CN116782921A

CN116782921A - NEUROD1 combination carrier

Info

Publication number: CN116782921A
Application number: CN202180080022.1A
Authority: CN
Inventors: J·许
Original assignee: Shenxi Revitalization Treatment Co
Current assignee: Shenxi Revitalization Treatment Co
Priority date: 2020-09-29
Filing date: 2021-09-28
Publication date: 2023-09-19

Abstract

The present disclosure relates to AAV vectors, compositions and methods relating to the conversion of glial cells to neurons by using NeuroD1 and Ascl1 or ISL1 or LHX3 coding sequences in AAV vectors.

Description

NEUROD1 combination carrier

Cross Reference to Related Applications

The present application claims the benefit of U.S. provisional application No. 63/084,971 filed on 9/29 in 2020 and U.S. provisional application No. 63/247,439 filed on 23 in 2021, both of which are incorporated herein by reference in their entirety.

Incorporation of the sequence Listing

Is included under the name ofThe file of P34840WO00_SL.TXT (37,040 bytes (in MS-Measurement of (r)) and created at 9.27 of 2021 are filed electronically herewith and incorporated by reference in their entirety.

Technical Field

The present disclosure includes methods and compositions for converting glial cells into neurons using AAV vectors comprising nucleic acid sequences encoding human NeuroD1 and other central and peripheral nervous system related factors.

Background

In subjects suffering from neurological disorders or following injury to the Central Nervous System (CNS) or Peripheral Nervous System (PNS), neurons are often killed or damaged and cannot regenerate.

Glial cells become reactive following CNS or PNS injury (such as brain injury) or neurological disorders.

There is currently no method available for regenerating functional new neurons in human subjects suffering from neurological disorders using adeno-associated virus (AAV).

Disclosure of Invention

In one aspect, the present disclosure provides and includes an adeno-associated virus (AAV) vector comprising: a human neurogenic differentiation factor 1 (hNeuroD 1) sequence, the hNeuroD1 sequence comprising the nucleic acid sequence of SEQ ID NO. 6, and a second sequence comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO. 11, 13 and 15, wherein the hNeuroD1 sequence and the second sequence are separated by: (i) a P2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID nos. 19 and 22, (ii) a T2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID nos. 20 and 23, or (iii) an Internal Ribosome Entry Site (IRES) sequence of an encephalomyocarditis virus comprising SEQ ID No. 3, wherein the hNeuroD1 sequence and the second sequence are operably linked to a regulatory element comprising: (a) A Glial Fibrillary Acidic Protein (GFAP) promoter comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs 4, 18 and 27; (b) An enhancer from the human elongation factor-1 alpha (EF 1-alpha) promoter comprising the nucleic acid sequence of SEQ ID NO. 2 or a Cytomegalovirus (CMV) enhancer comprising the nucleic acid sequence of SEQ ID NO. 17; (c) A chimeric intron comprising the nucleic acid sequence of SEQ ID NO. 5 or 28; (d) A woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs 7 and 30; and (e) an SV40 polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO. 8, an hGH polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO. 21, a synthetic polyadenylation signal comprising SEQ ID NO. 31 or a bGH polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO. 26.

In one aspect, the present disclosure provides and includes an adeno-associated virus (AAV) vector comprising: a nucleic acid coding sequence encoding a human neurogenic differentiation factor 1 (hNeuroD 1) protein, the hNeuroD1 protein comprising the amino acid sequence of SEQ ID NO. 10, and a second nucleic acid coding sequence encoding a second protein having an amino acid sequence selected from the group consisting of SEQ ID NO. 12, 14 and 16, wherein the hNeuroD1 coding sequence and the second coding sequence are separated by: (i) a P2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID nos. 19 and 22, (ii) a T2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID nos. 20 and 23, or (iii) an Internal Ribosome Entry Site (IRES) sequence of an encephalomyocarditis virus comprising SEQ ID No. 3, wherein the hNeuroD1 coding sequence and the second coding sequence are operably linked to a regulatory element comprising: (a) A Glial Fibrillary Acidic Protein (GFAP) promoter comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs 4, 18 and 27; (b) An enhancer from the human elongation factor-1 alpha (EF 1-alpha) promoter comprising the nucleic acid sequence of SEQ ID NO. 2 or a Cytomegalovirus (CMV) enhancer comprising the nucleic acid sequence of SEQ ID NO. 17; (c) A chimeric intron comprising the nucleic acid sequence of SEQ ID NO. 5 or 28; (d) A woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs 7 and 30; and (e) an SV40 polyadenylation signal having the nucleic acid sequence of SEQ ID NO. 8, an hGH polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO. 21, a synthetic polyadenylation signal comprising SEQ ID NO. 31 or a bGH polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO. 26.

In one aspect, the present disclosure provides and includes an adeno-associated virus (AAV) vector comprising a NeuroD1 nucleic acid coding sequence encoding a NeuroD1 (NeuroD 1) protein and a second nucleic acid coding sequence encoding a second protein, wherein the NeuroD1 coding sequence and the second protein coding sequence are separated by a linker sequence, wherein the NeuroD1 coding sequence and the second coding sequence are operably linked to a regulatory element comprising: (a) a Glial Fibrillary Acidic Protein (GFAP) promoter; (b) an enhancer; (c) a chimeric intron; (d) Woodchuck hepatitis virus posttranscriptional regulatory elements (WPREs); and (e) a polyadenylation signal.

In one aspect, the present disclosure provides and includes a composition comprising an adeno-associated virus (AAV) vector for converting human glial cells to functional neurons, wherein the AAV vector comprises: a human neurogenic differentiation factor 1 (hNeuroD 1) sequence, the hNeuroD1 sequence having the nucleic acid sequence of SEQ ID NO. 6, and a second sequence comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO. 11, 13 and 15, wherein the hNeuroD1 sequence and the second sequence are separated by: (i) a P2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID nos. 19 and 22, (ii) a T2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID nos. 20 and 23, or (iii) an Internal Ribosome Entry Site (IRES) sequence of an encephalomyocarditis virus comprising SEQ ID No. 3, wherein the hNeuroD1 sequence and the second sequence are operably linked to a regulatory element comprising: (a) A human Glial Fibrillary Acidic Protein (GFAP) promoter comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs 4, 18 and 27; (b) An enhancer from the human elongation factor-1 alpha (EF-1 alpha) promoter comprising the nucleic acid sequence of SEQ ID NO. 2 or a Cytomegalovirus (CMV) enhancer comprising the nucleic acid sequence of SEQ ID NO. 17; (c) A chimeric intron comprising the nucleic acid sequence of SEQ ID NO. 5 or 28; (d) A woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs 7 and 30; and (e) an SV40 polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO. 8, an hGH polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO. 21, a synthetic polyadenylation signal comprising SEQ ID NO. 31 or a bGH polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO. 26.

In one aspect, the present disclosure provides and includes a composition comprising an adeno-associated virus (AAV) vector for converting human glial cells to functional neurons, wherein the AAV vector comprises: a nucleic acid coding sequence encoding a human neurogenic differentiation factor 1 (hNeuroD 1) protein, the hNeuroD1 protein comprising the amino acid sequence of SEQ ID NO. 10, and a second nucleic acid coding sequence encoding a second protein having an amino acid sequence selected from the group consisting of SEQ ID NO. 12, 14 and 16, wherein the hNeuroD1 coding sequence and the second coding sequence are separated by: (i) a P2A linker comprising a nucleic acid selected from the group consisting of SEQ ID nos. 19 and 22, (ii) a T2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID nos. 20 and 23, or (iii) an Internal Ribosome Entry Site (IRES) sequence of an encephalomyocarditis virus comprising SEQ ID No. 3, wherein the hNeuroD1 coding sequence and the second coding sequence are operably linked to a regulatory element comprising: (a) A human Glial Fibrillary Acidic Protein (GFAP) promoter comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs 4, 18 and 27; (b) An enhancer from the human elongation factor-1 alpha (EF-1 alpha) promoter comprising the nucleic acid sequence of SEQ ID NO. 2 or a Cytomegalovirus (CMV) enhancer comprising the nucleic acid sequence of SEQ ID NO. 17; (c) A chimeric intron comprising the nucleic acid sequence of SEQ ID NO. 5 or 28; a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs 7 and 30; and (e) an SV40 polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO. 8, an hGH polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO. 21, a synthetic polyadenylation signal comprising SEQ ID NO. 31 or a bGH polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO. 26.

In one aspect, the present disclosure provides and includes a composition comprising an adeno-associated virus (AAV) vector for treating a subject in need thereof, wherein the AAV vector comprises a neurogenic differentiation factor 1 (NeuroD 1) sequence and a second protein sequence, wherein the NeuroD1 sequence and the second protein sequence are separated by a linker sequence, wherein the NeuroD1 sequence and the second sequence are operably linked to an expression control element comprising: (a) a Glial Fibrillary Acidic Protein (GFAP) promoter; (b) an enhancer; (c) a chimeric intron; (d) Woodchuck hepatitis virus posttranscriptional regulatory elements (WPREs); and (e) a polyadenylation signal.

In one aspect, the present disclosure provides and includes a method of converting reactive astrocytes into functional neurons in a living human brain, comprising: injecting an adeno-associated virus (AAV) into a subject in need thereof, wherein the AAV comprises a DNA vector construct comprising a human neurogenic differentiation factor 1 (hNeuroD 1) sequence, the hNeuroD1 sequence comprising a nucleic acid sequence of SEQ ID NO:6, and a second sequence comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 11, 13, and 15, wherein the hNeuroD1 sequence and the second sequence are separated by: (i) a P2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID nos. 19 and 22, (ii) a T2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID nos. 20 and 23, or (iii) an Internal Ribosome Entry Site (IRES) sequence of an encephalomyocarditis virus comprising SEQ ID No. 3, wherein the hNeuroD1 sequence and the second sequence are operably linked to a regulatory element comprising: (a) A human Glial Fibrillary Acidic Protein (GFAP) promoter comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs 4, 18 and 27; (b) An enhancer from the human elongation factor-1 alpha (EF-1 alpha) promoter comprising the nucleic acid sequence of SEQ ID NO. 2 or a Cytomegalovirus (CMV) enhancer comprising the nucleic acid sequence of SEQ ID NO. 17; (c) A chimeric intron comprising the nucleic acid sequence of SEQ ID NO. 5 or 28; (d) A woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs 7 and 30; and (e) an SV40 polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO. 8, an hGH polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO. 21, a synthetic polyadenylation signal comprising SEQ ID NO. 31 or a bGH polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO. 26.

In one aspect, the present disclosure provides and includes a method of converting reactive astrocytes into functional neurons in a living human brain, comprising: injecting an adeno-associated virus (AAV) into a subject in need thereof, wherein the AAV comprises a DNA vector construct comprising a nucleic acid encoding a human neurogenic differentiation factor 1 (hNeuroD 1) protein, the hNeuroD1 protein comprising an amino acid sequence of SEQ ID NO:10, and a second nucleic acid encoding a second protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 14, and 16, wherein the hNeuroD1 encoding sequence and the second encoding sequence are separated by: (i) a P2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID nos. 19 and 22, (ii) a T2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID nos. 20 and 23, or (iii) an Internal Ribosome Entry Site (IRES) sequence of an encephalomyocarditis virus comprising SEQ ID No. 3, wherein the hNeuroD1 coding sequence and the second nucleic acid coding sequence are operably linked to an expression control element comprising: (a) A human Glial Fibrillary Acidic Protein (GFAP) promoter comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs 4, 18 and 27; (b) An enhancer from the human elongation factor-1 alpha (EF-1 alpha) promoter comprising the nucleic acid sequence of SEQ ID NO. 2 or a Cytomegalovirus (CMV) enhancer comprising the nucleic acid sequence of SEQ ID NO. 17; (c) A chimeric intron comprising the nucleic acid sequence of SEQ ID NO. 5 or 28; (d) A woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs 7 and 30; and (e) an SV40 polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO. 8, an hGH polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO. 21, a synthetic polyadenylation signal comprising SEQ ID NO. 31 or a bGH polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO. 26.

In one aspect, the present disclosure provides and includes a method of converting glial cells into neurons in a subject in need thereof, comprising: delivering an adeno-associated virus (AAV) to a subject in need thereof, wherein the AAV comprises a DNA vector construct comprising a neurogenic differentiation factor 1 (NeuroD 1) sequence and a second protein sequence, wherein the NeuroD1 sequence and the second protein sequence are separated by a linker, wherein the NeuroD1 sequence and the second sequence are operably linked to an expression control element comprising: (a) a Glial Fibrillary Acidic Protein (GFAP) promoter; (b) an enhancer; (c) a chimeric intron; (d) Woodchuck hepatitis virus posttranscriptional regulatory elements (WPREs); and (e) a polyadenylation signal, wherein the vector is capable of converting at least one glial cell to a neuron in the subject in need thereof.

In one aspect, the present disclosure provides and includes a method of treating a neurological disorder in a subject in need thereof, comprising: delivering an adeno-associated virus (AAV) to a subject, wherein the AAV administered to the subject in need thereof comprises a DNA vector construct comprising a neurogenic differentiation factor 1 (NeuroD 1) sequence and a second sequence, wherein the NeuroD1 sequence and the second protein sequence are separated by a linker, wherein the NeuroD1 sequence and the second protein sequence are operably linked to an expression control element comprising: (a) a Glial Fibrillary Acidic Protein (GFAP) promoter; (b) An enhancer from the human elongation factor-1 alpha (EF-1 alpha) promoter; (c) a chimeric intron; (d) Woodchuck hepatitis virus posttranscriptional regulatory elements (WPREs); and (e) an SV40 polyadenylation signal.

In one aspect, the disclosure provides and includes an adeno-associated virus (AAV) vector comprising: a human neurogenic differentiation factor 1 (hNeuroD 1) sequence, the hNeuroD1 sequence comprising the nucleic acid sequence of SEQ ID No. 6, and a second sequence comprising a nucleic acid sequence selected from the group consisting of SEQ ID nos. 11, 13 and 15, wherein the hNeuroD1 sequence and the second sequence are separated by: (i) a P2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID nos. 19 and 22, (ii) a T2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID nos. 20 and 23, or (iii) an Internal Ribosome Entry Site (IRES) sequence of an encephalomyocarditis virus comprising SEQ ID No. 3, wherein said hNeuroD1 sequence and said second sequence are operably linked to a regulatory element comprising: (a) A Glial Fibrillary Acidic Protein (GFAP) promoter comprising the nucleic acid sequence of SEQ ID No. 27; (b) A Cytomegalovirus (CMV) enhancer comprising the nucleic acid sequence of SEQ ID No. 17; (c) a chimeric intron comprising the nucleic acid sequence of SEQ ID NO. 5; and (d) a bGH polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO. 26.

In one aspect, the disclosure provides and includes an adeno-associated virus (AAV) vector comprising: a human neurogenic differentiation factor 1 (hNeuroD 1) sequence, the hNeuroD1 sequence comprising the nucleic acid sequence of SEQ ID No. 6, and a second sequence comprising a nucleic acid sequence selected from the group consisting of SEQ ID nos. 11, 13 and 15, wherein the hNeuroD1 sequence and the second sequence are separated by: (i) a P2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID nos. 19 and 22, (ii) a T2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID nos. 20 and 23, or (iii) an Internal Ribosome Entry Site (IRES) sequence of an encephalomyocarditis virus comprising SEQ ID No. 3, wherein said hNeuroD1 sequence and said second sequence are operably linked to a regulatory element comprising: (a) A Glial Fibrillary Acidic Protein (GFAP) promoter comprising the nucleic acid sequence of SEQ ID No. 27; (b) A Cytomegalovirus (CMV) enhancer comprising the nucleic acid sequence of SEQ ID No. 17; (c) A chimeric intron comprising the nucleic acid sequence of SEQ ID NO. 28; and (d) a bGH polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO. 26.

In one aspect, the present disclosure provides and includes an adeno-associated virus (AAV) vector comprising a nucleic acid coding sequence encoding a human neuro-derived differentiation factor 1 (hNeuroD 1) protein, the hNeuroD1 protein comprising an amino acid sequence of SEQ ID No. 10, and a second nucleic acid coding sequence encoding a second protein having an amino acid selected from the group consisting of SEQ ID nos. 12, 14, and 16, wherein the hNeuroD1 coding sequence and the second coding sequence are separated by: (i) a P2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID nos. 19 and 22, (ii) a T2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID nos. 20 and 23, or (iii) an Internal Ribosome Entry Site (IRES) sequence of an encephalomyocarditis virus comprising SEQ ID No. 3, wherein the hNeuroD1 coding sequence and the second coding sequence are operably linked to a regulatory element comprising: (a) A Glial Fibrillary Acidic Protein (GFAP) promoter comprising the nucleic acid sequence of SEQ ID No. 27; (b) A Cytomegalovirus (CMV) enhancer comprising the nucleic acid sequence of SEQ ID No. 17; (c) a chimeric intron comprising the nucleic acid sequence of SEQ ID NO. 5; and (d) a bGH polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO. 26.

In one aspect, the present disclosure provides and includes an adeno-associated virus (AAV) vector comprising a nucleic acid coding sequence encoding a human neuro-derived differentiation factor 1 (hNeuroD 1) protein, the hNeuroD1 protein comprising an amino acid sequence of SEQ ID No. 10, and a second nucleic acid coding sequence encoding a second protein having an amino acid selected from the group consisting of SEQ ID nos. 12, 14, and 16, wherein the hNeuroD1 coding sequence and the second coding sequence are separated by: (i) a P2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID nos. 19 and 22, (ii) a T2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID nos. 20 and 23, or (iii) an Internal Ribosome Entry Site (IRES) sequence of an encephalomyocarditis virus comprising SEQ ID No. 3, wherein the hNeuroD1 coding sequence and the second coding sequence are operably linked to a regulatory element comprising: (a) A Glial Fibrillary Acidic Protein (GFAP) promoter comprising the nucleic acid sequence of SEQ ID No. 27; (b) A Cytomegalovirus (CMV) enhancer comprising the nucleic acid sequence of SEQ ID No. 17; (c) A chimeric intron comprising the nucleic acid sequence of SEQ ID NO. 28; and (d) a bGH polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO. 26.

In one aspect, the present disclosure provides and includes a composition comprising: (i) An adeno-associated virus (AAV) vector comprising a human neurogenic differentiation factor 1 (hNeuroD 1) sequence, the hNeuroD1 sequence comprising a nucleic acid sequence of SEQ ID NO:6, and (ii) an adeno-associated virus (AAV) comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 11, 13, and 15, wherein the hNeuroD1 sequence is operably linked to a regulatory element comprising: (a) A Glial Fibrillary Acidic Protein (GFAP) promoter comprising the nucleic acid sequence of SEQ ID No. 27; (b) An enhancer from the human elongation factor-1 alpha (EF 1-alpha) promoter comprising the nucleic acid sequence of SEQ ID NO. 2 or a Cytomegalovirus (CMV) enhancer comprising the nucleic acid sequence of SEQ ID NO. 17; (c) A chimeric intron comprising the nucleic acid sequence of SEQ ID NO. 28; (d) A woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) comprising the nucleic acid sequence of SEQ ID NO. 30; and (e) a bGH polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO. 26. In one aspect of the disclosure, (ii) comprises an AAV comprising a nucleic acid sequence comprising SEQ ID NO:13, the regulatory element comprising: (a) A Glial Fibrillary Acidic Protein (GFAP) promoter comprising the nucleic acid sequence of SEQ ID No. 27; (b) A Cytomegalovirus (CMV) enhancer comprising the nucleic acid sequence of SEQ ID No. 17; (c) A chimeric intron comprising the nucleic acid sequence of SEQ ID NO. 28; (d) A woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) comprising the nucleic acid sequence of SEQ ID NO. 30; and a bGH polyadenylation signal comprising the nucleic acid sequence of SEQ ID No. 26. In one aspect of the disclosure, (ii) comprises an AAV comprising a nucleic acid sequence comprising SEQ ID NO:11, the regulatory element comprising: (a) A Glial Fibrillary Acidic Protein (GFAP) promoter comprising the nucleic acid sequence of SEQ ID No. 27; (b) A Cytomegalovirus (CMV) enhancer comprising the nucleic acid sequence of SEQ ID No. 17; (c) A chimeric intron comprising the nucleic acid sequence of SEQ ID NO. 28; (d) A woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) comprising the nucleic acid sequence of SEQ ID NO. 30; and a bGH polyadenylation signal comprising the nucleic acid sequence of SEQ ID No. 26.

In one aspect, the present disclosure provides and includes a composition comprising: (i) An adeno-associated virus (AAV) vector comprising a nucleic acid coding sequence encoding a human neurogenic differentiation factor 1 (hNeuroD 1) protein, the hNeuroD1 protein comprising the amino acid sequence of SEQ ID NO:10, and (ii) an adeno-associated virus (AAV) vector comprising a nucleic acid coding sequence encoding a protein having an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 14, and 16, wherein the hNeuroD1 coding sequence is operably linked to a regulatory element comprising: (a) A Glial Fibrillary Acidic Protein (GFAP) promoter comprising the nucleic acid sequence of SEQ ID No. 27; (b) An enhancer from the human elongation factor-1 alpha (EF 1-alpha) promoter comprising the nucleic acid sequence of SEQ ID NO. 2 or a Cytomegalovirus (CMV) enhancer comprising the nucleic acid sequence of SEQ ID NO. 17; (c) A chimeric intron comprising the nucleic acid sequence of SEQ ID NO. 28; (d) A woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) comprising the nucleic acid sequence of SEQ ID NO. 30; and (e) a bGH polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO. 26. In one aspect, (ii) comprises an AAV vector comprising a nucleic acid encoding sequence encoding a protein having an amino acid sequence comprising SEQ ID No. 14 operably linked to a regulatory element comprising: (a) A Glial Fibrillary Acidic Protein (GFAP) promoter comprising the nucleic acid sequence of SEQ ID No. 27; (b) A Cytomegalovirus (CMV) enhancer comprising the nucleic acid sequence of SEQ ID No. 17; (c) A chimeric intron comprising the nucleic acid sequence of SEQ ID NO. 28; (d) A woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) comprising the nucleic acid sequence of SEQ ID NO. 30; and a bGH polyadenylation signal comprising the nucleic acid sequence of SEQ ID No. 26. In one aspect, (ii) comprises an AAV vector comprising a nucleic acid encoding sequence encoding a protein having an amino acid sequence comprising SEQ ID NO:12 operably linked to a regulatory element comprising: (a) A Glial Fibrillary Acidic Protein (GFAP) promoter comprising the nucleic acid sequence of SEQ ID No. 27; (b) A Cytomegalovirus (CMV) enhancer comprising the nucleic acid sequence of SEQ ID No. 17; (c) A chimeric intron comprising the nucleic acid sequence of SEQ ID NO. 28; (d) A woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) comprising the nucleic acid sequence of SEQ ID NO. 30; and a bGH polyadenylation signal comprising the nucleic acid sequence of SEQ ID No. 26.

Drawings

FIG. 1A depicts a diagram of CE Gfa681: neuroD1: P2A: ascl1: WPRE: SV 40.

FIG. 1B depicts a diagram of EF-1α: gfa681:NeuroD1:P2A:Ascl1:WPRE:SV 40.

FIG. 1C depicts a diagram of CE Gfa681:NeuroD1:GSG-P2A:Ascl1:WPRE:SV 40.

FIG. 1D depicts a diagram of EF-1α: gfa681:NeuroD1:GSG-P2A:Ascl1:WPRE:SV 40.

FIG. 2A depicts a diagram of CE Gfa681: neuroD1: P2A: ascl1: WPRE: hGH.

FIG. 2B depicts a diagram of EF-1α: gfa681:NeuroD1:P2A:Ascl 1:WPRE:hGH.

FIG. 2C depicts a diagram of CE Gfa681: neuroD1: GSG-P2A: ascl1: WPRE: hGH.

FIG. 2D depicts a diagram of EF-1α: gfa681:NeuroD1:GSG-P2A:Ascl 1:WPRE:hGH.

FIG. 3A depicts a diagram of CE Gfa681: neuroD1: T2A: ascl1: WPRE: SV 40.

FIG. 3B depicts a diagram of EF-1α: gfa681:NeuroD1:T2A:Ascl1:WPRE:SV 40.

FIG. 3C depicts a diagram of CE Gfa681:NeuroD1:GSG-T2A:Ascl1:WPRE:SV 40.

FIG. 3D depicts a diagram of EF-1α: gfa681:NeuroD1:GSG-T2A:Ascl1:WPRE:SV 40.

FIG. 4A depicts a diagram of CE Gfa681: neuroD1: T2A: ascl1: WPRE: hGH.

FIG. 4B depicts a diagram of EF-1α: gfa681:NeuroD1:T2A:Ascl 1:WPRE:hGH.

FIG. 4C depicts a diagram of CE Gfa681: neuroD1: GSG-T2A: ascl1: WPRE: hGH.

FIG. 4D depicts a diagram of EF-1α: gfa681:NeuroD1:GSG-T2A:Ascl 1:WPRE:hGH.

FIG. 5A depicts a diagram of CE Gfa681: neuroD1: P2A: isl1: WPRE: SV 40.

FIG. 5B depicts a diagram of EF-1α: gfa681:NeuroD1:P2A:Isl1:WPRE:SV 40.

FIG. 5C depicts a diagram of CE Gfa 681. NeuroD 1. GSG-P2A. Isl 1. WPRE. SV 40.

FIG. 5D depicts a diagram of EF-1α: gfa681:NeuroD1:GSG-P2A:Isl1:WPRE:SV 40.

FIG. 6A depicts a diagram of CE Gfa681: neuroD1: P2A: isl1: WPRE: hGH.

FIG. 6B depicts a diagram of EF-1α: gfa681:NeuroD 1:P2A:Isl1:WPRE:hGH.

FIG. 6C depicts a diagram of CE Gfa681: neuroD1: GSG-P2A: isl1: WPRE: hGH.

FIG. 6D depicts a diagram of EF-1α: gfa681:NeuroD1:GSG-P2 A:Isl1:hGH.

FIG. 7A depicts a diagram of CE Gfa681:NeuroD1:T2A:Isl1:WPRE:SV 40.

FIG. 7B depicts a diagram of EF-1α: gfa681:NeuroD1:T2A:Isl1:WPRE:SV 40.

FIG. 7C depicts a diagram of CE Gfa681:NeuroD1:GSG-T2A:Isl1:WPRE:SV 40.

FIG. 7D depicts a diagram of EF-1α: gfa681:NeuroD1:GSG-T2A:Isl1:WPRE:SV 40.

FIG. 8A depicts a diagram of CE Gfa681: neuroD1: T2A: isl1: WPRE: hGH.

FIG. 8B depicts a diagram of EF-1α: gfa681:NeuroD 1:T2A:Isl1:WPRE:hGH.

FIG. 8C depicts a diagram of CE Gfa681: neuroD1: GSG-T2A: isl1: WPRE: hGH.

FIG. 8D depicts a diagram of EF-1α: gfa681:NeuroD1:GSG-T2 A:Isl1:WPRE:hGH.

FIG. 9A depicts a diagram of CE Gfa 681. NeuroD 1. P2A LHX 3. WPRE. SV 40.

FIG. 9B depicts a diagram of EF-1α: gfa681:NeuroD1:P2A:LHX3:WPRE:SV 40.

FIG. 9C depicts a diagram of CE Gfa681 neuroD1 GSG-P2A LHX3 WPRE SV 40.

FIG. 9D depicts a diagram of EF-1α: gfa681:NeuroD1:GSG-P2A:LHX3:WPRE:SV 40.

FIG. 10A depicts a diagram of CE Gfa681: neuroD1: P2A: LHX3: WPRE: hGH.

FIG. 10B depicts a diagram of EF-1α: gfa681:NeuroD 1:P2A:LHX3:WPRE:hGH.

FIG. 10C depicts a diagram of CE Gfa681: neuroD1: GSG-P2A: LHX3: WPRE: hGH.

FIG. 10D depicts a diagram of EF-1α: gfa681:NeuroD1:GSG-P2 A:LHX3:WPRE:hGH.

FIG. 11A depicts a diagram of CE Gfa 681. NeuroD 1. T2A LHX 3. WPRE. SV 40.

FIG. 11B depicts a diagram of EF-1α: gfa681:NeuroD1:T2A:LHX3:WPRE:SV 40.

FIG. 11C depicts a diagram of CE Gfa681 neuroD1 GSG-T2A LHX3 WPRE SV 40.

FIG. 11D depicts a diagram of EF-1α: gfa681:NeuroD1:GSG-T2A:LHX3:WPRE:SV 40.

FIG. 12A depicts a diagram of CE Gfa681: neuroD1: T2A: LHX3: WPRE: hGH.

FIG. 12B depicts a diagram of EF-1α: gfa681:NeuroD 1:T2A:LHX3:WPRE:hGH.

FIG. 12C depicts a diagram of CE Gfa681: neuroD1: GSG-T2A: LHX3: WPRE: hGH.

FIG. 12D depicts a diagram of EF-1α: gfa681:NeuroD1:GSG-T2 A:LHX3:WPRE:hGH.

FIG. 13A depicts a diagram of CE Gfa681 ISL1 WPRE SV 40.

FIG. 13B depicts a diagram of EF-1α: gfa681:ISL1:WPRE:SV 40.

FIG. 13C depicts a diagram of CE: gfa1.6p: ISL1: WPRE: SV 40.

FIG. 13D depicts a diagram of EF-1α:Gfa1.6p:ISL1:WPRE:SV 40.

FIG. 13E depicts a diagram of CE: gfa2.2: ISL1: WPRE: SV 40.

FIG. 13F depicts a diagram of EF-1α:Gfa2.2:ISL1:WPRE:SV 40.

FIG. 14A depicts a diagram of CE Gfa681:ISL 1:WPRE:hGH.

FIG. 14B depicts a diagram of EF-1α: gfa 681:ISL1:WPRE:hGH.

FIG. 14C depicts a diagram of CE: gfa1.6p: ISL1: WPRE: hGH.

FIG. 14D depicts a diagram of EF-1α:Gfa1.6p:ISL 1:WPRE:hGH.

FIG. 14E depicts a diagram of CE: gfa2.2: ISL1: WPRE: hGH.

FIG. 14F depicts a diagram of EF-1α:Gfa2.2:ISL1:WPRE:hGH.

FIG. 15A depicts a diagram of CE Gfa681 LHX3 WPRE SV 40.

FIG. 15B depicts a diagram of EF-1α: gfa681:LHX3:WPRE:SV 40.

FIG. 15C depicts a diagram of CE: gfa1.6p: LHX3: WPRE: SV 40.

FIG. 15D depicts a diagram of EF-1α:Gfa1.6p:LHX3:WPRE:SV 40.

FIG. 15E depicts a diagram of CE: gfa2.2: LHX3: WPRE: SV 40.

FIG. 15F depicts a diagram of EF-1α: gfa2.2: LHX3: WPRE: SV 40.

FIG. 16A depicts a diagram of CE Gfa681: LHX3: WPRE: hGH.

FIG. 16B depicts a diagram of EF-1. Alpha.: gfa681: LHX3: WPRE: hGH.

FIG. 16C depicts a diagram of CE: gfa1.6p: LHX3: WPRE: hGH.

FIG. 16D depicts a diagram of EF-1α:Gfa1.6p:LHX3:WPRE:hGH.

FIG. 16E depicts a diagram of EE-1α:Gfa2.2:LHX3:WPRE:hGH.

FIG. 16F depicts a diagram of EF-1α: gfa2.2: LHX3: WPRE: hGH.

FIG. 17 measures AAV viral production of the P35 plasmid. Titer analysis was performed using the gene of interest (GOI) primer, ITR region primer and reverse package primer. The viral yield was calculated as vg/cell.

FIG. 18 depicts the establishment of a primary culture of rat astrocytes from Sprague-Dawley rat brain 3 days after birth. The leftmost panel presents images of GFAP stained cells. The middle left panel presents an image of SOX9 stained cells. The middle right panel presents images of DAPI stained cells. The rightmost panel presents a pooled image of GFAP, SOX9 and DAPI stained cells.

FIG. 19 depicts transfection of primary rat astrocytes with plasmid P5 (pEF-1α: hNeuroD1: GFP). The left panel presents images of NeuroD1 stained cells. The middle left panel presents images of GFP-expressing cells. The middle right panel represents DAPI stained cells. The right panel represents a pooled image of NeuroD1, GFP and DAPI stained cells.

FIG. 20 depicts expression of neuroD1 expression in plasmid transfected astrocytes. Primary rat astrocytes were transfected with P6 (pEF-1α: hNeuroD1: WPRE: SV 40) expression vector, P11 (CE: gfaABC1D: neuroD1: WPRE: SV 40) expression vector, P35 (EF-1α: gfaABC1D: neuroD1: WPRE: SV 40) expression vector, or P39 (EF-1α: gfa1.6: neuroD1: WPRE: SV 40). The upper panel shows the NeuroD1 staining of cells and the lower panel shows the combined NeuroD1 and DAPI staining of cells.

FIG. 21 depicts a comparison of AAV viral particle transduction at different doses using AAV9-P12 (pGfaABC 1D: GFP). Left figure shows 3x10 ¹⁰ Dose of vg per well. Middle diagram shows 1x10 ¹⁰ Dose of vg per well. The right figure shows 2.5x10 ⁹ Dose of vg per well.

Fig. 22A and 22B depict quantitative analysis of AAV particle transduction into primary rat astrocytes. FIG. 22A shows AAV9-P12 (pGfaABC 1D: GFP) and AAV5-P7 (pEF-1α: GFP) at an MOI of 5x10 ⁵ vg/cell, 2X10 ⁵ vg/cell and 5x10 ⁴ Percent transduction at vg/cell. FIG. 22B shows AAV9-P12 (pGfaABC 1D: GFP) in a series of densities (2X 10) ⁴ Individual cells/well, 1.5x10 ⁴ Individual cells/well, 1x10 ⁴ Individual cells/well and 5x10 ³ Individual cells/well) and inoculated with a series of amounts of virus (1 x10 in 100 μl of medium ¹³ 2. Mu.l, 1. Mu.l, 0.5. Mu.l, 0.25. Mu.l, 0.125. Mu.l) of vg/ml virus.

Figure 23 depicts transduction of AAV viral particles comprising NeuroD1 into primary rat astrocytes. Primary rat astrocytes were transduced with AAV5-P1 (AAV 5: pGfa2.2: cre) and AAV4-P4 (AAV 5: pCAG: flex: hNeuroD1: GFP) (left panel), AAV9-P9 (CE: gfaABC1D: neuroD1: GFP) (middle panel) or AAV9-P11 (CE: gfaABC1D: neuroD1: WPRE: SV 40) (right panel).

FIG. 24 depicts AAV9-P21 (CE-pGFA 681-CI-GFP-WPRE-SV40 pA) at 2X10 using a control plasmid ¹⁰ RCA three weeks after vg/ml transduction. Cells were immunostained with antibodies to the neuronal markers NeuN and MAP2, DAPI (nuclear dye). GFP fluorescence indicates the presence of cells transduced with the control plasmid.

FIG. 25 depicts RCA immunostained with anti-NeuroD 1 (ND 1) antibody and DAPI (nuclear dye) 24 hours after transfection with NXL-P134 (CE-pGfa 681-CRGI-hND 1-oPRE-bGHPA).

FIG. 26 depicts AAV9-P134 (CE-pGfa 681-CRGI-hND 1-oPRE-bGHPA) at 2X10 ¹⁰ RCA immunostained with anti-ND 1 antibody and DAPI (nuclear dye) 6 days after vg/ml transduction.

FIG. 27 depicts AAV9-P134 (CE-pGfa 681-CRGI-hND 1-oPRE-bGHPA) at 2X10 ¹⁰ RCA immunostained with anti-NeuN and anti-MAP 2 antibodies and DAPI (nuclear dye) 3 weeks after vg/ml transduction. Transduction was performed using ND 1-containing vectors to generate neurons (NeuN// MAP 2+) from astrocyte cultures.

FIG. 28 depicts RCA immunostained with anti-ND 1 antibody and DAPI (nuclear dye) 24 hours after transfection with NXL-P138 (EE-pGfa 681-CRGI-hND 1-oPRE-bGHPA).

FIG. 29 depicts the use of AAV9-P138 (EE-pGfa 681-CRGI-hND 1-oPRE-bGHPA) at 2X10 ¹⁰ RCA immunostained with anti-ND 1 antibody and DAPI (nuclear dye) 6 days after vg/ml transduction.

FIG. 30 depicts the use of AAV9-P138 (EE-pGfa 681-CRGI-hND 1-oPRE-bGHPA) at 2X10 ¹⁰ RCA immunostained with anti-NeuN and anti-MAP 2 antibodies and DAPI (nuclear dye) 3 weeks after vg/ml transduction. Transduction was performed using ND 1-containing vectors to generate neurons (NeuN// MAP 2+) from astrocyte cultures.

FIG. 31 depicts AAV9-P9 (CE-pGfa)681-CI-hND1-p2A-GFP-WPRE-SV40 pA) at 2X10 ¹⁰ RCA immunostained with anti-ND 1 antibody and DAPI (nuclear dye) 6 days after vg/ml transduction. GFP fluorescence indicates the presence of transduced cells.

FIG. 32 depicts AAV9-P9 (CE-pGfa 681-CI-hND1-P2A-GFP-WPRE-SV40 pA) at 2X10 ¹⁰ RCA immunostained with anti-NeuN and anti-MAP 2 antibodies and DAPI (nuclear dye) 3 weeks after vg/ml transduction. Transduction was performed using ND 1-containing vectors to generate neurons (NeuN// MAP 2+) from astrocyte cultures.

FIG. 33 depicts RCA immunostained with anti-ND 1 antibody and DAPI (nuclear dye) 24 hours after transfection with NXL-P22 (CE-pGfa 681-CI-hND1-WPRE-SV40 pA).

FIG. 34 depicts AAV9-P22 (CE-pGfa 681-CI-hND1 WPRE-SV40 pA) at 2X10 ¹⁰ RCA immunostained with anti-ND 1 antibody and DAPI (nuclear dye) 6 days after vg/ml transduction.

