Treatment of multiple sclerosis using NT-3 gene therapy

Abstract

This disclosure relates to a method for treating autoimmune diseases, such as multiple sclerosis, using rAAV-expressing NT-3 gene therapy. This disclosure provides a method for treating autoimmune multiple sclerosis. The method comprises administering a therapeutically effective dose of neurotrophin-3 (NT-3 or NTF-3, these terms used interchangeably in this application), pro-NT-3, or an effective fragment thereof, or nucleic acids encoding NT-3, pro-NT-3, or an effective fragment thereof, to a subject having multiple sclerosis or other autoimmune disease.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the priority of U.S. Provisional Application No. 63 / 463,501, filed on May 2, 2023, the entire disclosure of which is incorporated herein by reference.

[0002] Incorporation by Reference of Electronically Submitted Materials A computer - readable nucleotide / amino acid sequence listing, filed simultaneously with this specification and identified as follows, is incorporated herein by reference in its entirety: 59101_SeqListing.xml, size: 23,953 bytes, created on May 1, 2024.

[0003] The present disclosure relates to methods of using rAAV - expressing NT - 3 in gene therapy for treating multiple sclerosis. The present disclosure also relates to methods of using rAAV - expressing NT - 3 in gene therapy for treating autoimmune diseases.

Background Art

[0004] Multiple sclerosis (MS) is a common autoimmune demyelinating disease that affects over 2.5 million patients worldwide. Also known as disseminated encephalomyelitis, MS is an autoimmune disease in which the patient's immune system attacks the myelin of nerves in the brain and spinal cord, ultimately causing demyelination of the nerves that leads to secondary axonal loss. Unfortunately, the main cause of MS remains mostly unknown, and there is currently no cure for the disease.

[0005] Autoimmune diseases are conditions resulting from an abnormal immune response against functioning parts of the body, such as cells, tissues, and / or organs. At least 80 types of autoimmune diseases have been identified, and there is some evidence suggesting that there may be more than 100 types. Almost any part of the body can be involved. Common symptoms can be varied and transient, ranging from mild to severe, and generally include a low-grade fever and fatigue. As with MS, the cause of autoimmune diseases is unknown. Some autoimmune diseases, such as lupus, are hereditary, and certain cases can be triggered by infection or other environmental factors. Treatment depends on the type and severity of the condition. Nonsteroidal anti-inflammatory drugs (NSAIDs) and immunosuppressants are often used. Intravenous immunoglobulins may also be used occasionally. These treatments usually improve symptoms but typically do not cure the disease.

[0006] Recent studies have demonstrated that neurotrophin 3 (NT-3) is a versatile molecule with previously unknown or underestimated characteristics. In addition to its well-recognized effects on peripheral nerve regeneration and Schwann cells (SCs), NT-3 possesses anti-inflammatory and immunomodulatory effects (Yang et al., Mel Titer, 22(2):440-450 (2014)). NT-3 has recently been shown to attenuate spontaneous autoimmune peripheral polyneuropathy in a rodent model of chronic inflammatory demyelinating peripheral neuropathy occurring in humans (Yalvac et al., Gene therapy, 23(1):95-102 (2015)).

[0007] NT-3 is a trophic factor secreted by Schwann cells (SCs) that support nerve regeneration. SCs provide both growth factors and a scaffold that promotes the basal layer and axonal growth, so the ability of denervated SCs to survive is essential for nerve regeneration. Prolonged denervation results in reduced regenerative capacity associated with reduced expression of regeneration-related SC molecules (neurotrophic factors (NTFs) and their receptors), leading to atrophy of denervated SCs, division of Büngner's band, and loss of the SC basal layer scaffold.

[0008] Adeno-associated virus (AAV) is a replication-deficient parvovirus whose single-stranded DNA genome is approximately 4.7 kb long and contains a 145-nucleotide terminal inversion (ITR). Multiple serotypes of AAV exist. The nucleotide sequences of the serotype genomes are publicly known. For example, the complete genome of AAV-1 is provided to GenBank accession number NC_002077, the complete genome of AAV-2 is provided to GenBank accession number NC_001401 and Srivastava et al., J. Virol., 45:555-564 (1983), the complete genome of AAV-3 is provided to GenBank accession number NC_1829, the complete genome of AAV-4 is provided to GenBank accession number NC_001829, the genome of AAV-5 is provided to GenBank accession number AF085716, the complete genome of AAV-6 is provided to GenBank accession number NC_001862, at least portions of the genomes of AAV-7 and AAV-8 are provided to GenBank accession numbers AX753246 and AX753249 respectively, and the genome of AAV-9 is provided to Gao et al. The AAV-10 genome is provided in al., J. Virol., 78:6381-6388 (2004), the AAV-10 genome in Mol. Ther., 13(1):67-76 (2006), and the AAV-11 genome in Virology, 330(2):375-383 (2004). The Cis action sequence, which directs viral DNA replication (rep), capsid formation / packaging, and host cell chromosome integration, is contained within the AAV ITR. Three AAV promoters (named p5, p19, and p40 relative to their relative map locations) drive the expression of two AAV internal open reading frames encoding the rep and cap genes. Coupled with differential splicing of a single AAV intron (at nucleotides 2107 and 2227), two rep promoters (p5 and p19) produce four rep proteins (rep78, rep68, rep52, and rep40) from the rep gene. The rep proteins possess multiple enzymatic properties that ultimately contribute to the replication of the viral genome.The cap gene is expressed from the p40 promoter and encodes three capsid proteins: VP1, VP2, and VP3. Alternative splicing and non-consensus translation initiation sites are involved in the production of the three related capsid proteins. A single-consensus polyadenylation site is located at map position 95 of the AAV genome. The life cycle and genetics of AAV are outlined in Muzyczka, Current Topics in Microbiology and Immunology, 158:97-129 (1992).

[0009] AAV possesses unique characteristics that make it attractive, for example, as a vector for delivering foreign DNA to cells in gene therapy. AAV infection of cells in culture is non-cellular, and natural infection in humans and other animals is silent and asymptomatic. Furthermore, AAV infects many mammalian cells, allowing for the potential to target many different tissues in vivo. Additionally, AAV can transduce slow-dividing and non-dividing cells and persist essentially throughout the lifespan of those cells as a transcriptionally active nuclear episome (extrachromosomal factor). The AAV proviral genome is infectious as cloned DNA in a plasmid, enabling the construction of recombinant genomes. Furthermore, because the signals directing AAV replication, genomic capsid formation, and integration are contained within the ITR of the AAV genome, some or all of the internal approximately 4.3kb of genome (rep-cap, encoding replication and structural capsid proteins) can be replaced with foreign DNA. The rep and cap proteins can be supplied trans. Another important characteristic of AAV is that it is an extremely stable and robust virus. It readily withstands the conditions used to inactivate adenoviruses (56°C to 65°C for several hours), reducing the importance of chilling AAV. AAV can be freeze-dried. Finally, AAV-infected cells do not show resistance to co-infection. Therefore, there is a need to develop therapies for autoimmune diseases such as multiple sclerosis. This disclosure provides a gene therapy method for delivering NT-3 for the treatment of multiple sclerosis and other autoimmune diseases. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Yang et al.,Mel Titer,22(2):440-450(2014) [Non-Patent Document 2] Yalvac et al.,Gene therapy,23(1):95-102(2015) [Non-Patent Document 3] Gao et al., J. Virol., 78:6381-6388 (2004) [Non-Patent Document 4] Mol.Ther.,13(1):67-76(2006) [Non-Patent Document 5] Virology, 330(2):375-383(2004) [Non-Patent Document 6] Muzyczka,Current Topics in Microbiology and Immunology,158:97-129(1992) [Overview of the project]

[0011] This disclosure provides a method for treating autoimmune multiple sclerosis. The method comprises administering a therapeutically effective dose of neurotrophin-3 (NT-3 or NTF-3, these terms used interchangeably in this application), 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 having multiple sclerosis or other autoimmune disease.

[0012] NT-3, like other neurotrophins, was initially identified for its essential functions in the regulation of nervous system development, myelination, growth, axonal protection, and regeneration (12–16). However, the broader effects of NT-3 are now recognized and extend to a wide range of cell types, including involvement in immune cells and inflammatory responses. Studies have shown that NT-3 plays a crucial role in regulating or maintaining the T helper cell 1 and 2 (Th1 / Th2) balance through its interaction with its receptor, TrkC, which is expressed by Th2 cells (17). NT-3 acts as a trigger for IL-4 production in TrkC-expressing Th2 cells, influencing dendritic cell (DC) maturation and inducing regulatory DC formation. Thus, the NT-3 / TrkC system is thought to be involved in the induction or maintenance of Th2-dependent immunity. The efficacy of NT-3's immunomodulatory and anti-inflammatory properties has been previously demonstrated in a spontaneous autoimmune peripheral polyneuropathy (SAPP) mouse model (18) for chronic inflammatory polyradiculopathy (CIDP) in humans using an AAV1.NT-3 gene therapy approach (19-23). ​​This approach was developed due to the short half-life of the NT3 peptide in serum, requiring repeated subcutaneous delivery (24). In contrast, the gene therapy approach involves intramuscular (IM) delivery of the scAAV1.tMCK.NT-3 vector, which provides systemic effects after transduction into muscle to produce the NT-3 protein, which is continuously released into serum as detected by ELISA (18-23). Treated SAAP mice showed increased hindlimb grip strength, along with increased IL-10 and FoxP3 in the sciatic nerve, correlated with improved compound muscle action potentials, CMAP and increased remyelinate nerve fiber population, a reduced number of infiltrating CD3+ T cells, and reduced expression of tumor necrosis factor (TNF)-α and interleukin IL-1β (18). In addition, bone marrow-derived DCs showed significantly increased IL-10 secretion and decreased TNF-α when challenged with bacterial lipopolysaccharide (LPS) in the presence of NT-3, indicating that the DCs acquired tolerogenic characteristics (18).The anti-inflammatory effects of NT-3 have been previously demonstrated in an EAE model through the formation of an anti-inflammatory cytokine environment in the spinal cord by NT-3-transduced embryonic stem cell-derived microglia (25). Furthermore, NT-3 promotes oligodendrocyte precursor proliferation, survival, and differentiation, as well as myelin protein synthesis (26, 27). In addition, NT-3 possesses a remarkable ability to provide neuroprotection and reduce astrogliosis, which is crucial in MS plaque formation (28). Therefore, the success of NT-3 in neuroprotection and immunomodulation potentially fulfills the requirements necessary for the treatment of EAE and its clinically related chronic progressive MS.

[0013] This disclosure provides a method for treating an autoimmune disease in a human subject requiring treatment for an autoimmune disease, comprising the step of administering to the human subject a nucleic acid encoding an 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 an amino acid sequence of SEQ ID NO: 2. In some embodiments, the autoimmune disease is alopecia areata, Addison's disease, celiac disease, Crohn's disease, ulcerative colitis, autoimmune inflammatory myositis, Graves' disease, Hashimoto's thyroiditis, inflammatory bowel disease, multiple sclerosis, pemphigus, pernicious anemia, psoriasis, reactive arthritis, rheumatoid arthritis, Sjögren's syndrome, systemic lupus erythematosus, type 1 diabetes, or autoimmune hepatitis.

[0014] In one embodiment, the present disclosure provides a method for treating multiple sclerosis in a human subject requiring treatment for multiple sclerosis, comprising the step of administering a nucleic acid encoding an NT-3 polypeptide to the human subject, wherein a) the nucleic acid comprises a nucleotide sequence 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 an amino acid sequence of SEQ ID NO: 2.

[0015] This disclosure provides nucleic acids or compositions comprising said NT-3 polypeptides for use in treating autoimmune diseases in human subjects requiring treatment for autoimmune diseases, 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 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 2, or d) the nucleic acid comprises a nucleotide sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 2. For example, any of the disclosed viral vectors may be useful in treating autoimmune diseases such as alopecia areata, Addison's disease, celiac disease, Crohn's disease, ulcerative colitis, autoimmune inflammatory myositis, Graves' disease, Hashimoto's thyroiditis, inflammatory bowel disease, multiple sclerosis, pemphigus, pernicious anemia, psoriasis, reactive arthritis, rheumatoid arthritis, Sjögren's syndrome, systemic lupus erythematosus, type 1 diabetes, or autoimmune hepatitis.

[0016] In one exemplary embodiment, the present disclosure provides a nucleic acid or composition comprising said NT-3 polypeptide for use in the treatment of multiple sclerosis in human subjects requiring treatment for multiple sclerosis, wherein the viral vector comprises a nucleic acid encoding the NT-3 polypeptide, and 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 that is at least 90% identical to the amino acid sequence of SEQ ID NO: 2, or d) the nucleic acid comprises a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 2.

[0017] This disclosure also provides the use of nucleic acids encoding an NT-3 polypeptide for the preparation of a pharmaceutical product for treating an autoimmune disease in a human subject requiring treatment for an autoimmune disease, wherein the pharmaceutical product comprises a nucleic acid encoding an 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 an amino acid sequence of SEQ ID NO: 2. For example, any of the disclosed pharmaceutical products is useful for treating autoimmune diseases such as alopecia areata, Addison's disease, celiac disease, Crohn's disease, ulcerative colitis, autoimmune inflammatory myositis, Graves' disease, Hashimoto's thyroiditis, inflammatory bowel disease, multiple sclerosis, pemphigus, pernicious anemia, psoriasis, reactive arthritis, rheumatoid arthritis, Sjögren's syndrome, systemic lupus erythematosus, type 1 diabetes, or autoimmune hepatitis.

[0018] In addition, the Disclosure provides a use of nucleic acids encoding an NT-3 polypeptide for the preparation of a pharmaceutical product for the treatment of multiple sclerosis in human subjects requiring treatment for multiple sclerosis, wherein the pharmaceutical product comprises a nucleic acid encoding an 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 an amino acid sequence of SEQ ID NO: 2.

[0019] In any of the methods, uses, or nucleic acids or compositions disclosed herein, the viral vector, such as a recombinant adeno-associated virus vector, comprises a nucleic acid. In various embodiments, the viral vector is a recombinant adeno-associated virus (rAAV) vector. In relevant 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, AAVrh.10, or AAVrh.74. In a particular embodiment, the AAV capsid serotype is AAV-1.

[0020] Any of the methods, viral vectors, or compositions of this disclosure can be carried out with nucleic acids operably bound to muscle-specific transcriptional regulatory elements. The term “muscle-specific regulatory element” refers to a nucleotide sequence that modulates the expression of a coding sequence specific to expression in muscle tissue. These regulatory elements include enhancers and promoters. This disclosure provides constructs comprising the muscle-specific regulatory element MHCK7 promoter (muscle creatine kinase promoter (7) w hybrid intron), MCK promoter, and MCK enhancer / or alpha-myosin heavy chain (MHC) complex enhancer. In various embodiments, the muscle creatine kinase promoter sequence is shown in SEQ ID NO: 11.

[0021] Exemplary muscle-specific promoters include one or more of the following: human skeletal actin gene element, cardiac actin gene element, desmin promoter, skeletal alpha-actin (ASKA) promoter, troponin I (TNNI2) promoter, muscle cell-specific enhancer-binding factor (MEF) binding element, muscle creatine kinase (MCK) promoter, truncated MCK (tMCK) promoter, myosin heavy chain (MHC) promoter, hybrid α-myosin heavy chain enhancer / MHC enhancer promoter (MHCK7) promoter, C5-12 promoters, mouse creatine kinase enhancer element, skeletal fast-twitch muscle troponin C gene element, slow-twitch muscle cardiac troponin c gene element, slow-twitch muscle troponin I gene element, hypoxia-inducible nuclear factor (HIF) response element (HRE), steroid-inducible element, and glucocorticoid response element (GRE).

[0022] Any of the methods, compositions for use, or uses of this disclosure may be carried out with a nucleic acid sequence which, in 5' to 3' order, is (i) a first AAV2 inverted terminal repeat (ITR), (ii) a muscle creatine kinase promoter / enhancer sequence shown in nucleotides 147-860 of SEQ ID NO: 3, (iii) a nucleotide sequence encoding a human NT-3 polypeptide, and (iv) an rAAV genome sequence comprising a second AAV2 ITR sequence, wherein the human NT-3 polypeptide has an amino acid sequence that is at least 90% identical to or 100% identical to SEQ ID NO: 2, or a nucleotide sequence that is at least 90% identical to or 100% identical to nucleotides 1077-1850 of SEQ ID NO: 3.

[0023] Any of the methods, compositions for use, or uses of the present disclosure may be carried out with a nucleic acid further comprising the chimeric intron shown at nucleotides 892-1024 of SEQ ID NO: 3 on the 3' side of the promoter / enhancer. In addition, the nucleic acid of the present disclosure may further comprise the SV40 polyadenylation signal shown at nucleotides 1860-2059 of SEQ ID NO: 3 on the 3' side of the nucleotide sequence encoding the human NT-3 polypeptide.

[0024] In any of the methods, compositions for use, or uses of the Disclosure, the nucleic acids of the Disclosure may contain one or more inverted end repeat (ITR) sequences. For example, the nucleic acid may contain a first ITR shown in nucleotides 7-112 of SEQ ID NO: 3, and / or a second ITR shown in nucleotides 2121-2248 of SEQ ID NO: 3.

[0025] In some embodiments, the nucleic acid comprises the scAAV1.tMCK.NT-3 genome, which is at least 90% identical to SEQ ID NO: 9. In relevant embodiments, the nucleic acid comprising the scAAV1.tMCK.NT-3 genome is shown in SEQ ID NO: 9.

[0026] This disclosure provides a method for treating an autoimmune disease in a human subject requiring treatment for an autoimmune disease such as multiple sclerosis, comprising the step of administering to the human subject a certain dose of recombinant adeno-associated virus (rAAV) scAAV1.tMCK.NTF3 that results in sustained expression of a low concentration of NT-3 protein.

[0027] In any of the methods, compositions for use, or uses described herein, the nucleic acids or rAAVs disclosed herein are administered in doses that result in sustained expression of low concentrations of NT-3 polypeptides. In various embodiments, administration of the nucleic acids or rAAVs disclosed herein reduces inflammation in organs affected by autoimmune diseases, such as MS, in a subject. In various embodiments, administration of the nucleic acids or rAAVs disclosed herein modulates the immune response in a subject. In various embodiments, administration of the nucleic acids or rAAVs disclosed herein increases the percentage of regulatory T cells in organs affected by autoimmune diseases, such as MS, in a subject. In various embodiments, administration of the nucleic acids or rAAVs disclosed herein modulates cytokine expression in dendritic cells in a subject.

