex analysis, which reveals extensive cross-hybridization between serotypes along the length of the genome; and the presence of similar self-annealing segments corresponding to "inverted terminal repeats" (ITRs) at the ends. Similar patterns of infectivity also suggest that replication functions in each serotype are under similar regulatory control.

[0128] The term "vector" or "expression vector" refers to any type of genetic construct comprising a nucleic acid encoding an RNA that can be transcribed. An expression vector can contain a variety of control sequences, structural genes (e.g., target genes), and nucleic acid sequences that also provide other functions.

[0129] "Vector" means a DNA molecule, usually derived from a plasmid or bacteriophage into which a DNA fragment can be inserted or cloned. A recombinant vector will contain one or more unique restriction sites and may be capable of autonomous replication in a defined host or vector organism, making the cloned sequence reproducible. A vector contains a promoter operably linked to a gene or coding region, allowing expression of RNA when transfected into a recipient cell.

[0130] As used herein, "AAV vector" refers to a vector comprising one or more polynucleotides of interest (or transgenes) flanked by AAV terminal repeats (ITRs). Such AAV vectors can be replicated and packaged into infectious viral particles when present in host cells that have been transfected with vectors encoding and expressing rep and cap gene products.

[0131] "AAV virion" or "AAV viral particle" or "AAV vector particle" refers to a viral particle composed of at least one AAV capsid protein and an encapsidated polynucleotide AAV vector. 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 usually referred to as an "AAV vector particle" or simply "AAV vector". Therefore, the production of an AAV vector particle necessarily includes the production of an AAV vector, because such a vector is contained within the AAV vector particle.

[0132] As used herein, the term "about" refers to a + / - 10% deviation from a base value.

[0133] Unless defined otherwise, 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.

[0134] Gene therapy for peripheral neuropathy

[0135] In one aspect, the invention provides methods of treating a subject suffering from muscle atrophy using gene therapy.

[0136] Vectors that can be used to deliver therapeutic nucleic acids include viral vectors 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, adeno-associated virus vectors are most popular due to their low risk of insertional mutagenesis and immunogenicity, their lack of endogenous viral genes and their ability to produce at high titers. Kantor et al. reviewed a variety of methods for transferring genes to the central nervous system, while Goins et al. described gene therapy methods for treating chronic peripheral nervous system pain. See Kantor et al., Adv Genet. [Genetics Progress] 87, 125-197 (2014), and Goins et al., Neurobiol. Dis. [Neurobiology of Disease] 48 (2), 255-270 (2012), their disclosures are incorporated herein by reference. Specifically, it has been reported that a variety of viral vectors have been used to successfully deliver genes to Schwann cells, i.e., resident glial cells of peripheral nerves. Mason et al., Curr. Gene Ther. [Current Gene Therapy] 11, 75-89 (2011). If the vector is in a viral vector and the vector has been packaged, the virion can be used to infect the cell. If naked DNA is used, a transfection or transformation procedure suitable for a particular host cell can be used. Naked DNA formulations using polymers, liposomes or nanospheres can be used for gene delivery. The nucleic acid can be given in any desired form that provides a sufficiently effective delivery level, including in a viral particle, in a liposome, in a nanoparticle, and in a polymer complex.

[0137] A nucleic acid (e.g., cDNA or transgene) encoding a gene whose expression reduces peripheral neuropathy can be cloned into an expression cassette with a regulatory element such as a promoter (constitutive or regulatable) to drive transgene expression and a polyadenylation sequence downstream of the nucleic acid. For example, regulatory elements can be used that are 1) specific to a tissue or region of the body; 2) constitutive; and / or 3) inducible / regulatable.

[0138] In some embodiments, muscle-specific regulatory elements are used. Muscle-specific regulatory elements include muscle-specific promoters, including mammalian muscle creatine kinase (MCK) promoters, mammalian desmin promoters, mammalian troponin I (TNNI2) promoters, or mammalian skeletal α-actin (ASKA) promoters. Muscle-specific enhancers that can be used in the present invention are selected from the group consisting of: mammalian MCK enhancers, mammalian DES enhancers, and vertebrate troponin IIRE (TNI IRE, hereinafter referred to as FIRE) enhancers. One or more of these muscle-specific enhancer elements can be used in combination with the muscle-specific promoter of the present invention to provide tissue-specific regulatory elements.

[0139] A preferred vector for treating muscle atrophy by gene therapy is AAV. AAV-mediated gene delivery has become an effective and safe tool for both preclinical and clinical studies of neurological disorders. Ojala et al., Neuroscientist. [Neuroscientist], 21 (1): 84-98 (2015). Currently, AAV is the most widely used vector in clinical trials for neurological disorders, and no adverse effects associated with the use of this vector have been reported from clinical trials: Adeno-associated virus is a non-pathogenic dependent virus from the Parvoviridae family that requires auxiliary functions from other viruses (such as adenovirus or herpes simplex virus) to complete its life cycle. Wild-type (WT) AAV is characterized by a single-stranded DNA (ssDNA) genome of approximately 5 kb surrounded by a capsid, which has inverted terminal repeats (ITRs) at both ends.

[0140] Adenoviral vectors used to deliver transgenes to cells for applications such as in vivo gene therapy and in vitro research and / or production of transgenic products are often derived from adenoviruses that have deleted the early region 1 (E1) gene (Berkner, KL, Curr. Top. Micro. Immunol. [Current Topics in Microbiology and Immunology] 158 L39-66 1992). The deletion of the E1 gene renders such adenoviral vectors replication-defective and significantly reduces the expression of the remaining viral genes present in the vector. Recombinant adenoviral vectors have several advantages for use as gene delivery vehicles, including tropism for both dividing and non-dividing cells, minimal pathogenic potential, the ability to replicate to high titers to prepare vector stocks, and the potential to carry large inserts. However, it is believed that the presence of the remaining viral genes in adenoviral vectors may be harmful.

[0141] Therefore, in some embodiments, the adenoviral vector has a deletion of multiple adenoviral gene sequences. Specifically, a pseudoadenoviral vector (PAV) (also known as a 'gutless adenovirus') or a small adenoviral vector is an adenoviral vector derived from an adenoviral genome that contains the minimum cis-acting nucleotide sequence required for replication and packaging of the vector genome, and it may contain one or more transgenes (see U.S. Patent No. 5,882,877, incorporated herein by reference, which covers pseudoadenoviral vectors (PAVs) and methods for producing PAVs). Such PAVs that can accommodate up to about 36 kb of foreign nucleic acid are advantageous because the carrying capacity of the vector is optimized while reducing the possibility of a host immune response to the vector or the generation of replicative viruses. The PAV vector contains a 5' inverted terminal repeat (ITR) and a 3' ITR nucleotide sequence containing a replication origin and a cis-acting nucleotide sequence required for packaging the PAV genome, and can accommodate one or more transgenes with appropriate regulatory elements (e.g., promoters, enhancers, etc.).

[0142] AAV

[0143] The recombinant AAV genome of the invention comprises a nucleic acid molecule of the invention and one or more AAV ITRs flanking the nucleic acid molecule. The AAV DNA in the rAAV genome can be from any AAV serotype from which a recombinant virus can be derived, including but not limited to AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, and AAV-13 (see, e.g., Gao et al., PNAS, 99:11854-11859 (2002); and Viral Vectors for Gene Therapy: Methods and Protocols [Viral Vectors for Gene Therapy: Methods and Protocols], ed. Machida, Humana Press, 2003). In addition, pseudotyped AAV vectors can also be used in the methods described herein. Pseudotyped AAV vectors are those that 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. [Human Molecular Genetics], 10(26):3075-81). The generation of pseudotyped rAAV is disclosed in, for example, WO 01 / 83692. Other types of rAAV variants, such as rAAV with capsid mutations, are also contemplated. See, for example, Marsic et al., Molecular Genetics, 1996. Therapy, 22(11): 1900-1909 (2014). As described in the background technology section above, the nucleotide sequences of the genomes of various AAV serotypes are known in the art. In order to promote skeletal muscle-specific expression, AAV1, AAV6, AAV8 or AAVrh.74 can be used.

[0144] The DNA plasmid of the present invention comprises the rAAV genome of the present invention. The DNA plasmid is transferred to cells that allow infection by the helper virus of AAV (e.g., adenovirus, adenovirus or herpes virus deleted by E1) to assemble the rAAV genome into infectious viral particles. The technology for producing rAAV particles (wherein the AAV genome to be packaged, rep and cap genes, and helper virus functions are provided to cells) is standard in the art. The production of rAAV requires the presence of the following components in a single cell (referred to herein as a packaging cell): the rAAV genome, the AAV rep and cap genes separated from the rAAV genome (i.e., not in the rAAV genome), and the helper virus functions. The AAV rep and cap genes can be from any AAV serotype from which recombinant viruses can be derived, and can be from a different AAV serotype than the rAAV genome ITRs, including but not limited to AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAVrh.74, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, and AAV-13. The generation of pseudotyped rAAVs is disclosed, for example, in WO 01 / 83692, which is incorporated herein by reference in its entirety.

[0145] The method of generating packaging cells is to generate a cell line that stably expresses all the necessary components for AAV particle production. For example, a plasmid (or plasmids) comprising the following items is integrated into the genome of the cell: a rAAV genome lacking AAV rep and cap genes, an AAV rep and cap genes separated from the rAAV genome, and a selective marker (e.g., a neomycin resistance gene). The AAV genome has been introduced into bacterial plasmids by procedures such as GC tailing (Samulski et al., 1982, Proc. Natl. Acad. S6. USA [Proceedings of the National Academy of Sciences of the United States S6], 79: 2077-2081), adding synthetic linkers containing restriction endonuclease cleavage sites (Laughlin et al., 1983, Gene [Gene], 23: 65-73) or by direct blunt end ligation (Senapathy and Carter, 1984, J. Biol. Chem. [Journal of Biological Chemistry], 259: 4661-4666). The packaging cells are then infected with a helper virus (such as an adenovirus). The advantage of this method is that the cells are selectable and suitable for large-scale production of rAAV. Other examples of suitable methods use adenovirus or baculovirus instead of plasmids to introduce the rAAV genome and / or rep and cap genes into packaging cells.

[0146] The general principles of rAAV generation are reviewed in, for example, Carter, 1992, Current Opinions in Biotechnology, 1533-539; and Muzyczka, 1992, Curr. Topics in Microbial. and Immunol., 158:97-129). Various methods are described in the following literature: Ratschin et al., Mol. Cell. Biol. [Molecular and Cellular Biology] 4:2072 (1984); Hermonat et al., Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America], 81:6466 (1984); Tratschin et al., Mol. Cell. Biol. [Molecular and Cellular Biology] 5:3251 (1985); McLaughlin et al., J. Virol. [Journal of Virology], 62:1963 (1988); and Lebkowski et al., 1988 Mol. Cell. Biol. [Molecular and Cellular Biology], 7:349 (1988). Samulski et al. (1989, J. Virol., 63:3822-3828); U.S. Pat. No. 5,173,414; WO 95 / 13365 and corresponding U.S. Pat. No. 5,658,776; WO 95 / 13392; WO 96 / 17947; PCT / US98 / 18600; WO 97 / 09441 (PCT / US96 / 14423); WO 97 / 08298 (PCT / US96 / 13872); WO 97 / 21825 (PCT / US96 / 20777); WO 97 / 06243 (PCT / FR96 / 01064); WO 99 / 11764; Perrin et al. (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. Pat. No. 5,786,211; U.S. Pat. No. 5,871,982; and U.S. Pat. No. 6,258,595. The foregoing documents are hereby incorporated by reference in their entirety, with particular emphasis on those portions of the documents relating to rAAV production.

[0147] Therefore, the present invention provides packaging cells for producing infectious rAAV. In one embodiment, the packaging cells can be stably transformed cancer cells, such as HeLa cells, 293 cells and PerC.6 cells (homologous 293 cell lines). In another embodiment, the packaging cells are cells that are untransformed cancer cells, such as low-passage 293 cells (human fetal kidney cells transformed with E1 of adenovirus), MRC-5 cells (human fetal fibroblasts), WI-38 cells (human fetal fibroblasts), Vero cells (monkey kidney cells) and FRhL-2 cells (rhesus fetal lung cells).

[0148] The recombinant AAV of the present invention (i.e., infectious encapsidated rAAV particles) comprises the rAAV genome. In an exemplary embodiment, the genomes of both rAAVs lack AAVrep and capDNA, i.e., there is no AAVrep or capDNA between the ITRs of the genome. Examples of rAAVs that can be constructed to comprise nucleic acid molecules of the present invention are set forth in International Patent Application No. PCT / US2012 / 047999 (WO 2013 / 016352), which is incorporated by reference in its entirety.

[0149] rAAV can be purified by methods standard in the art, such as by column chromatography or cesium chloride gradients. Methods for purifying rAAV vectors from helper viruses are known in the art and include those disclosed, for example, in Clark et al., Hum. Gene Ther. [Human Gene Ther.], 10(6): 1031-1039 (1999); Schenpp and Clark, Methods Mol. Med. [Molecular Medicine Methods], 69427-443 (2002); U.S. Pat. No. 6,566,118 and WO 98 / 09657.

[0150] In another embodiment, the present invention contemplates a composition comprising the rAAV of the present invention. The composition of the present invention comprises rAAV and a pharmaceutically acceptable carrier. These compositions may also include other ingredients, such as diluents and adjuvants. Acceptable carriers, diluents, and adjuvants are nontoxic to the recipient at the doses and concentrations employed and are preferably inert, 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 polyvinyl pyrrolidone; 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, Pluronic, or polyethylene glycol (PEG).

[0151] The titer of rAAV to be administered in the methods of the invention will vary depending on, for example, the particular rAAV, the mode of administration, the therapeutic goal, the individual, and the cell type or cells being targeted, and can be determined by methods standard in the art. The titer of rAAV can be about 1 x 10 6 , about 1x 10 7 , about 1x10 8 , about 1x 10 9 , about 1x 10 10 , about 1x 10 11 , about 1x10 12 , about 1x 10 13 About 1x 10 14 The dosage may also be expressed in units of viral genomes (vg).

[0152] The present invention contemplates methods for transducing target cells with rAAV in vivo or in vitro. The in vivo method includes the step of administering an effective dose or multiple effective doses of a composition comprising rAAV of the present invention to an animal (including a human) in need thereof. If the dose is administered before the development of the disorder / disease, the administration is preventive. If the dose is administered after the development of the disorder / disease, the administration is therapeutic. In an embodiment of the present invention, an effective dose is a dose that alleviates (eliminates or reduces) at least one symptom associated with the disorder / disease state being treated, slows or prevents progression to the disorder / disease state, slows or prevents the progression of the disorder / disease state, reduces the extent of the disease, causes (partial or complete) remission of the disease and / or prolongs survival.

[0153] Specifically, the actual administration of the rAAV of the present invention can be accomplished by using any physical method for transporting the rAAV recombinant vector to the target tissue of the animal. Administration according to the present invention includes, but is not limited to, injection into muscle, bloodstream and / or direct injection into the liver. It has been shown that simply resuspending the rAAV in phosphate-buffered saline is sufficient to provide a medium that can be used for muscle tissue expression, and there are no known restrictions on the vectors or other components that can be co-administered with rAAV (but the conventional method of using rAAV should avoid compositions that can degrade DNA). The capsid protein of rAAV can be modified so that rAAV targets specific target tissues, such as muscle. See, for example, WO 02 / 053703, the disclosure of which is incorporated herein by reference. The pharmaceutical composition can be prepared as an injectable formulation or a topical formulation to be delivered to the muscle by transdermal transport. A variety of formulations for intramuscular injection and transdermal transport have been previously developed, and they can be used to practice the present invention. rAAV can be used with any pharmaceutically acceptable carrier to facilitate administration and handling.