FIG. 35 depicts AAV9-P22 (CE-pGfa 681-CI-hND1-WPRE-SV40 pA) at 2X10 ¹⁰ RCA immunostained with anti-NeuN and anti-MAP 2 antibodies and DAPI (nuclear dye) 3 weeks after vg/ml transduction. Transduction was performed using ND 1-containing vectors to generate neurons (NeuN// MAP 2+) from astrocyte cultures.

FIG. 36 depicts RCA immunostained with anti-ND 1 antibody and DAPI (nuclear dye) 24 hours after transfection with NXL-P35 (EE-pGfa 681-CI-hND1-WPRE-SV40 pA).

FIG. 37 depicts AAV9-P35 (EE-pGfa 681-CI-hND1 WPRE-SV40 pA) at 2X10 ¹⁰ RCA immunostained with anti-ND 1 antibody and DAPI (nuclear dye) 6 days after vg/ml transduction.

FIG. 38 depicts AAV9-P35 (EE-pGfa 681-CI-hND1-WPRE-SV40 pA) at 2X10 ¹⁰ RCA immunostained with anti-NeuN antibody and DAPI (nuclear dye) 3 weeks after vg/ml transduction. Transduction with ND 1-containing vectors resulted in the production of neurons (NeuN+) from astrocyte cultures.

FIG. 39 depicts RCA immunostained with anti-ND 1 antibody and DAPI (nuclear dye) 24 hours after transfection with NXL-P107 (CE-pGfa 681-CI-hND 1-bGHPA).

FIG. 40 depicts RCA immunostained with anti-ND 1 antibody and DAPI (nuclear dye) 24 hours after transfection with NXL-P108 (CE-pGfa 681-CI-hND 1-oPRE-bGHPA).

FIG. 41 depicts RCA immunostained with anti-ND 1 antibody and DAPI (nuclear dye) 24 hours after transfection with NXL-P109 (CE-pGfa 681-CRGI-hND 1-bGHPA).

FIG. 42 depicts cortical tissue 10 days post infection (dpi) in mice infected with AAV9-P12 (P12 control group), AAV9-P12+AAV9-P134 (P134 group), and AAV9-P12+AAV9-P138 (P138 group).

FIG. 43 depicts cortical tissue 30 days post infection (dpi) in mice infected with AAV9-P12+ AAV9-P134 (group P134) and AAV9-P12+ AAV9-P138 (group P138).

FIG. 44 depicts cortical tissue at 10dpi in mice infected with AAV9-P12 (P12 control group) and AAV9-P12+ AAV9-P134 (P134 group) (bilateral injury model).

FIG. 45 is a graph of measurements of AAV viral production by the P134, P130, P138 and P21 plasmids. Titer analysis was performed by qPCR using primers that amplify the gene of interest (GOI) and primers specific for the plasmid. The viral yield was calculated as vg/cell.

FIG. 46 depicts Lec2 cells immunostained with anti-Isl 1 antibody and DAPI (nuclear dye) 24 hours after transfection with NXL-P141 (CE-pGfa 681-CRGI-hIsl 1-oPRE-bGHPA).

FIG. 47 depicts Lec2 cells immunostained with anti-Isl 1 antibody, anti-ND 1 antibody and DAPI (nuclear dye) 24 hours after transfection with NXL-P142 (CE-pGfa 681-CI-hIsl1-P2A-ND 1-bGHPA).

FIG. 48 depicts Lec2 cells immunostained with anti-Isl 1 antibody, anti-ND 1 antibody and DAPI (nuclear dye) 24 hours after transfection with NXL-P143 (CE-pGfa 681-CI-hND1-P2A-hIsl 1-bGHPA).

FIG. 49 depicts Lec2 cells immunostained with anti-Isl 1 antibody, anti-ND 1 antibody and DAPI (nuclear dye) 24 hours after transfection with NXL-P144 (CE-pGfa 681-CI-hIsl1-IRES-ND 1-bGHPA).

FIG. 50 depicts a comparison of viral yields using NLX-P143 and NLX-P144.

FIG. 51 depicts AAV9-P143 (CE-pGfa 681-CI-hND1-P2A-hIsl 1-bGHPA) at 2X10 ¹⁰ C8-DIA cells immunostained with anti-Isl 1 antibody, anti-ND 1 antibody and DAPI (nuclear dye) 48 hours after vg/ml transduction.

FIG. 52 depicts AAV9-P144 (CE-pGfa 681-CI-hIsl1-IRES-hND 1-bGHPA) at 2X10 ¹⁰ C8-DIA cells immunostained with anti-Isl 1 antibody, anti-ND 1 antibody and DAPI (nuclear dye) 48 hours after vg/ml transduction.

FIG. 53 depicts Lec2 cells immunostained with anti-Ascl 1 antibody and DAPI (nuclear dye) 24 hours after transfection with NXL-P151 (CE-pGfa 681-CRGI-hASCl 1-oPRE-bGHPA).

FIG. 54 depicts Lec2 cells immunostained with anti-Ascl 1 antibody, anti-ND 1 antibody and DAPI (nuclear dye) 24 hours after transfection with NXL-P152 (CE-pGfa 681-CI-hASCl1-IRES-hND 1-bGHPA).

FIG. 55 depicts Lec2 cells immunostained with anti-Ascl 1 antibody and DAPI (nuclear dye) 48 hours after transduction with AAV9-P151 (CE-pGfa 681-CRGI-hASCl 1-oPRE-bGHPA).

FIG. 56 depicts Lec2 cells immunostained with anti-Ascl 1 antibody, anti-ND 1 antibody and DAPI (nuclear stain) 48 hours after transduction with AAV9-P152 (CE-pGfa 681-CI-hASCl1-IRES-hND 1-bGHPA).

Brief description of the sequence

A list of nucleic acid sequences and amino acid sequences is provided in table 1.

TABLE 1 nucleic acid sequences and amino acid sequences

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Detailed Description

Unless defined otherwise, all technical and scientific terms used have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Where a term is provided in the singular, the inventors of the present application also contemplate the various aspects of the present disclosure described in the plural of the term. Where there is a difference in terms and definitions used in the references incorporated by reference, the terms used in the present application shall have the definitions given herein. Other technical terms used have their ordinary meaning in the field in which they are used, as exemplified by various field-specific dictionaries, e.g. "AmericanScientific dictionary) ("edit of U.S. traditional dictionary (American Heritage Dictionaries)," 2011, boston and N.Y. Hoton Mivelin publishing company (Houghton Mifflin Harcourt, boston and New York)), "" McGraw-Hill Dictionary of Scientific and Technical Terms), "(6 th edition, 2002, N.Y. Magla-Hill, new York) or" [ oxford biology dictionary (Oxford Dictionary of Biology) ] (6 th edition, 2008, oxford and N.Y. oxford university publishing company (Oxford University Press, oxford and New York) ].

Any references cited herein, including, for example, all patents, published patent applications, and non-patent publications, are incorporated by reference in their entirety.

When a set of alternatives is presented, any and all combinations of the members making up the set of alternatives are specifically contemplated. For example, if the item is selected from the group consisting of A, B, C and D, the inventors will specifically contemplate each alternative individually (e.g., a alone, B alone, etc.) and such as A, B and D; a and C; b and C; etc. The term "and/or" when used in a list of two or more items means any one of the listed items by itself or in combination with any one or more other listed items. For example, the expression "a and/or B" is intended to mean either or both of a and B, i.e., a alone, B alone, or a combination of a and B. The expression "A, B and/or C" is intended to mean a alone, B alone, a combination of C, A and B alone, a combination of a and C, a combination of B and C, or a combination of A, B and C.

When numerical ranges are provided herein, the ranges are understood to include the edges of the ranges as well as any numbers between the defined edges of the ranges. For example, "between 1 and 10" includes any number between 1 and 10 as well as the numbers 1 and 10.

When the term "about" is used in reference to a number, it is understood to mean plus or minus 10%. For example, "about 100" will include from 90 to 110.

As used herein, "hND1" refers to a human neuro-derived differentiation factor (NeuroD 1) gene or protein.

As used herein, "CE" refers to a Cytomegalovirus (CMV) promoter enhancer sequence.

As used herein, "EE" refers to the Ef1 a promoter enhancer sequence.

As used herein, "pGfa681" refers to a human Glial Fibrillary Acidic Protein (GFAP) promoter truncated sequence of 681bp in size. As used herein, "pGfa681", "Gfa681", "pGfaABC1D" and "GfaABC1D" are used interchangeably.

As used herein, "CI" refers to a chimeric intron consisting of a 5 '-donor site from the first intron of the human β -globulin gene and a branching and 3' -acceptor site from the heavy chain variable region intron of the immunoglobulin gene.

As used herein, "CRGI" refers to chimeric introns of rabbit β -globulin and chicken β -actin that are similar in the CAG promoter.

As used herein, "GI" refers to the human Glial Fibrillary Acidic Protein (GFAP) first intron.

As used herein, "WPRE" refers to Woodchuck Hepatitis Virus (WHV) post-transcriptional regulatory elements.

As used herein, "oPRE" refers to an optimized version of WPRE.

As used herein, "SV40pA" refers to the polyadenylation signal of SV40 virus.

As used herein, "bGHpA" refers to the polyadenylation signal of bovine growth hormone.

As used herein, "SpA" represents a synthetic poly A signal.

As used herein, "vg" refers to the viral genome.

As used herein, "hls1" refers to the human insulin gene enhancer protein ISL-1.

As used herein, "hASC1" refers to human acetate-scan homolog 1.

Any of the compositions or carriers provided herein are specifically contemplated for use in any of the methods provided herein.

In one aspect, the methods and compositions provided herein comprise a carrier. As used herein, the term "vector" refers to a circular double stranded DNA molecule that is physically separated from chromosomal DNA. It should be noted that the term "vector" may be used interchangeably with the term "plasmid".

In one aspect, the vectors provided herein are recombinant vectors. As used herein, the term "recombinant vector" refers to a vector comprising a recombinant nucleic acid. As used herein, "recombinant nucleic acid" refers to a nucleic acid molecule formed by laboratory methods of gene recombination (such as, but not limited to, molecular cloning). The gene vector may be formed by laboratory methods of genetic recombination, such as, but not limited to, molecular cloning. Furthermore, the person skilled in the art can create recombinant vectors de novo by synthesizing plasmids from a single nucleotide or by splicing together nucleic acid molecules from different pre-existing vectors, but is not limited thereto.

Adeno-associated viruses (AAV) are replication-defective, non-enveloped, dependent parvovirus (dependoparvorrus) viruses that infect humans and other primate species. AAV is known not to cause disease in any species, but they cause a mild immune response. AAV can infect dividing cells and resting cells. AAV stably integrates into the human genome at a specific site on chromosome 19 known as the AAVs1 locus (nucleotides 7774-11429 of GenBank accession No. AC 010327.8), although random integration of other loci in the human genome is possible.

AAV comprises a linear genome having single stranded DNA of about 4700 nucleotides in length. The genome of AAV also includes 145 nucleotide long Inverted Terminal Repeats (ITRs) at each end of the genome. Two viral genes flank the ITR: rep (for replication, encoding a non-structural protein) and cap (for capsid, encoding a structural protein). The ITR contains all cis-acting elements required for AAV genome rescue, replication and packaging.

When used in gene therapy methods, the rep and cap genes of the AAV genomic sequences are removed and replaced with the DNA of interest located between the two AAV ITRs. As used herein, an "AAV vector construct" refers to a DNA molecule comprising a desired sequence inserted between two AAV ITR sequences. As used herein, "AAV vector" refers to AAV packaged with a DNA vector construct.

As used herein, the term "AAV vector serotype" refers primarily to variation within the capsid protein of an AAV vector.

In one aspect, the AAV vector is selected from the group consisting of: AAV vector serotype 1, AAV vector serotype 2, AAV vector serotype 3, AAV vector serotype 4, AAV vector serotype 5, AAV vector serotype 6, AAV vector serotype 7, AAV vector serotype 8, AAV vector serotype 9, AAV vector serotype 10, AAV vector serotype 11, and AAV vector serotype 12. In one aspect, the AAV vector is selected from the group consisting of AAV serotype 2, AAV serotype 5, and AAV serotype 9. In one aspect, the AAV vector is AAV serotype 1. In one aspect, the AAV vector is AAV serotype 2. In one aspect, the AAV vector is AAV serotype 3. In one aspect, the AAV vector is AAV serotype 4. In one aspect, the AAV vector is AAV serotype 5. In one aspect, the AAV vector is AAV serotype 6. In one aspect, the AAV vector is AAV serotype 7. In one aspect, the AAV vector is AAV serotype 8. In one aspect, the AAV vector is AAV serotype 9. In one aspect, the AAV vector is AAV serotype 10. In one aspect, the AAV vector is AAV serotype 11. In one aspect, the AAV vector is AAV serotype 12.

In one aspect, the AAV vector ITR is selected from the group consisting of: AAV serotype 1 ITRs, AAV serotype 2 ITRs, AAV serotype 3 ITRs, AAV serotype 4 ITRs, AAV serotype 5 ITRs, AAV serotype 6 ITRs, AAV serotype 7 ITRs, AAV serotype 8 ITRs, AAV serotype 9 ITRs, AAV serotype 10 ITRs, AAV serotype 11 ITRs, and AAV serotype 12 ITRs. In one aspect, the AAV vector ITRs are AAV serotype 1 ITRs. In one aspect, the AAV vector ITRs are AAV serotype 2 ITRs. In one aspect, the AAV vector ITRs are AAV serotype 3 ITRs. In one aspect, the AAV vector ITRs are AAV serotype 4 ITRs. In one aspect, the AAV vector ITRs are AAV serotype 5 ITRs. In one aspect, the AAV vector ITRs are AAV serotype 6 ITRs. In one aspect, the AAV vector ITRs are AAV serotype 7 ITRs. In one aspect, the AAV vector ITRs are AAV serotype 8 ITRs. In one aspect, the AAV vector ITRs are AAV serotype 9 ITRs. In one aspect, the AAV vector ITRs are AAV serotype 10 ITRs. In one aspect, the AAV vector ITRs are AAV serotype 11 ITRs. In one aspect, the AAV vector ITRs are AAV serotype 12 ITRs.

In one aspect, the at least one AAV vector ITR nucleic acid sequence is selected from the group consisting of: SEQ ID NOS 1 and 9. In one aspect, the at least one AAV vector ITR nucleic acid sequence is SEQ ID NO 1. In one aspect, the at least one AAV vector ITR nucleic acid sequence is SEQ ID NO 9.

In one aspect, the AAV ITR nucleic acid sequence comprises a sequence at least 70% identical to: SEQ ID NO. 1, or a complement thereof. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence at least 75% identical to: SEQ ID NO. 1, or a complement thereof. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence at least 80% identical to: SEQ ID NO. 1, or a complement thereof. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence at least 85% identical to: SEQ ID NO. 1, or a complement thereof. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence at least 90% identical to: SEQ ID NO. 1, or a complement thereof. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence at least 91% identical to: SEQ ID NO. 1, or a complement thereof. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence at least 92% identical to: SEQ ID NO. 1, or a complement thereof. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence at least 93% identical to: SEQ ID NO. 1, or a complement thereof. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence at least 94% identical to: SEQ ID NO. 1, or a complement thereof. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence at least 95% identical to: SEQ ID NO. 1, or a complement thereof. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence at least 96% identical to: SEQ ID NO. 1, or a complement thereof. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence at least 97% identical to: SEQ ID NO. 1, or a complement thereof. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence at least 98% identical to: SEQ ID NO. 1, or a complement thereof. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence at least 99% identical to: SEQ ID NO. 1, or a complement thereof. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence at least 99.5% identical to: SEQ ID NO. 1, or a complement thereof. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence at least 99.8% identical to: SEQ ID NO. 1, or a complement thereof. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence at least 99.9% identical to: SEQ ID NO. 1, or a complement thereof. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence 100% identical to: SEQ ID NO. 1, or a complement thereof.

In one aspect, the AAV ITR nucleic acid sequence comprises a sequence at least 70% identical to: SEQ ID NO. 9, or a complement thereof. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence at least 75% identical to: SEQ ID NO. 9, or a complement thereof. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence at least 80% identical to: SEQ ID NO. 9, or a complement thereof. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence at least 85% identical to: SEQ ID NO. 9, or a complement thereof. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence at least 90% identical to: SEQ ID NO. 9, or a complement thereof. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence at least 91% identical to: SEQ ID NO. 9, or a complement thereof. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence at least 92% identical to: SEQ ID NO. 9, or a complement thereof. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence at least 93% identical to: SEQ ID NO. 9, or a complement thereof. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence at least 94% identical to: SEQ ID NO. 9, or a complement thereof. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence at least 95% identical to: SEQ ID NO. 9, or a complement thereof. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence at least 96% identical to: SEQ ID NO. 9, or a complement thereof. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence at least 97% identical to: SEQ ID NO. 9, or a complement thereof. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence at least 98% identical to: SEQ ID NO. 9, or a complement thereof. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence at least 99% identical to: SEQ ID NO. 9, or a complement thereof. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence at least 99.5% identical to: SEQ ID NO. 9, or a complement thereof. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence at least 99.8% identical to: SEQ ID NO. 9, or a complement thereof. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence at least 99.9% identical to: SEQ ID NO. 9, or a complement thereof. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence 100% identical to: SEQ ID NO. 9, or a complement thereof.

As used herein, the term "percent identity" or "percent identity" with respect to two or more nucleotide or amino acid sequences is calculated by: (i) comparing the two optimally aligned sequences (nucleotides or amino acids) over a comparison window (one or more "alignable" regions), (ii) determining the number of positions at which the same nucleobase (for nucleotide sequences) or amino acid residue (for proteins and polypeptides) occurs in the two sequences to produce a number of matched positions, (iii) dividing the number of matched positions by the total number of positions in the comparison window, and then (iv) multiplying the quotient by 100% to produce a percentage of identity. If "percent identity" is calculated relative to the reference sequence without specifying a particular comparison window, the percent identity is determined by dividing the number of matching positions on the alignment area by the total length of the reference sequence. Thus, for the purposes of the present application, when two sequences (query and subject) are optimally aligned (gaps are allowed in their alignment), the "percent identity" of a query sequence is equal to the number of identical positions between the two sequences divided by the total number of positions of the query sequence over its length (or comparison window), and then multiplied by 100%.

When referring to amino acid usage sequence identity percentages, it is believed that the different residue positions typically differ by conservative amino acid substitutions, wherein the amino acid residue is replaced with other amino acid residues having similar chemical properties (e.g., charge or hydrophobicity) and thus do not alter the functional properties of the molecule. When conservative substitutions of sequences are different, the percent sequence identity may be adjusted upward to correct for the conservation of the substitution. Sequences that differ due to such conservative substitutions are said to have "sequence similarity" or "similarity".

To optimally align sequences to calculate their percent identity, various pairwise or multiplex sequence alignment algorithms and procedures are known in the art, such as ClustalW or Basic Local Alignment Search, which can be used to compare sequence identity or similarity between two or more nucleotide or amino acid sequences(BLAST ^TM ) Etc. Although other alignment and comparison methods are known in the art, the alignment and percent identity between two sequences (including the percent identity ranges described above) can beFor determination by the ClustalW algorithm, see, e.g., chenna et al, "Multiple sequence alignment with the Clustal series of programs," Nucleic Acids Research31:3497-3500 (2003); thompson et al, "Clustal W Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice," Nucleic Acids Research 22:4673-4680 (1994); larkin MA et al, "Clustal W and Clustal X version 2.0," Bioinformation 23:2947-48 (2007); and Altschul et al, "Basic local alignment search tool." J.mol. Biol.215:403-410 (1990), the entire contents and disclosures of which are incorporated herein by reference.

As used herein, the term "percent complementarity" or "percent complementarity" with respect to two nucleotide sequences is similar to the concept of percent identity, but refers to the percent of nucleotides of a query sequence that optimally base pair or hybridize to nucleotides of a subject sequence when the query sequence and the subject sequence are aligned and optimally base pair without a secondary folding structure (such as a loop, stem, or hairpin). Such percent complementarity may be between two DNA strands, two RNA strands, or one DNA strand and one RNA strand. "percent complementarity" may be calculated by: (i) optimally base pairing or hybridizing two nucleotide sequences in a linear and fully extended arrangement (i.e., without folding or secondary structure) over a comparison window, (ii) determining the number of base pair positions between the two sequences over the comparison window to produce a number of complementary positions, (iii) dividing the number of complementary positions by the total number of positions in the comparison window, and (iv) multiplying the quotient by 100% to yield the percent complementarity of the two sequences. The optimal base pairing of two sequences can be determined by hydrogen bonding based on known pairing of nucleotide bases, such as G-C, A-T and A-U. If the "percent complementarity" is calculated relative to the reference sequence without specifying a particular comparison window, the percent identity is determined by dividing the number of positions of complementarity between the two linear sequences by the total length of the reference sequence. Thus, for the purposes of the present application, when two sequences (query and subject) are optimally base paired (nucleotides that allow for mismatch or non-base pairing), the "percent complementarity" of the query sequence is equal to the number of base pairing positions between the two sequences divided by the total number of positions of the query sequence over its length, and then multiplied by 100%.

The use of the terms "polynucleotide", "nucleic acid sequence" or "nucleic acid molecule" is not intended to limit the present disclosure to polynucleotides comprising deoxyribonucleic acid (DNA). For example, ribonucleic acid (RNA) molecules are also contemplated. One of ordinary skill in the art will recognize that polynucleotides and nucleic acid molecules may comprise a combination of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogs. Polynucleotides of the present disclosure also encompass all forms of sequences, including but not limited to single stranded forms, double stranded forms, hairpins, stem loop structures, and the like. In one aspect, the nucleic acid molecules provided herein are DNA molecules. In one aspect, the nucleic acid molecules provided herein are RNA molecules. In one aspect, the nucleic acid molecules provided herein are single stranded. In one aspect, the nucleic acid molecules provided herein are double-stranded. The nucleic acid molecule may encode a polypeptide or a small RNA.

As used herein, the term "polypeptide" refers to a chain of at least two covalently linked amino acids. The polypeptide may be encoded by a polynucleotide provided herein. The proteins provided herein may be encoded by the nucleic acid molecules provided herein. The protein may comprise a polypeptide provided herein. As used herein, "protein" refers to a chain of amino acid residues capable of providing a cell with structural or enzymatic activity. As used herein, "coding sequence" refers to a nucleic acid sequence that encodes a protein.

As used herein, the term "CpG site" or "CG site" refers to a region of DNA sequence in which cytosine and guanine are separated by only one phosphate group.

As used herein, the term "CpG island" of a "CG island" refers to CpG sites that occur at a high frequency.

As used herein, the term "codon" refers to a sequence of three nucleotides.

As used herein, the term "codon optimized" refers to codons modified to enhance expression in a host cell of interest by replacing at least one codon of the sequence with a more or most frequently used codon in the gene of the host cell while maintaining the original amino acid sequence.

As used herein, the term "enhancer" refers to a region of DNA sequence that operates to initiate, assist, influence, cause and/or promote transcription and expression of an associated transcribable DNA sequence or coding sequence in at least some tissues, developmental stages and/or conditions. In one aspect, the enhancer is a cis-enhancer. In one aspect, the enhancer is a trans-enhancer.

Enhancer sequences can be identified by utilizing genomic techniques well known in the art. Non-limiting examples include the use of reporter genes and next generation sequencing methods such as chromatin co-immunoprecipitation sequencing (ChIP-seq), DNase I hypersensitivity sequencing (DNase-seq), micrococcus nuclease sequencing (MNase-seq), formaldehyde assisted separation regulatory element sequencing (faie-seq), and chromatin transposase accessibility sequencing assays (ATAC-seq).

As used herein, the term "operably linked" refers to a functional linkage between a promoter or other regulatory element and an associated transcribable DNA sequence or coding sequence of a gene (or transgene) such that the promoter or the like operates to initiate, assist, affect, cause and/or promote transcription and expression of the associated transcribable DNA sequence or coding sequence at least in certain tissues, developmental stages and/or conditions. As used herein, "regulatory element" refers to any sequence element that positively or negatively regulates expression of an operably linked sequence. "regulatory elements" include, but are not limited to, promoters, enhancers, leader sequences, transcription initiation sites (TSS), linkers, 5 'and 3' untranslated regions (UTRs), introns, polyadenylation signals and termination regions or sequences, and the like, which are suitable, necessary or preferred for regulating or allowing expression of a gene or transcribable DNA sequence in a cell. Such additional regulatory elements may be optional and used to enhance or optimize expression of the gene or transcribable DNA sequence.

As used herein, the term "promoter" refers to a DNA sequence that contains an RNA polymerase binding site, a transcription initiation site, and/or a TATA box and that assists or facilitates transcription and expression of an associated transcribable polynucleotide sequence and/or gene (or transgene). Promoters may be synthetically produced, altered, or derived from known or naturally occurring promoter sequences or other promoter sequences. Promoters may also include chimeric promoters comprising a combination of two or more heterologous sequences. Thus, promoters of the application may include variants of promoter sequences that are similar in composition to, but not identical to, other promoter sequences known or provided herein.

As used herein, "intron" refers to a nucleotide sequence that is removed by RNA splicing when messenger RNA (mRNA) is mature from a pre-mRNA.

As used herein, "mRNA" or "messenger RNA" refers to single stranded RNA corresponding to the genetic sequence of a gene.

mRNA expression can be measured using any suitable method known in the art. Non-limiting examples of measuring mRNA expression include quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), northern blotting (e.g., northern blotting), and RNA sequencing. The expression difference may be described as absolute quantization or relative quantization. See, e.g., livak and Schmittgen, methods,25:402-408 (2001).

As used herein, the term "glia" or "glial cell" refers to a non-neuronal cell in the CNS or PNS. In one aspect, the at least one glial cell is selected from the group consisting of: at least one oligodendrocyte, at least one astrocyte, at least one NG2 cell, at least one ependymal cell, and at least one microglial cell. In one aspect, the at least one glial cell is at least one oligodendrocyte. In one aspect, the at least one glial cell is at least one NG2 cell. In one aspect, the at least one glial cell is at least one ependymal cell. In one aspect, the at least one glial cell is at least one microglial cell. In one aspect, the at least one glial cell is at least one reactive astrocyte. In one aspect, the at least one astrocyte is at least one reactive astrocyte.

As used herein, the term "astrocyte" refers to a glial cell that is an important component of the brain. Astrocytes are involved in supporting neuronal functions such as synapse formation and plasticity, potassium buffering, nutrient supply, secretion and absorption of neural or glial transmitters, and maintenance of the blood brain barrier. As used herein, the term "reactive astrocytes" refers to an abnormal state of astrocytes after injury or disease.

As used herein, the term "NG2 cells" or "oligodendrocytes" refers to glial cells that express chondroitin sulfate proteoglycan (CSPG 4) and platelet derived growth factor alpha receptor (PDGFRA).

As used herein, the term "neuron" or "neuronal cell" refers to an electrically excitable cell that communicates with other neurons through synapses. In one aspect, the neuron is selected from the group consisting of a unipolar neuron, a bipolar neuron, a pseudounipolar neuron, and a multipolar neuron. In one aspect, the neuron is a unipolar neuron. In one aspect, the neuron is a bipolar neuron. In one aspect, the neuron is a pseudounipolar neuron. In one aspect, the neuron is a bipolar neuron. In one aspect, the neuron is selected from the group consisting of a sensory neuron, a motor neuron, and an interneuron. In one aspect, the neuron is a sensory neuron. In one aspect, the neuron is a motor neuron. In one aspect, the neuron is an interneuron.

As used herein, the term "functional neuron" refers to a neuron that can perform a biological process. Examples of biological processes include, but are not limited to, the processing and transmission of information and communication via chemical and electrical synapses.

As used herein, the term "glutamatergic neuron" refers to a subset of neurons that produce glutamate and establish excitatory synapses. As used herein, the term "excitatory synapse" refers to a synapse where an action potential in a presynaptic neuron increases the probability of an action potential occurring in a postsynaptic cell. As used herein, the term "action potential" or "nerve impulse" refers to an electrical pulse that passes through the axon membrane. As used herein, the term "axon" or "nerve fiber" refers to a neuron that conducts action potentials. As used herein, the term "gabaergic neuron" refers to a subset of neurons that produce GABA and establish inhibitory synapses. As used herein, the term "GABA" or "gamma-aminobutyric acid" refers to a compound that opens an ion channel to allow negatively charged chloride ions to flow into cells or positively charged potassium ions to flow out of cells. As used herein, the term "inhibitory synapse" refers to a synapse that brings the membrane potential of a post-synaptic neuron away from a threshold at which action potentials are generated. As used herein, the term "dopaminergic neuron" refers to a subset of neurons that produce dopamine. As used herein, the term "dopamine" refers to neurotransmitters. As used herein, the term "neurotransmitter" refers to endogenous chemicals that activate neurotransmission. As used herein, the term "neurotransmission" refers to the process of releasing neurotransmitters from the axon terminals of neurons. As used herein, the term "acetylcholine-producing neuron" or "cholinergic neuron" refers to a subset of neurons that secrete acetylcholine. As used herein, the term "acetylcholine" refers to neurotransmitters. As used herein, the term "serotonergic neuron" refers to a subset of neurons that synthesize serotonin. As used herein, the term "serotonin" refers to neurotransmitters. As used herein, "adrenergic neuron" refers to a neuron that releases epinephrine as a neurotransmitter. As used herein, the term "motor neuron" refers to a subset of neurons in which the cell body is located in the motor cortex, brain stem, or spinal cord and axons project outside the spinal cord or spinal cord and directly or indirectly control muscles and glands. As used herein, the term peptide energy neuron refers to a subset of neurons that utilize small peptide molecules as their neurotransmitters.

In one aspect, the neuron is a functional neuron. In one aspect, the functional neuron is selected from the group consisting of a glutamatergic neuron, a gabaergic neuron, a dopaminergic neuron, a cholinergic neuron, a serotonergic neuron, an adrenergic neuron, a motor neuron, and a peptidoergic neuron. In one aspect, the functional neuron is a glutamatergic neuron. In one aspect, the functional neuron is a gabaergic neuron. In one aspect, the functional neuron is a dopaminergic neuron. In one aspect, the functional neuron is a cholinergic neuron. In one aspect, the functional neuron is a serotonergic neuron. In one aspect, the functional neuron is an adrenergic neuron. In one aspect, the functional neuron is a motor neuron. In one aspect, the functional neuron is a peptide energy neuron.

As used herein, the term "transformed" or "transformed" refers to a cell type that changes its physical form and/or biological function to a different physical form and/or different biological function. In one aspect, the present disclosure provides for the conversion of at least one glial cell to at least one neuron. In one aspect, the conversion of at least one glial cell to at least one neuron occurs in the CNS or PNS. In one aspect, the conversion of at least one glial cell to at least one neuron occurs in the CNS. In one aspect, the conversion of at least one glial cell to at least one neuron occurs in PNS.

In one aspect, the present disclosure provides and includes a composition comprising an adeno-associated virus (AAV) vector for converting human glial cells to functional neurons, wherein the AAV vector comprises: a nucleic acid coding sequence encoding a human neurogenic differentiation factor 1 (hNeuroD 1) protein, the hNeuroD1 protein comprising the amino acid sequence of SEQ ID NO. 10, and a second nucleic acid coding sequence encoding a second protein having an amino acid sequence selected from the group consisting of SEQ ID NO. 12, 14 and 16, wherein the hNeuroD1 coding sequence and the second coding sequence are separated by: (i) a P2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID nos. 19 and 22, (ii) a T2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID nos. 20 and 23, or (iii) an Internal Ribosome Entry Site (IRES) sequence of an encephalomyocarditis virus comprising SEQ ID No. 3, wherein the hNeuroD1 coding sequence and the second coding sequence are operably linked to a regulatory element comprising: (a) A human Glial Fibrillary Acidic Protein (GFAP) promoter comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs 4, 18 and 27; (b) An enhancer from the human elongation factor-1 alpha (EF-1 alpha) promoter comprising the nucleic acid sequence of SEQ ID NO. 2 or a Cytomegalovirus (CMV) enhancer comprising the nucleic acid sequence of SEQ ID NO. 17; (c) A chimeric intron comprising the nucleic acid sequence of SEQ ID NO. 5 or 28; a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs 7 and 30; and (e) an SV40 polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO. 8, an hGH polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO. 21, a synthetic polyadenylation signal comprising SEQ ID NO. 31 or a bGH polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO. 26.

In one aspect, an AAV vector comprises a nucleic acid sequence encoding an AAV protein. In one aspect, the AAV vector comprises a nucleic acid sequence encoding a viral protein. Non-limiting examples of AAV proteins and viral proteins include rep and cap proteins.

NeuroD1 (also known as β2) is a basic helix-loop-helix (bHLH) transcription factor that forms heterodimers with other bHLH proteins to activate gene transcription containing a DNA sequence known as an E-cassette.

In one aspect, the NeuroD1 sequence is a human NeuroD1 (hNeuroD 1) sequence. In one aspect, the NeuroD1 sequence is selected from the group consisting of: a chimpanzee NeuroD1 sequence, a bonobo NeuroD1 sequence, a chimpanzee NeuroD1 sequence, a gorilla NeuroD1 sequence, a macaque NeuroD1 sequence, a marmoset NeuroD1 sequence, a pigtail monkey NeuroD1 sequence, a baboon NeuroD1 sequence, a gibbon NeuroD1 sequence, and a marmoset NeuroD1 sequence. In one aspect, the NeuroD1 sequence is a chimpanzee NeuroD1 sequence. In one aspect, the NeuroD1 sequence is a bonobo chimpanzee NeuroD1 sequence. In one aspect, the NeuroD1 sequence is a chimpanzee NeuroD1 sequence. In one aspect, the NeuroD1 sequence is a gorilla NeuroD1 sequence. In one aspect, the NeuroD1 sequence is a cynomolgus monkey NeuroD1 sequence. In one aspect, the NeuroD1 sequence is a marmoset NeuroD1 sequence. In one aspect, the NeuroD1 sequence is a cynomolgus monkey NeuroD1 sequence. In one aspect, the NeuroD1 sequence is a baboon NeuroD1 sequence. In one aspect, the NeuroD1 sequence is a gibbon NeuroD1 sequence. In one aspect, the NeuroD1 sequence is a cynomolgus monkey NeuroD1 sequence.

In one aspect, the NeuroD1 nucleic acid sequence comprises a sequence at least 70% identical to: SEQ ID NO. 6, or a complement thereof. In one aspect, the NeuroD1 nucleic acid sequence comprises a sequence at least 75% identical to: SEQ ID NO. 6, or a complement thereof. In one aspect, the NeuroD1 nucleic acid sequence comprises a sequence at least 80% identical to: SEQ ID NO. 6, or a complement thereof. In one aspect, the NeuroD1 nucleic acid sequence comprises a sequence at least 85% identical to: SEQ ID NO. 6, or a complement thereof. In one aspect, the NeuroD1 nucleic acid sequence comprises a sequence at least 90% identical to: SEQ ID NO. 6, or a complement thereof. In one aspect, the NeuroD1 nucleic acid sequence comprises a sequence at least 91% identical to: SEQ ID NO. 6, or a complement thereof. In one aspect, the NeuroD1 nucleic acid sequence comprises a sequence at least 92% identical to: SEQ ID NO. 6, or a complement thereof. In one aspect, the NeuroD1 nucleic acid sequence comprises a sequence at least 93% identical to: SEQ ID NO. 6, or a complement thereof. In one aspect, the NeuroD1 nucleic acid sequence comprises a sequence at least 94% identical to: SEQ ID NO. 6, or a complement thereof. In one aspect, the NeuroD1 nucleic acid sequence comprises a sequence at least 95% identical to: SEQ ID NO. 6, or a complement thereof. In one aspect, the NeuroD1 nucleic acid sequence comprises a sequence at least 96% identical to: SEQ ID NO. 6, or a complement thereof. In one aspect, the NeuroD1 nucleic acid sequence comprises a sequence at least 97% identical to: SEQ ID NO. 6, or a complement thereof. In one aspect, the NeuroD1 nucleic acid sequence comprises a sequence at least 98% identical to: SEQ ID NO. 6, or a complement thereof. In one aspect, the NeuroD1 nucleic acid sequence comprises a sequence at least 99% identical to: SEQ ID NO. 6, or a complement thereof. In one aspect, the NeuroD1 nucleic acid sequence comprises a sequence at least 99.5% identical to: SEQ ID NO. 6, or a complement thereof. In one aspect, the NeuroD1 nucleic acid sequence comprises a sequence at least 99.8% identical to: SEQ ID NO. 6, or a complement thereof. In one aspect, the NeuroD1 nucleic acid sequence comprises a sequence at least 99.9% identical to: SEQ ID NO. 6, or a complement thereof. In one aspect, the NeuroD1 nucleic acid sequence comprises a sequence 100% identical to: SEQ ID NO. 6, or a complement thereof.