[0028] In various embodiments, the nucleic acid viral vectors, rAAVs, compositions, or pharmaceuticals disclosed herein are administered intramuscularly. In relevant embodiments, the nucleic acids or rAAVs disclosed herein are administered by intramuscular injection.

[0029] In any of the methods, compositions for use, or uses described herein, the target subjects are elderly adult subjects. In some embodiments, the target subjects are pediatric subjects, e.g., subjects under 18 years of age. In some embodiments, the target subjects are adults (18 years and older). In particular, the target subjects are young adults (18–39 years), middle-aged adults (40–64 years), or elderly adults or elderly subjects (65 years and older), or old-age subjects (70 years and older). In some embodiments, MS typically begins in adults between 20 and 50 years of age. In some embodiments, MS is typically twice as common in women as in men.

[0030] This disclosure can be more readily understood by referring to the following drawings. [Brief explanation of the drawing]

[0031] [Figure 1] A schematic diagram of the cassette portion of the construct AAV.tMCK.NTF3 (shown in nucleotides 7-2248 of SEQ ID NO: 3, referred to herein as SEQ ID NO: 9) is provided. rAAV contains a muscle-specific tMCK promoter (SEQ ID NO: 11), a chimeric intron (SEQ ID NO: 5), a consensus Scozak sequence (SEQ ID NO: 6), NTF3 cDNA (SEQ ID NO: 1), and a polyadenylation signal (SEQ ID NO: 7). [Figure 2] This document provides restriction maps and ORF analysis for the cassette plasmid self-complementary pAAV.tMCK.NTF3 (SEQ ID NO: 3). [Figure 3-1] This provides the nucleotide sequence of the cassette-producing plasmid, self-complementary pAAV.tMCK.NTF3 (SEQ ID NO: 3). [Figure 3-2] Same as above. [Figure 4] This study demonstrates that AAV1.tMCK.NT-3 gene therapy induces detectable levels of NT-3 in serum. EAE mice were injected with a total of 1 × 10¹¹ vg of AAV1.tMCK.NT-3 into the right calf muscle. Blood samples were obtained from mice 7 weeks after injection, and NT-3 serum levels were determined by enzyme-linked immunosorbent assay. Data represent mean ± SEM (female cohort: n=3 for untreated, n=4 for NT-3 treated cohort; male cohort: n=3 for both treated and untreated). *p<0.05. [Figure 5] This study shows that NT-3 treatment improved the severity of experimental autoimmune encephalomyelitis (EAE) in mouse models compared to untreated mice. Higher clinical scores indicate a worse physiological state in mice. Overall scores in the NT-3 treated cohort were lower than in the untreated cohort. Data represent mean ± SEM (N=4 for the untreated cohort, N=5 for the NT-3 treated cohort). *p<0.05. [Figure 6](Figure 6A) Provides clinical scores for EAE mice (a, b) with and without AAV1.NT-3 treatment. Higher clinical scores indicate more severe symptoms in the mice. Red indicates the time of AAV1.NTF3 injection. Grip strength (c, d) and rotarod (e, f) analyses for EAE mice with and without AAV1.NT-3 treatment. Data represent mean ± SEM (female cohort: n=7 for untreated, n=9 for NT-3 treated; male cohort: n=5 for untreated, n=5 for NT-3 treated cohort). *p<0.05. (Figure 6B) Provides clinical scores for EAE mice (a, b) with and without AAV1.NT-3 treatment. Higher clinical scores indicate more severe symptoms in the mice. Red indicates the time of AAV1.NTF3 injection. Grip strength (c, d) and rotarod (e, f) analysis for EAE mice with and without AAV1.NT-3 treatment. Data represent mean ± SEM (female cohort: n=7 for untreated, n=9 for NT-3 treated; male cohort: n=5 for untreated, n=5 for NT-3 treated cohort). *p<0.05. (Figure 6C) Provides clinical scores for EAE mice (a, b) with and without AAV1.NT-3 treatment. Higher clinical scores indicate more severe symptoms in the mice. Red indicates the time of AAV1.NTF3 injection. Grip strength (c, d) and rotarod (e, f) analysis for EAE mice with and without AAV1.NT-3 treatment. Data represent mean ± SEM (female cohort: n=7 for untreated, n=9 for NT-3 treated; male cohort: n=5 for untreated, n=5 for NT-3 treated cohort). *p<0.05. (Figure 6D) Provides clinical scores for EAE mice (a, b) with and without AAV1.NT-3 treatment. Higher clinical scores indicate more severe symptoms in the mice. Red indicates the time of AAV1.NTF3 injection. Grip strength (c, d) and rotarod (e, f) analyses for EAE mice with and without AAV1.NT-3 treatment. Data represent mean ± SEM (female cohort: n=7 for untreated, n=9 for NT-3 treated; male cohort: n=5 for untreated, n=5 for NT-3 treated cohort). *p<0.05.(Figure 6E) Provides clinical scores for EAE mice (a, b) with and without AAV1.NT-3 treatment. Higher clinical scores indicate more severe symptoms in the mice. Red indicates the time of AAV1.NTF3 injection. Grip strength (c, d) and rotarod (e, f) analyses for EAE mice with and without AAV1.NT-3 treatment. Data represent mean ± SEM (female cohort: n=7 for untreated, n=9 for NT-3 treated; male cohort: n=5 for untreated, n=5 for NT-3 treated cohort). *p<0.05. (Figure 6F) Provides clinical scores for EAE mice (a, b) with and without AAV1.NT-3 treatment. Higher clinical scores indicate more severe symptoms in the mice. Red indicates the time of AAV1.NTF3 injection. Grip strength (c, d) and rotarod (e, f) analysis of EAE mice with and without AAV1 NT-3 treatment. Data represent mean ± SEM (female cohort: n=7 for untreated, n=9 for NT-3 treated; male cohort: n=5 for untreated, n=5 for NT-3 treated cohort). *p<0.05. [Figure 7A] The results of behavioral studies in NT-3-treated and untreated experimental autoimmune encephalomyelitis (EAE) mice are shown. Figure 7A shows the results from the rotarod assay, and Figure 7B shows the results from the grip strength assay. Data represent mean ± SEM (N=4 for the untreated cohort, N=5 for the NT-3-treated cohort). *p<0.05. [Figure 7B] Same as above. [Figure 8](Figure 8A) (a) Representative hematoxylin & eosin (H&E) stained sections from the lumbar spinal cord of untreated EAE mice showing multifocal meningeal lymphocytic inflammation, and (b) from the AAV.NT-3 injection cohort (arrows indicate inflammation). (c) Luxor Fast Blue stained paraffin sections of the lumbar spinal cord from untreated and (d) NT-3 treated EAEs, and (e) from wild-type mice showing the presence of subpia demyelination (asterisk) in untreated EAE mice. (f) Half-thick plastic sections from untreated mice showing subpia axonal loss (asterisk) contrast with (g) samples from the NT-3 treated cohort, revealing preservation of long white matter tracts in the descending anterolateral corticospinal tract 7 weeks post-treatment. Scale bars are 100 μm for a-e and 50 μm for f and g. (Figure 8B) (a) Representative hematoxylin & eosin (H&E) stained sections from the lumbar spinal cord of untreated EAE mice showing multifocal meningeal lymphocytic inflammation, and (b) from the AAV.NT-3 injection cohort (arrows indicate inflammation). (c) Luxor Fast Blue stained paraffin sections of the lumbar spinal cord from untreated and (d) NT-3 treated EAEs, and (e) from wild-type mice showing the presence of subpia demyelination (asterisk) in untreated EAE mice. (f) Half-thick plastic sections from untreated mice showing subpia axonal loss (asterisk) contrast with (g) samples from the NT-3 treated cohort, revealing preservation of long white matter pathways in the descending anterolateral corticospinal tract 7 weeks post-treatment. Scale bars are 100 μm for a-e and 50 μm for f and g. (Figure 8C) (a) Representative hematoxylin & eosin (H&E) stained sections from the lumbar spinal cord of untreated EAE mice showing multifocal meningeal lymphocytic inflammation, and (b) from the AAV.NT-3 injection cohort (arrows indicate inflammation). (c) Luxor Fast Blue stained paraffin sections of the lumbar spinal cord from untreated and (d) NT-3 treated EAEs, and (e) from wild-type mice showing the presence of subpia demyelination (asterisk) in untreated EAE mice.(f) Half-thick plastic sections from untreated mice showing subpia-axon loss (asterisk) contrast with (g) samples from the NT-3 treated cohort, revealing preservation of long white matter tracts in the descending anterolateral corticospinal tract 7 weeks post-treatment. Scale bars are 100 μm for a-e and 50 μm for f and g. (Figure 8D) (a) Representative hematoxylin & eosin (H&E) stained sections from the lumbar spinal cord of untreated EAE mice showing multifocal meningolymphocytic inflammation, and (b) from the AAV.NT-3 injection cohort (arrows indicate inflammation). (c) Luxor Fast Blue stained paraffin sections of the lumbar spinal cord from untreated and (d) NT-3 treated EAEs, as well as from wild-type mice showing the presence of subpia-demyelination (asterisk) in untreated EAE mice. (f) Half-thick plastic sections from untreated mice showing subpia-axon loss (asterisk) contrast with (g) samples from the NT-3 treated cohort, revealing preservation of long white matter tracts in the descending anterolateral corticospinal tract 7 weeks post-treatment. Scale bars are 100 μm for a-e and 50 μm for f and g. (Figure 8E) (a) Representative hematoxylin & eosin (H&E) stained sections from the lumbar spinal cord of untreated EAE mice showing multifocal meningolymphocytic inflammation, and (b) from the AAV.NT-3 injection cohort (arrows indicate inflammation). (c) Luxor Fast Blue stained paraffin sections of the lumbar spinal cord from untreated and (d) NT-3 treated EAEs, as well as from wild-type mice showing the presence of subpia-demyelination (asterisk) in untreated EAE mice. (f) Half-thick plastic sections from untreated mice showing subpia-axonal loss (asterisk) contrast with (g) samples from the NT-3 treatment cohort, revealing preservation of long white matter tracts in the descending anterolateral corticospinal tract 7 weeks post-treatment. Scale bars are 100 μm for a-e and 50 μm for f and g. (Figure 8F) (a) Representative hematoxylin & eosin (H&E) stained sections from untreated EAE mice showing multifocal meningolymphocytic inflammation, and (b) representative hematoxylin & eosin (H&E) stained sections from the lumbar spinal cord of the AAV.NT-3 injection cohort (arrows indicate inflammation).(c) Paraffin sections of lumbar spinal cord stained with Luxor Fast Blue from untreated and (d) NT-3 treated EAE mice, and (e) wild-type mice showing the presence of subpia-demyelination (asterisk) in untreated EAE mice. (f) Half-thick plastic sections from untreated mice showing subpia-axon loss (asterisk) contrast with (g) samples from the NT-3 treated cohort, revealing preservation of long white matter tracts in the descending anterolateral corticospinal tract 7 weeks post-treatment. Scale bars are 100 μm for a-e and 50 μm for f and g. (Figure 8G) (a) Representative hematoxylin & eosin (H&E) stained sections from lumbar spinal cord from untreated EAE mice showing multifocal meningolymphocytic inflammation, and (b) from the AAV.NT-3 injection cohort (arrows indicate inflammation). (c) Paraffin sections of lumbar spinal cord stained with Luxor Fast Blue from untreated and (d) NT-3 treated EAE mice, and (e) wild-type mice showing the presence of subpia-demyelination (asterisk) in untreated EAE mice. (f) Half-thick plastic sections from untreated mice showing subpia-axon loss (asterisk) contrast with (g) samples from the NT-3 treated cohort, revealing preservation of long white matter pathways in the descending anterolateral corticospinal tract 7 weeks post-treatment. Scale bars are 100 μm for a-e and 50 μm for f and g. [Figure 9](Figure 9A) (a) Representative immunofluorescence images of the anterior cortical spinal cord track from untreated (UT), (b) NT-3 treated (NT-3), and (c) wild-type (WT) mice are provided. (d) Bar graphs show the number of anti-neurofilament antibody-positive particles representing axons counted in a given region of white matter in the anterior cortical spinal cord track (UT, 52450.4±4851.3; NT-3, 71733.1±1778.0 axons / mm2; p=0.0203; n=3 for each cohort). Scale bar: 10 μm. Expression levels of (e, f) myelin basic protein (MBP) and (g, h) proteolipid protein (PLP) in the spinal cord from UT and NT-3 treated cohorts. MBP and PLP expression were significantly increased with treatment in females (e, g). (Figure 9B) (a) Representative immunofluorescence images of the anterior cortical spinal cord track from untreated (UT), (b) NT-3 treated (NT-3), and (c) wild-type (WT) mice are provided. (d) Bar graphs show the number of anti-neurofilament antibody-positive particles representing axons counted in a given region of white matter in the anterior cortical spinal cord track (UT, 52450.4±4851.3; NT-3, 71733.1±1778.0 axons / mm2; p=0.0203; n=3 for each cohort). Scale bar: 10 μm. Expression levels of (e, f) myelin basic protein (MBP) and (g, h) proteolipid protein (PLP) in the spinal cord from UT and NT-3 treated cohorts. MBP and PLP expression were significantly increased with treatment in females (e, g). (Figure 9C) (a) Representative immunofluorescence images of the anterior cortical spinal cord track from untreated (UT), (b) NT-3 treated (NT-3), and (c) wild-type (WT) mice are provided. (d) Bar graphs show the number of anti-neurofilament antibody-positive particles representing axons counted in a given region of white matter in the anterior cortical spinal cord track (UT, 52450.4±4851.3; NT-3, 71733.1±1778.0 axons / mm2; p=0.0203; n=3 for each cohort). Scale bar: 10 μm. Expression levels of (e, f) myelin basic protein (MBP) and (g, h) proteolipid protein (PLP) in the spinal cord from UT and NT-3 treated cohorts.MBP and PLP expression were significantly increased with treatment in females (e, g). (Figure 9D) (a) Representative immunofluorescence images of the anterior cortical spinal cord track from untreated (UT), (b) NT-3 treated (NT-3), and (c) wild-type (WT) mice are provided. (d) Bar graphs show the number of anti-neurofilament antibody-positive particles representing axons counted in a given region of white matter in the anterior cortical spinal cord track (UT, 52450.4±4851.3; NT-3, 71733.1±1778.0 axons / mm2; p=0.0203; n=3 for each cohort). Scale bar: 10 μm. Expression levels of (e, f) myelin basic protein (MBP) and (g, h) proteolipid protein (PLP) in the spinal cord from UT and NT-3 treated cohorts. MBP and PLP expression were significantly increased with treatment in females (e, g). (Figure 9E) (a) Representative immunofluorescence images of the anterior cortical spinal cord track from untreated (UT), (b) NT-3 treated (NT-3), and (c) wild-type (WT) mice are provided. (d) Bar graphs show the number of anti-neurofilament antibody-positive particles representing axons counted in a given region of white matter in the anterior cortical spinal cord track (UT, 52450.4±4851.3; NT-3, 71733.1±1778.0 axons / mm2; p=0.0203; n=3 for each cohort). Scale bar: 10 μm. Expression levels of (e, f) myelin basic protein (MBP) and (g, h) proteolipid protein (PLP) in the spinal cord from UT and NT-3 treated cohorts. MBP and PLP expression were significantly increased with treatment in females (e, g). (Figure 9F) (a) Representative immunofluorescence images of the anterior cortical spinal cord track from untreated (UT), (b) NT-3 treated (NT-3), and (c) wild-type (WT) mice are provided. (d) Bar graphs show the number of anti-neurofilament antibody-positive particles representing axons counted in a given region of white matter in the anterior cortical spinal cord track (UT, 52450.4±4851.3; NT-3, 71733.1±1778.0 axons / mm2; p=0.0203; n=3 for each cohort). Scale bar: 10 μm.(e, f) Expression levels of myelin basic protein (MBP) and (g, h) proteolipid protein (PLP) in the spinal cord from UT and NT-3 treated cohorts. MBP and PLP expression were significantly increased with treatment in females (e, g). (Figure 9G) (a) Representative immunofluorescence images of the anterior cortical spinal cord track from untreated (UT), (b) NT-3 treated (NT-3), and (c) wild-type (WT) mice are provided. (d) Bar graphs show the number of anti-neurofilament antibody-positive particles representing axons counted in a given region of white matter in the anterior cortical spinal cord track (UT, 52450.4±4851.3; NT-3, 71733.1±1778.0 axons / mm2; p=0.0203; n=3 for each cohort). Scale bar: 10 μm. (e, f) Expression levels of myelin basic protein (MBP) and (g, h) proteolipid protein (PLP) in the spinal cord from UT and NT-3 treated cohorts. MBP and PLP expression were significantly increased with treatment in females (e, g). (Figure 9H) (a) Representative immunofluorescence images of the anterior cortical spinal cord track from untreated (UT), (b) NT-3 treated (NT-3), and (c) wild-type (WT) mice are provided. (d) Bar graphs show the number of anti-neurofilament antibody-positive particles representing axons counted in a given region of white matter in the anterior cortical spinal cord track (UT, 52450.4±4851.3; NT-3, 71733.1±1778.0 axons / mm2; p=0.0203; n=3 for each cohort). Scale bar: 10 μm. Expression levels of (e, f) myelin basic protein (MBP) and (g, h) proteolipid protein (PLP) in the spinal cord from UT and NT-3 treatment cohorts. MBP and PLP expression were significantly increased with treatment in females (e, g). [Figure 10] This shows that NT-3 significantly reduced inflammatory markers in diseased organs in female mice. TNFα (Figure 10A), IL6 (Figure 10B), and IL1β (Figure 10C) were all reduced in the NT-3 treated cohort in both the brain and spinal cord. Data represent the mean ± SEM of mice in each group (N=2 for the untreated cohort, N=3 for the treated cohort). [Figure 11] This report provides expression levels of the inflammatory markers TNFα, IL1β, and IL6 in the brain and spinal cord from EAE mice, (a-c) female and (d-f) male. TNFα, IL1β, and IL6 were all significantly reduced in the NT-3 treatment cohort (NT-3) in both the brain and spinal cord compared to the untreated (UT) group. In males, IL1β expression was significantly reduced in the brain, and IL6 was significantly reduced in the spinal cord (e, f). Data represent the mean ± SEM of mice in each group (female cohort: n=4 for untreated, n=4 for treatment cohort; male cohort: n=3 for untreated, n=3 for treatment cohort). [Figure 12] This study demonstrates that NT-3 significantly increased the percentage of T regulatory cells (Treg cells) in experimental autoimmune encephalomyelitis (EAE) mice. Figure 12A shows lymph node-derived cells, and Figure 12B shows spleen-derived cells. Data represent the mean of mice in each group (N=2 for each cohort). [Figure 13](Figure 13A) Percentage of T regulatory cells (Treg cells) in lymph nodes and spleen from untreated and NT-3 treated EAE mice. (a) Representative flow cytometry image staining gated CD3+CD4+CD25+Foxp3+Treg cells from CD3+CD4+ cell population. (b) Population of Treg cells in spleen and lymph nodes from females and (c) males. Data represent mean ± SEM (female cohort: n=3 for untreated, n=4 for NT-3 treated cohort; male cohort: n=3 for untreated, n=3 for treated cohort). *p<0.05 (Figure 13B) Percentage of T regulatory cells (Treg cells) in lymph nodes and spleen from untreated and NT-3 treated EAE mice. (a) Representative flow cytometry image staining gated CD3+CD4+CD25+Foxp3+Treg cells from CD3+CD4+ cell population. (b) Population of Treg cells in the spleen and lymph nodes from females and (c) males. Data represent mean ± SEM (female cohort: n=3 for untreated, n=4 for NT-3 treated cohort; male cohort: n=3 for untreated, n=3 for treated cohort). *p<0.05 (Figure 13C) Percentage of T regulatory cells (Treg cells) in the lymph nodes and spleen from untreated and NT-3 treated EAE mice. (a) Representative flow cytometry image staining of gated CD3+CD4+CD25+Foxp3+Treg cells from CD3+CD4+ cell population. (b) Population of Treg cells in the spleen and lymph nodes from females and (c) males. Data represent mean ± SEM (female cohort: n=3 for untreated, n=4 for NT-3 treated cohort; male cohort: n=3 for untreated, n=3 for treated cohort). *p<0.05 [Figure 14]This study demonstrates that NT-3 significantly reduced TNFα expression in dendritic cells (DCs) challenged by Myobacterium. DCs extracted from NT-3-treated and untreated experimental autoimmune encephalomyelitis (EAE) mice were isolated from bone marrow. Cultured DCs were challenged with Myobacterium for 24 hours, and TNFα levels were determined as a marker of inflammation. Data represent mean ± SEM (N=4 for the untreated cohort, N=5 for the NT-3-treated cohort in combined female and male mice). *p<0.05. [Figure 15](Figure 15A) Demonstrating the tolerogenic characteristics of bone marrow-derived dendritic cells. (a) Dendritic cells isolated from the bone marrow of female EAE mice. Flow cytometry analysis showed that 74.1% of all cultured cells expressed the DC marker CD11c. DCs from NT-3 treated and untreated EAE (b) female and (c) male mice were challenged with Mycobacterium, and TNFα levels were measured by qPCR. Data represent mean ± SEM (female cohort: n=4 for untreated, n=4 for NT-3 treated cohort; male cohort: n=3 for untreated, n=3 for NT-3 treated cohort). *p<0.05. (Figure 15B) Demonstrating the tolerogenic characteristics of bone marrow-derived dendritic cells. (a) Dendritic cells isolated from the bone marrow of female EAE mice. Flow cytometry analysis showed that 74.1% of all cultured cells expressed the DC marker CD11c. DCs from NT-3-treated and untreated EAE (b) female and (c) male mice were challenged with Mycobacterium, and TNFα levels were measured by qPCR. Data represent mean ± SEM (female cohort: n=4 for untreated, n=4 for NT-3-treated cohort; male cohort: n=3 for untreated, n=3 for NT-3-treated cohort). *p<0.05. (Figure 15C) Dendritic cells isolated from bone marrow of (a) female EAE mice demonstrating the tolerogenic characteristics of bone marrow-derived dendritic cells. Flow cytometry analysis showed that 74.1% of all cultured cells expressed the DC marker CD11c. DCs from NT-3-treated and untreated EAE (b) female and (c) male mice were challenged with Mycobacterium, and TNFα levels were measured by qPCR. Data represent mean ± SEM (female cohort: n=4 for untreated, n=4 for NT-3 treated cohort; male cohort: n=3 for untreated, n=3 for NT-3 treated cohort). *p<0.05. [Modes for carrying out the invention]

