WO2021211713A1

WO2021211713A1 - Treatment of charcot-marie-tooth axonal type 2d using nt-3 gene therapy

Info

Publication number: WO2021211713A1
Application number: PCT/US2021/027291
Authority: WO
Inventors: Zarife SAHENK
Original assignee: Research Institute At Nationwide Children's Hospital
Priority date: 2020-04-14
Filing date: 2021-04-14
Publication date: 2021-10-21

Abstract

The present disclosure relates to methods of treating Charcot-Marie-Tooth axonal type 2D (CMT2D) using a rAAV vector for neurotrophin 3 (NT-3) gene therapy.

Description

TREATMENT OF CHARCOT-MARIE-TOOTH AXONAL TYPE 2D USING NT-3

GENE THERAPY

[0001] This applciation claims priority of U.S. Provisional Application No. 63/009,932, fild April 14, 2020, which is incorporated by reference herein in its entirety.

INCORPORATION BY REFERENCE OF THE SEQUENCE LISTING

[0002] This application contains, as a separate part of disclosure, a Sequence Listing in computer-readable form (filename: 55415_Seqlisting.txt; 20, 292 bytes-ASCII text file; created April 14, 2021) which is incorporated by reference herein in its entirety.

FIELD

[0003] The present disclosure relates to methods of using the rAAV for NT-3 gene therapy to treat Charcot-Marie-Tooth axonal type 2D (CMT2D).

BACKGROUND

[0004] Charcot-Marie-Tooth axonal type 2D (CMT2D) is an axonal type Charcot-Marie - Tooth disease (CMT) caused by autosomal dominant mutations in Glycyl tRNA synthetase (GARS) gene. CMT2D a peripheral sensorimotor neuropathy, characterized by distal weakness primarily and predominantly occurring in the upper limbs and tendon reflexes are absent or reduced in the arms and decreased in the legs.

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

[0006] Adeno-associated virus (AAV) is a replication-deficient parvovirus, the single-stranded DNA genome of which is about 4.7 kb in length including 145 nucleotide inverted terminal repeat (ITRs). There are multiple serotypes of AAV. The nucleotide sequences of the genomes of the 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., 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 is provided in GenBank Accession No. NC_00 1862; at least portions of 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). The sequence of the AAV-B1 genome is provided in Choudhury et al, Mol. Ther., 24(7): 1247-1257 (2016). Anc80 is an AAV vector that is of AAV1, AAV2, AAV8 and AAV9. The sequence of Anc80 is provided in Zinn et al., Cell Reports 12: 1056-1068, 2015, Vandenberghe et al, PCT/US2014/060163, both of which are incorporated by reference herein, in their entirety and GenBank Accession Nos. KT235804-KT235812.

[0007] Cis-acting sequences directing viral DNA replication (rep), encapsidation/packaging and host cell chromosome integration are contained within the AAV ITRs. Three AAV promoters (named p5, pl9, and p40 for their relative map locations) drive the expression of the two AAV internal open reading frames encoding rep and cap genes. The two rep promoters (p5 and pl9), coupled with the differential splicing of the single AAV intron (at nucleotides 2107 and 2227), result in the production of four rep proteins (rep 78, rep 68, rep 52, and rep 40) from the rep gene. Rep proteins possess multiple enzymatic properties that are ultimately responsible for replicating the viral genome. The cap gene is expressed from the p40 promoter and it encodes the three capsid proteins VP1, VP2, and VP3. Alternative splicing and non-consensus translational start sites are responsible for the production of the three related capsid proteins. A single consensus polyadenylation site is located at map position 95 of the AAV genome. The life cycle and genetics of AAV are reviewed in Muzyczka, Current Topics in Microbiology and Immunology, 158: 97-129 (1992).

[0008] AAV possesses unique features that make it attractive as a vector for delivering foreign DNA to cells, for example, in gene therapy. AAV infection of cells in culture is noncytopathic, and natural infection of humans and other animals is silent and asymptomatic. Moreover, AAV infects many mammalian cells allowing the possibility of targeting many different tissues in vivo. Moreover, AAV transduces slowly dividing and non-dividing cells, and can persist essentially for the lifetime of those cells as a transcriptionally active nuclear episome (extrachromosomal element). The AAV proviral genome is infectious as cloned DNA in plasmids which makes construction of recombinant genomes feasible. Furthermore, because the signals directing AAV replication, genome encapsidation and integration are contained within the ITRs of the AAV genome, some or all of the internal approximately 4.3 kb of the genome (encoding replication and structural capsid proteins, rep-cap) may be replaced with foreign DNA. The rep and cap proteins may be provided in trans. Another significant feature of AAV is that it is an extremely stable and hearty vims. It easily withstands the conditions used to inactivate adenovirus (56° to 65°C for several hours), making cold preservation of AAV less critical. AAV may even be lyophilized. Finally, AAV-infected cells are not resistant to superinfection.

[0009] There is a need for developing therapies for CMT2D. The disclosure provides gene therapy methods of delivering NT-3 for the treatment of CMT2D.

SUMMARY

[0010] The disclosure provides methods of treating Charcot-Marie-Tooth axonal type 2D (CMT2D). The method comprises administering a therapeutically effective amount of neurotrophin-3 (NT-3), pro-NT-3, or an effective fragment thereof, or a nucleic acid encoding NT-3, pro-NT-3, or an effective fragment thereof, to a subject with CMT2D.

[0011] In one embodiment, the disclosure provides for methods of treating CMT2D in a human subject in need thereof comprising the step of administering to the human subject a nucleic acid encoding a NT-3 polypeptide; wherein a) the nucleic acid comprises a nucleotide sequence that is at least 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 encoding an amino acid sequence that is at least 90% identical to SEQ ID NO: 2; or d) the nucleic acid comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2.

[0012] Any of the methods of the disclosure can be carried out with a nucleic acid that is operatively linked to a muscle- specific promoter, such as the muscle-specific creatine kinase (MCK) promoter. In various embodiments, the muscle creatine kinase promoter sequence is set out in nucleotides 147-860 of SEQ ID NO: 3. In any of the method of the disclosure, the nucleic acid is administered using a viral vector, such as adeno-associated virus vector. In various embodiments, the viral vector is a recombinant adeno-associated virus (rAAV) vector. In related embodiments, the rAAV further comprises a pharmaceutically acceptable carrier. In various embodiments, the rAAV capsid serotype is AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV- 10, AAV-11, AAV12, AAV13, Anc80, AAV-B1, AAVrh10, or AAVrh.74. In certain embodiments, the AAV capsid serotype is AAV-1.

[0013] Any of the methods of the disclosure can be carried out with a nucleic acid sequence that is the rAAV genome sequence comprising in order from 5' to 3': (i) a first AAV2 inverted terminal repeat sequence (ITR); (ii) a muscle creatine kinase promoter/enhancer sequence set out in nucleotides 147-860 of SEQ ID NO: 3; (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 is 100% identical to SEQ ID NO: 2, or is encoded by a nucleotide sequence at least 90% identical to nucleotides 1077-1850 of SEQ ID NO: 3 or 100% identical to nucleotides 1077-1850 of SEQ ID NO: 3.

[0014] Any of the methods of the disclosure can be carried out with a nucleic acid which further comprise 3’ to the promoter/enhancer, a chimeric intron set out in nucleotides 892-1024 of SEQ ID NO: 3. In addition, the nucleic acids of the disclosure can further comprise 3’ to said nucleotide sequence encoding a human NT-3 polypeptide, a SV40 polyadenylation signal set out in nucleotides 1860-2059 of SEQ ID NO: 3.

[0015] In any of the methods of the disclosure, the nucleic acids of the disclosure can comprise one or more inverted terminal repeat (ITR) sequences. For example, the nucleic can comprise a first ITR which is set out in nucleotides 7-112 of SEQ ID NO: 3, and/or a second ITR which is set out in nucleotides 2121-2248 of SEQ ID NO: 3. In some embodiments, the nucleic acids comprise an scAAV1.tMCK.NTF3 genome that is at least 90% identical to the nucleotide sequence set out in SEQ ID NO: 3. In related embodiments, the nucleic acid comprising the scAAV1.tMCK.NTF3 genome is set out in SEQ ID NO: 3. In some embodiments, the nucleic acids comprise an scAAV1.tMCK.NTF3 genome that is at least 90% identical to the nucleotide sequence set out in nucleotides 7-2248 of SEQ ID NO: 3. In related embodiments, the nucleic acid comprising the scAAV1.tMCK.NTF3 genome is set out in nucleotides 7-2248 of SEQ ID NO: 3. The scAAV1.tMCK.NTF3 genome is set out as SEQ ID NO: 9 which is identical to nucleotides 7-2248 of SEQ ID NO: 3.

[0016] The disclosure provides for methods of treating CMT2D 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 a low concentration of NT-3 protein. [0017] In any of the methods of the disclosure, the rAAV is administered at a dose that results in sustained expression of a low concentration of NT-3 polypeptide. In various embodiments, the 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. In related embodiments, the route of administration is intramuscular bilateral injection to the medial and lateral head of the gastrocnemius and tibialis anterior muscle.

[0018] In any of the methods of the disclosure, the subject may have a mutation in the GARS gene or a gene encoding an aminoacyl-tRNA synthetase. In various embodiments, the muscle strength improved in the subject is in the upper or lower extremities. In addition, in any of the method of the disclosure, the administration of the 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 results in improved muscle strength in the subject is in the upper or lower extremities, and for example the improvement in the muscle strength is measured as a decrease in composite score on CMT Pediatric scale (CMTPeds). In addition, in any of the method of the disclosure, the administration of the 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 results in a decrease or halt in disease progression over a two-year time period. Disease progression is measured by the CMTPeds. In related embodiments, the sciatic nerve conduction velocity is increased by 1- 100%.

[0019] The disclosure also provides for methods of administering AAV vector expressing NT- 3 as a surrogate gene therapy for treating CMT2D. NT-3 has a short half-life and the methods of the disclosure comprise administering an AAV vector for a sustained release of NT-3 protein, even though the subject expresses endogenous NT-3 protein. As a surrogate gene therapy, the administration of the AAV vector provides sustained delivery of the NT-3 protein by sustained secretion by muscle cells. This continuous sustained low circulating level of NT-3 protein provides a therapeutic effect with a minimal risk of toxicity. Systemic production of NT-3 by gene therapy is also a more convenient and cost effective therapy option when compared to repeated injections of a purified NT-3 peptide.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The present disclosure may be more readily understood by reference to the following figures, wherein: [0021] Figures 1-lB: (A)provide a schematic of the cassette portion of the construct AAV.tMCK.NTF3 (set out in nucleotides 7-2248 of SEQ ID NO: 3; referred to herein as SEQ ID NO: 9). The rAAV contains the muscle specific tMCK promoter (SEQ ID NO: 11), chimeric intron (SEQ ID NO: 5), consensus Kozak sequence (SEQ ID NO: 6), the NTF3 cDNA (SEQ ID No: 1), and a polyadenylation signal (SEQ ID NO: 7). (B) Gars^P278KY/+ and Gars^ΔETAQ/+ mice were injected with 1x10¹¹ vg of scAAV1.tMCK.NT-3 vector equally divided to right and left gastrocnemius muscles or with Ringer’s lactate as control. Serum samples were obtained from treated and untreated mice via cardiac puncture at corresponding endpoints, and NT-3 levels were determined by using enzyme-linked immunosorbent assay. Error bars are ± SEM; n=8 for UT.Gars^P278KY/+, n=12 for UT .Gars^ΔETAQ/+ , n=10 for NT-3.Gars^P278KY/+, n=ll for NT-3. Gars^ΔETAQ/+ .

[0022] Figure 2 provides a restriction map and ORF Analysis of a cassette plasmid sc pAAV.tMCK.NTF3 (SEQ ID NO: 3).

[0023] Figures 3A-3B provide graphs and images showing the direct effect of NT-3 on myotubes. Fig. 3A: Representative western blot images and analysis of Akt/mTOR pathway, Phospho (P)-Akt (Ser473), P-4EBP1 (Thr37/46), and P-S6 (Ser235/236) in myotubes incubated with recombinant human NT-3 (100 ng/ml) or PBS (control) for 30 minutes. Coomassie Blue stained membrane represents equal gel loading. Fig. 3B: Density values of phosphorylated protein bands were normalized to GAPDH and showed as percent of control group. The results shown are mean ± SEM from at least three independent experiment, *P <0.05, Student's paired t- test.

[0024] Figure 4 provides the nucleotide sequence of a cassette production plasmid, sc pAAV.tMCK.NTF3 (SEQ ID NO: 3).

[0025] Figure 5 provides a graph of rotarod data indicated an increase in the NT-3 treated Gars^P278KY/+ mice (n=10), a model for CMT2D compared to untreated group (n=6, p= 0.035).

[0026] Figures 6A-D provide photos and a graph demonstrating semiquantitative assessment of toe spread. Images represent toe spread levels defined as (A) good/full toe spread (score: 2), (B) partial toe spread with mild intermittent clasping (score: 1) and (C) no toe spread with clasping (score: 0). Average toe spreading grade improved significantly in the treated cohort ( AAV 1. NT-3: 1.5 ± 0.19, n=12 vs. untreated: 0.25 ± 0.15, n=8; p= 0.00024). (D) 58.3% of treated mice was graded with full toe spreading and 33.3% showed partial toe spreading. None of the untreated mice had full toe spread and 75% of them failed to exhibit toe spread.

[0027] Figures 7A-7C provide graphs of electrophysiology tests showing an increase in NCV and CMAP values in the treated Gars^P278KY/+ mice (p=0.02, 0.04). (D) Representative waveforms of the sciatic nerve motor nerve conduction from untreated (UT) and NT-3 cohorts are shown; base time is 2 ms for left, 1ms for right panel. Data represented as mean ± SEM, unpaired t-test.

[0028] Figure 8 provides a graph showing that NT-3 treatment increased the innervated NMJs. In NT-3 treated Gars^P278KY/+ mice, innervated NMJs increased 14.9% compared to the untreated (UT) group. In addition, with treatment partially innervated NMJs showed an increase of 8.3% while a 6.5% decrease in the denervated NMJs were observed.

[0029] Figures 9A-9G provide the morphological characteristics of peripheral nerves in Gars^P278KY/+ mice. Representative Imp -thick, toluidine blue-stained cross-sections of (A, B) ventral roots, (C, D) mid sciatic and (E, F) tibial nerves from WT (A, C, E) and Gars^P278KY/+ (B, C, F) mice. Peripheral nerves including ventral roots of Gars^P278KY/+ mutant display small axon size, uniformly thin myelin and increased myelinated fiber density. (G) Schematic depicting the endoneurial cross-sectional areas and MF densities of sciatic and tibial nerves from WT and Gars^P278KY/+ mice.

[0030] Figure 10 provides Myelinated fiber axon size distribution in sciatic and tibial nerves of Gars^P278KY/+ and WT mice. Composites of myelinated fiber axon size distribution (MF number per 0.1 mm² of endoneurial area) in the sciatic and tibial nerves from Gars^P278KY/+ mutant and age-matched WT mice. A narrow axon size distribution concentrating on the 2 and 3 pm axon diameters was observed in sciatic and tibial nerve of Gars^P278KY/+ mice. Data is represented as mean ± SEM; n=4 per group. A total of 0.0502 mm² of endoneurial area per mouse was analyzed (an average of myelinated fiber measurements = 1017 for sciatic, 564 for tibialis per WT; 701 for sciatic, 642 for tibialis per Gars ^P278KY/+ mouse).

[0031] Figure 11 provides a graph of the distribution of Fiber Size in Sciatic Nerve of NT-3 Treatment in Gars^P278KY/+ mice.

[0032] Figures 12A-12H demonstrate that NT-3 gene transfer improves myelin thickness of peripheral nerves in Gars^P278KY/+ mice. Representative 1 pm-thick, toluidine blue-stained cross- sections of (A, B) ventral roots and (C, D) sciatic nerves from (A, C) untreated and (B, D) treated Gars^P278KY/+ mice. Scale bar=10 μm . A notable increase of myelin thickness is seen with treatment (B, D) compared to samples from untreated (A, B). G ratios calculated for the sciatic nerve of the treated and untreated (E) Gars^P278KY/+ and (G) Gars^ΔETAQ/+ mice are shown as scatterplots against respective axon diameters (total number of analyzed MFs=1840 for untreated and 1792 for NT-3 treated Gars^P278KY/+ mice and 1270 for untreated and 1042 for NT-3 treated Gars^ΔETAQ/+ mice; n=4 per cohort). Lines indicate linear regression. The slopes are significantly different between the two groups for both mutants (E; NT-3, r²=0.1893; UT, r²=0.2770; Linear regression, p<0.0001 and G; NT-3, r²=0.3545; UT, r²=0.2279; Linear regression, p<0.0001). G- ratios shown as percent distribution indicate a shift to the left with an increase in the number of axons with thicker myelin with NT-3 gene transfer therapy in both (F) Gars^P278KY/+ and (H)

Gars ^ΔETAQ/+ mice.

[0033] Figures 13A-13C provide myelinated fiber axon size distribution in sciatic and tibial nerves of Gars^P278KY/+ and WT mice. Composites of myelinated fiber axon size distribution (MF number per 0.1 mm² of endoneurial area) in the sciatic and tibial nerves from Gars^P278KY/+ mutant and age-matched WT mice. A narrow axon size distribution concentrating on the 2 and 3 pm axon diameters was observed in sciatic and tibial nerve of Gars^P278KY/+ mice. Data is represented as mean ± SEM; n=4 per group. A total of 0.0502 mm² of endoneurial area per mouse was analyzed (an average of myelinated fiber measurements = 1017 for sciatic, 564 for tibialis per WT; 701 for sciatic, 642 for tibialis per Gars ^P278KY/+ mouse).

[0034] Figure 14 provides a graph of the distribution of G-ratio in Gars^P278KY/+ mice. A shift towards increased percent of fibers with smaller g ratio (thicker myelin) is present in the scAAV1.NTF3 treated cohort.