In one aspect, the nucleic acid sequence encodes a NeuroD1 protein comprising an amino acid sequence at least 70% identical or similar to: SEQ ID NO. 10. In one aspect, the nucleic acid sequence encodes a NeuroD1 protein comprising an amino acid sequence at least 75% identical or similar to: SEQ ID NO. 10. In one aspect, the nucleic acid sequence encodes a NeuroD1 protein comprising an amino acid sequence at least 80% identical or similar to: SEQ ID NO. 10. In one aspect, the nucleic acid sequence encodes a NeuroD1 protein comprising an amino acid sequence at least 85% identical or similar to: SEQ ID NO. 10. In one aspect, the nucleic acid sequence encodes a NeuroD1 protein comprising an amino acid sequence at least 90% identical or similar to: SEQ ID NO. 10. In one aspect, the nucleic acid sequence encodes a NeuroD1 protein comprising an amino acid sequence at least 91% identical or similar to: SEQ ID NO. 10. In one aspect, the nucleic acid sequence encodes a NeuroD1 protein comprising an amino acid sequence at least 92% identical or similar to: SEQ ID NO. 10. In one aspect, the nucleic acid sequence encodes a NeuroD1 protein comprising an amino acid sequence at least 93% identical or similar to: SEQ ID NO. 10. In one aspect, the nucleic acid sequence encodes a NeuroD1 protein comprising an amino acid sequence at least 94% identical or similar to: SEQ ID NO. 10. In one aspect, the nucleic acid sequence encodes a NeuroD1 protein comprising an amino acid sequence at least 95% identical or similar to: SEQ ID NO. 10. In one aspect, the nucleic acid sequence encodes a NeuroD1 protein comprising an amino acid sequence at least 96% identical or similar to: SEQ ID NO. 10. In one aspect, the nucleic acid sequence encodes a NeuroD1 protein comprising an amino acid sequence at least 97% identical or similar to: SEQ ID NO. 10. In one aspect, the nucleic acid sequence encodes a NeuroD1 protein comprising an amino acid sequence at least 98% identical or similar to: SEQ ID NO. 10. In one aspect, the nucleic acid sequence encodes a NeuroD1 protein comprising an amino acid sequence at least 99% identical or similar to: SEQ ID NO. 10. In one aspect, the nucleic acid sequence encodes a NeuroD1 protein comprising an amino acid sequence at least 99.5% identical or similar to: SEQ ID NO. 10. In one aspect, the nucleic acid sequence encodes a NeuroD1 protein comprising an amino acid sequence at least 99.8% identical or similar to: SEQ ID NO. 10. In one aspect, the nucleic acid sequence encodes a NeuroD1 protein comprising an amino acid sequence at least 99.9% identical or similar to: SEQ ID NO. 10. In one aspect, the nucleic acid sequence encodes a NeuroD1 protein comprising an amino acid sequence 100% identical or similar to: SEQ ID NO. 10.

Achaete-scale family BHLH transcription factor 1 (Ascl 1; also known as ASH1, HASH1, MASH-1, and bHLHa 46) encodes members of the basic helix-loop-helix family of transcription factors and is a gene that plays a role in neuronal commitment and differentiation.

In one aspect, the Ascl1 sequence is a human Ascl1 (hal 1) sequence. In one aspect, the Ascl1 sequence is selected from the group consisting of a chimpanzee Ascl1 sequence, a bonobo Ascl1 sequence, a chimpanzee Ascl1 sequence, a gorilla Ascl1 sequence, a macaque Ascl1 sequence, a marmoset Ascl1 sequence, a tail monkey Ascl1 sequence, a baboon Ascl1 sequence, a gibbon Ascl1 sequence, and a marmoset Ascl1 sequence. In one aspect, the Ascl1 sequence is a chimpanzee Ascl1 sequence. In one aspect, the Ascl1 sequence is a bonobo Ascl1 sequence. In one aspect, the Ascl1 sequence is a chimpanzee Ascl1 sequence. In one aspect, the Ascl1 sequence is a gorilla Ascl1 sequence. In one aspect, the Ascl1 sequence is a cynomolgus Ascl1 sequence. In one aspect, the Ascl1 sequence is marmoset Ascl1 sequence. In one aspect, the Ascl1 sequence is a cynomolgus Ascl1 sequence. In one aspect, the Ascl1 sequence is a baboon Ascl1 sequence. In one aspect, the Ascl1 sequence is a gibbon Ascl1 sequence. In one aspect, the Ascl1 sequence is a cynomolgus Ascl1 sequence.

In one aspect, the Ascl1 nucleic acid sequence comprises a sequence that is at least 70% identical to: SEQ ID NO. 11, or a complement thereof. In one aspect, the Ascl1 nucleic acid sequence comprises a sequence that is at least 75% identical to: SEQ ID NO. 11, or a complement thereof. In one aspect, the Ascl1 nucleic acid sequence comprises a sequence that is at least 80% identical to: SEQ ID NO. 11, or a complement thereof. In one aspect, the Ascl1 nucleic acid sequence comprises a sequence that is at least 85% identical to: SEQ ID NO. 11, or a complement thereof. In one aspect, the Ascl1 nucleic acid sequence comprises a sequence that is at least 90% identical to: SEQ ID NO. 11, or a complement thereof. In one aspect, the Ascl1 nucleic acid sequence comprises a sequence that is at least 91% identical to: SEQ ID NO. 11, or a complement thereof. In one aspect, the Ascl1 nucleic acid sequence comprises a sequence at least 92% identical to: SEQ ID NO. 11, or a complement thereof. In one aspect, the Ascl1 nucleic acid sequence comprises a sequence that is at least 93% identical to: SEQ ID NO. 11, or a complement thereof. In one aspect, the Ascl1 nucleic acid sequence comprises a sequence that is at least 94% identical to: SEQ ID NO. 11, or a complement thereof. In one aspect, the Ascl1 nucleic acid sequence comprises a sequence that is at least 95% identical to: SEQ ID NO. 11, or a complement thereof. In one aspect, the Ascl1 nucleic acid sequence comprises a sequence that is at least 911% identical to: SEQ ID NO. 11, or a complement thereof. In one aspect, the Ascl1 nucleic acid sequence comprises a sequence that is at least 97% identical to: SEQ ID NO. 11, or a complement thereof. In one aspect, the Ascl1 nucleic acid sequence comprises a sequence that is at least 98% identical to: SEQ ID NO. 11, or a complement thereof. In one aspect, the Ascl1 nucleic acid sequence comprises a sequence that is at least 99% identical to: SEQ ID NO. 11, or a complement thereof. In one aspect, the Ascl1 nucleic acid sequence comprises a sequence that is at least 99.5% identical to: SEQ ID NO. 11, or a complement thereof. In one aspect, the Ascl1 nucleic acid sequence comprises a sequence that is at least 99.8% identical to: SEQ ID NO. 11, or a complement thereof. In one aspect, the Ascl1 nucleic acid sequence comprises a sequence that is at least 99.9% identical to: SEQ ID NO. 11, or a complement thereof. In one aspect, the Ascl1 nucleic acid sequence comprises a sequence 100% identical to: SEQ ID NO. 11, or a complement thereof.

In one aspect, the nucleic acid coding sequence encodes an Ascl1 protein comprising an amino acid sequence that is at least 70% identical or similar to: SEQ ID NO. 12. In one aspect, the nucleic acid coding sequence encodes an Ascl1 protein comprising an amino acid sequence that is at least 75% identical or similar to: SEQ ID NO. 12. In one aspect, the nucleic acid coding sequence encodes an Ascl1 protein comprising an amino acid sequence that is at least 80% identical or similar to: SEQ ID NO. 12. In one aspect, the nucleic acid coding sequence encodes an Ascl1 protein comprising an amino acid sequence that is at least 85% identical or similar to: SEQ ID NO. 12. In one aspect, the nucleic acid coding sequence encodes an Ascl1 protein comprising an amino acid sequence that is at least 90% identical or similar to: SEQ ID NO. 12. In one aspect, the nucleic acid coding sequence encodes an Ascl1 protein comprising an amino acid sequence that is at least 91% identical or similar to: SEQ ID NO. 12. In one aspect, the nucleic acid coding sequence encodes an Ascl1 protein comprising an amino acid sequence that is at least 92% identical or similar to: SEQ ID NO. 12. In one aspect, the nucleic acid coding sequence encodes an Ascl1 protein comprising an amino acid sequence that is at least 93% identical or similar to: SEQ ID NO. 12. In one aspect, the nucleic acid coding sequence encodes an Ascl1 protein comprising an amino acid sequence that is at least 94% identical or similar to: SEQ ID NO. 12. In one aspect, the nucleic acid coding sequence encodes an Ascl1 protein comprising an amino acid sequence that is at least 95% identical or similar to: SEQ ID NO. 12. In one aspect, the nucleic acid coding sequence encodes an Ascl1 protein comprising an amino acid sequence that is at least 96% identical or similar to: SEQ ID NO. 12. In one aspect, the nucleic acid coding sequence encodes an Ascl1 protein comprising an amino acid sequence that is at least 97% identical or similar to: SEQ ID NO. 12. In one aspect, the nucleic acid coding sequence encodes an Ascl1 protein comprising an amino acid sequence that is at least 98% identical or similar to: SEQ ID NO. 12. In one aspect, the nucleic acid coding sequence encodes an Ascl1 protein comprising an amino acid sequence that is at least 99% identical or similar to: SEQ ID NO. 12. In one aspect, the nucleic acid coding sequence encodes an Ascl1 protein comprising an amino acid sequence that is at least 99.5% identical or similar to: SEQ ID NO. 12. In one aspect, the nucleic acid coding sequence encodes an Ascl1 protein comprising an amino acid sequence that is at least 99.8% identical or similar to: SEQ ID NO. 12. In one aspect, the nucleic acid coding sequence encodes an Ascl1 protein comprising an amino acid sequence that is at least 99.9% identical or similar to: SEQ ID NO. 12. In one aspect, the nucleic acid coding sequence encodes an Ascl1 protein comprising an amino acid sequence that is 120% identical or similar to: SEQ ID NO. 12.

Insulin gene enhancer proteins (ISL 1; also known as ISL LIM homeobox 1 and ISLET 1) are genes encoding transcription factors that contain two N-terminal LIM domains and one C-terminal homeodomain. The encoded protein plays a role in embryogenesis of langerhans islets.

In one aspect, the ISL1 sequence is a human ISL1 (hlsl 1) sequence. In one aspect, the ISL1 sequence is selected from the group consisting of a chimpanzee ISL1 sequence, a bonobo ISL1 sequence, a chimpanzee ISL1 sequence, a gorilla ISL1 sequence, a macaque ISL1 sequence, a marmoset ISL1 sequence, a pigtail ISL1 sequence, a baboon ISL1 sequence, a gibbon ISL1 sequence, and a lemma ISL1 sequence. In one aspect, the ISL1 sequence is a chimpanzee ISL1 sequence. In one aspect, the ISL1 sequence is a bonobo sylvestris ISL1 sequence. In one aspect, the ISL1 sequence is a chimpanzee ISL1 sequence. In one aspect, the ISL1 sequence is a gorilla ISL1 sequence. In one aspect, the ISL1 sequence is a cynomolgus ISL1 sequence. In one aspect, the ISL1 sequence is a marmoset ISL1 sequence. In one aspect, the ISL1 sequence is a cynomolgus ISL1 sequence. In one aspect, the ISL1 sequence is a baboon ISL1 sequence. In one aspect, the ISL1 sequence is a gibbon ISL1 sequence. In one aspect, the ISL1 sequence is a cynomolgus ISL1 sequence.

In one aspect, the ISL1 nucleic acid sequence comprises a sequence that is at least 70% identical to: SEQ ID NO. 13, or a complement thereof. In one aspect, the ISL1 nucleic acid sequence comprises a sequence that is at least 75% identical to: SEQ ID NO. 13, or a complement thereof. In one aspect, the ISL1 nucleic acid sequence comprises a sequence that is at least 80% identical to: SEQ ID NO. 13, or a complement thereof. In one aspect, the ISL1 nucleic acid sequence comprises a sequence that is at least 85% identical to: SEQ ID NO. 13, or a complement thereof. In one aspect, the ISL1 nucleic acid sequence comprises a sequence that is at least 90% identical to: SEQ ID NO. 13, or a complement thereof. In one aspect, the ISL1 nucleic acid sequence comprises a sequence that is at least 91% identical to: SEQ ID NO. 13, or a complement thereof. In one aspect, the ISL1 nucleic acid sequence comprises a sequence that is at least 92% identical to: SEQ ID NO. 13, or a complement thereof. In one aspect, the ISL1 nucleic acid sequence comprises a sequence that is at least 93% identical to: SEQ ID NO. 13, or a complement thereof. In one aspect, the ISL1 nucleic acid sequence comprises a sequence that is at least 94% identical to: SEQ ID NO. 13, or a complement thereof. In one aspect, the ISL1 nucleic acid sequence comprises a sequence that is at least 95% identical to: SEQ ID NO. 13, or a complement thereof. In one aspect, the ISL1 nucleic acid sequence comprises a sequence that is at least 913% identical to: SEQ ID NO. 13, or a complement thereof. In one aspect, the ISL1 nucleic acid sequence comprises a sequence that is at least 97% identical to: SEQ ID NO. 13, or a complement thereof. In one aspect, the ISL1 nucleic acid sequence comprises a sequence that is at least 98% identical to: SEQ ID NO. 13, or a complement thereof. In one aspect, the ISL1 nucleic acid sequence comprises a sequence that is at least 99% identical to: SEQ ID NO. 13, or a complement thereof. In one aspect, the ISL1 nucleic acid sequence comprises a sequence that is at least 99.5% identical to: SEQ ID NO. 13, or a complement thereof. In one aspect, the ISL1 nucleic acid sequence comprises a sequence that is at least 99.8% identical to: SEQ ID NO. 13, or a complement thereof. In one aspect, the ISL1 nucleic acid sequence comprises a sequence that is at least 99.9% identical to: SEQ ID NO. 13, or a complement thereof. In one aspect, the ISL1 nucleic acid sequence comprises a sequence that is 100% identical to: SEQ ID NO. 13, or a complement thereof.

In one aspect, the nucleic acid coding sequence encodes an ISL1 protein comprising an amino acid sequence that is at least 70% identical or similar to: SEQ ID NO. 14. In one aspect, the nucleic acid coding sequence encodes an ISL1 protein comprising an amino acid sequence that is at least 75% identical or similar to: SEQ ID NO. 14. In one aspect, the nucleic acid coding sequence encodes an ISL1 protein comprising an amino acid sequence that is at least 80% identical or similar to: SEQ ID NO. 14. In one aspect, the nucleic acid coding sequence encodes an ISL1 protein comprising an amino acid sequence that is at least 85% identical or similar to: SEQ ID NO. 14. In one aspect, the nucleic acid coding sequence encodes an ISL1 protein comprising an amino acid sequence that is at least 90% identical or similar to: SEQ ID NO. 14. In one aspect, the nucleic acid coding sequence encodes an ISL1 protein comprising an amino acid sequence that is at least 91% identical or similar to: SEQ ID NO. 14. In one aspect, the nucleic acid coding sequence encodes an ISL1 protein comprising an amino acid sequence that is at least 92% identical or similar to: SEQ ID NO. 14. In one aspect, the nucleic acid coding sequence encodes an ISL1 protein comprising an amino acid sequence that is at least 93% identical or similar to: SEQ ID NO. 14. In one aspect, the nucleic acid coding sequence encodes an ISL1 protein comprising an amino acid sequence that is at least 94% identical or similar to: SEQ ID NO. 14. In one aspect, the nucleic acid coding sequence encodes an ISL1 protein comprising an amino acid sequence that is at least 95% identical or similar to: SEQ ID NO. 14. In one aspect, the nucleic acid coding sequence encodes an ISL1 protein comprising an amino acid sequence that is at least 96% identical or similar to: SEQ ID NO. 14. In one aspect, the nucleic acid coding sequence encodes an ISL1 protein comprising an amino acid sequence that is at least 97% identical or similar to: SEQ ID NO. 14. In one aspect, the nucleic acid coding sequence encodes an ISL1 protein comprising an amino acid sequence that is at least 98% identical or similar to: SEQ ID NO. 14. In one aspect, the nucleic acid coding sequence encodes an ISL1 protein comprising an amino acid sequence that is at least 99% identical or similar to: SEQ ID NO. 14. In one aspect, the nucleic acid coding sequence encodes an ISL1 protein comprising an amino acid sequence that is at least 99.5% identical or similar to: SEQ ID NO. 14. In one aspect, the nucleic acid coding sequence encodes an ISL1 protein comprising an amino acid sequence that is at least 99.8% identical or similar to: SEQ ID NO. 14. In one aspect, the nucleic acid coding sequence encodes an ISL1 protein comprising an amino acid sequence that is at least 99.9% identical or similar to: SEQ ID NO. 14. In one aspect, the nucleic acid coding sequence encodes an ISL1 protein comprising an amino acid sequence that is 140% identical or similar to: SEQ ID NO. 14.

LIM-homeobox 3 (LHX 3; also referred to as LIM3 and CPHD 3) genes encode proteins from a family of proteins with unique cysteine-rich zinc binding domains (LIM domains).

In one aspect, the LHX3 sequence is a human LHX3 (hLHX 3) sequence. In one aspect, the LHX3 sequence is selected from the group consisting of a chimpanzee LHX3 sequence, a bonobo LHX3 sequence, a chimpanzee LHX3 sequence, a gorilla LHX3 sequence, a macaque LHX3 sequence, a marmoset LHX3 sequence, a pigtail LHX3 sequence, a baboon LHX3 sequence, a gibbon LHX3 sequence, and a lemma LHX3 sequence. In one aspect, the LHX3 sequence is a chimpanzee LHX3 sequence. In one aspect, the LHX3 sequence is a bonobo chimpanzee LHX3 sequence. In one aspect, the LHX3 sequence is a chimpanzee LHX3 sequence. In one aspect, the LHX3 sequence is a gorilla LHX3 sequence. In one aspect, the LHX3 sequence is a cynomolgus LHX3 sequence. In one aspect, the LHX3 sequence is a marmoset LHX3 sequence. In one aspect, the LHX3 sequence is a tail monkey LHX3 sequence. In one aspect, the LHX3 sequence is a baboon LHX3 sequence. In one aspect, the LHX3 sequence is a gibbon LHX3 sequence. In one aspect, the LHX3 sequence is a lemma LHX3 sequence.

In one aspect, the LHX3 nucleic acid sequence comprises a sequence at least 70% identical to: SEQ ID NO. 15, or a complement thereof. In one aspect, the LHX3 nucleic acid sequence comprises a sequence at least 75% identical to: SEQ ID NO. 15, or a complement thereof. In one aspect, the LHX3 nucleic acid sequence comprises a sequence at least 80% identical to: SEQ ID NO. 15, or a complement thereof. In one aspect, the LHX3 nucleic acid sequence comprises a sequence at least 85% identical to: SEQ ID NO. 15, or a complement thereof. In one aspect, the LHX3 nucleic acid sequence comprises a sequence at least 90% identical to: SEQ ID NO. 15, or a complement thereof. In one aspect, the LHX3 nucleic acid sequence comprises a sequence at least 91% identical to: SEQ ID NO. 15, or a complement thereof. In one aspect, the LHX3 nucleic acid sequence comprises a sequence at least 92% identical to: SEQ ID NO. 15, or a complement thereof. In one aspect, the LHX3 nucleic acid sequence comprises a sequence at least 93% identical to: SEQ ID NO. 15, or a complement thereof. In one aspect, the LHX3 nucleic acid sequence comprises a sequence at least 94% identical to: SEQ ID NO. 15, or a complement thereof. In one aspect, the LHX3 nucleic acid sequence comprises a sequence at least 95% identical to: SEQ ID NO. 15, or a complement thereof. In one aspect, the LHX3 nucleic acid sequence comprises a sequence at least 96% identical to: SEQ ID NO. 15, or a complement thereof. In one aspect, the LHX3 nucleic acid sequence comprises a sequence at least 97% identical to: SEQ ID NO. 15, or a complement thereof. In one aspect, the LHX3 nucleic acid sequence comprises a sequence at least 98% identical to: SEQ ID NO. 15, or a complement thereof. In one aspect, the LHX3 nucleic acid sequence comprises a sequence at least 99% identical to: SEQ ID NO. 15, or a complement thereof. In one aspect, the LHX3 nucleic acid sequence comprises a sequence at least 99.5% identical to: SEQ ID NO. 15, or a complement thereof. In one aspect, the LHX3 nucleic acid sequence comprises a sequence at least 99.8% identical to: SEQ ID NO. 15, or a complement thereof. In one aspect, the LHX3 nucleic acid sequence comprises a sequence at least 99.9% identical to: SEQ ID NO. 15, or a complement thereof. In one aspect, the LHX3 nucleic acid sequence comprises a sequence 100% identical to: SEQ ID NO. 15, or a complement thereof.

In one aspect, the nucleic acid coding sequence encodes an LHX3 protein comprising an amino acid sequence at least 70% identical or similar to: SEQ ID NO. 16. In one aspect, the nucleic acid coding sequence encodes an LHX3 protein comprising an amino acid sequence at least 75% identical or similar to: SEQ ID NO. 16. In one aspect, the nucleic acid coding sequence encodes an LHX3 protein comprising an amino acid sequence at least 80% identical or similar to: SEQ ID NO. 16. In one aspect, the nucleic acid coding sequence encodes an LHX3 protein comprising an amino acid sequence at least 85% identical or similar to: SEQ ID NO. 16. In one aspect, the nucleic acid coding sequence encodes an LHX3 protein comprising an amino acid sequence at least 90% identical or similar to: SEQ ID NO. 16. In one aspect, the nucleic acid coding sequence encodes an LHX3 protein comprising an amino acid sequence at least 91% identical or similar to: SEQ ID NO. 16. In one aspect, the nucleic acid coding sequence encodes an LHX3 protein comprising an amino acid sequence at least 92% identical or similar to: SEQ ID NO. 16. In one aspect, the nucleic acid coding sequence encodes an LHX3 protein comprising an amino acid sequence at least 93% identical or similar to: SEQ ID NO. 16. In one aspect, the nucleic acid coding sequence encodes an LHX3 protein comprising an amino acid sequence at least 94% identical or similar to: SEQ ID NO. 16. In one aspect, the nucleic acid coding sequence encodes an LHX3 protein comprising an amino acid sequence at least 95% identical or similar to: SEQ ID NO. 16. In one aspect, the nucleic acid coding sequence encodes an LHX3 protein comprising an amino acid sequence at least 96% identical or similar to: SEQ ID NO. 16. In one aspect, the nucleic acid coding sequence encodes an LHX3 protein comprising an amino acid sequence at least 97% identical or similar to: SEQ ID NO. 16. In one aspect, the nucleic acid coding sequence encodes an LHX3 protein comprising an amino acid sequence at least 98% identical or similar to: SEQ ID NO. 16. In one aspect, the nucleic acid coding sequence encodes an LHX3 protein comprising an amino acid sequence at least 99% identical or similar to: SEQ ID NO. 16. In one aspect, the nucleic acid coding sequence encodes an LHX3 protein comprising an amino acid sequence at least 99.5% identical or similar to: SEQ ID NO. 16. In one aspect, the nucleic acid coding sequence encodes an LHX3 protein comprising an amino acid sequence at least 99.8% identical or similar to: SEQ ID NO. 16. In one aspect, the nucleic acid coding sequence encodes an LHX3 protein comprising an amino acid sequence at least 99.9% identical or similar to: SEQ ID NO. 16. In one aspect, the nucleic acid coding sequence encodes an LHX3 protein comprising an amino acid sequence 100% identical to: SEQ ID NO. 16.

In one aspect, the AAV vector comprises a second protein sequence, wherein the second protein is selected from the group consisting of Ascl1, ISL1, and LHX3. In one aspect, the AAV vector comprises a second protein coding sequence, wherein the second protein is Ascl1. In one aspect, the AAV vector comprises a second protein coding sequence, wherein the second protein is ISL1. In one aspect, the AAV comprises a second protein coding sequence, wherein the second protein is LHX3.

In one aspect, the functionality of the AAV vectors provided herein is measured by assessing transcript levels and protein levels of NeuN, bisdermatan (DCX), β3-tubulin, (neurofilament 200) NF-200, (microtubule-associated protein 2) MAP2, ionized calcium binding adapter molecule (Iba 1).

As used herein, the term "NeuN" or "Fox-3" or "Rbfox2" or "hexaribonucleotide binding protein-3" refers to a protein that is homologous to the protein product of a sex determining gene in caenorhabditis elegans (Caenorhabditis elegans) and is a neuronal nuclear antigen.

As used herein, the term "DCX" or "doubering" or "lissencephalin-X" refers to microtubule-associated proteins expressed by neuronal precursor cells and immature neurons in embryonic and adult cortical structures.

As used herein, the term "β3-tubulin" or "class III β -tubulin" or "β -tubulin III" refers to the tubulin family of tubulin elements found in neurons.

As used herein, the term "NF-200" refers to a class of proteins that are type IV intermediate filars found in the cytoplasm of neurons.

As used herein, the term "MAP2" refers to a protein that belongs to the microtubule-associated protein family and that plays a role in determining and stabilizing neuronal morphology during neuronal development.

As used herein, the term "Iba1" refers to microglial macrophage-specific calcium binding protein.

In one aspect, the AAV vector comprises a NeuroD1 coding sequence, an Als1 coding sequence. In one aspect, the AAV vector comprises a NeuroD1 coding sequence. In one aspect, the AAV comprises an Als1 coding sequence.

In one aspect, the AAV vector comprises a NeuroD1 coding sequence, an ISL1 coding sequence. In one aspect, the AAV vector comprises a NeuroD1 coding sequence. In one aspect, the AAV comprises an ISL1 coding sequence.

In one aspect, the AAV vector comprises a NeuroD1 coding sequence, an LHX3 coding sequence. In one aspect, the AAV vector comprises a NeuroD1 coding sequence. In one aspect, the AAV comprises an LHX3 coding sequence.

As used herein, a "linker" or "spacer" is a short sequence that separates multiple proteins and coding domains. The linker may be cleavable or non-cleavable and facilitates the co-expression of multiple genes in a single vector.

As used herein, a "2A self-cleaving peptide" or "2A peptide" is a class of linkers that can induce cleavage of a recombinant protein in a cell.

As used herein, "P2A linker" refers to a porcine teschovirus-1 (P2A) linker, which is a member of the 2A self-cleaving peptide.

As used herein, "IRES" refers to an internal ribosome entry site of an encephalomyocarditis virus (EMCV).

In one aspect, the P2A linker has a nucleic acid sequence selected from the group consisting of: SEQ ID NOS.19 and 22. In one aspect, the P2A linker has the following nucleic acid sequence: SEQ ID NO. 19. In one aspect, the P2A linker has the following nucleic acid sequence: SEQ ID NO. 19. In one aspect, the P2A protein has the following nucleic acid coding sequence: SEQ ID NO. 24.

As used herein, a "T2A linker" is a linker that designates the vein flatworm virus 2A (thosea asigna virus a, T2A), which is a member of the 2A self-cleaving peptide.

In one aspect, the T2A linker has a nucleic acid sequence selected from the group consisting of: SEQ ID NOS.20 and 23. In one aspect, the T2A linker has the following nucleic acid sequence: SEQ ID NO. 20. In one aspect, the T2A linker has the following nucleic acid sequence: SEQ ID NO. 23. In one aspect, the T2A protein has the following nucleic acid coding sequence: SEQ ID NO. 25.

As used herein, "E2A linker" refers to a equine rhinitis a virus (E2A) linker, which is a member of the 2A self-cleaving peptide.

As used herein, "F2A linker" refers to a hand-foot-and-mouth virus (F2A) linker, which is a member of the 2A self-cleaving peptide.

In one aspect, the linker is selected from the group consisting of a P2A linker, a T2A linker, an E2A linker, and an F2A linker. In one aspect, the linker is a P2A linker. In one aspect, the linker is a T2A linker. In one aspect, the linker is an E2A linker. In one aspect, the linker is an F2A linker.

In one aspect, the linker sequence is a P2A linker. In one aspect, the P2A linker nucleic acid sequence comprises a sequence that is at least 70% identical to: SEQ ID NO. 19, or a complement thereof. In one aspect, the P2A linker nucleic acid sequence comprises a sequence that is at least 75% identical to: SEQ ID NO. 19, or a complement thereof. In one aspect, the P2A linker nucleic acid sequence comprises a sequence at least 80% identical to: SEQ ID NO. 19, or a complement thereof. In one aspect, the P2A linker nucleic acid sequence comprises a sequence that is at least 85% identical to: SEQ ID NO. 19, or a complement thereof. In one aspect, the P2A linker nucleic acid sequence comprises a sequence at least 90% identical to: SEQ ID NO. 19, or a complement thereof. In one aspect, the P2A linker nucleic acid sequence comprises a sequence that is at least 91% identical to: SEQ ID NO. 19, or a complement thereof. In one aspect, the P2A linker nucleic acid sequence comprises a sequence at least 92% identical to: SEQ ID NO. 19, or a complement thereof. In one aspect, the P2A linker nucleic acid sequence comprises a sequence at least 93% identical to: SEQ ID NO. 19, or a complement thereof. In one aspect, the P2A linker nucleic acid sequence comprises a sequence at least 94% identical to: SEQ ID NO. 19, or a complement thereof. In one aspect, the P2A linker nucleic acid sequence comprises a sequence at least 95% identical to: SEQ ID NO. 19, or a complement thereof. In one aspect, the P2A linker nucleic acid sequence comprises a sequence that is at least 915% identical to: SEQ ID NO. 19, or a complement thereof. In one aspect, the P2A linker nucleic acid sequence comprises a sequence at least 97% identical to: SEQ ID NO. 19, or a complement thereof. In one aspect, the P2A linker nucleic acid sequence comprises a sequence that is at least 98% identical to: SEQ ID NO. 19, or a complement thereof. In one aspect, the P2A linker nucleic acid sequence comprises a sequence that is at least 99% identical to: SEQ ID NO. 19, or a complement thereof. In one aspect, the P2A linker nucleic acid sequence comprises a sequence that is at least 99.5% identical to: SEQ ID NO. 19, or a complement thereof. In one aspect, the P2A linker nucleic acid sequence comprises a sequence that is at least 99.8% identical to: SEQ ID NO. 19, or a complement thereof. In one aspect, the P2A linker nucleic acid sequence comprises a sequence that is at least 99.9% identical to: SEQ ID NO. 19, or a complement thereof. In one aspect, the P2A linker nucleic acid sequence comprises a sequence 100% identical to: SEQ ID NO. 19, or a complement thereof.

In one aspect, the linker sequence is a P2A linker. In one aspect, the P2A linker nucleic acid sequence comprises a sequence that is at least 70% identical to: SEQ ID NO. 22, or a complement thereof. In one aspect, the P2A linker nucleic acid sequence comprises a sequence that is at least 75% identical to: SEQ ID NO. 22, or a complement thereof. In one aspect, the P2A linker nucleic acid sequence comprises a sequence at least 80% identical to: SEQ ID NO. 22, or a complement thereof. In one aspect, the P2A linker nucleic acid sequence comprises a sequence that is at least 85% identical to: SEQ ID NO. 22, or a complement thereof. In one aspect, the P2A linker nucleic acid sequence comprises a sequence at least 90% identical to: SEQ ID NO. 22, or a complement thereof. In one aspect, the P2A linker nucleic acid sequence comprises a sequence that is at least 91% identical to: SEQ ID NO. 22, or a complement thereof. In one aspect, the P2A linker nucleic acid sequence comprises a sequence at least 92% identical to: SEQ ID NO. 22, or a complement thereof. In one aspect, the P2A linker nucleic acid sequence comprises a sequence at least 93% identical to: SEQ ID NO. 22, or a complement thereof. In one aspect, the P2A linker nucleic acid sequence comprises a sequence at least 94% identical to: SEQ ID NO. 22, or a complement thereof. In one aspect, the P2A linker nucleic acid sequence comprises a sequence at least 95% identical to: SEQ ID NO. 22, or a complement thereof. In one aspect, the P2A linker nucleic acid sequence comprises a sequence that is at least 915% identical to: SEQ ID NO. 22, or a complement thereof. In one aspect, the P2A linker nucleic acid sequence comprises a sequence at least 97% identical to: SEQ ID NO. 22, or a complement thereof. In one aspect, the P2A linker nucleic acid sequence comprises a sequence that is at least 98% identical to: SEQ ID NO. 22, or a complement thereof. In one aspect, the P2A linker nucleic acid sequence comprises a sequence that is at least 99% identical to: SEQ ID NO. 22, or a complement thereof. In one aspect, the P2A linker nucleic acid sequence comprises a sequence that is at least 99.5% identical to: SEQ ID NO. 22, or a complement thereof. In one aspect, the P2A linker nucleic acid sequence comprises a sequence that is at least 99.8% identical to: SEQ ID NO. 22, or a complement thereof. In one aspect, the P2A linker nucleic acid sequence comprises a sequence that is at least 99.9% identical to: SEQ ID NO. 22, or a complement thereof. In one aspect, the P2A linker nucleic acid sequence comprises a sequence 100% identical to: SEQ ID NO. 22, or a complement thereof.

In one aspect, the nucleic acid encoding sequence encodes a P2A protein comprising an amino acid sequence that is at least 70% identical or similar to: SEQ ID NO. 24. In one aspect, the nucleic acid encoding sequence encodes a P2A protein comprising an amino acid sequence that is at least 75% identical or similar to: SEQ ID NO. 24. In one aspect, the nucleic acid encoding sequence encodes a P2A protein comprising an amino acid sequence that is at least 80% identical or similar to: SEQ ID NO. 24. In one aspect, the nucleic acid encoding sequence encodes a P2A protein comprising an amino acid sequence that is at least 85% identical or similar to: SEQ ID NO. 24. In one aspect, the nucleic acid encoding sequence encodes a P2A protein comprising an amino acid sequence that is at least 90% identical or similar to: SEQ ID NO. 24. In one aspect, the nucleic acid encoding sequence encodes a P2A protein comprising an amino acid sequence that is at least 91% identical or similar to: SEQ ID NO. 24. In one aspect, the nucleic acid encoding sequence encodes a P2A protein comprising an amino acid sequence at least 92% identical or similar to: SEQ ID NO. 24. In one aspect, the nucleic acid encoding sequence encodes a P2A protein comprising an amino acid sequence that is at least 93% identical or similar to: SEQ ID NO. 24. In one aspect, the nucleic acid encoding sequence encodes a P2A protein comprising an amino acid sequence that is at least 94% identical or similar to: SEQ ID NO. 24. In one aspect, the nucleic acid encoding sequence encodes a P2A protein comprising an amino acid sequence at least 95% identical or similar to: SEQ ID NO. 24. In one aspect, the nucleic acid encoding sequence encodes a P2A protein comprising an amino acid sequence at least 96% identical or similar to: SEQ ID NO. 24. In one aspect, the nucleic acid encoding sequence encodes a P2A protein comprising an amino acid sequence that is at least 97% identical or similar to: SEQ ID NO. 24. In one aspect, the nucleic acid encoding sequence encodes a P2A protein comprising an amino acid sequence that is at least 98% identical or similar to: SEQ ID NO. 24. In one aspect, the nucleic acid encoding sequence encodes a P2A protein comprising an amino acid sequence that is at least 99% identical or similar to: SEQ ID NO. 24. In one aspect, the nucleic acid encoding sequence encodes a P2A protein comprising an amino acid sequence that is at least 99.5% identical or similar to: SEQ ID NO. 24. In one aspect, the nucleic acid encoding sequence encodes a P2A protein comprising an amino acid sequence that is at least 99.8% identical or similar to: SEQ ID NO. 24. In one aspect, the nucleic acid encoding sequence encodes a P2A protein comprising an amino acid sequence that is at least 99.9% identical or similar to: SEQ ID NO. 24. In one aspect, the nucleic acid encoding sequence encodes a P2A protein comprising an amino acid sequence 100% identical or similar to: SEQ ID NO. 24.

In one aspect, the linker sequence comprises a T2A linker. In one aspect, the T2A linker nucleic acid sequence comprises a sequence that is at least 70% identical to: SEQ ID NO. 20, or a complement thereof. In one aspect, the T2A linker nucleic acid sequence comprises a sequence that is at least 75% identical to: SEQ ID NO. 20, or a complement thereof. In one aspect, the T2A linker nucleic acid sequence comprises a sequence at least 80% identical to: SEQ ID NO. 20, or a complement thereof. In one aspect, the T2A linker nucleic acid sequence comprises a sequence at least 85% identical to: SEQ ID NO. 20, or a complement thereof. In one aspect, the T2A linker nucleic acid sequence comprises a sequence at least 90% identical to: SEQ ID NO. 20, or a complement thereof. In one aspect, the T2A linker nucleic acid sequence comprises a sequence that is at least 91% identical to: SEQ ID NO. 20, or a complement thereof. In one aspect, the T2A linker nucleic acid sequence comprises a sequence at least 92% identical to: SEQ ID NO. 20, or a complement thereof. In one aspect, the T2A linker nucleic acid sequence comprises a sequence at least 93% identical to: SEQ ID NO. 20, or a complement thereof. In one aspect, the T2A linker nucleic acid sequence comprises a sequence at least 94% identical to: SEQ ID NO. 20, or a complement thereof. In one aspect, the T2A linker nucleic acid sequence comprises a sequence at least 95% identical to: SEQ ID NO. 20, or a complement thereof. In one aspect, the T2A linker nucleic acid sequence comprises a sequence at least 917% identical to: SEQ ID NO. 20, or a complement thereof. In one aspect, the T2A linker nucleic acid sequence comprises a sequence at least 97% identical to: SEQ ID NO. 20, or a complement thereof. In one aspect, the T2A linker nucleic acid sequence comprises a sequence that is at least 98% identical to: SEQ ID NO. 20, or a complement thereof. In one aspect, the T2A linker nucleic acid sequence comprises a sequence that is at least 99% identical to: SEQ ID NO. 20, or a complement thereof. In one aspect, the T2A linker nucleic acid sequence comprises a sequence that is at least 99.5% identical to: SEQ ID NO. 20, or a complement thereof. In one aspect, the T2A linker nucleic acid sequence comprises a sequence that is at least 99.8% identical to: SEQ ID NO. 20, or a complement thereof. In one aspect, the T2A linker nucleic acid sequence comprises a sequence that is at least 99.9% identical to: SEQ ID NO. 20, or a complement thereof. In one aspect, the T2A linker nucleic acid sequence comprises a sequence 100% identical to: SEQ ID NO. 20, or a complement thereof.