[0032] This disclosure relates to a method for treating autoimmune diseases such as multiple sclerosis (MS). The method disclosed herein includes administering a therapeutically effective dose of neurotrophin-3 (NT-3), pro-NT-3, or an effective fragment thereof, or nucleic acids encoding NT-3, pro-NT-3, or an effective fragment thereof, to a subject having multiple sclerosis.

[0033] NT-3 is a key growth factor that stimulates glial cell survival and differentiation, as well as axonal growth and myelin formation. NT-3 is also known to possess immunomodulatory and anti-inflammatory properties, as previously demonstrated in a spontaneous autoimmune peripheral polyneuropathy (SAPP) mouse model for chronic inflammatory polyradiculopathy (CIDP) in humans.

[0034] In one embodiment, the present disclosure provides a gene therapy method for treating autoimmune diseases such as MS, in which the NT-3 coding sequence of the NTF3 gene is delivered to a subject using autocomplementary adeno-associated virus (scAAV) type 1 under the control of a muscle-specific tMCK promoter.

[0035] Preclinical studies (Sahenk, Zarife, et al. "AAV1.NT-3 gene therapy for Charcot-Marie-Tooth neuropathy." Molecular Therapy 22.3(2014):511-521) showed that tremor J mice (Tr J ), we demonstrated that delivery of the construct AAV1.tMCK.NTF3 to the gastrocnemius muscle of a naturally occurring mouse model for CMT1 improved nerve regeneration, myelin formation, myelinated fiber density, sciatic nerve complex muscle action potential amplitude, and functional performance in rotarod testing and hindlimb grip strength (see Example 1 of PCT / US2021 / 027279).

[0036] The primary therapeutic goal in MS is to block destructive immune effector cells while enhancing immunosuppressive regulatory cells. In the study described herein, a gene therapy approach was administered to an EAE mouse model of MS via IM delivery of the AAV1.tMCK.NT-3 vector. The data provided herein demonstrate the systemic effects after transduction of muscle to produce the NT-3 protein, as detected by ELISA. This approach provided histopathological evidence of remyelination supported by increased myelin protein gene expression and axonal protection, and reduced clinical severity. The data herein also demonstrate that functional and histopathological improvements occurred in conjunction with an anti-inflammatory and immunomodulatory environment. Six to seven weeks after AAV1.NT-3 treatment, there was an increase in the Treg cell population in both peripheral lymphocytes and splenic cells from both female and male cohorts. Inflammatory markers TNFα, IL1β, and IL6 were also significantly reduced or showed a tendency to decrease in the treatment cohort, thus the increased Treg population was associated with a suppressed inflammatory state in both the spinal cord and brain. In MS patients, Th1 and Th17 cells were increased along with their associated cytokines IL-1, IL-6, IL-17, IFNγ, and TNFα, but no significant difference in Treg frequency was reported compared to healthy controls (Danikowski et al., J Neuroinflammation;14:117, 2017). However, Tregs from these patients were reported to have lower suppressive capacity (Goswami et al., Hum Vaccin Immunother;18:2035117, 2022), suggesting that functional deficiencies in Tregs may contribute to the pathogenesis of MS. In addition, studies have shown that Tregs may be restricted from migrating to the site of neuroinflammation or may undergo apoptosis upon arrival (Danikowski et al., J Neuroinflammation;14:117,2017).

[0037] Conceptually, CNS-derived dendritic cells (DCs) may be associated with Treg cell proliferation and thereby contribute to the resolution of CNS inflammation. DCs can modulate the proliferation and function of Treg cells during CNS inflammation. However, in MS patients, DCs exhibit a modified phenotype, with dysfunctional interactions between DCs and Treg cells, leading to loss of effector T cell suppression, myelin destruction, nerve damage, and neuroinflammation (Attfield et al., Nat Rev Immunol.; 22:734-750, 2022). Therefore, modulating DC tolerogenic tolerance may be important in treating MS. The data provided herein demonstrate that NT-3 suppressed the DC tolerogenic response in an in vitro assay by isolating DCs from bone marrow and then challenging them with Mycobacterium. TNFα levels were significantly suppressed in NT-3-treated DCs compared to untreated DCs. Collectively, these data demonstrate the immunomodulatory role of NT-3 in suppressing autoimmune responses in EAE mice.

[0038] Multiple sclerosis Multiple sclerosis (MS) is the most common demyelinating disease affecting more than 2.5 million people worldwide. Also known as disseminated encephalomyelitis, MS is an autoimmune disease in which the patient's immune system attacks the myelin of nerves in the brain and spinal cord, ultimately causing demyelination of nerves that leads to secondary axonal loss. In aspects, this disclosure describes the use of AAV1.NT-3 gene therapy for the treatment of multiple sclerosis.

[0039] In this embodiment, the type of multiple sclerosis is relapsing, primary progressive, or secondary progressive.

[0040] In this embodiment, multiple sclerosis is chronic progressive multiple sclerosis.

[0041] As used herein, terms such as “treatment” and “to treat” refer to obtaining a desired pharmacological or physiological effect. The effect may be therapeutic in terms of partial or complete cure of a disease, or partial or complete inhibition or prevention of adverse effects resulting from the disease. Terms such as “treatment” and “to treat” may also refer to improving clinical symptoms, delaying or preventing more harmful clinical symptoms, reducing or preventing increased inflammation, and / or modulating an immune response. As used herein, “treatment” encompasses any treatment of a disease in mammals, particularly humans, and may include inhibiting a disease or condition, i.e., stopping its onset, and alleviating a disease, i.e., causing disease regression.

[0042] As used herein, “prevention” refers to any action that provides a benefit to a person at risk of developing a condition such as multiple sclerosis or an autoimmune disease.

[0043] As used herein, “pharmaceutically acceptable” means that, in light of the severity of the disease and the need for treatment, the compound or composition is suitable for administration to a subject in the manner described herein without undue adverse side effects.

[0044] The terms “therapeutically effective” and “pharmacologically effective” are intended to qualitatively define the amount of drug that achieves the goal of improvement in disease severity and incidence. Therapeutic effectiveness may be measured by evaluating the reduction of symptoms in the subject in response to the administration of NT-3.

[0045] The term “effective fragment” refers to a portion of a 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 activity includes supporting the survival and differentiation of existing neurons, as well as inducing and supporting the growth and differentiation of new neurons and synapses. In addition, NT-3 activity includes stimulating muscle growth and muscle function, activating mTOR signaling, increasing muscle fiber diameter, and / or increasing muscle contractility. In other embodiments, NT-3 activity includes reducing inflammation in organs affected by MS or autoimmune disease in a subject, where the organ is the brain, spinal cord, joints, muscles, skin, pancreas, liver, or kidney, reducing inflammation, modulating the immune response, and increasing the percentage of regulatory T cells in organs affected by MS or autoimmune disease, where the organ is a lymph node, spleen, thymus, or peripheral blood, or modulating cytokine expression in dendritic cells.

[0046] As used herein, the term “diagnosis” may encompass determining the likelihood that a subject will develop a disease (e.g., MS or another autoimmune disease, without limitation) or determining the presence or nature of a disease in a subject. As used herein, the term “diagnosis” may also encompass determining the severity and possible outcomes of a disease or episode of disease, or the likelihood of recovery, commonly referred to as prognosis. “Diagnosis” may also encompass diagnosis in the context of rational therapy, which includes guiding therapy, such as initial selection of therapy, modification of therapy (e.g., adjustment of dose or medication regimen, without limitation).

[0047] As used herein, “subject” can be any animal and may also be referred to as a patient. Preferably, the subject is a vertebrate, and more preferably, the subject is a mammal, such as a domestic animal (e.g., cattle, horses, pigs, sheep) or a pet (e.g., dogs, cats, guinea pigs). In some embodiments, the subject is a human. In some embodiments, the subject is an adult (18 years of age or older). In particular, the subject is a young adult (18–39 years of age), a middle-aged adult (40–64 years of age), or an elderly adult or elderly subject (65 years of age or older), or an elderly subject (70 years of age or older). In some embodiments, MS typically begins in an adult between the ages of 20 and 50.

[0048] The terms “polynucleotide” or “nucleic acid molecule” refer to polymeric forms of nucleotides that are at least 10 bases long. The terms include DNA molecules (e.g., cDNA or genomic or synthetic DNA) and RNA molecules (e.g., mRNA or synthetic RNA), as well as DNA or RNA analogs containing unnatural nucleotide analogs, unnatural nucleoside bonds, or both. Nucleic acids can have any topological three-dimensional structure. For example, nucleic acids can have single-stranded, double-stranded, triple-stranded, quadruple-stranded, partially double-stranded, branched, hairpin, cyclic, or padlock three-dimensional structures.

[0049] As used herein, the term “gene” refers to a nucleotide sequence that can direct the synthesis of an enzyme or other polypeptide molecule (for example, which may include coding sequences, e.g., consecutive open reading frames (ORFs) that code for a polypeptide) or that may be functional in itself in an organism. Genes in an organism may be clustered within operons as defined herein, and operons are separated from other genes and / or operons by intergenetic DNA. Individual genes contained within an operon may be duplicated among individual genes without intergenetic DNA.

[0050] As used herein, the term "AAV" is a common abbreviation for adeno-associated virus. Adeno-associated viruses are single-stranded DNA parvoviruses that grow only in cells, provided with certain functions by co-infecting helper viruses. Currently, there are 13 serotypes of AAV that have been characterized. General information and an overview of AAV can be found, for example, in Carter, 1989, Handbook of Parvoviruses, Vol. 1, pp. 169–228, and Berns, 1990, Virology, pp. 1743–1764, Raven Press, (New York). However, since it is well known that various serotypes are very closely related both structurally and functionally, even at the genetic level, it is quite expected that these same principles may apply to additional AAV serotypes. (See, for example, Blacklowe, 1988, pp. 165-174 of Parvoviruses and Human Disease, JR Pattison, ed., and Rose, Comprehensive Virology 3:1-61 (1974)). For example, all AAV serotypes clearly exhibit very similar replication characteristics mediated by homologous rep genes, all possessing three related capsid proteins, such as those expressed in AAV2. The degree of relatedness is further suggested by heteroduplex analysis revealing extensive cross-hybridization between serotypes along genome length and the presence of similar self-annealing segments at the terminals corresponding to "terminal inversion sequences" (ITRs). Similar infectivity patterns also suggest that replication function in each serotype is under similar regulatory control.

[0051] The terms "vector" or "expression vector" refer to any type of gene construct containing nucleic acid that encodes transcribed RNA. Expression vectors can contain various regulatory sequences, structural genes (e.g., the gene of interest), and nucleic acid sequences that also perform other functions.

[0052] A "vector" typically refers to a DNA molecule derived from a plasmid or bacteriophage into which a fragment of DNA can be inserted or cloned. Recombinant vectors contain one or more unique restriction sites and may be capable of autonomous replication in a defined host or vehicle organism so that the cloned sequence is reproducible. The vector contains a promoter operably bound to a gene or coding region so that RNA is expressed upon transfection into recipient cells.

[0053] As used herein, “recombinant AAV (rAAV)” refers to a viral vector containing one or more polynucleotides (or transgenes) of interest adjacent to the AAV terminal repeat sequence (ITR). Such rAAVs can replicate and package into infectious viral particles when present in host cells transfected with vectors encoding and expressing rep and cap gene products.

[0054] An "rAAV virion," "rAAV virus particle," or "rAAV vector particle" refers to a viral particle composed of at least one AAV capsid protein and a capsidized polynucleotide rAAV vector. If the particle contains heterologous polynucleotides (i.e., polynucleotides other than the wild-type AAV genome, such as a transgene delivered to a mammalian cell), it is typically referred to as an "rAAV vector particle" or simply an "rAAV particle." Thus, since such an rAAV genome is contained within the rAAV vector particle, the production of an rAAV vector particle necessarily involves the production of rAAV.

[0055] As used herein, the term "approximately" refers to a deviation of + / - 10% from the baseline value.

[0056] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which this disclosure pertains.

[0057] autoimmune disease In one embodiment, the Disclosure provides a method for treating a subject having an autoimmune disease using the gene therapy of the Disclosure. "Autoimmune disease" means a condition, disease, or disorder in which the subject's immune response targets its own functioning cells, tissues, and / or organs.

[0058] In embodiments, several common diseases generally considered autoimmune include celiac disease, type 1 diabetes, Graves' disease, inflammatory bowel disease, multiple sclerosis, alopecia areata, Addison's disease, pernicious anemia, psoriasis, rheumatoid arthritis, and systemic lupus erythematosus. The presence and severity of symptoms of an autoimmune disease will depend on the location and type of autoimmune response that occurs. A person may have two or more autoimmune diseases simultaneously and exhibit symptoms of each. The signs and symptoms presented, as well as the disease itself, may depend on age, hormones, environment, and other factors. Generally, common symptoms include fatigue, low-grade fever, generally feeling unwell (sick), muscle and joint pain, and rashes.

[0059] Since multiple sclerosis is also a type of autoimmune disease, certain information provided in the previous section (i.e., under "Multiple Sclerosis") is also applicable to this current section (i.e., under "Autoimmune Diseases").

[0060] In other embodiments, autoimmune diseases include alopecia areata, Addison's disease, celiac disease, Crohn's disease, ulcerative colitis, autoimmune inflammatory myositis, Graves' disease, Hashimoto's thyroiditis, inflammatory bowel disease, multiple sclerosis, pemphigus, pernicious anemia, psoriasis, reactive arthritis, rheumatoid arthritis, Sjögren's syndrome, systemic lupus erythematosus, type 1 diabetes, or autoimmune hepatitis.

[0061] As used herein, terms such as “treatment” and “to treat” mean, when used in reference to an autoimmune disease, to obtain a desired pharmacological or physiological effect. The effect may be therapeutic in the sense of partial or complete cure of the disease, or partial or complete inhibition or prevention of adverse effects resulting from the autoimmune disease. Terms such as “treatment” and “to treat” may also mean improving clinical symptoms, delaying or preventing more adverse clinical symptoms, reducing the severity of an autoimmune disease or preventing symptoms or prevalence, or modulating, reducing, or preventing an autoimmune response or inflammation.

[0062] As used herein, “treatment” encompasses any treatment of a disease in mammals, particularly humans, and may include inhibiting a disease or condition, i.e., stopping its onset, and alleviating a disease, i.e., causing disease regression, or causing regression of an autoimmune response or inflammation.

[0063] Gene therapy for multiple sclerosis and / or autoimmune diseases In one embodiment, the disclosure provides a method for treating subjects with autoimmune diseases, such as multiple sclerosis, using gene therapy.