[0035] Figures 15A-15C provide graphs demonstrating Rotarod performance and sciatic nerve conduction studies in Gars^ΔETAQ/+ mice. (A) Rotarod performance test and sciatic nerve conduction studies measuring (B) NCV and (C) CMAP of treated Gars^ΔETAQ/+ mice at endpoint showed no significant change compared to untreated cohort. Data represented as mean ± SEM, unpaired t-test.

[0036] Figures 16A-16N demonstrate NT-3 gene transfer improves neuromuscular junction assembly and neuromyopathy in Gars mutants. Representative images showing (A) innervated, (B) partially innervated and (C) denervated NMJs from the lumbrical muscles of the Gars^P278KY/+ mice, stained with IHC techniques. Scale bar=10 pm. (D) Innervated NMJs increased 14.9% in the treated mice. A total of 243 NMJs from NT-3-treated and 229 NMJs from UT-mice were evaluated (n=6 mice per group). (E) Representative H&E stained images of gastrocnemius muscle from the Gars^P278KY/+ mice at postnatal day 10 showing small fibers with mild size variability and rare fibers with central nuclei (arrow) and (H-J) at 4 months of age. (E) Scale bar=20 pm. Muscles from 4 months old Gars^P278KY/+ mice (H-J) showed marked fiber size variability with atrophic angular fibers (H; arrows), rare fibers undergoing necrosis (I; arrow) and exceedingly small fibers with prominent central nuclei (J). Scale bar=10 pm. Muscle fiber size histograms of the gastrocnemius muscle from (F) Gars^P278KY/+ (3699 fibers, derived from n=6) and age-matched WT (1364 fibers, n=4), and (G) Gars^ΔETAQ/+ (1412 fibers, n=4) and age- matched WT (1287 fibers, n=4). There is a significant increase in small fiber population in both mutants, which is more prominent in Gars^P278KY/+ mice. Representative H&E images of gastrocnemius muscle from (K) untreated and (L) AAVl.NT-3-treated Gars^P278KY/+ mice showing a decrease in the number of abnormally small or hypertrophied fibers with treatment (L). Scale bar=30 pm. (M) Muscle fiber size distribution graph comparing the treated (3788 fibers, n=7) and untreated (3699 fibers, n=6) Gars^P278KY/+ mice showed a shift to larger diameter subgroups with NT-3 gene therapy. (N) Bar graph showing a decrease in the number of internal nuclei in the gastrocnemius muscle from the treated (n=7) compared to UT Gars^P278KY/+ mice (n=6). Data is represented as mean± SEM; Two-way ANOVA, Sidak’s multiple comparisons test.

[0037] Figure 17 demonstrates gender-based muscle fiber size distribution in treated and untreated Gars^P278KY/+ mice. Gender-based muscle fiber size distribution bar graphs of gastrocnemius muscle from Gars^P278KY/+ mice at 12 weeks post NT-3 gene transfer. Data shows the gender-based distribution of the Figure 4M. Data is represented as mean ± SEM; n=4 for NT- 3-treated females and n=3 for each remainder male and female groups; p=0.0204, Two-way ANOVA, Tukey’s multiple comparisons test.

[0038] Figures 18A-18F demonstrate abnormalities in muscle histochemistry using SDH and COX reactions in Gars mutants. (A, B, E) SDH and (C, D, F) COX staining in the gastrocnemius muscles from (A - D) Gars^P278KY/+ and (E, F) Gars ^ΔETAQ/+ mutants showed reduced SDH and COX activities in fibers, most prominent at the superficial zones of the muscles of Gars^P278KY/+ mice (arrows). Scale bar=25 pm for A, B and F; 50 pm for C; 20 pm for D; 30 pm for E. Numerous ragged blue/brown fibers indicating increased mitochondria content in SDH and COX stains, respectively were also noted (arrows). (D - G) Bar graphs represent relative expression levels of COX1 and COX3, and Atp5d and mtDNA copy number/genomic DNA in the treated and untreated Gars^P278KY/+ mice and age-matched WT mice showing both combined and gender- based data. (H - K) Bar graphs represent relative expression levels of COX1 and COX3, and Atp5d and mtDNA copy number/genomic DNA in the untreated Gars^ΔETAQ/+ mice and age- matched WT mice showing both combined and gender-based data. Data is represented as mean ± SEM; Two-way ANOVA, Tukey’s multiple comparisons test. (n=8, NT-3.Gars^P278KY/+ mice; n=6, UT .Gars^p278KY/+ mice; n=5, WT (4 mo); n=12, Gars^ΔETAQ/+ mice; n=8, WT (10 mo).

[0039] Figures 19A-19K provide muscle histochemistry in the NT-3 treated Gars^P278KY/+ mice and expression levels of mitochondrial proteins. (A, B) Representative images of COX-reacted sections from (A) superficial and (B) deep zones of gastrocnemius muscle in the treated Gars^P278KY/+ mice and from (C) WT control. Scale bar = 30 pm. NT-3 gene transfer therapy improved intensity and distribution of COX activity towards normalization. Data is represented as mean ± SEM; Two-way ANOVA, Tukey’s multiple comparisons test. (n=8, NT- 3.Gars^p278KY/+ mice; n=6, UT .Gars^P278KY/+ mice; n=5, WT (4 mo); n=12, Gars^ΔETAQ/+ mice; n=8, WT (10 mo).

[0040] Figures 20A-20D provide myoblast fusion in Gars^P278KY/+ mice. Fluorescent images of myotubes differentiated from primary myoblast cultures isolated from one-month-old (A) Gars^P278KY/+ and (B) WT mice. Myotubes were stained with an anti-MHC antibody (red) and DAPI for nucleus (green), scale bar=50 μm. Randomly selected areas were photographed at 20X magnification. (Nikon Eclipse Ti2-E, Japan). A total of 710 myotubes from Gars^P278KY/+ mice and 659 myotubes from C57BL/6 mice were analyzed (25-26 images derived from n=2 mice in each group). (C) Fusion index was significantly reduced in the Gars^P278KY/+ myoblasts (65.8%) compared to WT mice (86.1%). Error bars are ± SEM, p<0.0001, unpaired t-test. (D)

Scattergram showing the myotube length and number of nuclei. Overall, Gars^P278KY/+ myotubes were shorter and had fewer nuclei than WT.

DETAILED DESCRIPTION

[0041] The present disclosure relates to methods of treating Charcot-Marie-Tooth axonal type 2D (CMT2D). The method comprises administering a therapeutically effective amount of neurotrophin-3 (NT-3), pro-NT-3, or an effective fragment thereof, or a nucleic acid encoding NT-3, pro-NT-3, or an effective fragment thereof, to a subject with CMT2D. The disclosure also provides gene therapy methods of increasing muscle strength in subjects in with CMT2D.

[0042] In one embodiment, the disclosure provides for gene therapy methods of treating CMT2D wherein the NT-3 encoding sequence of the NTF3 gene is delivered to the subject using self-complementary adeno-associated virus (scAAV) type 1 under control of a muscle-specific tMCK promoter.

[0043] Pre-clinical studies demonstrated that delivery of the construct AAVl.tMCK.NTF3 to the gastrocnemius muscle of the trembler J mice (Tr^J), a naturally occurring mouse model for CMT1, improved nerve regeneration, myelination, myelinated fiber density, sciatic nerve compound muscle action potential amplitude and functional performance on rotarod testing and hindlimb grip strength (see Example 1). In addition, these studies demonstrate improvements in the denervated status of NMJs as well as increases in muscle fiber size along with attenuation of myopathic changes. Improvements in the milder phenotype Gars^ΔETAQ/+ was less pronounced. In addition, no evidence of length-dependent axonal loss at distal tibial nerves was observed, therefore, furthering the characterization of the neuropathic process as distal-terminal axonopathy. Additional characterization of these Gars mutants using routine histopathology and oxidative enzyme histochemistry in muscle revealed the presence of a primary myopathic process associated with alterations in the content and distribution of oxidative enzymes in muscle and increased expression levels of PGCla, which is an important transcriptional coactivator of mitochondrial biogenesis and respiration (Handschin et al. Endocrine reviews 2006;27:728-735). Moreover, complex I, III, and Atp5d transcripts were significantly decreased suggesting that the muscle phenotype might be related to mitochondrial dysfunction. NT-3 gene therapy attenuated these abnormalities in muscle, which we report here as a novel finding.

Charcot-Marie-Tooth axonal type 2D

[0044] Charcot-Marie-Tooth axonal type 2D (CMT2D) is an axonal type CMT caused by autosomal dominant mutations in Glycyl tRNA synthetase (GARS)¹ gene. CMT2D is also known as GARS 1 -associated axonal neuropathy. CMT2D is characterized by adolescent or early-adult onset of weakness in the hands that may be preceded by transient cramping and pain in the hands on exposure to cold and cramping in calf muscles on exertion. This is followed by progressive weakness and atrophy of thenar and first dorsal interosseus muscles; hypothenar eminence is spared until later in the course of illness. Previous genetic and physical mapping efforts localized the responsible gene(s) to a well-defined region on human chromosome 7p¹.

[0045] As used herein, the terms "treatment", "treating", and the like, refer to obtaining a desired pharmacologic or physiologic effect. The effect may be therapeutic in terms of a partial or complete cure for a disease or an adverse effect attributable to the disease. "Treatment", as used herein, covers any treatment of a disease in a mammal, particularly in a human, and can include inhibiting the disease or condition, i.e., arresting its development; and relieving the disease, i.e., causing regression of the disease.

[0046] Prevention, as used herein, refers to any action providing a benefit to a subject at risk of being afflicted with Charcot-Marie-Tooth axonal type 2D (CMT2D).

[0047] "Pharmaceutically acceptable" as used herein means that the compound or composition or viral vector is suitable for administration to a subject for the methods described herein, without unduly deleterious side effects in light of the severity of the disease and necessity of the treatment.

[0048] The terms "therapeutically effective" and "pharmacologically effective" are intended to qualify the amount of an agent which will achieve the goal of improvement in disease severity and the frequency of incidence. The effectiveness of treatment may be measured by evaluating a reduction in symptoms in a subject in response to the administration of NT-3.

[0049] The term “effective fragment” refers to a portion of the polynucleotide sequence encoding a functional fragment of the NT-3 polypeptide. The term “effective fragment” also refers to a portion of the NT-3 polypeptide amino acid sequence that retains NT-3 growth factor activity. Exemplary NT-3 growth factor 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 activity includes stimulating muscle growth and muscle function.

[0050] As used herein, the term "diagnosis" can encompass determining the likelihood that a subject will develop a disease, or the existence or nature of disease in a subject. The term diagnosis, as used herein also encompasses determining the severity and probable outcome of disease or episode of disease or prospect of recovery, which is generally referred to as prognosis). "Diagnosis" can also encompass diagnosis in the context of rational therapy, in which the diagnosis guides therapy, including initial selection of therapy, modification of therapy (e.g., adjustment of dose or dosage regimen), and the like.

[0051] A "subject," as used herein, can be any animal, and may also be referred to as the patient. Preferably the subject is a vertebrate animal, and more preferably the subject is a mammal, such as a domesticated farm animal (e.g., cow, horse, pig) or pet (e.g., dog, cat). In some embodiments, the subject is a human.

[0052] 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 analogs of DNA or RNA containing non-natural nucleotide analogs, non-native inter-nucleoside bonds, or both. The nucleic acid can be in any topological conformation. For instance, the nucleic acid can be single- stranded, double- stranded, triple-stranded, quadruplexed, partially double-stranded, branched, hair-pinned, circular, or in a padlocked conformation.

[0053] The term "gene" as used herein refers to a nucleotide sequence that can direct synthesis of an enzyme or other polypeptide molecule (e.g., can comprise coding sequences, for example, a contiguous open reading frame (ORF) which encodes a polypeptide) or can itself be functional in the organism. A gene in an organism can be clustered within an operon, as defined herein, wherein the operon is separated from other genes and/or operons by intergenic DNA. Individual genes contained within an operon can overlap without intergenic DNA between the individual genes.

[0054] As used herein, the term "AAV" is a standard abbreviation for adeno-associated virus. Adeno-associated virus is a single- stranded DNA parvovirus that grows only in cells in which certain functions are provided by a co-infecting helper virus. There are currently thirteen serotypes of AAV that have been characterized. General information and reviews of AAV can be found in, for example, Carter, 1989, Handbook of Parvoviruses, Vol. 1, pp. 169-228, and Bems, 1990, Virology, pp. 1743-1764, Raven Press, (New York). However, it is fully expected that these same principles will be applicable to additional AAV serotypes since it is well known that the various serotypes are quite closely related, both structurally and functionally, even at the genetic level. (See, for example, Blacklowe, 1988, pp. 165-174 of Parvoviruses and Human Disease, J. R. 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 bear three related capsid proteins such as those expressed in AAV2. The degree of relatedness is further suggested by heteroduplex analysis which reveals extensive cross-hybridization between serotypes along the length of the genome; and the presence of analogous self-annealing segments at the termini that correspond to "inverted terminal repeat sequences" (ITRs). The similar infectivity patterns also suggest that the replication functions in each serotype are under similar regulatory control.

[0055] The term "vector" or "expression vector" refers to any type of genetic construct comprising a nucleic acid coding for an RNA capable of being transcribed. Expression vectors can contain a variety of control sequences, structural genes (e.g., genes of interest), and nucleic acid sequences that serve other functions as well.

[0056] By "vector" is meant a DNA molecule, usually derived from a plasmid or bacteriophage, into which fragments of DNA may 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 vehicle organism such that the cloned sequence is reproducible. A vector contains a promoter operably linked to a gene or coding region such that, upon transfection into a recipient cell, an RNA is expressed.

[0057] A “recombinant AAV (rAAV)” as used herein refers to a viral vector comprising one or more polynucleotides of interest (or transgenes) that are flanked by AAV terminal repeat sequences (ITRs). Such rAAV can be replicated and packaged into infectious viral particles when present in a host cell that has been transfected with a vector encoding and expressing rep and cap gene products.

[0058] A "rAAV virion" or "rAAV viral particle" or "rAAV vector particle" refers to a viral particle composed of at least one AAV capsid protein and an encapsidated polynucleotide rAAV vector. If the particle comprises a heterologous polynucleotide (i.e. a polynucleotide other than a wild-type AAV genome such as a transgene to be delivered to a mammalian cell), it is typically referred to as a “rAAV vector particle” or simply “rAAV particle.” Thus, production of AAV vector particle necessarily includes production of rAAV, as such a rAAV genome is contained within a rAAV vector particle.

[0059] As used herein, the term "about" refers to +/- 10% deviation from the basic value. [0060] 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 disclosure belongs.

Gene Therapy of Peripheral Neuropathy

[0061] In one aspect, the present disclosure provides methods of treating a subject having muscular atrophy using gene therapy.

[0062] Vectors which can be used to deliver a therapeutic nucleic acid include viral and non- viral vectors. Suitable vectors which can be used include adenovirus, adeno-associated vims, retrovirus, lentivims, HSV (herpes simplex virus) and plasmids. An advantage of Herpes simplex virus vectors is their natural tropism for sensory neurons. However, adenovirus associated viral vectors are most popular, due to their low risk of insertional mutagenesis and immunogenicity, their lack of endogenous viral genes, and their ability to be produced at high titer. Kantor et al. review a variety of methods of gene transfer to the central nervous system, while Goins et al. describe methods of gene therapy for the treatment of chronic peripheral nervous system pain. See Kantor et al., Adv Genet. 87, 125-197 (2014), and Goins et al., Neurobiol. Dis. 48(2), 255- 270 (2012), the disclosures of which are incorporated herein by reference. In particular, successful gene delivery to Schwann cells, the resident glia cells of peripheral nerves, has been reported using various viral vectors. Mason et al., Curr. Gene Ther.ll, 75-89 (2011). If the vector is in a viral vector and the vector has been packaged, then the virions can be used to infect cells. If naked DNA is used, then transfection or transformation procedures as are appropriate for the particular host cells can be used. Formulations of naked DNA utilizing polymers, liposomes, or nanospheres can be used for gene delivery. Nucleic acids can be administered in any desired format that provides sufficiently efficient delivery levels, including in virus particles, in liposomes, in nanoparticles, and complexed to polymers.

[0063] The nucleic acid (e.g., cDNA or transgene) encoding a gene whose expression decreases peripheral neuropathy can be cloned into an expression cassette that has 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 that are 1) specific to a tissue or region of the body; 2) constitutive; and/or 3) inducible/regulatable can be used. [0064] In some embodiments, muscle-specific regulatory elements are used. Muscle-specific regulatory elements include muscle-specific promoters including mammalian muscle creatine kinase (MCK) promoter, mammalian desmin promoter, mammalian troponin I (TNNI2) promoter, or mammalian skeletal alpha-actin (ASKA) promoter. Muscle-specific enhancers useful in the present disclosure are selected from the group consisting of mammalian MCK enhancer, mammalian DES enhancer, and vertebrate troponin I IRE (TNI IRE, herein after referred to as FIRE) enhancer. One or more of these muscle-specific enhancer elements may be used in combination with a muscle-specific promoter of the disclosure to provide a tissue- specific regulatory element.

[0065] A preferred viral vector for use in treating muscular atrophy by gene therapy is AAV. AAV-mediated gene delivery has emerged as an effective and safe tool for both preclinical and clinical studies of neurological disorders (Ojala et al., Neuroscientist., 21(l):84-98 (2015). Currently, rAAV is the most widely used vector for clinical trials for neurological disorders, and no adverse effects linked to the use of this vector have ever been reported from clinical trials: Adeno- associated virus (AAV) is a non-pathogenic dependovirus from the parvoviridae family requiring helper functions from other viruses, such as adenovirus or herpes simplex virus, to fulfill its life cycle. The wild-type (WT) AAV is characterized by a single-stranded DNA (ssDNA) genome, with inverted terminal repeats (ITR) at both ends, of approximately 5 kb surrounded by a capsid.

[0066] Adenoviral vectors for use to deliver transgenes to cells for applications such as in vivo gene therapy and in vitro study and/or production of the products of transgenes, commonly are derived from adenoviruses by deletion of the early region 1 (El) genes (Berkner, K. L., Curr.