In one aspect, the linker sequence comprises a T2A linker. In one aspect, the T2A linker nucleic acid sequence comprises a sequence that is at least 70% identical to: SEQ ID NO. 23, or a complement thereof. In one aspect, the T2A linker nucleic acid sequence comprises a sequence that is at least 75% identical to: SEQ ID NO. 23, or a complement thereof. In one aspect, the T2A linker nucleic acid sequence comprises a sequence at least 80% identical to: SEQ ID NO. 23, or a complement thereof. In one aspect, the T2A linker nucleic acid sequence comprises a sequence at least 85% identical to: SEQ ID NO. 23, or a complement thereof. In one aspect, the T2A linker nucleic acid sequence comprises a sequence at least 90% identical to: SEQ ID NO. 23, or a complement thereof. In one aspect, the T2A linker nucleic acid sequence comprises a sequence that is at least 91% identical to: SEQ ID NO. 23, or a complement thereof. In one aspect, the T2A linker nucleic acid sequence comprises a sequence at least 92% identical to: SEQ ID NO. 23, or a complement thereof. In one aspect, the T2A linker nucleic acid sequence comprises a sequence at least 93% identical to: SEQ ID NO. 23, or a complement thereof. In one aspect, the T2A linker nucleic acid sequence comprises a sequence at least 94% identical to: SEQ ID NO. 23, or a complement thereof. In one aspect, the T2A linker nucleic acid sequence comprises a sequence at least 95% identical to: SEQ ID NO. 23, or a complement thereof. In one aspect, the T2A linker nucleic acid sequence comprises a sequence at least 917% identical to: SEQ ID NO. 23, or a complement thereof. In one aspect, the T2A linker nucleic acid sequence comprises a sequence at least 97% identical to: SEQ ID NO. 23, or a complement thereof. In one aspect, the T2A linker nucleic acid sequence comprises a sequence that is at least 98% identical to: SEQ ID NO. 23, or a complement thereof. In one aspect, the T2A linker nucleic acid sequence comprises a sequence that is at least 99% identical to: SEQ ID NO. 23, or a complement thereof. In one aspect, the T2A linker nucleic acid sequence comprises a sequence that is at least 99.5% identical to: SEQ ID NO. 23, or a complement thereof. In one aspect, the T2A linker nucleic acid sequence comprises a sequence that is at least 99.8% identical to: SEQ ID NO. 23, or a complement thereof. In one aspect, the T2A linker nucleic acid sequence comprises a sequence that is at least 99.9% identical to: SEQ ID NO. 23, or a complement thereof. In one aspect, the T2A linker nucleic acid sequence comprises a sequence 100% identical to: SEQ ID NO. 23, or a complement thereof.

In one aspect, the nucleic acid encoding sequence encodes a T2A protein comprising an amino acid sequence that is at least 70% identical or similar to: SEQ ID NO. 25. In one aspect, the nucleic acid encoding sequence encodes a T2A protein comprising an amino acid sequence that is at least 75% identical or similar to: SEQ ID NO. 25. In one aspect, the nucleic acid encoding sequence encodes a T2A protein comprising an amino acid sequence at least 80% identical or similar to: SEQ ID NO. 25. In one aspect, the nucleic acid encoding sequence encodes a T2A protein comprising an amino acid sequence that is at least 85% identical or similar to: SEQ ID NO. 25. In one aspect, the nucleic acid encoding sequence encodes a T2A protein comprising an amino acid sequence that is at least 90% identical or similar to: SEQ ID NO. 25. In one aspect, the nucleic acid encoding sequence encodes a T2A protein comprising an amino acid sequence that is at least 91% identical or similar to: SEQ ID NO. 25. In one aspect, the nucleic acid encoding sequence encodes a T2A protein comprising an amino acid sequence at least 92% identical or similar to: SEQ ID NO. 25. In one aspect, the nucleic acid encoding sequence encodes a T2A protein comprising an amino acid sequence that is at least 93% identical or similar to: SEQ ID NO. 25. In one aspect, the nucleic acid encoding sequence encodes a T2A protein comprising an amino acid sequence at least 94% identical or similar to: SEQ ID NO. 25. In one aspect, the nucleic acid encoding sequence encodes a T2A protein comprising an amino acid sequence at least 95% identical or similar to: SEQ ID NO. 25. In one aspect, the nucleic acid encoding sequence encodes a T2A protein comprising an amino acid sequence at least 96% identical or similar to: SEQ ID NO. 25. In one aspect, the nucleic acid encoding sequence encodes a T2A protein comprising an amino acid sequence at least 97% identical or similar to: SEQ ID NO. 25. In one aspect, the nucleic acid encoding sequence encodes a T2A protein comprising an amino acid sequence that is at least 98% identical or similar to: SEQ ID NO. 25. In one aspect, the nucleic acid encoding sequence encodes a T2A protein comprising an amino acid sequence that is at least 99% identical or similar to: SEQ ID NO. 25. In one aspect, the nucleic acid encoding sequence encodes a T2A protein comprising an amino acid sequence that is at least 99.5% identical or similar to: SEQ ID NO. 25. In one aspect, the nucleic acid encoding sequence encodes a T2A protein comprising an amino acid sequence that is at least 99.8% identical or similar to: SEQ ID NO. 25. In one aspect, the nucleic acid encoding sequence encodes a T2A protein comprising an amino acid sequence that is at least 99.9% identical or similar to: SEQ ID NO. 25. In one aspect, the nucleic acid encoding sequence encodes a T2A protein comprising an amino acid sequence 100% identical or similar to: SEQ ID NO. 25.

In one aspect, an AAV or vector of the disclosure comprises an Internal Ribosome Entry Site (IRES) sequence of an encephalomyocarditis virus. In one aspect, the IRES sequence comprises SEQ ID NO. 3. In one aspect, the IRES sequence comprises a sequence at least 70% identical to: SEQ ID NO. 3, or a complement thereof. In one aspect, the IRES sequence comprises a sequence at least 75% identical to: SEQ ID NO. 3, or a complement thereof. In one aspect, the IRES sequence comprises a sequence at least 80% identical to: SEQ ID NO. 3, or a complement thereof. In one aspect, the IRES sequence comprises a sequence at least 85% identical to: SEQ ID NO. 3, or a complement thereof. In one aspect, the IRES sequence comprises a sequence at least 90% identical to: SEQ ID NO. 3, or a complement thereof. In one aspect, the IRES sequence comprises a sequence at least 91% identical to: SEQ ID NO. 3, or a complement thereof. In one aspect, the IRES sequence comprises a sequence at least 92% identical to: SEQ ID NO. 3, or a complement thereof. In one aspect, the IRES sequence comprises a sequence at least 93% identical to: SEQ ID NO. 3, or a complement thereof. In one aspect, the IRES sequence comprises a sequence at least 94% identical to: SEQ ID NO. 3, or a complement thereof. In one aspect, the IRES sequence comprises a sequence at least 95% identical to: SEQ ID NO. 3, or a complement thereof. In one aspect, the IRES sequence comprises a sequence at least 96% identical to: SEQ ID NO. 3, or a complement thereof. In one aspect, the IRES sequence comprises a sequence at least 97% identical to: SEQ ID NO. 3, or a complement thereof. In one aspect, the IRES sequence comprises a sequence at least 98% identical to: SEQ ID NO. 3, or a complement thereof. In one aspect, the IRES sequence comprises a sequence at least 99% identical to: SEQ ID NO. 3, or a complement thereof. In one aspect, the IRES sequence comprises a sequence at least 99.5% identical to: SEQ ID NO. 3, or a complement thereof. In one aspect, the IRES sequence comprises a sequence at least 99.8% identical to: SEQ ID NO. 3, or a complement thereof. In one aspect, the IRES sequence comprises a sequence at least 99.9% identical to: SEQ ID NO. 3, or a complement thereof. In one aspect, the IRES sequence comprises a sequence 100% identical to: SEQ ID NO. 3, or a complement thereof.

Glial fibrillary acidic protein (glial fibrillary acid protein, GFAP); also known as glial fibrillary acidic protein (glial fibrillary acidic protein) is a member of the class III intermediate filamin family, which is expressed in the central nervous system and plays a role in cellular communication and blood brain barrier function.

In one aspect, the promoter is selected from the group consisting of: GFAP promoter, sox9 promoter, S100b promoter, aldh1l1 promoter, lipocalin 2 (Lcn 2) promoter, glutamine synthetase promoter, aquaporin-4 (AQP 4) promoter, oligodendrocyte transcription factor (Olig 2) promoter and synaptoprotein promoter, NG2 promoter, ionized calcium binding adapter molecule 1 (Iba 1) promoter, cluster of differentiation 86 (CD 86) promoter, platelet Derived Growth Factor Receptor Alpha (PDGFRA) promoter, platelet Derived Growth Factor Receptor Beta (PDGFRB) promoter, elongation factor 1-alpha (EF 1 a) promoter, CAG promoter, cytomegalovirus (CMV) promoter, ubiquitin promoter. In one aspect, the promoter is a GFAP promoter. In one aspect, the promoter is a truncated GFAP promoter. In one aspect, the promoter is the Sox9 promoter. In one aspect, the promoter is an S100b promoter. In one aspect, the promoter is an Aldh1l1 promoter. In one aspect, the promoter is an Lcn2 promoter. In one aspect, the promoter is a glutamine synthetase promoter. In one aspect, the promoter is the AQP4 promoter. In one aspect, the promoter is an Olig2 promoter. In one aspect, the promoter is a synapsin promoter. In one aspect, the promoter is the Iba1 promoter. In one aspect, the promoter is a CD86 promoter. In one aspect, the promoter is a PDGFRA promoter. In one aspect, the promoter is a PDGFRB promoter. In one aspect, the promoter is an EF1a promoter. In one aspect, the promoter is a CAG promoter. In one aspect, the promoter is a CMV promoter. In one aspect, the promoter is a ubiquitin promoter.

In one aspect, the GFAP promoter is a promoter that directs astrocyte-specific expression of a protein called Glial Fibrillary Acidic Protein (GFAP) in a cell. In one aspect, the GFAP promoter sequence is a human GFAP (hGFAP) promoter sequence. In one aspect, the GFAP promoter is selected from the group consisting of GfaABC1D (also referred to as "pGfa 681"), gfa1.6 and hgfa2.2. In one aspect, the GFAP promoter is GfaABC1D (also referred to as "pGfa 681"). In one aspect, the GFAP promoter is gfa1.6. In one aspect, the GFAP promoter is hgfa2.2. In one aspect pGfa681 is SEQ ID NO 27. In one aspect, GFAP Gfa1.6 is SEQ ID NO. 4. In one aspect, hGFa2.2 is SEQ ID NO. 18. In one aspect, the GFAP promoter is selected from the group consisting of: SEQ ID NOS 4, 18 and 27. In one aspect, the GFAP promoter is SEQ ID NO. 4. In one aspect, the GFAP promoter is SEQ ID NO. 18. In one aspect, the GFAP promoter is SEQ ID NO. 27.

In one aspect, the GFAP promoter sequence is selected from the group consisting of: a chimpanzee GFAP promoter sequence, a bonobo GFAP promoter sequence, a chimpanzee GFAP promoter sequence, a gorilla GFAP promoter sequence, a macaque GFAP promoter sequence, a marmoset GFAP promoter sequence, a pigtail GFAP promoter sequence, a baboon GFAP promoter sequence, a gibbon GFAP promoter sequence, and a marmoset GFAP promoter sequence. In one aspect, the GFAP promoter sequence is a chimpanzee GFAP promoter sequence. In one aspect, the GFAP promoter sequence is a bonobo GFAP promoter sequence. In one aspect, the GFAP promoter sequence is a chimpanzee GFAP promoter sequence. In one aspect, the GFAP promoter sequence is a gorilla GFAP promoter sequence. In one aspect, the GFAP promoter sequence is a macaque GFAP promoter sequence. In one aspect, the GFAP promoter sequence is a marmoset GFAP promoter sequence. In one aspect, the GFAP promoter sequence is a monkey GFAP promoter sequence. In one aspect, the GFAP promoter sequence is a baboon GFAP promoter sequence. In one aspect, the GFAP promoter sequence is a gibbon GFAP promoter sequence. In one aspect, the GFAP promoter sequence is a cynomolgus GFAP promoter sequence.

In one aspect, the GFAP promoter sequence comprises at least 100 nucleotides. In one aspect, the GFAP promoter comprises at least 500 nucleotides. In another aspect, the GFAP promoter comprises at least 1000 nucleotides. In yet another aspect, the GFAP promoter comprises at least 1500 nucleotides.

It is understood in the art that fragments of a promoter sequence may function to drive transcription of an operably linked nucleic acid molecule. For example, but not limited to, if a 1000 nucleotide promoter is truncated to 500 nucleotides and the 500 nucleotide fragment is capable of driving transcription, the 500 nucleotide fragment is referred to as a "functional fragment".

In one aspect, the promoter comprises at least 10 nucleotides. In one aspect, the promoter comprises at least 50 nucleotides. In one aspect, the promoter comprises at least 100 nucleotides. In one aspect, the intron comprises at least 150 nucleotides. In one aspect, the promoter comprises at least 200 nucleotides. In one aspect, the promoter comprises at least 250 nucleotides. In one aspect, the promoter comprises at least 300 nucleotides. In one aspect, the promoter comprises at least 350 nucleotides. In one aspect, the promoter comprises at least 400 nucleotides. In one aspect, the promoter comprises at least 450 nucleotides. In one aspect, the promoter comprises at least 500 nucleotides. In one aspect, the promoter comprises between 50 nucleotides and 7500 nucleotides. In one aspect, the promoter comprises between 50 nucleotides and 5000 nucleotides. In one aspect, the promoter comprises between 50 nucleotides and 2500 nucleotides. In one aspect, the promoter comprises between 50 nucleotides and 1000 nucleotides. In one aspect, the promoter comprises between 50 nucleotides and 500 nucleotides. In one aspect, the promoter comprises between 10 nucleotides and 7500 nucleotides. In one aspect, the promoter comprises between 10 nucleotides and 5000 nucleotides. In one aspect, the promoter comprises between 10 nucleotides and 2500 nucleotides. In one aspect, the promoter comprises between 10 nucleotides and 1000 nucleotides. In one aspect, the promoter comprises between 10 nucleotides and 500 nucleotides

In one aspect, the GFAP promoter nucleic acid sequence comprises a sequence at least 70% identical to: SEQ ID NO. 4, 18, 27 or a functional fragment thereof. In one aspect, the GFAP promoter nucleic acid sequence comprises a sequence at least 75% identical to: SEQ ID NO. 4, 18, 27 or a functional fragment thereof. In one aspect, the GFAP promoter nucleic acid sequence comprises a sequence at least 80% identical to: SEQ ID NO. 4, 18, 27 or a functional fragment thereof. In one aspect, the GFAP promoter nucleic acid sequence comprises a sequence at least 85% identical to: SEQ ID NO. 4, 18, 27 or a functional fragment thereof. In one aspect, the GFAP promoter nucleic acid sequence comprises a sequence at least 90% identical to: SEQ ID NO. 4, 18, 27 or a functional fragment thereof. In one aspect, the GFAP promoter nucleic acid sequence comprises a sequence at least 91% identical to: SEQ ID NO. 4, 18, 27 or a functional fragment thereof. In one aspect, the GFAP promoter nucleic acid sequence comprises a sequence at least 92% identical to: SEQ ID NO. 4, 18, 27 or a functional fragment thereof. In one aspect, the GFAP promoter nucleic acid sequence comprises a sequence at least 93% identical to: SEQ ID NO. 4, 18, 27 or a functional fragment thereof. In one aspect, the GFAP promoter nucleic acid sequence comprises a sequence at least 94% identical to: SEQ ID NO. 4, 18, 27 or a functional fragment thereof. In one aspect, the GFAP promoter nucleic acid sequence comprises a sequence at least 95% identical to: SEQ ID NO. 4, 18, 27 or a functional fragment thereof. In one aspect, the GFAP promoter nucleic acid sequence comprises a sequence at least 96% identical to: SEQ ID NO. 4, 18, 27 or a functional fragment thereof. In one aspect, the GFAP promoter nucleic acid sequence comprises a sequence at least 97% identical to: SEQ ID NO. 4, 18, 27 or a functional fragment thereof. In one aspect, the GFAP promoter nucleic acid sequence comprises a sequence at least 98% identical to: SEQ ID NO. 4, 18, 27 or a functional fragment thereof. In one aspect, the GFAP promoter nucleic acid sequence comprises a sequence at least 99% identical to: SEQ ID NO. 4, 18, 27 or a functional fragment thereof. In one aspect, the GFAP promoter nucleic acid sequence comprises a sequence at least 99.5% identical to: SEQ ID NO. 4, 18, 27 or a functional fragment thereof. In one aspect, the GFAP promoter nucleic acid sequence comprises a sequence at least 99.8% identical to: SEQ ID NO. 4, 18, 27 or a functional fragment thereof. In one aspect, the GFAP promoter nucleic acid sequence comprises a sequence at least 99.9% identical to: SEQ ID NO. 4, 18, 27 or a functional fragment thereof. In one aspect, the GFAP promoter nucleic acid sequence comprises a sequence 100% identical to: SEQ ID NO. 4, 18, 27 or a functional fragment thereof.

As used herein, the term "brain" refers to an organ that serves as a central nervous system. In one aspect, the brain comprises the brain, cortex, cerebellum, and/or brain stem.

As used herein, the term "cortex" refers to the outer layer of brain nerve tissue.

As used herein, the term "striatum" or "striatum" (or "striatum") refers to clusters of neurons in the basal ganglia under the anterior cerebral cortex, and includes the ventral striatum and the dorsal striatum.

As used herein, the term "substantia nigra" refers to the clusters of neurons in the basal ganglia under the mesocortex and comprises dense and reticular portions.

As used herein, the term "forebrain" refers to the forefront of the brain.

As used herein, the term "putamen" refers to the circular structure of the forebrain base and is an integral part of the dorsal striatum.

As used herein, the term "caudate nucleus" refers to the structure of the anterior brain base and is an integral part of the dorsal striatum.

As used herein, the term "subcortical basal ganglia" refers to clusters of neurons in the deep brain hemisphere.

As used herein, the term "spinal cord" refers to structures that function in transmitting nerve signals from the motor cortex to the body.

As used herein, the term "motor cortex" refers to the area of the frontal lobe of the cerebral cortex involved in the planning, control and execution of voluntary movements.

In one aspect, the methods provided herein convert reactive astrocytes into functional neurons in the brain. In one aspect, the methods provided herein convert reactive astrocytes into functional neurons in the cerebral cortex of the brain. In one aspect, the methods provided herein convert reactive astrocytes into functional neurons in the striatum of the brain. In one aspect, the methods provided herein convert reactive astrocytes into functional neurons in the dorsal striatum of the brain. In one aspect, the methods provided herein convert reactive astrocytes into functional neurons in the spinal cord of the brain. In one aspect, the methods provided herein convert reactive astrocytes into functional neurons in the putamen of the brain. In one aspect, the methods provided herein convert reactive astrocytes into functional neurons in the caudate nucleus of the brain. In one aspect, the methods provided herein convert reactive astrocytes into functional neurons in the substantia nigra of the brain.

Elongation factor 1 alpha (EF-1 alpha; also known as eEF1a 1) is an isoform of the alpha subunit of the elongation factor 1 complex. The complex is involved in the enzymatic delivery of aminoacyltRNA to the ribosome. EF-1 alpha isoforms are expressed in brain, placenta, lung, liver, kidney and pancreas.

In one aspect, the enhancer sequence from the EF-1 alpha promoter is a human enhancer sequence from the EF-1 alpha promoter. In one aspect, the enhancer sequence from the EF-1 alpha promoter is selected from the group consisting of: a chimpanzee enhancer sequence from an EF-1 a promoter, a bonobo enhancer sequence from an EF-1 a promoter, a chimpanzee enhancer sequence from an EF-1 a promoter, a gorilla enhancer sequence from an EF-1 a promoter, a macaque enhancer sequence from an EF-1 a promoter, a marmoset enhancer sequence from an EF-1 a promoter, a pigtail enhancer sequence from an EF-1 a promoter, a baboon enhancer sequence from an EF-1 a promoter, a gibbon enhancer sequence from an EF-1 a promoter, and a marmoset enhancer sequence from an EF-1 a promoter. In one aspect, the enhancer sequence from the EF-1 alpha promoter is a chimpanzee enhancer sequence from the EF-1 alpha promoter. In one aspect, the enhancer sequence from the EF-1. Alpha. Promoter is a bonobo enhancer sequence from the EF-1. Alpha. Promoter. In one aspect, the enhancer sequence from the EF-1 alpha promoter is a chimpanzee enhancer sequence from the EF-1 alpha promoter. In one aspect, the enhancer sequence from the EF-1 alpha promoter is a gorilla enhancer sequence from the EF-1 alpha promoter. In one aspect, the enhancer sequence from the EF-1. Alpha. Promoter is a cynomolgus monkey enhancer sequence from the EF-1. Alpha. Promoter. In one aspect, the enhancer sequence from the EF-1 alpha promoter is a marmoset enhancer sequence from the EF-1 alpha promoter. In one aspect, the enhancer sequence from the EF-1. Alpha. Promoter is a monkey enhancer sequence from the EF-1. Alpha. Promoter. In one aspect, the enhancer sequence from the EF-1. Alpha. Promoter is a baboon enhancer sequence from the EF-1. Alpha. Promoter. In one aspect, the enhancer sequence from the EF-1 alpha promoter is a gibbon enhancer sequence from the EF-1 alpha promoter. In one aspect, the enhancer sequence from the EF-1 alpha promoter is a cynomolgus monkey enhancer sequence from the EF-1 alpha promoter.

In one aspect, the enhancer from the EF-1 alpha promoter nucleic acid sequence comprises a sequence at least 70% identical to: SEQ ID NO. 2, or a complement thereof. In one aspect, the enhancer from the EF-1 alpha promoter nucleic acid sequence comprises a sequence at least 75% identical to: SEQ ID NO. 2, or a complement thereof. In one aspect, the enhancer from the EF-1 alpha promoter nucleic acid sequence comprises a sequence at least 80% identical to: SEQ ID NO. 2, or a complement thereof. In one aspect, the enhancer from the EF-1 alpha promoter nucleic acid sequence comprises a sequence at least 85% identical to: SEQ ID NO. 2, or a complement thereof. In one aspect, the enhancer from the EF-1 alpha promoter nucleic acid sequence comprises a sequence at least 90% identical to: SEQ ID NO. 2, or a complement thereof. In one aspect, the enhancer from the EF-1 alpha promoter nucleic acid sequence comprises a sequence at least 91% identical to: SEQ ID NO. 2, or a complement thereof. In one aspect, the enhancer from the EF-1 alpha promoter nucleic acid sequence comprises a sequence at least 92% identical to: SEQ ID NO. 2, or a complement thereof. In one aspect, the enhancer from the EF-1 alpha promoter nucleic acid sequence comprises a sequence at least 93% identical to: SEQ ID NO. 2, or a complement thereof. In one aspect, the enhancer from the EF-1 alpha promoter nucleic acid sequence comprises a sequence at least 94% identical to: SEQ ID NO. 2, or a complement thereof. In one aspect, the enhancer from the EF-1 alpha promoter nucleic acid sequence comprises a sequence at least 95% identical to: SEQ ID NO. 2, or a complement thereof. In one aspect, the enhancer from the EF-1 alpha promoter nucleic acid sequence comprises a sequence at least 96% identical to: SEQ ID NO. 2, or a complement thereof. In one aspect, the enhancer from the EF-1 alpha promoter nucleic acid sequence comprises a sequence at least 97% identical to: SEQ ID NO. 2, or a complement thereof. In one aspect, the enhancer from the EF-1 alpha promoter nucleic acid sequence comprises a sequence at least 98% identical to: SEQ ID NO. 2, or a complement thereof. In one aspect, the enhancer from the EF-1 alpha promoter nucleic acid sequence comprises a sequence at least 99% identical to: SEQ ID NO. 2, or a complement thereof. In one aspect, the enhancer from the EF-1 alpha promoter nucleic acid sequence comprises a sequence at least 99.5% identical to: SEQ ID NO. 2, or a complement thereof. In one aspect, the enhancer from the EF-1 alpha promoter nucleic acid sequence comprises a sequence at least 99.8% identical to: SEQ ID NO. 2, or a complement thereof. In one aspect, the enhancer from the EF-1 alpha promoter nucleic acid sequence comprises a sequence at least 99.9% identical to: SEQ ID NO. 2, or a complement thereof. In one aspect, the enhancer from the EF-1 alpha promoter nucleic acid sequence comprises a sequence 100% identical to: SEQ ID NO. 2, or a complement thereof.

Cytomegalovirus (CMV) is a genus of viruses in the order herpesviridae (Herpesvirale).

In one aspect, the enhancer sequence from CMV is a human enhancer sequence from CMV. In one aspect, the enhancer sequence from CMV is selected from the group consisting of: a chimpanzee enhancer sequence from CMV, a bonobo enhancer sequence from CMV, a chimpanzee enhancer sequence from CMV, a gorilla enhancer sequence from CMV, a macaque enhancer sequence from CMV, a marmoset enhancer sequence from CMV, a pigtail enhancer sequence from CMV, a baboon enhancer sequence from CMV, a gibbon enhancer sequence from CMV, and a marmoset enhancer sequence from CMV. In one aspect, the enhancer sequence from CMV is a chimpanzee enhancer sequence from CMV. In one aspect, the enhancer sequence from CMV is a bonobo enhancer sequence from CMV. In one aspect, the enhancer sequence from CMV is a chimpanzee enhancer sequence from CMV. In one aspect, the enhancer sequence from CMV is a gorilla enhancer sequence from CMV. In one aspect, the enhancer sequence from CMV is a cynomolgus monkey enhancer sequence from CMV. In one aspect, the enhancer sequence from CMV is a marmoset enhancer sequence from CMV. In one aspect, the enhancer sequence from CMV is a monkey enhancer sequence from CMV. In one aspect, the enhancer sequence from CMV is a baboon enhancer sequence from CMV. In one aspect, the enhancer sequence from CMV is a gibbon enhancer sequence from CMV. In one aspect, the enhancer sequence from CMV is a armpit enhancer sequence from CMV.

In one aspect, the enhancer from the CMV nucleic acid sequence comprises a sequence at least 70% identical to: SEQ ID NO. 17, or a complement thereof. In one aspect, the enhancer from the CMV nucleic acid sequence comprises a sequence at least 75% identical to: SEQ ID NO. 17, or a complement thereof. In one aspect, the enhancer from the CMV nucleic acid sequence comprises a sequence at least 80% identical to: SEQ ID NO. 17, or a complement thereof. In one aspect, the enhancer from the CMV nucleic acid sequence comprises a sequence at least 85% identical to: SEQ ID NO. 17, or a complement thereof. In one aspect, the enhancer from the CMV nucleic acid sequence comprises a sequence at least 90% identical to: SEQ ID NO. 17, or a complement thereof. In one aspect, the enhancer from the CMV nucleic acid sequence comprises a sequence at least 91% identical to: SEQ ID NO. 17, or a complement thereof. In one aspect, the enhancer from the CMV nucleic acid sequence comprises a sequence at least 911% identical to: SEQ ID NO. 17, or a complement thereof. In one aspect, the enhancer from the CMV nucleic acid sequence comprises a sequence at least 93% identical to: SEQ ID NO. 17, or a complement thereof. In one aspect, the enhancer from the CMV nucleic acid sequence comprises a sequence at least 94% identical to: SEQ ID NO. 17, or a complement thereof. In one aspect, the enhancer from the CMV nucleic acid sequence comprises a sequence at least 95% identical to: SEQ ID NO. 17, or a complement thereof. In one aspect, the enhancer from the CMV nucleic acid sequence comprises a sequence at least 96% identical to: SEQ ID NO. 17, or a complement thereof. In one aspect, the enhancer from the CMV nucleic acid sequence comprises a sequence at least 97% identical to: SEQ ID NO. 17, or a complement thereof. In one aspect, the enhancer from the CMV nucleic acid sequence comprises a sequence at least 98% identical to: SEQ ID NO. 17, or a complement thereof. In one aspect, the enhancer from the CMV nucleic acid sequence comprises a sequence at least 99% identical to: SEQ ID NO. 17, or a complement thereof. In one aspect, the enhancer from the CMV nucleic acid sequence comprises a sequence at least 99.5% identical to: SEQ ID NO. 17, or a complement thereof. In one aspect, the enhancer from the CMV nucleic acid sequence comprises a sequence at least 99.8% identical to: SEQ ID NO. 17, or a complement thereof. In one aspect, the enhancer from the CMV nucleic acid sequence comprises a sequence at least 99.9% identical to: SEQ ID NO. 17, or a complement thereof. In one aspect, the enhancer from the CMV nucleic acid sequence comprises a sequence 100% identical to: SEQ ID NO. 17, or a complement thereof.

In one aspect, the vectors of the present disclosure comprise a chimeric intron. In one aspect, the chimeric intron consists of a 5 '-donor site from the first intron of the human β -globulin gene and a branching and 3' -acceptor site from the heavy chain variable region intron of the immunoglobulin gene. In one aspect, the chimeric intron is a chimeric intron of rabbit β -globulin and chicken β -actin that are similar in the CAG promoter. In one aspect, the vectors of the present disclosure comprise a Glial Fibrillary Acidic Protein (GFAP) intron. In one aspect, the vectors of the present disclosure comprise a Glial Fibrillary Acidic Protein (GFAP) first intron.

Introns can be divided into at least five classes, including: splicing the introns; transferring the RNA intron; group I introns; group II introns; and group III introns. Introns may be synthetically generated, altered, or derived from known or naturally occurring intron sequences or other intron sequences. Introns may also include chimeric introns comprising a combination of two or more heterologous sequences. Thus, introns of the application may include variants of intron sequences that are similar in composition to, but not identical to, other intron sequences known or provided herein. In one aspect, the intron comprises at least 10 nucleotides. In one aspect, the intron comprises at least 50 nucleotides. In one aspect, the intron comprises at least 100 nucleotides. In one aspect, the intron comprises at least 150 nucleotides. In one aspect, the intron comprises at least 200 nucleotides. In one aspect, the intron comprises at least 250 nucleotides. In one aspect, the intron comprises at least 300 nucleotides. In one aspect, the intron comprises at least 350 nucleotides. In one aspect, the intron comprises at least 400 nucleotides. In one aspect, the intron comprises at least 450 nucleotides. In one aspect, the intron comprises at least 500 nucleotides. In one aspect, the intron comprises between 50 nucleotides and 7500 nucleotides. In one aspect, the intron comprises between 50 nucleotides and 5000 nucleotides. In one aspect, the intron comprises between 50 nucleotides and 2500 nucleotides. In one aspect, the intron comprises between 50 nucleotides and 1000 nucleotides. In one aspect, the intron comprises between 50 nucleotides and 500 nucleotides. In one aspect, the intron comprises between 10 nucleotides and 7500 nucleotides. In one aspect, the intron comprises between 10 nucleotides and 5000 nucleotides. In one aspect, the intron comprises between 10 nucleotides and 2500 nucleotides. In one aspect, the intron comprises between 10 nucleotides and 1000 nucleotides. In one aspect, the intron comprises between 10 nucleotides and 500 nucleotides.

In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 70% identical to: SEQ ID NO. 5, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 75% identical to: SEQ ID NO. 5, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 80% identical to: SEQ ID NO. 5, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 85% identical to: SEQ ID NO. 5, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 90% identical to: SEQ ID NO. 5, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 91% identical to: SEQ ID NO. 5, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 92% identical to: SEQ ID NO. 5, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 93% identical to: SEQ ID NO. 5, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 94% identical to: SEQ ID NO. 5, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 95% identical to: SEQ ID NO. 5, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 96% identical to: SEQ ID NO. 5, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 97% identical to: SEQ ID NO. 5, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 98% identical to: SEQ ID NO. 5, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 99% identical to: SEQ ID NO. 5, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 99.5% identical to: SEQ ID NO. 5, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 99.8% identical to: SEQ ID NO. 5, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 99.9% identical to: SEQ ID NO. 5, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence 100% identical to: SEQ ID NO. 5, or a complement thereof.

In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 70% identical to: SEQ ID NO. 28, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 75% identical to: SEQ ID NO. 28, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 80% identical to: SEQ ID NO. 28, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 85% identical to: SEQ ID NO. 28, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 90% identical to: SEQ ID NO. 28, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 91% identical to: SEQ ID NO. 28, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 92% identical to: SEQ ID NO. 28, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 93% identical to: SEQ ID NO. 28, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 94% identical to: SEQ ID NO. 28, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 95% identical to: SEQ ID NO. 28, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 96% identical to: SEQ ID NO. 28, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 97% identical to: SEQ ID NO. 28, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 98% identical to: SEQ ID NO. 28, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 99% identical to: SEQ ID NO. 28, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 99.5% identical to: SEQ ID NO. 28, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 99.8% identical to: SEQ ID NO. 28, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 99.9% identical to: SEQ ID NO. 28, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence 100% identical to: SEQ ID NO. 28, or a complement thereof.

In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 70% identical to: SEQ ID NO. 29, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 75% identical to: SEQ ID NO. 29, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 80% identical to: SEQ ID NO. 29, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 85% identical to: SEQ ID NO. 29, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 90% identical to: SEQ ID NO. 29, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 91% identical to: SEQ ID NO. 29, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 92% identical to: SEQ ID NO. 29, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 93% identical to: SEQ ID NO. 29, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 94% identical to: SEQ ID NO. 29, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 95% identical to: SEQ ID NO. 29, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 96% identical to: SEQ ID NO. 29, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 97% identical to: SEQ ID NO. 29, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 98% identical to: SEQ ID NO. 29, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 99% identical to: SEQ ID NO. 29, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 99.5% identical to: SEQ ID NO. 29, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 99.8% identical to: SEQ ID NO. 29, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 99.9% identical to: SEQ ID NO. 29, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence 100% identical to: SEQ ID NO. 29, or a complement thereof.

The woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) is a DNA sequence that creates a tertiary structure that enhances expression of a delivery gene in a viral vector.

In one aspect, the WPRE nucleic acid sequence is an optimized version of WPRE.

In one aspect, the WPRE nucleic acid sequence is selected from the group consisting of: SEQ ID NOS.7 and 30. In one aspect, the WPRE nucleic acid sequence is SEQ ID NO. 7. In one aspect, the WPRE nucleic acid sequence is SEQ ID NO. 30.

In one aspect, the WPRE nucleic acid sequence comprises a sequence at least 70% identical to: SEQ ID NO. 7, or a complement thereof. In one aspect, the WPRE nucleic acid sequence comprises a sequence at least 75% identical to: SEQ ID NO. 7, or a complement thereof. In one aspect, the WPRE nucleic acid sequence comprises a sequence at least 80% identical to: SEQ ID NO. 7, or a complement thereof. In one aspect, the WPRE nucleic acid sequence comprises a sequence at least 85% identical to: SEQ ID NO. 7, or a complement thereof. In one aspect, the WPRE nucleic acid sequence comprises a sequence at least 90% identical to: SEQ ID NO. 7, or a complement thereof. In one aspect, the WPRE nucleic acid sequence comprises a sequence at least 91% identical to: SEQ ID NO. 7, or a complement thereof. In one aspect, the WPRE nucleic acid sequence comprises a sequence at least 92% identical to: SEQ ID NO. 7, or a complement thereof. In one aspect, the WPRE nucleic acid sequence comprises a sequence at least 93% identical to: SEQ ID NO. 7, or a complement thereof. In one aspect, the WPRE nucleic acid sequence comprises a sequence at least 94% identical to: SEQ ID NO. 7, or a complement thereof. In one aspect, the WPRE nucleic acid sequence comprises a sequence at least 95% identical to: SEQ ID NO. 7, or a complement thereof. In one aspect, the WPRE nucleic acid sequence comprises a sequence at least 96% identical to: SEQ ID NO. 7, or a complement thereof. In one aspect, the WPRE nucleic acid sequence comprises a sequence at least 97% identical to: SEQ ID NO. 7, or a complement thereof. In one aspect, the WPRE nucleic acid sequence comprises a sequence at least 98% identical to: SEQ ID NO. 7, or a complement thereof. In one aspect, the WPRE nucleic acid sequence comprises a sequence at least 99% identical to: SEQ ID NO. 7, or a complement thereof. In one aspect, the WPRE nucleic acid sequence comprises a sequence at least 99.5% identical to: SEQ ID NO. 7, or a complement thereof. In one aspect, the WPRE nucleic acid sequence comprises a sequence at least 99.8% identical to: SEQ ID NO. 7, or a complement thereof. In one aspect, the WPRE nucleic acid sequence comprises a sequence at least 99.9% identical to: SEQ ID NO. 7, or a complement thereof. In one aspect, the WPRE nucleic acid sequence comprises a sequence 100% identical to: SEQ ID NO. 7, or a complement thereof.

In one aspect, the WPRE nucleic acid sequence comprises a sequence at least 70% identical to: SEQ ID NO. 30, or a complement thereof. In one aspect, the WPRE nucleic acid sequence comprises a sequence at least 75% identical to: SEQ ID NO. 30, or a complement thereof. In one aspect, the WPRE nucleic acid sequence comprises a sequence at least 80% identical to: SEQ ID NO. 30, or a complement thereof. In one aspect, the WPRE nucleic acid sequence comprises a sequence at least 85% identical to: SEQ ID NO. 30, or a complement thereof. In one aspect, the WPRE nucleic acid sequence comprises a sequence at least 90% identical to: SEQ ID NO. 30, or a complement thereof. In one aspect, the WPRE nucleic acid sequence comprises a sequence at least 91% identical to: SEQ ID NO. 30, or a complement thereof. In one aspect, the WPRE nucleic acid sequence comprises a sequence at least 92% identical to: SEQ ID NO. 30, or a complement thereof. In one aspect, the WPRE nucleic acid sequence comprises a sequence at least 93% identical to: SEQ ID NO. 30, or a complement thereof. In one aspect, the WPRE nucleic acid sequence comprises a sequence at least 94% identical to: SEQ ID NO. 30, or a complement thereof. In one aspect, the WPRE nucleic acid sequence comprises a sequence at least 95% identical to: SEQ ID NO. 30, or a complement thereof. In one aspect, the WPRE nucleic acid sequence comprises a sequence at least 96% identical to: SEQ ID NO. 30, or a complement thereof. In one aspect, the WPRE nucleic acid sequence comprises a sequence at least 97% identical to: SEQ ID NO. 30, or a complement thereof. In one aspect, the WPRE nucleic acid sequence comprises a sequence at least 98% identical to: SEQ ID NO. 30, or a complement thereof. In one aspect, the WPRE nucleic acid sequence comprises a sequence at least 99% identical to: SEQ ID NO. 30, or a complement thereof. In one aspect, the WPRE nucleic acid sequence comprises a sequence at least 99.5% identical to: SEQ ID NO. 30, or a complement thereof. In one aspect, the WPRE nucleic acid sequence comprises a sequence at least 99.8% identical to: SEQ ID NO. 30, or a complement thereof. In one aspect, the WPRE nucleic acid sequence comprises a sequence at least 99.9% identical to: SEQ ID NO. 30, or a complement thereof. In one aspect, the WPRE nucleic acid sequence comprises a sequence 100% identical to: SEQ ID NO. 30, or a complement thereof.