[0154] Transduction can be performed using a gene cassette containing tissue-specific control elements. For example, one embodiment of the present invention provides a method for transducing muscle cells and muscle tissue directed by muscle-specific control elements, including but not limited to those derived from the actin and myosin gene families (e.g., from 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 [Molecular Cell Biology] 11:4854-4862 (1991)], control elements derived from the human skeletal actin gene [Muscat et al., Mol Cell Biol [Molecular Cell Biology], 7:4089-4099 (1987)], the cardiac actin gene, the muscle creatine kinase sequence element [see Johnson et al., Mol Cell Biol [Molecular Cell Biology], 7:4089-4099 (1987)], the myosin gene ... Cell Biol [Molecular Cell Biology], 9:3393-3399 (1989)] and the mouse creatine kinase enhancer (mCK) element, control elements derived from the skeletal fast-twitch troponin C gene, the slow-twitch cardiac troponin C gene, and the slow-twitch troponin I gene: hypoxia-inducible nuclear factor (Semenza et al., Proc Natl Acad Sci USA [Proceedings of the National Academy of Sciences of the United States of America], 88:5680-5684 (1991)), steroid-inducible elements and promoters (including the glucocorticoid response element (GRE) (see Mader and White, Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 90:5603-5607 (1993))), and other control elements.

[0155] Muscle tissue is not a vital organ in vivo and is easily accessible, making it an attractive target for in vivo DNA delivery. The present invention contemplates sustained expression of miRNA from transduced myofibers.

[0156] "Muscle cell" or "muscle tissue" means a cell or cell population derived from any kind of muscle (e.g., skeletal muscle and smooth muscle, such as from the digestive tract, bladder, blood vessels, or heart tissue). Such muscle cells can be differentiated or undifferentiated, such as myoblasts, myocytes, myotubes, cardiomyocytes, and cardiomyoblasts.

[0157] The term "transduction" is used to refer to the administration / delivery of the coding region of NT-3 to recipient cells in vivo or in vitro via the replication-defective rAAV of the present invention, thereby resulting in the expression of NT-3 by the recipient cells.

[0158] In one embodiment, gene therapy is NT-3 gene therapy delivered via recombinant adeno-associated virus (AAV). The inventors have developed an AAV expression cassette carrying a human NT-3 cDNA coding sequence under the control of a CMV promoter or a three-muscle specific creatine kinase (tMCK) promoter. The inventors have previously shown that using a recombinant AAV1 vector to increase neurotrophin-3 expression in trembling (Try) mice (which are models of Charcot-Marie-Tooth disease variant CMT1A) can achieve improvements in motor function, histopathology, and electrophysiology of peripheral nerves. See Sahenk et al., Mol Ther. [Molecular Therapy] 22 (3): 511-21 (2014), the disclosure of which is incorporated herein by reference.

[0159] Thus, the present invention provides methods of administering an effective dose (or doses administered substantially simultaneously or doses provided at intervals) of rAAV encoding NT-3 to a patient in need thereof.

[0160] Dosage and route of administration

[0161] The present invention provides local and systemic administration of effective doses of rAAV and compositions of the present invention, including the combination therapy of the present invention. For example, systemic administration is administration into the circulatory system so that the whole body is affected. Systemic administration includes enteral administration (such as absorption through the gastrointestinal tract) and parenteral administration by injection, infusion or implantation.

[0162] Thus, routes of administration of rAAV contemplated in the foregoing methods include, but are not limited to, intraperitoneal (IP), intramuscular (IM), and intravascular [including, for example, inter-arterial limb perfusion (ILP) and intravenous (IV) routes.

[0163] The dose of rAAV to be administered in the methods disclosed herein will vary depending on, for example, the particular rAAV, the mode of administration, the therapeutic goal, the individual, and the cell type or cells being targeted, and can be determined by methods standard in the art. More than one dose may be administered, for example, one, two, three, or more doses. The titer of the rAAV in a dose may be about 1 x 10 6 , about 1x 10 7 , about 1x10 8 , about 1x 10 9 , about 1x 10 10 , about 1x 1 11 , about 1x 10 12 , about 1.5x10 12 , about 1x 10 12 , about 3x 10 12 , about 4x 10 12 , about 5x 10 12, about 6x 10 12 , about 6.5x 10 12 , about 7x 10 12 , 1x10 13 , about 1x 10 14 , or to about 1x 10 15 or more DNase-resistant particles (DRP) / ml. Doses can also be expressed in units of viral genomes (vg) (i.e., 1x10 7 vg, 1x 10 8 vg, 1x 10 9 vg, 1x 10 10 vg, 1x10 11 vg, 1x 10 12 vg, about 1.5x 10 12 vg, about 1x 10 12 vg, about 3x 10 12 vg, about 4x 10 12 vg, about 5x 10 12 vg, about 6x 10 12 vg, about 6.5x 10 12 vg, about 7x 10 12 vg, 1x 10 13 vg, 1x 10 14 vg, 1x 10 15 ). Methods for titrating AAV are described in Clark et al., Hum. Gene Ther., 10: 1031-1039 (1999).

[0164] In some embodiments of the foregoing methods where the route of administration is the IM route, the dose of rAAV administered is about 1.5 x 10 12 to at least about 6.5 x 10 12 vg / kg. (All ranges herein are intended to represent each individual value within those ranges, as well as the individual upper and lower limits of each range.) In some embodiments of the foregoing methods where the route of administration is IM, the dose of rAAV administered is 2 x 10 12 In some embodiments of the aforementioned methods where the route of administration is IM, the dose of rAAV administered is 4 x 10 12 In some embodiments of the aforementioned methods where the route of administration is IM, the dose of rAAV administered is 6 x 10 12 vg / kg.

[0165] Human patients are subjects contemplated for treatment herein.Human patients are subjects contemplated for treatment by IM delivery herein.Such patients include those patients, for example: i) adult subjects (>18 years old) diagnosed with CMT1A, ii) 1.5Mb duplication is shown at 17p11.2 including the peripheral myelin protein 22 (PMP22) gene, iii) males and females of any race or ethnic group, iv) ankle dorsiflexor weakness is shown (complete ROM should be present against gravity, but complete dorsiflexion against gravity cannot be maintained or heel standing for 3 seconds or longer (Northstar criteria)), iv) abnormal nerve conduction velocity, v) ability to cooperate with clinical evaluation and repeated nerve conduction studies, and vi) sexually active subjects are willing to adopt reliable contraceptive methods during the study. Suitable patients may not include, for example, patients with: 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 trial entry (e.g., corticosteroids, cyclosporine, tacrolimus, methotrexate, cyclophosphamide, intravenous immunoglobulin), iii) persistent leukopenia or leukocytosis (WBC ≤ 3.5K / μL or ≥ 20.0K / μL) or absolute neutrophil count < 1.5K / μL, iv) AAV1 binding antibody titer ≥ 1:50 as determined by ELISA immunoassay, v) concomitant illness or need for long-term medication that the PI believes poses an unnecessary risk of gene transfer, vi) ankle contracture or surgery that precludes appropriate muscle strength testing, vii) pregnancy, breastfeeding, or attempted pregnancy, viiii) other causes of neuropathy, and / or ix) limb surgery within the past six months. In an exemplary clinical protocol, CMT1A patients received a total dose of the vector scAAV1.tMCK.NTF3, which was distributed to the medial and lateral heads of the gastrocnemius and tibialis anterior (TA) muscles of the leg, which preferentially causes ankle weakness and instability in CMT. Subjects received one of the following: i) a low dose of 2 x 10 12 vg / kg carrier (total dose) or ii) high dose of 6x10 12 vg / kg vehicle (total dose).

[0166] In one embodiment, the vector is administered by IM injection in the absence of a diluent. In an alternative embodiment, the composition for intramuscular injection includes an adjuvant, for example, sesame oil or peanut oil can be used or in a propylene glycol aqueous solution, and a sterile aqueous solution. If necessary, such aqueous solutions can be buffered, and the liquid diluent is first made isotonic with saline or glucose. rAAV can be prepared by mixing appropriately with a surfactant (such as hydroxypropyl cellulose) in water as a solution of a free acid (DNA contains an acidic phosphate group) or a pharmacologically acceptable salt. Dispersions of rAAV can 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 the growth of microorganisms. In this regard, the sterile aqueous media used can all be easily obtained by standard techniques well known to those skilled in the art.

[0167] The pharmaceutical carrier, diluent or excipient suitable for injection purposes include sterile aqueous solution or dispersion and the sterile powder for the temporary preparation of sterile injectable solution or dispersion. In all cases, the form must be sterile and must have the fluidity reaching the degree of easy injection. It must be stable and must be preserved against the contamination of microorganisms (such as bacteria and fungi) under the conditions of manufacture and storage. The carrier can be a solvent or dispersion medium comprising the following: for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, etc.), its applicable mixture and vegetable oil. Appropriate fluidity can be maintained (for example) by using coating (such as lecithin), maintained by maintaining the required particle size in the case of dispersion, and maintained by using a surfactant. The effect of microorganisms can be prevented by various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it will be preferred to include isotonic agents, such as sugar or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0168] Sterile injectable solutions are prepared by incorporating rAAV in the desired amount in an appropriate solvent with the various other ingredients enumerated above as needed, followed by filtered sterilization. In general, dispersions are prepared by incorporating the sterilized active ingredient into a sterile vehicle containing a basic dispersion medium and the required other ingredients from those enumerated above. In the case of 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 ingredients from a previously sterile filtered solution thereof.

[0169] rAAV transduction can also be performed in vitro. In one embodiment, the desired target muscle cells are removed from a subject, transduced with rAAV and reintroduced into the subject. Alternatively, isogenic or xenogeneic muscle cells can be used, where those cells do not generate an inappropriate immune response in the subject.

[0170] On the other hand, rAAV genomes are provided herein. The rAAV genomes administered contain NT-3 polynucleotides under the control of transcription control sequences. The rAAV genome lacks AAVrep and capDNA. The AAVDNA in the rAAV genome can be from any AAV serotype from which recombinant viruses can be derived, including but not limited to AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, and AAVrh.74. The nucleotide sequences of the genomes of these AAV serotypes are known in the art, as described in the background technology section above.

[0171] In some embodiments, the transcriptional control sequences of the rAAV genome are muscle-specific control elements, including but not limited to those derived from the actin and myosin gene families (e.g., from the myoD gene family [see Weintraub et al., Science e [Science], 251:761-766 (1991)]), myocyte-specific enhancer binding factor MEF-2 [Cserjesi and Olson, Mol. Cell. Biol. [Molecular and Cellular Biology], 11:4854-4862 (1991)], control elements from the human skeletal actin gene [Muscat et al., Mol. Cell. Biol. [Molecular and Cellular Biology], 7:4089-4099 (1987)], cardiac actin gene, muscle creatine kinase (MCK) promoter [Johnson et al., Mol. Cell. Biol. [Molecular and Cellular Biology], 9:3393-3399 (1989)] and MCK enhancer, MHCK7 promoter (a modified form of the MCK promoter incorporating an enhancer from the myosin heavy chain (Salva et al., Mol. Ther. [Molecular Therapy], 15:320-329 (2007)), the desmin promoter, control elements derived from the skeletal fast-twitch troponin C gene, the slow-twitch cardiac troponin C gene, and the slow-twitch troponin I gene: hypoxia-inducible nuclear factor (Semenza et al., Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America], 88:5680-5684 (1991)), steroid-inducible elements and promoters (including glucocorticoid response elements (GRE) (see Mader and White, Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America], 90:5603-5607 (1993))), and other control elements. In some embodiments, the transcriptional control element includes the MCK promoter. In some embodiments, the transcriptional control element includes the MHCK7 promoter.

[0172] In some embodiments, the NT-3 polynucleotide in the rAAV genome is the NT-3 cDNA listed in SEQ ID NO: 1 (corresponding to nucleotides 1077-1850 of SEQ ID NO: 11). In some embodiments, the NT-3 polynucleotide in the rAAV genome is the NT-3 cDNA listed in Genbank Accession No. NM_001102654 or the NT-3 cDNA sequence listed in SEQ ID NO: 1, or is a variant polynucleotide having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the NT-3 cDNA. In some embodiments, the variant NT-3 polynucleotide encodes the same NT-3 polypeptide as the polypeptide encoded by the NT-3 cDNA of SEQ ID NO: 1. The amino acid sequence of the NT-3 polypeptide encoded by the NT-3 cDNA listed in SEQ ID NO: 1 or provided by Genbank Accession No. NM_001102654 is listed in SEQ ID NO: 2. In some embodiments, the variant NT-3 polynucleotide encodes a variant 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 listed in SEQ ID NO: 1 or provided by Genbank Accession No. NM_001102654 (SEQ ID NO: 2). The amino acid sequence change can be, for example, a substitution, deletion or insertion of one or more amino acids, preferably a conservative substitution. The variant NT-3 polypeptide can have any combination of amino acid substitutions, deletions or insertions that retain the activity of the polypeptide. In one aspect, the variant NT-3 polypeptide can have a number of amino acid changes such that its amino acid sequence is at least 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 99.5% identical to the amino acid sequence set forth in SEQ ID NO: 1 or encoded by the NT-3 cDNA provided by Genbank Accession No. NM_001102654 (SEQ ID NO: 2).

[0173] In some embodiments, the rAAV genome is an AAV.tMCK.NTF3 genome, the sequence of which is the NT-3 gene cassette set forth in SEQ ID NO: 11 and annotated in Table 4 (see Example 3).

[0174] In yet another aspect, an isolated nucleic acid is provided comprising the nucleotide sequence set forth in SEQ ID NO: 11. In some embodiments, the isolated nucleic acid consists of the nucleotide sequence set forth in SEQ ID NO:11.

[0175] Also provided is an isolated nucleic acid comprising, in order from 5' to 3': (i) a first AAV2 inverted terminal repeat (ITR) sequence (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 has an amino acid sequence that is at least 90% identical to SEQ ID NO:2, 100% identical to SEQ ID NO:2, or encoded by nucleotides 1077-1850 of SEQ ID NO:11.

[0176] Recombinant AAVs comprising the aforementioned nucleic acid as well as rAAVs comprising a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 1 are contemplated.

[0177] A DNA plasmid comprising the rAAV genome of the present disclosure is provided. The DNA plasmid comprises the rAAV genome envisioned herein. The DNA plasmid is transferred to cells that are allowed to be infected by the helper virus of AAV (e.g., adenovirus, adenovirus or herpes virus deleted by E1) to assemble the rAAV genome into infectious viral particles. The technology for producing rAAV particles (wherein the AAV genome to be packaged, rep and cap genes, and helper virus functions are provided to cells) is standard in the art. The production of rAAV requires the presence of the following components in a single cell (referred to herein as a packaging cell): the rAAV genome, the AAV rep and cap genes separated from the rAAV genome (i.e., not in the rAAV genome), and the helper virus functions. The AAV rep and cap genes can be from any AAV serotype from which recombinant viruses can be derived, and can be from different AAV serotypes other than the rAAV genome ITRs, including but not limited to AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, and AAVrh74. The generation of pseudotyped rAAVs is disclosed, for example, in WO 01 / 83692. Other types of rAAV variants, such as rAAVs with capsid mutations, are also contemplated. See, for example, Marsic et al., Molecular Therapy, 22(11): 1900-1909 (2014).