Top. Micro. Immunol. 158 L39-66 1992). Deletion of El genes renders such adenoviral vectors replication defective and significantly reduces expression of the remaining viral genes present within 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, ability to replicate to high titer for preparation of vector stocks, and the potential to carry large inserts. However, it is believed that the presence of the remaining viral genes in adenoviral vectors can be deleterious.

[0067] Accordingly, in some embodiments, adenoviral vectors with deletions of various adenoviral gene sequences. In particular, pseudoadenoviral vectors (PAVs), also known as 'gutless adenovirus' or mini- adenoviral vectors, are adenoviral vectors derived from the genome of an adenovirus that contain minimal cis-acting nucleotide sequences required for the replication and packaging of the vector genome and which can contain one or more transgenes (See, U.S. Pat. No. 5,882,877 which covers pseudoadenoviral vectors (PAV) and methods for producing PAV, incorporated herein by reference). Such PAVs, which can accommodate up to about 36 kb of foreign nucleic acid, are advantageous because the carrying capacity of the vector is optimized, while the potential for host immune responses to the vector or the generation of replication-competent viruses is reduced. PAV vectors contain the 5' inverted terminal repeat (ITR) and the 3' ITR nucleotide sequences that contain the origin of replication, and the cis acting nucleotide sequence required for packaging of the PAV genome, and can accommodate one or more transgenes with appropriate regulatory elements, e.g. promoter, enhancers, etc.

AAV

[0068] Recombinant AAV (rAAV) genomes of the disclosure comprise nucleic acid molecule of the disclosure and one or more AAV ITRs flanking a nucleic acid molecule. AAV DNA in the rAAV genomes may be from any AAV serotype for 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, AAV- 13, Anc80, AAVrh.74, AAVrh10 and AAV-B1 (see, e.g., Gao et al., PNAS, 99:11854-11859 (2002)); and Viral Vectors for Gene Therapy: Methods and Protocols, ed. Machida, Humana Press, 2003). Furthermore, pseudotyped rAAV vectors may also be utilized in the methods described herein. Pseudotyped rAAV vectors are those which contain the genome of one AAV serotype in the capsid of a second AAV serotype; for example, a rAAV vector that contains the AAV2 capsid and the AAV1 genome or an rAAV vector that contains the AAV5 capsid and the AAV 2 genome. (Auricchio et al., (2001) Hum. Mol. Genet., 10 (26):3075-81). Production of pseudotyped rAAV is disclosed in, for example, WO 01/83692. Other types of rAAV variants, for example rAAV with capsid mutations, are also contemplated. See, for example, Marsic et al., Molecular Therapy, 22(11): 1900-1909 (2014). As noted in the Background section above, the nucleotide sequences of the genomes of various AAV serotypes are known in the art. To promote skeletal muscle specific expression, AAV1, AAV6, AAV8 or AAVrh.74 may be used.

[0069] DNA plasmids of the disclosure comprise rAAV genomes of the disclosure. The DNA plasmids are transferred to cells permissible for infection with a helper virus of AAV (e.g., adenovirus, El-deleted adenovirus or herpes vims) for assembly of the rAAV genome into infectious viral particles. Techniques to produce rAAV particles, in which a rAAV genome to be packaged, rep and cap genes, and helper virus functions are provided to a cell, are standard in the art. Production of rAAV requires that the following components are present within a single cell (denoted herein as a packaging cell): a rAAV genome, AAV rep and cap genes separate from (i.e., not in) the rAAV genome, and helper virus functions. The AAV rep and cap genes may be from any AAV serotype for which recombinant virus can be derived and may 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, Anc80, AAV-B1, AAVrh.74, AAVrh.10, AAV-8, AAV-9, AAV- 10, AAV-11, AAV-12 and AAV-13. Production of pseudotyped rAAV is disclosed in, for example, WO 01/83692 which is incorporated by reference herein in its entirety.

[0070] A method of generating a packaging cell is to create a cell line that stably expresses all the necessary components for rAAV particle production. For example, a plasmid (or multiple plasmids) comprising a rAAV genome lacking AAV rep and cap genes, AAV rep and cap genes separate from the rAAV genome, and a selectable marker, such as a neomycin resistance gene, are integrated into the genome of a cell. rAAV genomes have been introduced into bacterial plasmids by procedures such as GC tailing (Samulski et al., 1982, Proc. Natl. Acad. S6. USA, 79:2077-2081), addition of synthetic linkers containing restriction endonuclease cleavage sites (Laughlin et al., 1983, Gene, 23:65-73) or by direct, blunt -end ligation (Senapathy & Carter, 1984, J. Biol. Chem., 259:4661-4666). The packaging cell line is then infected with a helper virus such as adenovims. The advantages of this method are that the cells are selectable and are suitable for large-scale production of rAAV. Other examples of suitable methods employ adenovirus or baculovirus rather than plasmids to introduce rAAV genomes and/or rep and cap genes into packaging cells.

[0071] General principles of rAAV production are reviewed in, for example, Carter, 1992, Current Opinions in Biotechnology, 1533-539; and Muzyczka, 1992, Curr. Topics in Microbial. and Immunol., 158:97-129). Various approaches are described in Ratschin et al., Mol. Cell.

Biol. 4:2072 (1984); Hermonat et al., Proc. Natl. Acad. Sci. USA, 81:6466 (1984); Tratschin et al., Mol. Cell. Biol. 5:3251 (1985); McLaughlin et al., J. Virol., 62:1963 (1988); and Lebkowski et al., 1988 Mol. Cell. Biol., 7:349 (1988). Samulski et al. (1989, J. Virol., 63:3822-3828); U.S. Patent No. 5,173,414; WO 95/13365 and corresponding U.S. Patent No. 5,658,776 ; WO 95/13392; WO 96/17947; PCT/US98/18600; WO 97/09441 (PCT/US 96/ 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. Patent. No. 5,786,211; U.S. Patent No. 5,871,982; and U.S. Patent. No. 6,258,595. The foregoing documents are hereby incorporated by reference in their entirety herein, with particular emphasis on those sections of the documents relating to rAAV production.

[0072] The disclosure thus provides packaging cells that produce infectious rAAV. In one embodiment packaging cells may be stably transformed cancer cells such as HeLa cells, 293 cells and PerC.6 cells (a cognate 293 line). In another embodiment, packaging cells are cells that are not transformed cancer cells, such as low passage 293 cells (human fetal kidney cells transformed with El 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).

[0073] Recombinant AAV particles (i.e., infectious encapsidated rAAV particles) of the disclosure comprise a rAAV genome. In exemplary embodiments, the genomes of both rAAV lack AAV rep and cap DNA, that is, there is no AAV rep or cap DNA between the ITRs of the genomes. Examples of rAAV that may be constructed to comprise the nucleic acid molecules of the disclosure are set out in International Patent Application No. PCT/US2012/047999 (WO 2013/016352) incorporated by reference herein in its entirety.

[0074] The rAAV may be purified by methods standard in the art such as by column chromatography or cesium chloride gradients. Methods for purifying rAAV from helper vims are known in the art and include methods disclosed in, for example, Clark et al., Hum. Gene Ther., 10(6): 1031-1039 (1999); Schenpp and Clark, Methods Mol. Med., 69427-443 (2002); U.S. Patent No. 6,566,118 and WO 98/09657.

[0075] In another embodiment, the disclosure contemplates compositions comprising viral vectors or rAAVs of the present disclosure. Compositions of the disclosure comprise rAAV and a pharmaceutically acceptable carrier. The viral vectors may also comprise other ingredients such as diluents and adjuvants. Acceptable carriers, diluents and adjuvants are nontoxic to recipients and are preferably inert at the dosages and concentrations employed, and include buffers such as phosphate, citrate, or other organic acids; antioxidants such as ascorbic acid; low molecular weight polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counter ions such as sodium; and/or nonionic surfactants such as Tween, pluronics or polyethylene glycol (PEG).

[0076] Titers of rAAV to be administered in methods of the disclosure will vary depending, for example, on the particular rAAV, the mode of administration, the treatment goal, the individual, and the cell type(s) being targeted, and may be determined by methods standard in the art. Titers of rAAV may range from about 1x10⁶, about 1x10⁷, about 1x10⁸, about 1x10⁹, about 1x10¹⁰, about 1x10¹¹, about 1x10¹², about 1x10¹³ to about 1x10¹⁴ or more DNase resistant particles (DRP) per ml. Dosages may also be expressed in units of viral genomes (vg).

[0077] Methods of transducing a target cell with rAAV, in vivo or in vitro , are contemplated by the disclosure. The in vivo methods comprise the step of administering an effective dose, or effective multiple doses, of a composition comprising an rAAV of the disclosure to an animal (including a human being) in need thereof. If the dose is administered prior to development of a disorder/disease, the administration is prophylactic. If the dose is administered after the development of a disorder/disease, the administration is therapeutic. In embodiments of the disclosure, an effective dose is a dose that alleviates (eliminates or reduces) at least one symptom associated with the disorder/disease state being treated, that slows or prevents progression to a disorder/disease state, that slows or prevents progression of a disorder/disease state, that diminishes the extent of disease, that results in remission (partial or total) of disease, and/or that prolongs survival.

[0078] In particular, actual administration of rAAV of the present disclosure may be accomplished by using any physical method that will transport the rAAV recombinant vector into the target tissue of an animal. Administration according to the disclosure includes, but is not limited to, injection into muscle, the bloodstream and/or directly into the liver. Simply resuspending a rAAV in phosphate buffered saline has been demonstrated to be sufficient to provide a vehicle useful for muscle tissue expression, and there are no known restrictions on the carriers or other components that can be co-administered with the rAAV (although compositions that degrade DNA should be avoided in the normal manner with rAAV). Capsid proteins of a rAAV may be modified so that the rAAV is targeted to a particular target tissue of interest such as muscle. See, for example, WO 02/053703, the disclosure of which is incorporated by reference herein. Pharmaceutical compositions can be prepared as injectable formulations or as topical formulations to be delivered to the muscles by transdermal transport. Numerous formulations for both intramuscular injection and transdermal transport have been previously developed and can be used in the practice of the disclosure. The rAAV can be used with any pharmaceutically acceptable carrier for ease of administration and handling.

[0079] Transduction may be carried out with gene cassettes comprising tissue specific control elements. For example, one embodiment of the disclosure provides methods of transducing muscle cells and muscle tissues directed by muscle specific control elements, including, but not limited to, those derived from the actin and myosin gene families, such as from the myoD gene family [See Weintraub et al, Science, 251: 761-766 (1991)], the myocyte- specific enhancer binding factor MEF-2 [Cserjesi and Olson, Mol Cell Biol 11: 4854-4862 (1991)], control elements derived from the human skeletal actin gene [Muscat et al, Mol Cell Biol, 7: 4089-4099 (1987)], the cardiac actin gene, muscle creatine kinase sequence elements [See Johnson et al, Mol Cell Biol, 9: 3393-3399 (1989)] and the murine 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 factors (Semenza et al, Proc Natl Acad Sci USA, 88: 5680-5684 (1991)), steroid-inducible elements and promoters including the glucocorticoid response element (GRE) (See Mader and White, Proc. Natl. Acad. Sci. USA 90: 5603-5607 (1993)), and other control elements.

[0080] Muscle tissue is an attractive target for in vivo DNA delivery, because it is not a vital organ and is easy to access. The disclosure contemplates sustained expression of NT-3from transduced myofibers.

[0081] By “muscle cell” or “muscle tissue” is meant a cell or group of cells derived from muscle of any kind (for example, skeletal muscle and smooth muscle, e.g. from the digestive tract, urinary bladder, blood vessels or cardiac tissue). Such muscle cells may be differentiated or undifferentiated, such as myoblasts, myocytes, myotubes, cardiomyocytes and cardiomy oblasts.

[0082] The term “transduction” is used to refer to the administration/delivery of the coding region of NT-3 to a recipient cell either in vivo or in vitro, via a replication-deficient rAAV of the disclosure resulting in expression of NT-3 by the recipient cell. [0083] In one embodiment, the gene therapy is NT-3 gene therapy via rAAV delivery. An AAV expression cassette carrying human NTF3 cDNA coding sequence under the control of the triple muscle- specific creatine kinase (tMCK) promoter is disclosed herein. It has been previously shown that an improvement in motor function, histopathology, and electrophysiology of peripheral nerves can be achieved using the recombinant AAV 1 to increase neurotrophin-3 expression in the tremble (Try) mouse, which is a model for the Charcot-Marie-Tooth disease variant CMT1A. See Sahenk et al., Mol Ther. 22(3):511-21 (2014), the disclosure of which is incorporated herein by reference.

[0084] Thus, the disclosure provides methods of administering an effective dose (or doses, administered essentially simultaneously or doses given at intervals) of rAAV that encode NT-3 to a patient in need thereof.

Doses and Routes of Administration

[0085] The disclosure provides for local administration and systemic administration of an effective dose of rAAV and compositions of the disclosure including combination therapy of the disclosure. For example, systemic administration is administration into the circulatory system so that the entire body is affected. Systemic administration includes enteral administration such as absorption through the gastrointestinal tract and parental administration through injection, infusion or implantation.

[0086] Routes of administration for the rAAV contemplated in the foregoing methods therefore include, but are not limited to, intraperitoneal (IP), intramuscular (IM) and intravascular including, for example, inter- arterial limb perfusion (ILP) and intravenous (IV) routes.

[0087] The dose of rAAV to be administered in methods disclosed herein will vary depending, for example, on the particular rAAV, the mode of administration, the treatment goal, the individual, and the cell type(s) being targeted, and may be determined by methods standard in the art. More than one dose may be administered, for example, one, two, three or more doses. Titers of rAAV in a dose may range from about 1x10⁶, about 1x10⁷, about 1x10⁸, about 1x10⁹, about 1x10¹⁰, about 1xl¹¹, about 1x10¹², about 1.5x10¹², about 1x10¹², about 3x10¹², about 4x10¹², about 5x10¹², about 6x10¹², about 6.5 x10¹², about 7x10¹², 1x10¹³, about 1x10¹⁴, or to about 1x10¹⁵ or more DNase resistant particles (DRP) per ml. Dosages may also be expressed in units of viral genomes (vg) (i.e., about 1x10⁷ vg, about 1x10⁸ vg, about 1x10⁹ vg, about 1x10¹⁰ vg, about 1x10¹¹ vg, about 1.5x10¹¹ vg, about 3x10¹¹ vg, about 4x10¹¹ vg, about 5x10¹¹ vg, about 6x10¹¹vg, about 6.5 x10¹¹ vg, about 7x10¹¹ vg, about 1x10¹² vg, about 1.5x10¹²vg, about 1x10¹² vg, about 3x10¹² vg, about 4x10¹² vg, about 5x10¹² vg, about 6x10¹² vg, about 6.5 x10¹² vg, about 7x10¹² vg, 1x10¹³ vg, 1x10¹⁴ vg, 1x10¹⁵ respectively). Methods for titering rAAV are described in Clark et al., Hum. Gene Ther., 10: 1031-1039 (1999).

[0088] In some embodiments of the foregoing methods in which the route of administration is an IM route, the dose of the rAAV administered is from about 1.5x10¹² to at least about 6.5x10¹² vg/kg. (All ranges herein are intended to represent each individual value in the ranges, as well as the individual upper and lower values of each range.) In some embodiments of the foregoing methods in which the route of administration is IM, the dose of the rAAV administered is 2x10¹² vg/kg. In some embodiments of the foregoing methods in which the route of administration is IM, the dose of the rAAV administered is 4x10¹² vg/kg. In some embodiments of the foregoing methods in which the route of administration is IM, the dose of the rAAV administered is 6x10¹² vg/kg.

[0089] Human patients are subjects contemplated herein for treatment. Human patients are subjects contemplated herein for treatment by IM delivery. Such patients include those patients that, e.g.: i) children (<10 years), adolescent or adult subjects (>18 years) diagnosed with CMT2D (Sivakumar et al., Brain (2005), 128, 2304-2314), ii) exhibit a mutation in the GARS gene or a gene encoding an aminoacyl-tRNA synthetase, iii) males and females of any ethnic or racial group, iv) exhibit atrophy and or weakness of the thenar and first dorsal interosseus muscles or the extensor digitorum brevi or the toe dorsiflexors; v) exhibits muscular atrophy with foot drop; vi) ability to cooperate for clinical evaluation and repeat nerve conduction studies, and vii) willingness of sexually active subjects to practice a reliable method of contraception during the study. In various embodiments the mutation in the GARS gene includes, but is not limited to, one or more mutations from Table 1 or a p.Ser265Tyr mutations as described in Yalcouye, et ah, Mol Genet Genomic Med. 2019; 7(7): e00782. In various embodiments, the mutation in a gene encoding an aminoacyl-tRNA synthetase includes, but is not limited to, C157R, P234KY, G240R or mutations in genes encoding an aminoacyl-tRNA synthetase described in Wei et al., J. Biol. Chem. (2019) 294(14) 5321-5339 and Storkebaum et al., Bioessays. 2016 Sep; 38(9): 818- 829. Aminoacyl-tRNA synthetases are essential enzymes that catalyze the first reaction in protein biosynthesis, namely the charging of transfer RNAs (tRNAs) with their cognate amino acids. Suitable patients may not include, e.g., those with i) active viral infection based on clinical observations or serological evidence of HIV, or Hepatitis A, B or C infection, ii) ongoing immunosuppressive therapy or immunosuppressive therapy within 6 months of starting the trial (e.g., corticosteroids, cyclosporine, tacrolimus, methotrexate, cyclophosphamide, intravenous immunoglobulin), iii) persistent leukopenia or leukocytosis (WBC < 3.5 K/μL or > 20.0 K/μL) or an absolute neutrophil count < 1.5K/μL, iv) AAV1 binding antibody titers > 1:50 as determined by ELISA immunoassay, v) concomitant illness or requirement for chronic drug treatment that in the opinion of the PI creates unnecessary risks for gene transfer, vi) ankle contractures or surgeries preventing proper muscle strength testing, vii) pregnancy, breast feeding, or plans to become pregnant, viii) other causes of neuropathy, and/or ix) limb surgery in the past six months. In an exemplary clinical protocol, CMT2D patients receive a total dose of scAAV1.tMCK.NTF3 divided into medial and lateral heads of the gastrocnemius and tibialis anterior (TA) muscles of legs which are preferentially causing ankle weakness and instability in CMT. Subjects receive one of the following: i) low dose of rAAV of 2x10¹² vg/kg (total dose) or ii) a high dose of rAAV of 6x10¹² vg/kg (total dose).