SV40 polyadenylation signals (also known as SV40 PolyA; simian Virus 40PolyA; and PolyA) are DNA sequences that terminate transcription and add PolyA tails at the 3' end of messenger RNA (mRNA).

The hGH polyadenylation signal (also known as hGH PolyA) is a DNA sequence that can terminate transcription and add the PolyA tail at the 3' end of the messenger RNA (mRNA).

The bGH polyadenylation signal (also known as bGH PolyA or bGHpA) refers to the PolyA signal or PolyA tail of bovine growth hormone.

As used herein, "PolyA tail" refers to a stretch of RNA that contains only nucleobase adenine. In one aspect, an RNA molecule transcribed from an AAV vector construct provided herein comprises a poly a tail. In one aspect, the PolyA tail comprises at least two adenine. In one aspect, the PolyA tail comprises at least ten adenine. In one aspect, the PolyA tail comprises at least 50 adenine. In one aspect, the polyA tail comprises at least 100 adenine. In one aspect, the polyA tail comprises at least 150 adenine. In one aspect, the PolyA tail comprises at least 200 adenine. In one aspect, the polyA tail comprises at least 250 adenine. In one aspect, the PolyA tail comprises between 50 adenine and 300 adenine.

In one aspect, the SV40 polyadenylation signal nucleic acid sequence comprises a sequence at least 70% identical to: SEQ ID NO. 8, or a complement thereof. In one aspect, the SV40 polyadenylation signal nucleic acid sequence comprises a sequence at least 75% identical to: SEQ ID NO. 8, or a complement thereof. In one aspect, the SV40 polyadenylation signal nucleic acid sequence comprises a sequence at least 80% identical to: SEQ ID NO. 8, or a complement thereof. In one aspect, the SV40 polyadenylation signal nucleic acid sequence comprises a sequence at least 85% identical to: SEQ ID NO. 8, or a complement thereof. In one aspect, the SV40 polyadenylation signal nucleic acid sequence comprises a sequence at least 90% identical to: SEQ ID NO. 8, or a complement thereof. In one aspect, the SV40 polyadenylation signal nucleic acid sequence comprises a sequence at least 91% identical to: SEQ ID NO. 8, or a complement thereof. In one aspect, the SV40 polyadenylation signal nucleic acid sequence comprises a sequence at least 92% identical to: SEQ ID NO. 8, or a complement thereof. In one aspect, the SV40 polyadenylation signal nucleic acid sequence comprises a sequence at least 93% identical to: SEQ ID NO. 8, or a complement thereof. In one aspect, the SV40 polyadenylation signal nucleic acid sequence comprises a sequence at least 94% identical to: SEQ ID NO. 8, or a complement thereof. In one aspect, the SV40 polyadenylation signal nucleic acid sequence comprises a sequence at least 95% identical to: SEQ ID NO. 8, or a complement thereof. In one aspect, the SV40 polyadenylation signal nucleic acid sequence comprises a sequence at least 96% identical to: SEQ ID NO. 8, or a complement thereof. In one aspect, the SV40 polyadenylation signal nucleic acid sequence comprises a sequence at least 97% identical to: SEQ ID NO. 8, or a complement thereof. In one aspect, the SV40 polyadenylation signal nucleic acid sequence comprises a sequence at least 98% identical to: SEQ ID NO. 8, or a complement thereof. In one aspect, the SV40 polyadenylation signal nucleic acid sequence comprises a sequence at least 99% identical to: SEQ ID NO. 8, or a complement thereof. In one aspect, the SV40 polyadenylation signal nucleic acid sequence comprises a sequence at least 99.5% identical to: SEQ ID NO. 8, or a complement thereof. In one aspect, the SV40 polyadenylation signal nucleic acid sequence comprises a sequence at least 99.8% identical to: SEQ ID NO. 8, or a complement thereof. In one aspect, the SV40 polyadenylation signal nucleic acid sequence comprises a sequence at least 99.9% identical to: SEQ ID NO. 8, or a complement thereof. In one aspect, the SV40 polyadenylation signal nucleic acid sequence comprises a sequence 100% identical to: SEQ ID NO. 8, or a complement thereof.

In one aspect, the hGH polyadenylation signal nucleic acid sequence comprises a sequence at least 70% identical to: SEQ ID NO. 17, or a complement thereof. In one aspect, the hGH polyadenylation signal nucleic acid sequence comprises a sequence at least 75% identical to: SEQ ID NO. 17, or a complement thereof. In one aspect, the hGH polyadenylation signal nucleic acid sequence comprises a sequence at least 80% identical to: SEQ ID NO. 17, or a complement thereof. In one aspect, the hGH polyadenylation signal nucleic acid sequence comprises a sequence at least 85% identical to: SEQ ID NO. 17, or a complement thereof. In one aspect, the hGH polyadenylation signal nucleic acid sequence comprises a sequence at least 90% identical to: SEQ ID NO. 17, or a complement thereof. In one aspect, the hGH polyadenylation signal nucleic acid sequence comprises a sequence at least 91% identical to: SEQ ID NO. 17, or a complement thereof. In one aspect, the hGH polyadenylation signal nucleic acid sequence comprises a sequence at least 92% identical to: SEQ ID NO. 17, or a complement thereof. In one aspect, the hGH polyadenylation signal nucleic acid sequence comprises a sequence at least 93% identical to: SEQ ID NO. 17, or a complement thereof. In one aspect, the hGH polyadenylation signal nucleic acid sequence comprises a sequence at least 94% identical to: SEQ ID NO. 17, or a complement thereof. In one aspect, the hGH polyadenylation signal nucleic acid sequence comprises a sequence at least 95% identical to: SEQ ID NO. 17, or a complement thereof. In one aspect, the hGH polyadenylation signal nucleic acid sequence comprises a sequence at least 96% identical to: SEQ ID NO. 17, or a complement thereof. In one aspect, the hGH polyadenylation signal nucleic acid sequence comprises a sequence at least 97% identical to: SEQ ID NO. 17, or a complement thereof. In one aspect, the hGH polyadenylation signal nucleic acid sequence comprises a sequence at least 917% identical to: SEQ ID NO. 17, or a complement thereof. In one aspect, the hGH polyadenylation signal nucleic acid sequence comprises a sequence at least 99% identical to: SEQ ID NO. 17, or a complement thereof. In one aspect, the hGH polyadenylation signal nucleic acid sequence comprises a sequence at least 99.5% identical to: SEQ ID NO. 17, or a complement thereof. In one aspect, the hGH polyadenylation signal nucleic acid sequence comprises a sequence at least 99.17% identical to: SEQ ID NO. 17, or a complement thereof. In one aspect, the hGH polyadenylation signal nucleic acid sequence comprises a sequence at least 99.9% identical to: SEQ ID NO. 17, or a complement thereof. In one aspect, the hGH polyadenylation signal nucleic acid sequence comprises a sequence 100% identical to: SEQ ID NO. 17, or a complement thereof.

In one aspect, the bGH polyadenylation signal nucleic acid sequence comprises a sequence at least 70% identical to: SEQ ID NO. 26, or a complement thereof. In one aspect, the hGH polyadenylation signal nucleic acid sequence comprises a sequence at least 75% identical to: SEQ ID NO. 26, or a complement thereof. In one aspect, the hGH polyadenylation signal nucleic acid sequence comprises a sequence at least 80% identical to: SEQ ID NO. 26, or a complement thereof. In one aspect, the hGH polyadenylation signal nucleic acid sequence comprises a sequence at least 85% identical to: SEQ ID NO. 26, or a complement thereof. In one aspect, the hGH polyadenylation signal nucleic acid sequence comprises a sequence at least 90% identical to: SEQ ID NO. 26, or a complement thereof. In one aspect, the hGH polyadenylation signal nucleic acid sequence comprises a sequence at least 91% identical to: SEQ ID NO. 26, or a complement thereof. In one aspect, the hGH polyadenylation signal nucleic acid sequence comprises a sequence at least 92% identical to: SEQ ID NO. 26, or a complement thereof. In one aspect, the hGH polyadenylation signal nucleic acid sequence comprises a sequence at least 93% identical to: SEQ ID NO. 26, or a complement thereof. In one aspect, the hGH polyadenylation signal nucleic acid sequence comprises a sequence at least 94% identical to: SEQ ID NO. 26, or a complement thereof. In one aspect, the hGH polyadenylation signal nucleic acid sequence comprises a sequence at least 95% identical to: SEQ ID NO. 26, or a complement thereof. In one aspect, the hGH polyadenylation signal nucleic acid sequence comprises a sequence at least 96% identical to: SEQ ID NO. 26, or a complement thereof. In one aspect, the hGH polyadenylation signal nucleic acid sequence comprises a sequence at least 97% identical to: SEQ ID NO. 26, or a complement thereof. In one aspect, the hGH polyadenylation signal nucleic acid sequence comprises a sequence at least 98% identical to: SEQ ID NO. 26, or a complement thereof. In one aspect, the hGH polyadenylation signal nucleic acid sequence comprises a sequence at least 99% identical to: SEQ ID NO. 26, or a complement thereof. In one aspect, the hGH polyadenylation signal nucleic acid sequence comprises a sequence at least 99.5% identical to: SEQ ID NO. 26, or a complement thereof. In one aspect, the hGH polyadenylation signal nucleic acid sequence comprises a sequence at least 99.31% identical to: SEQ ID NO. 26, or a complement thereof. In one aspect, the hGH polyadenylation signal nucleic acid sequence comprises a sequence at least 99.9% identical to: SEQ ID NO. 26, or a complement thereof. In one aspect, the hGH polyadenylation signal nucleic acid sequence comprises a sequence 100% identical to: SEQ ID NO. 26, or a complement thereof.

As used herein, the term "central nervous system" or "CNS" refers to the brain and spinal cord of bilaterally symmetric animals. The CNS also includes retina, optic nerve, olfactory nerve and olfactory epithelial cells.

As used herein, the term "peripheral nervous system" or "PNS" refers to nerves and ganglia outside the brain and spinal cord, excluding retina, optic nerve, olfactory nerve and olfactory epithelial cells. In one aspect, the peripheral nervous system is divided into the somatic nervous system and the autonomic nervous system.

As used herein, the term "somatic nervous system" refers to the portion of PNS that is associated with the autonomous control of body movement.

As used herein, the term "autonomic nervous system" refers to the PNS portion that regulates internal organ function

As used herein, the term "GFAP positive" refers to a cell having an accumulation of protein of human Glial Fibrillary Acidic Protein (GFAP) detectable using techniques standard in the art or an accumulation of mRNA expression of GFAP detectable using techniques standard in the art. In one aspect, the glial cells are GFAP positive.

As used herein, the term "detectable" refers to the accumulation of a recognizable protein or mRNA.

Antibodies can be used to identify protein accumulation. Non-limiting examples of measuring protein accumulation include Western blotting, enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, and immunofluorescence. The antibodies provided herein may be polyclonal or monoclonal. Antibodies provided herein having specific binding affinity for a protein can be generated using methods well known in the art. The antibodies provided herein can be attached to a solid support (such as a microtiter plate) using methods known in the art.

As used herein, the terms "multiplicity of infection" and "MOI" refer to the number of virions added per cell during infection.

As used herein, the term "virion" refers to a form of viral infection outside of a host cell.

As used herein, the term "neurological disorder" refers to a disorder, disease, condition, injury, or disease in the central or peripheral nervous system. Non-limiting examples of neurological disorders can be found in Neurological Disorders: course and treatment, version 2 (2002) (Academic Press Inc.) and Christopher Goetz, textbook of Clinical Neurology, version 3 (2007) (Saunders).

As used herein, the term "injury" refers to an injury to the central or peripheral nervous system.

In one aspect, the neurological disorder is selected from the group consisting of: alzheimer's disease, parkinson's disease, amyotrophic Lateral Sclerosis (ALS), huntington's disease, epilepsy, physical injury, stroke, cerebral aneurysms, traumatic brain injury, concussion, tumors, inflammation, infection, ataxia, brain atrophy, spinal cord atrophy, multiple sclerosis, traumatic spinal cord injury, ischemic or hemorrhagic myelopathy (myelopathy), global cerebral ischemia, hypoxic ischemic encephalopathy, embolism, fibrocartilage embolic myelopathy, thrombosis, kidney disease, chronic inflammatory diseases, meningitis, and cerebral venous sinus thrombosis. In one aspect, the neurological disorder is alzheimer's disease. In one aspect, the neurological disorder is parkinson's disease. In one aspect, the neurological disorder is ALS. In one aspect, the neurological disorder is huntington's disease. In one aspect, the neurological disorder is epilepsy. In one aspect, the neurological condition is a physical injury. In one aspect, the neurological condition is stroke. In one aspect, the neurological disorder is ischemic stroke. In one aspect, the neurological disorder is hemorrhagic stroke. In one aspect, the neurological disorder is a cerebral aneurysm. In one aspect, the neurological disorder is traumatic brain injury. In one aspect, the neurological disorder is concussion. In one aspect, the neurological disorder is a tumor. In one aspect, the neurological disorder is inflammation. In one aspect, the neurological disorder is an infection. In one aspect, the neurological disorder is ataxia. In one aspect, the neurological disorder is brain atrophy. In one aspect, the neurological disorder is spinal atrophy. In one aspect, the neurological disorder is multiple sclerosis. In one aspect, the neurological disorder is traumatic spinal cord injury. In one aspect, the neurological disorder is ischemic or hemorrhagic myelopathy (myelopathy). In one aspect, the neurological disorder is global cerebral ischemia. In one aspect, the neurological disorder is hypoxic ischemic encephalopathy. In one aspect, the neurological disorder is embolism. In one aspect, the neurological disorder is fibrocartilage embolic spinal cord disease. In one aspect, the neurological disorder is thrombosis. In one aspect, the neurological disorder is kidney disease. In one aspect, the neurological disorder is a chronic inflammatory disease. In one aspect, the neurological disorder is meningitis. In one aspect, the neurological disorder is cerebral sinus thrombosis.

In one aspect, the neurological disorder includes damage to the CNS or PNS. In one aspect, the neurological condition includes damage to the CNS. In one aspect, the neurological condition includes damage to PNS.

In one aspect, the present disclosure provides and includes a method of converting reactive astrocytes into functional neurons in a living human brain, comprising: injecting an adeno-associated virus (AAV) into a subject in need thereof, wherein the AAV comprises a DNA vector construct comprising a human neurogenic differentiation factor 1 (hNeuroD 1) sequence, the hNeuroD1 sequence comprising a nucleic acid sequence of SEQ ID NO:6, and a second sequence comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 11, 13, and 15, wherein the hNeuroD1 sequence and the second sequence are separated by: a P2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID nos. 19 and 22, or a T2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID nos. 20 and 23, or an Internal Ribosome Entry Site (IRES) sequence of an encephalomyocarditis virus comprising SEQ ID No. 3, wherein the hNeuroD1 sequence and the second sequence are operably linked to a regulatory element comprising: (a) A human Glial Fibrillary Acidic Protein (GFAP) promoter comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs 4, 18 and 27; (b) An enhancer from the human elongation factor-1 alpha (EF-1 alpha) promoter comprising the nucleic acid sequence of SEQ ID NO. 2 or a Cytomegalovirus (CMV) enhancer comprising the nucleic acid sequence of SEQ ID NO. 17; (c) A chimeric intron comprising the nucleic acid sequence of SEQ ID NO. 5 or 28; (d) A woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs 7 and 30; and (e) an SV40 polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO. 8, an hGH polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO. 21, a synthetic polyadenylation signal comprising SEQ ID NO. 31 or a bGH polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO. 26.

In one aspect, the present disclosure provides and includes a method of converting reactive astrocytes into functional neurons in the brain of a living human brain, comprising: injecting an adeno-associated virus (AAV) into a subject in need thereof, wherein the AAV comprises a DNA vector construct comprising a nucleic acid coding sequence encoding a human neurogenic differentiation factor 1 (hNeuroD 1) protein comprising the amino acid sequence of SEQ ID NO:10, and a second nucleic acid coding sequence encoding a second protein having an amino acid selected from the group consisting of SEQ ID NOs: 12, 14 and 16, wherein the hNeuroD1 coding sequence and the second coding sequence are selected from the group consisting of SEQ ID NOs: 19 and 22, or a T2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 20 and 23, or an Internal Ribosome Entry Site (IRES) sequence of an encephalomyocarditis virus comprising SEQ ID NO:3, wherein the hNeuroD1 coding sequence and the second nucleic acid coding sequence are operably linked to an expression control element comprising: (a) A human Glial Fibrillary Acidic Protein (GFAP) promoter comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs 4, 18 and 27; (b) An enhancer from the human elongation factor-1 alpha (EF-1 alpha) promoter comprising the nucleic acid sequence of SEQ ID NO. 2 or a Cytomegalovirus (CMV) enhancer comprising the nucleic acid sequence of SEQ ID NO. 17; (c) A chimeric intron comprising the nucleic acid sequence of SEQ ID NO. 5 or 28; (d) A woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs 7 and 30; and (e) an SV40 polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO. 8, an hGH polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO. 21, a synthetic polyadenylation signal comprising SEQ ID NO. 31 or a bGH polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO. 26.

In one aspect, the methods provided herein are capable of converting at least one glial cell into a neuron. In one aspect, the methods provided herein convert at least one glial cell to a neuron.

In one aspect, the compositions provided herein are capable of converting at least one glial cell into a neuron. In one aspect, the compositions provided herein convert at least one glial cell to a neuron.

In one aspect, the compositions provided herein comprise an AAV vector for treating a subject in need thereof, wherein the AAV vector comprises a first NeuroD1 sequence and a second NeuroD1 sequence, wherein the first NeuroD1 sequence and the second NeuroD1 sequence are separated by a linker sequence, wherein the first NeuroD1 sequence and the second NeuroD1 sequence are operably linked to an expression control element comprising a first promoter, an enhancer, a chimeric intron, a WPRE, and a polyadenylation signal.

In one aspect, the compositions provided herein comprise an AAV vector for treating a subject in need thereof, wherein the AAV vector comprises a first NeuroD1 sequence and a second NeuroD1 sequence, wherein the first NeuroD1 sequence is operably linked to a first promoter, and the second NeuroD1 sequence is operably linked to a second promoter, wherein the first and second NeuroD1 sequences are further operably linked to an enhancer, a chimeric intron, a WPRE, and a polyadenylation signal.

As used herein, the term "mammal" refers to any species classified as a class of mammals.

As used herein, the term "human" refers to Homo sapiens. In one aspect, the human suffers from a neurological disorder.

As used herein, the term "living human" refers to a person having cardiac, respiratory and brain activity.

As used herein, the term "non-human primate" refers to any species or subspecies classified as primates but not homo sapiens. Non-limiting examples of non-human primates include chimpanzees, bonobo, red chimpanzees, gorillas, macaques, marmosets, pigtails, baboons, gibbons, and lemkeys.

As used herein, the term "delivery" or "delivery" refers to the treatment of a mammal with an AAV vector or composition provided herein. In one aspect, an AAV vector or composition provided herein is delivered to a subject in need thereof. In one aspect, an AAV vector or composition provided herein is formulated for delivery to a subject in need thereof. In one aspect, delivering includes local delivery. In one aspect, an AAV vector or composition provided herein is formulated for topical delivery. In one aspect, the delivery comprises systemic delivery. In one aspect, an AAV vector or composition provided herein is formulated for systemic delivery. In one aspect, delivery comprises injecting an AAV vector or composition provided herein into a subject in need thereof. In one aspect, the delivery is selected from the group consisting of: intraperitoneal, intramuscular, intravenous, intrathecal, intracerebral, intracranial, lateral cerebral compartments of the brain, intracavitary, intravitreal, subretinal, intraparenchymal, intranasal, or oral administration. In one aspect, the delivery comprises intraperitoneal delivery. In one aspect, the delivery comprises intramuscular delivery. In one aspect, the delivery comprises intravenous delivery. In one aspect, the delivery comprises intrathecal delivery. In one aspect, the delivery comprises intra-brain delivery. In one aspect, the delivery comprises intracranial delivery. In one aspect, the delivery comprises lateral intraventricular delivery of the brain. In one aspect, the delivery comprises intracavitary delivery. In one aspect, the delivery comprises intravitreal delivery. In one aspect, the delivery comprises subretinal intradelivery. In one aspect, the delivery comprises intraparenchymal delivery. In one aspect, the delivery comprises intranasal delivery. In one aspect, the delivery comprises oral administration.

As used herein, the term "injection" refers to delivery of an AAV vector or composition provided herein under pressure and effort. By way of non-limiting example, injection may include the use of a syringe and needle.

In one aspect, an AAV vector or composition provided herein is injected into the brain of a subject. In one aspect, the AAV vector or composition is injected into the cerebral cortex of a subject. In one aspect, an AAV vector or composition provided herein is injected into the striatum of a subject. In one aspect, an AAV vector or composition provided herein is injected into the spinal cord or subject. In one aspect, the AAV vector or composition is injected into the dorsal striatum of a subject. In one aspect, the AAV vector or composition is injected into the putamen of a subject. In one aspect, the AAV vector or composition is injected into the caudate nucleus of a subject. In one aspect, the AAV vector or composition is injected into the substantia nigra of a subject.

In one aspect, an AAV vector or composition provided herein has spread between about 1% and about 100% in the brain. In one aspect, an AAV vector or composition provided herein has spread in the brain between about 1% and about 10%, between 1% and about 20%, between 1% and about 30%, between 10% and about 20%, between 10% and about 30%, between about 10% and about 40%, between about 20% and about 30%, between about 20% and about 40%, between about 20% and about 50%, between about 30% and about 40%, between about 30% and about 50%, between about 30% and about 60%, between about 40% and about 50%, between about 40% and about 60%, between about 40% and about 70%, between about 50% and about 60%, between about 50% and about 70%, between about 60% and about 80%, between about 60% and about 90%, between about 70% and about 80%, between about 70% and about 90%, between about 100% and about 100%, or between about 100%.

In one aspect, an AAV vector or composition provided herein has spread between about 1% and about 100% in the cerebral cortex. In one aspect of the present invention, AAV vectors or compositions provided herein have spread between about 1% and about 10%, between 1% and about 20%, between 1% and about 30%, between 10% and about 20%, between 10% and about 30%, between about 10% and about 40%, between about 20% and about 30%, between about 20% and about 40%, between about 20% and about 50%, between about 30% and about 40%, between about 30% and about 50%, between about 30% and about 60%, between about 40% and about 50%, between about 40% and about 60%, between about 40% and about 70%, between about 50% and about 60%, between about 50% and about 70%, between about 50% and about 80%, between about 60% and about 90%, between about 70% and about 80%, between about 70% and about 70%, between about 90% and about 100%, between about 100% and about 100% or between about 100%.

In one aspect, an AAV vector or composition provided herein has spread between about 1% and about 100% in the spinal cord. In one aspect, an AAV vector or composition provided herein has spread between about 1% and about 10%, between 1% and about 20%, between 1% and about 30%, between 10% and about 20%, between 10% and about 30%, between about 10% and about 40%, between about 20% and about 30%, between about 20% and about 40%, between about 20% and about 50%, between about 30% and about 40%, between about 30% and about 50%, between about 30% and about 60%, between about 40% and about 50%, between about 40% and about 60%, between about 40% and about 70%, between about 50% and about 60%, between about 50% and about 70%, between about 60% and about 80%, between about 60% and about 90%, between about 70% and about 80%, between about 70% and about 90%, between about 100% and about 100%, or between about 100%.

In one aspect, an AAV vector or composition provided herein has spread between about 1% and about 100% in the striatum. In one aspect of the present invention, AAV vectors or compositions provided herein have diffused in the striatum between about 1% and about 10%, between 1% and about 20%, between 1% and about 30%, between 10% and about 20%, between 10% and about 30%, between about 10% and about 40%, between about 20% and about 30%, between about 20% and about 40%, between about 20% and about 50%, between about 30% and about 40%, between about 30% and about 50%, between about 30% and about 60%, between about 40% and about 50%, between about 40% and about 60%, between about 40% and about 70%, between about 50% and about 60%, between about 50% and about 70%, between about 60% and about 80%, between about 60% and about 90%, between about 70% and about 80%, between about 70%, about 40% and about 90% and about 100%, or between about 100% and about 100%.

In one aspect, an AAV vector or composition provided herein has spread between about 1% and about 100% in the dorsal striatum. In one aspect of the present invention, AAV vectors or compositions provided herein have diffused between about 1% and about 10%, between 1% and about 20%, between 1% and about 30%, between 10% and about 20%, between 10% and about 30%, between about 10% and about 40%, between about 20% and about 30%, between about 20% and about 40%, between about 20% and about 50%, between about 30% and about 40%, between about 30% and about 50%, between about 30% and about 60%, between about 40% and about 50%, between about 40% and about 60%, between about 40% and about 70%, between about 50% and about 60%, between about 50% and about 70%, between about 60% and about 80%, between about 60% and about 90%, between about 70% and about 80%, between about 70% and about 90%, between about 100% and about 100%, or between about 100% and about 100%.

In one aspect, an AAV vector or composition provided herein has been spread between about 1% and about 100% in the putamen. In one aspect, an AAV vector or composition provided herein has diffused between about 1% and about 10%, between 1% and about 20%, between 1% and about 30%, between 10% and about 20%, between 10% and about 30%, between about 10% and about 40%, between about 20% and about 30%, between about 20% and about 40%, between about 20% and about 50%, between about 30% and about 40%, between about 30% and about 50%, between about 30% and about 60%, between about 40% and about 50%, between about 40% and about 60%, between about 40% and about 70%, between about 50% and about 60%, between about 50% and about 70%, between about 60% and about 80%, between about 60% and about 90%, between about 70% and about 70%, between about 90% and about 100%, between about 100% and about 100%.

In one aspect, an AAV vector or composition provided herein has been spread between about 1% and about 100% in the caudate nucleus. In one aspect of the present invention, AAV vectors or compositions provided herein have diffused between about 1% and about 10%, between 1% and about 20%, between 1% and about 30%, between 10% and about 20%, between 10% and about 30%, between about 10% and about 40%, between about 20% and about 30%, between about 20% and about 40%, between about 20% and about 50%, between about 30% and about 40%, between about 30% and about 50%, between about 30% and about 60%, between about 40% and about 50%, between about 40% and about 60%, between about 40% and about 70%, between about 50% and about 60%, between about 50% and about 70%, between about 50% and about 80%, between about 60% and about 90%, between about 70% and about 80%, between about 70% and about 70%, between about 90% and about 100%, between about 100% and about 100% or between about 100%.

In one aspect, an AAV vector or composition provided herein diffuses between about 1% and about 100% from the injection site. In one aspect, an AAV vector or composition provided herein has diffused from an injection site between about 1% and about 10%, between 1% and about 20%, between 1% and about 30%, between 10% and about 20%, between 10% and about 30%, between about 10% and about 40%, between about 20% and about 30%, between about 20% and about 40%, between about 20% and about 50%, between about 30% and about 40%, between about 30% and about 50%, between about 30% and about 60%, between about 40% and about 50%, between about 40% and about 60%, between about 40% and about 70%, between about 50% and about 60%, between about 50% and about 70%, between about 60% and about 80%, between about 60% and about 90%, between about 70% and about 80%, between about 70% and about 90%, between about 100% and about 100%, or between about 100%.

In one aspect, an AAV vector or composition provided herein has between about 1% and about 100% spread in the substantia nigra. In one aspect, an AAV vector or composition provided herein has diffused between about 1% and about 10%, between 1% and about 20%, between 1% and about 30%, between 10% and about 20%, between 10% and about 30%, between about 10% and about 40%, between about 20% and about 30%, between about 20% and about 40%, between about 20% and about 50%, between about 30% and about 40%, between about 30% and about 50%, between about 30% and about 60%, between about 40% and about 50%, between about 40% and about 60%, between about 40% and about 70%, between about 50% and about 60%, between about 50% and about 70%, between about 60% and about 80%, between about 60% and about 90%, between about 70% and about 70%, between about 90% and about 100%, between about 100% and about 100%.

As used herein, the term "AAV particle" refers to the packaged capsid form of an AAV virus that delivers its nucleic acid genome to a cell.

In one aspect, a composition comprising AAV particles encoded by an AAV vector provided herein is provided to be between 10 ¹⁰ AAV particles/mL and 10 ¹⁴ Concentration between AAV particles/mL. In one aspect, a composition comprising AAV particles encoded by an AAV vector provided herein is provided to be between 10 ¹⁰ AAV particles/mL and 10 ¹¹ Between 10 AAV particles/mL ¹⁰ AAV particles/mL and 10 ¹² Between 10 AAV particles/mL ¹⁰ AAV particles/mL and 10 ¹³ Between 10 AAV particles/mL ¹¹ AAV particles/mL and 10 ¹² AAV particles/mL is between 10 ¹¹ AAV particles/mL and 10 ¹³ Between 10 AAV particles/mL ¹¹ AAV particles/mL and 10 ¹⁴ Between 10 AAV particles/mL ¹² AAV particles/mL and 10 ¹³ Between 10 AAV particles/mL ¹² AAV particles/mL and 10 ¹⁴ Between or between 10 AAV particles/mL ¹³ AAV particles/mL and 10 ¹⁴ Concentration between AAV particles/mL.

In one aspect, a composition comprising AAV particles encoded by an AAV vector provided herein is injected in a volume of between 10 μl and 1000 μl. In one aspect of the present invention, a composition comprising AAV particles encoded by an AAV vector provided herein is injected at a volume between 10 μl and 100 μl, between 10 μl and 200 μl, between 10 μl and 300 μl, between 100 μl and 200 μl, between 100 μl and 300 μl, between 100 μl and 400 μl, between 200 μl and 300 μl, between 200 μl and 400 μl, between 200 μl and 500 μl, between 300 μl and 400 μl, between 300 μl and 500 μl, between 300 μl and 600 μl, between 400 μl and 500 μl, between 400 μl and 600 μl, between 400 μl and 700 μl, between 500 μl and 600 μl, between 500 μl and 700 μl, between 500 μl and 900 μl, between 500 μl and 800 μl, between 600 μl and 900 μl, between 700 μl and 700 μl, between 700 μl and 900 μl, between 1000 μl and 900 μl, between 700 μl and 900 μl, between 1000 μl, between 700 μl and 900 μl, between 400 μl and 900 μl).

As used herein, the term "subject" refers to any animal subject. Non-limiting examples of animal subjects include humans, laboratory animals (e.g., primates, rats, mice), domestic animals (e.g., cows, sheep, goats, pigs, turkeys, chickens), and domestic pets (e.g., dogs, cats, rodents, etc.).

As used herein, "a subject in need thereof" refers to a subject suffering from a neurological disorder. In one aspect, a subject in need thereof has a neurological disorder selected from the group consisting of: alzheimer's disease, parkinson's disease, amyotrophic Lateral Sclerosis (ALS), huntington's disease, epilepsy, physical injury, stroke, cerebral aneurysms, traumatic brain injury, concussion, tumors, inflammation, infection, ataxia, brain atrophy, spinal cord atrophy, multiple sclerosis, traumatic spinal cord injury, ischemic or hemorrhagic myelopathy (myelopathy), global cerebral ischemia, hypoxic ischemic encephalopathy, embolism, fibrocartilage embolic myelopathy, thrombosis, kidney disease, chronic inflammatory diseases, meningitis, and cerebral venous sinus thrombosis. In one aspect, a subject in need thereof suffers from alzheimer's disease. In one aspect, a subject in need thereof suffers from parkinson's disease. In one aspect, a subject in need thereof has ALS. In one aspect, a subject in need thereof suffers from huntington's disease. In one aspect, a subject in need thereof suffers from epilepsy. In one aspect, a subject in need thereof suffers from physical injury. In one aspect, a subject in need thereof suffers from stroke. In one aspect, a subject in need thereof has ischemic stroke. In one aspect, a subject in need thereof suffers from hemorrhagic stroke. In one aspect, a subject in need thereof has a cerebral aneurysm. In one aspect, a subject in need thereof suffers from traumatic brain injury. In one aspect, a subject in need thereof suffers from concussion. In one aspect, a subject in need thereof has a tumor. In one aspect, a subject in need thereof suffers from inflammation. In one aspect, a subject in need thereof suffers from an infection. In one aspect, a subject in need thereof suffers from ataxia. In one aspect, a subject in need thereof suffers from brain atrophy. In one aspect, a subject in need thereof suffers from spinal atrophy. In one aspect, a subject in need thereof suffers from multiple sclerosis. In one aspect, a subject in need thereof suffers from a traumatic spinal cord injury. In one aspect, a subject in need thereof suffers from ischemic or hemorrhagic myelopathy (myelopathy). In one aspect, a subject in need thereof suffers from global cerebral ischemia. In one aspect, a subject in need thereof suffers from hypoxic ischemic encephalopathy. In one aspect, a subject in need thereof suffers from embolism. In one aspect, a subject in need thereof suffers from fibrocartilage embolic myelopathy. In one aspect, a subject in need thereof suffers from thrombosis. In one aspect, a subject in need thereof suffers from kidney disease. In one aspect, a subject in need thereof suffers from a chronic inflammatory disease. In one aspect, a subject in need thereof has meningitis. In one aspect, a subject in need thereof is suffering from cerebral venous sinus thrombosis.

In one aspect, the subject in need thereof is a mammal. In one aspect, the subject in need thereof is a human. In one aspect, a subject in need thereof is a non-human primate. In one aspect, a subject in need thereof is selected from the group consisting of chimpanzees, bonobes, chimpanzees, gorillas, macaques, marmosets, pigtails, baboons, gibbons, and lemkeys. In one aspect, the subject in need thereof is a chimpanzee. In one aspect, the subject in need thereof is a bonobo. In one aspect, the subject in need thereof is an gorilla. In one aspect, the subject in need thereof is a gorilla. In one aspect, the subject in need thereof is cynomolgus monkey. In one aspect, the subject in need thereof is a marmoset. In one aspect, the subject in need thereof is a cynomolgus monkey. In one aspect, the subject in need thereof is a baboon. In one aspect, the subject in need thereof is a gibbon. In one aspect, the subject in need thereof is a cynomolgus monkey.