[0178] The method of generating packaging cells is to generate a cell line that stably expresses all the necessary components for AAV particle production. For example, a plasmid (or plasmids) comprising the following items is integrated into the genome of the cell: a rAAV genome lacking AAV rep and cap genes, an AAV rep and cap genes separated from the rAAV genome, and a selective marker (e.g., a neomycin resistance gene). The AAV genome has been introduced into bacterial plasmids by procedures such as GC tailing (Samulski et al., 1982, Proc. Natl. Acad. S6. USA [Proceedings of the National Academy of Sciences of the United States S6], 79: 2077-2081), adding synthetic linkers containing restriction endonuclease cleavage sites (Laughlin et al., 1983, Gene [Gene], 23: 65-73) or by direct blunt end ligation (Senapathy and Carter, 1984, J. Biol. Chem. [Journal of Biological Chemistry], 259: 4661-4666). The packaging cells are then infected with a helper virus (such as an adenovirus). The advantage of this method is that the cells are selectable and suitable for large-scale production of rAAV. Other examples of suitable methods use adenovirus or baculovirus rather than plasmids to introduce the rAAV genome and / or rep and cap genes into packaging cells. Methods for producing rAAV with a self-complementary genome are also known in the art.

[0179] The general principles of rAAV generation are reviewed in, for example, Carter, 1992, Current Opinions in Biotechnology, 1533-539; and Muzyczka, 1992, Curr. Topics in Microbial. and Immunol., 158:97-129). Various methods are described in the following literature: Ratschin et al., Mol. Cell. Biol. [Molecular and Cellular Biology] 4:2072 (1984); Hermonat et al., Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America], 81:6466 (1984); Tratschin et al., Mol. Cell. Biol. [Molecular and Cellular Biology] 5:3251 (1985); McLaughlin et al., J. Virol., [Journal of Virology] 62:1963 (1988); and Lebkowski et al., 1988 Mol. Cell. Biol. [Molecular and Cellular Biology], 7:349 (1988). Samulski et al. (1989, J. Virol., 63:3822-3828); U.S. Pat. No. 5,173,414; WO 95 / 13365 and corresponding U.S. Pat. No. 5,658,776; WO 95 / 13392; WO 96 / 17947; PCT / US98 / 18600; WO 97 / 09441 (PCT / US96 / 14423); WO 97 / 08298 (PCT / US96 / 13872); WO 97 / 21825 (PCT / US96 / 20777); WO 97 / 06243 (PCT / FR96 / 01064); WO 99 / 11764; Perrin et al. (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. Pat. No. 5,786,211; U.S. Pat. No. 5,871,982; and U.S. Pat. No. 6,258,595. The foregoing documents are hereby incorporated by reference in their entirety, with particular emphasis on those portions of the documents relating to rAAV production.

[0180] In another aspect, the present disclosure therefore provides packaging cells for producing infectious rAAV. In one embodiment, the packaging cells can be stably transformed cancer cells, such as HeLa cells, 293 cells, and PerC.6 cells (homologous 293 cell lines). In another embodiment, the packaging cells are cells that are untransformed cancer cells, such as low-passage 293 cells (human fetal kidney cells transformed with E1 of adenovirus), MRC-5 cells (human fetal fibroblasts), WI-38 cells (human fetal fibroblasts), Vero cells (monkey kidney cells), and FRhL-2 cells (rhesus fetal lung cells).

[0181] rAAV can be purified by methods standard in the art, such as by column chromatography or cesium chloride gradients. Methods for purifying rAAV vectors from helper viruses are known in the art and include those disclosed, for example, in Clark et al., Hum. Gene Ther. [Human Gene Ther.], 10(6): 1031-1039 (1999); Schenpp and Clark, Methods Mol. Med. [Molecular Medicine Methods], 69427-443 (2002); U.S. Pat. No. 6,566,118 and WO 98 / 09657.

[0182] Thus, in another aspect, the disclosure contemplates rAAV comprising NT-3 polynucleotides. In some embodiments, the rAAV comprises an AAV rh74 capsid and NT-3 polynucleotides. In some embodiments, the genome of the rAAV lacks AAV rep and capDNA. In some embodiments of these methods, the rAAV is rAAVrh7.4.tMCK.NTF3. In some embodiments, the rAAV is a self-complementary genome.

[0183] On the other hand, the present disclosure contemplates compositions comprising rAAV as described herein. The compositions of the present disclosure comprise rAAV in a pharmaceutically acceptable carrier. These compositions may also comprise other ingredients, such as diluents. Acceptable carriers and diluents are nontoxic to the recipient at the doses and concentrations employed and are preferably inert, 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 dextrins; 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, Pluronic, or polyethylene glycol (PEG). In some embodiments, rAAV is formulated in Tris, MgCl2, NaCl, and Pluronic F68. In some embodiments, rAAV is formulated in 20 mM Tris (pH 8.0), 1 mM MgCl2, and 200 mM NaCl containing 0.001% Pluronic F68.

[0184] Combination therapy is also contemplated herein. Combinations used herein include simultaneous therapy or sequential therapy. Combinations of the methods of the present disclosure with standard drug therapy (e.g., corticosteroids and / or immunosuppressive drugs), as well as combinations with novel treatments are particularly contemplated. In various embodiments, the subject is treated with a corticosteroid before, during, or after (or in any arrangement of a combination of two or more of the three possibilities) treatment of the subject according to the methods contemplated herein. For example, these combinations include administering a corticosteroid, such as prednisolone, before, during, and / or after administration of the rAAV vector.

[0185] Sterile injectable solutions are prepared by incorporating rAAV in the desired amount in an appropriate solvent with the various other ingredients enumerated above as needed, followed by filtered sterilization. In general, dispersions are prepared by incorporating the sterilized active ingredient into a sterile vehicle containing a basic dispersion medium and the required other ingredients from those enumerated above. In the case of 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 ingredients from a previously sterile filtered solution thereof.

[0186] Stimulates muscle growth

[0187] One aspect of the present invention provides a method for stimulating muscle growth in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of neurotrophin-3 (NT-3), pre-NT-3 or an effective fragment thereof, or a nucleic acid encoding NT-3, pre-NT-3 or an effective fragment thereof

[0188] In some embodiments, the methods of the invention can be used to increase muscle strength, muscle mass, or muscle endurance and reduce muscle fatigue in a subject.

[0189] Muscles can be divided into three types: skeletal muscle, cardiac muscle and smooth muscle. Skeletal muscle is the muscle tissue that is able to generate force and transmit that force to the skeleton, enabling breathing, movement and posture maintenance. Cardiac muscle is the muscle of the heart. Smooth muscle is the muscle tissue of the arteries and intestinal walls. The methods and compositions of the present invention are primarily applicable to skeletal muscle, but may additionally have a positive effect on smooth muscle. "A skeletal muscle" and "skeletal muscles" are defined as muscles that interact with bones, tendons and joints.

[0190] In some embodiments, the present invention provides methods of treating conditions, diseases, disorders and conditions that cause decreased muscle strength (also referred to herein as musculoskeletal diseases, as well as muscle dysfunction and muscle wasting diseases). The major categories of musculoskeletal diseases are muscular dystrophy and muscle atrophy.

[0191] In some embodiments, the present invention provides methods for treating musculoskeletal diseases, including muscle dysfunction and muscle wasting diseases or disorders (including hereditary myopathy, neuromuscular disease, muscle atrophy, drug-induced myopathy, or illness, disease, disorder or condition that causes reduced muscle strength). The present invention also provides methods for treating neuropathy (such as CMT-hereditary and CMT1A) and axonal and demyelinating polyneuropathy (such as chronic inflammatory demyelinating polyneuropathy). The treatment method includes administering a therapeutically effective amount of neurotrophin-3 (NT-3), pre-NT-3 or an effective fragment thereof, or a nucleic acid encoding NT-3, pre-NT-3 or an effective fragment thereof to a patient in need. In some embodiments, 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 disorders associated with impaired regenerative radial growth phases.

[0192] In some embodiments, NT-3 can be used to treat muscle atrophy. Muscle atrophy is a general term used to describe a condition characterized by the consumption or loss of muscle tissue caused by a variety of diseases, disorders, other conditions or events. Muscle atrophy may be the result of, but is not limited to, long-term immobilization caused by recovery from severe burns, major joint replacement surgery, neuropathic pain, peripheral neuropathy, necrotizing vasculitis, zero gravity environments (e.g., astronauts and spacemen), long-term hospitalizations, degenerative diseases (e.g., amyotrophic lateral sclerosis) and organ transplants as well as spinal cord injuries, chronic hemodialysis and stroke.

[0193] In some embodiments, NT-3 can be used to treat disuse muscle atrophy. Disuse muscle atrophy is a condition characterized by wasting or loss of muscle tissue due to long-term inactivity. Disuse muscle atrophy may be the result of, but is not limited to, long-term immobilization due to recovery from severe burns, major joint replacement surgery, neuropathic pain, zero gravity environments (e.g., astronauts and cosmonauts), long-term hospitalization, anorexia and organ transplantation as well as spinal cord injury, chronic hemodialysis and stroke.

[0194] In some embodiments, NT-3 can be used to treat age-related muscle atrophy. Age-related muscle atrophy is a condition characterized by wasting or loss of muscle tissue and replacement of muscle tissue by fibrotic tissue as a subject ages.

[0195] In some embodiments, NT-3 can be used to treat sarcopenia. Sarcopenia is a condition characterized by wasting or loss of muscle tissue and replacement of muscle tissue with fibrotic tissue as a subject ages.

[0196] In some embodiments, NT-3 can be used to treat muscle wasting in cachexia. Cachexia is weight loss, muscle atrophy, fatigue, weakness, and significant loss of appetite in a person (not an active attempt to lose weight but a result of chronic disease). The muscle wasting component of cachexia may be the result of, but is not limited to, cancer, multiple sclerosis, tuberculosis, acquired immunodeficiency syndrome, human immunodeficiency virus, malnutrition, Parkinson's disease, emphysema, heart failure, motor neuron disease, cystic fibrosis, dementia, sarcopenia, chronic obstructive pulmonary disease, kidney disease, and renal failure.

[0197] In some embodiments, NT-3 can be used to treat muscle wasting caused by viral infections (e.g., HIV, Epstein-Barr virus), bacterial infections (e.g., mycobacteria and rickettsia), post-polio syndrome, and parasitic infections (e.g., trypanosoma and schistosomes) in subjects at risk for developing muscle wasting.

[0198] Neurotrophin-3

[0199] In some embodiments, a therapeutically effective amount of NT-3, pre-NT-3, or a NT-3 analog thereof is administered to a subject to stimulate muscle growth. Neurotrophin 3 (NT-3) is a neurotrophic factor in the NGF (nerve growth factor) family of neurotrophic factors. NT-3 is a protein growth factor that is active on certain neurons of the peripheral and central nervous systems; it is known to help support the survival and differentiation of existing neurons, and encourages the growth and differentiation of new neurons and synapses.

[0200] The disclosure includes blocking peptides that are substantially similar to at least a portion of the amino acid sequence of the extracellular domain of Cx26. As used herein, the term "portion" refers to an amino acid sequence in the extracellular domain of Cx26 that includes at least 4 amino acids. In other embodiments, a portion refers to an amino acid sequence of at least 6 amino acids in length, an amino acid sequence of at least 8 amino acids in length, or an amino acid sequence of at least 10 amino acids in length. Therefore, the blocking peptide is composed of at least 4, 6, 8, or 10 amino acids. Similarly, the blocking peptides described herein may have a maximum size. The maximum size of the blocking peptide is related to the overall size of the peptide and includes any additional sequence connected to the peptide, such as a protein transduction domain. In some embodiments, the blocking peptide has a maximum size of less than about 200 amino acids, while in other embodiments, the blocking peptide has a maximum size of less than about 100 amino acids. In other embodiments, the 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.

[0201] As used herein, the term "polypeptide" refers to an oligopeptide, a peptide or a protein sequence, or to a fragment, part or subunit of any of these, as well as to a naturally occurring or synthetic molecule. 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 and analogs, glycosylated polypeptides, all "simulated" and "peptoid" polypeptide forms, and retro-inversion peptides (also referred to as all-D-retro or mtro-enantiomer peptides).

[0202] "Substantially similar" means that a given amino acid (or nucleic acid) sequence has at least 85%, more preferably at least 90%, and even more preferably at least 95% identity to a reference sequence. Identity or homology with respect to such sequences is defined herein as the percentage of amino acid residues in the candidate sequence that are identical to the known peptide, after aligning the sequences and introducing gaps (if necessary) to achieve the maximum homology percentage and not considering any conservative substitutions as part of the sequence identity. N-terminal, C-terminal or internal extensions, deletions or insertions into the peptide sequence should not be construed as affecting homology.

[0203] Substantially similar peptides include those that differ by one or more amino acid changes, where these changes, such as substitutions, additions or deletions of amino acid residues, do not eliminate the properties of the related peptides, such as their ability to associate with FAK or NANOG. In addition, sequences that only describe or encode proteins in which conservative substitutions are made only in conserved regions are generally substantially similar. Preferably, substantially similar sequences also retain the unique activity of the polypeptide.

[0204] Examples of conservative substitutions include: substitutions between non-polar (hydrophobic) residues such as isoleucine, leucine or methionine. Similarly, the present invention contemplates substitutions of a polar (hydrophilic) residue, such as substitutions between arginine and lysine, between glutamine and asparagine, and between glycine and serine. In addition, substitutions of a basic residue (such as lysine, arginine or histidine) for another, or substitutions of an acidic residue (such as aspartic acid or glutamic acid) for another are also contemplated. Examples of non-conservative substitutions include substitutions of a non-polar (hydrophobic) residue (such as isoleucine, valine, leucine, alanine, methionine) for a polar (hydrophilic) residue (such as cysteine, glutamine, glutamic acid, lysine), and / or substitutions of a polar residue for a non-polar residue.

[0205] The phrase "conservative substitution" also includes the use of chemically derived residues to replace non-derivatized residues, as long as the peptide retains the necessary ability to associate with NT-3. Substantially similar peptides also include the presence of additional amino acids or the absence of one or more amino acids, which do not affect the necessary ability to associate with NT-3. For example, substantially similar peptides may contain N or C terminal cysteine, through which the peptide can be covalently attached to a carrier protein (e.g., albumin) if desired. This attachment can reduce the clearance of the peptide from the blood, and can also reduce the proteolysis rate of the peptide. In addition, for the purposes 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 clearance of the peptide.

[0206] In some embodiments, a pro-neurotrophin-3 protein (pro-NT-3) is administered to a subject. The pro-neurotrophin-3 is an approximately 30 kDa NT-3 pro-form that can be 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 [Journal of Neuroscience], 33 (4), 622-631 (2011).