[0064] In embodiments, the methods, viral vectors, compositions for use, or uses of the present disclosure are carried out in combination with one or more other therapies (e.g., without limitation, therapies commonly used to treat multiple sclerosis and / or autoimmune diseases).

[0065] Vectors that can be used to deliver therapeutic nucleic acids include viral and nonviral vectors. Suitable vectors that can be used include adenoviruses, adeno-associated viruses, retroviruses, lentiviruses, HSV (herpes simplex virus), and plasmids. The advantage of herpes simplex virus vectors is their natural tropism to sensory neurons. However, adenovirus-associated virus vectors are the most popular due to their low risk of insertional mutagenesis and immunogenicity, lack of endogenous viral genes, and ability to be produced at high titers. Kantor et al. outline various methods of gene transfer into the central nervous system, while Goins et al. describe methods of gene therapy for the treatment of chronic peripheral nerve pain. See Kantor et al., Adv Genet. 87, 125-197 (2014) and Goins et al., Neurobiol. Dis. 48(2), 255-270 (2012), whose disclosures are incorporated herein by reference. In particular, successful gene delivery to Schwann cells and resident glial cells of peripheral nerves has been reported using various viral vectors. (Mason et al., Curr. Gene Ther. 11, 75-89 (2011)). If the vector is in a viral vector and the vector is packaged, cells can be infected using virions. If naked DNA is used, an appropriate transfection or transformation procedure for a specific host cell 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 form that provides a sufficiently efficient level of delivery, for example, in viral particles, liposomes, or nanoparticles, and can be complexed with polymers.

[0066] A nucleic acid (e.g., cDNA or transgene) that codes for a gene whose expression treats (e.g., reduces or prevents, without limitation) an autoimmune disease such as multiple sclerosis can be cloned into an expression cassette having regulatory elements such as a promoter (constitutive or regulated) that drives the transgene expression, and a polyadenylated 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 / regulated can be used.

[0067] In some embodiments, muscle-specific transcriptional regulatory elements are used. Muscle-specific regulatory elements include muscle-specific promoters, such as the mammalian muscle creatine kinase (MCK) promoter, the truncated MCK (tMCK) promoter, the myosin heavy chain (MHC) promoter, the hybrid α-myosin heavy chain enhancer / MHC enhancer-promoter (MHCK7) promoter, the C5-12 promoter, the mammalian desmin promoter, the mammalian troponin I (TNNI2) promoter, or the mammalian skeletal alpha-actin (ASKA) promoter. The development and improvement of muscle-specific promoters are generally based on the skeletal muscle α-actin, muscle creatine kinase, and desmin genes, as well as other genes expressed in muscle. The muscle-specific enhancers useful in this disclosure are selected from the group consisting of mammalian MCK enhancers, mammalian DES enhancers, and vertebrate troponin I IRE (TNI IRE, hereafter referred to as FIRE) enhancers, skeletal fast-twitch muscle troponin C gene elements, slow-twitch muscle cardiac troponin c gene elements, slow-twitch muscle troponin I gene elements, hypoxia-inducible nuclear factor (HIF) response elements (HREs), steroid-inducible elements, and glucocorticoid response elements (GREs). One or more of these muscle-specific enhancer elements may be used in combination with the muscle-specific promoters of this disclosure to provide tissue-specific regulatory elements.

[0068] The preferred viral vector for use in treating multiple sclerosis or autoimmune diseases by gene therapy is AAV. AAV-mediated gene delivery has emerged as an effective and safe tool for both preclinical and clinical trials of neurological disorders. Ojala et al., Neuroscientist., 21(1):84-98 (2015). Currently, rAAV is the most widely used vector for clinical trials of neurological disorders, and no adverse effects associated with the use of this vector have been reported from clinical trials to date. Adeno-associated viruses (AAVs) are nonpathogenic dependent viruses of the Parvoviridae family that require helper functions from other viruses, such as adenoviruses or herpes simplex viruses, to accomplish their life cycle. Wild-type (WT) AAV is characterized by a single-stranded DNA (ssDNA) genome of approximately 5 kb surrounded by a capsid, with inverted terminal repeats (ITRs) at both ends.

[0069] Adenovirus vectors for delivering transgenes to cells for applications such as in vivo gene therapy and in vitro testing and / or production of transgene products are generally derived from adenoviruses by deletion of the initial region 1(El) gene (Berkner, KL, Curr. Top. Micro. Immunol. 158 L39-66 1992). Deletion of the El gene results in a loss of replication of such adenovirus vectors and significantly reduces the expression of the remaining viral genes present in the vector. Recombinant adenovirus vectors have several advantages for use as gene delivery vehicles, including tropism in both dividing and non-dividing cells, minimal pathogenicity potential, the ability to replicate to high titers for the preparation of vector stocks, and the potential to carry large inserts. However, the presence of remaining viral genes in adenovirus vectors is considered potentially detrimental.

[0070] Therefore, in some embodiments, adenovirus vectors having deletions of various adenovirus gene sequences. In particular, pseudoadenovirus vectors (PAVs), also known as "gutless adenovirus" or miniadenovirus vectors, are adenovirus vectors derived from the genome of an adenovirus that contain the minimal cis-acting nucleotide sequence necessary for the replication and packaging of the vector genome and can contain one or more transgenes (see U.S. Patent No. 5,882,877, which is incorporated herein by reference, encompassing pseudoadenovirus vectors (PAVs) and methods for producing PAVs). Such PAVs, capable of accommodating up to approximately 36 kb of exogenous nucleic acid, are advantageous because the vector's carrying capacity is optimized while the potential for a host immune response to the vector or the generation of a replicable virus is reduced. A PAV vector contains 5' inverted terminal repeat (ITR) and 3' ITR nucleotide sequences that include the origin of replication, as well as cis-acting nucleotide sequences necessary for packaging the PAV genome, and can accommodate one or more transgenes with appropriate regulatory elements, such as promoters and enhancers.

[0071] AAV The recombinant AAV (rAAV) genomes of this disclosure include the nucleic acid molecule and one or more AAV ITRs adjacent to the nucleic acid molecule. The AAV DNA in the rAAV genome may be derived from any AAV serotype capable of inducing recombinant viruses, 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, AAVrh.10, and AAV-B1 (see, e.g., Gao et al., PNAS, 99:11854-11859 (2002)) and their variants (Viral Vectors for Gene Therapy: Methods and Protocols, ed. Machida, Humana Press, 2003). Furthermore, pseudotyped rAAV vectors may also be utilized in the manner described herein. Pseudotyped rAAV vectors contain the genome of one AAV serotype in the capsid of a second AAV serotype, for example, an rAAV vector containing an AAV2 capsid and an AAV1 genome, or an rAAV vector containing an AAV5 capsid and an AAV2 genome (Auricchio et al., (2001) Hum. Mol. Genet., 10(26):3075-81). The generation of pseudotyped rAAV is disclosed, for example, in WO01 / 83692. Other types of rAAV variants, such as rAAV with capsid mutations, are also intended. See, for example, Marsic et al., Molecular Therapy, 22(11):1900-1909 (2014). As described in the background technology section above, the nucleotide sequences of various AAV serotype genomes are known in the art. AAV1, AAV6, AAV8, or AAVrh.74 may be used to promote skeletal muscle-specific expression.

[0072] The DNA plasmid of this disclosure comprises the rAAV genome of this disclosure. To assemble the rAAV genome into infectious viral particles, the DNA plasmid is transferred to cells that are tolerant of infection with an AAV helper virus (e.g., adenovirus, E1-deficient adenovirus, or herpesvirus). Techniques for producing rAAV particles, in which the rAAV genome, rep and cap genes, and helper virus functions are provided to the cell, are standard in the art. The production of rAAV requires that the following components, the rAAV genome, the AAV rep and cap genes isolated from (i.e., not present in) the rAAV genome, and the helper virus functions, be present in a single cell (referred to herein as the packaging cell). The AAV rep and cap genes may originate from any AAV serotype capable of inducing recombinant virus, but are 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, or AAVrh.74, and their variants, and may originate from AAV serotypes different from the rAAV genome ITR. The production of pseudotyped rAAV is disclosed, for example, in WO01 / 83692, which is incorporated in its entirety herein by reference.

[0073] The method for generating packaging cells involves creating a cell line that stably expresses all the components necessary for rAAV particle production. For example, a plasmid (or multiple plasmids) containing selectable markers, such as an rAAV genome lacking AAV rep and cap genes, AAV rep and cap genes isolated from an rAAV genome, and a neomycin resistance gene, is incorporated into the cell genome. The rAAV genome has been introduced into bacterial plasmids by procedures such as GC tailing (Samulski et al., 1982, Proc. Natl. Acad. S6. USA, 79:2077-2081), addition of a synthetic linker containing restriction endonuclease cleavage sites (Laughlin et al., 1983, Gene, 23:65-73), or direct blunt-end ligation (Senapathy & Carter, 1984, J. Biol. Chem., 259:4661-4666). Next, the packaging cell line is infected with a helper virus such as adenovirus. The advantage of this method is that the cells are selectable and it is suitable for large-scale production of rAAV. Another example of a preferred method is to use adenovirus or baculovirus instead of plasmids to introduce the rAAV genome and / or rep and cap genes into the packaging cells.

[0074] The general principles of rAAV production are outlined, for example, in Carter, 1992, Current Opinions in Biotechnology, 1533-539, and Muzyczka, 1992, Curr. Topics in Microbial. and Immunol., 158:97-129). Various 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, WO95 / 13365 and corresponding U.S. Patent No. 5,658,776, WO95 / 13392, WO96 / 17947, PCT / US98 / 18600, WO97 / 09441 (PCT / US96 / 14423), WO97 / 08298 (PCT / US96 / 13872), WO97 / 21825 (PCT / US96 / 20777), WO97 / 06243 (PCT / FR96 / 01064), WO99 / 11764, Perrin et al. (1995) Vaccine 13:1244-1250, Paul et al. This is described in al. (1993) Human Gene Therapy 4:609-615, Clark et al. (1996) Gene Therapy 3:1124-1132, U.S. Patent Nos. 5,786,211, 5,871,982, and 6,258,595. The aforementioned documents are incorporated herein by reference in their entirety, with particular emphasis on the portions relating to rAAV production.

[0075] Accordingly, this disclosure provides packaging cells that produce infectious rAAV. In one embodiment, the packaging cells may be stably transformed cancer cells such as HeLa cells, 293 cells, and PerC.6 cells (allogeneic 293 strain). In another embodiment, the packaging cells may be non-transformed cancer cells, such as low-passage 293 cells (human fetal kidney cells transformed with adenovirus E1), MRC-5 cells (human fetal fibroblasts), WI-38 cells (human fetal fibroblasts), Vero cells (monkey kidney cells), and FRhL-2 cells (rhesus macaque fetal lung cells).

[0076] The recombinant AAV particles of this disclosure (i.e., infectious capsidized rAAV particles) comprise an rAAV genome. In exemplary embodiments, the genomes of both rAAVs lack AAV rep and cap DNA, i.e., there is no AAV rep or cap DNA between the ITRs of the genome. Examples of rAAVs that can be constructed to comprise the nucleic acid molecules of this disclosure are described in International Patent Application No. PCT / US2012 / 047999 (WO2013 / 016352), which is incorporated herein by reference in its entirety.

[0077] rAAV can be purified by methods standard in the art, for example, by column chromatography or a cesium chloride gradient. Methods for purifying rAAV from helper viruses are known in the art and include, for example, the methods disclosed in Clark et al., Hum. Gene Ther., 10(6):1031-1039 (1999), Schenpp and Clark, Methods Mol. Med., 69 427-443 (2002), U.S. Patent No. 6,566,118, and WO98 / 09657.

[0078] In another embodiment, the present disclosure contemplates a composition comprising the rAAV of the present disclosure. The composition of the present disclosure comprises rAAV and a pharmaceutically acceptable carrier. The composition may also include other components such as diluents and adjuvants. The acceptable carrier, diluent, and adjuvant are non-toxic to the recipient and preferably inert at the dosages and concentrations used, and include buffers such as phosphates, citrates, or other organic acids; antioxidants such as ascorbic acid; low molecular weight polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as Tween®, Pluronic®, or polyethylene glycol (PEG).

[0079] The titer of rAAV administered by the method of the present disclosure may vary, for example, depending on the particular rAAV, mode of administration, treatment goal, individual, and targeted cell type(s), and can be determined by standard methods in the art. The titer of rAAV can range from about 1×10 6 to about 1×10 7 to about 1×10 8 [[ID=[]] 9 to about 1×10 10 to about 1×10 11 to about 1×10 12 to about 1×10 13 [[ID=[]]to about 1×10 14 or more DNase-resistant particles (DRP). The dosage may also be expressed in units of viral genome (vg).

[0080] ​A method for transducing rAAV into target cells in vivo or in vitro is contemplated by this disclosure. An in vivo method comprises the step of administering an effective dose, or multiple effective doses, of the rAAV-containing composition of this disclosure to an animal (including, but not limited to, humans) that requires it. If the dose is administered before the onset of a disorder / disease, the administration is prophylactic. If the dose is administered after the onset of a disorder / disease, the administration is therapeutic. In embodiments of this disclosure, an effective dose is a dose that alleviates (e.g., eliminates or reduces, but not limited to) at least one symptom associated with the disorder / disease condition being treated, slows or prevents progression to a disorder / disease condition, reduces the extent of the disease, results in remission (partial or complete) of the disease, and / or prolongs survival.

[0081] In particular, the practical administration of rAAV according to this disclosure can be achieved by using any physical method for delivering rAAV to target tissue in an animal. Administration according to this disclosure includes, but is not limited to, intramuscular injection, bloodstream injection, and / or direct injection into the liver. Simply resuspending rAAV in phosphate-buffered saline has been demonstrated to be sufficient to provide a vehicle useful for muscle tissue expression, and there are no known limitations on carriers or other components that may be co-administered with rAAV (however, compositions that degrade DNA should be avoided in the usual manner with rAAV). The capsid protein of rAAV may be modified so that rAAV is targeted to a specific target tissue of interest, such as muscle. See, for example, WO02 / 053703, which is incorporated herein by reference. Pharmaceutical compositions can be prepared as injectable formulations or as topical formulations delivered to muscle by transdermal transport. Numerous formulations for both intramuscular injection and transdermal transport have been previously developed and can be used in the implementation of the disclosure. rAAV can be used with any pharmaceutically acceptable carrier to facilitate administration and handling.

[0082] Transduction can be performed using gene cassettes containing tissue-specific regulatory elements. For example, one embodiment of the present disclosure provides a method for transducing muscle cells and muscle tissue directed by muscle-specific regulatory elements, but is not limited to, those derived from actin and myosin gene families, e.g., the myoD gene family [see Weintraub et al., Science, 251:761-766 (1991)], muscle cell-specific enhancer binding factor MEF-2 (Cserjesi and Olson, Mol Cell Biol 11:4854-4862 (1991)), regulatory elements derived from human skeletal actin genes (Muscat et al., Mol Cell Biol, 7:4089-4099 (1987)), cardiac actin genes, muscle creatine kinase sequence elements (Johnson et al., Mol Cell See Biol, 9:3393-3399 (1989), as well as regulatory elements derived from the mouse creatine kinase enhancer (mCK) element, the skeletal fast-twitch muscle troponin C gene, the slow-twitch muscle cardiac troponin C gene, and the slow-twitch muscle troponin I gene: hypoxia-inducible nuclear factor (Semenza et al., Proc Natl Acad Sci USA, 88:5680-5684 (1991)), 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 regulatory elements.

[0083] Muscle tissue is an attractive target for in vivo DNA delivery because it is easily accessible, gene expression can be restricted to muscle tissue which has muscle-specific promoters, and NT-3 is naturally produced by muscle. This disclosure aims to achieve sustained expression of NT-3 from transduced muscle fibers.

[0084] "Muscle cells" or "muscle tissue" means cells or groups of cells derived from any type of muscle (e.g., skeletal muscle and smooth muscle, e.g., gastrointestinal tract, bladder, blood vessels, or cardiac tissue). Such muscle cells may be differentiated or undifferentiated, including myoblasts, myocytes, myotubes, cardiomyocytes, and cardiac muscle cells.

[0085] The term “transduction” is used to refer to the administration / delivery of the coding region of NT-3 to recipient cells, either in vivo or in vitro, via the replication-deficient rAAV of this disclosure, resulting in NT-3 expression by the recipient cells.

[0086] In one embodiment, the gene therapy is NT-3 gene therapy via rAAV delivery. An AAV expression cassette carrying a human NT-3 cDNA coding sequence under the control of a triple muscle-specific creatine kinase (tMCK) promoter is disclosed herein. It has been previously shown that improvements in peripheral nerve motor function, histopathology, and electrophysiology can be achieved by using AAV1 to increase neurotrophin-3 expression in tremor (Try) mice, a model for the Charcot-Marie-Tooth disease variant CMT1A. See Sahenk et al., Mol Ther. 22(3):511-21 (2014), whose disclosure is incorporated herein by reference.

[0087] Therefore, this disclosure provides a method for administering an effective dose (or essentially a dose administered simultaneously or at intervals) of rAAV encoding NT-3 to a patient in need.

[0088] Dosage and route of administration This disclosure provides effective doses of rAAV and compositions of this disclosure, including combination therapies, for topical and systemic administration. For example, systemic administration means administration to the circulatory system so that the whole body is affected. Systemic administration includes enteral administration, such as absorption through the gastrointestinal tract, and parenteral administration through injection, infusion, or transplantation.

[0089] Therefore, the routes of administration for rAAV intended in the manner described above are not limited to these, but include intraperitoneal (IP), intramuscular (IM), and intravascular routes, such as interarterial perfusion (ILP) and intravenous (IV) routes.