In one aspect, the subject in need thereof is a male. In one aspect, the subject in need thereof is a female. In one aspect, the subject in need thereof is neutral. In one aspect, the subject in need thereof is a premature infant. In one aspect, the premature infant is born before 36 weeks of gestation. In one aspect, the subject in need thereof is a term infant. In one aspect, the term infant is less than about 2 months of age. In one aspect, the subject in need thereof is a neonate. In one aspect, the neonate is less than about 1 month old. In one aspect, the subject in need thereof is an infant. In one aspect, the infant is between 2 months of age and 24 months of age. In one aspect of the present invention, infants between 2 and 3 months of age, between 2 and 4 months of age, between 2 and 5 months of age, between 3 and 4 months of age, between 3 and 5 months of age, between 3 and 6 months of age, between 4 and 5 months of age, between 4 and 6 months of age, between 4 and 7 months of age, between 5 and 6 months of age, between 5 and 7 months of age between 5 and 8 months of age, between 6 and 7 months of age, between 6 and 8 months of age, between 6 and 9 months of age, between 7 and 8 months of age, between 7 and 9 months of age, between 7 and 10 months of age, between 8 and 9 months of age, between 8 and 10 months of age, between 8 and 11 months of age, between 9 and 10 months of age between 9 and 11 months of age, between 9 and 12 months of age, between 10 and 11 months of age, between 10 and 12 months of age, between 10 and 13 months of age, between 11 and 12 months of age, between 11 and 13 months of age, between 11 and 14 months of age, between 12 and 13 months of age, between 12 and 14 months of age, between 12 and 15 months of age, between 13 and 14 months of age, between 13 and 15 months of age, between 13 and 16 months of age, between 14 and 15 months of age, between 14 and 16 months of age, between 14 and 17 months of age, between 15 and 16 months of age, between 15 and 17 years of age, between 15 and 18 years of age, between 16 and 17 months of age, between 16 and 17 years of age, between, between 16 and 18 months of age, between 16 and 19 months of age, between 17 and 18 months of age, between 17 and 19 months of age, between 17 and 20 months of age, between 18 and 19 months of age, between 18 and 20 months of age, between 18 and 21 years of age, between 19 and 20 months of age, between 19 and 21 months of age, between 19 and 22 months of age, between 20 and 21 months of age, between 20 and 22 months of age, between 20 and 23 months of age, between 21 and 22 months of age, between 21 and 23 months of age, between 21 and 24 months of age, between 22 and 23 months of age, and between 23 and 24 years of age. In one aspect, the subject in need thereof is a pediatric subject. In one aspect, the young child is between 1 year and 4 years old. In one aspect, the young child is between 1 and 2 years old, between 1 and 3 years old, between 1 and 4 years old, between 2 and 3 years old, between 2 and 4 years old, and between 3 and 4 years old. In one aspect, the subject in need thereof is a pediatric subject. In one aspect, the child is between 2 and 5 years of age. In one aspect, the child is between 2 and 3 years old, between 2 and 4 years old, between 2 and 5 years old, between 3 and 4 years old, between 3 and 5 years old, and between 4 and 5 years old. In one aspect, the subject in need thereof is a child. In one aspect, the child is between 6 and 12 years old. In one aspect, the child is between 6 and 7 years old, between 6 and 8 years old, between 6 and 9 years old, between 7 and 8 years old, between 7 and 9 years old, between 7 and 10 years old, between 8 and 9 years old, between 8 and 10 years old, between 8 and 11 years old, between 9 and 10 years old, between 9 and 11 years old, between 9 and 12 years old, between 10 and 11 years old, between 10 and 12 years old, and between 11 and 12 years old. In one aspect, the subject in need thereof is an adolescent. In one aspect, the teenager is between 13 and 19 years of age. In one aspect, teenagers are between 13 and 14 years old, between 13 and 15 years old, between 13 and 16 years old, between 14 and 15 years old, between 14 and 16 years old, between 14 and 17 years old, between 15 and 16 years old, between 15 and 17 years old, between 15 and 18 years old, between 16 and 17 years old, between 16 and 18 years old, between 16 and 19 years old, between 17 and 18 years old, between 17 and 19 years old, and between 18 and 19 years old. In one aspect, the subject in need thereof is a pediatric subject. In one aspect, the pediatric subject is between 1 day of age and 18 years of age. In one aspect of the present invention, pediatric subjects are between 1 day old and 1 year old, between 1 day old and 2 years old, between 1 day old and 3 years old, between 1 year old and 2 years old, between 1 year old and 3 years old, between 1 year old and 4 years old, between 2 years old and 3 years old, between 2 years old and 4 years old, between 2 years old and 5 years old, between 3 years old and 4 years old, between 3 years old and 5 years old, between 3 years old and 6 years old between 4 and 5 years old, between 4 and 6 years old, between 4 and 7 years old, between 5 and 6 years old, between 5 and 7 years old, between 5 and 8 years old, between 6 and 7 years old, between 6 and 8 years old, between 6 and 9 years old, between 7 and 8 years old, between 7 and 9 years old, between 7 and 10 years old, between 8 and 9 years old between 4 and 5 years, between 4 and 6 years, between 4 and 7 years, between 5 and 6 years, between 5 and 7 years, between 5 and 8 years, between 6 and 7 years between 6 and 8 years, between 6 and 9 years, between 7 and 8 years, between 7 and 9 years, between 7 and 10 years, between 8 and 9 years, between 16 and 18 years and between 17 and 18 years. In one aspect, the subject in need thereof is an elderly subject. In one aspect, the elderly subject is between 65 and 95 years old or higher. In one aspect, an elderly subject is between 65 and 70 years old, between 65 and 75 years old, between 65 and 80 years old, between 70 and 75 years old, between 70 and 80 years old, between 70 and 85 years old, between 75 and 80 years old, between 75 and 85 years old, between 75 and 90 years old, between 80 and 85 years old, between 80 and 90 years old, between 80 and 95 years old, between 85 and 90 years old, and between 85 and 95 years old. In one aspect, the subject in need thereof is an adult subject. In one aspect, the adult subject is between 20 years and 95 years or more. In one aspect of the present invention, an adult subject is between 20 and 25 years old, between 20 and 30 years old, between 20 and 35 years old, between 25 and 30 years old, between 25 and 35 years old, between 25 and 40 years old, between 30 and 35 years old, between 30 and 40 years old, between 30 and 45 years old, between 35 and 40 years old, between 35 and 45 years old, between 35 and 50 years old, between 40 and 45 years old, between 40 and 50 years old, between 40 and 55 years old, between 45 and 50 years old, between 45 and 55 years old, between 45 and 60 years old, between 50 and 55 years old, between 50 and 60 years old between 50 and 65, between 55 and 60, between 55 and 65, between 55 and 70, between 60 and 65, between 60 and 70, between 60 and 75, between 65 and 70, between 65 and 75, between 65 and 80, between 70 and 75, between 70 and 80, between 70 and 85, between 75 and 80, between 75 and 85, between 75 and 90, between 80 and 95, between 85 and 90. In one aspect, a subject in need thereof is between 1 and 5 years old, between 2 and 10 years old, between 3 and 18 years old, between 21 and 50 years old, between 21 and 40 years old, between 21 and 30 years old, between 50 and 90 years old, between 60 and 90 years old, between 70 and 90 years old, between 60 and 80 years old, or between 65 and 75 years old. In one aspect, the subject in need thereof is a young aged subject (65 to 74 years old). In one aspect, the subject in need thereof is an elderly subject (75 to 84 years old). In one aspect, the subject in need thereof is an elderly subject (> 85 years).

As used herein, the term "flow rate" refers to the rate of delivery of an AAV vector or composition. In one aspect, the flow rate is between 0.1 μl/min and 5.0 μl/min. In one aspect, the flow rate is between 0.1 and 0.2 μL/min, between 0.1 and 0.3 μL/min, between 0.1 and 0.4 μL/min, between 0.2 and 0.3 μL/min, between 0.2 and 0.4 μL/min, between 0.2 and 0.5 μL/min, between 0.3 and 0.4 μL/min, between 0.3 and 0.5 μL/min, between 0.3 and 0.6 μL/min, between 0.3 and 0.5 μL/min, between 0.4 and 0.6 μL/min, between 0.4 and 0.7 μL/min, between 0.5 and 6 μL/min, between 0.5 and 0.6 μL/min, between 0.7 μL/min, between 0.3 and 0.6 μL/min, between 0.7 and 0.8 and 7 μL/min, between 0.7 and 7 μL/min, between 0.3 and 0.6 μL/min, between 0.7 and 0.8.7 μL/min, between 0.3 and 0.6 μL/min, between 0.7 and 0.5 μL/min, between 0.8 and 1.1. Mu.L/min, between 0.9 and 1.0. Mu.L/min, between 0.9 and 1.1. Mu.L/min, between 0.9 and 1.2. Mu.L/min, between 1.0 and 1.1. Mu.L/min, between 1.0 and 1.2. Mu.L/min, between 1.0 and 1.3. Mu.L/min, between 1.1 and 1.2. Mu.L/min, between 1.3 and 1.3. Mu.L/min, between 1.1 and 1.1. Mu.L/min, between 1.2 and 1.2. Mu.L/min, between 1.2 and 1.4. Mu.L/min, between 1.2 and 1.5. Mu.5 and 1.5 and 6.1.1 and 6.1.1.1 and 6.1.1.1.4. Mu.L/min, between 1.3 and 1.1.1 and 1.1.4. Mu.L/min, between 1.1 and 1.1.1 and 1.1.1.1 and 6. Mu.L/min, between 1.1.1 and 6.1.1.1 and 6. Mu.L/1.L/min Between 1.6 and 1.9. Mu.L/min, between 1.7 and 1.8. Mu.L/min, between 1.7 and 1.9. Mu.L/min, between 1.7 and 2.0. Mu.L/min, between 1.8 and 1.9. Mu.L/min, between 1.8 and 2.0. Mu.L/min, between 1.8 and 2.1. Mu.L/min, between 1.9 and 2.0. Mu.L/min, between 1.9 and 2.9. Mu.L/min, between 1.9 and 2.2. Mu.L/min, between 2.0 and 2.0. Mu.L/min, between 2.0 and 2.2. Mu.L/min, between 2.0 and 2.3. Mu.3 and 2.3, between 2.4 and 2.4. Mu.4. Mu.L/min, between 1 and 2.4. Mu.3, between 1 and 2.4. Mu.L/min, between 1.9 and 2.2.2.2 and 2.2. Mu.L/min, between 1 and 2.4. Mu.L/min Between 2.4 and 2.7 μL/min, between 2.5 and 2.6 μL/min, between 2.5 and 2.7 μL/min, between 2.5 and 2.8 μL/min, between 2.6 and 2.7 μL/min, between 2.6 and 2.8 μL/min, between 2.6 and 2.9 μL/min, between 2.7 and 2.8 μL/min, between 2.7 and 2.9 μL/min, between 2.7 and 3.0 μL/min, between 2.8 and 2.9 μL/min, between 2.8 and 3.0 μL/min between 2.8 and 3.1 μL/min, between 2.9 and 3.0 μL/min, between 2.9 and 3.1 μL/min, between 2.9 and 3.2 μL/min, between 3.0 and 3.1 μL/min, between 3.0 and 3.2 μL/min, between 3.0 and 3.3 μL/min, between 3.1 and 3.2 μL/min, between 3.1 and 3.3 μL/min, between 3.1 and 3.4 μL/min, between 3.2 and 3.3 μL/min, between 3.2 and 3.2 μL/min, between 3.4 μL/min, between 3.2 and 3.4 μL/min, between 3.2 and 3.5 μL/min, between 3.3 and 3.4 μL/min, between 3.3 and 3.5 μL/min, between 3.3 and 3.6 μL/min, between 3.4 and 3.5 μL/min, between 3.4 and 3.6 μL/min, between 3.4 and 3.7 μL/min, between 3.5 and 3.6 μL/min, between 3.5 and 3.7 μL/min, between 3.5 and 3.8 μL/min, between 3.6 and 3.7 μL/min, between 3.6 and 3.8 μL/min between 3.6 and 3.9 μL/min, between 3.7 and 3.8 μL/min, between 3.7 and 3.9 μL/min, between 3.7 and 4.0 μL/min, between 3.8 and 3.9 μL/min, between 3.8 and 4.0 μL/min, between 3.8 and 4.1 μL/min, between 3.9 and 4.0 μL/min, between 3.9 and 4.1 μL/min, between 3.9 and 4.2 μL/min, between 4.0 and 4.1 μL/min, between 4.0 and 4.0 μL/min, between 4.2 μL/min, between 3.9 and 4.2 μL/min, between 4.0 and 4.3 μL/min, between 4.1 and 4.2 μL/min, between 4.1 and 4.3 μL/min, between 4.1 and 4.4 μL/min, between 4.2 and 4.3 μL/min, between 4.2 and 4.4 μL/min, between 4.2 and 4.2 μL/min, between 4.2 and 4.5 μL/min, between 4.3 and 4.4 μL/min, between 4.3 and 4.5 μL/min, between 4.3 and 4.6 μL/min, between 4.4 and 4.5 μL/min, between 4.4 and 4.6 μL/min, between 4.4 and 4.7 μL/min, between 4.7 and 4.7 μL/min, between 4.5 and 4.6 μL/min, between 4.7 and 4.6 μL/min, between 4.3 and 4.4 and 4.6 μL/min, between 4.3 and 4.5 μL/min, between 4.3 and 4.6 μL/min, between 4.6 μL/min and 4.6 μL/min, between 4.7 and 4.7 μL/min, between 4.7 and 4.6 μL/min Between 4.8 and 5.0 or between 4.9 and 5.0. Mu.L/min.

As used herein, the term "therapeutically effective dose" or "pharmaceutically active dose" refers to an amount of an AAV particle or composition provided herein that is effective to treat a neurological disorder. In one aspect, the AAV particles or compositions provided herein may be provided with a pharmaceutically acceptable carrier. As used herein, a "pharmaceutically acceptable carrier" refers to a non-toxic solvent, dispersant, excipient, adjuvant, or other material that is mixed with an AAV particle or composition provided herein.

Non-limiting examples of pharmaceutically acceptable carriers include diluents, adjuvants, excipients or acid resistant encapsulating ingredients in liquid (e.g., saline), gel, nanoparticle, exosome, lipid vesicles or solid form. Non-limiting examples of suitable diluents and excipients include pharmaceutical grade physiological saline, dextrose, glycerol, mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like, and combinations thereof. In one aspect, the therapeutically effective dose contains auxiliary substances such as wetting or emulsifying agents, stabilizing agents, or pH buffering agents. In one aspect, a therapeutically effective dose of an AAV particle or composition provided herein is injected into a subject. In one aspect, a therapeutically effective dose of an AAV particle or composition provided herein is delivered to a subject. In one aspect, a therapeutically effective dose is administered with at least one pharmaceutically acceptable carrier. In one aspect, a therapeutically effective dose contains between about 1% and about 5%, between about 5% and about 10%, between about 10% and about 15%, between about 15% and about 20%, between about 20% and about 25%, between about 25% and about 30%, between about 30% and about 35%, between about 40 and about 45%, between about 50% and about 55%, between about 1% and about 95%, between about 2% and about 95%, between about 5% and about 95%, between about 10% and about 95%, between about 15% and about 95%, between about 20% and about 95%, between about 25% and about 95%, between about 30% and about 95%, between about 35% and about 95%, between about 40% and about 95%, between about 45% and about 95%, between about 50% and about 95%, between about 55% and about 95%, between about 60% and about 95%, between about 45% and about 95%, between about 95% and about 95%, between about 80% and about 95%, or between about 95% and about 80% of the AAV.

In one aspect, a therapeutically effective dose is delivered to a subject in need thereof at least once daily or at least once weekly for at least two consecutive days or weeks. In one aspect, a therapeutically effective dose is delivered to a subject in need thereof at least once daily or at least once weekly for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive days or weeks. In one aspect, a therapeutically effective dose is delivered to a subject in need thereof at least once daily or at least once weekly for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 consecutive weeks. In one aspect, a therapeutically effective dose is delivered to a subject in need thereof at least once daily or at least once weekly for up to 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive days or weeks. In one aspect, a therapeutically effective dose is delivered to a subject in need thereof at least once daily or at least once weekly for up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 consecutive weeks or months. In one aspect, a therapeutically effective dose is delivered to a subject in need thereof for at least one administration for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 consecutive months or years, for a prolonged period of time throughout the subject's life cycle, or for an indefinite period of time. In one aspect, a therapeutically effective dose is delivered once a year to a subject in need thereof for 2 years, 3 years, or 5 years. In one aspect, a therapeutically effective dose is delivered once a year to a subject in need thereof for 2 consecutive years. In one aspect, a therapeutically effective dose is delivered once a year to a subject in need thereof for 3 consecutive years. In one aspect, a therapeutically effective dose is delivered once a year to a subject in need thereof for 5 consecutive years.

As used herein, the terms "alleviating", "cure" or "resolution" refer to the percentage of a subject in need thereof that is cured of or achieves relief or complete resolution of a neurological disorder in response to a therapeutically effective dose.

As used herein, the term "response rate" refers to the percentage of a therapeutically effective dose that a subject in need thereof responds positively (e.g., the severity or frequency of one or more symptoms decreases).

In one aspect, a therapeutically effective dose achieves at least about 50% relief, cure, response rate, or resolution of the neurological disorder. In one aspect, a therapeutically effective dose eliminates, reduces, slows or delays symptoms of one or more neurological disorders. Non-limiting examples of neurological disorder symptoms include tremors, bradykinesia (bradykinesia), muscle stiffness, impaired posture and balance, automatic loss of motion, uncoordinated motion, uncontrolled motion, spontaneous jerky motion, speech changes, numbness, and writing changes. In one aspect, the symptom of the neurological disorder is a motor symptom. Non-limiting examples of movement symptoms include involuntary movement disorders or autonomic movement disorders. In one aspect, the symptom of the neurological disorder is a cognitive symptom. Non-limiting examples of cognitive symptoms include fine motor skills, tremors, epilepsy, chorea, dystonia, dyskinesia, slow or abnormal eye movements, impaired gait, impaired posture, impaired balance, speech difficulties, dysphagia, tissue difficulties, difficulty in prioritizing (difficulty prioritizing), difficulty concentrating on tasks, lack of flexibility, lack of impulse control, outbreak, lack of consciousness on their own behavior and/or ability, slow processing ideas, difficulty learning new information, difficulty memory things, difficulty communicating, difficulty following commands, difficulty performing tasks.

In one aspect, the symptom of the neurological disorder is a psychotic symptom. Non-limiting examples of psychotic symptoms include depression, irritability, sadness or apathy, social withdrawal, insomnia, fatigue, lack of energy, obsessive compulsive disorder, mania, bipolar disorders and weight loss. In one aspect, the symptom of the neurological disorder is an impairment of at least one blood vessel. In one aspect, the symptom of the neurological disorder is an impaired blood brain barrier. In one aspect, the symptom of the neurological disorder is impaired blood flow. Non-limiting examples of tests for evaluating the elimination, reduction, slowing or delay of neurological condition symptoms include the Unified Huntington's Disease Rating Scale (UHDRS) score, the UHDRS Total Functional Capacity (TFC), the UHDRS functional assessment, the UHDRS gait score, the UHDRS Total Motor Score (TMS), the hamilton depression scale (HAM-D), the columbian suicide severity rating scale (C-SSRS), the montreal cognitive assessment (MoCA), the modified rank scale (mRS), the National Institutes of Health Stroke Scale (NIHSS) and the pap index (BI), the rising-walking timing Test (TUG), the Chedoke upper limb and hand functional activity scale (CAHAI), the symbolic digital modality test, the controlled oral word association task (Controlled Oral Word Association tasks), magnetic Resonance Imaging (MRI), functional magnetic resonance imaging (fMRI) and Positron Emission Tomography (PET) scans.

In one aspect, a therapeutically effective dose achieves a relief, cure, response rate, or resolution of between about 10% and about 99% or more of the neurological disorder. In one aspect, a therapeutically effective dose achieves between 10% and 100% relief, cure, response rate, or regression rate of the neurological condition, such as between 10% and 15%, between 10% and 20%, between 10% and 25%, between 15% and 20%, between 15% and 25%, between 15% and 30%, between 20% and 25%, between 20% and 30%, between 20% and 35%, between 25% and 30%, between 25% and 35%, between 25% and 40%, between 30% and 35%, between 30% and 40%, between 35% and 45%, between 35% and 50%, between 40% and 45%, between 40% and 50%, between 40% and 55%, between 45% and 60%, between 50% and 55%, between 50% and 60%, between between 50% and 65%, between 55% and 60%, between 55% and 65%, between 55% and 70%, between 60% and 65%, between 60% and 70%, between 60% and 75%, between 65% and 70%, between 65% and 75%, between 65% and 80%, between 70% and 75%, between 70% and 80%, between between 70% and 85%, between 75% and 80%, between 75% and 85%, between 75% and 90%, between 80% and 85%, between 80% and 90%, between 80% and 95%, between 85% and 90%, between 85% and 95%, between 85% and 100%, between 90% and 95%, between, between 90% and 100% or between 95% and 100%.

In one aspect, the therapeutically effective dose eliminates, reduces, slows or delays between 10% and 100% of the symptoms of one or more neurological disorders, such as between 10% and about 15%, between 10% and 20%, between 10% and 25%, between 15% and 20%, between 15% and 25%, between 15% and 30%, between 20% and 25%, between 20% and 30%, between 20% and 35%, between 25% and 30%, between 25% and 35%, between 25% and 40%, between 30% and 35%, between 30% and 40%, between 35% and 45%, between 35% and 50%, between 40% and 45%, between 40% and 50%, between 40% and 55%, between 45% and 50%, between 45% and 55%, between 45% and 60%, between 50% and 55%, between between 50% and 60%, between 50% and 65%, between 55% and 60%, between 55% and 65%, between 55% and 70%, between 60% and 65%, between 60% and 70%, between 60% and 75%, between 65% and 70%, between 65% and 75%, between 65% and 80%, between 70% and 75%, between between 70% and 80%, between 70% and 85%, between 75% and 80%, between 75% and 85%, between 75% and 90%, between 80% and 85%, between 80% and 90%, between 80% and 95%, between 85% and 90%, between 85% and 95%, between 85% and 100%, between 90% and 95%, between, between 90% and 100% or between 95% and 100%.

In one aspect, the symptoms of the neurological disorder are assessed annually on the day of treatment, 1 day after treatment, 3 months after treatment, 6 months after treatment, 1 year after treatment, and after treatment.

In one aspect, the symptoms of the neurological disorder are assessed between 1 day after treatment and 7 days after treatment. In one aspect, symptoms can be assessed between 1 day and 2 days post-treatment, between 1 day and 3 days post-treatment, between 1 day and 4 days post-treatment, between 2 days post-treatment and 3 days post-treatment, between 2 days post-treatment and 4 days post-treatment, between 2 days post-treatment and 5 days post-treatment, between 3 days post-treatment and 4 days post-treatment, between 3 days post-treatment and 5 days post-treatment, between 3 days post-treatment and 6 days post-treatment, between 4 days post-treatment and 5 days post-treatment, between 4 days post-treatment and 6 days post-treatment, between 4 days post-treatment and 7 days post-treatment, between 5 days post-treatment and 6 days post-treatment, between 5 days post-treatment and 7 days post-treatment, or between 6 days post-treatment and 7 days post-treatment. In one aspect, symptoms can be assessed between 1 week after treatment and 4 weeks after treatment. In one aspect, symptoms can be assessed between 1 week and 2 weeks after treatment, between 1 week and 3 weeks after treatment, between 1 week and 4 weeks after treatment, between 2 weeks and 3 weeks after treatment, between 2 weeks and 4 weeks after treatment, or between 3 weeks and 4 weeks after treatment. In one aspect, symptoms can be assessed between 1 month after treatment and 12 months after treatment. In one aspect of the present invention, symptoms can be between 1 month and 2 months after treatment, between 1 month and 3 months after treatment, between 1 month and 4 months after treatment, between 2 months and 3 months after treatment, between 2 months and 4 months after treatment, between 2 months and 5 months after treatment, between 3 months and 4 months after treatment, between 3 months and 5 months after treatment, between 3 months and 6 months after treatment, between 4 months and 5 months after treatment, between 4 months and 6 months after treatment, between 4 months and 7 months after treatment, between 5 months and 6 months after treatment, between 5 months and 7 months after treatment, between 5 months and between 5 months and 8 months, between 6 months and 7 months, between 6 months and 8 months, between 6 months and 9 months, between 7 months and 8 months, between 7 months and 9 months, between 7 months and 10 months, between 8 months and 9 months, between 8 months and 10 months, between 8 months and 11 months, between 9 months and 10 months, between 9 months and 11 months, between 9 months and 12 months, between 10 months and 11 months Assessment between 10 months and 12 months after treatment or between 11 months and 12 months after treatment. In one aspect, symptoms can be assessed between 1 year after treatment and about 20 years after treatment. In one aspect, symptoms may be assessed between 1 year and 5 years post-treatment, between 1 year and 10 years post-treatment, between 1 year and 15 years post-treatment, between 5 years post-treatment and 10 years post-treatment, between 5 years post-treatment and 15 years post-treatment, between 5 years post-treatment and 20 years post-treatment, between 10 years post-treatment and 15 years post-treatment, between 10 years post-treatment and 20 years post-treatment, or between 15 years post-treatment and 20 years post-treatment.

As used herein, the term "survival rate" refers to the survival of a subject cohort in a treatment group after a given period of time following diagnosis of a neurological disorder.

In one aspect, a therapeutically effective dose achieves an increase in survival rate of between about 10% and 99% or more. In one aspect, a therapeutically effective dose achieves an increase in survival rate of between 10% and 100%, such as between 10% and 15%, between 10% and 20%, between 10% and 25%, between 15% and 20%, between 15% and 25%, between 15% and 30%, between 20% and 25%, between 20% and 30%, between 20% and 35%, between 25% and 30%, between 25% and 35%, between 25% and 40%, between 30% and 35%, between 30% and 40%, between 35% and 45%, between 35% and 50%, between 40% and 45%, between 40% and 50%, between 40% and 55%, between 45% and 60%, between 50% and 55%, between 50% and 60%, between between 50% and 65%, between 55% and 60%, between 55% and 65%, between 55% and 70%, between 60% and 65%, between 60% and 70%, between 60% and 75%, between 65% and 70%, between 65% and 75%, between 65% and 80%, between 70% and 75%, between 70% and 80%, between between 70% and 85%, between 75% and 80%, between 75% and 85%, between 75% and 90%, between 80% and 85%, between 80% and 90%, between 80% and 95%, between 85% and 90%, between 85% and 95%, between 85% and 100%, between 90% and 95%, between, between 90% and 100% or between 95% and 100%.

As used herein, the term "life expectancy" refers to the period of time in which the subject is expected to survive.

In one aspect, a therapeutically effective dose increases the life expectancy by between about 10% and 99% or more. In one aspect, a therapeutically effective dose increases the life expectancy by between 10% and 100%, such as between 10% and 15%, between 10% and 20%, between 10% and 25%, between 15% and 20%, between 15% and 25%, between 15% and 30%, between 20% and 25%, between 20% and 30%, between 20% and 35%, between 25% and 30%, between 25% and 35%, between 25% and 40%, between 30% and 35%, between 30% and 40%, between 35% and 45%, between 35% and 50%, between 40% and 45%, between 40% and 50%, between 40% and 55%, between 45% and 60%, between 50% and 55%, between 50% and 60%, between between 50% and 65%, between 55% and 60%, between 55% and 65%, between 55% and 70%, between 60% and 65%, between 60% and 70%, between 60% and 75%, between 65% and 70%, between 65% and 75%, between 65% and 80%, between 70% and 75%, between 70% and 80%, between between 70% and 85%, between 75% and 80%, between 75% and 85%, between 75% and 90%, between 80% and 85%, between 80% and 90%, between 80% and 95%, between 85% and 90%, between 85% and 95%, between 85% and 100%, between 90% and 95%, between, between 90% and 100% or between 95% and 100%.

In one aspect, the therapeutically effective dose reduces the amount of atrophy in the brain of a subject in need thereof by between about 10% and 99% or more. In one aspect, the therapeutically effective dose reduces the amount of atrophy in the brain of a subject in need thereof by between 10% and 100%, such as between 10% and 15%, between 10% and 20%, between 10% and 25%, between 15% and 20%, between 15% and 25%, between 15% and 30%, between 20% and 25%, between 20% and 30%, between 20% and 35%, between 25% and 30%, between 25% and 35%, between 25% and 40%, between 30% and 35%, between 30% and 40%, between 35% and 45%, between 35% and 50%, between 40% and 45%, between 40% and 50%, between 40% and 55%, between 45% and 60%, between 50% and 55%, between 50% and 60%, between between 50% and 65%, between 55% and 60%, between 55% and 65%, between 55% and 70%, between 60% and 65%, between 60% and 70%, between 60% and 75%, between 65% and 70%, between 65% and 75%, between 65% and 80%, between 70% and 75%, between 70% and 80%, between between 70% and 85%, between 75% and 80%, between 75% and 85%, between 75% and 90%, between 80% and 85%, between 80% and 90%, between 80% and 95%, between 85% and 90%, between 85% and 95%, between 85% and 100%, between 90% and 95%, between, between 90% and 100% or between 95% and 100%.

In one aspect, the amount of atrophy in the brain of a subject in need thereof is assessed annually on the day of treatment, 1 day after treatment, 3 months after treatment, 6 months after treatment, 1 year after treatment, and after treatment.

In one aspect, the amount of atrophy in the brain of a subject in need thereof is assessed between 1 day post-treatment and 7 days post-treatment. In one aspect, symptoms can be assessed between 1 day and 2 days post-treatment, between 1 day and 3 days post-treatment, between 1 day and 4 days post-treatment, between 2 days post-treatment and 3 days post-treatment, between 2 days post-treatment and 4 days post-treatment, between 2 days post-treatment and 5 days post-treatment, between 3 days post-treatment and 4 days post-treatment, between 3 days post-treatment and 5 days post-treatment, between 3 days post-treatment and 6 days post-treatment, between 4 days post-treatment and 5 days post-treatment, between 4 days post-treatment and 6 days post-treatment, between 4 days post-treatment and 7 days post-treatment, between 5 days post-treatment and 6 days post-treatment, between 5 days post-treatment and 7 days post-treatment, or between 6 days post-treatment and 7 days post-treatment. In one aspect, symptoms can be assessed between 1 week after treatment and 4 weeks after treatment. In one aspect, symptoms can be assessed between 1 week and 2 weeks after treatment, between 1 week and 3 weeks after treatment, between 1 week and 4 weeks after treatment, between 2 weeks and 3 weeks after treatment, between 2 weeks and 4 weeks after treatment, or between 3 weeks and 4 weeks after treatment. In one aspect, symptoms can be assessed between 1 month after treatment and 12 months after treatment. In one aspect of the present invention, symptoms can be between 1 month and 2 months after treatment, between 1 month and 3 months after treatment, between 1 month and 4 months after treatment, between 2 months and 3 months after treatment, between 2 months and 4 months after treatment, between 2 months and 5 months after treatment, between 3 months and 4 months after treatment, between 3 months and 5 months after treatment, between 3 months and 6 months after treatment, between 4 months and 5 months after treatment, between 4 months and 6 months after treatment, between 4 months and 7 months after treatment, between 5 months and 6 months after treatment, between 5 months and 7 months after treatment, between 5 months and between 5 months and 8 months, between 6 months and 7 months, between 6 months and 8 months, between 6 months and 9 months, between 7 months and 8 months, between 7 months and 9 months, between 7 months and 10 months, between 8 months and 9 months, between 8 months and 10 months, between 8 months and 11 months, between 9 months and 10 months, between 9 months and 11 months, between 9 months and 12 months, between 10 months and 11 months Assessment between 10 months and 12 months after treatment or between 11 months and 12 months after treatment. In one aspect, symptoms can be assessed between 1 year after treatment and about 20 years after treatment. In one aspect, symptoms may be assessed between 1 year and 5 years post-treatment, between 1 year and 10 years post-treatment, between 1 year and 15 years post-treatment, between 5 years post-treatment and 10 years post-treatment, between 5 years post-treatment and 15 years post-treatment, between 5 years post-treatment and 20 years post-treatment, between 10 years post-treatment and 15 years post-treatment, between 10 years post-treatment and 20 years post-treatment, or between 15 years post-treatment and 20 years post-treatment.

Non-limiting examples of tests to assess the amount of atrophy in the brain of a subject in need thereof include Nissle staining, MRI, functional magnetic resonance fMRI, and PET scanning.

Although the present disclosure has been described with reference to preferred embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof to accommodate specific cases without departing from the scope of the present disclosure. Therefore, it is intended that the disclosure not be limited to the particular embodiment disclosed as the best mode contemplated for carrying out this disclosure, but that the disclosure will include all embodiments falling within the scope and spirit of the appended claims.

The examples set forth herein illustrate several embodiments of the disclosure, but should not be construed as limiting the scope of the disclosure in any way.

Examples

Example 1 AAV vector constructs

One hundred ninety two AAV vector constructs were constructed:

EF-1α: gfa681:NeuroD1:P2A:Ascl1:WPRE:SV40 (FIG. 1B);

EF-1α:Gfa1.6:NeuroD1:P2A:Ascl1:WPRE:SV40；

EF-1α:GFA2.2:NeuroD1:P2A:Ascl1:WPRE:SV40；

EF-1α: gfa681:NeuroD1:P2A:Ascl1:WPRE:hGH (FIG. 2B);

EF-1α:Gfa1.6:NeuroD1:P2A:Ascl1:WPRE:hGH；

EF-1α:GFA2.2:NeuroD1:P2A:Ascl1:WPRE:hGH；

CE Gfa681: neuroD1: P2A: ascl1: WPRE: SV40 (FIG. 1A);

CE:Gfa1.6:NeuroD1:P2A:Ascl1:WPRE:SV40；

CE:GFA2.2:NeuroD1:P2A:Ascl1:WPRE:SV40；

CE Gfa681: neuroD1: P2A: ascl1: WPRE: hGH (FIG. 2A);

CE:Gfa1.6:NeuroD1:P2A:Ascl1:WPRE:hGH；

CE:GFA2.2:NeuroD1:P2A:Ascl1:WPRE:hGH；

EF-1α: gfa681:NeuroD1:T2A:Ascl1:WPRE:SV40 (FIG. 3B);

EF-1α:Gfa1.6:NeuroD1:T2A:Ascl1:WPRE:SV40；

EF-1α:GFA2.2:NeuroD1:T2A:Ascl1:WPRE:SV40；

EF-1α: gfa681:NeuroD1:T2A:Ascl1:WPRE:hGH (FIG. 4B);

EF-1α:Gfa1.6:NeuroD1:T2A:Ascl1:WPRE:hGH；

EF-1α:GFA2.2:NeuroD1:T2A:Ascl1:WPRE:hGH；

CE Gfa681: neuroD1: T2A: ascl1: WPRE: SV40 (FIG. 3A);

CE:Gfa1.6:NeuroD1:T2A:Ascl1:WPRE:SV40；

CE:GFA2.2:NeuroD1:T2A:Ascl1:WPRE:SV40；

CE Gfa681: neuroD1: T2A: ascl1: WPRE: hGH (FIG. 4A);

CE:Gfa1.6:NeuroD1:T2A:Ascl1:WPRE:hGH；

CE:GFA2.2:NeuroD1:T2A:Ascl1:WPRE:hGH；

EF-1α: gfa681:NeuroD1:P2A:ISL1:WPRE:SV40 (FIG. 5B);

EF-1α:Gfa1.6:NeuroD1:P2A:ISL1:WPRE:SV40；

EF-1α:GFA2.2:NeuroD1:P2A:ISL1:WPRE:SV40；

EF-1α: gfa681:NeuroD1:P2A:ISL1:WPRE:hGH (FIG. 6B);

EF-1α:Gfa1.6:NeuroD1:P2A:ISL1:WPRE:hGH；

EF-1α:GFA2.2:NeuroD1:P2A:ISL1:WPRE:hGH；

CE Gfa681: neuroD1: P2A: ISL1: WPRE: SV40 (FIG. 5A);

CE:Gfa1.6:NeuroD1:P2A:ISL1:WPRE:SV40；

CE:GFA2.2:NeuroD1:P2A:ISL1:WPRE:SV40；

CE Gfa681 neuroD 1P 2A ISL1 WPRE Hgh (FIG. 6A);

CE:Gfa1.6:NeuroD1:P2A:ISL1:WPRE:hGH；

CE:GFA2.2:NeuroD1:P2A:ISL1:WPRE:hGH；

EF-1α: gfa681:NeuroD1:T2A:ISL1:WPRE:SV40 (FIG. 7B);

EF-1α:Gfa1.6:NeuroD1:T2A:ISL1:WPRE:SV40；

EF-1α:GFA2.2:NeuroD1:T2A:ISL1:WPRE:SV40；

EF-1α: gfa681:NeuroD1:T2A:ISL1:WPRE:hGH (FIG. 8B);

EF-1α:Gfa1.6:NeuroD1:T2A:ISL1:WPRE:hGH；

EF-1α:GFA2.2:NeuroD1:T2A:ISL1:WPRE:hGH；

CE Gfa681 neuroD 1T 2A ISL1 WPRE SV40 (FIG. 7A);

CE:Gfa1.6:NeuroD1:T2A:ISL1:WPRE:SV40；

CE:GFA2.2:NeuroD1:T2A:ISL1:WPRE:SV40；

CE Gfa681: neuroD1: T2A: ISL1: WPRE: hGH (FIG. 8A);

CE:Gfa1.6:NeuroD1:T2A:ISL1:WPRE:hGH；

CE:GFA2.2:NeuroD1:T2A:ISL1:WPRE:hGH；

EF-1α: gfa681:NeuroD1:P2A:LHX3:WPRE:SV40 (FIG. 9B);

EF-1α:Gfa1.6:NeuroD1:P2A:LHX3:WPRE:SV40；

EF-1α:GFA2.2:NeuroD1:P2A:LHX3:WPRE:SV40；

EF-1α: gfa681:NeuroD1:P2A:LHX3:WPRE:hGH (FIG. 10B);

EF-1α:Gfa1.6:NeuroD1:P2A:LHX3:WPRE:hGH；

EF-1α:GFA2.2:NeuroD1:P2A:LHX3:WPRE:hGH；

CE Gfa681: neuroD1: P2A: LHX3: WPRE: SV40 (FIG. 9A);

CE:Gfa1.6:NeuroD1:P2A:LHX3:WPRE:SV40；

CE:GFA2.2:NeuroD1:P2A:LHX3:WPRE:SV40；

CE Gfa681: neuroD1: P2A: LHX3: WPRE: hGH (FIG. 10A);

CE:Gfa1.6:NeuroD1:P2A:LHX3:WPRE:hGH；

CE:GFA2.2:NeuroD1:P2A:LHX3:WPRE:hGH；

EF-1α: gfa681:NeuroD1:T2A:LHX3:WPRE:SV40 (FIG. 11B);

EF-1α:Gfa1.6:NeuroD1:T2A:LHX3:WPRE:SV40；

EF-1α:GFA2.2:NeuroD1:T2A:LHX3:WPRE:SV40；

EF-1α: gfa681:NeuroD1:T2A:LHX3:WPRE:hGH (FIG. 12B);

EF-1α:Gfa1.6:NeuroD1:T2A:LHX3:WPRE:hGH；

EF-1α:GFA2.2:NeuroD1:T2A:LHX3:WPRE:hGH；

CE Gfa681 neuroD 1T 2A LHX3 WPRE SV40 (FIG. 11A);

CE:Gfa1.6:NeuroD1:T2A:LHX3:WPRE:SV40；

CE:GFA2.2:NeuroD1:T2A:LHX3:WPRE:SV40；

CE Gfa681: neuroD1: T2A: LHX3: WPRE: hGH (FIG. 12A);

CE:Gfa1.6:NeuroD1:T2A:LHX3:WPRE:hGH；

CE:GFA2.2:NeuroD1:T2A:LHX3:WPRE:hGH；

EF-1α: gfa681:NeuroD1:GSG-P2A:Ascl1:WPRE:SV40 (FIG. 1D);

EF-1α:Gfa1.6:NeuroD1:GSG-P2A:Ascl1:WPRE:SV40；

EF-1α:GFA2.2:NeuroD1:GSG-P2A:Ascl1:WPRE:SV40；

EF-1α: gfa681:NeuroD1:GSG-P2A:Ascl1:WPRE:hGH (FIG. 2D);

EF-1α:Gfa1.6:NeuroD1:GSG-P2A:Ascl1:WPRE:hGH；

EF-1α:GFA2.2:NeuroD1:GSG-P2A:Ascl1:WPRE:hGH；

CE Gfa681: neuroD1: GSG-P2A: ascl1: WPRE: SV40 (FIG. 1C);

CE:Gfa1.6:NeuroD1:GSG-P2A:Ascl1:WPRE:SV40；

CE:GFA2.2:NeuroD1:GSG-P2A:Ascl1:WPRE:SV40；

CE Gfa681: neuroD1: GSG-P2A: ascl1: WPRE: hGH (FIG. 2C);

CE:Gfa1.6:NeuroD1:GSG-P2A:Ascl1:WPRE:hGH；

CE:GFA2.2:NeuroD1:GSG-P2A:Ascl1:WPRE:hGH；

EF-1α: gfa681:NeuroD1:GSG-T2A:Ascl1:WPRE:SV40 (FIG. 3D);