[0207] Treatment of muscular dystrophy

[0208] The present invention provides a method for treating a subject suffering from peripheral muscle atrophy. Pyruvic acid compounds can be used to provide preventive and / or therapeutic treatment. Pyruvic acid compounds can be administered to a subject prophylactically, for example, before peripheral neuropathy occurs. Prophylactic (i.e., preventive) administration effectively reduces the likelihood of subsequent peripheral neuropathy in a subject, or reduces the severity of subsequent peripheral neuropathy. Preventive treatment can be provided to a subject with an elevated risk of developing peripheral neuropathy (such as a subject with a family history of peripheral neuropathy). The expression of myelin protein 22 (PMP22) mutations represents 70%-80% of all Charcot-Marie-Tooth neuropathy that occurs, and therefore, their presence can be used as a standard for selecting patients receiving treatment using pyruvic acid compounds described herein.

[0209] Alternatively, the compounds of the invention may be administered therapeutically to a subject already afflicted with peripheral neuropathy. In one embodiment of therapeutic administration, administration of these compounds is effective to eliminate peripheral neuropathy; in another embodiment, administration of the pyruvate compound is effective to reduce the severity of peripheral neuropathy or prolong the life of a subject afflicted with the neuropathy. In some embodiments, the method of treatment consists of administering to a subject a therapeutically effective amount of a pyruvate compound in a pharmaceutically acceptable formulation over a substantial period of time.

[0210] CMT variants

[0211] Charcot-Marie-Tooth (CMT) hereditary neuropathies refer to a group of disorders characterized by chronic motor and sensory polyneuropathy, also known as hereditary motor and sensory neuropathies. The autosomal dominant CMT neuropathies are: demyelinating (also known as CMT1), axonal nondemyelinating (also known as CMT2), and dominant intermediate CMT (DI-CMT). Other neuropathies that are also equivalent to CMT are distal hereditary motor neuropathy (9dHMN) and distal spinal muscular atrophy (DSMA) and Dejerine-Sottas syndrome (DSS). The description and classification of CMT neuropathies are described in Bird, Gene Reviews, Seattle, WA: University of Washington, Seattle, PIM20301532, updated on June 28, 2018.

[0212] Currently, there are more than 70 known gene variants known for genes associated with CMT. These gene variants are provided in Table 1 below using the classification system of Magy et al. (Neurology 90: e870-6, 2018). The inherent pattern of each gene variant associated with CMT is autosomal dominant (AD), autosomal recessive (AR) or X-linked (XL). The neuropathy for each gene variant associated with CMT is axonal (Ax), demyelinating (De) and intermediate (In). The "other names" provided in Table 1 are names used in other classification systems, including dominant intermediate CMT (DI-CMT), distal spinal muscular dystrophy (DSMA), hereditary sensory and autonomic neuropathy (HSAN) and distal hereditary motor neuropathy (dHMN).

[0213] Table 1

[0214]

[0215]

[0216]

[0217]

[0218]

[0219] MOI = Mode of Inheritance AD ​​= Autosomal Dominant AR = Autosomal Recessive XL = X-Linked Ax = Axonal De = Demyelination In = Intermediate UMN = Upper Motor Neuron DI-CMT = Dominant Intermediate CMT DSMA = Distal Spinal Muscular Atrophy ALS = Amyotrophic Lateral Sclerosis

[0220] HSAN = hereditary sensory and autonomic neuropathy

[0221] dHMN = distal hereditary motor neuropathy

[0222] Administration and preparations

[0223] The carrier or peptide used together with some embodiments of the present invention can be incorporated into a pharmaceutical composition suitable for administration to a subject. In some specific embodiments, the pharmaceutical composition comprises a carrier of the present invention and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delay agents that are physiologically compatible. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, etc., and combinations thereof. In many cases, isotonic agents, such as sugar, polyols (such as mannitol, sorbitol), or sodium chloride may be preferably included in the composition. Pharmaceutically acceptable carriers may further include a small amount of auxiliary substances (such as wetting agents or emulsifiers, preservatives or buffers), which extend the shelf life of the carrier or pharmaceutical composition or enhance its effectiveness.

[0224] These The vector or peptide can be administered acutely (i.e., during the course of disease or shortly after the event leading to muscle atrophy), or can be administered prophylactically (e.g., before a planned 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 time and interval of administration varies according to the subject's symptoms, and can be administered at intervals of several hours to several days, over the course of several hours, days, weeks, or longer, as will be determined by those skilled in the art.

[0225] Compositions containing carriers or peptides are usually administered intravenously. When administered intravenously, these compositions may be combined with other ingredients (such as carriers and / or adjuvants). The peptides may also be covalently attached to protein carriers (such as albumin) to minimize the clearance of the peptides. There is no restriction on the nature of the other ingredients, but such ingredients must be pharmaceutically acceptable, effective for their intended administration and not reduce the activity of the active ingredients of the composition.

[0226] The pharmaceutical form suitable for injection includes sterile aqueous solution or dispersion and sterile powder for the temporary preparation of sterile injectable solution or dispersion. In all cases, the final solution form must be sterile and liquid. Typical carriers include solvents or dispersion media, which include, for example, aqueous buffer solutions (i.e., biocompatible buffers), ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, suitable mixtures thereof), surfactants or vegetable oils. Sterilization can be completed by any art-recognized technology, which includes but is not limited to filtering or adding antibacterial or antifungal agents, such as parabens, chlorobutanol (chlorobutano), phenol, sorbic acid or thimerosal. In addition, isotonic agents such as sugar or sodium chloride can be incorporated into the subject composition.

[0227] The production of sterile injectable solutions containing the subject peptides is by mixing these compounds in the required amount in an appropriate solvent with the above-mentioned various ingredients, and then sterilizing as required, preferably filtration sterilization. In order to obtain sterile powders, the above solutions are vacuum dried or freeze-dried as required.

[0228] When the peptide of the present invention is administered orally, its pharmaceutical composition containing an effective dose of the peptide may also contain an inert diluent as an assimilable edible carrier, etc., in a hard or soft shell gelatin capsule, compressed into tablets, or may be in an elixir, suspension, syrup, etc. Therefore, the peptide of the present invention is mixed in a pharmaceutically effective amount with a therapeutically effective amount of a suitable pharmaceutically acceptable carrier for convenient and effective administration.

[0229] As used herein, the expression "effective amount" or "therapeutically effective amount" refers to an amount of a pharmaceutical agent sufficient to stimulate muscle growth or reduce or prevent muscle atrophy. The exact amount required will vary from subject to subject, depending on the species, age and general condition of the subject, the specific therapeutic agent, its mode and / or route of administration, etc. However, it should be understood that the total daily dosage of the compounds and compositions of the present invention can be determined by the attending physician within the scope of reasonable medical judgment. The specific therapeutically effective dosage level for any particular patient or organism will depend on a variety of factors, including the disorder being treated and the severity of the disorder; the activity of the specific compound used; the specific composition used; the age, weight, general health, sex and diet of the subject; the time of administration, route of administration and excretion rate of the specific composition used; the duration of treatment; drugs used in combination or coincidentally with the specific composition used; and factors well known in the medical field.

[0230] The carrier or peptide can be administered in a manner compatible with the dosage formulation and in such an amount that is also therapeutically effective. The systemic dose depends on the patient's age, weight and condition and the route of administration. For example, a suitable dose of the peptide administered 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, more preferably at most about 500 mg to about 10 g per dose. Since the peptide compositions of the present invention are eventually cleared from the bloodstream, it is indicated and preferably these compositions are re-administered.

[0231] Examples

[0232] Therefore, aspects and embodiments of the present invention are illustrated by the following examples. However, there are various other embodiments within the scope of the present invention and it should not be limited to the specific examples provided herein.

[0233] Example 1

[0234] AAV1.NT-3 gene therapy increases muscle fiber diameter via activation of the mTOR pathway and metabolic remodeling in a CMT mouse model

[0235] NT-3 has well-established effects on peripheral nerves and Schwann cells, namely promoting axonal regeneration and associated myelination. The effects of AAV.NT-3 gene therapy on the oxidative state of neurogenic muscles in trembling (TrJ) mice at 16 weeks after gene injection were evaluated, and it was found that the increase in muscle fiber size was associated with a change in the oxidative state of muscle fibers toward the normalization of the fiber type ratio in the wild type. The NT-3-induced increase in fiber size was most prominent for the fast-glycolytic fiber population. These changes in TrJ muscles were accompanied by increased levels of 4E-BP1 and S6 protein phosphorylation, which served as evidence of mTORC1 activation. At the same time, the expression levels of the mitochondrial biogenesis regulator PGC 1α and glycolytic markers (HK1 and PK1) were increased in TrJ muscles. In vitro studies showed that recombinant NT-3 can induce Akt / mTOR pathway activation directly in TrkC-expressing myotubes but not in myoblasts. In addition, myogenin expression levels were significantly higher and p75NTR expression was downregulated in myotubes compared with myoblasts, indicating that NT-3-induced myoblast differentiation was associated with mTORC1 activation. These studies show for the first time that NT-3 increases muscle fiber diameter in neurogenic muscle by directly activating the mTOR pathway, and the increase in fiber size is more prominent for fast-twitch fibers.

[0236] method

[0237] Animals, treatment regimen, and histopathology: TrJ mice (B6.D2-Pmp22Tr-J / J) and C57BL / 6 wild-type mice were obtained from Jackson Laboratory (Bar Harbor, ME). TrJ mice aged 9 to 12 weeks were injected in the left gastrocnemius muscle with PBS (n=6) or 3×10 10 vg self-complementary (sc) AAV1.tMCK.NT-3 vector (n=6). Another group of TrJ mice was injected with 1x10 11 vg of single-stranded AAV1.CMV.NT-3 to induce high NT-3 expression levels for comparison (n=6). WT mice received PBS (n=6) or 3x 10 10 vg of scAAV1.tMCK.NT-3 vector (n=4). Each group of mice was euthanized, and their muscles were harvested at 16 weeks after gene injection and processed for cryostat sections. Succinate dehydrogenase (SDH) enzyme histochemistry was used to evaluate metabolic fiber type differentiation using a standard protocol established in-house. Muscle fiber type specific diameter measurements were obtained from 12[tin thick-SDH stained sections. The three images represent three different areas of the gastrocnemius muscle along the midline axis (each section of each area of ​​each animal) (a deep area mainly composed of STO, an intermediate area showing a checkerboard appearance of STO and FTO or FTG, and a superficial area mainly composed of FTG fibers), which were taken using an Olympus BX41 microscope and a SPOT camera at X20 magnification. This method was selected to capture changes in fiber oxidation status in each area in response to metabolic changes caused by treatment. The diameters of dark (STO), intermediate (FTO) and light FTG fibers were determined by measuring the shortest distance across muscle fibers using Zeiss Axiovision LE4 software and expressed as a percentage of total fibers. The average fiber diameter (mean ± SEM) was derived from the combination of all 3 fiber types in each group. An average of 1250 fibers (between 960 and 1486) were measured for each group.

[0238] Production and efficacy of AAV.NT-3 vectors: The design of self-complementary AAV viral vectors with serotype 1 containing NT-3 under the tMCK or CMV promoter was previously described and generated at the Viral Vector Core at Nationwide Children's Hospital, Columbus. Sahenk et al., Mol Ther [Molecular Therapy], 22(3):511-521 (2014). Aliquots of virus were kept at -80°C until use. Blood samples were collected from treated and untreated mice by eye bleeding under anesthesia at 6 and 16 weeks after injection, and serum NT-3 levels were determined using capture ELISA.

[0239] C2C12 myoblast culture and myotube formation: C2C12 myoblasts were cultured in growth medium (GM) consisting of DMEM medium (Gibco-Invitrogen, #10569010, Carlsbad, CA) supplemented with 10% FBS (Fisher Scientific, #26-140-079, Carlsbad, CA) and 1% penicillin / streptomycin solution (Gibco-Invitrogen, #15640055, Carlsbad, CA) in a humidified chamber at 37°C and 5% CO2. Myotube formation was induced on confluent cultured cells by switching GM to differentiation medium [DMEM medium (Gibco-Invitrogen, #11965092, Carlsbad, CA) supplemented with 5% horse serum (Gibco-Invitrogen, #26050088, Carlsbad, CA) and 1% penicillin / streptomycin solution (Gibco-Invitrogen, #15640055, Carlsbad, CA). All subsequent assays, including QPCR, Western blot, and ELISA, were performed with myotubes starting 3 days after induction of myotube formation. Both myoblasts and myotubes were exposed to recombinant human NT-3 (Pepprotech, Rocky Hill, NJ) at a concentration of 100 ng / ml in 6-well plates. The culture medium was collected to detect glucose consumption and lactate formation by using a glucose and lactate assay kit (Eton Bioscience, San Diego, CA) according to the manufacturer's instructions.

[0240] QPCR experiments: Total RNA was isolated from gastrocnemius muscle of NT-3 treated and untreated control mice at the end point. 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, Texas, USA) followed by the Trascriptor First Strand cDNA synthesis kit (Roche, #04379012001, Roche, Switzerland, USA) according to the manufacturer's instructions. Other qPCR experiments were performed by using the iTaqTm Universal Greensupermix (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) are found in the literature. Other primer sequences are found in primer bands. Wang et 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-assisted software (ABI).

[0241] Protein extraction and Western blotting experiments: Frozen gastrocnemius muscle blocks were cut into 20 μin thickness, placed in 2 ml small plastic tubes (15-20 slices per block), and homogenized in lysis buffer [RIPA lysis buffer (ThermoFisher, #89900, USA) with 1x Halt protease inhibitor (ThermoFisher, #78429, USA) and 1x phosphatase inhibitor (Sigma, #P0044, USA)] using an automatic particle mixer and a disposable pestle for 20 seconds each time, three times. For in vitro signal transduction assays, myoblasts and myotubes were collected in 2 ml small test tubes after incubation with NT-3 (100 ng / ml) for 30 minutes and lysed in the same manner as described above. The lysate was centrifuged at 13,000 rpm for 10 minutes at 4°C, and the supernatant was carefully collected. Protein concentration was measured by using a BCA protein assay kit (Thermo Fisher Scientific, #23252, Waltham, MA, USA). Protein samples (10-40 tg) were concentrated to 4%-12% Bis-Tris Plus precast 10 or 15 well polyacrylamide gel (Thermo Fisher Scientific, #NW04120BOX) was run and transferred to PDVF membrane (GE Healthcare, #10600021, Pittsburgh, USA). The membrane was blocked with 5% bovine serum albumin (BSA, Bedford, Massachusetts, USA) in TBS buffer with 0.05% Tween 20 (TBS-T, Amresco, Ohio, USA) for 2 hours at room temperature and incubated with appropriate primary antibodies in TBS-T buffer with 5% BSA at 4°C in a cold room overnight. The primary antibodies used in this study were as follows: anti-phospho S6 Ser235 / 236 (#4858), anti-S6 (#2217), anti-phospho Akt Ser473 (#4060), anti-Akt (#9272), anti-phospho 4E-BP1 thr37 / 46 (#2855), anti-4E-BP1 (#9644), anti-GAPDH (Santa Cruz, #sc365062). After washing five times with TBS-T for 5 min on an orbital shaker, the membranes were incubated with secondary antibodies from R&D Systems, Minneapolis, MN, USA [HRP-conjugated anti-rabbit (#HAF008), HRP-conjugated anti-mouse (HAF007)] in 5% dry milk in TBS-T buffer for 1 h. The membrane was washed again with TBS-T using the same method as described above, then incubated with ECL Prime western detection reagent (Amersham, #RPN2232, New Jersey, USA) for 1-3 minutes, and then exposed to X-ray film (Denville, #E3018, Massachusetts, USA) using multiple exposure times. The protein bands on the film were photographed using a camera (Sony A600, Japan), and the band intensity was quantified using (Quantity-One software, Bio-Rad, v.4.6.9). The relative content of the protein analyzed in each sample was determined by normalizing the band intensity to the content of GAPDH in the same sample. The membrane was stained with 0.1% Coomassie Brilliant Blue R stain (Thermo Fisher Scientific, USA), rinsed and photographed to confirm that the protein loading in each lane was equal.