[0090]

Table 1 Clinical and molecular features of mutations in the GARS gene.

^a C: Catalytic domain; DI: dimer interface; W: WHEP domain; Ins I: Insertion I domain; Ins II: Insertion II domain; Ins III: Insertion III domain; ACBD: anti-codon binding domain b * average age of disease onset c CMT: Charcot-Marie-Tooth disease; SMA: spinal muscular atrophy; dSMA: distal SMA; d AD: autosomal dominant; NA: not available

[0091] In one embodiment, the rAAV is administered by IM injection without diluent. In alternative embodiments, compositions for intramuscular injection include an adjuvant such as sesame or peanut oil or in aqueous propylene glycol can be employed, as well as sterile aqueous solutions. Such aqueous solutions can be buffered, if desired, and the liquid diluent first rendered isotonic with saline or glucose. Solutions of rAAV as a free acid (DNA contains acidic phosphate groups) or a pharmacologically acceptable salt can be prepared in water suitably mixed with a surfactant such as hydroxpropylcellulose. A dispersion of rAAV can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. In this connection, the sterile aqueous media employed are all readily obtainable by standard techniques well-known to those skilled in the art.

[0092] The pharmaceutical carriers, diluents or excipients suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating actions of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of a dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0093] Sterile injectable solutions are prepared by incorporating rAAV in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filter sterilization. Generally, dispersions are prepared by incorporating the sterilized active ingredient into a sterile vehicle which contains the 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, the preferred methods of preparation are vacuum drying and the freeze drying technique that yield a powder of the active ingredient plus any additional desired ingredient from the previously sterile-filtered solution thereof.

[0094] Transduction with rAAV may also be carried out in vitro. In one embodiment, desired target muscle cells are removed from the subject, transduced with rAAV and reintroduced into the subject. Alternatively, syngeneic or xenogeneic muscle cells can be used where those cells will not generate an inappropriate immune response in the subject.

[0095] In another aspect, rAAV genomes are provided herein. The genomes of the rAAV administered comprise a NTF3 polynucleotide under the control of transcription control sequences. The rAAV genomes lack AAV rep and cap DNA. AAV DNA in the rAAV genomes may be from any AAV serotype for 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, AAV12, AAV13, Anc80, AAV-B1, AAVrh.10, and AAVrh.74.

The nucleotide sequences of the genomes of these AAV serotypes are known in the art as noted in the Background Section above.

[0096] In some embodiments, the transcription control sequences of the rAAV genomes are muscle- specific control elements, including, but not limited to, those derived from the actin and myosin gene families, such as from the myoD gene family [See Weintraub et al., Science , 251: 761-766 (1991)], the myocyte- specific enhancer binding factor MEF-2 [Cserjesi and Olson, Mol. Cell. Biol., 11: 4854-4862 (1991)], control elements derived from the human skeletal actin gene [Muscat et al., Mol. Cell. Biol., 7: 4089-4099 (1987)], the cardiac actin gene, muscle creatine kinase (MCK) promoter [Johnson et al., Mol. Cell. Biol., 9:3393-3399 (1989)] and the MCK enhancer, MHCK7 promoter (a modified version of MCK promoter that incorporates an enhancer from myosin heavy chain (Salva et al, Mol. Ther., 15: 320-329 (2007)), 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 factors (Semenza et al., Proc. Natl. Acad. Sci. USA, 88: 5680-5684 (1991)), steroid-inducible elements and promoters including the glucocorticoid response element (GRE) (See Mader and White, Proc. Natl. Acad. Sci. USA, 90: 5603-5607 (1993)), and other control elements. In some embodiments, the transcription control elements include the MCK promoter/enhancer which is included in the AAV.tMCK.NTF3 genome disclosed herein. The MCK promoter/enhancer is composed of the muscle creatine kinase promoter with an added enhancer element (enh358MCK, 584-bp) fused to it. A triple tandem of the MCK enhancer (206-bp) was ligated to the 87-bp basal promoter in the tMCK promoter/enhancer. In some embodiments, the transcription control elements and the tMCK promoter/enhancer is included in the AAV.tMCK.NTF3 genome as set out in SEQ ID NO: 9. In some embodiments, the tMCK promoter/enhancer is according to the nucleotide sequence of SEQ ID NO: 11.

[0097] In some embodiments, the NTF3 polynucleotide in a rAAV genome is the NTF3 cDNA set out in SEQ ID NO: 1 (corresponding to nucleotides 1077-1850 of SEQ ID NO: 3). In some embodiments, the NTF3 polynucleotide in a rAAV genome is the NTF3 cDNA set out in GenBank Accession # NM_001102654 or the NTF3 cDNA sequence set out as SEQ ID NO: 1, or is a variant polynucleotide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the NTF3 cDNA sequence set out as SEQ ID NO: 1. In some embodiments, the variant NTF3 polynucleotide encodes the same NTF3 polypeptide as the polypeptide encoded by the NTF3 cDNA of SEQ ID NO: 1. The amino acid sequence of the NTF3 polypeptide encoded by the NTF3 cDNA set out as SEQ ID NO: 1 or provided as GenBank Accession # NM_001102654 is set out in SEQ ID NO:2. In some embodiments, the variant NTF3 polynucleotide encodes a variant NTF3 polypeptide with at least one amino acid sequence alteration as compared to the amino acid sequence of the polypeptide (SEQ ID NO: 2) encoded by NTF3 cDNA set out in SEQ ID NO: 1 or provided as GenBank Accession # NM_001102654. An amino acid sequence alteration can be, for example, a substitution, a deletion, or an insertion of one or more amino acids, preferably conservative substitutions. A variant NTF3 polypeptide can have any combination of amino acid substitutions, deletions or insertions where activity of the polypeptide is retained. In one aspect, a variant NTF3 polypeptide can have a number of amino acid alterations such that its amino acid sequence shares at least 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity with the amino acid sequence (SEQ ID NO: 2) encoded by NTF3 cDNA set out as SEQ ID NO: 1 or provided as GenBank Accession # NM_001102654.

[0098] In some embodiments, the rAAV genome is the AAV.tMCK.NTF3 genome, the sequence of the NT-3 gene cassette of which is set out in nucleotides 7-2248 of SEQ ID NO: 3 and is annotated in Table 3 (see Example 2).

[0099] In yet another aspect, an isolated nucleic acid comprising the nucleotide sequence set out in nucleotides 7-2248 of SEQ ID NO: 3 is provided. In some embodiments, the isolated nucleic acid consists of the nucleotide sequence set out in nucleotides 7-2248 of SEQ ID NO: 3.

[00100] Also provided is an isolated nucleic acid comprising, in order from 5' to 3': (i) a first AAV2 inverted terminal repeat sequence (ITR) (SEQ ID NO: 4); (ii) a muscle creatine kinase promoter sequence (SEQ ID NO: 11 and as set out in nucleotides 147-860 of 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, is 100% identical to SEQ ID NO:2, or is encoded by nucleotides 1077-1850 of SEQ ID NO: 3. [00101] Recombinant AAV comprising the foregoing nucleic acids are contemplated as well as rAAV comprising a nucleotide sequence that is at least 90% identical to the nucleotide sequence depicted in SEQ ID NO: 1.

[00102] DNA plasmids comprising rAAV genomes of the disclosure are provided. The DNA plasmids comprise rAAV genomes contemplated herein. An exemplary DNA plasmid is provided as SEQ ID NO: 3 and annotated in Table 3 (see Example 2). The DNA plasmids are transferred to cells permissible for infection with a helper virus of AAV (e.g., adenovirus, El- deleted adenovirus or herpesvirus) for assembly of the rAAV genome into infectious viral particles. Techniques to produce rAAV particles, in which an AAV genome to be packaged, rep and cap genes, and helper virus functions are provided to a cell are standard in the art.

Production of rAAV requires that the following components are present within a single cell (denoted herein as a packaging cell): a rAAV genome, AAV rep and cap genes separate from (i.e., not in) the rAAV genome, and helper virus functions. The AAV rep and cap genes may be from any AAV serotype for which recombinant virus can be derived and may 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, AAV-8, AAV-9, AAV- 10, AAV- 11, Anc80, AAV-B1, AAVrh.10, and AAVrh74. Production of pseudotyped rAAV is disclosed in, for example, WO 01/83692. Other types of rAAV variants, for example rAAV with capsid mutations, are also contemplated. See, for example, Marsic et al., Molecular Therapy, 22(11): 1900-1909 (2014).

[00103] A method of generating a packaging cell is to create a cell line that stably expresses all the necessary components for rAAV particle production. For example, a plasmid (or multiple plasmids) comprising a rAAV genome lacking AAV rep and cap genes, AAV rep and cap genes separate from the rAAV genome, and a selectable marker, such as a neomycin resistance gene, are integrated into the genome of a cell. rAAV genomes have been introduced into bacterial plasmids by procedures such as GC tailing (Samulski et al., 1982, Proc. Natl. Acad. S6. USA, 79:2077-2081), addition of synthetic linkers containing restriction endonuclease cleavage sites (Laughlin et al., 1983, Gene, 23:65-73) or by direct, blunt-end ligation (Senapathy & Carter, 1984, J. Biol. Chem., 259:4661-4666). The packaging cell line is then infected with a helper virus such as adenovirus. The advantages of this method are that the cells are selectable and are suitable for large-scale production of rAAV. Other examples of suitable methods employ adenovirus or baculovirus rather than plasmids to introduce rAAV genomes and/or rep and cap genes into packaging cells. Methods for producing rAAV with self-complementary genomes are also known in the art.

[00104] General principles of rAAV production are reviewed in, for example, Carter, 1992, Current Opinions in Biotechnology, 1533-539; and Muzyczka, 1992, Curr. Topics in Microbial. and Immunol., 158:97-129). Various approaches are described in Ratschin et al., Mol. Cell.

Biol. 4:2072 (1984); Hermonat et al., Proc. Natl. Acad. Sci. USA, 81:6466 (1984); Tratschin et al., Mol. Cell. Biol. 5:3251 (1985); McLaughlin et al., J. Virol., 62:1963 (1988); and Lebkowski et al., 1988 Mol. Cell. Biol., 7:349 (1988). Samulski et al. (1989, J. Virol., 63:3822-3828); U.S. Patent No. 5,173,414; WO 95/13365 and corresponding U.S. Patent No. 5,658,776; WO 95/13392; WO 96/17947; PCT/US98/18600; WO 97/09441 (PCT/US 96/ 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. Patent. No. 5,786,211; U.S. Patent No. 5,871,982; and U.S. Patent. No. 6,258,595. The foregoing documents are hereby incorporated by reference in their entirety herein, with particular emphasis on those sections of the documents relating to rAAV production.

[00105] In a further aspect, the disclosure thus provides packaging cells that produce infectious rAAV. In one embodiment packaging cells may be stably transformed cancer cells such as HeLa cells, 293 cells and PerC.6 cells (a cognate 293 line). In another embodiment, packaging cells are cells that are not transformed cancer cells, such as low passage 293 cells (human fetal kidney cells transformed with El 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).

[00106] The rAAV may be purified by methods standard in the art such as by column chromatography or cesium chloride gradients. Methods for purifying rAAV from helper vims are known in the art and include methods disclosed in, for example, Clark et al., Hum. Gene Ther., 10(6): 1031-1039 (1999); Schenpp and Clark, Methods Mol. Med., 69427-443 (2002); U.S. Patent No. 6,566,118 and WO 98/09657.

[00107] Thus, in another aspect, the disclosure contemplates a rAAV comprising a NTF3 polynucleotide. In some embodiments, the rAAV comprises AAV rh74 capsid and a NTF3 polynucleotide. In some embodiments, the genome of the rAAV lacks AAV rep and cap DNA. In some embodiments of the methods, the rAAV is rAAVrh7.4.tMCK.NTF3. In some embodiments, the rAAV is a self-complementary genome.

[00108] In another aspect, the disclosure contemplates compositions comprising a rAAV described herein. Compositions of the disclosure comprise rAAV in a pharmaceutically acceptable carrier. The compositions may also comprise other ingredients such as diluents. Acceptable carriers and diluents are nontoxic to recipients and are preferably inert at the dosages and concentrations employed, and include buffers such as phosphate, citrate, or other organic acids; antioxidants such as ascorbic acid; low molecular weight polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or 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, pluronics or polyethylene glycol (PEG). In some embodiments, the rAAV is formulated in Tris, MgCE_. NaCl and pluronic F68. In some embodiments, the rAAV is formulated in 20 mM Tris (pH 8.0), 1 mM MgCE and 200 mM NaCl containing 0.001% pluronic F68.

[00109] Combination treatments are also contemplated herein. Combinations as used herein include simultaneous treatment or sequential treatments. Combinations of methods of the disclosure with standard medical treatments (e.g., corticosteroids and/or immunosuppressive drugs) are specifically contemplated, as are combinations with novel treatments. In various embodiments, subjects are treated with corticosteroids before, during or after (or with any permutation of combinations of two or more of the three possibilities), the subject is treated according to a method contemplated herein. For example, the combinations include administering a corticosteroid, e.g. prednisolone, before, during and/or after administration of the rAAV.

[00110] Sterile injectable solutions are prepared by incorporating rAAV in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filter sterilization. Generally, dispersions are prepared by incorporating the sterilized active ingredient into a sterile vehicle which contains the 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, the preferred methods of preparation are vacuum drying and the freeze drying technique that yield a powder of the active ingredient plus any additional desired ingredient from the previously sterile-filtered solution thereof.

Stimulating Muscle Growth

[00111] One aspect of the disclosure provides a method of stimulating muscle growth in a subject with Charcot-Marie-Tooth axonal type 2D (CMT2D), comprising administering a therapeutically effective amount of neurotrophin-3 (NT-3), pro-NT-3, or an effective fragment thereof, or a nucleic acid encoding NT-3, pro-NT-3, or an effective fragment thereof; to a subject in need thereof

[00112] In some embodiments, the methods of the disclosure may be used to increase muscle strength, muscle mass, or muscle endurance and decrease muscle fatigue in a subject.

[00113] Muscle can be divided into three types: skeletal muscle, cardiac muscle, and smooth muscle. Skeletal muscle is muscle tissue capable of generating force and transferring that force to the skeleton enables breathing, movement, and posture maintenance. Cardiac muscle is muscle of the heart. Smooth muscle is muscle tissue of the arterial and bowel walls. The methods and compositions of the present disclosure apply primarily to skeletal muscle and, but may additionally positively affect smooth muscles. "Skeletal muscle" and "skeletal muscles" are defined as muscles with interactions with bones, tendons, and joints.

[00114] In some embodiments, the present disclosure provides a method of treatment of illnesses, diseases, disorders, and conditions that cause a decrease in muscle strength (also referred to herein as musculoskeletal diseases, and as muscle dysfunction and muscle-wasting diseases). The main categories of musculoskeletal diseases are muscular dystrophies and muscular atrophy.

[00115] In some embodiments, the disclosure provides methods for the treatment of musculoskeletal diseases, including muscle dysfunction and muscle-wasting diseases or disorders, including hereditary myopathy, neuromuscular disease, muscular atrophy, drug- induced. myopathy, or an illness, disease, disorder or condition that causes a decrease in muscle strength. The disclosure also provides for methods for the treatment CMT2D including the muscle atrophy and weakness exhibited by subjects having a mutation in the GARS gene. The method of treatment includes administering to a patient in need thereof a therapeutically effective amount of neurotrophin-3 (NT-3), pro-NT-3, or an effective fragment thereof, or a nucleic acid encoding NT-3, pro-NT-3, or an effective fragment thereof.

Neurotophin-3

[00116] In some embodiments, a therapeutically effective amount of NT-3, pro-NT-3, or an NT-3 analog thereof is administered to the subject to stimulate muscle growth. Neurotrophin 3 (NT-3) is a neurotrophic factor in the NGF (Nerve Growth Factor) family of neurotrophins. NT- 3 is a protein growth factor which has activity on certain neurons of the peripheral and central nervous system; it is best known for helping to support the survival and differentiation of existing neurons, and encourages the growth and differentiation of new neurons and synapses.

[00117] As used herein, the term "polypeptide" refers to an oligopeptide, peptide, or protein sequence, or to a fragment, portion, or subunit of any of these, and to naturally occurring or synthetic molecules. The term "polypeptide" also includes amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and may contain any type of modified amino acids, The term "polypeptide" also includes peptides and polypeptide fragments, motifs and the like, glycosylated polypeptides, all "mimetic" and "peptidomimctic" polypeptide forms, and retro-inversion peptides (also referred to as all-D-retro or mtro-enantio peptides).

[00118] "Substantially similar" means that a given amino acid (or nucleic acid) sequence shares at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with a reference sequence. In various embodiments, "substantially similar" means that a given amino acid (or nucleic acid) sequence shares at least 85%, more preferably at least 90%, and even more preferably at least 95% identity with a reference sequence. Identity or homology with respect to such sequences is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with the known peptides, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology, 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 shall not be construed as affecting homology.

[00119] Substantially similar peptides include those that differ by one or more amino acid alterations, where the alterations, e.g., substitutions, additions or deletions of amino acid residues, do not abolish the properties of the relevant peptides, such as their ability to associate with FAK or NANOG. Furthermore, only sequences describing or encoding proteins in which only conservative substitutions are made in the conserved regions are substantially similar overall. Preferable, substantially similar sequences also retain the distinctive activity of the poly peptide.

[00120] Examples of conservative substitutions include the substitution of a non-polar (hydrophobic) residue such as isoleucine, leucine, or methionine for another. Likewise, the present disclosure contemplates the substitution of one polar (hydrophilic) residue such as between arginine and lysine, between glutamine and asparagine, and between glycine and serine. Additionally, the substitution of a basic residue such as lysine, arginine or histidine for another or the substitution of one acidic residue such as aspartic acid or glutamic acid for another is also contemplated. Examples of non-conservative substitutions include the substitution of a non-polar ;hydrophobic) residue such as isoleucine, valine, leucine, alanine, methionine for a polar (hydrophilic) residues such as cysteine, glutamine, glutamic acid, lysine, and/or a polar residue for a non-polar residue.