EF-1α:Gfa1.6:NeuroD1:GSG-T2A:Ascl1:WPRE:SV40；

EF-1α:GFA2.2:NeuroD1:GSG-T2A:Ascl1:WPRE:SV40；

EF-1α: gfa681:NeuroD1:GSG-T2A:Ascl1:WPRE:hGH (FIG. 4D);

EF-1α:Gfa1.6:NeuroD1:GSG-T2A:Ascl1:WPRE:hGH；

EF-1α:GFA2.2:NeuroD1:GSG-T2A:Ascl1:WPRE:hGH；

CE Gfa681: neuroD1: GSG-T2A: ascl1: WPRE: SV40 (FIG. 3C);

CE:Gfa1.6:NeuroD1:GSG-T2A:Ascl1:WPRE:SV40；

CE:GFA2.2:NeuroD1:GSG-T2A:Ascl1:WPRE:SV40；

CE Gfa681: neuroD1: GSG-T2A: ascl1: WPRE: hGH (FIG. 4C);

CE:Gfa1.6:NeuroD1:GSG-T2A:Ascl1:WPRE:hGH；

CE:GFA2.2:NeuroD1:GSG-T2A:Ascl1:WPRE:hGH；

EF-1α: gfa681:NeuroD1:GSG-P2A:ISL1:WPRE:SV40 (FIG. 5D);

EF-1α:Gfa1.6:NeuroD1:GSG-P2A:ISL1:WPRE:SV40；

EF-1α:GFA2.2:NeuroD1:GSG-P2A:ISL1:WPRE:SV40；

EF-1α Gfa681 neuroD1 GSG-P2A ISL1 WPRE hGH; (FIG. 6D)

EF-1α:Gfa1.6:NeuroD1:GSG-P2A:ISL1:WPRE:hGH；

EF-1α:GFA2.2:NeuroD1:GSG-P2A:ISL1:WPRE:hGH；

CE Gfa681: neuroD1: GSG-P2A: ISL1: WPRE: SV40 (FIG. 5C);

CE:Gfa1.6:NeuroD1:GSG-P2A:ISL1:WPRE:SV40；

CE:GFA2.2:NeuroD1:GSG-P2A:ISL1:WPRE:SV40；

CE Gfa681: neuroD1: GSG-P2A: ISL1: WPRE: hGH (FIG. 6C);

CE:Gfa1.6:NeuroD1:GSG-P2A:ISL1:WPRE:hGH；

CE:GFA2.2:NeuroD1:GSG-P2A:ISL1:WPRE:hGH；

EF-1α: gfa681:NeuroD1:GSG-T2A:ISL1:WPRE:SV40 (FIG. 7D);

EF-1α:Gfa1.6:NeuroD1:GSG-T2A:ISL1:WPRE:SV40；

EF-1α:GFA2.2:NeuroD1:GSG-T2A:ISL1:WPRE:SV40；

EF-1α: gfa681:NeuroD1:GSG-T2A:ISL1:WPRE:hGH (FIG. 8D);

EF-1α:Gfa1.6:NeuroD1:GSG-T2A:ISL1:WPRE:hGH；

EF-1α:GFA2.2:NeuroD1:GSG-T2A:ISL1:WPRE:hGH；

CE Gfa681: neuroD1: GSG-T2A: ISL1: WPRE: SV40 (FIG. 7C);

CE:Gfa1.6:NeuroD1:GSG-T2A:ISL1:WPRE:SV40；

CE:GFA2.2:NeuroD1:GSG-T2A:ISL1:WPRE:SV40；

CE Gfa681: neuroD1: GSG-T2A: ISL1: WPRE: hGH (FIG. 8C);

CE:Gfa1.6:NeuroD1:GSG-T2A:ISL1:WPRE:hGH；

CE:GFA2.2:NeuroD1:GSG-T2A:ISL1:WPRE:hGH；

EF-1α: gfa681:NeuroD1:GSG-P2A:LHX3:WPRE:SV40 (FIG. 9D);

EF-1α:Gfa1.6:NeuroD1:GSG-P2A:LHX3:WPRE:SV40；

EF-1α:GFA2.2:NeuroD1:GSG-P2A:LHX3:WPRE:SV40；

EF-1α: gfa681:NeuroD1:GSG-P2A:LHX3:WPRE:hGH (FIG. 10D);

EF-1α:Gfa1.6:NeuroD1:GSG-P2A:LHX3:WPRE:hGH；

EF-1α:GFA2.2:NeuroD1:GSG-P2A:LHX3:WPRE:hGH；

CE Gfa681: neuroD1: GSG-P2A: LHX3: WPRE: SV40 (FIG. 9C);

CE:Gfa1.6:NeuroD1:GSG-P2A:LHX3:WPRE:SV40；

CE:GFA2.2:NeuroD1:GSG-P2A:LHX3:WPRE:SV40；

CE Gfa681: neuroD1: GSG-P2A: LHX3: WPRE: hGH (FIG. 10C);

CE:Gfa1.6:NeuroD1:GSG-P2A:LHX3:WPRE:hGH；

CE:GFA2.2:NeuroD1:GSG-P2A:LHX3:WPRE:hGH；

EF-1α: gfa681:NeuroD1:GSG-T2A:LHX3:WPRE:SV40 (FIG. 11D);

EF-1α:Gfa1.6:NeuroD1:GSG-T2A:LHX3:WPRE:SV40；

EF-1α:GFA2.2:NeuroD1:GSG-T2A:LHX3:WPRE:SV40；

EF-1α: gfa681:NeuroD1:GSG-T2A:LHX3:WPRE: hgh (FIG. 12D);

EF-1α:Gfa1.6:NeuroD1:GSG-T2A:LHX3:WPRE:hGH；

EF-1α:GFA2.2:NeuroD1:GSG-T2A:LHX3:WPRE:hGH；

CE Gfa681: neuroD1: GSG-T2A: LHX3: WPRE: SV40 (FIG. 11C);

CE:Gfa1.6:NeuroD1:GSG-T2A:LHX3:WPRE:SV40；

CE:GFA2.2:NeuroD1:GSG-T2A:LHX3:WPRE:SV40；

CE Gfa681: neuroD1: GSG-T2A: LHX3: WPRE: hGH (FIG. 12C);

CE, gfa1.6, neuroD1, GSG-T2A, LHX3, WPRE, hGH; and

CE:GFA2.2:NeuroD1:GSG-T2A:LHX3:WPRE:hGH

EF-1α:Gfa681:NeuroD1:WPRE:SV40；

EF-1α:Gfa1.6:NeuroD1:WPRE:SV40；

EF-1α:GFA2.2:NeuroD1::WPRE:SV40；

EF-1α:Gfa681:NeuroD1WPRE:hGH；

EF-1α:Gfa1.6:NeuroD1:WPRE:hGH；

EF-1α:GFA2.2:NeuroD1:WPRE:hGH；

CE:Gfa681:NeuroD1:WPRE:SV40；

CE:Gfa1.6:NeuroD1:WPRE:SV40；

CE:GFA2.2:NeuroD1:WPRE:SV40；

CE:Gfa681:NeuroD1:WPRE:hGH；

CE:Gfa1.6:NeuroD1:WPRE:hGH；

CE:GFA2.2:NeuroD1:WPRE:hGH；

CE Gfa681 ISL1 WPRE SV40 (FIG. 13A);

EF-1α: gfa681:ISL1:WPRE:SV40 (FIG. 13B);

CE: gfa1.6p: ISL1: WPRE: SV40 (FIG. 13C);

EF-1α: gfa1.6p: ISL1: WPRE: SV40 (FIG. 13D);

CE Gfa2.2:ISL1:WPRE:SV40 (FIG. 13E);

EF-1α: gfa2.2: ISL1: WPRE: SV40 (FIG. 13F);

CE Gfa681 ISL1 WPRE hGH (FIG. 14A);

EF-1α: gfa681:ISL1:WPRE:hGH (FIG. 14B);

CE: gfa1.6p: ISL1: WPRE: hGH (FIG. 14C);

EF-1α: gfa1.6p: ISL1: WPRE: hGH (FIG. 14D);

CE: gfa2.2: ISL1: WPRE: hGH (FIG. 14E);

EF-1α: gfa2.2: ISL1: WPRE: hGH (FIG. 14F);

CE Gfa681 LHX3 WPRE SV40 (FIG. 15A);

EF-1α: gfa681:LHX3:WPRE:SV4 (FIG. 15B);

CE Gfa1.6p LHX3 WPRE SV4 (FIG. 15C);

EF-1α: gfa1.6p: LHX3: WPRE: SV40 (FIG. 15D);

CE Gfa2.2 LHX3 WPRE SV40 (FIG. 15E);

EF-1α: gfa2.2: LHX3: WPRE: SV40 (FIG. 15F);

CE Gfa681 LHX3 WPRE hGH (FIG. 16A);

EF-1α: gfa681:LHX3:WPRE:hGH (FIG. 16B);

CE: gfa1.6p: LHX3: WPRE: hGH (FIG. 16C);

EF-1α: gfa1.6p: LHX3: WPRE: hGH (FIG. 16D);

EF-1α: gfa2.2: LHX3: WPRE: hGH (FIG. 16E); and

EF-1α: gfa2.2: LHX3: WPRE: hG (FIG. 16F).

All 192 vector constructs used pHSG-299 (Takara, mountain View, calif.), a pUC-based vector construct containing the replication origin, kanamycin resistance gene and multiple cloning site (MSC), and lacZ gene as the backbone.

The 5 'end of the expression cassette is an enhancer from the human elongation factor 1 alpha promoter (EF-1 alpha enhancer; SEQ ID NO: 2) or the cytomegalovirus enhancer (CMV enhancer; SEQ ID NO: 17) which places the 5' on the following promoters: 758 nucleotide GFAP promoter (GfaABC 1D; SEQ ID NO: 3), 1667 nucleotide GFAP promoter (Gfa1.6; SEQ ID NO: 4), or 2214 nucleotide GFAP promoter (GFA 2.2 SEQ ID NO: 18).

After the EF-1 a enhancer/GFAP promoter (e.g., 3' of the EF-1 a enhancer/GFAP promoter), several additional sequences are introduced into the expression cassette in a 5' to 3' direction, including: chimeric intron (SEQ ID NO: 5); human NeuroD1 coding sequence (hNeuroD 1; SEQ ID NO: 6); and the human Ascl1 coding sequence (hASCl 1; SEQ ID NO: 11), the human ISL1 coding sequence (hISL 1; SEQ ID NO: 13) or the human LHX3 coding sequence (hLHX 3; SEQ ID NO: 15); linker sequences (P2A; SEQ ID NO: 19), (GSG-P2A; SEQ ID NO: 22), (T2A; SEQ ID NO: 20) or (GSG-T2A; SEQ ID NO: 23); and woodchuck hepatitis virus posttranscriptional regulatory elements (WPRE; SEQ ID NO: 7). These sequences are both operably linked to the SV40 poly (A) signal (SEQ ID NO: 8) or the hGH poly (A) signal (SEQ ID NO: 21). Enhancers, GFAP promoters, chimeric introns, hNeuroD1 coding sequence, hASCl1 coding sequence, or hISL1 coding sequence, or hLHX3 coding sequence, adaptors, WPRE, and poly (A) signals are flanked by two AAV ITR sequences.

Example 2 AAV Virus production

Each of the 192 plasmids was co-transfected into 293AAV cells using polyethylenimine together with a Rep-Cap plasmid (a plasmid comprising a promoter driving AAV Rep and Cap gene expression) and a helper plasmid (a plasmid comprising a promoter driving adenovirus E2A, E and VA RNA expression) to produce recombinant AAV virions (Cell Biolabs, inc.)

The transfected cells were scraped and centrifuged 72 hours after transfection. The cell pellet was frozen and thawed, i.e., placed in a dry ice/ethanol mixture, then placed in a 37 ℃ water bath. The freeze/thaw cycle was repeated three more times. AAV lysates were purified (e.g., cell debris removed) by ultracentrifugation at 350,000g for 1 hour in a discontinuous iodixanol gradient. The virus-containing layer was collected and then concentrated using a Millipore Amicon ultrafiltration centrifuge. Viral titers were then determined by qPCR using primers or gene/expression cassette specific sequences that amplified the ITR region.

EXAMPLE 3 astrocyte cultures

Human cortical astrocytes (HA 1800; scienCell Research Laboratories, inc., carlsbad, california) were subcultured at confluence exceeding 90%. For subculture, trypLE was used ^TM Select (Invitrogen, carlsbad, california) trypsinized cells, centrifuged at 200×g for 5 min, then resuspended and inoculated in a culture medium containing DMEM/F12 (Gibco); 10% fetal bovine serum (Gibco); penicillin/streptomycin (Gibco); 3.5mM glucose (Sigma-Aldrich); b27 (Gibco); 10ng/mL epidermal growth factor (Invitrogen); and 10ng/mL fibroblast growth factor 2 (Invitrogen). Astrocytes were cultured on poly-D-lysine (Sigma-Aldrich) coated coverslips (12 mm) of 24 well plates (BD Biosciences) at a density of about 50,000 cells per coverslip.

Rat primary astrocytes (isolated from Sprague Dawley rat cortex or striatum) in a culture medium containing DMEM/F12 (Gibco); 10% fetal bovine serum (Gibco), penicillin/streptomycin (Gibco); cultured in a medium of 3.5mM glucose (Gibco).

All cells were kept in humid air containing 5% carbon dioxide at 37 ℃.

Example 4 testing of AAV vectors in astrocyte cultures (in vitro)

10 using the recombinant AAV obtained from the method of example 2 ¹⁰ Individual particles/mL and 10 ¹⁴ The concentration range of individual particles/mL infects human cortical astrocytes and rat primary astrocytes from example 3. Twenty-four hours after infection of the cells, the medium was replaced with one containing DMEM/F12 (Gibco); n2 additive (Gibco); and 20ng/mL of a differentiation medium of brain-derived neurotrophic factor (Invitrogen). Differentiation medium was added to the cell culture every four days. See Song et al, nature,417:39-44 (2002).

The voids in the cell culture are filled with additional human astrocytes to support functional development of the transformed neurons when astrocytes or rat primary astrocytes are transformed into neurons.

Example 5 test of AAV vector efficacy

10 using the recombinant AAV obtained from the method of example 2 ¹⁰ Individual particles/mL and 10 ¹⁴ The concentration range of individual particles/mL infects the 4 th to 7 th passages of the human cortical astrocytes and rat primary astrocytes (or astrocytes of other brain regions or spinal cord) of example 3. qPCR, enzyme-linked immunosorbent assay (ELISA) and western blot were performed to determine expression of NeuroD1, ascl1, ISL1 or LHX3 transcripts and protein levels.

Expression of NeuN, bisdermatan (DCX), β3-tubulin, NF-200, and MAP2 was assessed by qPCR, ELISA, western blotting and immunostaining to determine the functional output of recombinant AAV.

Example 6 test of AAV vector titres and infection Rate

The purified AAV vectors were treated with DNase I to eliminate residuesPlasmid contamination. AAV vectors were serially diluted 100-fold, 500-fold, 2500-fold and 12500-fold. AAV plasmid backbone was diluted to generate a standard curve by serial dilution. Plasmid dilution to 10 ⁴ 、10 ⁵ 、10 ⁶ 、10 ⁷ And 10 ⁸ molecule/uL. qPCR was performed on diluted AAV vector and diluted AAV plasmid. The primers used were directed to the ITR region (forward ITR primer 5'-GGAACCCCTAGTGATGGAGTT (SEQ ID NO: 33), reverse ITR primer 5' -CGGCCTCAGTGAGCGA (SEQ ID NO: 34)). The qPCR mixture contained 10uL Universal SYBR Master Mix X, 2uL 5uM forward ITR primer, 2uL 5uM reverse ITR primer, 5uL test sample or dilution standard and 1uL H ₂ O. The qPCR procedure was 95℃for 10 minutes, followed by 40 cycles of 95℃for 15 seconds, 60℃for 30 seconds, and then the melting curve. The data were analyzed using qPCR cycler software. The physical titer (viral genome (vg)/ml) of AAV samples was calculated from the standard curve.

AAV vector infection rates were tested by a 50% tissue culture infection dose (TCID 50) assay using standard protocols of the american type culture collection (ATCC; manassas, VA).

Example 7 testing of AAV dose ranges (in vivo)

Recombinant AAV obtained from the method of example 2 was injected into C57/BL6 mice by bilateral intracranial injection into the motor cortex. Each AAV is 1x10 ¹¹ 、3x10 ¹¹ 、1x10 ¹² 、3x10 ¹² 、1x10 ¹² 、3x10 ¹² 、1x10 ¹³ The dose of each viral genome/mL was injected in a volume of 1 uL. Each dose was evaluated 4, 20 and 60 days post injection to determine the Optimal Effective Dose (OED), maximum Tolerated Dose (MTD) and Minimum Effective Dose (MED) at the cellular and tissue level. Three mice per time point. OED, MTD and MED were determined by assessing astrocyte to neuron conversion efficiency and potential toxicity via immunostaining of NeuroD1, acl1, ISL1, LHX3, GFAP, neuN and Iba 1. If the first dose range is insufficient to determine OED, MTD and MED, then 1x10 ¹⁰ Individual viral genomes/mL to 1x10 ¹⁴ GC/mL was performed in a second dose range at a volume of 1 uL.

Example 8 comparison of neuronal conversion of recombinant AAV obtained from various AAV vectors in human cell culture (in vitro)

AAV vector constructs were designed as described in example 1 to express NeuroD1 alone or Ascl1 alone or ISL1 alone or LHX3 alone. The following recombinant AAV was obtained as described in example 2: (1) an AAV vector construct expressing NeuroD1 alone; (2) AAV vector constructs expressing Ascl1 alone; (3) AAV vector constructs expressing ISL1 alone; (4) AAV vector constructs expressing LHX3 alone; (5) A combination of AAV vector constructs (1), (2), (3) and (4); (6) AAV vector constructs expressing NeuroD1 and linker and Ascl 1; (7) AAV vector constructs expressing NeuroD1 and adaptors and ISL 1; and (8) AAV vector constructs expressing NeuroD1 and linker and LHX3. The resulting recombinant AAV was used to infect human cortical astrocytes and human primary microglia of example 3. Twenty-four hours after infection of the cells, the medium was replaced with one containing DMEM/F12 (Gibco); n2 additive (Gibco); and 20ng/mL of a differentiation medium of brain-derived neurotrophic factor (Invitrogen). Differentiation medium was added to the cell culture every four days. See Song et al, nature,417:39-44 (2002). The voids in the cell culture are filled with additional human astrocytes to support functional development of the transformed neurons when astrocytes or rat primary astrocytes are transformed into neurons. Neuronal conversion levels for each treatment were measured and compared.

Example 9 testing of AAV vectors in human subjects (in vivo)

Human cortical astrocytes were infected in vivo using the recombinant AAV obtained from example 2. Recombinant AAV at 10 ¹⁰ Individual particles/mL and 10 ¹⁴ The concentration range of individual particles/mL is injected into the cerebral cortex of a human subject suffering from a neurological disorder in a volume range of 10 μl to 1 mL. The symptoms of neurological disorders, brain imaging (including MRI, PET scan, or a combination of MRI and PET) and behavioral indicators of the human subject are observed before, during, and after injection. Post-injection observations were made weekly until the first month post-injection. After the first month following injection, observations were made once a month for the next 11 months and could be extended to viral injection2 years after the injection.

Example 10 dose-scale determination of non-human primate

The volume of NeuroD1 expressing brain tissue from example 7 divided by the number of vector genomes (mm ³ Vector genome) is used to determine the viral infection rate of brain tissue. Calculation of specific brain region volume to be treated (mm) in non-human primate ³ ) And the dose range of the vector genome was scaled according to the infection rate obtained in example 7. Dose-range studies were performed as in example 7 and OED, MTD and MED were determined by assessing astrocyte to neuron conversion efficiency and potential toxicity via immunostaining of NeuroD1, ascl1, ISL1, LHX3, GFAP, neuN and Iba 1.

Example 11 treatment (in vivo) of a subject suffering from Parkinson's disease in need thereof

Subjects suffering from parkinson's disease are treated with recombinant AAV obtained from the method of example 2. Neurological symptoms in subjects include speech changes, tremors, uncontrolled movements, impaired cognitive function, and writing changes. Recombinant AAV at 10 ¹⁰ Individual particles/mL and 10 ¹⁴ The concentration range of individual particles/mL is injected into the cerebral cortex of a human subject suffering from a neurological disorder in a volume range of 10 μl to 1000 μl. Symptoms and behavior indicators of neurological disorders in human subjects were observed before, during and after injection. Post-injection observations were made weekly until the first month post-injection. After the first month after injection, observations were made once a month for the next 11 months and can be extended to 2 years after virus injection.

Example 12 treatment (in vivo) of a subject in need thereof suffering from Stroke

A subject suffering from stroke is treated with a recombinant AAV comprising a first NeuroD1 sequence and a second NeuroD1 sequence obtained from the method of example 2. The neurological symptoms of the subject include speech changes and writing changes. Recombinant AAV at 10 ¹⁰ Individual particles/mL and 10 ¹⁴ The concentration range of individual particles/mL is injected into the cerebral cortex of a human subject suffering from a neurological disorder in a volume range of 10 μl to 1000 μl. Observation before, during and after injection Symptoms of neurological disorders and behavioral indicators of a human subject. Post-injection observations were made weekly until the first month post-injection. After the first month after injection, observations were made once a month for the next 11 months, and can be extended to 2 years after virus injection

Example 13 treatment (in vivo) of a subject in need thereof suffering from spinal cord injury

Subjects suffering from spinal cord injury were treated with recombinant AAV obtained from the method of example 2. The neurological symptoms of the subject include impaired voluntary movement. Recombinant AAV at 10 ¹⁰ Individual particles/mL and 10 ¹⁴ Individual particles/mL are injected into the spinal cord of a human subject suffering from a neurological disorder in a volume ranging from 10 μl to 1000 μl. The symptoms of neurological disorders, spinal cord imaging (including MRI, PET scan, or a combination of MRI and PET) and behavioral indicators of a human subject are observed before, during, and after injection. Post-injection observations were made weekly until the first month post-injection. After the first month after injection, observations were made once a month for the next 11 months and can be extended to 2 years after virus injection.

Example 14 AAV Virus production of P35

Recombinant AAV was obtained as described in example 2. The P35 plasmid was co-transfected into AAV293 cells with the Rep-Cap plasmid expressing serotype 9 capsid protein and Helper plasmid P40Helper (P40H) or pALD-X80 (X80) to produce recombinant AAV viral particles (P35-P40H or P35-X80). Viral titers were determined by qPCR using primers that amplify the gene of interest (GOI), primers specific for the P34 plasmid and the ITR region. Reverse package primers were used to evaluate non-specific packages. An increase in viral yield was observed for the X80 helper plasmid compared to the P40H helper plasmid (FIG. 17). EXAMPLE 15 successful establishment of Primary culture of rat astrocytes

Cortex and striatal tissue were isolated from Sprague-Dawley rat brains 3 days postnatal. Tissues were treated with papain to generate a single cell suspension and inoculated into poly-D-lysine coated flasks. Cells were immunostained with GFAP antibody and SOX9 antibody. Cells were counterstained with DAPI antibody. Over 95% of the cells were astrocytes identified by GFAP and SOX9 staining (fig. 18). The leftmost panel presents images of GFAP stained cells. The middle left panel presents an image of SOX9 stained cells. The middle right panel presents images of DAPI stained cells. The rightmost panel presents a pooled image of GFAP, SOX9 and DAPI stained cells.

EXAMPLE 16 successful transfection of rat astrocytes

Primary rat astrocytes were seeded in 24-well plates, glass coverslips coated with poly-D-lysine, and transfected with plasmid P5 (pEF-1α: hNeuroD1: GFP), control plasmid and Lipofectamine LTX reagent using standard protocols of Thermo Fisher Scientific. Forty-eight hours after transfection, cells were fixed and immunostained with anti-NeuroD 1 antibody, followed by immunostaining with secondary antibody labeled Alexa-568. Cells were counterstained with DAPI to reveal all nuclei (fig. 19). The left panel presents images of cells stained for ND 1. The middle left panel presents images of GFP-expressing cells. The middle right panel represents DAPI stained cells. The right panel represents a pooled image of NeuroD1, GFP and DAPI stained cells.

EXAMPLE 17 comparison of plasmid transfection

Primary rat astrocytes were inoculated and transfected with expression vectors P6 (pEF-1α: hNeuroD1: WPRE: SV 40), P11 (CE: gfaABC1D: neuroD1: SV 40), P35 (EF-1α: gfaABC1D: neuroD1: WPRE: SV 40) and P39 (EF-1α: gfa1.6: neuroD1: WPRE: SV 40) as described in example 16 to test the efficiency of NeuroD1 transfection of cells. P11 resulted in the highest expression of neuroD1 shown by the neuroD1 staining of the cells (FIG. 20; upper panel shows neuroD1 staining of the cells, lower panel shows pooled neuroD1 and DAPI staining of the cells).

Example 18 successful transduction of AAV viral particles into Primary rat astrocytes

Control virus particles from AAV9-P12 (pGfaABC 1D: GFP) were used at 3X10 ¹⁰ vg/well, 1x10 ¹⁰ vg/well, 2.5x10 ¹⁹ Dose of vg/well recombinant AAV obtained from the method of example 2 was transduced into primary rat astrocytes in 100ul medium in 96-well plates. 24 hours prior to transduction, RCA of generations 5-7 were inoculated onto poly-D-lysine (PDL) -coated glass coverslips in 24-well plates, pooledThe degree was 50%. Cells were transduced with the indicated titers of virus in fresh astrocyte medium. The medium was refreshed the next day and every 3-4 days. Images obtained six days after GFP positive cell transduction showed higher transduction rates when the viral titer was higher (figure 21).

Example 19 quantitative analysis of transduction of aav viral particles into primary rat astrocytes.

The recombinant AAV obtained from the method of example 2 was transduced into primary rat astrocytes seeded in 24-well or 96-well plates with the viral particles AAV9-P12 (pGfaABC 1D: GFP) and AAV5-P7 (pEF-1. Alpha.: GFP). Cells were harvested 7 days after infection by trypsinization. Cells were fixed, washed and suspended in PBS. Viral transduction was analyzed using flow cytometry to count GFP positive cells compared to all cells (fig. 22A-22B). FIG. 22A shows AAV9-P12 (pGfaABC 1D: GFP) and AAV5-P7 (pEF-1α: GFP) at an MOI of 5x10 ⁵ vg/cell, 2X10 ⁵ vg/cell and 5x10 ⁴ Transduction at vg/cell. FIG. 22B shows AAV9-P12 (pGfaABC 1D: GFP) at a range of densities (2X 10) ⁴ Individual cells/well, 1.5x10 ⁴ Individual cells/well, 1x10 ⁴ Individual cells/well and 5x10 ³ Individual cells/well) and inoculated with a series of amounts of virus (1 x10 in 100 μl of medium ¹³ 2. Mu.l, 1. Mu.l, 0.5. Mu.l, 0.25. Mu.l, 0.125. Mu.l) of vg/ml virus.

Example 20 successful transduction of AAV virions containing NeuroD1 to primary rat astrocytes.

The recombinant AAV obtained from the method of example 2 was transduced into primary rat astrocytes seeded in 24-well plates with viral particles 1) AAV5-P1 (AAV 5: pGfa2.2: cre) and AAV5-P4 (AAV 5: pCAG: flex: hNeuroD1: GFP); 2) AAV9-P9 (CE: gfaABC1D: neuroD1: GFP); and 3) AAV9-P11 (CE: gfaABC1D: neuroD1: WPRE: SV 40). At 1x10 ⁶ MOI of vg/cells infected with viral particles were fixed six days post infection and immunostained for NeuroD 1. Cells were counterstained with DAPI to show all cells (fig. 23).

Example 21 neurod1 vector-induced transgene expression in vitro and astrocyte to neuron transformation.

Materials and methods

Primary rat astrocyte culture:rat Cortical Astrocytes (RCA) were isolated from Sprague Dawley rat cortical tissue 3 days after birth. Cells were maintained in Astrocyte Medium (AM) consisting of DMEM supplemented with 10% FBS, 2.5mM glutamine, 3.5mM glucose, penicillin/streptomycin. The first two passages of cells were subcultured at a ratio of 1:3-1:4 at low cell density to promote residual progenitor cell differentiation. When 90% -100% confluence is reached, the ratio of subsequent subcultures is 1:2 or 1:3. The 5 th-7 th generation cells were used for transfection and transduction. Immunostaining with GFAP antibodies showed>90% of the cells were GFAP positive astrocytes. The 6 th generation astrocyte cultures were immunostained with astrocyte markers GFAP and Sox9 (fig. 18).

Carrier body: AAV is produced from selected vectors and tested in vitro using rat astrocytes ：

·NXL-P9(CE-pGfa681-CI-hND1-p2A-GFP-WPRE-SV40pA)

·NXL-P22(CE-pGfa681-CI-hND1-WRPE-SV40pA)

·NXL-P35(EE-pGfa681-CI-hND1-WRPE-SV40pA)

·NXL-P37(EE-pGfa681-CI-hND1-p2A-GFP-WPRE-SV40pA

·NXL-P107(CE-pGfa681-CI-hND1-bGHpA)

·NXL-P108(CE-pGfa681-CI-hND1-oPRE-bGHpA)

·NXL-P109(CE-pGfa681-CRGI-hND1-bGHpA)

·NXL-P130(CE-pGfa681-GI-hND1-oPRE-bGHpA)

·NXL-P134(CE-pGfa681-CRGI-hND1-oPRE-bGHpA)

·NXL-P136(EE-Gfa681-CRGI-hND1-bGHpA)

·NXL-P138(EE-Gfa681-CRGI-hND1-oPRE-bGHpA)

Virus production:viruses for in vitro studies utilize polyethylene subunits by triple transfection (GOI, helper and Rep/Cap plasmids) using adherent AAV293 cellsAmine (PEI) production. Virus recovery and purification is achieved by ultracentrifugation or using commercial purification kits.

Specifically, AAV293 cells (Cell Biolabs, cat# AAV-100) were seeded in 15-cm dishes 24 hours prior to transfection. Cells with 70% -85% confluence were transfected with 10ug GOI, 10ug Rep/Cap and 14ug pALD-X80 (Alveron) or pHelper (Cell Biolabs) in each dish using Polyethylenimine (PEI) at a DNA to PEI ratio of 1:4. Multiple dishes were transfected for production according to the desired scale. The medium was refreshed daily. Seventy-two hours after transfection, cells were collected and lysed to harvest virus using AAVpro purification kit (Takara, cat #6666,6675,6235) according to the manufacturer's protocol.

Viral titers were determined by real-time quantitative PCR using primer pairs in the ITR region, primers to amplify the gene of interest (GOI), or vector-specific primers. Plasmid DNA was used as a standard. Production yield of 10 ³ To 10 ⁴ vg/cell level. FIG. 45 depicts how each of the P134, P130, P138 and P21 plasmids co-transfected into AAV293 cells with the Rep-Cap plasmid expressing serotype 9 capsid protein and helper plasmid pALD-X80 (X80) produced recombinant AAV viral particles as measured by qPCR.

Transfection and immunofluorescence:24-48 hours prior to transfection, 5-7 th generation Rat Cortical Astrocytes (RCA) were seeded onto poly-D-lysine (PDL) -coated glass coverslips in 24-well plates at a confluency of 30% -50%. Cells were transfected with 300ng vector DNA using Lipofectamine reagent (Thermo Fisher Cat # 15338) according to the manufacturer's protocol. 24-48 hours after transfection, cells were fixed with 4% paraformaldehyde in PBS, followed by washing and immunostaining with anti-NeuroD 1 (anti-ND 1) antibody (Abcam cat#ab 60704) followed by immunostaining with a secondary antibody conjugated with a fluorescent dye (Invitrogen, alexa Fluor). Images were taken under a fluorescence microscope (Zeiss Axiovert A1, zen Blue). The gene expression level was evaluated by comparing the fluorescence intensities.

Transduction and immunofluorescence:24-48 hours prior to transduction, RCA of generations 5-7 was inoculated onto poly-D-lysine (PDL) -coated glass coverslips in 24-well plates at a confluency of 30% -50%. With 2-6X10 in fresh astrocyte medium ¹⁰ AAV transduced cells were isolated from each viral genome (vg)/ml. The medium was refreshed the next day and every 3-4 days. Three to six days post transduction, cells were fixed with 4% paraformaldehyde in PBS, followed by washing and immunostaining with anti-ND 1 antibody (Abcam cat#ab 60704), followed by immunostaining with a fluorescent dye conjugated secondary antibody (Invitrogen, alexafluor) for viewing and image taking under a fluorescent microscope (Zeiss Axiovert A1, zen Blue). The gene expression level was evaluated by comparing the fluorescence intensities.

Astrocyte to neuron transformation assessment24-48 hours prior to transduction, RCA of generations 5-7 were inoculated onto poly-D-lysine (PDL) -coated glass coverslips in 24-well plates at a confluency of 30% -50%. 2-6X10 in 500ul fresh astrocyte medium (DMEM supplemented with 10% FBS, 2.5mM glutamine, 3.5mM glucose, penicillin/streptomycin) ¹⁰ vg/ml of virus transduced cells. At 48 hours post transduction, the medium was replaced with 5% FBS astrocyte medium. Subsequently, 100ul of transformation medium (DMEM/F12+1% FBS+B27+N2 and 1uM Rock inhibitor and 10ng/ml BDNF) was added daily for 4 days. After 4 days, the medium was completely replaced with transformation medium.

Cells were fixed with 4% paraformaldehyde in PBS at different desired time points (three days, one to five weeks post transduction), followed by washing and immunostaining with antibodies to ND1 (Abcam cat#ab 60704), to NeuN (Millipore, cat#abn 78), to Map2 (Invitrogen, cat#pa 5-17646), followed by immunostaining with a secondary antibody conjugated with a fluorescent dye (Invitrogen, alexafluor) for viewing and imaging under a fluorescent microscope (Zeiss Axiovert A1, zen Blue).

Results of in vitro studies:

all the NeuroD1 (ND 1) plasmids tested were effective in driving expression of NeuroD1 (fig. 25-41). The expression level of NeuroD1 is affected by the elements in the vector. Among the three versions of the GFA promoter, the 681bp promoter showed the highest expression level of neuroD1, and the 1.6kb promoter showed the weakest expression level of neuroD 1. Promoter enhancer elements significantly affect the expression level of NeuroD 1. The CMV enhancer increases the expression level of NeuroD1 over the ef1α enhancer. Chimeric introns and WPRE also increased expression levels of NeuroD 1.

As shown by positive staining of NeuN and/or MAP2 (fig. 27, 30, 32, 35 and 38), all AAVs tested containing ND1 were effective in driving expression of ND1 and inducing astrocyte to neuronal conversion in cultured rat astrocytes. When astrocytes were transduced with vectors driving higher ND1 expression, the conversion was higher. Vectors NXL-P134 and NXL-P138, and viruses produced using these vectors (i.e., AAV9-P134 and AAV9-P138, respectively) are most effective in driving ND1 expression and inducing astrocyte to neuronal conversion, with AAV-P134 being the most effective (FIGS. 25-30). Plasmid AAV9-P21 (CE-pGFA 681-CI-GFP-WPRE-SV40 pA) without ND1 sequence served as a control and it did not induce astrocyte to neuronal transformation as shown by the lack of positive staining for NeuN and/or Map2 (FIG. 24).

NeuN/RBFOX3 (neuronal nucleoprotein) is a marker of neuronal differentiation that stains the nuclear and perinuclear cytoplasm of neurons. MAP2 (microtubule-associated protein 2) is another neuronal marker for chromatin microtubules including dendrites in neurons.

One week after transduction with AAV containing ND1, a small number of NeuN and MAP2 positive cells (neurons) were observed. After two and three weeks, more NeuN/MAP2 positive cells were observed. Some NeuN/MAP2 positive cells showed typical neuronal morphology.

Example 22 neurod1 viral vector-induced transgene expression in vivo and astrocyte to neuron transformation.

AAV9-P134 and AAV9-P138 viruses were used for in vivo studies. AAV9-P12, driving GFP expression alone (no ND 1) under GFAP promoter, was used for control and to identify GFAP expressing cells (astrocytes).