[0242] Statistics: For comparison of muscle fiber size between treated and untreated groups, statistical analysis was performed using one-way analysis of variance (Anova) in GraphpadPrism6 software. If applicable to other statistical analyses, Student's t-test or one-way analysis of variance was performed. The significance level was set at P < 0.05. Results are given as mean ± SEM in all experiments.

[0243] result

[0244] AAV1.NT-3-induced remodeling of TrJ muscle fiber types

[0245] Previously in the TrJ muscle, a shift from fast to slow fibers was observed as part of the neuropathic phenotype. Nicks et al., J Neuropathol Exp Neurol, 72(10):942-954 (2013). In the present study, SDH staining was used to assess metabolic fiber type differentiation in the gastrocnemius muscle of the TrJ and age-matched WT by sampling from deep, intermediate, and superficial regions of the muscle. At 16 weeks after gene injection, STO fibers were significantly reduced compared to the untreated (PBS) group showing neurogenic changes, small angle fibers, and type groupings, and in particular, increased fiber size of the FTO and FTG types ( Figure 1 A and Figure 1 B). Quantitative studies showed that both the number and percentage of STO fibers per unit area were significantly higher in TrJ-PBS muscles than in WT muscles, which is consistent with previous studies. Figure 4 ) of the two promoters 1x 10 11 The vg dose of AAV1.NT-3 showed a shift in fiber type from STO to FTO / FTG fibers. The mean density or percentage of STO in both treatment groups (derived from n=3-5 mice in each group) was not significantly different from WT muscle, indicating a shift toward normalization of fiber type distribution in the case of NT-3 ( Figure 1 C) Furthermore, AAV1.NT-3 treated WT muscles showed no significant changes in the fiber type distribution profile.

[0246] NT-3 treatment in TrJ mice showed different effects on the increase in muscle fiber size (Table 2). When NT-3 was expressed under the control of the tMCK promoter, significant diameter increases were observed only in FTG fibers. In the second treatment group, in which high NT-3 expression was obtained using the CMV promoter, the diameters of all fiber types increased significantly. Interestingly, this dose-dependent NT-3 effect was only visible in neurogenic TrJ muscles; at the same time point 16 weeks after gene injection, no significant diameter changes were seen in any fiber type in WT muscles treated with NT-3 gene therapy.

[0247] Table 2 shows that for fast-twitch fibers, the increase in fiber size in neurogenic TrJ muscles after NT-3 gene therapy was more pronounced.

[0248] Average fiber diameter

[0249]

[0250] *, p<0.001

[0251] AAV-NT-3 improves mTOR signaling and metabolic markers in TrJ muscles

[0252] The histological findings of TrJ muscles in response to AAV1.NT-3 treatment shown above prompted the investigation of whether mTORC1 activation plays a role in NT-3-induced radial growth of muscle fibers. The activity of mTORC1 was assessed by the phosphorylation levels of its downstream substrates 4EBP-1 and ribosomal S6P in muscle samples from each group. In AAV1.NT-3-treated TrJ muscles, the levels of phosphorylated 4EBP-1 and S6P were significantly increased compared to the untreated counterparts obtained from the TrJ-PBS control ( Figure 2 In contrast, NT-3 treatment was found to have no significant effect on the phosphorylation levels of 4EBP-1 and S6P in WT muscle ( Figure 2 B).

[0253] mTORC1 regulates the synthesis of nuclear-encoded mitochondrial proteins via 4E-BP, controlling mitochondrial activity and biogenesis, thereby coordinating energy consumption and production

[17] . Thus, in NT-3-treated TrJ muscles, PGC 1α, a master regulator of mitochondrial biogenesis, was upregulated, indicating that NT-3 is able to reverse the defective expression levels of PGC 1α seen in neurogenic muscles ( Figure 2 C). Consistent with the lack of mTORC1 activation, no changes in PGC 1α expression levels were observed in WT muscles treated with NT-3 ( Figure 2 C). The increase in fiber size in TrJ muscles treated with NT-3 gene therapy occurred mainly in FTO and FTG fibers, which have higher glycolytic activity than slow fibers

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

[0254] NT-3 activates the Akt / mTORC1 pathway via the TrkC receptor in C2C12 myotubes

[0255] The in vivo studies described here show that the increase in fiber size and fiber type remodeling induced by AAV1.NT-3 treatment in TrJ muscles is associated with mTORC1 activation. However, the question needs to be answered whether this change is solely a result of reinnervation or whether NT-3 can directly alter muscle protein synthesis and cellular metabolism independently of nerve regeneration. Next, the direct effect of NT-3 on the mTOR pathway in an in vitro system without the influence of nerves was investigated by exposing C2C12 myoblasts and myotubes to recombinant NT-3. The results indicate that NT-3 can induce activation of the Akt / mTOR pathway in myotubes but not in myoblasts ( Figure 3 A). Treatment of myotubes with 100 ng of recombinant NT-3 for 30 min resulted in significantly higher phosphorylation of Akt, 4EBP1, and S6P compared to the control group ( Figure 3 A). In another set of experiments, it was found that NT-3 significantly enhanced the expression of mitochondrial biogenesis marker PGC 1α and glycolysis marker PK1 in myotubes after 48 hours of incubation ( Figure 3 B). Thus, analysis of supernatants at this time point revealed increased glucose consumption and increased lactate production in NT-3 treated myotubes compared to controls ( Figure 3 C) Under the conditions in which these experiments were performed, no effect of NT-3 on HK1 expression levels was observed.

[0256] The expression of p75NTR and TrkC receptors and myogenin (which is a marker in the differentiation pathway of myoblasts into myoblasts and myotube cultures) was analyzed. NT-3 exerts its biological effects by binding to its preferred receptor TrkC or to the low affinity neurotrophic factor receptor p75NTR. Schecterson LC, Bothwell M, Neuron, 9(3): 449-463 (1992). p75NTR is expressed in C2C12 myoblasts and is downregulated during myogenic differentiation. Seidl et al., Journal of cellularphysiology, 176(1): 10-21 (1998). It has been shown that the neurotrophic factor NGF affects myogenic differentiation and cell growth via p75NTR, and the downregulation of p75NTR is considered to be essential for myogenic differentiation. Similar to the effects of NGF, NT-3 was found to also enhance myogenin expression in myotubes, and as expected, this was associated with significantly higher expression of p75NTR in myoblasts than in myotubes, whereas TrkC expression levels did not differ between the two groups ( Figure 3 D) There was no difference in the effect of NT-3 on the expression levels of these receptors in myoblasts or myotubes.

[0257] discuss

[0258] The evidence provided herein is that NT-3 can directly affect neurogenic muscle metabolism, thereby causing fiber size increase and fiber type remodeling towards normalization via mTORC1 activation. The increase in fiber size is most obvious for type II fibers, especially for FTO subtypes. In addition, it has been shown that NT-3, as an important contributor to its in vivo effect in neurogenic muscles, can directly induce Akt / mTOR pathway activation in myotubes rather than in myoblasts. Interestingly, NT-3 gene therapy at the same dose in WT muscle did not affect these characteristics, but the effect of NT-3 on denervated WT muscle when using the gene therapy paradigm was not studied. It has been previously shown that with or without local delivery of NT-3 to the nerve crush site, NT-3 has a differential effect on type II muscle fiber subtypes in rat gastrocnemius 8 months after nerve repair, and it is found that the proportion and size of type IIb fibers return to normal. Sterne et al., J Cell Biol [Journal of Cell Biology], 139 (3): 709-715 (1997). However, this effect is interpreted as enhanced axonal regeneration by NT-3 with beneficial consequences for motor target organs, and the possibility that NT-3 may specifically affect a subgroup of motor neurons that may determine the type IIb muscle fiber phenotype. It can be argued that the findings of our in vivo studies may represent a combined action of NT-3 on nerves and muscles. However, our in vitro data underscore the evidence that NT-3 has a direct effect on muscle metabolism via activation of Akt / mTORC1, and that this direct effect may be important in neurogenic muscles, leading to a preferential increase in the size of FTG, i.e., Erb-type fibers.

[0259] In conditional transgenic mice, expression of a constitutively active form of Akt leads to muscle hypertrophy due to the growth of type III muscle fibers. Izumiya et al., Cell metabolism, 7(2):159-172 (2008). This is associated with upregulation of transcripts involved in glycolysis, increased glucose consumption and lactate production, which are associated with lower insulin levels, increased glucagon levels in the blood, and resistance to obesity induced by a high-fat diet. In contrast, mTOR inactivation is associated with a reduction in the glycolytic enzymes PK1 and HK1. Risson et al., J Cell Biol, 187(6):859-874 (2009). In our study, AAV.NT-3 treatment in TrJ muscle increased the size of FTG fibers and the expression of the glycolytic enzymes PK-1 and HK-1. In addition, in vitro studies revealed that NT-3 increased glucose uptake and lactate formation in myotubes and upregulation of PK-1. Although these results suggest that NT-3 may be involved in controlling systemic metabolism by regulating fast / glycolytic fibers, further studies are needed to characterize its role in detail. In addition, AAV.NT-3 treatment was able to reverse the defective expression levels of PGC1α and the elevated levels of activated 4E-BP1 seen in TrJ neurogenic muscles. It has been previously shown that in skeletal muscle, mTOR regulates mitochondrial biogenesis and metabolism through 4E-BP1 / PGC1α. Tsai et al., J Clin Invest [Journal of Clinical Research], 125(8): 2952-2964 (2015). NT-3 may also play a role in promoting oxidative phosphorylation by activating 4EBP1 and PGC lα in muscle.

[0260] NT-3 was first found at high levels in the central nervous system (CNS) during fetal development and is reduced in the adult brain, suggesting that it plays an important role in early neuronal development [28,29]. NT-3 is also important in peripheral nerves and has a positive role at multiple stages of neuromuscular development. In Xenopus neuromuscular co-cultures, muscle-derived NT-3 significantly enhances the maturation of synaptic transmission at neuromuscular junctions [30-33]. In addition, NT-3 increases the survival of SCs, a key component of the neuromuscular system

[34] . NT-3 expressed in SCs promotes nerve regeneration and is an important component of the autocrine survival loop that ensures the survival and differentiation of SCs in adult nerves [35-39]. In studies of the CMT1A mouse model, several important biological effects of NT-3 were observed: (i) increased SC number, (ii) increased number of myelinated fibers, and (iii) normalization of the axonal neurofilament cytoskeleton [5,40]. Another NT-3 effect of particular interest here is the increase in myelin thickness, which is viewed as morphological evidence that NT-3 may affect myelin protein production.

[0261] Earlier studies have shown a large body of evidence that mTORC1 plays a role in regulating myelination in the CNS. Transgenic overexpression of constitutively active Akt kinase is sufficient to enhance myelin membrane growth in the CNS via mTOR signaling [41, 42], and protein synthesis stimulated by IGF-1 in oligodendrocyte progenitor cells requires the PI3KJAkt and MER / ERK pathways

[43] . The ability of NT-3, which targets the translation machinery, to stimulate myelin protein synthesis was first shown in primary cultures of oligodendrocytes

[44] . NT-3 was found to upregulate 4EBP1 phosphorylation in oligodendrocytes via the PI3K / mTOR pathway. Using a genetic inactivation approach to remove mTOR function, particularly in SCs, affected the ability of the SCs to form myelin normally

[45] . Indeed, in mutants, myelin was found to be thin, internode length was short, and radial growth of axons was impaired. In addition, the phosphorylation of mTOR downstream targets S6 and 4E-BP1 was low

[45] .

[0262] Based on these previous studies, the effects of NT-3 on increases in muscle fiber diameter are likely through the same mechanism, namely a direct effect via mTORC1 activation. It is important to note that NT-3 gene therapy in WT muscle at the same doses used in TrJs did not induce significant changes in fiber type size or type distribution. These observations in WT muscle suggest that NT-3 does not act on well-differentiated or normally functioning cells, but rather on cellular metabolism that may be remodeled by pathological processes. A supporting piece of evidence is that NT-3 did not alter functional recovery after crush injury in WT animals, resulting in only slightly more axons than control or NGF-treated animals

[46] . Our toxicology studies evaluating scAAV1.tMCK.NT-3 are consistent with these observations, showing no significant changes in fiber type size or type distribution after 1X 10 13 vg / kg (10-fold higher than the highest dose recommended for human trials of CMT1A treatment) in C57BL / 6 or TrJ mice.

[0263] During muscle development, p75NTR is transiently expressed on myoblasts that form myotubes / muscle fibers or differentiate into satellite cells

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

[23] . Similar to the action of NGF

[21] , NT-3 was found to enhance the expression of myogenin in myotubes, and as expected, this was associated with significantly higher expression of p75NTR in myoblasts, while the expression of p75NTR was significantly downregulated in myotubes. p75NTR and TrkC expression in TrJ and WT muscle samples were examined, and it was found that both of these were expressed at significantly higher levels in neurogenic TrJ muscles compared to WT, and these levels decreased in response to NT-3 ( Figure 5 Interestingly, this observation does not allow conclusions to be drawn about which cell type expresses these receptors, or whether they are expressed in all muscle fibers or only a subset of muscle fibers, 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, to date, recent studies combining histological studies of muscle biopsies with molecular and cellular analyses of primary muscle precursor cells have shown that p75NTR is expressed in the majority of satellite cells in vivo and is a marker for fiber regeneration in inflamed and dystrophic muscles [47,48]. Our findings in neurogenic muscle are of particular interest and warrant more comprehensive investigation of the mechanisms involved in different disease processes.

[0264] As a conserved Ser / Thr kinase, mTOR is a central regulator of cell growth by integrating signals from nutrients, growth factors, energy status, and environmental stress. The importance of mTOR in cell biology and pathobiology, particularly in muscle, which has remarkable metabolic and morphological adaptability, is currently of great interest. mTOR is associated with the formation of mTORC1 in raptor, and muscle-specific inactivation in raptor has been shown to result in muscle atrophy, impaired oxidative capacity, and increased glycogen stores, leading to dystrophic features that are most evident in oxidative muscle

[49] . On the other hand, loss of mTOR activity exacerbates myopathic features in both slow-oxidative and fast-glycolytic muscles and displays metabolic changes similar to those observed in muscle-deficient raptors, including impaired oxidative metabolism, altered mitochondrial regulation, and glycogen accumulation

[26] . In studies using a cardiotoxin-induced muscle necrosis / regeneration cycle paradigm, the inventors recently demonstrated impaired regeneration in a mouse model of limb-girdle muscular dystrophy type 2A and showed that calpain-3-deficient muscle is associated with perturbed mTORC1 signaling and defective mitochondrial biogenesis (reviewed in). In summary, the findings described herein have many implications for the potential use of NT-3, not only for the treatment of neuropathies, with benefits for both nerves and muscles, but also for muscle wasting disorders, including aging, cancer cachexia or type II myofiber atrophy, and inherited or acquired autoimmune primary muscle disorders associated with impaired regenerative radial growth phases [50-52]. Understanding the role of perturbations in mTOR signaling in these disorders should allow the development of new combinatorial therapeutic strategies, in which NT-3 may play an important role.