[0090] The dose of rAAV administered by the methods disclosed herein may vary depending, for example, on the specific rAAV, mode of administration, therapeutic target, individual, and targeted cell type(s), and may be determined by standard methods in the art. More than one dose, e.g., one, two, three, or more doses, may be administered. The potency of rAAV at a given dose is approximately 1 × 10⁶ per ml. 6 , about 1×10 7 , about 1×10 8 , about 1×10 9 , about 1×10 10 , about 1×1 11 , about 1×10 12 , about 1.5×10 12 , about 1×10 12 , about 3×10 12 , about 4×10 12 , about 5×10 12 , about 6×10 12 , about 6.5×10 12 , about 7×10 12 , 1 x 10 13 , about 1×10 14 , or approximately 1 x 10 15 The above range may apply to DNase-resistant particles (DRPs). The dosage can also be expressed in units of viral genome (vg) (i.e., 1 × 10⁻⁶ each). 7 vg, 1×10 8 vg, 1×10 9 vg, 1×10 10 vg, 1×10 11 vg, 1×10 12 vg, approx. 1.5×10 12 vg, approx. 1×10 12 vg, approx. 3×10 12 vg, approx. 4×10 12 vg, approx. 5×10 12 vg, approx. 6×10 12 vg, approx. 6.5×1012 vg, approx. 7×10 12 vg, 1×10 13 vg, 1×10 14 vg, 1×10 15 A method for titrating rAAV is described in Clark et al., Hum. Gene Ther., 10:1031-1039 (1999). In embodiments, the dosage may also be expressed in units of viral genome (vg / kg) (i.e., 1 × 10⁻⁶ each). 7 vg / kg, 1 × 10 8 vg / kg, 1 × 10 9 vg / kg, 1 × 10 10 vg / kg, 1 × 10 11 vg / kg, 1 × 10 12 vg / kg, approximately 1.5×10 12 vg / kg, approximately 1×10 12 vg / kg, approx. 3×10 12 vg / kg, approx. 4×10 12 vg / kg, approx. 5×10 12 vg / kg, approximately 6×10 12 vg / kg, approximately 6.5×10 12 vg / kg, approximately 7×10 12 vg / kg, 1 × 10 13 vg / kg, 1 × 10 14 vg / kg, 1 × 10 15 (vg / kg).

[0091] In some embodiments of the aforementioned method, where the route of administration is the IM route, the dose of rAAV administered is approximately 1.5 × 10⁻⁶. 12 ~At least approximately 6.5 × 10 12 The value is vg / kg. (All ranges in this specification are intended to represent individual values ​​within a range, as well as individual upper and lower limits within each range.) In some embodiments of the aforementioned method, where the route of administration is IM, the dose of rAAV administered is 2 × 10⁻⁶ 12 The value is vg / kg. In some embodiments of the aforementioned method, where the route of administration is IM, the dose of rAAV administered is 4 × 10⁻⁶ 12The value is vg / kg. In some embodiments of the aforementioned method, where the route of administration is IM, the dose of rAAV administered is 6 × 10⁻⁶ 12 It is vg / kg.

[0092] Human patients are subjects intended herein for treatment by IM delivery. Such patients include i) adult subjects (18 years or older), particularly elderly adults (>65 years), diagnosed with multiple sclerosis or autoimmune disease, and ii) males and females of any ethnic or racial group. In embodiments, subjects are pediatric subjects, e.g., subjects under 18 years of age. For example, some patients may experience age-related slowing, difficulty climbing stairs or standing up from a seated position, slow walking, easy fatigue, difficulty maintaining balance, difficulty raising arms above shoulder height, or difficulty maintaining an upright posture. Suitable patients may not include, for example, i) active viral infection based on clinical observation or serological evidence of HIV or hepatitis A, B, or C infection; ii) continuous immunosuppressive therapy or immunosuppressive therapy (e.g., corticosteroids, cyclosporine, tacrolimus, methotrexate, cyclophosphamide, intravenous immunoglobulin) within 6 months prior to the start of the study; iii) persistent leukopenia or leukocytosis (WBC ≤ 3.5 K / μL or ≥ 20.0 K / μL), or absolute neutrophil count < 1.5 K / μL; iv) AAV1-binding antibody titer ≥ 1:50 as determined by ELISA immunoassay; v) in the opinion of the PI, a requirement for comorbidity or chronic drug treatment that would create an unnecessary risk of gene transfer; vi) ankle contracture or surgery that would prevent appropriate muscle strength testing; vii) pregnancy, lactation, or planning of pregnancy; viiii) other causes of neuropathy; and / or ix) limb surgery in the past 6 months. In an exemplary clinical protocol, patients with multiple sclerosis or autoimmune disease receive a total dose of scAAV1, tMCK, and NTF3, split into the medial and lateral heads of the gastrocnemius and tibialis anterior (TA) muscles of the leg, which are predominantly showing muscle weakness and instability. The subjects are as follows: i) 2 × 10 12 ii) Low dose rAAV (total dose) in vg / kg or 6 × 10 12Receive one of the high-dose rAAVs (total dose) in vg / kg.

[0093] In one embodiment, rAAV is administered by IM injection without a diluent. In alternative embodiments, the composition for intramuscular injection may contain an adjuvant such as sesame or peanut oil, or it may be aqueous propylene glycol and a sterile aqueous solution. Such aqueous solutions may be buffered as needed, and the liquid diluent may be first isotonicized with physiological saline or glucose. Solutions of rAAV as a free acid (DNA contains acidic phosphate groups) or a pharmacokinetically acceptable salt may be prepared in water suitably mixed with a surfactant such as hydroxypropyl cellulose. Dispersions of rAAV may also be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof, as well as in oil. Under normal storage and use conditions, these formulations contain preservatives to prevent microbial growth. In this regard, all sterile aqueous media used are readily available by standard techniques well known to those skilled in the art.

[0094] Suitable pharmaceutical carriers, diluents, or excipients for injection applications include sterile aqueous solutions or dispersions, and sterile powders for the immediate preparation of sterile injection solutions or dispersions. In all cases, these forms must be sterile and fluid enough to allow for easy syringe injection. They must be stable under manufacturing and storage conditions and protected against microbial contamination, such as bacteria and fungi. Carriers may be solvents or dispersion media containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Adequate fluidity can be maintained, for example, by the use of coating agents such as lecithin, by maintaining the required particle size in the case of dispersants, and by the use of surfactants. Prevention of microbial action can be provided by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it would be preferable to include isotonic agents, such as sugars or sodium chloride. The prolonged absorption of injectable compositions can be achieved by using absorption-delaying agents, such as aluminum monostearate and gelatin.

[0095] Sterile injectable solutions are prepared by combining the required amount of rAAV in a suitable solvent, along with various other components listed above as needed, and then sterilizing by filtration. Generally, dispersions are prepared by mixing the sterilized active ingredient into a sterile vehicle containing a basic dispersion medium and other required components from those listed above. For sterile powders for the preparation of sterile injectable solutions, preferred methods of preparation are vacuum drying and freeze-drying techniques, which yield a powder of the active ingredient plus any additional desired components from those solutions that have been previously sterilized and filtered.

[0096] Transduction with rAAV can also be performed in vitro. In one embodiment, desired target muscle cells are isolated from the target, transduced with rAAV, and reintroduced into the target. Alternatively, syngeneic or heterologous muscle cells may be used if those cells do not produce an inappropriate immune response in the target.

[0097] In another embodiment, an rAAV genome is provided herein. The rAAV genome to be administered contains NT-3 polynucleotides under the control of a transcriptional regulatory sequence. The rAAV genome lacks AAV rep and cap DNA. The AAV DNA in the rAAV genome may be derived from any AAV serotype capable of inducing recombinant viruses, including, but not limited to, 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, AAVrh.74, or variants thereof. The nucleotide sequences of the genomes of these AAV serotypes are known in the art as described in the background section above.

[0098] In some embodiments, the transcriptional regulatory sequences of the rAAV genome are, but are not limited to, muscle-specific regulatory elements, derived from the actin and myosin gene families, e.g., the myoD gene family [see Weintraub et al., Science, 251:761-766 (1991)], muscle cell-specific enhancer-binding factor MEF-2 [Cserjesi and Olson, Mol. Cell. Biol., 11:4854-4862 (1991)], regulatory elements derived from human skeletal actin genes [Muscat et al., Mol. Cell. Biol., 7:4089-4099 (1987)], cardiac actin genes, muscle creatine kinase (MCK) promoters [Johnson et al.] al., Mol. Cell. Biol., 9:3393-3399 (1989)), and MCK enhancer, MHCK7 promoter (modified version of the MCK promoter incorporating the enhancer from the myosin heavy chain (Salva et al., Mol. Ther., 15:320-329 (2007)), regulatory elements derived from the skeletal fast muscle troponin C gene, the slow muscle cardiac troponin C gene, and the slow muscle troponin I gene: hypoxia-inducible nuclear factor (Semenza et al., Proc. Natl. Acad. Sci. USA, 88:5680-5684 (1991)), steroid-inducible elements and promoters including the glucocorticoid response element (GRE) (Mader and See White, Proc. Natl. Acad. Sci. USA, 90:5603-5607 (1993), as well as other regulatory elements. In some embodiments, the transcriptional regulatory elements include an MCK promoter / enhancer contained in the AAV.tMCK.NTF3 genome disclosed herein. The MCK promoter / enhancer consists of a muscle creatine kinase promoter to which a fused enhancer element (enh358MCK, 584 bp) is added. A triple tandem of the MCK enhancer (206 bp) was ligated to an 87 bp basal promoter in the tMCK promoter / enhancer.In some embodiments, the transcriptional regulatory element and the tMCK promoter / enhancer are contained within the AAV.tMCK.NTF3 genome shown in SEQ ID NO: 9. In some embodiments, the tMCK promoter / enhancer follows the nucleotide sequence of SEQ ID NO: 11.

[0099] In some embodiments, the NT-3 polynucleotide in the rAAV genome is the NT-3 cDNA shown in SEQ ID NO: 1 (corresponding to nucleotides 1077-1850 of SEQ ID NO: 3). In some embodiments, the NT-3 polynucleotide in the rAAV genome is either the NT-3 cDNA shown in GenBank accession number NM_001102654 or the NTF3 cDNA sequence shown in SEQ ID NO: 1, or 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 with the NT-3 cDNA sequence shown in SEQ ID NO: 1. In some embodiments, the variant NT-3 polynucleotide encodes the same NT-3 polypeptide as the polypeptide encoded by the NT-3 cDNA of SEQ ID NO: 1. The amino acid sequence of the NT-3 polypeptide encoded by the NT-3 cDNA shown as SEQ ID NO: 1 or provided as GenBank accession number NM_001102654 is shown in SEQ ID NO: 2. In some embodiments, the variant NT-3 polynucleotide encodes a variant NT-3 polypeptide having at least one amino acid sequence modification compared to the amino acid sequence of the polypeptide encoded by the NT-3 cDNA shown as SEQ ID NO: 1 or provided as GenBank accession number NM_001102654 (SEQ ID NO: 2). The amino acid sequence modification may be, for example, a substitution, deletion, or insertion of one or more amino acids, preferably conservative substitutions. The variant NT-3 polypeptide may have any combination of amino acid substitutions, deletions, or insertions in which polypeptide activity is preserved.In one embodiment, the variant NT-3 polypeptide may have a number of amino acid changes, and its amino acid sequence may share 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 the NT-3 cDNA, which is shown as SEQ ID NO: 1 or provided as GenBank accession number NM_001102654.

[0100] In some embodiments, the rAAV genome is the AAV.tMCK.NTF3 genome, and the sequence of its NT-3 gene cassette is shown in Sequence ID No. 9.

[0101] In yet another embodiment, an isolated nucleic acid is provided that contains the nucleotide sequence shown in nucleotides 7-2248 of SEQ ID NO: 3. In some embodiments, the isolated nucleic acid consists of the nucleotide sequence shown in nucleotides 7-2248 of SEQ ID NO: 3.

[0102] Isolated nucleic acids are also provided, comprising, in 5' to 3' order, (i) a first AAV2 inverted terminal repeat sequence (ITR) (SEQ ID NO: 4), (ii) a muscle creatine kinase promoter sequence (shown 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, or 100% identical to SEQ ID NO: 2, or is encoded by nucleotides 1077-1850 of SEQ ID NO: 3.

[0103] The goal is to create recombinant AAVs containing the aforementioned nucleic acids, and rAAVs containing nucleotide sequences that are at least 90% identical to the nucleotide sequence shown in Sequence ID No. 1.

[0104] A DNA plasmid containing the rAAV genome of this disclosure is provided. The DNA plasmid contains the rAAV genome contemplated herein. An exemplary DNA plasmid is provided as Sequence ID No. 3 and noted in Table 3 (see Example 2). The DNA plasmid is transferred to a cell tolerant of infection with an AAV helper virus (e.g., adenovirus, E1 deletion adenovirus, or herpesvirus) for assembly of the rAAV genome into infectious viral particles. Techniques for producing rAAV particles, in which the AAV genome, rep and cap genes, and helper virus functions to be packaged are provided to the cell, are standard in the art. The production of rAAV requires that the following components, the rAAV genome, the AAV rep and cap genes isolated from (i.e., not present in) the rAAV genome, and the helper virus functions, be present in a single cell (referred to herein as the packaging cell). The AAV rep and cap genes may originate from any AAV serotype capable of inducing recombinant viruses, but are not limited to, 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, AAVrh.74, or variants thereof, from an AAV serotype different from the rAAV genome ITR. The generation of pseudotyped rAAV is disclosed, for example, in WO01 / 83692. Other types of rAAV variants, e.g., rAAV with capsid mutations, are also considered. See, for example, Marsic et al., Molecular Therapy, 22(11):1900-1909 (2014).

[0105] The method for generating packaging cells involves creating a cell line that stably expresses all the components necessary for rAAV particle production. For example, a plasmid (or multiple plasmids) containing selectable markers, such as an rAAV genome lacking AAV rep and cap genes, AAV rep and cap genes isolated from an rAAV genome, and a neomycin resistance gene, is incorporated into the cell genome. The rAAV genome has been introduced into bacterial plasmids by procedures such as GC tailing (Samulski et al., 1982, Proc. Natl. Acad. S6. USA, 79:2077-2081), addition of a synthetic linker containing restriction endonuclease cleavage sites (Laughlin et al., 1983, Gene, 23:65-73), or direct blunt-end ligation (Senapathy & Carter, 1984, J. Biol. Chem., 259:4661-4666). Next, the packaging cell line is infected with a helper virus such as an adenovirus. The advantage of this method is that the cells are selectable and it is suitable for large-scale production of rAAV. Another example of a suitable method is the use of an adenovirus or baculovirus rather than a plasmid to introduce the rAAV genome and / or rep and cap genes into the packaging cells. Methods for producing rAAV with a self-complementary genome are also known in the art.

[0106] The general principles of rAAV production are outlined, for example, in Carter, 1992, Current Opinions in Biotechnology, 1533-539, and Muzyczka, 1992, Curr. Topics in Microbial. and Immunol., 158:97-129). Various 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, WO95 / 13365 and corresponding U.S. Patent No. 5,658,776, WO95 / 13392, WO96 / 17947, PCT / US98 / 18600, WO97 / 09441 (PCT / US96 / 14423), WO97 / 08298 (PCT / US96 / 13872), WO97 / 21825 (PCT / US96 / 20777), WO97 / 06243 (PCT / FR96 / 01064), WO99 / 11764, Perrin et al. (1995) Vaccine 13:1244-1250, Paul et al. This is described in al. (1993) Human Gene Therapy 4:609-615, Clark et al. (1996) Gene Therapy 3:1124-1132, U.S. Patent Nos. 5,786,211, 5,871,982, and 6,258,595. The aforementioned documents are incorporated herein by reference in their entirety, with particular emphasis on the portions relating to rAAV production.

[0107] In a further embodiment, the disclosure provides packaging cells that produce infectious rAAV. In one embodiment, the packaging cells may be stably transformed cancer cells such as HeLa cells, 293 cells, and PerC.6 cells (allogeneic 293 strain). In another embodiment, the packaging cells may be non-transformed cancer cells, such as low-passage 293 cells (human fetal kidney cells transformed with adenovirus E1), MRC-5 cells (human fetal fibroblasts), WI-38 cells (human fetal fibroblasts), Vero cells (monkey kidney cells), and FRhL-2 cells (rhesus macaque fetal lung cells).

[0108] rAAV can be purified by methods standard in the art, for example, by column chromatography or a cesium chloride gradient. Methods for purifying rAAV from helper viruses are known in the art and include, for example, the methods disclosed in Clark et al., Hum. Gene Ther., 10(6):1031-1039 (1999), Schenpp and Clark, Methods Mol. Med., 69 427-443 (2002), U.S. Patent No. 6,566,118, and WO98 / 09657.

[0109] Therefore, in other embodiments, the disclosure intends an rAAV comprising an NT-3 polynucleotide. In some embodiments, the rAAV comprises an AAVrh74 capsid and an NT-3 polynucleotide. In some embodiments, the rAAV genome lacks AAV rep and cap DNA. In some embodiments of the method, the rAAV is rAAVrh.74.tMCK.NTF3. In some embodiments, the rAAV is a self-complementary genome.

[0110] In another embodiment, the Disclosure envisions compositions comprising rAAV as described herein. The compositions of the Disclosure comprise rAAV in a pharmaceutically acceptable carrier. The compositions may also comprise other components such as diluents. Acceptable carriers and diluents are nontoxic to the recipient and preferably inactive at the dosage and concentration used, and comprise buffers such as phosphates, citrates, or other organic acids; antioxidants such as ascorbic acid; low molecular weight polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as Tween®, Pluronic®, or polyethylene glycol (PEG). In some embodiments, rAAV is formulated in Tris, MgCl2, NaCl, and Pluronic® F68. In some embodiments, rAAV is formulated in 20 mM Tris (pH 8.0), 1 mM MgCl2, and 200 mM NaCl containing 0.001% Pluronic® F68.

[0111] Combination therapy is also intended herein. Combinations as used herein include concurrent or sequential therapy. Combinations of the methods disclosed herein with standard medical treatments (e.g., corticosteroids and / or immunosuppressants) are particularly intended, as are combinations with novel therapies. In various embodiments, the subject is treated with a corticosteroid before, during, or after (or in any permutation of two or more combinations of the three potentials), and the subject is treated according to the methods intended herein. For example, a combination includes administering a corticosteroid, such as prednisolone, before, during, and / or after administration of rAAV.

[0112] Sterile injectable solutions are prepared by incorporating the required amount of rAAV into a suitable solvent, along with various other components listed above as needed, and then sterilizing by filtration. Generally, dispersions are prepared by mixing the sterilized active ingredient into a sterile vehicle containing a basic dispersion medium and other required components from those listed above. For sterile powders for the preparation of sterile injectable solutions, preferred preparation methods are vacuum drying and freeze-drying techniques, which yield a powder of the active ingredient and any additional desired components from its previously sterilized filtered solution.

[0113] Neurotofin-3 In some embodiments, therapeutically effective doses of NT-3, pro-NT-3, or their NT-3 analogs are administered to subjects 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 that has activity on certain neurons in the peripheral and central nervous systems, best known for helping to support the survival and differentiation of existing neurons and promoting the growth and differentiation of new neurons and synapses.