[00121] The phrase "conservative substitution" also includes the use of chemically derivatized residues in place of a non-derivatized residues as long as the peptide retains the requisite ability to associate with NT-3. Substantially similar peptides also include the presence of additional amino acids or the deletion of one. or more amino acids which do not affect the requisite ability to associate with NT-3, For example, substantially similar peptides can contain an N- or C-. terminal cysteine, by which, if desired, the peptide may be covalently attached to a carrier protein, e.g., albumin. Such attachment can decrease clearing of the peptide from the blood and also decrease the rate of proteolysis of the peptides. In addition, for purposes of the present, disclosure, peptides containing D-amino acids in place of L-amino acids are also included in the term "conservative substitution." The presence of such D-isomers can. help minimize proteolytic activity and clearing of the peptide.

[00122] In some embodiments, a pro-neurotrophin-3 protein (pro-NT-3) is administered to the subject. The pro form of neurotrophin-3 is a -30 kDa precursor form of NT-3 which is converted to the mature NT by enzymatic cleavage and removal of a -15 kDa N-terminal prodomain. See Tauris et al., Eur. J Neurosci, 33(4), 622-631 (2011).

Treatment of Muscular Atrophy

[00123] The present disclosure provides a method of treating a subject having periperhal muscle atrophy. Pyruvate compounds can be used to provide prophylactic and/or therapeutic treatment. Pyruvate compounds can, for example, be administered prophylactically to a subject in advance of the occurrence of peripheral neuropathy. Prophylactic (i.e., preventive) administration is effective to decrease the likelihood of the subsequent occurrence of peripheral neuropathy in the subject, or decrease the severity of peripheral neuropathy that subsequently occurs. Prophylactic treatment may be provided to a subject that is at elevated risk of developing peripheral neuropathy, such as a subject with a family history of peripheral neuropathy.

[00124] Alternatively, the compounds of the disclosure can be administered therapeutically to a subject that is already afflicted by peripheral neuropathy. In one embodiment of therapeutic administration, administration of the compounds is effective to eliminate the peripheral neuropathy; in another embodiment, administration of the pyruvate compounds is effective to decrease the severity of the peripheral neuropathy or lengthen the lifespan of the subject so afflicted. In some embodiments, the method of treatment consists of administering a therapeutically effective amount of a pyruvate compound in a pharmaceutically acceptable formulation to the subject over a substantial period of time.

Administration and Formulation

[00125] The vector or peptide used with some embodiments of the present disclosure can be incorporated into pharmaceutical compositions suitable for administration to a subject. In some particular embodiments, the pharmaceutical composition comprises the vector of the disclosure and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. In many cases, it can be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Pharmaceutically acceptable carriers can further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the vector or pharmaceutical composition.

[00126] The vectors or peptides can be administered acutely (i.e., during the onset or shortly after events leading to muscular atrophy), or can be administered prophylactically (e.g., before scheduled surgery, or before the appearance of signs or symptoms), or administered during the course of muscular atrophy to reduce or ameliorate the progression of symptoms that would otherwise occur. The timing and interval of administration is varied according to the subject's symptoms, and can be administered at an interval of several hours to several days, over a time course of hours, days, weeks or longer, as would be determined by one skilled in the art.

[00127] The compositions containing the vectors or peptides are generally administered intravenously. When administered intravenously, the compositions may be combined with other ingredients, such as carriers and/or adjuvants. Peptides may also be covalently attached to a protein carrier, such as albumin, so as to minimize clearing of the peptides. There are no limitations on the nature of the other ingredients, except that such ingredients must be pharmaceutically acceptable, efficacious for their intended administration and cannot degrade the activity of the active ingredients of the compositions.

[00128] The pharmaceutical forms suitable for injection include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the ultimate solution form must be sterile and fluid. Typical carriers include a solvent or dispersion medium containing, for example, water buffered aqueous solutions (i.e., biocompatible buffers), ethanol, polyols such as glycerol, propylene glycol, polyethylene glycol, suitable mixtures thereof, surfactants or vegetable oils. Sterilization can be accomplished by any art-recognized technique, including but not limited to, filtration or addition of antibacterial or antifungal agents, for example, paraben, chlorobutano, phenol, sorbic acid or thimerosal. Further, isotonic agents such as sugars or sodium chloride may be incorporated in the subject compositions.

[00129] Production of sterile injectable solutions containing the subject peptides is accomplished_by incorporated these compounds in the required amount in the appropriate solvent with various_ingredients enumerated above, as required, followed by sterilization, preferably filter_sterilization. To obtain a sterile powder, the above solutions are vacuum-dried or freeze- dried as_necessary.

[00130] When the peptides of the disclosure are administered orally, the pharmaceutical compositions thereof containing an effective dose of the peptide can also contain an inert diluent, as assimilable edible carrier and the like, be in hard or soft shell gelatin capsules, be compressed into tablets, or may be in an elixir, suspension, syrup or the like. The subject peptides are thus compounded for convenient and effective administration in pharmaceutically effective amounts with a suitable pharmaceutically acceptable carrier in a therapeutically effective amount.

[00131] The expressions "effective amount" or "therapeutically effective amount," as used herein, refers to a sufficient amount of agent to stimulate muscle growth or decrease 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 particular therapeutic agent, its mode and/or route of administration, and the like. It will be understood, however, that the total daily usage of the compounds and compositions of the present disclosure can be decided by an attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject or organism will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific composition employed; the duration of the treatment; drugs used in combination or coincidental with the specific composition employed; and like factors well known in the medical arts.

[00132] The vectors or peptides can be administered in a manner compatible with the dosage formulation and in such amount as well be therapeutically effective. Systemic dosages depend on the age, weight and conditions of the patient and on the administration route. For example, a suitable dose of peptide for. the administration to adult humans ranges from about 0.001 to about 20.0 mg per kilogram of body weight. The peptides should preferably be administered in an amount of at least about 50 mg per dose, more preferably in an amount up to about 500 mg to about 1 gram per dose. Since the peptide compositions of this disclosure will eventually be cleared from the bloodstream, re-administration of the compositions is indicated and preferred.

Examples

[00133] Thus, aspects and embodiments of the disclosure are illustrated by the following examples. Additional data relating to the scAAV1.tMCK.NTF3 vector described in the examples below is provided in International Application No. PCT/US2018/056765, which is incorporated by reference. However, there are a wide variety of other embodiments within the scope of the present disclosure, which should not be limited to the particular examples provided herein. Example 1

NT-3 Delivery using scAAV1.tMCK.NTF3

[00134] The composition administered is a non-replicating recombinant adeno-associated virus termed scAAV1.tMCK.NTF3, a diagram of the cassette plasmid of which is shown in Figure 2. The rAAV contains the human NTF3 gene under the control of a tMCK muscle- specific promoter. In vivo biopotency was tested following the intramuscular injection of the rAAV (1x10¹¹ vg) into gastrocnemius muscles of C57B16 mice followed by quantification of circulating NT-3 in the serum by ELISA at 4 to 6 weeks after gene injection.

[00135] Firstly it was demonstrated that scAAV1.CMV.NTF3 delivered to gastrocnemius muscle produced prolonged and therapeutic NT-3 blood levels sufficient to provide functional, electrophysiological and histopathological improvement in TrJ nerves. It was then investigated if it was possible to produce the required rAAVdose and achieve same level of expression by packaging the expression cassette by scAAV1. A dose- response study was performed on C57BL/6 mice comparing serum NT-3 ELISA data following intramuscular injection of scAAV1.tMCK.NTF3 and scAAV1.CMV.NTF3 at 3 doses (3 x 10⁹ vg, 1 x 10¹⁰ vg and 3 x 10¹⁰ vg). Adminstraton of scAAV1.CMV.NTF3 at 1x10¹¹ vg produced significantly higher NT-3 levels than the single-stranded rAAV at the same dose consistent with greater potency using selfcomplementary vectors. At a half-log less dose (3 x 10¹⁰ vg), rAAV comprising either CMV and tMCK produced comparable NT-3 serum levels to those obtained from mice that received scAAV1. CMV.NTF3 at 1x10¹¹ vg dose, which produced a biological response. The NT-3 levels (mean ± SEM) were measured from TrJ mice at 24 weeks post injection. There is significant difference in NT-3 levels among all 7 groups, p value<0.0001. A significant difference in NT-3 levels was observed for highest and intermediate doses of rAAVs for both promoters and control. However, analysis failed to find significant difference for lower doses for both rAAV. Kruskal- Wallis test is used to compare serum NT-3 among all groups (PBS, CMV 3E+09/1E+10/3E+10 and tMCK 3E+09/1E+10/3E+10). Mann-Whitney U test is used to compare NT-3 between each group and PBS (control) group, and Bonferroni correction is used to adjust for multiple comparisons. See Sahenk et al., Mol. Ther. 22(3): 511-521, 2014, which is incorporated by reference herein in its entirety.

[00136] Muscle diameter increases at 40 weeks posttreatment: The effects of NT-3 gene therapy was assessed in TrJ mice upon muscle fiber size at 40 weeks postinjection in a subset of animals injected with scAAV1.CMV.NTF3 (1 x 10¹¹ vg) compared to PBS. Neurogenic changes characterized by atrophic angular fibers and group atrophy were evident in the muscles from untreated mice while evidence for reinnervation as fiber type groupings and an overall fiber size increase were recognizable as treatment effect. Muscle fiber size histograms generated from contralateral anterior and posterior compartment muscles of the left lower limb (tibialis anterior and gastrocnemius) showed an increase in fiber diameter.

[00137] Additional studies have shown that NT-3 stimulates Akt/mTOR pathway in SCs cells giving rise to improved myelination and radial growth of axons in the nerve and NT-3 also has a direct stimulatory effect on myotubes through Trk-C receptors indicating its role in fiber diameter increase in muscles of TrJ mice. Figures 3A-3B show that NT-3 increased the phosphorylation of Akt (P-Akt) and mTOR targets, 4EBP-1 (P-4EBP1) and PS6K (P-S6K) in SC and myotube cultures. These studies provide evidence for the choice of anterior and posterior muscles of the lower leg for rAAV delivery in this clinical trial.

Studies with Self-complementary (sc) AAV1 and the Use of a Muscle Specific Truncated Creatine Kinase ttMCK) Promoter

[00138] scAAV permits lower dosing that adds up to enhanced safety and dosing levels that will meet production standards. The use of tMCK promoter is a valued objective again offering greater safety by avoiding off target effects. In the following set of experiments the efficacy of scAAV1.NTF3 under control of the CMV promoter was compared to the muscle specific tMCK promoter both given at three doses, within a half-log range (3 x 10⁹ vg, 1 x 10¹⁰ vg and 3 x 10¹⁰ vg). The efficacy AAV1.NTF3 gene transfer in TrJ mice peripheral nerves were assessed by electrophysiological (Table 2) and morphological studies 24 weeks post gene transfer. The evidence of transgene expression was assessed by measuring serum NT-3 levels using ELISA (Fig. IB). [00139] Table 2. CMAP and Conduction Velocity in the TrJ Sciatic Nerve.

[00140] The examiner during electrodiagnostic studies was blinded to the treatment groups. There is no statistical difference between AAV1.NTF3.CMV (high dose, HD) and AAVl.NTF3.tMCK (high dose, HD) on CMAP and it was preferred to use the muscle specific tMCK promoter. This is further supported by the NT-3 levels in ELISA Assay where a significant difference in NT-3 levels was observed for highest and intermediate doses of rAAV for both promoters and control.

Example 2

Construction of NT-3 Expressing AAV Construct

[00141] Design of self-complementary rAAV viral vectors with serotype 1 containing NTF3 cDNA under tMCK was described previously in Sahenk et al., Mol Ther, 22(3):511-521 (2014), which is incorporated by reference herein in its entirety. Aliquoted viruses were kept in -80°C until use. Blood samples were collected from treated and non-treated mice by eye bleeding under anesthesia at 6 and 16 weeks post injection and serum was assayed for NT-3 levels using a capture ELISA (Fig. IB). The construct is referred to herein as scAAV1.tMCK.NTF3 (Fig. 1A). [00142] A tMCK promoter/enhancer sequence was used to drive muscle-specific gene expression and is composed of the muscle creatine kinase promoter with an added enhancer element (enh358MCK, 584-bp) fused to it. A triple tandem of the MCK enhancer (206-bp) was ligated to the 87-bp basal promoter in the tMCK promoter/enhancer.

[00143] The scAAV1.tMCK.NTF3 drug product was produced by 3 plasmid DNA transfection of human HEK293 Master Cell Bank cells with: (i) the pAAV.tMCK.NTF3- vector plasmid (see Figure 2), (ii) an AAV1 helper plasmid termed R88/C1 containing the AAV rep2 and Capl wild-type genes and (iii) the helper adenovirus plasmid

[00144] A schematic representation of the plasmid with molecular features and open reading frames is shown in Figure 2. The rAAV genome derived from pAAV.tMCK.NTF3 plasmid is a self-complementary DNA genome containing the human NTF3 cDNA expression cassette flanked by AAV2 inverted terminal repeat sequences (ITR). It is this sequence that is encapsulated into AAV1 virions. Plasmid pAAV.tMCK.NTF3 was constructed by inserting the tMCK expression cassette driving a NTF3 gene sequence into the AAV cloning vector psub201. The human NTF3 gene is expressed from the mouse triple tandem MCK promoter which is a modification of the previously described CK6 promoter and contains a triple E box sequence.

An SV40 polyadenylation signal is used for efficient transcription termination. The cassette also contains a chimeric intron for increased gene expression and is composed of the 5' donor site from the first intron of the human b-globin gene and the branchpoint and 3 ' splice acceptor site from the intron that is between the leader and the body of an immunoglobulin gene heavy chain variable region. The NTF3 expression cassette has a consensus Kozak immediately in front of the ATG start and 200 bp SV40 polyA signal for efficient mRNA termination. The NTF3 cDNA is included in its entirety (NCBI Reference Sequence: NM_001102654). The only viral sequences included in this vector are the inverted terminal repeats of AAV2, which are required for both viral DNA replication and packaging. The AAV ITRs are sequences that are nearly identical on both ends, but in opposite orientation. The “left” (mutated) ITR has the terminal resolution site deleted to allow hairpin formation of the genome. The identity of all DNA plasmid elements are confirmed by DNA plasmid sequencing on the plasmid source stock.

[00145] Shown in Table 3 are the base pair locations of relevant molecular features within the rAAV vector DNA plasmid of SEQ ID NO: 3.

Example 3

NT-3 Gene Therapy in a CMT2D Model Gars^P278KY

[00146] Glycyl tRNA synthetase (GARS) mutations are associated with CMT2D. The mouse model Gars^p278KY (also referred to as GarsNmf^49/+ ) represents severe early onset CMT2D with findings including reduced axon size and number, slow conduction velocities, abnormal neuromuscular junction (NMJ) morphology and muscle denervation^{2, 3}.

Experimental cohorts

[00147] The efficacy of scAAV1.tMCK.NTF3 gene therapy was tested in 4-6 weeks old Gars^{p278KY +} mice (n=10, both genders) via intramuscular injection of rAAV at lE+11 vg dose, divided in equal doses between the gastrocnemius muscles. Untreated age and sex matched Gars^p278KY mice served as controls (n=6). Mice were sacrificed 12 weeks post-injection. The outcome measures were evaluated using well established protocols to include rotarod test for assessment of motor function and coordination, sciatic nerve electrophysiology and nerve and muscle histopathology (morphometric evaluation of myelinated fiber size and densities and G ratios at the mid-sciatic level, analysis of neuromuscular junction status using immunohistochemistry in whole mount lumbrical muscles and quantitative assessment of muscle fiber size and internal nuclei status in gastrocnemius muscle). The serum NT-3 levels were also determined at endpoint using a capture ELISA as previously reported in Sahenk et al. (Mol. Therapy 22:511-521, 2014) and are provided in Fig. IB. Functional study-Rotarod test

[00148] Rotarod test was performed twice, at 4 weeks-post gene injection as initial evaluation and at the endpoint; the test included an acclimation run followed by three test runs.

[00149] Acclimation Run: The mice were placed on the rotarod which was set at a speed of 5 rpm, with an acceleration of 0.2 rpm/s. The time at which each mouse fell from the rod was recorded. If the mouse turned to the wrong direction, fell from any human error, or failed to reach a minimum 15 seconds of run, the mouse was rerun at the end. If the mouse was never able to reach 15 seconds, data of that mouse was not used. After at least 30 seconds of rest and recover time, the mice were run again to reach three runs in total.

[00150] Test Runs and Data Interpretation: The mice were let to recover for at least 24 hours after the acclimation day. The test runs were performed as given in the acclimation run protocol. If any mice failed to run for 15 seconds minimum or fell due to turning around, they were run fourth time to replace the data from that incomplete run. If the mouse had more than two failures, data of that mouse was not included in the final data. The average of the two best runs was calculated for each mouse and used for further analyses.

[00151] Results: Rotarod analyses indicated a significant improvement (33%) in the treated mice compared to the untreated cohort at the endpoint (p=0.0035) when mice reached 17-18 weeks of age, three months after NT-3 gene transfer (NT-3: 71.54 sec, n=12 vs. UT: 47.94 sec, n=8; p= 0.0008). There was no statistical difference between UT and NT-3 treated cohorts at baseline (47.06 sec vs 56.31 sec, p=0.30). Compared to the initial evaluation at 4 weeks post- injection (56.31 ± 5.54 seconds), the endpoint performance in the scAAV1.NTF3 group increased 27% (p=0.044), while performance of the untreated group remained unchanged (p=0.90). Over a 12 week-time period, the performance of the treated mice improved (56.31 sec vs 71.54 sec, p=0.044) while in the UT mice no change was observed (47.06 sec vs 47.94 sec, p=0.90). See Figure 5.