[0265] Example 2

[0266] NT-3 delivery using the scAAV1.tMCK.NFT3 vector

[0267] The composition administered is a non-replicating recombinant adeno-associated virus called scAAV1.tMCK.NTF3, such as Figure 3 The vector contains the human NT-3 gene under the control of the tMCK muscle-specific promoter. 11 vg) In vivo biopotency will be tested after intramuscular injection into the gastrocnemius muscle of C57B16 mice, followed by quantification of circulating NT-3 in serum by ELISA at 4 to 6 weeks after gene injection.

[0268] We first demonstrated that ssAAV1.CMV.NTF3 delivered to the gastrocnemius muscle produced prolonged and therapeutic NT-3 blood levels sufficient to provide functional, electrophysiological, and histopathological improvements in the TrJ nerve. We then investigated whether it was possible to generate the required vector doses and achieve the same level of expression by packaging the expression cassette with scAAV1. A dose-response study was performed in C57BL / 6 mice, comparing the effects of ssAAV1.CMV.NTF3 on NT-3 expression at 3 doses (3x10 9 vg, 1x 10 10 vg and 3x 10 10 vg) Serum NT-3 ELISA data after intramuscular injection of scAAV1.tMCK.NTF3 and scAAV1.CMV.NTF3. 11 vg administration of the sc.rAAV1.CMV.NTF3 vector produced significantly higher levels of NT-3 than the single-stranded vector at the same dose, consistent with the greater potency of the self-complementary vector. 10 vg), CMV and tMCK vectors were both generated from cells receiving 1 x 10 11 The NT-3 serum levels comparable to those obtained by mice of ss.AAV1.CMV.NTF3 at a vg dose produced a biological response.NT-3 levels of TrJ mice at 24 weeks after injection (mean ± SEM).In all 7 groups, there were significant differences in NT-3 levels, with p values ​​<0.0001.For both promoters and controls, significant differences in the NT-3 levels of the highest and intermediate doses of the vectors were observed.However, for the lower doses of the two vectors, no significant differences were found in the analysis.The serum NT-3 in all groups (PBS, CMV 3E+09 / 1E+10 / 3E+10 and tMCK3E+09 / 1E+10 / 3E+10) was compared using the Kruskal-Wallis test.NT-3 between each group and the PBS (control) group was compared 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.

[0269] Increased muscle diameter at 40 weeks after treatment: Compared with PBS, the muscle diameter increased significantly after injection of ssAAV1.CMV.NTF3 (1x 10 11The effect of NT-3 gene therapy on muscle fiber size was evaluated in TrJ mice at 40 weeks post-injection in a subgroup of animals treated with NT-3 (vg). Neurogenic changes characterized by atrophic angular fibers and grouped atrophy were evident in the muscles of untreated mice, while evidence of reinnervation in the form of increased fiber type groups and overall fiber size was considered a therapeutic effect. Muscle fiber size histograms generated from the contralateral anterior and posterior compartment muscles (tibialis anterior and gastrocnemius) of the left lower limb showed increased fiber diameter.

[0270] Additional studies in our laboratory have shown that NT-3 stimulates the Akt / mTOR pathway in SC cells, thereby improving myelination and radial outgrowth of axons in nerves, and that NT-3 also has a direct stimulatory effect on myotubes through the Trk-C receptor, indicating a role in the increase in fiber diameter in the muscles of TrJ mice. Figure 3 A shows that NT-3 increases phosphorylation of Akt (P-Akt) and mTOR targets 4EBP-1 (P-4EBP1) and PS6K (P-S6K) in SC and myotube cultures. These studies provide evidence to support our rationale for selecting the anterior and posterior calf muscles for vector delivery in this clinical trial.

[0271] Studies using self-complementary (sc) AAV1 and the muscle-specific truncated creatine kinase (tMCK) promoter

[0272] scAAV allows for lower dosing, thus enhancing safety, as well as dosing levels that meet manufacturing standards. The use of the tMCK promoter is a valuable goal, again providing increased safety by avoiding off-target effects. In the next set of experiments, the efficacy of scAAV1.NTF3 under the control of the CMV promoter was compared with the muscle-specific tMCK promoter, with both being administered at three doses over a half-log range (3x10 9 vg, 1x 10 10 vg and 3x 10 10 vg) administration. At 24 weeks after gene transfer, the efficacy of AAV1.NTF3 gene transfer in peripheral nerves of TrJ mice was evaluated by electrophysiological (Table 3) and morphological studies. Evidence of transgene expression was assessed by measuring serum NT-3 levels using ELISA.

[0273] Table 3. CMAP and conduction velocity in the TrJ sciatic nerve

[0274]

[0275] During the electrodiagnostic study, the examiner was blinded to the treatment group. CMAP was not statistically different 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 highest and intermediate doses of the vector for both the promoter and the control.

[0276] The complete disclosures of all patents, patent applications, and publications cited herein, as well as electronically available materials, are incorporated by reference. The above detailed description and examples are given only for the sake of clarity of understanding. They should not be construed as unnecessary limitations. The present invention is not limited to the precise details shown and described, as variations obvious to those skilled in the art will be included within the present invention defined by the claims.

[0277] Example 3

[0278] Construction of AAV constructs expressing NT-3

[0279] The design of a serotype 1 self-complementary AAV viral vector containing NT-3 cDNA under the control of the tMCK or CMV promoter was previously described in Sahenk et al., Mol Ther [Molecular Therapy], 22(3): 511-521 (2014), which is incorporated herein by reference in its entirety. Aliquots of virus were kept at -80°C until use. Blood samples were collected from treated and untreated mice by eye bleeding under anesthesia at 6 and 16 weeks after injection, and serum NT-3 levels were determined using capture ELISA. The construct is referred to herein as scAAV1.tMCK.NTF3.

[0280] The tMCK promoter / enhancer sequence is used to drive muscle-specific gene expression and consists of the muscle creatine kinase promoter and an additional enhancer element (enh358MCK, 584-bp) fused thereto. The triple tandem MCK enhancer (206-bp) is connected to the 87-bp basal promoter in the tMCK promoter / enhancer.

[0281] The scAAV1.tMCK.NTF3 drug product is produced by transfecting human HEK293 Master Cell Bank cells with three plasmid DNAs: (i) pAAV.tMCK.NTF3-vector plasmid (see Figure 7 ), (ii) an AAV1 helper plasmid called R88 / C1 containing the AAVrep2 and Cap1 wild-type genes, and (iii) a helper adenovirus plasmid

[0282] Schematic diagram of the plasmid with molecular features and open reading frames is shown in Figure 7 As shown. The AAV vector genome derived from the pAAV.tMCK.NTF3 plasmid is a self-complementary DNA genome containing a human NTF3 cDNA expression cassette flanked by AAV2 inverted terminal repeats (ITRs). It is this sequence that is packaged into the AAV1 virion. The plasmid pAAV.tMCK.NTF3 is constructed by inserting the tMCK expression cassette driving the NTF3 gene sequence into the AAV cloning vector psub201. The human NTF3 gene is expressed by a mouse triple tandem MCK promoter, which is a modified form 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 increasing gene expression, and is composed of a 5' donor site of the first intron of the human β-globin gene and a branch point and a 3' splice acceptor site of the intron between the leader sequence and the main body of the variable region of the heavy chain of the immunoglobulin gene. The NTF3 expression cassette has a consensus Kozak immediately before the ATG start and a 200 bp SV40 poly A signal that effectively terminates the mRNA. The NTF3 cDNA is included in its entirety (NCBI reference sequence: NM_001102654). The only viral sequence contained in this vector is the inverted terminal repeat sequence of AAV2, which is required for viral DNA replication and packaging. AAV ITRs are sequences that are almost identical at both ends but in opposite orientations. The terminal resolution site of the "left" (mutant) ITR is deleted to allow genomic hairpin formation. The identity of all DNA plasmid elements was confirmed by DNA plasmid sequencing of the plasmid source stock.

[0283] Table 4 shows the base pair positions of relevant molecular features within the AAV vector DNA plasmid of SEQ ID NO:11.

[0284]

[0285] Example 4

[0286] Phase I Intramuscular Study

[0287] For the initial Phase I intramuscular (IM) safety study, sustained NT-3 transgene expression is the outcome measure proposed in this first phase, which is important for a number of reasons. A true assessment of toxicity relies on demonstrating gene expression. If muscle is not transduced, toxicity testing (adverse effects) will be more difficult to interpret (transduction failure vs. loss of gene expression). This study provides an opportunity to establish a method to clearly identify transgene expression and distinguish it from endogenous gene expression.

[0288] This clinical trial is an open-label, one-shot ascending dose study in which scAAV1.tMCK.NTF3 is administered by intramuscular injection into the gastrocnemius and tibialis anterior muscles in both legs of CMT1A subjects with PMP22 gene duplication. Nine adult patients with CMT1A over the age of 18 were enrolled in one of two cohorts in the trial. The first three subjects received the lowest effective dose (2.0×10 12 vg / kg) were enrolled in Cohort 1. An additional six subjects were enrolled in Cohort 2 with a 3-fold dose escalation (6.0X 10 12 vg / kg). Monitoring after gene transfer includes follow-up at intervals of 7, 14, 30, 60, 90, 120 days and 3 months, and the rest of the follow-up of the trial is extended to 2 years. Safety is the primary endpoint of this clinical gene transfer trial. Stop criteria are based on the development of unacceptable toxicity, which is defined as any grade II ocular or systemic toxicity or the occurrence of any grade III or higher toxicity that has not subsided after two weeks. The secondary endpoint is the following efficacy, which is defined as the cessation of the ability decline measured by the CMT Pediatric Scale (CMTPedS) at 2 years after gene transfer. CMTPedS is a scale of 11 items, which consists of the following: functional agility test, nine-hole column test (9HPT), hand grip, plantar flexion and dorsiflexion strength (using a handheld myometer), pinprick and vibration sensation, Bruininks Oseretsky test-balance assessment, gait assessment, long jump and six-minute walk test (6MWT). Exploratory outcome measures will include a timed 100-meter test (100M), peroneal and ulnar CMAP amplitudes and sensory and motor conduction velocities, a revised sensory test for increased sensitivity to pinprick, touch testing, and vibration assessment, visual analog scales for pain and fatigue, the Brief Health Questionnaire (SF-36) as a measure of quality of life, and circulating NT-3 levels.

[0289] Patients included in the trial include any racial, ethnic, or gender background. The criteria for this disease have been defined and will follow the guidelines previously established by Shy et al. (Neurology 64: 1209-1214, 2001 and Neurology 70: 378-383, 2008).

[0290] The inclusion criteria for this study were as follows:

[0291] Adult subjects (>18 years old) diagnosed with CMT1A

[0292] ●Must exhibit a 1.5Mb duplication at 17p11.2 that includes the peripheral myelin protein 22 (PMP22) gene

[0293] ●Men and women of any race or ethnicity

[0294] ●Must demonstrate ankle dorsiflexor weakness (should have full ROM against gravity but cannot maintain full dorsiflexion against gravity or be able to stand on the heels for 3 seconds or longer (Northstar criteria)]

[0295] Abnormal nerve conduction velocity

[0296] ·Ability to cooperate with clinical evaluation and repeat nerve conduction studies

[0297] Sexually active subjects are willing to use reliable contraceptive methods during the study

[0298] The exclusion criteria for this study were as follows:

[0299] Active viral infection based on clinical observation or serologic evidence of HIV or hepatitis A, B, or C infection

[0300] Ongoing immunosuppressive therapy or immunosuppressive therapy within 6 months of trial entry (e.g., corticosteroids, cyclosporine, tacrolimus, methotrexate, cyclophosphamide, intravenous immunoglobulin)

[0301] Persistent leukocytosis or leukocytosis (WBC ≤ 3.5K / μL or ≥ 20.0K / μL) or absolute neutrophil count < 1.5K / μL

[0302] AAV1 binding antibody titer ≥ 1:50, as determined by ELISA immunoassay

[0303] Comorbidities or the need for long-term drug treatment, which the PI believes will pose an unnecessary risk of gene transfer

[0304] Ankle contracture or surgery that prevents appropriate muscle strength testing

[0305] Pregnant, breastfeeding or planning to become pregnant

[0306] Other causes of neuropathy

[0307] Have had limb surgery in the past six months

[0308] Table 5 Study design

[0309]

[0310] * Table 5 provides an overview of the dosing regimen based on a 70 kg patient weight as an example. The dose volume is based on approximately 1.5 x 10 13 Target product concentration in vg / ml.

[0311] Baseline measurements before injection (Day -30 to Day -1)

[0312] After obtaining written informed consent and completing hospital enrollment procedures, the following baseline medical procedures and measurements were performed.

[0313] Medical history

[0314] Taking concomitant medications

[0315] Physical examination

[0316] Chest X-ray

[0317] Echocardiography

[0318] EKG

[0319] Hematology Blood Laboratory (CBC)

[0320] Coagulation parameters: platelets, PT / INR, PTT

[0321] Clinical chemistry blood laboratory: bilirubin, blood urea nitrogen (BUN), GGT,

[0322] Alkaline phosphatase, alpha-fetoprotein (AFP), creatinine, amylase, serum protein electrophoresis, electrolytes, glucose, creatine kinase

[0323] Urinalysis

[0324] Viral screening (hepatitis and HIV)

[0325] Pregnancy test (women of childbearing age only)

[0326] Blood immunology: neutralizing antibodies (AAV1) and ELISpots

[0327] (NT-3 and AAV1)

[0328] ELISA serum (circulating NT-3)

[0329] Efficacy measures

[0330] Exploratory metrics

[0331] Photos of the injection site

[0332] Pre-injection dose of prednisolone

[0333] Prior to gene transfer, each patient will receive a 1 mg / kg / day oral dose of prednisone, up to a maximum of 60 mg / day, and then a second dose on the day of gene transfer. Subjects will receive prednisone 24 hours and 48 hours after gene transfer, for a total of 4 doses.

[0334] Gene transfer protocol

[0335] Self-complementary AAV1 carrying the human NTF3 gene under the control of the tMCK promoter (scAAV1.tMCK.NTF3) was administered to the medial and lateral heads of the tibialis anterior (TA) muscle in a single bilateral intramuscular injection.

[0336] Gene transfer procedures

[0337] The gene transfer procedure is as follows:

[0338] The gene transfer infusion procedure was performed under sterile conditions.

[0339] • Deliver vehicle without diluent for a total of approximately 10 mL to 28 mL per patient (5 to 14 mL per limb, divided among 3 muscles).

[0340] • The vector will be delivered to the operating room in pre-labeled syringes sealed in double leak-proof bags carried in a designated labeled cooler.

[0341] A total of 5 mL to 14 mL of vehicle will be administered to each and lateral heads of the gastrocnemius and TA muscles, for a total of 3 to 6 injections per muscle (each injection volume will be 0.5 to 1.0 ml). Fig. 8A and Figure 8B Schematic diagram of the injection site.

[0342] The injection is at least 0.25 cm below the fascia and along the longitudinal axis of the muscle under ultrasound guidance.

[0343] The interval between each injection is about 1.5 cm.