[0114] As used herein, the term “polypeptide” refers to an oligopeptide, peptide, or protein sequence, or any fragment, part, or subunit thereof, as well as naturally occurring or synthetic molecules. The term “polypeptide” also includes amino acids linked to each other by peptide bonds or modified peptide bonds, i.e., peptide isostea, and may contain any type of modified amino acids. The term “polypeptide” also includes peptides and polypeptide fragments, motifs, glycosylated polypeptides, all “mimicking” and “peptide-mimicking” polypeptide forms, and retro-inversion peptides (also referred to as whole D-retro or mtro-enantiopeptides).

[0115] "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 a candidate sequence that is identical to a known peptide after the sequences have been aligned and gaps introduced as necessary to achieve the maximum homology percentage, and no conservative substitutions are considered as part of sequence identity. N-terminus, C-terminus, or internal extensions, deletions, or insertions into a peptide sequence should not be interpreted as affecting homology.

[0116] Substantially similar peptides include those that differ by one or more amino acid modifications, where the modification, e.g., substitution, addition, or deletion of amino acid residues, does not disrupt the properties of the relevant peptide, e.g., its ability to associate with FAK or NANOG. Furthermore, only sequences describing or encoding proteins in which only conservative substitutions are made in conserved regions are substantially similar overall. Preferred, substantially similar sequences also retain the unique activity of the polypeptide.

[0117] Examples of conservative substitutions include substitutions of nonpolar (hydrophobic) residues such as isoleucine, leucine, or methionine with another residue. Similarly, this disclosure intends to substitute a single polar (hydrophilic) residue, such as between arginine and lysine, between glutamine and asparagine, and between glycine and serine. In addition, substitutions of basic residues such as lysine, arginine, or histidine with another, or substitutions of a single acidic residue such as aspartic acid or glutamic acid with another, are also intended. Examples of non-conservative substitutions include substitutions of nonpolar (hydrophobic) residues such as isoleucine, valine, leucine, alanine, and methionine with polar (hydrophilic) residues such as cysteine, glutamine, glutamic acid, and lysine, and / or substitutions of polar residues with nonpolar residues.

[0118] The term “conservative substitution” also includes the use of chemically derivatized residues instead of non-derivatized residues, as long as the peptide retains the ability necessary 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 that do not affect the ability necessary to associate with NT-3. For example, substantially similar peptides may contain an N-terminal or C-terminal cysteine, thereby allowing the peptide to covalently bind to a carrier protein, such as albumin, if desired. Such binding can reduce the removal of the peptide from the blood and also reduce the rate of peptide proteolysis. In addition, for the purposes of this disclosure, peptides containing D-amino acids instead of L-amino acids are also included in the term “conservative substitution.” The presence of such D-isomers can help minimize proteolytic activity and peptide removal.

[0119] In some embodiments, the pro-neurotrophin-3 protein (pro-NT-3) is administered to the subject. The pro-form of neurotrophin-3 is an approximately 30 kDa NT-3 precursor form, which is converted to mature NT by enzymatic cleavage and removal of the approximately 15 kDa N-terminal pro-domain. See Tauris et al., Eur. J Neurosci, 33(4), 622-631 (2011).

[0120] Administration and Formulation Vectors or peptides used in conjunction with some embodiments of this disclosure can be incorporated into pharmaceutical compositions suitable for administration to a subject. In some specific embodiments, the pharmaceutical composition comprises the vector of this disclosure and a pharmaceutically acceptable carrier. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antimicrobial and antifungal agents, isotonic agents, and absorption retarders that are physiologically compatible. Examples of pharmaceutically acceptable carriers include water, saline, phosphate-buffered saline, dextrose, glycerol, ethanol, and one or more combinations thereof. Often, it may be preferable to include isotonic agents, such as sugars, polyhydric alcohols, such as mannitol, sorbitol, or sodium chloride, in the composition. The pharmaceutically acceptable carrier may further include small amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives, or buffers, that improve the shelf life or efficacy of the vector or pharmaceutical composition.

[0121] Vectors or peptides may be administered acutely (i.e., during the onset of the condition or immediately after an event leading to muscle atrophy), prophylactically (e.g., before a planned surgery or before the appearance of signs or symptoms), or during the course of muscle atrophy to reduce or improve the progression of symptoms that would otherwise occur. The timing and intervals of administration may vary depending on the condition being treated and may be determined by those skilled in the art, with intervals ranging from several hours to several days, or over periods of several hours, several days, or even several weeks.

[0122] Compositions containing vectors or peptides are generally administered intravenously. When administered intravenously, the composition may be combined with other components, such as carriers and / or adjuvants. Peptides may also be covalently bound to protein carriers, such as albumin, to minimize peptide removal. The properties of the other components are not limited, except that such components must be pharmaceutically acceptable, effective for their intended administration, and not reduce the activity of the active component of the composition.

[0123] Pharmaceutical forms suitable for injection include sterile aqueous solutions or dispersions and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. In all cases, the final solution form must be sterile or fluid. Typical carriers include solvents or dispersion media containing, for example, aqueous buffered water (i.e., biocompatible buffers), ethanol, polyols, such as glycerol, propylene glycol, polyethylene glycol, suitable mixtures thereof, surfactants, or vegetable oils. Sterilization can be achieved by any technique recognized in the art, including, but not limited to, filtration or addition of antimicrobial or antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, or thimerosal. Furthermore, isotonic agents such as sugars or sodium chloride may be incorporated into the composition.

[0124] The production of a sterile, injectable solution containing the target peptide is achieved by incorporating the required amounts of these compounds into a suitable solvent, along with the various components listed above as needed, followed by sterilization, preferably by filtration. To obtain a sterile powder, the solution is vacuum-dried or freeze-dried as needed.

[0125] When the peptides of this disclosure are administered orally, the pharmaceutical composition containing an effective dose of the peptide may also contain an inert diluent, an absorbable food carrier, etc., and may be contained in a hard or soft shell gelatin capsule, which can be compressed into a tablet, or in an elixir, suspension, syrup, etc. Thus, the target peptide is formulated with a therapeutically effective amount of a suitable pharmaceutically acceptable carrier for convenient and effective administration in a pharmaceutically effective amount.

[0126] As used herein, the terms “effective dose” or “therapeutic effective dose” refer to an amount of drug sufficient to stimulate muscle growth or to reduce or prevent muscle atrophy. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the specific therapeutic agent, the mode and / or route of administration, etc. However, it will be understood that the total daily dose of the compounds and compositions of this disclosure may be determined by the attending physician within the bounds of sound medical judgment. A specific therapeutic effective dose level for any particular subject or organism will depend on a variety of factors, including the disorder being treated and its severity, the activity of the particular compound used, the particular composition used, the subject’s age, weight, overall health, sex, and diet, the timing of administration, route of administration, and excretion rate of the particular composition used, the duration of treatment, drugs used in combination with or concurrently with the particular composition used, and similar factors well known in the medical field.

[0127] The vector or peptide can be administered in a manner suitable for the drug formulation and in a therapeutically effective amount. The systemic dose depends on the patient's age, weight, and condition, as well as the route of administration. For example, a suitable dose of the peptide for administration to an adult human is in the range of about 0.001 to about 20.0 mg per kilogram of body weight. The peptide should preferably be administered in an amount of at least about 50 mg per dose, and more preferably in an amount of up to about 500 mg to about 1 gram per dose. Since the peptide composition of this disclosure is eventually removed from the bloodstream, re-administration of the composition is indicated and preferred. [Examples]

[0128] Accordingly, aspects and embodiments of this disclosure are illustrated by the following examples. However, there are a wide variety of other embodiments within the scope of this disclosure that should not be limited to the specific examples provided herein.

[0129] Example 1 NT-3 delivery using scAAV1.tMCK.NTF3 The administered composition is a non-replicating recombinant adeno-associated virus called scAAV1.tMCK.NTF3, and a diagram of its cassette plasmid is shown in Figure 2. rAAV contains the human NT-3 gene under the control of the tMCK muscle-specific promoter. The in vivo biological efficacy of rAAV is (1 × 10⁻¹⁶). 11 Intramuscular injection into the gastrocnemius muscle of C57B16 mice (vg), followed by gene injection, was used to quantify circulating NT-3 in the serum by ELISA 4–6 weeks later.

[0130] Firstly, it was demonstrated that scAAV1.CMV.NTF3 delivered to the gastrocnemius muscle resulted in long-term therapeutic NT-3 serum levels sufficient to provide functional, electrophysiological, and histopathological improvements in TrJ neurons. Next, we investigated whether it was possible to achieve the same level of expression by producing the required rAAV dose and packaging the expression cassette with scAAV1. Dose-response studies were conducted in C57BL / 6 mice, with three doses (3 × 10⁶). 9 vg, 1×10 10 vg, and 3×10 10 We compared serum NT-3 ELISA data after intramuscular injection of scAAV1.tMCK.NTF3 and scAAV1.CMV.NTF3 in vg). 1×10 11 Administration of scAAV1, CMV, NTF3 rAAV in vg resulted in significantly higher NT-3 levels than single-stranded rAAV at the same dose, consistent with greater potency, using an autocomplementary vector. (Semi-log lower dose (3 × 10)) 10 In vg), rAAV containing either CMV or tMCK is 1 × 10 11The NT-3 serum levels obtained from mice treated with scAAV1, CMV, and NTF3 at a dose of vg were equivalent to those obtained from mice that received scAAV1, CMV, and NTF3, indicating a biological response. NT-3 levels (mean ± SEM) were measured from TrJ mice 24 weeks after injection. Significant differences in NT-3 levels were observed among all 7 groups, p-value < 0.0001. Significant differences in NT-3 levels were observed for the highest and intermediate doses of rAAV for both promoter and control groups. However, analysis did not find significant differences for lower doses of both rAAV groups. The Kruskal-Wallis test was 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). The Mann-Whitney U test was used to compare NT-3 between each group and the PBS (control) group, and Bonferroni correction was used to adjust for multiple comparisons. See Sahenk et al., Mol.Ther.22(3):511-521, 2014, which is incorporated herein by reference in its entirety.

[0131] Muscle diameter increases 40 weeks after treatment: The effect of NT-3 gene therapy is compared to PBS with scAAV1.CMV.NTF3 (1×10 11 Muscle fiber size at 40 weeks post-injection was evaluated in TrJ mice against a subset of animals injected with vg). Neurogenic changes characterized by atrophied keratinized fibers and group atrophy were evident in the muscles of untreated mice, but evidence of nerve regeneration as fiber type grouping and overall fiber size increase was recognizable as a therapeutic effect. Muscle fiber size histograms generated from the contralateral anterior and posterior compartment muscles of the left lower limb (tibialis anterior and gastrocnemius) showed an increase in fiber diameter.

[0132] Additional studies have shown that NT-3 stimulates the Akt / mTOR pathway in SC cells, resulting in improved myelination and axonal radial growth in nerves. NT-3 also has a direct stimulating effect on myotubes via Trk-C receptors, demonstrating its role in increasing fiber diameter in the muscles of TrJ mice.

[0133] Testing with autocomplementary (sc)AAV1 and the use of muscle-specific cleavage-type creatine kinase (tMCK) promoters scAAV allows for lower doses and is added to the enhanced safety and dosage levels that will meet production standards. The use of the tMCK promoter is again an important objective, providing greater safety by avoiding off-target effects. In the following series of experiments, the efficacy of scAAV1.NTF3 under the control of the CMV promoter was measured in the semi-log range (3 × 10⁻¹⁰). 9 vg, 1×10 10 vg, and 3×10 10 The results were compared with muscle-specific tMCK promoters administered at three doses within the same range (vg). The efficacy of AAV1.NTF3 gene translocation in TrJ mouse peripheral nerves was evaluated by electrophysiological (Table 2) and morphological tests 24 weeks after translocation. Evidence of transgene expression was assessed by measuring serum NT-3 levels using ELISA. [Table 2]

[0134] Investigators during electrodiagnostic testing were blinded to the treatment group. No statistically significant differences were observed between AAV1.NTF3.CMV (high dose, HD) and AAV1.NTF3.tMCK (high dose, HD) on CMAP, and the use of the muscle-specific tMCK promoter was preferred. This was further supported by NT-3 levels in the ELISA assay, where significant differences in NT-3 levels were observed for the highest and intermediate doses of rAAV for both the promoter and control groups.

[0135] Example 2 Construction of an NT-3 expressing AAV construct The design of a self-complementary rAAV virus vector having serotype 1 containing NT-3 cDNA under tMCK was previously described in Sahenk et al., Mol Ther, 22(3):511-521 (2014), which is incorporated herein by reference in its entirety. Ali-coated viruses were kept at -80°C until use. Blood samples were collected from treated and untreated mice by ocular hemorrhage under anesthesia at 6 and 16 weeks post-injection, and serum was assayed for NT-3 levels using capture ELISA. The construct is referred herein to as scAAV1.tMCK.NTF3.

[0136] The tMCK promoter / enhancer sequence is used to drive muscle-specific gene expression and consists of a muscle creatine kinase promoter to which a fused enhancer element (enh358MCK, 584bp) is added. A triple tandem of MCK enhancers (206bp) was ligated to the 87bp basal promoter in the tMCK promoter / enhancer.

[0137] The scAAV1.tMCK.NTF3 drug product was produced by three plasmid DNA transfections of human HEK293 master cell bank cells with (i) the pAAV.tMCK.NTF3 vector plasmid (see Figure 2), (ii) an AAV1 helper plasmid called R88 / C1 containing AAV rep2 and Cap1 wild-type genes, and (iii) a helper adenovirus plasmid.

[0138] Figure 2 shows a schematic diagram of the plasmid with molecular features and an open reading frame. The rAAV genome derived from the pAAV.tMCK.NTF3 plasmid is a self-complementary DNA genome containing a human NT-3 cDNA expression cassette flanked by an AAV2 inverted terminal repeat (ITR). This sequence is encapsulated in the AAV1 virion. The plasmid pAAV.tMCK.NTF3 was constructed by inserting a tMCK expression cassette driving the NT-3 gene sequence into the AAV cloning vector psub201. The human NT-3 gene is expressed from a mouse triple tandem MCK promoter containing a triple E-box sequence, which is a modification of the aforementioned CK6 promoter. The SV40 polyadenylation signal is used for efficient transcription termination. The cassette also contains chimeric introns for increased gene expression, consisting of a 5' donor site and branch point from the first intron of the human β-globin gene, as well as a 3' splice acceptor site from an intron located between the leader and body of the immunoglobulin gene heavy chain variable region. The NT-3 expression cassette has a consensus scozak immediately before the ATG start and a 200 bp SV40 polyA signal for efficient mRNA termination. The NT-3 cDNA is included in its entirety (NCBI reference sequence: NM_001102654). The only viral sequence included in this vector is the inverted end sequence of AAV2, which is necessary for both viral DNA replication and packaging. The AAV ITR is a sequence that is nearly identical at both ends but has opposite orientation. The "left" (mutant) ITR has a deleted end degradation site to allow for genomic hairpin formation. The identity of all DNA plasmid elements is confirmed by DNA plasmid sequencing on plasmid source stocks.

[0139] Table 3 shows the base pair positions of relevant molecular features within the rAAV vector DNA plasmid of Sequence ID No. 3. [Table 3-1]

[0140] Example 3 An experimental autoimmune encephalomyelitis (EAE) mouse model that mimics MS disease. Experimental autoimmune encephalomyelitis (EAE) is the most commonly used experimental model for human inflammatory demyelinating disease, multiple sclerosis (MS). (Bjelobaba, Ivana, et al. "Animal models of multiple sclerosis: Focus on experimental autoimmune encephalomyelitis." Journal of Neuroscience Research 96.6(2018):1021-1042). EAE is a complex condition in which interactions between various immunopathological and neuropathological mechanisms lead to approximations of the major pathological features of MS: inflammation, demyelination, axonal loss, and gliosis. The counter-regulatory mechanisms of inflammation and remyelination resolution also occur in EAE and therefore can serve as a model for these processes. EAE possesses complex neuropharmacology, and many of the drugs currently or soon to be used in MS have been developed, tested, or validated based on EAE trials.

[0141] This disclosure uses a mouse model of chronic relapsing experimental autoimmune encephalomyelitis (EAE) to mimic the disease progression and clinical state of MS in human patients. The EAE model is the most common and widely accepted mouse model to mimic MS due to its shared histopathological and immunological similarities. We investigated the efficacy of an AAV NT-3 gene therapy approach treating EAE mice as an indicator for using this approach to treat MS.

[0142] The examples provided in this disclosure investigate the potential of using NT-3 as a therapeutic approach to treat multiple sclerosis and other autoimmune diseases. Preliminary data show that AAV1.tMCK.NT-3 gene therapy improved the clinical severity of EAE, possibly by modulating the immune system, without being constrained by theory. The potential of using AAV1.tMCK.NT-3 gene therapy via intramuscular delivery to EAE models to attenuate the disease process may demonstrate that it is a useful therapeutic approach. Overall, NT-3 exhibits immune system modulating potential and therefore has potential for use as a treatment for chronic progressive MS and potentially other autoimmune diseases.

[0143] Example 4 Treatment with NT-3 gene therapy improved clinical scores and behavioral outcomes in EAE mice. EAE induction was performed using the protocol published by Bittner, Stefan, et al. "Myelin oligodendrocyte glycoprotein (MOG35-55) induced experimental autoimmune encephalomyelitis (EAE) in C57BL / 6 mice." Journal of Visualized Experiments: JoVE 86 (2014). Briefly, both male (n=4) and female (n=5) C57BL / 6 mice aged 8–12 weeks were induced with synthetic myelin oligodendrocyte glycoprotein peptides 35–55 (MOG35-55) in a complete Freund's adjuvant (CFA) containing 4 mg / mL of Mycobacterium tuberculosis H37RA. 35-55 Immunized with MEVGWYRSPFSRVVHLYRNGK). Total 200 μg of MOG 35-55Peptides and 200 μg of H37RA were emulsified in CFA and subcutaneously (sc) injected into the flanks of mice. Mice were also intraperitoneally (IP) injected with 400 ng of pertussis toxin in 200 μl of PBS. A second dose of pertussis toxin was administered 48 hours later. AAV1.tMCK.NTF3 was delivered via intramuscular (IM) injection into the right gastrocnemius muscle 21 days after EAE induction (1 week after peak disease activity) (1.0 × Ringer's lactate). 11 (vg, 50 μl volume), mice were closely monitored and sacrificed approximately 70 days after EAE induction. EAE induction resulted in a predicted peak of disease activity at 2 weeks; therefore, to evaluate therapeutic efficacy during the chronic phase, NT-3 gene therapy was delivered at the plateau phase of disease progression, 1 week after peak disease activity. This approach ensures systemic NT-3 effects through secretion from muscle into circulation. Endpoint blood samples from terminally anesthetized EAE-induced and untreated mice were collected by cardiac puncture, and serum was assayed for NT-3 levels using capture ELISA as previously reported in Yalvac et al. (Mol. Ther. 22:1353-1363, 2014). Serum NT-3 was readily detectable in AAV1.NT-3 treated mice, while levels in the untreated cohort were below the detection threshold of the assay (Figure 3).