[00152] Improvements in the rotarod test in the treated group were associated with clinical observations of improved toe spreading (Fig. 6A-6D). Sciatic nerve conduction studies in Gars^P278KY/+ mice performed at the endpoint. NCV was significantly improved with treatment corresponding to a 42% increase compared to UT cohort (p=0.024) (Fig. 7A). CMAP was also increased in the NT-3 cohort compared to UT (p=0.013) (Fig. 7B). Representative waveforms of the sciatic nerve motor nerve conduction from UT and NT-3 cohorts are shown; base time is 2 ms for left, 1ms for right panel (Fig. 1C). Data represented as mean ± SEM, unpaired t-test.

Electrophysiology studies

[00153] Electrophysiological studies were done in all cohorts at endpoint. Mice under isoflurane anesthesia were placed on a heating pad to maintain body temperature around 37°C. Left sciatic nerve conduction studies was obtained using a Nicolet Viasys Viking Select EMG EP System (USA) and disposable subdermal needle recording electrodes (for both stimulation and recording). A pair of stimulating electrodes were first positioned at the distal location medial of the gastroc muscle. A pair of recording electrodes are positioned in the foot pad and parallel to the gastroc. For the first test run, the starting stimulation was at 3-5 mV for duration of 0.1ms. which was later increased until receiving highest amplitude and clearest take off. The location of the stimulating electrodes was marked. The stimulating electrodes was then moved about 10 mm up the sciatic nerve proximally (sciatic notch). Another test was run to find the best location that provides maximum distance between locations, free of stimulation artifacts. The distance between the two stimulation locations was measured. There were 4 total markers in the program. The marker tool was used to indicate the following points in the recording: the first two markers were at initiation of the response, followed by maximum positive peak, and the last marker was at the return to resting position. Returned results were latency, Compound Muscle Action Potential (CMAP) amplitude, negative area under curve, duration, and motor nerve conduction velocity (NCV).

[00154] Sciatic nerve conduction outcome studies supported the efficacy of NT-3 gene transfer therapy. The NCVs were significantly improved with treatment resulting in a 42% increase in the sciatic NCV compared to the untreated cohort (Fig. 7 A). Mean CMAP amplitude was also increased significantly in the AAV1.NT-3 treated Gars^P278KY/+ mice (Fig. 7B, 1C), correlating with functional outcome.

Histopatholosical Studies

[00155] Mice were perfused via cardiac approach with 4% paraformaldehyde followed by 3% glutaraldehyde or paraformaldehyde alone to prior to removal of sciatic nerves, lumbar and sacral spinal cord segments, lumbar dorsal root ganglia and lumbrical muscles for appropriate further processing using well established protocols for plastic embedding or immunohistochemistry. Gastroc and tibialis anterior muscles were removed immediately prior to perfusion for obtaining fresh frozen muscle samples.

[00156] Immunohistochemical Analysis of neuromuscular junctions (NMJ). The Gars^P278KY/+ mice were reported to have abnormal neuromuscular junction morphology³.

Immunofluorescence protocol was adapted from ^4,5. Lumbrical muscles were fixed in 2% PFA in 0.1 M phosphate buffer (pH 7.3) at 4°C overnight. After incubating in PBS for one hour, tissues were blocked overnight at 4°C in blocking buffer (1% Triton X-100, 4% BSA in PBS). Tissues were then incubated in primary antibodies in blocking solution (Acetylchloline receptor antibody, a-Bungarotoxin, T1175, 1:500; Anti-Neurofilament 200 antibody, N4142, 1:500; SV2 antibody, AB_2315387, 1:50) for at least 24 hours, followed by washing at least three times in PBS. Next, the tissueswere incubated in secondary antibodies in blocking buffer for 24 hours (Alexa Fluor 488-conjugated anti-rabbit and anti-mouse secondary IgG, 1:500), followed by washing at least three times in PBS. Tissues were placed between two slides and pressed with a binder clip for ten minutes. Samples were mounted in anti-fade mounting media and imaged using a confocal microscope. Images were interpreted as follows: NMJs were termed as innervated when nerve completely overlapped the acetylchloline receptors (AChRs), as partially innervated when some parts of the AChRs were not overlapping with nerve and as denervated when there was not any nerve co-localizing with AChRs3.

[00157] Using the parameters described, more than 200 NMJs derived from six mice for each cohort were analyzed. This analysis showed that NT-3 treatment resulted a 14.9% increase in the innervated NMJs (Figure 8 or Figure 15D).

Sciatic Nerves

[00158] Light microscopic examination of the 1μm-thick, plastic embedded and toluidine blue stained cross sections from sciatic nerves in the scAAV1.NTF3 injected mice revealed an increase of axon diameter size along with an increase of myelin thickness which were notable for the large fiber population. Representative lpm-thick, toluidine blue stained cross sections of sciatic nerves from scAAV1.NTF3 treated and untreated Gars^{P278KY/+ /+} mice were analyzed. An increase of axon diameter size and myelin thickness, notable for the large fiber population, was observed. Figure 9 demonstrates that compared to WT (Fig. 9A-C), peripheral nerves from Gars^P278KY/+ mice showed strikingly small axon size along with an increase in the density of MFs as illustrated in samples from ventral roots, mid sciatic, and distal tibial nerves (Fig. 9D-F). [00159] To assess the efficacy of NT-3 gene therapy, it was important to have a detailed understanding of the disease process according to the anatomical site of involvement in peripheral nerves of the Gars^P278KY/+ model. One possibility is that the neuropathic process may be a length-dependent distal axonopathy causing axonal loss in distal nerves (“dying back” process), a feature of many neuropathic conditions including classical CMT phenotype. To investigate this, detailed quantification of MFs (MF density per unit area of endoneurium, MF- axon size distribution, and actual MF number per nerve) at mid- sciatic (proximal) and tibial nerves (distal) from Gars mutants and age-matched WT controls were carried out and provided in Tables 4 and 5 below.

Table 4: Analysis of myelinated fibers in sciatic and tibial nerves or Gars^P278KY/+ and Gars^ΔETAQ/+ mice and age matched WT mice

Table 5: Myelinated fiber density in the sciatic and tibial nerves from Gars^P278KY/+ and wild type mice.

[00160] In WT nerves, compared to sciatic nerve (proximal), the endoneurial cross-sectional area-decrease at the tibial level was about 70% and the mean density of MFs was 1.5 times higher than that of the proximal level. This density increase can be explained by the fact that in WT, axon size decreases along the nerve from proximal-distal direction²⁹. In Gars^P278KY/+ nerves, we found significantly smaller endoneurial cross-sectional areas; there was also an increase in MF density about 2.6 fold at the sciatic level and 2 fold at the tibial level compared to WT nerves (Tables 4 and 5). Interestingly, however, the decrease in endoneurial cross-sectional area at tibial level (about 54%) was not as prominent as in WT, and proximal to distal MF density change was not significant, 1.15 times higher than that of the proximal level (Fig. 9G, depicting morphometric values from Table 4). The analysis of proximo-distal MF-axon size distribution in WT sciatic nerves show a wide range of axon size distribution with diameters >6 pm, constituting 48% of all fibers; in the tibial nerve, this subpopulation was only 7%, confirming previous observations of decrease in proximodistal axon size as described in Medori et al. (Proc. Natl. Acad. Sci. USA 8:7716-7720,1985) (Fig. 10). In Gars^P278KY/+ nerves, however, due to a much narrower distribution of axon size and the absence of large-diameter axons, the proximodistal axon size change and resulting MF density increase appeared less pronounced. Moreover, the total number of MFs in the tibial nerve from Gars^P278KY/+ was not statistically different than that was obtained from the WT tibial nerve (Table 4 and 5). In accordance with these qualitative data as well as our microscopic observations on the tibial nerve and intramuscular nerve bundles of lumbrical muscles, which showed no evidence of acute Wallerian degeneration or myelin ovoids, it was concluded that there is no length dependent MF loss in Gars^P278KY/+ mice in the age group studied. In the presence of well-defined abnormal NMJ morphology (Seburn et al. Neuron 51:715-726, 2006, Spaulding et al. J. Neurosci. 36:3254-3267. 2016) and the current findings described herein, the disease process in this model can best be classified as a distal terminal axonopathy.

[00161] The axon size distribution of the myelinated fiber histograms confirmed this observation showing an increase in the number of myelinated fibers with diameter >3 μm corresponding to 29.2% of the total fiber population while the same subgroup constituted 17.3% in the untreated mice (Error! Reference source not found.11, data derived from n=4 mice, 2 females and two males in each group; approximately 1000 measurements were obtained from 0.02 mm² cross sectional area in each mice using Bioquant 2014 software).

[00162] Similar improvements in the axon diameter and myelin thickness were observed in the roots as well as the distally in the tibial nerves. Examination of the peripheral nerve samples from roots to intramuscular nerve bundles did not reveal findings of ongoing axonal degeneration suggesting a distal terminal axonopathy as the underlying pathologic process. Axonal sprouting was not observed.

[00163] G-ratio analysis : G- ratio is a measure used to quantify myelin thickness typically determined by dividing the inner diameter by the outer diameter of individual fibers, however, this can only be accurately measured when the fibers are circular. To remediate, myelin interior and exteriors were outlined in Axiovision (AxioVs40x64 V 4.9.1.0) to determine area, which was used to derive diameters to yield g-ratio, similar to the methods used in MRI estimation^6,7.

[00164] Results: Data was scatter-plotted, and the slopes of scAAV1.NTF3 treated vs untreated were compared in Graphpad (Graphpad Prism 8.2.0). G-ratio determinations in sciatic nerves showed an increase of myelin thickness in treated mice with treated (n=4) and untreated groups (n=4) having significantly different slopes of scatterplots (Linear regression analysis: p<0.0001, Figure 12E). Treated mice showed trends toward optimal peripheral nervous system G-ratio of 0.6^{8, 9} (Table 6, Figure 13).

[00165] Table 6. Average G-ratios of Gars^P278KY/+ mice.

Muscle histopathology

[00166] Muscle fiber diameter analysis: Histological analyses were performed on skeletal muscles to analyze myofiber size changes in the treated and untreated Gars^P278KY/+ mice. Hematoxylin and eosin (H&E) staining was used to measure fiber diameter. H&E was performed on 12 μm-thick sections cut from fresh frozen muscle tissues. Three random images representing inner, intermediate and outer parts of the muscle were taken under 20X magnification. Using Axiovision (AxioVs40x64 V 4.9.1.0), diameters were taken by measuring the shortest distance between the sarcolemma and at a position that the fiber was split into equal parts. Due to the large amount of fiber shape possibilities, the fiber diameters were determined based on the previous criteria and adjusted to best fit the shape. Data from all three images were combined to complete a data set for each individual mouse. In addition, the fibers with central nuclei (s) were quantified.

[00167] In summary, gastrocnemius muscles from treated mice (n=4) displayed a significant decrease in the small fiber population (with diameter < 10 pm) which was 11% of total fibers while in the untreated group (n=4) this population constituted 31%. The right-skewed distribution of fiber size in the untreated group shifted to a normal Gaussian distribution (A total of 2603 fibers in the untreated and 2386 fibers in the treated mice were analyzed). Treatment also resulted in a decrease of fibers with central nuclei (Figures 16M and 16N).

[00168] Intramuscular delivery of scAAV1.NTF3 in the mouse model Gars^P278KY/+ at 12 weeks post treatment resulted significant functional and electrophysiological improvements compared to the untreated control cohort. This was supported with quantitative histopathology showing increases in axon size and myelin thickness and improvements in the denervated status of NMJs as well as muscle fiber diameter size.

[00169] These data shows that scAAV1.NTF3 gene therapy has clear disease modifying effects in a mouse model for CMT2D and provides additional support for the utility of scAAV1.NTF3 for gene therapy in patients having CMT2D.

Example 4

NT-3 Gene Therapy in a CMT2D Model Gars ^ΔETAQ/+

[00170] The initial characterization of Gars^ΔETAQ/+ mice have been described previously (Seburn et al. Neuron 51:715-726, 2006; Morelli et al, J. Clin. Invest. 129:5568-5583, 2019). Heterozygous Gars^ΔETAQ/+ mice were outbred with C57BL/6 mice and heterozygous offspring were used. Genotypes were established by PCR analysis of genomic DNA isolated from tail clips. Eight- to ten- weeks old Gars^ΔETAQ/+ (6 females and 5 males, n=l 1) were injected with 1x10 ¹ vg (supercoil titer) of scAAV1.tMCK.NT-3 vector equally divided into right and left gastrocnemius muscles. Age-matched Gars^ΔETAQ/+ (5 females and 7 males, n=12) mice injected with Ringer’s lactate served as controls. Over-dosage of xylazine/ketamine anesthesia was used to euthanize Gars^ΔETAQ/+ mice at twelve weeks-, and Gars^ΔETAQ/+ mice at 30 weeks-post gene treatment.

[00171] The efficacy of scAAV1.tMCK.NT-3 delivery in the Gars^ΔETAQ/+ mutant mice was assessed when mice reached 10 (38-40 weeks) months of age, at 30 weeks post gene delivery. Rotarod performance, CMAP, and sciatic NCV values in the treated cohort were not statistically different from the untreated, which might be related to the milder manifestation of the phenotype in Gars^ΔETAQ/+ mice (Fig. 15). Quantitative studies carried out in Gars^ΔETAQ/+ mice, which were similar to those described in Example 3, revealed that MF density increase per unit area of peripheral nerves was less conspicuous compared to Gars^P278KY/+ mice. Example 5

NT-3 Improved Myelin Thickness in GARS Mutant Mice

[00172] The following paragraphs are a further discussion on the cross sections of the nerves in Gars^P278KY/+ mice and provide analysis of Gars ^ΔETAQ/+ mice. Examination of 1 pm thick cross- sections from roots, sciatic and tibial nerves revealed an apparent increase in myelin thickness in the scAAV1.tMCK.NT-3 injected Gars^P278KY/+ and Gars^ΔETAQ/+ mice compared to the untreated samples (Fig. 12A-D). At 12-week post NT-3 gene transfer, g ratio (axon diameter/fiber diameter) of the myelinated fibers in the sciatic nerves from Gars^P278KY/+ mice showed an increase in myelin thickness corroborating the electrophysiological studies (Fig. 12E). The average g ratio in the Ringer’s lactate injected Gars^P278KY/+ mice (0.715 ± 0.002) is significantly greater than that obtained from age-matched WT data (0.61 ± 0.002, p<0.0001), reflecting the presence of thinner myelin in this model. In the AAVl.tMCK.NT-3 injected Gars^P278KY/+ mice, the g ratio was significantly reduced ( AAV 1. NT-3: 0.649 ± 0.002, vs. untreated: 0.715 ± 0.002, p< 0.0001) and the percent of fibers with g ratio greater than 0.6 was down to 31% which constituted about 59% of MFs in the untreated group (Fig. 12F). G ratio values obtained from Gars^ΔETAQ/+ mutant (Gars ^ΔETAQ/+: 0.691 ± 0.003 vs. Gars^P278KY/+ : 0.715 ± 0.002; p<0.0001), confirmed the microscopic observations that the extent of hypomyelination is more severe in the Gars^P278KY/+ mice. At 6 months post- AAV l.tMCK.NT-3 vector injection in the Gars^ΔETAQ/+ mice the myelin thickness showed an increase for the axon size (Fig. 12G) with significantly reduced g ratio (AAV1.NT-3: 0.638 ± 0.003 vs. untreated: 0.691 ± 0.003, p<0.0001) and the percent fibers with g ratio greater than 0.6 reduced down to 23.4% which constituted 48% of the MF population in the untreated cohort (Fig. 12H).

[00173] MF axon-size distribution histograms from sciatic nerves of Gars^P278KY/+ and Gars^ΔETAQ/+ mice showed NT-3 gene therapy had no effect on MF-axon size or number per unit area compared to the untreated cohort (Figure 13A-C).

Example 6

Efficacy of NT-3 gene transfer therapy in muscles of Gars^P278KY/+ and Gars ^ΔETAQ/+ mice NT-3 improved neuromxovathx in Gars ^P278KY/+ and Gars ^ΔETAQ/+ mice

[00174] Muscles from Gars^P278KY/+ mice at postnatal day 10 revealed overall uniformly small fibers with mild size variability and rare small fibers with central nuclei as previously shown (Fig. 16E). Muscles from older mice of both mutants, however, displayed marked fiber size variability (Fig. 16F, G), atrophic angular (denervated) fibers, rare fibers undergoing necrosis, and exceedingly small fibers with prominent central nuclei, compatible with necrosis/regeneration cycles, illustrated here in the Gars^P278KY/+ mutant (Fig. 16H-J). It is proposed that that these small round fibers with diameters around 5 pm displaying prominent central nuclei are reminiscent of myotubes and their occurrence in small clusters may suggest impaired fusion events. Fiber size distribution histograms from Gars^P278KY/+ muscle revealed that over 30% of fibers had diameters <10 pm while in the milder Gars^ΔETAQ/+ phenotype this subpopulation was much smaller (Fig. 16F, G).

[00175] With NT-3 treatment, we found notable improvements in muscle histopathology in both mutants with apparent decreases in fiber subpopulations composed of abnormally small or hypertrophied ones (Fig. 16K, L). In the gastrocnemius muscle from Gars^P278KY/+ mice, the smallest fiber population (with diameter <10 pm), constituting 33% of the total in the untreated cohort decreased down to 9.4% with NT-3 gene therapy (Fig. 16M). In addition, the NT-3 gene transfer resulted in a significant decrease in muscle fibers with internal nuclei (Fig. 16N). Further analysis upon gender stratification revealed that the largest percentage of small fibers is in the untreated males (Fig. 17). Collectively, these findings show that the muscle pathology developed later in life in these Gars mutants is compatible with an ongoing neuromyopathic process and that NT-3 gene therapy attenuates these histopathological findings significantly.