[0344] Hospitalization monitoring

[0345] After the gene transfer injection, the subject returns to the designated inpatient bed and closely monitors vital signs and respiratory function. Vital signs are monitored approximately every hour for the first 4 hours, and then every 4 hours until discharge. Safety is assessed by physical examination and laboratory evaluation. The subject is given a third dose of oral prednisone (Day 1), and the fourth and final dose of prednisone is given on the second day (48 hours after injection, Day 2). Approximately 48 hours after gene transfer, the patient will be discharged (if no side effects are observed).

[0346] Outpatient monitoring

[0347] Following discharge, patients return for follow-up at 7, 14, 30, 60, 90, 120, and 3-month intervals, with the remainder of the trial's follow-up extending to 2 years after gene transfer. Blood samples obtained at all visits will be evaluated for NT-3 protein expression as shown in a serum ELISA with anti-NT-3 antibody. Additionally, efficacy and exploratory measures will be tested in patients at visits 7, 9, 11, 13, and 14. Serum ELISA is a direct measure of functional gene expression, and secondary outcome measures show efficacy of the circulating transgene.

[0348] Primary endpoint:

[0349] Safety was the primary endpoint of this clinical gene transfer trial, which was evaluated based on the development of unacceptable toxicity (defined as the occurrence of any grade III or higher, unexpected treatment-related toxicity).

[0350] Security Metrics

[0351] Safety will be measured at each study visit with a collection of height and weight, vital signs, physical examination and system examinations, and a battery of blood laboratory work. Laboratory work will include CBC, platelets, blood urea nitrogen (BUN), GGT, bilirubin, alkaline phosphatase, alpha-fetoprotein, creatinine, amylase, serum protein electrophoresis, electrolytes, glucose, PT / INR and PTT, CK, urinalysis. Immunology will consist of the following: ELISA for neutralizing anti-AAV1 antibodies and for detecting anti-NT-3 antibodies, ELISpot for detecting T cell responses to AAV1 and NT-3. All adverse events will be recorded and assessed for association with gene transfer.

[0352] Secondary End Points

[0353] A secondary endpoint of the clinical gene transfer trial was efficacy, defined as cessation of decline in performance as measured by the CMT Pediatric Rating Scale (CMTPedS) at 2 years after gene transfer.

[0354] Upper and lower extremity muscle strength was tested using a handheld dynamometer for distal movements of the upper extremity (hand grip) and leg (dorsiflexion of the foot). The limb being tested was immobilized for testing, which significantly improved the reliability of the isometric contraction of the foot (29). Upper extremity function was measured using the 9-hole post test (9HPT). Lower extremity function was measured using the timed 10-meter run / walk (T10MW) test.

[0355] Exploratory outcome measures

[0356] Exploratory outcomes included a timed 100-meter test (100M), peroneal and ulnar CMAP amplitudes and sensory and motor conduction velocities, a revised sensory test for pinprick sensitivity, touch testing, and vibration assessment, visual analog scales for pain and fatigue, the Brief Health Questionnaire (SF-36) as a measure of quality of life, and circulating NT-3 levels.

[0357] Electrophysiological testing includes ulnar sensory nerve amplitude and compound muscle action potential (CMAP) amplitude of the ulnar nerve (recorded from the abductor digiti minimi muscle) and peroneal nerve (recorded from the tibialis anterior muscle) and measurement of sensory and motor conduction velocity. The peroneal CMAP amplitude of the tibialis anterior muscle has been shown to be a useful outcome measure for clinical trials in patients with CMT1A (30); and for upper limb motor symptoms, the ulnar CMAP amplitude has been shown to be the most useful parameter (31). During these testing procedures, the temperature of the feet and hands will be maintained at 32°C-34°C. A visual analog scale will be used for the measurement of pain and fatigue (32). The Brief Health Questionnaire (SF-36) is used as a quality of life document to monitor and compare disease burden before and after treatment.

[0358] Patients are offered a financial incentive to undergo an optional fascicular sural nerve biopsy. If chosen, biopsies are obtained before the start of treatment (from the left sural) and at the end of the study (from the right sural). The biopsies are performed as outpatient examinations under local anesthesia. The tissue will be processed and examined to evaluate the effect of NT-3 on myelinated fiber regeneration. In order for the level of pre- and post-treatment material to match the length of the entire nerve, the proximal end of the incision (2.5 cm in length) will be placed exactly 10 cm above the Achilles tendon (8).

[0359] Statistical analysis

[0360] Safety is the primary endpoint of this clinical gene transfer trial. This will be evaluated based on the development of unacceptable toxicity (defined as the occurrence of any grade III or higher, unexpected treatment-related toxicity). Safety will be measured at each study with a collection of height and weight, vital signs, physical examination and system examination, and a series of blood laboratory work. Laboratory work will include CBC, platelets, blood urea nitrogen (BUN), GGT, bilirubin, alkaline phosphatase, alpha-fetoprotein, creatinine, amylase, serum protein electrophoresis, electrolytes, B12, glucose, PT / INR and PTT, CK, urinalysis. Immunology will consist of the following: neutralizing anti-AAV1 antibodies and ELISA for detecting anti-NT-3 antibodies, ELISpot for detecting T cell responses to AAV1 and NT-3. All adverse events will be recorded and assessed for association with gene transfer.

[0361] The main secondary outcome measure was defined as no decrease in disease severity on the CMT Disease Pediatric Rating Scale (CMTPedS) score. The computerized scoring system of the CMTPedS uses z-scores from a normative sample to determine individual scores based on the number of standard deviations that the patient's performance is compared to the healthy population. Throughout the study, all patients were scored on the CMTPeedS at a fixed age of 20 years, which was necessary for two reasons. The first reason is that the original CMTPeedS scale relies on normative values ​​for individual scale items when calculating the final score, but normative values ​​do not exist for individuals over 20 years of age. The second reason is that although scoring a child to compare with healthy peers is necessary for children who are still developing, it is limited in that it will result in functional decline when the child passes another birthday. Even if a child's original score on the test does not change, the scoring criteria will become more stringent as they age, reflecting the expected improvement in motor skills that is usually seen in healthy children. However, in clinical trials of degenerative diseases, stopping actual progression will constitute a successful trial. Keeping the age of the reference control data consistent throughout the study allows the use of the validated scoring system built into the CMTPeedS. It also allows observation of actual changes in assessment, allowing detection of cessation of progress.

[0362] The proportion of patients whose CMTPeedS scores improved or remained unchanged over a two-year period was estimated with 95% confidence intervals using the binomial test. As an exploratory purpose, the proportion of patients who did not decline in each of the 11 items of the CMTPeedS, as well as in the 100-meter timed test (100M), CMAP amplitude, visual analog scale (VAS) for pain intensity, Short Form Health Questionnaire score (SF-36), and circulating NT-3 levels were estimated separately. In addition, the association between circulating NT-3 levels and each outcome measure was assessed using Pearson or Spearman correlation coefficients at each measurement point. Because the study is preliminary, p values ​​for multiple comparisons will not be adjusted. However, based on the Bonferroni-Holm mitigation procedure, sensitivity analyses were performed and indicated which associations were statistically significant after adjustment.

[0363] Based on longitudinal natural history data ( 31 , 32 ), secondary outcome measures of success were defined as cessation of the rate of deterioration in a standardized composite score of handgrip strength and ankle dorsiflexion strength, time to complete the 9-hole column test, and time to walk / run 10 meters.

[0364] Example 5

[0365] Toxicology studies

[0366] The aim of this study was to evaluate the effects of a single intramuscular injection of 50 μg / ml of WT (C57BL / 6) and 1.5 μg / ml of Trembler (Tr J Safety of a self-complementary adeno-associated viral (scAAV) vector expressing human neurotrophin 3 (NTF3) cDNA under the control of the muscle-specific tMCK promoter (scAAV1.tMCK.NTF3) in WT and trembler mice. The results reported here demonstrate that a single intramuscular injection is safe and well tolerated for 48 weeks after injection in wild-type and trembler mice.

[0367] Test animals

[0368] To wild-type C57BL / 6 (negative control) and Tr J (Test article) Mice were injected intramuscularly with vehicle (0.9% sterile saline) or 1 x 10 13 vg / kg of scAAV1.tMCK.NTF3 in a total volume of 50 μl. This vector mouse strain is available from Jackson Laboratory (stock #000664) or Charles River (stock #027). J Mice are available from The Jackson Laboratory (stock #002504).

[0369] Trembler mice were 8-10 weeks of age at the time of injection. Due to animal availability and known breeding issues with this animal strain, a subset of animals were treated at 12 weeks of age.

[0370] A total of 88 (44 male / 44 female) animals were included in the study. 44 animals (22C57BL / 6, 22TrJ) were treated with saline control, while 44 animals (22C57BL / 6, 22TrJ) were treated with scAAV1.tMCK.NTF3 test article. Of these numbers, half of the animals in each group were euthanized at 24 weeks after treatment, and the remaining animals were euthanized at 48 weeks after treatment. Animals were uniquely identified by ear tags.

[0371] Study Design

[0372] To evaluate the safety of the scAAV1.tMCK.NTF3 test article, 4-6 week old wild-type C57BL / 6 and 8-12 week old Tr mice were injected with 10% paraformaldehyde (P < 0.05) via a single IM injection into the left gastrocnemius muscle. J Mice were injected with vehicle (0.9% sterile saline) or 1 x 10 13vg / kg of scAAV1.tMCK.NTF3 in a total volume of 50 μl. The animals were observed daily for general health and morbidity throughout the study. Mortality was checked twice daily. Body weight was measured every 2 weeks. Functional tests of hypersensitivity (hot plate and acoustic startle tests) were performed every 8 weeks. Behavioral tests (rotarod and wire suspension) were performed every 2 weeks.

[0373] Thermosensitivity testing was performed using the Rodent hot plate test starting 2 weeks before injection, then on day 0, and every 8 weeks. For testing, animals were placed on a flat surface maintained at 55°C and movement was restricted to 100 cm using a 15 cm high Plexiglas wall surrounding the flat surface. 2 area. Start the timer immediately after placing the animal on the heated surface and record the latency of the reaction time by a stopwatch to 0.1 s. The following activities are considered as responses to the thermal stimulus: licking or flicking / flapping of the hind paws, attempts to escape by jumping are also acceptable responses. When such a response is observed, the mouse is removed immediately.

[0374] Acoustic sensitivity was tested two weeks before injection, then at day 0 (baseline), at the peak of serum NT-3 levels (8 to 12 weeks), and every 8 weeks thereafter after all animals were tested for auditory hypersensitivity using the acoustic startle test (AST). Startle reactivity was measured using a single startle chamber (SR-Lab, San Diego Instruments, San Diego, California) as previously described in Beigneux et al., Behavioral Brain Res. 171: 295-302, 2006.

[0375] The chamber consists of a transparent, non-restrictive Plexiglas cylinder placed on a platform in a ventilated chamber. A high-frequency speaker in the chamber produces a continuous background noise of 65 dB and various auditory stimuli. The vibration of the Plexiglas cylinder caused by the animal's systemic startle response is converted into an analog signal by a piezoelectric unit attached to the platform. The signal is then digitized and stored by a computer. Sixty-five readings are taken at intervals of 1 ms from the onset of the stimulus, and the average amplitude (V avg) to determine the auditory startle response. A background noise level of 65dB was presented during a 5min adaptation period and continued throughout the test period. All prepulse inhibition (PPI) test sessions consisted of startle trials (pulse only), prepulse trials (prepulse + pulse) and no stimulation trials (no stimulation). Pulse only trials consisted of 40ms 120dB broadband noise pulses. PPI was measured by prepulse + pulse trials (consisting of 20ms noise prepulse, 100ms delay, and then 40ms 120dB startle pulse). The acoustic prepulse intensity was 69, 73 and 81dB. No stimulation trials consisted of background noise only. The test period began and ended with five presentations of pulse trials alone; in between, each trial type appeared 10 times in pseudo-random order. An average of 15s (ranging from 12-30s) was between two trials.

[0376] For the rotarod analysis, at least one week of training was required to ensure that all subjects had learned the task to the same extent. In the accelerating rotation protocol, the animal was placed on the rod, which accelerated to 5 rpm and then increased at 5 rpm / sec7. Animals received an average of three trials per session.

[0377] For the wire hanging assay, the four paws of the animal were placed on a 2 mm diameter wire held horizontally 35 cm above a thick cushion layer. The length of time until the mouse fell from the wire was recorded, and the mouse was given 1 minute of recovery time after each fall. Each test session consisted of three trials, from which the average score was taken.

[0378] At 22 and 46 weeks after injection (2 weeks before euthanasia), blood was collected from the retro-orbital sinus for hematological studies (erythrocyte count, hematocrit, hemoglobin, leukocyte count, sum difference, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, mean corpuscular volume, mean platelet volume, platelet count, reticulocyte count). The following parameters of clinical chemistry were analyzed: alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase, bilirubin (total and direct), blood urea nitrogen, creatinine, creatine kinase, glucose, total protein.

[0379] At 24 and 48 weeks post-injection, blood was collected by intracardiac puncture and serum was used for immunization studies. Serum samples were collected for anti-AAV1 and anti-NT-3 antibody titration for all animals in each group (regardless of treatment or sex). Serum samples were also used for ELISA determination of circulating NT-3 levels.

[0380] At 24 and 48 weeks after injection, Tr JThe following tissues of the animals were subjected to histopathological analysis: gonads, brain, spleen, kidney, jejunum, colon, pancreas, heart, lung, stomach, liver, inguinal lymph nodes, spinal cord, gastrocnemius (left and right) and gross lesions (if any). Tissues were fixed in 10% neutral buffered formalin, sectioned and stained with hematoxylin and eosin. Histological analysis was performed by SNBLUSA.

[0381] Tissues for internal histopathology were collected from 6 animals per group (3M, 3F) at 24 and 48 weeks post-injection, but Tr J Control groups (n=5, 3M, 2F) were excluded. Mice were sacrificed and perfused transcardially with 4% paraformaldehyde in phosphate buffer (0.1 M, pH 7.4). Lumbar spinal cords, dorsal root ganglia (DRGs), and entire sciatic nerve segments (sciatic notch to popliteal fossa) were dissected. The proximal half of the sciatic nerve segment and one lumbar DRG in situ length were transferred to glutaraldehyde fixative, processed for plastic embedding and preparation of 1 μm thick sections. The remaining sciatic nerve tissue and lumbar DRG were cryoprotected in 30% sucrose, frozen in isopentane cooled in liquid nitrogen, and cut into 12 μm sections for immunohistochemistry to detect CGRP positivity of DRG neurons and axons (the spinal cord and brain of TrJ mice were placed in 10% neutral buffered formalin).

[0382]

[0383] Animal dosing, observation, and analysis

[0384] Indicated animals were dosed once with a volume of 50 μL via a single IM injection into the left gastrocnemius muscle on day 0. For accurate dosing, animals were anesthetized with isoflurane inhalation for at least 15 minutes. Dosing was performed by direct injection into the left gastrocnemius muscle. Note that the entire vehicle dose was accurately injected into the muscle. After dosing, the animals were observed until they were able to walk and return to the cage. Each animal was observed daily throughout the study. Body weight was measured every 2 weeks. Mortality checks were performed twice a day.