[0144] Mice were monitored every other day after EAE induction. Each mouse's clinical score was defined as shown in Table 3, with 0 indicating no clinical signs and 10 indicating death (Terry et al., Methods Mol Biol; 1304:145-160, 2016). Higher scores indicated more severe symptoms, and thus a worse clinical state for the mouse. Overall, females were more severely affected than males (data not shown). Combining males and females, preliminary data showed that NT-3 treatment significantly reduced the clinical score of mice, thus improving the severity of EAE (Figure 4). Clinical scores showed substantial improvement in treated mice compared to untreated mice.

[0145] Additional trials also showed a significant and steady reduction in clinical scores, from a mean 3 at day 15 to a mean 1.5 at day 30 and thereafter, with the difference from the untreated cohort being statistically significant between 25 and 70 days after EAE induction. In the male cohort, a significant reduction in clinical scores was observed in the treatment group within 25–35 days after EAE induction, but no further improvement was observed at 40 days (Figure 6b). [Table 3-2]

[0146] In addition to clinical scores, behavioral tests were performed in EAE mice (Figure 7). Rotarod data (Figure 7A) showed that the NT-3 treated cohort exhibited significantly better motor coordination compared to the untreated group. As shown in Figure 7B, grip strength in treated mice showed little change until approximately 5.5 weeks after NT-3 treatment, after which the treated cohort began to show a trend of increasing grip strength. These data correlated with reduced expression of pro-inflammatory cytokines, interleukin-1β (IL-1β), tumor necrosis factor (TNF-α), and interleukin-6 (IL-6), in both the brain and spinal cord in the NT-3 treated cohort, as shown in Example 5.

[0147] Additional tests were consistent with improvements in clinical scores, and the performance of the treated EAE cohort was better than that of the untreated counterpart in grip strength (Figure 6c, d) and rotarod testing (Figure 6e, f). To investigate the functional outcomes of treatment in EAE mice, hindlimb grip strength and rotarod function were assessed weekly, starting on the day of AAV1.NTF3 injection (3 weeks after EAE induction). Grip strength in AAV1.NTF3-treated females (Figure 6c) remained relatively stable over the 6-week period (mean 0.095 kg / m2 at week 0 to 0.097 kg / m2 at week 6). In contrast, the decline in this function was approximately 40% in the untreated female cohort during the same period (0.096 kg / m2 at week 6). 2 ~0.058 kg / m 2 Observed at 5 weeks (NT-3: 0.10±0.008 kg / m²) from the average,2 , n = 7 for UT: 0.071 ± 0.009 kg / m 2 , p = 0.027) and at week 6 (NT-3: 0.097 ± 0.015 kg / m 2 for UT: 0.058 ± 0.021 kg / m 2 , p = 0.032) compared to untreated counterparts, resulted in significantly higher force generation in the treatment cohort. Females showed higher grip strength performance than female counterparts at baseline (Figure 6d). For the untreated male cohort, grip strength remained relatively stable (average of 0.118 kg / m at week 0 2 and 0.117 kg / m at week 6 2 average), while the AAV.NTF3 treatment cohort showed a slight increase in performance of about 14% (from an average of 0.123 kg / m 2 to an average of 0.142 kg / m 2 average). Similar to females, AAV1.NTF3 treatment in males started one week after gene delivery and resulted in overall higher grip strength compared to their untreated counterparts, which was statistically significant at week 6 (NT-3: 0.143 ± 0.008 kg / m 2 for UT: 0.117 ± 0.005 kg / m 2 n = 5 per cohort, p = 0.023).

[0148] In the rotarod trial, the treated female cohort (Figure 6e) showed overall better performance from week 1 to week 6 after treatment, while the untreated control showed a gradual decline, resulting in a 48% higher performance at week 6 (38.8±3.8 seconds vs. 20.2±12.5 seconds, p=0.02). The treatment response to rotarod performance in males was more modest (Figure 6f), showing significant differences at weeks 4 and 6 (40.0±4.3 seconds vs. 27.7±4.7 seconds at week 4, p=0.045, and 34.9±2.2 seconds vs. 26.6±3.7 seconds at week 6, p=0.047). At the endpoint, the untreated cohort showed a 13% decrease in rotarod time from baseline (30.3 seconds at week 0 to 26.6 seconds at week 6), while the AAV1.NTF3-treated cohort showed a 10% increase in rotarod time compared to baseline (31.7 seconds at week 0 to 34.9 seconds at week 6), corresponding to a 23.8% improvement in the endpoint with treatment compared to the untreated counterpart. In summary, these data indicate that AAV1.NTF3 gene therapy in EAE mice results in significant improvements in clinical scores, stronger hindlimb grip strength tests, and better sensorimotor function in terms of rotarod performance. Note that the NT-3 effect in both sexes was equal and normalized to WT, and the larger percentage change in functional tests or clinical scores in females was associated with disease progression and was more severe in females than in males.

[0149] Example 5 AAV1.NTF3-treated EAE mice exhibit reduced inflammation, improved remyelination, and axonal protection in the spinal cord. Inflammation was assessed by examining hematoxylin & eosin (H&E) stained sections from the sacral, lumbar, mesothoracic, and upper thoracic spinal cord segments of untreated and treated mice (n=4 per cohort) 10 weeks after EAE induction. Multifocal perivascular subpiatric inflammation was present at different levels in all four spinal cord segments examined in all untreated mice (Figure 8a). In the AAV1.NTF3 treatment group (Figure 8b), only a few small areas of perivascular inflammation were observed in one of the four segments from two mice. Furthermore, Luxor Fast Blue (LFB) stained paraffin sections (Figures 8c-e) and toluidine blue stained half-thick plastic sections from treated mice revealed preservation of long white matter tracts, such as those shown in the descending anterolateral corticospinal tract, 7 weeks post-treatment (Figures 8f, g). Specifically, both preserved myelinated fibers and abundant, thin, remyelinate axons were observed in high-resolution plastic-embedded sections after treatment. Furthermore, quantitative immunofluorescence testing using anti-neurofilament antibodies as axonal markers revealed that AAV1.NTF3 gene therapy significantly attenuated axonal loss in the long tract of the spinal cord (Figures 9a-d). We also showed that improved remyelination and axonal protection were clearly associated with NT-3-induced increases in myelin basic protein (MBP) and proteolipid protein (PLP) expression levels (Figures 9e-h). This highlights the NT-3 effect as increased remyelination and its protective effect on myelinated fiber integrity. In summary, these observations indicate that NT-3 gene therapy significantly reduced subpia inflammatory response in the white matter of the spinal cord, improved remyelination, and reduced axonal loss. No pathological changes were detected in the roots, sciatic nerve, or muscles. Example 6: Characteristic Analysis of the Immunomodulatory Effects of NT-3 Gene Therapy in EAE Mice

[0150] Since EAE is a chronic autoinflammatory disease affecting the brain and spinal cord, we investigated the inflammatory state in these tissues in mice. We measured the levels of inflammatory markers TNFα, IL1β, and IL6 expression in the brain and spinal cord of treated and untreated mice. The collected experimental data showed that all three inflammatory markers were significantly reduced in the NT-3 treated cohort (Figure 10).

[0151] In additional tests characterizing the immunomodulatory effects of the treatment, RNA was isolated from fresh frozen brain and spinal cord tissue samples, and real-time PCR was performed to determine the expression levels of inflammatory markers, including TNFα, IL1β, and IL6. In the female cohort, the NT-3 treatment group showed significantly reduced expression levels of all three markers in both the brain and spinal cord compared to samples from untreated EAE mice (Figure 11a-c). The male cohort, exhibiting mild disease, showed less pronounced therapeutic efficacy. AAV1.NTF3 gene delivery in male EAE mice resulted in significantly reduced IL1β expression in the brain and IL6 in the spinal cord, although TNFα and IL1β expression levels in the spinal cord, as well as IL6 levels in the brain, decreased without reaching statistically significant levels (Figure 11d-f).

[0152] Regulatory T cells (Treg cells) are thought to play a crucial role in maintaining peripheral immune tolerance. Tregs are thought to function by suppressing effector CD4+ T cell subsets that mediate autoimmune responses. Dysregulation of suppression and migration markers on Tregs is associated with the pathogenesis of MS37. To investigate whether AAV1.NTF3 treatment increased Tregs, we measured the percentage of CD4+CD25+Foxp3+ Treg cells in lymph nodes and spleens from treated and untreated EAE mice. Treg cells generally function as immunosuppressants and are important in preventing autoimmune diseases. As shown in Figure 12, NT-3 gene therapy increased the percentage of CD3+CD4+CD25+ and Foxp3+ Treg cells in total cells extracted from lymph nodes and spleens in both male and female EAE mice exhibiting the immunomodulatory effects of NT-3. Since MS patients are known to have persistently low Treg cell counts, the observed immunomodulatory effect of NT-3 in the EAE model is significant, suggesting the potential efficacy of NT-3 gene therapy in chronic progressive MS cases in humans.

[0153] In additional studies, the percentage of CD4+CD25+Foxp3+Treg cells in EAE mice was analyzed in cell populations from lymph nodes and spleens by flow cytometry after gene therapy treatment (Figure 13a). AAV1.NTF3 treatment significantly increased the percentage of Treg cell population in lymph nodes compared to samples from untreated counterparts in both female and male cohorts (Figures 13b, c). We also found a significant increase in Treg cell population in spleen cells from treated females (approximately 52%) (Figure 13b), but no significant increase was observed in males (Figure 13c). These results are consistent with the immunomodulatory role of NT-3, which is favorable to attenuating the severity of EAE.

[0154] Circulating bone marrow-derived dendritic cells (DCs) are important antigen (Ag)-presenting cells (APCs) that accumulate in the CNS during EAE (Clarkson et al., Journal of neuroimmunology; 277:39-4, 2014). These migratory cells present major histocompatibility complex (MHC) restriction Ag to CD4+ T cells and CD8+ T cells (cross-presentation), and thus can play a central role in priming the adaptive immune response. To investigate whether NT-3 treatment imparts tolerance characteristics to dendritic cells (DCs), bone marrow-derived DCs were extracted from treated and untreated EAE mice. Cultured DCs were challenged with Mycobacterium for 24 hours, and TNFα levels were then determined as a marker of inflammation. 24 hours after incubation with Mycobacterium, DCs from the NT-3 treatment cohort demonstrated lower levels of TNFα (i.e., lower induction of TNFα) compared with the untreated cohort (Figure 14).

[0155] In an additional study, bone marrow dendritic cells (DCs) were isolated from the femurs of a treatment-naive cohort treated with AAV1.NTF3, and the DC population was determined by positive signaling from the DC marker CD11c. We first determined that approximately 74.1% of all cells analyzed were positive for the CD11c marker (Figure 15a). To investigate whether AAV1.NTF3 treatment induces tolerogenic features in DCs, bone marrow-derived DCs from both treated and treatment-naive cohorts were exposed to mycobacteria. DCs from treatment-naive females showed a significant increase in the expression of the pro-inflammatory marker TNFα by qPCR at 24 hours of incubation with mycobacteria (Figure 15b). However, this increase in TNFα was significantly lower in DCs exposed to mycobacteria from the AAV1.NTF3-treated group compared to the untreated counterpart, which suggests that NT-3 has the potential to induce tolerogenic features in DCs. DCs from untreated males also showed increased TNFα levels in response to mycobacterial challenge, although this did not reach statistical significance (Figure 15c), but no change in TNFα levels was observed from the treatment group 6 weeks after gene injection. Overall, these results support the immunomodulatory role of NT-3 in providing neuroprotective and safeguards against EAE disease processes, thereby favoring tolerance.

[0156] conclusion The provided data demonstrate that NT-3 plays an immunomodulatory role in regulating the autoimmune system in a mouse model of EAE in MS. NT-3 therapy successfully improved the clinical severity of EAE in the mouse model by regulating its immune system. AAV1.NTF3 gene therapy reduced the severity of the EAE model in MS, likely through the regulation of the immune system.

[0157] The data demonstrate that circulating NT-3 induces a tolerance-induced immune response by reducing inflammation and increasing Treg cells in both the spleen and lymph nodes after transduction into muscle via AAV1.tMCK.NTF3 vector injection. This proof-of-principle study demonstrates the potential of a clinical translational pathway for AAV-delivered NT-3 for the treatment of chronic progressive MS.

[0158] All patents, patent applications, and publications cited herein, as well as the complete disclosure of electronically available materials, are incorporated by reference. The modes and examples for carrying out the above inventions are provided solely for the purpose of facilitating understanding and should not be read as unnecessary limitations, for this disclosure is not limited to the exact details shown and described herein, and variations that are obvious to those skilled in the art are included within the scope of the disclosure as defined by the claims.

[0159] While this disclosure describes specific embodiments, it will be understood by those skilled in the art that changes and modifications may occur. Therefore, only such limitations as those found in the claims should be imposed on this disclosure.

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[0161] Sequence Listing Sequence No. 1 <210>1 <211>774 <212>DNA <213>Homo sapiens 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 SEQ ID NO: 2 <210>2 <211>270 <212>PRT <213>Homo sapiens Met Val Thr Phe Ala Thr Ile Leu Gln Val Asn Lys Val Met Ser Ile 1 5 10 15 Leu Phe Tyr Val Ile Phe Leu Ala Tyr Leu Arg Gly Ile Gln Gly Asn 20 25 30 Asn Met Asp Gln Arg Ser Leu Pro Glu Asp Ser Leu Asn Ser Leu Ile 35 40 45 Ile Lys Leu Ile Gln Ala Asp Ile Leu Lys Asn Lys Leu Ser Lys Gln 50 55 60 Met Val Asp Val Lys Glu Asn Tyr Gln 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 Gln 85 90 95 Pro Val Ile Ala Met Asp Thr Glu Leu Leu Arg Gln Gln 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 Gln Val Thr Val Leu Gly Glu Ile 180 185 190 Lys Thr Gly Asn Ser Pro Val Lys Gln Tyr Phe Tyr Glu Thr Arg Cys 195 200 205 Lys Glu Ala Arg Pro Val Lys Asn Gly Cys Arg Gly Ile Asp Asp Lys 210 215 220 His Trp Asn Ser Gln Cys Lys Thr Ser Gln Thr Tyr Val Arg Ala Leu 225 230 235 240 Thr Ser Glu Asn Asn Lys Leu Val Gly Trp Arg Trp Ile Arg Ile Asp 245 250 255 Thr Ser Cys Val Cys Ala Leu Ser Arg Lys Ile Gly Arg Thr 260 265 270 Sequence number 3 <210>3 <211>5884 <212>DNA <213>Artificial sequence <223>Full sequence of sc pAAV.tMCK.NTF3 plasmid genome <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 agtcatcggc catcgacatt cggggacacc aggtcacggt gctgggggag atcaaaacgg 1620. gcaactctcc cgtcaaaca tatttttatg aaacgcgatg tagcc aggccggtca aaaacggttg caggggtatt gatgataac actggaactc tcagtgcaaa acatcccaaa cctacgtccg agcactgact tcagagaaca ataaactcgt gggctggcgg tggatacgga tagacacgtc ctgtgtgtgt gccttgtcga gaaaaatcgg aagaacatga ggcggccgcg gggatccaga catgataaga tacattgatg agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta accattata gctgcaataa acaagttaac aacaacaatt gcattcattt tatgtttcag gttcaggggg aggtgtggga ggttttttcg gcgcgcctct aggcatggc tacgtagata agtagcatgg cgggttaatc attack aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccggggcgacc 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 ctatctcggt ctattctttt gattattaag ggattttgcc gattcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa tttaaatatt tgcttataca atcttcctgt ttttggggct tttctgatta tcaaccgggg 3240. tacatatgat tgacatgcta gttttacgat taccgttcat cgattctctt gtttgctcca gactctcagg caatgacctg atagccttttg tagagacctc tcaaaaatag ctaccctctc cggcatgaat ttatcagcta gaacggttga atatcatatt gatggtgatt tgactgtctc cggcctttct cacccgtttg aatctttacc tacacattac tcaggcattg catttaaaat atatgagggt tctaaaaatt tttatccttg cgttgaata aaggcttctc ccgcaaaagt attacagggt cataatgttt ttggtacaac cgatttagct ttatgctctg aggctttatt gcttaatttt gctaattctt tgccttgcct gtatgattta ttggatgttg gaattcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatatgg tgcactctca 3660 gtacaatctg ctctgatgcc ccatagttaa 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 caccagaaa cgctggtgaa agtaaaagat gctgaagatc 4140 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga 4200 gttttcgcc 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 Sequence number 4 <210>4 <211>106 <212>DNA <213>Artificial sequence <220> <223>Synthetic polynucleotide <220> <221>Other features <223>5’ ITR <400>4 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtgg 106 Sequence number 5 <210>5 <211>133 <212>DNA <213>Artificial sequence <220> <223>Synthetic polynucleotide <220> <221>Other features <223>Chimeric intron <400>5 gtaagtatca aggttacaag acaggtttaa ggagaccaat agaaactggg cttgtcgaga 60 cagagaagac tcttgcgttt ctgataggca cctattggtc ttactgacat ccactttgcc 120 tttctctcca cag 133 Sequence number 6 <210>6 <211>5 <212>DNA <213>Artificial sequence <220> <223> Synthetic polynucleotides <220> <221> Other features <223> Kozak Array <400> 6 ccacc 5 Sequence ID 7 <210> 7 <211> 200 <212> DNA <213> Artificial arrangement <220> <223> Synthetic polynucleotides <220> <221> Other features <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 Sequence ID 8 <210> 8 <211> 128 <212> DNA <213> Artificial arrangement <220> <223> Synthetic polynucleotides <220> <221> Other features <223> 3'ITR <400> 8 aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 60 ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc 120 gagcgcgc 128 SEQ ID NO: 9 <210>9 <211>2248 <212>DNA <213>Artificial sequence <220> <223>Synthetic polynucleotide <220> <221>Other features <223>AAV.tMCK.NTF3 genomic 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 atttttgaa aaacaagctc tccaagcaga tggtggacgt tagged taccagagca ccctgcccaa agctgaggct ccccgagagc cggagcgggg agggcccgcc aagtcagcat 1320 tccagccggt gattgcaatg gacaccgac tgctgcgaca acagagacgc tacaactcac cgcgggtcct gctgagcgac agcaccccct tggagcccc gcccttgtat ctcatggagg 1440 attackgtggg cagccccgtg gtggcgaaca gaacatcacg gcggaaacgg tacgcggagc ataagagtca ccgaggggag tactcggtat gtgacagtga gagtctgtgg gtgaccgaca agtcatcggc catcgacatt cggggacacc aggtcacggt gctgggggag atcaaaacgg 1620. gcaactctcc cgtcaaaca tatttttatg aaacgcgatg tagcc aggccggtca aaaacggttg caggggtatt gatgataac actggaactc tcagtgcaaa acatcccaaa cctacgtccg agcactgact tcagagaaca ataaactcgt gggctggcgg tggatacgga tagacacgtc ctgtgtgtgt gccttgtcga gaaaaatcgg aagaacatga ggcggccgcg gggatccaga catgataaga tacattgatg agtttggaca aaccacaact agaatgcagt 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 Sequence ID 10 <210> 10 <211> 668 <212> DNA <213> Artificial arrangement <220> <223> Synthetic polynucleotides <220> <221> Other features <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 Sequence ID 11 <210> 11 <211> 714 <212> DNA <213> Artificial arrangement <220> <223> Synthetic polynucleotides <220> <221> Other features <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 SEQ ID NO: 12 <210>12 <211>861 <212>DNA <213>Artificial Sequence <220> <223>Synthetic polynucleotide <220> <221>Other features <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 gttattgct gataaatctg gagccggtga gcgtgggtct 720 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac 780 acgacgggga gtcaggcaac tatggatgaa cgaatagac agatcgctga gataggtgcc 840 tcactgatta agcattggta a 861

Claims

1. A method for treating multiple sclerosis (MS) in a human subject requiring treatment for multiple sclerosis (MS), comprising the step of administering a nucleic acid encoding an NT-3 polypeptide to the human subject, a) The nucleic acid contains a nucleotide sequence that is at least 90% identical to the nucleotide sequence of Sequence ID No. 1, b) The nucleic acid contains the nucleotide sequence of SEQ ID NO: 1, c) The nucleic acid contains a nucleotide sequence that encodes an amino acid sequence that is at least 90% identical to SEQ ID NO: 2, or d) A method wherein the nucleic acid comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:

2.