Decreased oxidative phosphorylation markers in mutant Gars muscle is reversed with NT-3 sene therapy

[00176] Muscle histochemistry using SDH and COX reactions in the untreated Gars^P278KY/+ mice revealed severely reduced SDH and COX activities in fibers, most prominent at the superficial zones of the gastrocnemius muscle, which are predominantly composed of type 2 or fast-twitch glycolytic fibers. Interestingly, there were numerous fibers with generous mitochondria content giving rise to the appearance of ragged blue/brown in SDH and COX stains respectively (Fig. 18A-D). Compared to Gars^P278KY/+ muscle, these findings were less prominent in the Gars^ΔETAQ/+ mutant. GAS muscle from 10-month old Gars^ΔETAQ/+ mice showed hypertrophied fibers with decreased SDH activity, fibers with focal areas of loss of COX activity, or ragged brown fibers (Fig. 18E, F). A reversal of these findings was observed with AAV 1. NT-3 gene therapy as illustrated in muscles from Gars^P278KY/+ mice at 3 months post gene delivery at the superficial and deep zones of gastrocnemius muscle in Fig. 19A, B (Fig. 19C represents WT gastrocnemius muscle).

[00177] Abnormalities in muscle enzyme histochemistry in Gars mutants suggested that mitochondrial dysfunction may play a role in muscle phenotype. To explore this further, the expression levels of C 0X1 and COX3 were measured, which are mtDNA encoded subunits of cytochrome c oxidase of respiratory complex IV and Atp5d. Real-time qPCR in untreated Gars^P278KY/+ muscle (female and male combined) showed that COX1 and COX 3 transcripts were approximately half and 1/3^rd the levels of age-matched WT controls, respectively (Fig. 19D, E). Atp5d transcripts were also significantly low, down to 1/6^th of the WT (Fig. 19F). No sex difference for these transcripts was observed in the Gars^P278KY/+ mutant (Fig. 19D-F). In WT, however, COX1 transcripts were found higher in females while males had 4.3-fold higher Atp5d transcripts than females (Fig 19F). Interestingly, compared to WT females, Atp5d expression in female Gars^P278KY/+ muscle did not change; in contrast, males showed significantly low expression, 1/5^th of that in WT males. Most notably, the reversal of reduced COX staining in Gars^P278KY/+ muscle (Fig. 19A, B) was dramatically reflected in COX1, COX3, and Atp5d expression levels following NT-3 gene transfer showing over 70, 40, and 50-fold increases in these transcripts respectively, without sex difference (Fig. 19D-F). Interestingly, the mitochondria copy number appeared normalized with treatment showing a pattern observed in WT, males having higher mitochondria content than females (Fig. 6G).

[00178] Similar studies conducted in the untreated Gars^ΔETAQ/+ muscle at 10 months of age, showed prominent decreases in relative expression levels of COX1, COX3, and Atp5d without sex influence compared to age-matched WT muscle (Fig. 6H-J). Interestingly, Atp5d transcripts from aged WT muscles did not show gender difference favoring males as seen in young WT muscle at 4 months of age although there was a trend for males having higher expression levels than females without reaching statistical difference (Fig. 6F, J). No sex difference for mitochondria content in Gars^ΔETAQ/+ muscle or age-matched/old WT muscle was noted (Fig. 6K). Collectively, these findings indicate that the primary myopathic process in these Gars mutants is associated with decreased oxidative phosphorylation markers compatible with mitochondria dysfunction. Moreover, we found that NT-3 gene therapy resulted in normalization, a reversal of mitochondria content of muscle towards WT patterns for both males and females. Example 7

Defective Myoblast Fusion in Gars^P278KY/+ Mice

[00179] The histopathological studies provided strong clues that myotubes expressing dominant GARS mutation display impaired fusion events and altered development of multinucleated myotubes. To explore this directly, myoblasts isolated from Gars^P278KY/+ mice muscles and WT controls were induced to differentiate to form in vitro myotubes. Three days after the induction, the myotubes were stained with an anti-MHC antibody and a nuclear stain to count the number of nuclei that had been incorporated into multinucleated myotubes. Overall, a large fraction of Gars^P278KY/+ myotubes were shorter, ovoid shape with single nucleus or multiple clumped nuclei instead of the elongated shape seen in WT cells (Fig. 20A, B). A significant reduction of fusion competence between WT (86.1%) and Gars^P278KY/+ (65.8%) myoblasts (p< 0.00001; Fig. 20C) was observed. In addition, shorter Gars^P278KY/+ myotubes on average had more nuclei than WT myotubes; the opposite relationship was present for the longer WT myotubes having more nuclei than the mutant (Fig. 20D). Taken together, myoblast fusion assays demonstrate that both Gars^P278KY/+ mature myocytes and myoblast progenitors are fusion defective. This defect likely contributes to the muscle phenotype in the Gars^P278KY/+ model.

[00180] The complete disclosure of all patents, patent applications, and publications, and electronically available material cited herein are incorporated by reference. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The disclosure is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the disclosure defined by the claims.

[00181] While the present disclosure has been described in terms of specific embodiments, it is understood that variations and modifications will occur to those skilled in the art.

Accordingly, only such limitations as appear in the claims should be placed on the disclosure.

References

1. Antonellis A, Ellsworth RE, Sambuughin N, et al. Glycyl tRNA synthetase mutations in Charcot-Marie-Tooth disease type 2D and distal spinal muscular atrophy type V. American journal of human genetics 2003;72:1293-1299.

2. Liao YC, Liu YT, Tsai PC, et al. Two Novel De Novo GARS Mutations Cause Early- Onset Axonal Charcot-Marie-Tooth Disease. PloS one 2015;10:e0133423. 3. Sebum KL, Nangle LA, Cox GA, Schimmel P, Burgess RW. An active dominant mutation of glycyl-tRNA synthetase causes neuropathy in a Charcot-Marie-Tooth 2D mouse model. Neuron 2006;51:715-726.

4. Lin W, Burgess RW, Dominguez B, Pfaff SL, Sanes JR, Lee KF. Distinct roles of nerve and muscle in postsynaptic differentiation of the neuromuscular synapse. Nature 2001;410:1057- 1064.

5. Valdez G, Heyer MP, Feng G, Sanes JR. The role of muscle microRNAs in repairing the neuromuscular junction. PLoS One 2014;9:e93140.

6. West KL, Kelm ND, Carson RP, Does MD. A revised model for estimating g-ratio from MRI. Neuroimage 2016;125:1155-1158.

7. Mohammadi S, Carey D, Dick F, et al. Whole-Brain In-vivo Measurements of the Axonal G-Ratio in a Group of 37 Healthy Volunteers. Front Neurosci 2015;9:441.

8. Chomiak T, Hu B. What is the optimal value of the g-ratio for myelinated fibers in the rat CNS? A theoretical approach. PLoS One 2009;4:e7754.

9. Sahenk Z, Galloway G, Edwards C, et al. TrkB and TrkC agonist antibodies improve function, electrophysiologic and pathologic features in Trembler J mice. Exp Neurol 2010;224:495-506.

SEQUENCE LISTING

<110> The Research Institute at Nationwide Children's Hospital

<120> TREATMENT OF CHARCOT -MARIE-TOOTH AXONAL TYPE 2D USING NT-3 GENE THERAPY

<130> 28335/55415P1

<160> 12

<170> Patentln version 3.5

<210> 1 <211> 774 <212> DNA <213> Homo sapiens

<220>

<221> misc_feature <223> hNTF3

<400> 1 atgtccatct tgttttatgt gatatttctc gcttatctcc gtggcatcca aggtaacaac 60 atggatcaaa ggagtttgcc agaagactcg ctcaattccc tcattattaa gctgatccag 120 gcagatattt tgaaaaacaa gctctccaag cagatggtgg acgttaagga aaattaccag 180 agcaccctgc ccaaagctga ggctccccga gagccggagc ggggagggcc cgccaagtca 240 gcattccagc cggtgattgc aatggacacc gaactgctgc gacaacagag acgctacaac 300 tcaccgcggg tcctgctgag cgacagcacc cccttggagc ccccgccctt gtatctcatg 360 gaggattacg tgggcagccc cgtggtggcg aacagaacat cacggcggaa acggtacgcg 420 gagcataaga gtcaccgagg ggagtactcg gtatgtgaca gtgagagtct gtgggtgacc 480 gacaagtcat cggccatcga cattcgggga caccaggtca cggtgctggg ggagatcaaa 540 acgggcaact ctcccgtcaa acaatatttt tatgaaacgc gatgtaagga agccaggccg 600 gtcaaaaacg gttgcagggg tattgatgat aaacactgga actctcagtg caaaacatcc 660 caaacctacg tccgagcact gacttcagag aacaataaac tcgtgggctg gcggtggata 720 cggatagaca cgtcctgtgt gtgtgccttg tcgagaaaaa tcggaagaac atga 774

<210> 2 <211> 270 <212> PRT <213> Homo sapiens

<220>

<221> MIS C_FE ATURE <223> NT-3 amino acid sequence

<400> 2

Met Val Thr Phe Ala Thr lie Leu Gin Val Asn Lys Val Met Ser lie 1 5 10 15

Leu Phe Tyr Val lie Phe Leu Ala Tyr Leu Arg Gly Ile Gin Gly Asn 20 25 30

Asn Met Asp Gin Arg Ser Leu Pro Glu Asp Ser Leu Asn Ser Leu Ile 35 40 45 lie Lys Leu Ile Gin Ala Asp Ile Leu Lys Asn Lys Leu Ser Lys Gin

50 55 60

Met Val Asp Val Lys Glu Asn Tyr Gin Ser Thr Leu Pro Lys Ala Glu

65 70 75 80

Ala Pro Arg Glu Pro Glu Arg Gly Gly Pro Ala Lys Ser Ala Phe Gin

85 90 95

Pro Val lie Ala Met Asp Thr Glu Leu Leu Arg Gin Gin Arg Arg Tyr

100 105 110

Asn Ser Pro Arg Val Leu Leu Ser Asp Ser Thr Pro Leu Glu Pro Pro

115 120 125

Pro Leu Tyr Leu Met Glu Asp Tyr Val Gly Ser Pro Val Val Ala Asn

130 135 140

Arg Thr Ser Arg Arg Lys Arg Tyr Ala Glu His Lys Ser His Arg Gly

145 150 155 160

Glu Tyr Ser Val Cys Asp Ser Glu Ser Leu Trp Val Thr Asp Lys Ser

165 170 175

Ser Ala Ile Asp Ile Arg Gly His Gin Val Thr Val Leu Gly Glu Ile

180 185 190

Lys Thr Gly Asn Ser Pro Val Lys Gin Tyr Phe Tyr Glu Thr Arg Cys

195 200 205

Lys Glu Ala Arg Pro Val Lys Asn Gly Cys Arg Gly lie Asp Asp Lys

210 215 220

His Trp Asn Ser Gin Cys Lys Thr Ser Gin Thr Tyr Val Arg Ala Leu

225 230 235 240 Thr Ser Glu Asn Asn Lys Leu Val Gly Trp Arg Trp lie Arg Ile Asp 245 250 255

Thr Ser Cys Val Cys Ala Leu Ser Arg Lys lie Gly Arg Thr 260 265 270

<210> 3 <211> 5884 <212> DNA

<213> Artificial Sequence <220>

<223> Synthetic Polynucleotide

<220>

<221> misc_feature

<223> scpAAVl.tMCK.NTF3 plasmid genome full Sequence <400> 3 cagcagctgc gcgctcgctc gctcactgag gccgcccggg caaagcccgg gcgtcgggcg 60 acctttggtc gcccggcctc agtgagcgag cgagcgcgca gagagggagt ggggttaacc 120 aattggcggc cgcaaacttg catgccccac tacgggtcta ggctgcccat gtaaggaggc 180 aaggcctggg gacacccgag atgcctggtt ataattaacc ccaacacctg ctgccccccc 240 ccccccaaca cctgctgcct gagcctgagc ggttacccca ccccggtgcc tgggtcttag 300 gctctgtaca ccatggagga gaagctcgct ctaaaaataa ccctgtccct ggtggatcca 360 ctacgggtct atgctgccca tgtaaggagg caaggcctgg ggacacccga gatgcctggt 420 tataattaac cccaacacct gctgcccccc cccccccaac acctgctgcc tgagcctgag 480 cggttacccc accccggtgc ctgggtctta ggctctgtac accatggagg agaagctcgc 540 tctaaaaata accctgtccc tggtggacca ctacgggtct aggctgccca tgtaaggagg 600 caaggcctgg ggacacccga gatgcctggt tataattaac cccaacacct gctgcccccc 660 ccccccaaca cctgctgcct gagcctgagc ggttacccca ccccggtgcc tgggtcttag 720 gctctgtaca ccatggagga gaagctcgct ctaaaaataa ccctgtccct ggtcctccct 780 ggggacagcc cctcctggct agtcacaccc tgtaggctcc tctatataac ccaggggcac 840 aggggctgcc cccgggtcac ctgcagaagt tggtcgtgag gcactgggca ggtaagtatc 900 aaggttacaa gacaggttta aggagaccaa tagaaactgg gcttgtcgag acagagaaga 960 ctcttgcgtt tctgataggc acctattggt cttactgaca tccactttgc ctttctctcc 1020 acaggtgtcc actcccagtt caattacagc gcgtggtacc tgcagggata tccaccatgt 1080 ccatcttgtt ttatgtgata tttctcgctt atctccgtgg catccaaggt aacaacatgg 1140 atcaaaggag tttgccagaa gactcgctca attccctcat tattaagctg atccaggcag 1200 atattttgaa aaacaagctc tccaagcaga tggtggacgt taaggaaaat taccagagca 1260 ccctgcccaa agctgaggct ccccgagagc cggagcgggg agggcccgcc aagtcagcat 1320 tccagccggt gattgcaatg gacaccgaac tgctgcgaca acagagacgc tacaactcac 1380 cgcgggtcct gctgagcgac agcaccccct tggagccccc gcccttgtat ctcatggagg 1440 attacgtggg cagccccgtg gtggcgaaca gaacatcacg gcggaaacgg tacgcggagc 1500 ataagagtca ccgaggggag tactcggtat gtgacagtga gagtctgtgg gtgaccgaca 1560 agtcatcggc catcgacatt cggggacacc aggtcacggt gctgggggag atcaaaacgg 1620 gcaactctcc cgtcaaacaa tatttttatg aaacgcgatg taaggaagcc aggccggtca 1680 aaaacggttg caggggtatt gatgataaac actggaactc tcagtgcaaa acatcccaaa 1740 cctacgtccg agcactgact tcagagaaca ataaactcgt gggctggcgg tggatacgga 1800 tagacacgtc ctgtgtgtgt gccttgtcga gaaaaatcgg aagaacatga ggcggccgcg 1860 gggatccaga catgataaga tacattgatg agtttggaca aaccacaact agaatgcagt 1920 gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta accattataa 1980 gctgcaataa acaagttaac aacaacaatt gcattcattt tatgtttcag gttcaggggg 2040 aggtgtggga ggttttttcg gcgcgcctct agagcatggc tacgtagata agtagcatgg 2100 cgggttaatc attaactaca aggaacccct agtgatggag ttggccactc cctctctgcg 2160 cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg 2220 ggcggcctca gtgagcgagc gagcgcgcca gctggcgtaa tagcgaagag gcccgcaccg 2280 atcgcccttc ccaacagttg cgcagcctga atggcgaatg gaattccaga cgattgagcg 2340 tcaaaatgta ggtatttcca tgagcgtttt tcctgttgca atggctggcg gtaatattgt 2400 tctggatatt accagcaagg ccgatagttt gagttcttct actcaggcaa gtgatgttat 2460 tactaatcaa agaagtattg cgacaacggt taatttgcgt gatggacaga ctcttttact 2520 cggtggcctc actgattata aaaacacttc tcaggattct ggcgtaccgt tcctgtctaa 2580 aatcccttta atcggcctcc tgtttagctc ccgctctgat tctaacgagg aaagcacgtt 2640 atacgtgctc gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg 2700 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt 2760 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc 2820 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg 2880 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga 2940 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc 3000 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa 3060 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa 3120 tttaaatatt tgcttataca atcttcctgt ttttggggct tttctgatta tcaaccgggg 3180 tacatatgat tgacatgcta gttttacgat taccgttcat cgattctctt gtttgctcca 3240 gactctcagg caatgacctg atagcctttg tagagacctc tcaaaaatag ctaccctctc 3300 cggcatgaat ttatcagcta gaacggttga atatcatatt gatggtgatt tgactgtctc 3360 cggcctttct cacccgtttg aatctttacc tacacattac tcaggcattg catttaaaat 3420 atatgagggt tctaaaaatt tttatccttg cgttgaaata aaggcttctc ccgcaaaagt 3480 attacagggt cataatgttt ttggtacaac cgatttagct ttatgctctg aggctttatt 3540 gcttaatttt gctaattctt tgccttgcct gtatgattta ttggatgttg gaattcctga 3600 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatatgg tgcactctca 3660 gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg 3720 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct 3780 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg 3840 gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt 3900 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac 3960 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa 4020 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat 4080 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc 4140 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga 4200 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg 4260 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc 4320 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag 4380 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc 4440 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg 4500 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg 4560 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac 4620 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac 4680 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg 4740 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg 4800 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg 4860 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac 4920 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg 4980 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg 5040 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc 5100 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc 5160 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt 5220 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc 5280 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact 5340 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac 5400 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag 5460 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg 5520 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg 5580 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga 5640 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt 5700 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct 5760 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg 5820 aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt 5880 aatg 5884

<210> 4 <211> 106 <212> DNA

<213> Artificial Sequence <220>

<223> Synthetic Polynucleotide

<220>

<221> misc_feature <223> 5'ITR

<400> 4 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtgg 106 <210> 5 <211> 133 <212> DNA

<213> Artificial Sequence <220>

<223> Synthetic Polynucleotide

<220>

<221> misc_feature <223> Chimeric Intron

<400> 5 gtaagtatca aggttacaag acaggtttaa ggagaccaat agaaactggg cttgtcgaga 60 cagagaagac tcttgcgttt ctgataggca cctattggtc ttactgacat ccactttgcc 120 tttctctcca cag 133