[0385] At the appropriate age, mice were overdosed with a ketamine / xylazine mixture (200 mg / kg / 20 mg / kg). Blood was collected via cardiac puncture. Tissues were then collected and sent for analysis.

[0386] Body weight, mouse hematology, and clinical chemistry were plotted for each time point for each group. ELISA assays for anti-AAV1 circulating antibodies, anti-NT-3 circulating antibodies, and circulating NT-3 levels were performed for all endpoints. All animals underwent hypersensitivity functional testing using a rodent hot plate and acoustic startle apparatus every 8 weeks. All animals underwent behavioral testing, i.e., rotarod and wire suspension tests, every 2 weeks. Formal histopathology of organs was performed on all animals. Internal histopathology of lumbar spinal cord and DRG was performed on 6 mice per group (3M, 3F), but Tr was included in 5 animals. J Groups (3M, 2F) were excluded. Histopathological evaluation included immunocytochemical distribution of CGRP positivity (in lumbar DRG neurons and spinal cord) and plastic embedding of sciatic nerve and DRG neurons to analyze pathological changes.

[0387] result

[0388] Morbidity and mortality

[0389] All mice survived the injection procedure and passed the initial observation period without any signs of discomfort. J Animal deaths. No deaths were associated with the test article.

[0390] weight

[0391] Body weights of all mice in each group were measured every two weeks throughout the study. All treatment groups maintained stable weight gains throughout the study.

[0392] In C57BL / 6 males, there were no significant differences between test article-injected animals and controls. In females, control animals had slightly higher body weights compared to test article-injected animals. J In male animals, the saline-treated group was significantly heavier than the NT-3-treated group. J There were no significant differences in body weight among mice.

[0393] Hematology and Clinical Chemistry

[0394] There were no test article-related changes in hematology parameters at the 22 and 46 weeks or serum chemistries at the 24 and 48 week time points.

[0395] ELISA assay

[0396] ELISA assays for anti-AAV1 antibodies, anti-NT-3 antibodies, and circulating NT-3 were performed using serum samples collected at 24 and 48 week necropsies.

[0397] To measure circulating anti-AAV1 antibodies, serum samples were collected at 24 and 48 weeks after vector administration and antibody titers were determined by binding enzyme-linked immunosorbent assay (ELISA), see Table 7 below. Animals treated with scAAV1.tMCK.NTF3 had elevated circulating anti-AAV1 capsid antibodies. Antibody titers were similar in males and females at both time points. No positive titers (>1:50) were detected in animals treated with saline controls.

[0398]

[0399] To measure anti-NT-3 circulating antibodies, serum samples were collected at 24 and 48 weeks after vector administration, and the antibody titers against NT-3 were determined by combining enzyme-linked immunosorbent assay (ELISA). At 24 and 48 weeks after vector administration, the antibody titers of all mice were <1:50 (considered negative). See Table 8.

[0400]

[0401]

[0402] Tr collected at 24 and 48 weeks after vector administration were measured by standard binding ELISA assay. J Circulating NT-3 levels in serum samples of mice. Intramuscular injection of scAAV1.tMCK.NTF3 vector resulted in increased Tr levels at 24 and 48 weeks after vector administration. J NT-3 was robustly expressed and secreted in mice. Table 9 shows the expression of NT-3 in saline and vehicle injected groups. J Mean and standard deviation of serum NT-3 levels 24 and 48 hours after injection. Gender had no effect on circulating NT-3 levels.

[0403]

[0404] Hypersensitivity Test

[0405] In C57BL / 6 and TrJ mice, thermosensitivity testing was performed at baseline (before test article treatment with control saline) and every 8 weeks until 48 weeks post-injection. There were no differences between control and vehicle-injected mice in the C57BL / 6 and TrJ groups. There were also no significant differences in withdrawal latency between male and female mice in each group.

[0406] Auditory sensitivity measurements in the acoustic startle test demonstrated that AST was significantly inhibited by prepulses at all intensities (69, 73, and 81 dB), and the level of inhibition depended on the intensity of the prepulse in both wild-type and TrJ mice. Treatment with the test article did not alter the PPI response in C57BL / 6 wild-type mice. In control-treated TrJ animals, a lower %-PPI was detected, which improved toward normalization in test article (NT-3)-treated TrJ animals. NT-3-treated TrJ animals showed a decrease in %-PPI, which was significantly decreased in control-treated TrJ animals. J The improvements in the animals were primarily seen in males.

[0407] Behavioral testing

[0408] For rotarod assessment, animals in the C57BL / 6 group did not show any differences in rotarod function tests regardless of treatment. Animals in the TrJ NT-3 treated group showed significant improvements in rotarod performance starting 16 weeks after treatment compared to control TrJ animals, which persisted until endpoint.

[0409] For the wire hanging assessment, animals in the C57BL / 6 group showed no differences in wire hanging functional testing regardless of treatment. Animals in the TrJ NT-3 treated group showed significant improvements in wire hanging ability beginning 28 weeks after treatment compared to control TrJ animals, which persisted to endpoint.

[0410] Histopathology

[0411] Formal histopathology was performed. A list of organs and tissues analyzed is presented in Table 10 below.

[0412]

[0413]

[0414] After euthanasia, the lumbar spinal cord, dorsal root ganglia (DRG) and the entire sciatic nerve (from the sciatic notch to the popliteal fossa) were removed and processed for histopathological evaluation. In-house histopathology A list of the individual animals included is shown in Table 11.

[0415]

[0416]

[0417] Immunocytochemical analysis of CGRP distribution

[0418] For immunocytochemical analysis of CGRP distribution, cross-sections of lumbar spinal cord segments were examined at 24 and 48 weeks after injection. This analysis revealed that TrJ There was no increase in CGRP reactivity in either C57BL / 6 or Drosophila animals treated with NT-3.

[0419] Plastic-embedded sections were analyzed for pathological changes. In C57BL / 6 animals, plastic-embedded left sciatic nerves and left lumbar DRG neurons examined at 24 and 48 weeks post-injection showed no pathological changes. At 48 weeks post-treatment, age-related changes such as wrinkling / infolding and outfolding of myelin sheaths, indicating axonal atrophy, were observed in both treatment groups of C57BL / 6 animals. J Animals showed significant loss of myelinated fibers and many hypomyelinated or bare axons. J Animals displayed small myelinated fibers, a marked increase in myelin thickness, and a reduction in exposed axons. NT-3 treatment did not have any adverse effects in TrJ mice, as evidenced by the absence of axonal sprouting within the DRG.

[0420] In conclusion, throughout this study, C57BL / 6 and Tr J No adverse treatment-related findings occurred in any of the animals, indicating that the treatment was well tolerated up to 48 weeks post-injection.

[0421] discuss

[0422] To evaluate the safety of scAAV1.tMCK.NTF3 delivered via a single intramuscular injection, a toxicology study was designed that included a total of 88 (44 male / 44 female) animals. 44 animals (22C57BL / 6, 22TrJ) were treated with 0.9% sterile saline control, while 44 animals (22C57BL / 6, 22TrJ) were treated with 1x 10 13 vg / kg of scAAV1.tMCK.NTF3 test article. Among these animals, half of the animals in each group were euthanized at 24 weeks after treatment, and the remaining animals were euthanized at 48 weeks after treatment.

[0423] The data in this report demonstrate that treatment of animals at a dose 10 times higher than the recommended clinical dose did not result in adverse safety effects associated with the test article. Throughout the study, the general health and morbidity of the animals were observed daily, and mortality checks were performed twice a day. Animal weights were measured every 2 weeks. Functional tests of hypersensitivity (thermosensitivity and acoustics) were performed every 8 weeks. Behavioral tests were performed every 2 weeks. All animals survived the injection procedure and the initial observation period without signs of discomfort. There were 4 deaths in TrJ animals due to a water system failure, which was unrelated to the administration of the test article. No other deaths were recorded in this study.

[0424] Following necropsy, hematology and clinical chemistry were measured and did not show any differences related to the test article. Anti-AAV1 serum ELISA was performed and showed the expected increase in circulating antibodies in animals treated with the test article that was non-sex specific. Anti-NT-3 serum ELISA was performed on all animals and showed negative antibody titers at all time points. Circulating NT-3 levels were also measured for all treatment groups and were elevated only in test article treated animals. There were no sex specific effects on circulating NT-3 levels. Thermal hypersensitivity testing revealed that C57BL / 6 or Tr J There were no treatment or sex specific changes in the animals. Auditory sensitivity measurements revealed no test article related changes in C57BL / 6 animals but did show a significant difference in Tr with control article treatment. J Defects in animals, and Tr treated with test articles J The animals showed an improvement towards normalization. This improvement was mainly in male Tr J Rotarod assessment of motor control in C57BL / 6 animals revealed no differences associated with test article treatment. J TrJ animals treated with the test article showed significant improvements compared to C57BL / 6 animals. Likewise, wire suspension assessment revealed no test article-related differences in the C57BL / 6 group. J Animals began to show significant improvements in wire hanging time 28 weeks after treatment and continued to the endpoint.

[0425] Tissues and organs collected from all groups at 24 weeks and 48 weeks after treatment were evaluated. In summary, at the 24-week autopsy, minimal mononuclear cell infiltration was observed in the left gastrocnemius injected with the test article in 3 / 4 of the first group of males and 4 / 5 of the first group of females. However, similar infiltration was also observed in the uninjected gastrocnemius in the remaining first group of females (animal 3942). At the 24-week autopsy, no such change was observed in the left gastrocnemius injected with saline or the uninjected right gastrocnemius in any of the second group (control) animals. At the 48-week autopsy, minimal mononuclear cell infiltration was observed in the left gastrocnemius injected with the test article in 2 / 5 of the first group of males and 1 / 6 of the first group of females. All other microscopic findings were randomly distributed in the control and treated animals, were background findings of the species, or were considered to be related to the administration of the test article.

[0426] In-house histology was performed on lumbar spinal cord segments, dorsal root ganglia, and sciatic nerves of a subset of animals (3M, 3F per group). Examination of cross-sections of lumbar spinal cord segments revealed no increase in CGRP activity in animals treated with NT-3, regardless of treatment. Pathological evaluation of plastic-embedded sciatic nerves and lumbar DRG neurons showed no changes in C57BL / 6 animals, except for age-related differences. J In animals, treatment with the test article resulted in improved pathology as evidenced by an increase in small myelinated fibers, increased myelin thickness, and a decrease in bare axons. The lack of axonal sprouting in DRGs further supports the safety of the test article.

[0427] In summary, the collected data presented in this study indicate that in male and female C57BL / 6 wild-type and Tr J In mice, 1 x 10 13 The test article scAAV1.tMCK.NTF3 injected directly into the gastrocnemius muscle at a dose of vg / kg was well tolerated for 48 weeks post-injection as demonstrated by the multiple measures described above.

[0428] References

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[0430] 2. Saporta AS, Sottile SL, Miller LJ, Feely SM, Siskind CE, Shy ME. Charcot-Marie-Tooth disease subtypes and genetic testing strategies. AnnNeurol 2011;69:22-33.

[0431] 3.Sahenk Z.Abnormal Schwann cell-axon interactions in CMTneuropathies.The effects of mutant Schwann cells on the axonal cytoskeletonand regeneration-associated myelination.AnnN Y Acad Sci 1999;883:415-426.

[0432] 4.Verhamme C,de Haan RJ,Vermeulen M,Baas F,de Visser M,vanSchaikIN.Oral high dose ascorbic acid treatment for one year in young CMT1Apatients:arandomised,double-blind,placebo-controlledphaseIItrial.BMCmedicine2009;7:70.

[0433] 5.MicallefJ,Attarian S,Dubourg O,etal.Effectofascorbic acidinpatientswith Charcot-Marie-Tooth disease type 1A:a multicentre,randomised,double-blind,placebo-controlledtrial.LancetNeurol 2009;8:1103-1110.

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[0462] Although the present invention has been described in terms of specific embodiments, it is understood that numerous changes and modifications will occur to those skilled in the art. Therefore, only such limitations as appear in the claims shall apply to the present invention.

Claims

1. An adeno-associated virus (AAV) vector comprising: (i) a 5'ITR comprising SEQ ID NO: 4; (ii) an MCK promoter comprising SEQ ID NO: 3; (iii) an intron comprising SEQ ID NO: 5; (iv) a Kozak sequence comprising SEQ ID NO: 6; (v) a nucleotide sequence encoding a human NT-3 polypeptide; (vi) SV40 polyadenylation signal; and (vii) a 3′ ITR comprising SEQ ID NO:

8.

2. The AAV vector of claim 1, wherein the SV40 polyadenylation signal comprises SEQ ID NO:

7.

3. The AAV vector of claim 1, comprising a sequence that is at least 99% identical to nucleotides 7-2248 of SEQ ID NO:

11.

4. The AAV vector of claim 3, comprising a sequence that is 100% identical to nucleotides 7-2248 of SEQ ID NO:

11.

5. The AAV vector of 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 variants thereof.

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

74.

7. A composition comprising the AAV vector according to claim 1 and a pharmaceutically acceptable carrier.

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

9. Use of the AAV vector according to claim 1 for the manufacture of a medicament for treating a muscle wasting disorder or a neuropathy in a human subject.

10. The use according to claim 9, wherein the medicament is formulated to result in sustained expression of the NT-3 polypeptide.

11. The use according to claim 9, wherein the subject suffers from Charcot-Marie-Tooth (CMT) neuropathy.

12. The use according to claim 11, wherein the subject has the gene variant Val30Met, Ile107Val or Ser77Tyr.

13. The method of claim 9, wherein the subject suffers from transthyretin amyloid neuropathy; acquired neuropathy caused by cancer, diabetes, human immunodeficiency virus (HIV) infection, thyroid disorder, hypothyroidism, hypoglycemia, uremia, renal insufficiency, liver dysfunction, liver failure, polycythemia, connective tissue disorder, Lyme disease, celiac disease, leprosy, porphyria, Sjögren's syndrome, poliomyelitis, acromegaly, lipid / glycolipid metabolism disorder, West Nile syndrome, amyloidosis, mitochondrial disorder, dysproteinemia disorder, monoclonal gammopathy of undetermined significance (MGUS), POEMS syndrome, nutritional / vitamin deficiency, vitamin B 12 deficiency, vitamin E deficiency, copper deficiency, hereditary myopathy, peripheral neuropathy, toxic neuropathy, autoimmune peripheral polyneuropathy, acute inflammatory demyelinating polyradiculoneuropathy (AIDP), chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), vasculitic mononeuritis multiplex, paraneuropathy, idiopathic ganglionitis, amyotrophic lateral sclerosis, multifocal motor block neuropathy, lower motor neuron syndrome, neuromuscular disease, muscular dystrophy, drug-induced myopathy, sarcopenia, cachexia, type II muscle fiber dystrophy, age-related muscular dystrophy, or acquired autoimmune primary muscle disorder.

14. Use of the AAV vector according to claim 1 for the manufacture of a medicament for improving muscle strength or stimulating muscle growth in a human subject.

15. The use according to claim 14, wherein the drug administration route is intramuscular injection.

16. The use according to claim 14, wherein the improvement in muscle strength is measured as a decrease in the composite score on the CMT Pediatric Rating Scale (CMTPeds) or as a decrease in disease progression over a two-year period.

17. The use according to claim 14, wherein the nucleic acid is a recombinant adeno-associated virus (rAAV) genome and the medicament is formulated to result in sustained expression of the NT-3 polypeptide.

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