2. A method for treating an autoimmune disease in a human subject requiring treatment for the autoimmune disease, comprising the step of administering a nucleic acid encoding an NT-3 polypeptide to the human subject, a) The nucleic acid contains a nucleotide sequence that is at least 90% identical to the nucleotide sequence of Sequence ID No. 1, b) The nucleic acid contains the nucleotide sequence of SEQ ID NO: 1, c) The nucleic acid contains a nucleotide sequence that encodes an amino acid sequence that is at least 90% identical to SEQ ID NO: 2, or d) A method wherein the nucleic acid comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:

2.

3. The method according to claim 2, wherein the autoimmune disease is alopecia areata, Addison's disease, celiac disease, Crohn's disease, ulcerative colitis, autoimmune inflammatory myositis, Graves' disease, Hashimoto's thyroiditis, inflammatory bowel disease, multiple sclerosis, pemphigus, pernicious anemia, psoriasis, reactive arthritis, rheumatoid arthritis, Sjögren's syndrome, systemic lupus erythematosus, type 1 diabetes, or autoimmune hepatitis.

4. The method according to any one of claims 1 to 3, wherein the nucleic acid encoding the NT-3 polypeptide is operably bound to a muscle-specific promoter.

5. The method according to claim 4, wherein the muscle-specific promoter is a muscle-specific creatine kinase promoter (MCK).

6. The method according to claim 5, wherein the muscle creatine kinase promoter has the nucleotide sequence shown in SEQ ID NO:

11.

7. The method according to any one of claims 1 to 6, wherein the nucleic acid is administered using a viral vector.

8. The method according to claim 7, wherein the viral vector is recombinant adeno-associated virus (rAAV).

9. The method according to claim 8, 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.

10. The method according to claim 9, wherein the rAAV capsid serotype is AAV-1.

11. The rAAV genome sequence is in the order from 5' to 3'. (i) The first AAV2 inverted terminal repeat sequence (ITR), (ii) Muscle creatine kinase promoter / enhancer sequence shown at nucleotides 147-860 of Sequence ID No. 3, (iii) Nucleotide sequences encoding human NT-3 polypeptides, and (iv) Contains a second AAV2 ITR sequence, The method according to any one of claims 8 to 10, wherein the human NT-3 polypeptide has an amino acid sequence that is at least 90% identical to SEQ ID NO: 2, or 100% identical to SEQ ID NO: 2, or is encoded by a nucleotide sequence that is at least 90% identical to nucleotides 1077 to 1850 of SEQ ID NO: 3, or 100% identical to nucleotides 1077 to 1850 of SEQ ID NO:

3.

12. The method according to claim 11, wherein the nucleic acid sequence further comprises a chimeric intron shown in nucleotides 892-1024 of SEQ ID NO: 3 on the 3' side of the promoter / enhancer.

13. The method according to claim 11 or 12, wherein the nucleic acid sequence further comprises an SV40 polyadenylation signal, shown in nucleotides 1860-2059 of SEQ ID NO: 3, on the 3' side of the nucleotide sequence encoding a human NT-3 polypeptide.

14. The method according to any one of claims 11 to 13, wherein the first ITR is shown in nucleotides 7 to 112 of SEQ ID NO: 3, and / or the second ITR is shown in nucleotides 2121 to 2248 of SEQ ID NO:

3.

15. The method according to any one of claims 1 to 14, wherein the nucleic acid comprises an scAAV1.tMCK.NTF3 rAAV genome which is at least 90% identical to SEQ ID NO:

9.

16. The method according to any one of claims 1 to 14, wherein the nucleic acid comprising the scAAV1.tMCK.NTF3 genome is shown in Sequence ID No.

9.

17. The method according to any one of claims 1 to 16, wherein the nucleic acid is administered in a dose that results in sustained expression of a low concentration of NT-3 polypeptide.

18. The method according to any one of claims 1 to 17, wherein administering the nucleic acid or rAAV reduces inflammation in organs affected by MS or autoimmune disease in the subject.

19. The method according to claim 18, wherein the organ affected by MS or autoimmune disease is the brain, spinal cord, joints, muscles, skin, pancreas, liver, or kidney.

20. The reduction of inflammatory markers and inflammatory cytokines such as TNFα, IL1β, IL6, IL17, or IL22, according to claim 18.

21. The method according to any one of claims 1 to 20, wherein administration of the nucleic acid or rAAV modulates the immune response in the subject.

22. The method according to claim 21, wherein the percentage of regulatory T cells is increased in organs affected by MS or autoimmune disease.

23. The method according to claim 22, wherein the organ affected by MS or autoimmune disease is a lymph node, spleen, thymus, or peripheral blood.

24. The method according to any one of claims 1 to 23, wherein administration of the nucleic acid or rAAV modulates cytokine expression in dendritic cells in the subject.

25. The method according to any one of claims 1 to 24, wherein the nucleic acid or rAAV is administered by intramuscular injection.

26. The method according to any one of claims 1 to 25, wherein the subject is an elderly adult.

27. A viral vector for use in the treatment of multiple sclerosis (MS) in human subjects requiring treatment for multiple sclerosis (MS), wherein the composition comprises a nucleic acid encoding an NT-3 polypeptide, a) The nucleic acid contains a nucleotide sequence that is at least 90% identical to the nucleotide sequence of Sequence ID No. 1, b) The nucleic acid contains the nucleotide sequence of SEQ ID NO: 1, c) The nucleic acid contains a nucleotide sequence that encodes an amino acid sequence that is at least 90% identical to SEQ ID NO: 2, or d) A viral vector wherein the nucleic acid comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:

2.

28. A viral vector for use in treating autoimmune diseases in human subjects requiring treatment for autoimmune diseases, wherein the composition comprises a nucleic acid encoding an NT-3 polypeptide, a) The nucleic acid contains a nucleotide sequence that is at least 90% identical to the nucleotide sequence of Sequence ID No. 1, b) The nucleic acid contains the nucleotide sequence of SEQ ID NO: 1, c) The nucleic acid contains a nucleotide sequence that encodes an amino acid sequence that is at least 90% identical to SEQ ID NO: 2, or d) A viral vector wherein the nucleic acid comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:

2.

29. The viral vector according to claim 28, wherein the autoimmune disease is alopecia areata, Addison's disease, celiac disease, Crohn's disease, ulcerative colitis, autoimmune inflammatory myositis, Graves' disease, Hashimoto's thyroiditis, inflammatory bowel disease, multiple sclerosis, pemphigus, pernicious anemia, psoriasis, reactive arthritis, rheumatoid arthritis, Sjögren's syndrome, systemic lupus erythematosus, type 1 diabetes, or autoimmune hepatitis.

30. The viral vector according to any one of claims 27 to 29, wherein the nucleic acid encoding the NT-3 polypeptide is operably bound to a muscle-specific promoter.

31. The viral vector according to claim 30, wherein the muscle-specific promoter is a muscle-specific creatine kinase promoter (MCK).

32. The viral vector according to claim 31, wherein the muscle creatine kinase promoter has the nucleotide sequence shown in SEQ ID NO:

11.

33. The viral vector according to any one of claims 27 to 32, wherein the nucleic acid is administered using a viral vector.

34. The viral vector according to claim 33, wherein the viral vector is recombinant adeno-associated virus (rAAV).

35. The viral vector according to claim 34, 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.

36. The viral vector according to claim 34, wherein the rAAV capsid serotype is AAV-1.

37. The aforementioned rAAV genome sequence is in the order from 5' to 3', (i) The first AAV2 inverted terminal repeat sequence (ITR), (ii) Muscle creatine kinase promoter / enhancer sequence shown at nucleotides 147-860 of Sequence ID No. 3, (iii) Nucleotide sequences encoding human NT-3 polypeptides, and (iv) Contains a second AAV2 ITR sequence, The viral vector according to any one of claims 34 to 36, wherein the human NT-3 polypeptide has an amino acid sequence that is at least 90% identical to SEQ ID NO: 2, or 100% identical to SEQ ID NO: 2, or is encoded by a nucleotide sequence that is at least 90% identical to nucleotides 1077 to 1850 of SEQ ID NO: 3, or 100% identical to nucleotides 1077 to 1850 of SEQ ID NO:

3.

38. The viral vector according to claim 37, wherein the nucleic acid sequence further comprises a chimeric intron shown in nucleotides 892-1024 of SEQ ID NO: 3 on the 3' side of the promoter / enhancer.

39. The viral vector according to claim 37 or 38, wherein the nucleic acid sequence further comprises an SV40 polyadenylation signal, shown in nucleotides 1860-2059 of SEQ ID NO: 3, on the 3' side of the nucleotide sequence encoding a human NT-3 polypeptide.

40. The viral vector according to any one of claims 37 to 39, wherein the first ITR is represented by nucleotides 7 to 112 of SEQ ID NO: 3, and / or the second ITR is represented by nucleotides 2121 to 2248 of SEQ ID NO:

3.

41. A viral vector according to any one of claims 27 to 40, comprising the scAAV1.tMCK.NTF3rAAV genome, wherein the nucleic acid is at least 90% identical to SEQ ID NO:

9.

42. The viral vector according to any one of claims 27 to 40, wherein the nucleic acid comprising the scAAV1.tMCK.NTF3 genome is shown in Sequence ID No.

9.

43. The viral vector according to any one of claims 27 to 42, wherein the nucleic acid is administered in a dose that results in sustained expression of a low concentration of NT-3 polypeptide.

44. The viral vector according to any one of claims 27 to 43, wherein administration of the nucleic acid or rAAV reduces inflammation in organs affected by MS or autoimmune disease in the subject.

45. The viral vector according to claim 44, wherein the organ affected by MS or autoimmune disease is the brain, spinal cord, joints, muscles, skin, pancreas, liver, or kidney.

46. The reduction of inflammatory markers and inflammatory cytokines such as TNFα, IL1β, IL6, IL17, or IL22, according to claim 44, the viral vector.

47. The viral vector according to any one of claims 27 to 46, wherein administration of the nucleic acid or rAAV modulates the immune response in the subject.

48. The viral vector according to claim 47, wherein the percentage of regulatory T cells is increased in organs affected by MS or autoimmune disease.

49. The viral vector according to claim 48, wherein the organ affected by MS or autoimmune disease is a lymph node, spleen, thymus, or peripheral blood.

50. The viral vector according to any one of claims 27 to 49, wherein administration of the nucleic acid or rAAV modulates cytokine expression in dendritic cells in the subject.

51. The viral vector according to any one of claims 27 to 50, wherein the composition is formulated for administration by intramuscular injection.

52. The viral vector according to any one of claims 27 to 51, wherein the subject is an elderly adult.

53. The use of nucleic acids encoding NT-3 polypeptides for the preparation of pharmaceuticals for the treatment of multiple sclerosis (MS) in human subjects requiring treatment for multiple sclerosis (MS), a) The nucleic acid contains a nucleotide sequence that is at least 90% identical to the nucleotide sequence of Sequence ID No. 1, b) The nucleic acid contains the nucleotide sequence of SEQ ID NO: 1, c) The nucleic acid contains a nucleotide sequence that encodes an amino acid sequence that is at least 90% identical to SEQ ID NO: 2, or d) Use wherein the nucleic acid comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:

2.

54. The use of nucleic acids encoding NT-3 polypeptides for the preparation of pharmaceuticals for treating autoimmune diseases in human subjects requiring treatment of autoimmune diseases, a) The nucleic acid contains a nucleotide sequence that is at least 90% identical to the nucleotide sequence of Sequence ID No. 1, b) The nucleic acid contains the nucleotide sequence of SEQ ID NO: 1, c) The nucleic acid contains a nucleotide sequence that encodes an amino acid sequence that is at least 90% identical to SEQ ID NO: 2, or d) Use wherein the nucleic acid comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:

2.

55. The use according to claim 54, wherein the autoimmune disease is alopecia areata, Addison's disease, celiac disease, Crohn's disease, ulcerative colitis, autoimmune inflammatory myositis, Graves' disease, Hashimoto's thyroiditis, inflammatory bowel disease, multiple sclerosis, pemphigus, pernicious anemia, psoriasis, reactive arthritis, rheumatoid arthritis, Sjögren's syndrome, systemic lupus erythematosus, type 1 diabetes, or autoimmune hepatitis.

56. The use according to any one of claims 53 to 55, wherein the nucleic acid encoding the NT-3 polypeptide is operably bound to a muscle-specific promoter.

57. The use according to claim 56, wherein the muscle-specific promoter is a muscle-specific creatine kinase promoter (MCK).

58. The use according to claim 57, wherein the muscle creatine kinase promoter has the nucleotide sequence shown in SEQ ID NO:

11.

59. The use according to any one of claims 53 to 58, wherein the nucleic acid is administered using a viral vector.

60. The use according to claim 59, wherein the viral vector is recombinant adeno-associated virus (rAAV).

61. The use according to claim 60, 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, AAV-12, AAV-13, Anc80, AAV-B1, AAVrh. 10, or AAVrh.

74.

62. The use according to claim 60, wherein the rAAV capsid serotype is AAV-1.

63. The aforementioned rAAV genome sequence is in the order from 5' to 3', (i) The first AAV2 inverted terminal repeat sequence (ITR), (ii) Muscle creatine kinase promoter / enhancer sequence shown at nucleotides 147-860 of Sequence ID No. 3, (iii) Nucleotide sequences encoding human NT-3 polypeptides, and (iv) Contains a second AAV2 ITR sequence, The use according to any one of claims 60 to 62, wherein the human NT-3 polypeptide has an amino acid sequence that is at least 90% identical to SEQ ID NO: 2, or 100% identical to SEQ ID NO: 2, or is encoded by a nucleotide sequence that is at least 90% identical to nucleotides 1077 to 1850 of SEQ ID NO: 3, or 100% identical to nucleotides 1077 to 1850 of SEQ ID NO:

3.

64. The use according to claim 63, wherein the nucleic acid sequence further comprises a chimeric intron shown in nucleotides 892-1024 of SEQ ID NO: 3 on the 3' side of the promoter / enhancer.

65. The use according to claim 63 or 64, wherein the nucleic acid sequence further comprises an SV40 polyadenylation signal, shown in nucleotides 1860-2059 of SEQ ID NO: 3, on the 3' side of the nucleotide sequence encoding the human NT-3 polypeptide.

66. The use according to any one of claims 63 to 65, wherein the first ITR is shown in nucleotides 7 to 112 of SEQ ID NO: 3, and / or the second ITR is shown in nucleotides 2121 to 2248 of SEQ ID NO:

3.

67. The use according to any one of claims 53 to 66, wherein the nucleic acid comprises an scAAV1.tMCK.NTF3 rAAV genome which is at least 90% identical to SEQ ID NO:

9.

68. The use according to any one of claims 53 to 66, wherein the nucleic acid comprising scAAV1.tMCK.NTF3 genome is shown in Sequence ID No.

9.

69. The use according to any one of claims 53 to 68, wherein the nucleic acid is administered in a dose that results in sustained expression of a low concentration of NT-3 polypeptide.

70. The use according to any one of claims 53 to 69, wherein the administration of the nucleic acid or rAAV reduces inflammation in organs affected by MS or autoimmune disease in the subject.

71. The use according to claim 70, wherein the organ affected by MS or autoimmune disease is the brain, spinal cord, joints, muscles, skin, pancreas, liver, or kidney.

72. The reduction of inflammatory markers and inflammatory cytokines such as TNFα, IL1β, IL6, IL17, or IL22, as described in claim 70.

73. The use according to any one of claims 53 to 72, wherein the administration of the nucleic acid or rAAV modulates the immune response in the subject.

74. The use according to claim 73, wherein the percentage of regulatory T cells is increased in organs affected by MS or autoimmune disease.

75. The use according to claim 74, wherein the organ affected by MS or autoimmune disease is a lymph node, spleen, thymus, or peripheral blood.

76. The use according to any one of claims 53 to 75, wherein administration of the nucleic acid or rAAV modulates cytokine expression in dendritic cells in the subject.

77. The use according to any one of claims 53 to 76, wherein the composition is formulated for administration by intramuscular injection.

78. The use according to any one of claims 53 to 77, wherein the subject is an elderly adult.

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