<210> 6 <211> 5 <212> DNA

<213> Artificial Sequence <220>

<223> Synthetic Polynucleotide

<220>

<221> misc_feature <223> Kozak Sequence

<400> 6 ccacc 5

<210> 7 <211> 200 <212> DNA

<213> Artificial Sequence <220>

<223> Synthetic Polynucleotide <220>

<221> misc_feature <223> POLY A sequence

<400> 7 ggggatccag acatgataag atacattgat gagtttggac aaaccacaac tagaatgcag 60 tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt aaccattata 120 agctgcaata aacaagttaa caacaacaat tgcattcatt ttatgtttca ggttcagggg 180 gaggtgtggg aggttttttc 200

<210> 8 <211> 128 <212> DNA

<213> Artificial Sequence <220>

<223> Synthetic Polynucleotide

<220>

<221> misc_feature <223> 3'ITR

<400> 8 aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 60 ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc 120 gagcgcgc 128

<210> 9 <211> 2248 <212> DNA

<213> Artificial Seuqence <220>

<223> Synthetic Polynucleotide

<220>

<221> misc_feature

<223> AAVl.tMCK.NTF3 genome sequence <400> 9 cagcagctgc gcgctcgctc gctcactgag gccgcccggg caaagcccgg gcgtcgggcg 60 acctttggtc gcccggcctc agtgagcgag cgagcgcgca gagagggagt ggggttaacc 120 aattggcggc cgcaaacttg catgccccac tacgggtcta ggctgcccat gtaaggaggc 180 aaggcctggg gacacccgag atgcctggtt ataattaacc ccaacacctg ctgccccccc 240 ccccccaaca cctgctgcct gagcctgagc ggttacccca ccccggtgcc tgggtcttag 300 gctctgtaca ccatggagga gaagctcgct ctaaaaataa ccctgtccct ggtggatcca 360 ctacgggtct atgctgccca tgtaaggagg caaggcctgg ggacacccga gatgcctggt 420 tataattaac cccaacacct gctgcccccc cccccccaac acctgctgcc tgagcctgag 480 cggttacccc accccggtgc ctgggtctta ggctctgtac accatggagg agaagctcgc 540 tctaaaaata accctgtccc tggtggacca ctacgggtct aggctgccca tgtaaggagg 600 caaggcctgg ggacacccga gatgcctggt tataattaac cccaacacct gctgcccccc 660 ccccccaaca cctgctgcct gagcctgagc ggttacccca ccccggtgcc tgggtcttag 720 gctctgtaca ccatggagga gaagctcgct ctaaaaataa ccctgtccct ggtcctccct 780 ggggacagcc cctcctggct agtcacaccc tgtaggctcc tctatataac ccaggggcac 840 aggggctgcc cccgggtcac ctgcagaagt tggtcgtgag gcactgggca ggtaagtatc 900 aaggttacaa gacaggttta aggagaccaa tagaaactgg gcttgtcgag acagagaaga 960 ctcttgcgtt tctgataggc acctattggt cttactgaca tccactttgc ctttctctcc 1020 acaggtgtcc actcccagtt caattacagc gcgtggtacc tgcagggata tccaccatgt 1080 ccatcttgtt ttatgtgata tttctcgctt atctccgtgg catccaaggt aacaacatgg 1140 atcaaaggag tttgccagaa gactcgctca attccctcat tattaagctg atccaggcag 1200 atattttgaa aaacaagctc tccaagcaga tggtggacgt taaggaaaat taccagagca 1260 ccctgcccaa agctgaggct ccccgagagc cggagcgggg agggcccgcc aagtcagcat 1320 tccagccggt gattgcaatg gacaccgaac tgctgcgaca acagagacgc tacaactcac 1380 cgcgggtcct gctgagcgac agcaccccct tggagccccc gcccttgtat ctcatggagg 1440 attacgtggg cagccccgtg gtggcgaaca gaacatcacg gcggaaacgg tacgcggagc 1500 ataagagtca ccgaggggag tactcggtat gtgacagtga gagtctgtgg gtgaccgaca 1560 agtcatcggc catcgacatt cggggacacc aggtcacggt gctgggggag atcaaaacgg 1620 gcaactctcc cgtcaaacaa tatttttatg aaacgcgatg taaggaagcc aggccggtca 1680 aaaacggttg caggggtatt gatgataaac actggaactc tcagtgcaaa acatcccaaa 1740 cctacgtccg agcactgact tcagagaaca ataaactcgt gggctggcgg tggatacgga 1800 tagacacgtc ctgtgtgtgt gccttgtcga gaaaaatcgg aagaacatga ggcggccgcg 1860 gggatccaga catgataaga tacattgatg agtttggaca aaccacaact agaatgcagt 1920 gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta accattataa 1980 gctgcaataa acaagttaac aacaacaatt gcattcattt tatgtttcag gttcaggggg 2040 aggtgtggga ggttttttcg gcgcgcctct agagcatggc tacgtagata agtagcatgg 2100 cgggttaatc attaactaca aggaacccct agtgatggag ttggccactc cctctctgcg 2160 cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg 2220 ggcggcctca gtgagcgagc gagcgcgc 2248

<210> 10 <211> 668 <212> DNA

<213> Artificial Sequence <220>

<223> Synthetic Polynucleotide

<220>

<221> misc_feature <223> pBR322 Ori

<400> 10 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg 60 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact 120 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg 180 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg 240 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac 300 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca 360 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga 420 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc 480 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct 540 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg 600 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct 660 tttgctca 668

<210> 11 <211> 714 <212> DNA

<213> Artificial Sequence <220>

<223> Synthetic Polynucleotide

<220>

<221> misc_feature <223> tMCK Promoter

<400> 11 ccactacggg tctaggctgc ccatgtaagg aggcaaggcc tggggacacc cgagatgcct 60 ggttataatt aaccccaaca cctgctgccc cccccccccc aacacctgct gcctgagcct 120 gagcggttac cccaccccgg tgcctgggtc ttaggctctg tacaccatgg aggagaagct 180 cgctctaaaa ataaccctgt ccctggtgga tccactacgg gtctatgctg cccatgtaag 240 gaggcaaggc ctggggacac ccgagatgcc tggttataat taaccccaac acctgctgcc 300 cccccccccc caacacctgc tgcctgagcc tgagcggtta ccccaccccg gtgcctgggt 360 cttaggctct gtacaccatg gaggagaagc tcgctctaaa aataaccctg tccctggtgg 420 accactacgg gtctaggctg cccatgtaag gaggcaaggc ctggggacac ccgagatgcc 480 tggttataat taaccccaac acctgctgcc cccccccccc aacacctgct gcctgagcct 540 gagcggttac cccaccccgg tgcctgggtc ttaggctctg tacaccatgg aggagaagct 600 cgctctaaaa ataaccctgt ccctggtcct ccctggggac agcccctcct ggctagtcac 660 accctgtagg ctcctctata taacccaggg gcacaggggc tgcccccggg tcac 714

<210> 12 <211> 861 <212> DNA

<213> Artificial Sequence <220>

<223> Synthetic Polynucleotide

<220>

<221> misc_feature <223> AMP R Sequence

<400> 12 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct 60 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca 120 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc 180 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc 240 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg 300 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta 360 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc 420 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt 480 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg 540 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct 600 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc 660 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct 720 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac 780 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc 840 tcactgatta agcattggta a 861

Claims

WHAT IS CLAIMED:

1. A method of treating Charcot-Marie-Tooth axonal type 2D (CMT2D) in a human subject in need thereof comprising the step of administering to the human subject a nucleic acid encoding a NT-3 polypeptide; wherein a) the nucleic acid comprises a nucleotide sequence that is at least 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 encoding an amino acid sequence that is at least 90% identical to SEQ ID NO: 2; or d) the nucleic acid comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2.

2. The method of claim 1, wherein the nucleic acid encoding the NT-3 polypeptide is operatively linked to a muscle- specific promoter.

3. The method of claim 2, wherein the muscle- specific promoter is muscle-specific creatine kinase promoter (MCK).

4. The method of claim 3, wherein the muscle creatine kinase promoter has the nucleotide sequence set out in SEQ ID NO: 11.

5. The method of any one of claims 1-4, wherein the nucleic acid is administered using a viral vector.

6. The method of claim 5, wherein the viral vector is a recombinant adeno- associated virus (rAAV).

7. The method of claim 6, wherein the rAAV capsid serotype is AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV- 10, AAV-11, AAV12, AAV13, Anc80, AAV-B1, AAVrh.10, or AAVrh.74.

8. The method of claim 7, wherein the rAAV capsid serotype is AAV-1.

9. The method of any one of claims 6-8, wherein the rAAV genome sequence comprises in order from 5' to 3':

(i) a first AAV2 inverted terminal repeat sequence (ITR);

(ii) a muscle creatine kinase promoter/enhancer sequence set out in nucleotides 147-860 of SEQ ID NO: 3;

(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 is 100% identical to SEQ ID NO: 2, or is encoded by a nucleotide sequence at least 90% identical to nucleotides 1077-1850 of SEQ ID NO: 3 or 100% identical to nucleotides 1077-1850 of SEQ ID NO: 3.

10. The method of claim 9, wherein the nucleic acid sequence further comprises 3’ to said promoter/enhancer, a chimeric intron set out in nucleotides 892-1024 of SEQ ID NO: 3.

11. The method of claims 9 or 10, wherein the nucleic acid sequence further comprises 3’ to said nucleotide sequence encoding a human NT-3 polypeptide, a SV40 polyadenylation signal set out in nucleotides 1860-2059 of SEQ ID NO: 3.

12. The method of any one of claims 9-11, wherein said first ITR is set out in nucleotides 7-112 of SEQ ID NO: 3, and/or said second ITR is set out in nucleotides 2121-2248 of SEQ ID NO: 3.

13. The method of any one of claims 1-12, wherein the nucleic acid comprises the scAAV1.tMCK.NTF3 rAAV genome that is at least 90% identical to SEQ ID NO: 9.

14. The method of any one of claims 1-13, wherein the nucleic acid comprising the scAAV1.tMCK.NTF3 genome is set out in SEQ ID NO: 9.

15. The method of any one of claims 1-14, wherein the nucleic acid is administered at a dose that results in sustained expression of a low concentration of NT-3 polypeptide.

16. The method of any one of claims 1-15, wherein the route of administration is intramuscular injection.

17. The method of any one of claims 1-16, wherein the subject has a mutation in the

GARS gene or a gene encoding an aminoacyl-tRNA synthetase.

18. The method of any one of claims 1-17, wherein the route of administration is intramuscular bilateral injection to the medial and lateral head of the gastrocnemius and tibialis anterior muscle.

19. The method of any one of claims 1-18, wherein administration of the nucleotide sequence encoding a NT-3 polypeptide improves muscle strength in the upper or lower extremities in the subject.

20. The method of any one of claims 1-19, wherein the improvement in the muscle strength is measured as a decrease in composite score on the CMT Pediatric Scale (CMTPeds) or as a decrease in disease progression over a two year time period.

21. The method of any one of claims 1-20, wherein sciatic nerve conduction velocity is increased by 1-100%.

22. Use of a nucleic acid encoding an NT-3 polypeptide in the manufacture of a medicament for treating Charcot-Marie-Tooth axonal type 2D (CMT2D) in a human subject, wherein: a) the nucleic acid comprises a nucleotide sequence that is at least 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 encoding an amino acid sequence that is at least 90% identical to SEQ ID NO:2; or d) the nucleic acid comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2.

23. The use of claim 22, wherein the nucleic acid encoding the NT-3 polypeptide is operatively linked to a muscle- specific promoter.

24. The use of claim 23, wherein the muscle-specific promoter is muscle- specific creatine kinase promoter (MCK).

25. The use of claim 24, wherein the muscle creatine kinase promoter has the nucleotide sequence set out in SEQ ID NO: 11.

26. The use of any one of claims 22-25, wherein the nucleic acid is in a viral vector.

27. The use of claim 26, wherein the viral vector is a recombinant adeno-associated virus (rAAV).

28. The use of claim 27, wherein the rAAV capsid serotype is AAV-1, AAV-2, AAV- 3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV- 10, AAV-11, AAV12, AAV13, Anc80, AAV-B1, AAVrh.10, or AAVrh.74.

29. The use of claim 28, wherein the rAAV capsid serotype is AAV-1.

30. The use of any one of claims 27-29, wherein the the rAAV genome sequence comprises in order from 5' to 3':

(i) a first AAV2 inverted terminal repeat sequence (ITR);

(iii) a nucleotide sequence encoding a human NT-3 polypeptide; and

31. The use of claim 30, wherein the nucleic acid sequence further comprising 3’ to said promoter/enhancer, a chimeric intron set out in nucleotides 892-1024 of SEQ ID NO: 3.

32. The use of claims 30 or 31, wherein the nucleic acid sequence further comprising 3’ to said nucleotide sequence encoding a human NT-3 polypeptide, a SV40 polyadenylation signal set out in nucleotides 1860-2059 of SEQ ID NO: 3.

33. The use of any one of claims 30-32, wherein said first ITR is set out in nucleotides 7-112 of SEQ ID NO: 3, and/or said second ITR is set out in nucleotides 2121-2248 of SEQ ID NO: 3.

34. The use of any one of claims 22-33, wherein the nucleic acid comprises the scAAV1.tMCK.NTF3 rAAV genome that is at least 90% identical to the nucleotide sequence set out in SEQ ID NO: 3.

35. The use of any one of claims 22-34, wherein the nucleic acid comprising the scAAV1.tMCK.NTF3 genome set out in SEQ ID NO: 9.

36. The use of any one of claims 22-35, wherein the nucleic acid is administered at a dose that results in sustained expression of a low concentration of NT-3 polypeptide.

37. The use of any one of claims 22-36, wherein the medicament is formulated for administration by intramuscular injection.

38. The use of any one of claims 22-37, wherein the subject has a mutation in the

GARS gene or a gene encoding an aminoacyl-tRNA synthetase.

39. The use of any one of claims 22-38, wherein the medicament is formulated for administration by intramuscular bilateral injection to the medial and lateral head of the gastrocnemius and tibialis anterior muscle.

40. The use of any one of claims 22-39, wherein administration of the medicament improves the muscle strength improved in the subject is in the upper or lower extremities.

41. The use of any one of claims 22-40, wherein the improvement in the muscle strength is measured as a decrease in composite score on the CMT Pediatric Scale (CMTPeds) or as a decrease in disease progression over a two year time period.

42. The use of any one of claims 22-41, wherein sciatic nerve conduction velocity is increased by 1-100%.

43. A viral vector for use in the treatment of Charcot-Marie-Tooth axonal type 2D (CMT2D) in a human subject, wherein the viral vector comprises a nucleic acid encoding an NT- 3 polypeptide, wherein the nucleic acid comprises: a) a nucleotide sequence that is at least 90% identical to the nucleotide sequence of SEQ ID NO: 1; b) the nucleotide sequence of SEQ ID NO: 1; c) a nucleotide sequence encoding an amino acid sequence that is at least 90% identical to SEQ ID NO: 2; or d) a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2.

44. The viral vector of claim 43, wherein the nucleic acid encoding the NT-3 polypeptide is operatively linked to a muscle-specific promoter.

45. The viral vector of claim 44, wherein the muscle- specific promoter is muscle- specific creatine kinase promoter (MCK).

46. The viral vector of claim 45, wherein the muscle creatine kinase promoter has the nucleotide sequence set out in SEQ ID NO: 11.

47. The viral vector of any one of claims 43-46, wherein the nucleic acid is in a viral vector.

48. The viral vector of claim 47, wherein the viral vector is a recombinant adeno- associated virus (rAAV).

49. The viral vector of claim 48, wherein the rAAV capsid serotype is AAV-1, AAV- 2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV12, AAV13, Anc80, AAV-B1, AAVrh.10, or AAVrh.74.

50. The viral vector of claim 49, wherein the rAAV capsid serotype is AAV-1.

51. The viral vector of any one of claims 48-50, wherein the rAAV genome sequence comprises in order from 5' to 3':

(i) a first AAV2 inverted terminal repeat sequence (ITR);

(iii) a nucleotide sequence encoding a human NT-3 polypeptide; and

52. The viral vector of claim 51, wherein the nucleic acid sequence further comprising 3’ to said promoter/enhancer, a chimeric intron set out in nucleotides 892-1024 of SEQ ID NO: 3.

53. The viral vector of claims 51 or 52, wherein the nucleic acid sequence further comprising 3’ to said nucleotide sequence encoding a human NT-3 polypeptide, a SV40 polyadenylation signal set out in nucleotides 1860-2059 of SEQ ID NO: 3.

54. The viral vector of any one of claims 51-53, wherein said first ITR is set out in nucleotides 7-112 of SEQ ID NO: 3, and/or said second ITR is set out in nucleotides 2121-2248 of SEQ ID NO: 3.

55. The viral vector of any one of claims 43-54, wherein the nucleic acid comprises the scAAV1.tMCK.NTF3 rAAV genome that is at least 90% identical to the nucleotide sequence set out in SEQ ID NO: 3.

56. The viral vector of any one of claims 43-55, wherein the nucleic acid comprising the scAAV1.tMCK.NTF3 genome set out in SEQ ID NO: 9.

57. The viral vector of any one of claims 43-56, wherein the nucleic acid is administered at a dose that results in sustained expression of a low concentration of NT-3 polypeptide.

58. The viral vector of any one of claims 43-57, wherein the viral vector is formulated for administration by intramuscular injection.

59. The viral vector of any one of claims 43-58, wherein the subject has a mutation in the GARS gene or a gene encoding an aminoacyl-tRNA synthetase.

60. The viral vector of any one of claims 43-59, wherein viral vector is formulated for administration byintramuscular bilateral injection to the medial and lateral head of the gastrocnemius and tibialis anterior muscle.

61. The viral vector of any one of claims 43-60, wherein administration of the viral vector improves the muscle strength improved in the subject is in the upper or lower extremities.

62. The viral vector of any one of claims 43-61, wherein the improvement in the muscle strength is measured as a decrease in composite score on the CMT Pediatric Scale (CMTPeds) or as a decrease in disease progression over a two year time period.

63. The viral vector of any one of claims 43-62, wherein sciatic nerve conduction velocity is increased by 1-100%.