Single-stranded adeno-associated virus (ssAAV, abbreviated AAV) vectors NXL-P12, NXL-P134 and NXL-P138 were packaged into AAV serotype 9 (AAV 9) and then subjected to subsequent iodixanol gradient ultracentrifugation and concentration. Titration of purified AAV virus was performed using a quantitative PCR-based method. All AAV used in this study were prepared in 0.001% Pluronic F-68 (Poloxamer 188 solution, PFL01-100ML,Caisson Laboratories,Smithfield,UT,USA) in PBS (pH 7.4)

Normal C57BL/6J mice over 8 weeks of age were injected with AAV9-P134, AAV-P138 and AAV9-P12 viruses as follows:

p12 control group: AAV9-P12 5x10 ¹¹ GC/ml,1 μl, cortical injection 1 time (unilateral) (n=6)

Group P134: AAV 9-P12.5x10 ¹¹ GC/ml+AAV9-P134 2.5x10 ¹¹ GC/ml,1 μl, cortical injection 1 time (unilateral) (n=6)

P138 group: AAV 9-P12.5x10 ¹¹ GC/ml+AAV9-P138 2.5x10 ¹¹ GC/ml,1 μl, cortical injection 1 time (unilateral) (n=6)

Mice were sacrificed 10 days (dpi) and 30dpi post infection and analyzed for cortical tissue. Animals were anesthetized with 1.25% avetin and then sequentially perfused intracardially with saline (0.9% NaCl) followed by 4% Paraformaldehyde (PFA). Brains were collected and, after overnight fixation in 4% PFA, placed in 20% and 30% sucrose at 4 ℃ in sequence until the tissues subsided. Embedding dehydrated brain into optimal cutting temperature (Tissue-O.C.T. compounds, +.>Finetek, torrance, CA, USA) and then serially sectioned at 30 μm thickness on the coronal plane on a cryostat (Thermo Scientific, shanghai, china). For immunofluorescence, free-floating brain sections were first washed with PBS and blocked for 1 hour in 5% normal donkey serum, 3% bovine serum albumin and 0.3% triton x-100 prepared in PBS at Room Temperature (RT), then incubated overnight at 4 ℃ with primary antibody diluted in blocking solution. After additional washing with 0.2% PBST (0.2% tween-20 in PBS), the samples were washed with 0.5. Mu.g/. Mu.L of 4', 6-diamidino-2-phenylindole (DAPI; F. Hoffmann-La Roche, natley, NJ, USA) and the appropriate donkey anti-mouse/rabbit secondary antibody conjugated to Alexa Fluor 555, mountain conjugated to Alexa Fluor 488 Goat anti-chicken secondary antibody (1:1000,Life technologies,Carlsbad,CA,USA) and goat anti-rat conjugated to Alexa Fluor 647 (Life technologies)/guinea pig (Jackson immune research) secondary antibody (1:500) were incubated for 2 hours at room temperature and then washed extensively with PBS. Sample end use->The caplets (VECTOR Laboratories, burlingame, CA, USA) were capped and sealed with nail polish. Representative images were taken with a confocal microscope (LSM 880, zeiss, jena, germany). The primary antibodies used were as follows: rat anti-GFAP (astrocyte marker, 1:1000, cat#13-0300, invitrogen), guinea pig anti-NeuN (neuronal marker, 1:1000, cat#ABN90, millipore), mouse anti-neuroD 1 (1:500, cat#ab60704, abcam) and chicken anti-GFP (1:1000, cat#ab13970, abcam). The fluorescence microscope (Axio image Z2, zeiss, & gt was used by Zeiss Axioplan>Germany) or confocal microscopy (LSM 880, zeiss, jena, germany). Quantitative analysis was performed from 3 brain sections per mouse (3 mice per group) based on 4 randomly selected fields (212 μm x μ, 212 μm, obtained from LSM880 confocal microscopy at 400 x magnification). Data are shown as mean ± SEM.

Control virus P12 expressing GFP reporter alone was first compared to NeuroD1 expressing viruses P134 and P138 (both P12 were added together to track transformed neurons). When control virus P12 was injected into the uninjured mouse cortex, the infected cells were predominantly astrocytes without NeuroD1 expression at 10dpi (days post injection) (fig. 42). In contrast, neuroD1 expression was clearly detected in both P134 and P138 groups. While most of the NeuroD 1-expressing cells in the P138 group were still astrocytes at 10dpi, some of the NeuroD 1-expressing cells in the P134 group were already neun+ neurons, indicating that P134 may have better conversion capacity than P138. Furthermore, analysis of cortical brain tissue at 10dpi in group P134 mice showed high levels of astrocyte conversion to neurons, as evidenced by morphological changes in GFP positive cells such as the presence of long projections (fig. 42). Group P138 showed lower levels of conversion.

The infected cells in the control group (P12) remained astrocytes 30 days after virus injection, but most GFP-positive cells in the P134 group were neurons expressing NeuN. However, the conversion rate was lower for group P138 than for group P134. Most of the infected cells in this stage P138 group remain astrocytes and GFP signal in the transformed neurons is weak. Furthermore, analysis of cortical brain tissue at 30dpi in group P134 mice showed even higher levels of astrocyte conversion to neurons, as evidenced by the presence of long projections in GFP positive cells (FIG. 43)

AAV9-P134 virus was also effective in a bilateral lesion mouse model. In normal C57BL/6J mice (over 8 weeks old), ischemic stroke was induced by injecting 1. Mu.L of Endothelin 1,1-31aa (1. Mu.g/. Mu.L) on each side of the cortex. Mice were anesthetized with 20mg/kg of 1.25% Avertin (a mixture of 12.5mg/mL 2, 2-tribromoethanol and 25 μl/mL 2-methyl-2-butanol, sigma, st.Louis, MO, USA) by intraperitoneal injection, and then placed on a stereotactic frame in the prone position. Endothelin-1 (ET-1) and virus were injected by a glass pipette into the motor cortex at +0.2mm anterior and posterior (AP, from the bregma), -1.5mm medial-lateral (ML, from the bregma, left), -0.7mm dorsal-ventral (DV, from the dura). The injection rate was 80nL/min. The pipette is kept in place for about 10 minutes after injection and then slowly withdrawn. Seven days after injection of Endothelin 1, mice were injected with AAV9-P12 and AAV9-P134 viruses as follows:

Group P12: AAV9-P12 5x10 ¹¹ GC/ml, 1. Mu.L, 1 injection per side of cortex (bilateral)

Group P14: AAV 9-P12.5x10 ¹¹ GC/ml+AAV9-P134 2.5x10 ¹¹ GC/ml, 1. Mu.L, 1 injection per side of cortex (bilateral)

Mice were sacrificed 10 days (dpi) after virus injection and cortical tissue was analyzed. When control virus P12 was injected into the cortex of ET-1 injured mice, the infected cells were predominantly astrocytes without NeuroD1 expression at 10dpi (days post injection) (fig. 44). In contrast, neuroD1 expression was detected in the P134 group. Analysis of cortical brain tissue from group P134 mice at 10dpi showed high levels of astrocyte conversion to neurons, as evidenced by the morphological changes observed in GFP positive cells, such as the presence of long projections (fig. 44).

Example 23 expression of transgenes induced by neuroD1/Isl1 and neuroD1/Ascl1 vectors in vitro.

Materials and methods

Cell culture:C8-DIA cells

CD8-DIA cells (ATCC, cat#CRL-2541) are a clonal, permanent cell line with astrocyte or microglial properties, which have been established from explant cultures of mouse brains 8 days postnatal after spontaneous transformation in vitro. (Alliot F, pessac B.brain Res.306:283-291,1984. PubMed:6466977). C8-DIA cells were maintained at 5% CO ₂ In a 37℃incubator, the medium consisted of DMEM supplemented with 10% FBS, 2.5mM glutamine and penicillin/streptomycin. When 90% -100% confluence was reached, cells were subcultured at a ratio of 1:5. The transfection and transduction rates of C8-DIA cells were higher than primary rat astrocytes and were good alternatives for assessing vector gene expression.

Cell culture: lec2 cells

Lec2 cells (ATCC cat#crl1736) are mutant clones of epithelial cell lines derived from CHO (chinese hamster ovary) cell lines. (Stanley P, sininovich L.systemic Cell Genet.3:391-405,1977.PubMed: 60679). Lec2 cells were maintained at 5% CO ₂ In a 37℃incubator, the medium consisted of an alpha MEM supplemented with 10% FBS, 2.5mM glutamine and penicillin/streptomycin. When 90% -100% confluence was reached, cells were subcultured at a ratio of 1:5. Lec2 cells can be transfected and transduced with high efficiency. They are good substitutes for astrocytes evaluating vector gene expression.

And (3) a carrier:vectors were tested by transfection of C8-DIA cells or Lec2 cells. Furthermore, AAV is produced with the selected vector and tested in vitro by transduction:

·NXL-P141(CE-pGfa681-CRGI-hIsl1-oPRE-bGHpA)

·NXL-P142(CE-pGfa681-CI-hIsl1-p2A-hND1-bGHpA)

·NXL-P143(CE-pGfa681-CI-hND1-p2A-hIsl1-bGHpA)

·NXL-P144(CE-pGfa681-CI-hIsl1-IRES-hND1-bGHpA)

·NXL-P181(CE-pGfa681-hIsl1-SpA)

·NXL-P143(CE-pGfa681-CI-hND1-p2A-hIsl1-bGHpA)

·NXL-P144(CE-pGfa681-CI-hIsl1-IRES-hND1-bGHpA)

·NXL-P151(CE-pGfa681-CRGI-hAscl1-oPRE-bGHpA)

·NXL-P152(CE-pGfa681-CI-hAscl1-IRES-hND1-bGHpA)

Virus production:viral use adherence for in vitro studiesAAV293Cells were generated by triple transfection (GOI, helper and Rep/Cap plasmids) using Polyethylenimine (PEI). Virus recovery and purification is achieved by ultracentrifugation or using commercial purification kits.

Specifically, AAV293 cells (Cell Biolabs, cat# AAV-100) were seeded in 15-cm dishes 24 hours prior to transfection at a confluency of 70% -85%. Cells were transfected with 10ug GOI, 10ug Rep/Cap, 14ug pALD-X80 (Alveron) or pHelper (Cell Biolabs) in each dish using PEI at a DNA to PEI ratio of 1:4. Multiple dishes were transfected for production according to the desired scale. The medium was refreshed daily. 72 hours after transfection, cells were collected and lysed to harvest virus using AAVpro purification kit (Takara, cat#6666,6675,6235) according to the manufacturer's protocol. Viral titers were determined by real-time quantitative PCR using primer pairs in the ITR region or vector-specific primers. Plasmid DNA was used as a standard. Virus production yield of about 10 ³ To 10 ⁴ vg/cell level.

Transfection and immunofluorescence: lec2 cells were seeded on 24-well plate glass coverslips at a confluency of 30% -50% 24 hours prior to transfection. Each cell was transfected with 500ng of plasmid DNA using Lipofectamine reagent (Thermo Fisher Cat # 15338) according to the manufacturer's protocol. 24-48 hours after transfection, cells were fixed with 4% paraformaldehyde in PBS, followed by washing and washing Immunostaining was performed with anti-ND 1 (Abcam cat#ab 60704) antibodies and/or anti-Isl 1 (DSHB, 40.2D6) and/or anti-Ascl 1 (BD Bioscience, cat# 556604) followed by immunostaining with a secondary antibody conjugated with a fluorescent dye (Invitrogen, alexa Fluor). Images were taken under a fluorescence microscope (Zeiss Axiovert A1, zen Blue). The gene expression level was evaluated by comparing the fluorescence intensities.

Transduction and immunofluorescence:24 hours prior to transduction, C8-DIA cells were seeded on 24-well plate glass coverslips at a confluency of 30% -50%. With 2-6X10 in fresh medium ¹⁰ vg/ml of virus transduced cells. The medium was refreshed the next day and every 3-4 days. Three to six days post-transfection, cells were fixed with 4% paraformaldehyde in PBS, followed by washing and immunostaining with anti-ND 1 (Abcam cat#ab 60704) antibodies and/or anti-Isl 1 (DSHB, 40.2D6) and/or anti-Ascl 1 (BD Bioscience, cat# 556604), followed by immunostaining with a secondary antibody conjugated with a fluorescent dye (Invitrogen, alexafluor). Images were taken under a fluorescence microscope (Zeiss Axiovert A1, zen Blue). The gene expression level was evaluated by comparing the fluorescence intensities.

Results of in vitro studies:

the hIs and hlsl 1/hND1 constructs tested effectively driven expression of hlsl 1 and/or hND1 by transfection and/or transduction of cultured cells in which positive staining of hlsl 1 and hND1 demonstrated this (fig. 46-49, 51 and 52). The tested hASCl1 and hASCl1/hND1 constructs effectively driven expression of hASCl1 and/or hND1 by transfection and/or transduction of cultured cells in which positive staining of hASCl1 and hND1 demonstrated this (FIGS. 53-56).

Various further modifications and improvements to the compositions and methods of the present disclosure will be apparent to those skilled in the art. The following non-limiting examples are contemplated:

1. an adeno-associated virus (AAV) vector comprising: a human neurogenic differentiation factor 1 (hNeuroD 1) sequence, the hNeuroD1 sequence comprising a nucleic acid sequence of SEQ ID No. 6, and a second sequence comprising a nucleic acid sequence selected from the group consisting of SEQ ID nos. 11, 13, and 15, wherein the hNeuroD1 sequence and the second sequence are separated by: (i) a P2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID nos. 19 and 22, (ii) a T2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID nos. 20 and 23, or (iii) an Internal Ribosome Entry Site (IRES) sequence of an encephalomyocarditis virus comprising SEQ ID No. 3, wherein the hNeuroD1 sequence and the second sequence are operably linked to a regulatory element comprising:

(a) A Glial Fibrillary Acidic Protein (GFAP) promoter comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs 4, 18 and 27;

(b) An enhancer from the human elongation factor-1 alpha (EF 1-alpha) promoter comprising the nucleic acid sequence of SEQ ID NO. 2 or a Cytomegalovirus (CMV) enhancer comprising the nucleic acid sequence of SEQ ID NO. 17;

(c) A chimeric intron comprising the nucleic acid sequence of SEQ ID NO. 5 or 28;

(d) A woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs 7 and 30; and

(e) An SV40 polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO. 8, an hGH polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO. 21, a synthetic polyadenylation signal comprising SEQ ID NO. 31 or a bGH polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO. 26.

2. An adeno-associated virus (AAV) vector comprising: a nucleic acid coding sequence encoding a human neurogenic differentiation factor 1 (hNeuroD 1) protein, said hNeuroD1 protein comprising the amino acid sequence of SEQ ID No. 10, and a second nucleic acid coding sequence encoding a second protein having an amino acid selected from the group consisting of SEQ ID nos. 12, 14 and 16, wherein said hNeuroD1 coding sequence and said second coding sequence are separated by: (i) a P2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID nos. 19 and 22, (ii) a T2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID nos. 20 and 23, or (iii) an Internal Ribosome Entry Site (IRES) sequence of an encephalomyocarditis virus comprising SEQ ID No. 3, wherein the hNeuroD1 coding sequence and the second coding sequence are operably linked to a regulatory element comprising:

(e) An SV40 polyadenylation signal having the nucleic acid sequence of SEQ ID NO. 8, an hGH polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO. 21, a synthetic polyadenylation signal comprising SEQ ID NO. 31 or a bGH polyadenylation signal comprising the nucleic acid sequence of SEQ ID NO. 26.

3. An adeno-associated virus (AAV) vector comprising a NeuroD1 nucleic acid coding sequence encoding a NeuroD1 (NeuroD 1) protein and a second nucleic acid coding sequence encoding a second protein, wherein the NeuroD1 coding sequence and the second protein coding sequence are separated by a linker sequence, wherein the NeuroD1 coding sequence and the second coding sequence are operably linked to a regulatory element comprising:

(a) Glial Fibrillary Acidic Protein (GFAP) promoter;

(b) An enhancer;

(c) Chimeric introns;

(d) Woodchuck hepatitis virus posttranscriptional regulatory elements (WPREs); and

(e) Polyadenylation signals.

4. A composition comprising an adeno-associated virus (AAV) vector for converting human glial cells to functional neurons, wherein the AAV vector comprises: a human neurogenic differentiation factor 1 (hNeuroD 1) sequence, the hNeuroD1 sequence having the nucleic acid sequence of SEQ ID No. 6, and a second sequence comprising a nucleic acid sequence selected from the group consisting of SEQ ID nos. 11, 13 and 15, wherein the hNeuroD1 sequence and the second sequence are separated by: (i) a P2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID nos. 19 and 22, (ii) a T2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID nos. 20 and 23, or (iii) an Internal Ribosome Entry Site (IRES) sequence of an encephalomyocarditis virus comprising SEQ ID No. 3, wherein the hNeuroD1 sequence and the second sequence are operably linked to a regulatory element comprising:

(a) A human Glial Fibrillary Acidic Protein (GFAP) promoter comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs 4, 18 and 27;

(b) An enhancer from the human elongation factor-1 alpha (EF-1 alpha) promoter comprising the nucleic acid sequence of SEQ ID NO. 2 or a Cytomegalovirus (CMV) enhancer comprising the nucleic acid sequence of SEQ ID NO. 17;

5. A composition comprising an adeno-associated virus (AAV) vector for converting human glial cells to functional neurons, wherein the AAV vector comprises: a nucleic acid coding sequence encoding a human neurogenic differentiation factor 1 (hNeuroD 1) protein, said hNeuroD1 protein comprising the amino acid sequence of SEQ ID No. 10, and a second nucleic acid coding sequence encoding a second protein having an amino acid selected from the group consisting of SEQ ID nos. 12, 14 and 16, wherein said hNeuroD1 coding sequence and said second coding sequence are separated by: (i) a P2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID nos. 19 and 22, (ii) a T2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID nos. 20 and 23, or (iii) an Internal Ribosome Entry Site (IRES) sequence of an encephalomyocarditis virus comprising SEQ ID No. 3, wherein the hNeuroD1 coding sequence and the second coding sequence are operably linked to a regulatory element comprising:

6. A composition comprising an adeno-associated virus (AAV) vector for treating a subject in need thereof, wherein the AAV vector comprises a neurogenic differentiation factor 1 (NeuroD 1) sequence and a second protein sequence, wherein the NeuroD1 sequence and the second protein sequence are separated by a linker sequence, wherein the NeuroD1 sequence and the second sequence are operably linked to an expression control element, the expression control element

The piece comprises:

(a) Glial Fibrillary Acidic Protein (GFAP) promoter;

(b) An enhancer;

(c) Chimeric introns;

(e) Polyadenylation signals.

7. The AAV vector of any one of embodiments 1-3 or composition of any one of embodiments 4-6, wherein the AAV vector is selected from the group consisting of AAV serotype 2, AAV serotype 5, and AAV serotype 9.

8. The AAV vector or composition of embodiment 7, wherein the AAV vector is AAV serotype 2.

9. The AAV vector or composition of embodiment 7, wherein the AAV vector is AAV serotype 5.

10. The AAV vector or composition of embodiment 7, wherein the AAV vector is AAV serotype 9.

11. The composition of embodiment 4 or 5, wherein the glial cell is a reactive astrocyte.

12. The composition of embodiment 4 or 5, wherein the functional neuron is selected from the group consisting of a glutamatergic neuron, a gabaergic neuron, a dopaminergic neuron, a cholinergic neuron, a serotonergic neuron, an adrenergic neuron, a motor neuron, and a peptidoergic neuron.

13. The composition of embodiments 4 or 5, wherein the human suffers from a neurological disorder.

14. The AAV vector of example 3 or the composition of example 6, wherein the NeuroD1 is human NeuroD1 (hNeuroD 1).

15. The AAV vector of example 3 or the composition of example 6, wherein the second protein is selected from the group consisting of acetate-scale family BHLH transcription factor 1 (Ascl 1), insulin gene enhancer protein (ISL 1), and LIM-homeobox 3 (LHX 2).

16. The AAV vector or composition of embodiment 15, wherein the second protein is Ascl1.

17. The AAV vector or composition of embodiment 16, wherein the Ascl1 is human Ascl1 (hal 1).

18. The AAV vector or composition of embodiment 15, wherein the second protein is ISL1.

19. The AAV vector or composition of embodiment 18, wherein the ISL1 is human ISL1 (hsl 1).

20. The AAV vector or composition of embodiment 15, wherein the second protein is LHX3.

21. The AAV vector or composition of embodiment 20, wherein the LHX3 is human LHX3 (hLHX 3).

22. The AAV vector of example 3 or the composition of example 6, wherein the NeuroD1 is selected from the group consisting of chimpanzee NeuroD1, bonobo NeuroD1, red-chimpanzee NeuroD1, gorilla NeuroD1, macaque NeuroD1, marmoset NeuroD1, pigtail NeuroD1, baboon NeuroD1, gibbon NeuroD1, and marmoset NeuroD 1.

23. The AAV vector or composition of embodiment 14, wherein the hNeuroD1 comprises a nucleic acid encoding sequence encoding an amino acid sequence at least 80% identical or similar to: SEQ ID NO. 10.

24. The AAV vector or composition of embodiment 17, wherein the hAscl1 comprises a nucleic acid encoding sequence encoding an amino acid sequence at least 80% identical or similar to: SEQ ID NO. 12.

25. The AAV vector or composition of embodiment 19, wherein the hsl 1 comprises a nucleic acid coding sequence encoding an amino acid sequence at least 80% identical or similar to: SEQ ID NO. 14.

26. The AAV vector or composition of embodiment 21, wherein the hLHX3 comprises a nucleic acid coding sequence encoding an amino acid sequence that is at least 80% identical or similar to: SEQ ID NO. 16.

27. The AAV vector or composition of embodiment 14, wherein the hNeuroD1 sequence comprises a nucleic acid sequence that is at least 80% identical to: SEQ ID NO. 6, or a complement thereof.

28. The AAV vector or composition of embodiment 17, wherein the hAscl1 sequence comprises a nucleic acid sequence that is at least 80% identical to: SEQ ID NO. 11, or a complement thereof.

29. The AAV vector or composition of embodiment 19, wherein the hsl 1 sequence comprises a nucleic acid sequence that is at least 80% identical to: SEQ ID NO. 13, or a complement thereof.

30. The AAV vector or composition of embodiment 21, wherein the hLHX3 sequence comprises a nucleic acid sequence that is at least 80% identical to: SEQ ID NO. 15, or a complement thereof.

31. The AAV vector of example 3 or the composition of example 6, wherein the linker is selected from the group consisting of P2A and T2A.

32. The AAV vector or composition of embodiment 31, wherein the linker is the P2A.

33. The AAV vector or composition of embodiment 31, wherein the linker is the T2A.

34. The AAV vector or composition of embodiment 31, wherein the P2A linker comprises a nucleic acid sequence that is at least 80% identical to a sequence selected from the group consisting of: SEQ ID NOS 19 and 22, or the complements thereof.

35. The AAV vector or composition of embodiment 31, wherein the T2A linker comprises a nucleic acid sequence that is at least 80% identical to a sequence selected from the group consisting of: SEQ ID NOS 20 and 23, or the complements thereof.

36. The AAV vector of example 3 or composition of example 6, wherein the GFAP promoter is a human GFAP (hGFAP) promoter.

37. The AAV vector of example 3 or the composition of example 6, wherein the GFAP promoter is selected from the group consisting of a chimpanzee GFAP promoter, a bonoban GFAP promoter, a chimpanzee GFAP promoter, a macaque GFAP promoter, a marmoset GFAP promoter, a pigtail GFAP promoter, a baboon GFAP promoter, a gibbon GFAP promoter, and a lemur GFAP promoter.

38. The AAV vector or composition of any one of the preceding embodiments, wherein the IRES sequence comprises a nucleic acid sequence at least 80% identical to: SEQ ID NO. 3, or a complement thereof.

39. The AAV vector or composition of embodiment 36, wherein the hGFAP promoter comprises a nucleic acid sequence that is at least 80% identical to: SEQ ID NO. 4, or a complement thereof.

40. The AAV vector or composition of embodiment 36, wherein the hGFAP promoter comprises a nucleic acid sequence that is at least 80% identical to: 18, or the complement thereof.

41. The AAV vector or composition of embodiment 36, wherein the hGFAP promoter comprises a nucleic acid sequence that is at least 80% identical to: SEQ ID NO. 27, or a complement thereof.

42. The AAV vector of example 3 or the composition of example 6, wherein the enhancer is selected from the group consisting of an enhancer from the human elongation factor-1 alpha (EF 1-alpha) promoter and a Cytomegalovirus (CMV) enhancer.

43. The AAV vector or composition of embodiment 42, wherein the EF 1-a comprises a nucleic acid sequence that is at least 80% identical to: SEQ ID NO. 2, or a complement thereof.

44. The AAV vector or composition of embodiment 42, wherein the CMV enhancer comprises a nucleic acid sequence at least 80% identical to: SEQ ID NO. 17, or a complement thereof.

45. The AAV vector of example 3 or composition of example 6, wherein the chimeric intron comprises a nucleic acid sequence that is at least 80% identical to: SEQ ID NO. 5, or a complement thereof.

46. The AAV vector of example 3 or composition of example 6, wherein the chimeric intron comprises a nucleic acid sequence that is at least 80% identical to: SEQ ID NO. 28, or a complement thereof.

47. The AAV vector of embodiment 3 or composition of embodiment 6, wherein the WPRE comprises a nucleic acid sequence at least 80% identical to a nucleic acid sequence selected from the group consisting of: SEQ ID NOS.7 and 30, or the complements thereof.

48. The AAV vector of example 3 or composition of example 6, wherein the polyadenylation signal is selected from the group consisting of SV40 polyadenylation signal, hGH polyadenylation signal, synthetic polyadenylation signal, and bGH polyadenylation signal.

49. The AAV vector or composition of embodiment 48, wherein the SV40 polyadenylation signal comprises a nucleic acid sequence that is at least 80% identical to: SEQ ID NO. 8, or a complement thereof.

50. The AAV vector or composition of embodiment 48, wherein the hGH polyadenylation signal comprises a nucleic acid sequence that is at least 80% identical to: SEQ ID NO. 21, or a complement thereof.

51. The AAV vector or composition of embodiment 48, wherein the hGH polyadenylation signal comprises a nucleic acid sequence that is at least 80% identical to: SEQ ID NO. 26, or a complement thereof.

52. The AAV vector of example 3, or composition of example 6, wherein the AAV vector further comprises a nucleic acid sequence encoding an AAV protein sequence.

53. The AAV vector of any one of embodiments 1-3, or composition of any one of embodiments 4-6, wherein the AAV vector comprises an AAV serotype 2 Inverted Terminal Repeat (ITR).

54. The AAV vector of any one of embodiments 1-3, or composition of any one of embodiments 4-6, wherein the AAV vector comprises an AAV serotype 5 Inverted Terminal Repeat (ITR).

55. The AAV vector of any one of embodiments 1-3, or composition of any one of embodiments 4-6, wherein the AAV vector comprises an AAV serotype 9 Inverted Terminal Repeat (ITR).

56. The AAV vector of any one of embodiments 1-3, or composition of any one of embodiments 4-6, wherein the AAV vector comprises at least one ITR nucleic acid sequence at least 80% identical to: SEQ ID NO. 1.

57. The AAV vector of any one of embodiments 1-3, or composition of any one of embodiments 4-6, wherein the AAV vector comprises at least one ITR nucleic acid sequence at least 80% identical to: SEQ ID NO. 9.

58. The composition of embodiment 6, wherein the subject in need thereof is a mammal.

59. The composition of embodiment 58 wherein the mammal is a human.

60. The composition of embodiment 58, wherein the mammal is a non-human primate.

61. The composition of embodiment 6, wherein the subject in need thereof has a neurological disorder.

62. The composition of embodiments 13 or 61, wherein the neurological disorder comprises an injury to the Central Nervous System (CNS) or peripheral nervous system.

63. The composition of embodiments 13 or 61, wherein the neurological condition comprises injury to the CNS.

64. The composition of embodiments 13 or 61, wherein the neurological disorder is selected from the group consisting of: alzheimer's disease, parkinson's disease, amyotrophic Lateral Sclerosis (ALS), huntington's disease, epilepsy, physical injury, stroke, cerebral aneurysms, traumatic brain injury, concussion, tumors, inflammation, infection, ataxia, brain atrophy, spinal cord atrophy, multiple sclerosis, traumatic spinal cord injury, ischemic or hemorrhagic myelopathy (myelopathy), global cerebral ischemia, hypoxic ischemic encephalopathy, embolism, fibrocartilage embolic myelopathy, thrombosis, kidney disease, chronic inflammatory diseases, meningitis, and cerebral venous sinus thrombosis.

65. The composition of embodiment 13 or 61, wherein the neurological disorder is alzheimer's disease.

66. The composition of embodiment 13 or 61, wherein the neurological disorder is parkinson's disease.

67. The composition of embodiment 13 or 61, wherein the neurological disorder is ALS.

68. The composition of embodiment 13 or 61, wherein the neurological disorder is huntington's disease.

69. The composition of embodiment 13 or 61, wherein the neurological disorder is stroke.

70. The composition of embodiment 69, wherein the stroke is ischemic stroke.

71. The composition of embodiment 69, wherein the stroke is hemorrhagic stroke.

72. The composition of embodiment 61, wherein the composition is capable of converting at least one glial cell into neurons.

73. The composition of embodiment 72, wherein the glial cell is selected from the group consisting of an astrocyte and a NG2 cell.

74. The composition of embodiment 72, wherein the glial cell is an astrocyte.

75. The composition of embodiment 74, wherein the astrocytes are reactive astrocytes.

76. The composition of embodiment 72, wherein the glial cells are GFAP positive.

77. The composition of embodiment 72, wherein the neuron is a functional neuron.

78. The composition of embodiment 72, wherein the functional neuron is selected from the group consisting of a glutamatergic neuron, a gabaergic neuron, a dopaminergic neuron, a cholinergic neuron, a serotonergic neuron, an adrenergic neuron, a motor neuron, and a peptidoergic neuron.

79. The composition of embodiment 78, wherein the functional neuron is a glutamatergic neuron.

80. The composition of embodiment 6, wherein the composition is formulated for delivery to a subject in need thereof.

81. The composition of embodiment 80, wherein the composition is formulated for topical delivery.

82. The composition of embodiment 80, wherein the composition is formulated for systemic delivery.

83. The composition of any one of embodiments 80-82, wherein the composition is formulated for delivery via intraperitoneal, intramuscular, intravenous, intrathecal, intracerebral, intracranial, intraventricular of the brain, intracavitary of the cerebellum, intravitreal, subretinal, intraparenchymal, intranasal, or oral administration.

84. A method comprising delivering the composition of example 6 to the subject in need thereof.

85. The method of embodiment 84, wherein the composition is formulated for delivery to a subject in need thereof.

86. The method of embodiment 84, wherein the delivering comprises topical administration.

87. The method of embodiment 84, wherein said delivering comprises systemic administration.

88. The method of any one of embodiments 84-87, wherein the delivering comprises intraperitoneal, intramuscular, intravenous, intrathecal, intracerebral, intracranial, lateral intracerebroventricular, intravitreal, intraretinal, intraparenchymal, intranasal, or oral administration.

89. A method of converting reactive astrocytes in a living human brain into functional neurons, comprising: injecting an adeno-associated virus (AAV) into a subject in need thereof, wherein the AAV comprises a DNA vector construct comprising: a human neurogenic differentiation factor 1 (hNeuroD 1) sequence, the hNeuroD1 sequence comprising a nucleic acid sequence of SEQ ID No. 6, and a second sequence comprising a nucleic acid sequence selected from the group consisting of SEQ ID nos. 11, 13, and 15, wherein the hNeuroD1 sequence and the second sequence are separated by: (i) a P2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID nos. 19 and 22, (ii) a T2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID nos. 20 and 23, or (iii) an Internal Ribosome Entry Site (IRES) sequence of an encephalomyocarditis virus comprising SEQ ID No. 3, wherein the hNeuroD1 sequence and the second sequence are operably linked to a regulatory element comprising:

90. A method of converting reactive astrocytes in a living human brain into functional neurons, comprising: injecting an adeno-associated virus (AAV) into a subject in need thereof, wherein the AAV comprises a DNA vector construct comprising: a nucleic acid coding sequence encoding a human neurogenic differentiation factor 1 (hNeuroD 1) protein, said hNeuroD1 protein comprising the amino acid sequence of SEQ ID No. 10, and a second nucleic acid coding sequence encoding a second protein having an amino acid selected from the group consisting of SEQ ID nos. 12, 14 and 16, wherein said hNeuroD1 coding sequence and said second coding sequence are separated by: (i) a P2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID nos. 19 and 22, (ii) a T2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID nos. 20 and 23, or (iii) an Internal Ribosome Entry Site (IRES) sequence of an encephalomyocarditis virus comprising SEQ ID No. 3, wherein the hNeuroD1 coding sequence and the second nucleic acid coding sequence are operably linked to an expression control element comprising:

91. A method of converting glial cells into neurons in a subject in need thereof, comprising: delivering an adeno-associated virus (AAV) to the subject in need thereof, wherein the AAV comprises a DNA vector construct comprising a neurogenic differentiation factor 1 (NeuroD 1) sequence and a second protein sequence, wherein the NeuroD1 sequence and the second protein sequence are separated by a linker, wherein the NeuroD1 sequence and the second sequence are operably linked to an expression control element comprising:

(a) Glial Fibrillary Acidic Protein (GFAP) promoter;

(b) An enhancer;

(c) Chimeric introns;

(e) And a polyadenylation signal, which is a polyadenylation signal,

wherein the vector is capable of converting at least one glial cell to a neuron in the subject in need thereof.

92. A method of treating a neurological disorder in a subject in need thereof, comprising: delivering an adeno-associated virus (AAV) to the subject, wherein the AAV administered to the subject in need thereof comprises a DNA vector construct comprising a neurogenic differentiation factor 1 (NeuroD 1) sequence and a second sequence, wherein the NeuroD1 sequence and the second protein sequence are separated by a linker, wherein the NeuroD1 sequence and the second protein sequence are operably linked to an expression control element, the method comprising

The expression control element comprises:

(a) Glial Fibrillary Acidic Protein (GFAP) promoter;

(b) An enhancer from the human elongation factor-1 alpha (EF-1 alpha) promoter;

(c) Chimeric introns;

(e) SV40 polyadenylation signal.

93. The method of any one of embodiments 89-92, wherein the AAV vector is selected from the group consisting of AAV serotype 2, AAV serotype 5, and AAV serotype 9.

94. The method of embodiment 93, wherein the AAV is AAV serotype 2.

95. The method of embodiment 93, wherein the AAV is AAV serotype 5.

96. The method of embodiment 93, wherein the AAV is AAV serotype 9.

97. The method of embodiment 89 or 90, wherein the functional neuron is a glutamatergic neuron, a gabaergic neuron, a dopaminergic neuron, a cholinergic neuron, a serotonergic neuron, an adrenergic neuron, a motor neuron, and a peptidoergic neuron.

98. The method of embodiment 91 or 92, wherein the NeuroD1 is human NeuroD1 (hNeuroD 1).

99. The method of embodiment 91 or 92, wherein the second protein is selected from the group consisting of actete-scale family BHLH transcription factor 1 (Ascl 1), insulin gene enhancer protein (ISL 1), and LIM-homeobox 3 (LHX 2).

100. The method of embodiment 99, wherein the second protein is Ascl1.

101. The method of embodiment 100, wherein the Ascl1 is human Ascl1 (hAscl 1).

102. The method of embodiment 99, wherein the second protein is ISL1.

103. The method of embodiment 102, wherein the ISL1 is human ISL1 (hISL 1)

104. The method of embodiment 99 wherein the second protein is LHX3.

105. The method of embodiment 104 wherein the LHX3 is human LHX3 (hLHX 3).

106. The method of embodiment 91 or 92, wherein the NeuroD1 is selected from the group consisting of chimpanzee NeuroD1, bonobo NeuroD1, chimpanzee NeuroD1, gorilla NeuroD1, macaque NeuroD1, marmoset NeuroD1, pigtail NeuroD1, baboon NeuroD1, gibbon NeuroD1, and marmoset NeuroD 1.

107. The method of embodiment 98, wherein the hNeuroD1 comprises an amino acid coding sequence encoding an amino acid sequence at least 80% identical or similar to: SEQ ID NO. 10.

108. The method of embodiment 98, wherein the fscl 1 comprises an amino acid coding sequence that encodes an amino acid sequence that is at least 80% identical or similar to: SEQ ID NO. 12.

109. The method of embodiment 103, wherein the hsl 1 comprises an amino acid coding sequence encoding an amino acid sequence at least 80% identical or similar to: SEQ ID NO. 14.

110. The method of embodiment 105, wherein the hLHX3 comprises an amino acid coding sequence that encodes an amino acid sequence that is at least 80% identical or similar to: SEQ ID NO. 16. The method of embodiment 98, wherein the hNeuroD1 coding sequence comprises a nucleic acid sequence that is at least 80% identical to: SEQ ID NO. 6, or a complement thereof.

111. The method of embodiment 98, wherein the fscl 1 coding sequence comprises a nucleic acid sequence that is at least 80% identical to: SEQ ID NO. 11, or a complement thereof.

112. The method of embodiment 103, wherein the hsl 1 coding sequence comprises a nucleic acid sequence that is at least 80% identical to: SEQ ID NO. 13, or a complement thereof.

113. The method of embodiment 105 wherein the hLHX3 coding sequence comprises a nucleic acid sequence that is at least 80% identical to: SEQ ID NO. 15, or a complement thereof.

114. The method of embodiment 91 or 92, wherein the GFAP promoter is a human GFAP (hGFAP) promoter.

115. The method of embodiment 91 or 92, wherein the GFAP promoter is selected from the group consisting of a chimpanzee GFAP promoter, a bonobo GFAP promoter, a chimpanzee GFAP promoter, a gorilla GFAP promoter, a macaque GFAP promoter, a marmoset GFAP promoter, a pigtail monkey GFAP promoter, a baboon GFAP promoter, a gibbon ape GFAP promoter, and a marmoset GFAP promoter.

116. The method of any one of embodiments 89 to 115, wherein the IRES sequence comprises a nucleic acid sequence that is at least 80% identical to: SEQ ID NO. 3, or a complement thereof.

117. The method of embodiment 114, wherein the hGFAP promoter comprises a nucleic acid sequence that is at least 80% identical to: SEQ ID NO. 4, or a complement thereof.

118. The method of embodiment 114, wherein the hGFAP promoter comprises a nucleic acid sequence that is at least 80% identical to: 18, or the complement thereof.

119. The method of embodiment 114, wherein the hGFAP promoter comprises a nucleic acid sequence that is at least 80% identical to: SEQ ID NO. 27, or a complement thereof.

120. The method of embodiment 91 or 92, wherein the linker is selected from the group consisting of P2A or T2A.

121. The method of embodiment 120, wherein the P2A linker comprises a nucleic acid sequence that is at least 80% identical to a sequence selected from the group consisting of: SEQ ID NOS 19 and 22, or the complements thereof.

122. The method of embodiment 120, wherein the T2A linker comprises a nucleic acid sequence that is at least 80% identical to a sequence selected from the group consisting of: SEQ ID NOS 20 and 23, or the complements thereof.

123. The method of embodiment 91 or 92, wherein the enhancer comprises a nucleic acid sequence that is at least 80% identical to: SEQ ID NO. 2, or a complement thereof.

124. The method of embodiment 91 or 92, wherein the chimeric intron comprises a nucleic acid sequence that is at least 80% identical to: SEQ ID NO. 5 or 28, or a complement thereof.

125. The method of embodiments 91 or 92, wherein the WPRE comprises a nucleic acid sequence at least 80% identical to a nucleic acid sequence selected from the group consisting of: SEQ ID NOS.7 and 30, or the complements thereof.

126. The method of embodiment 91 or 92, wherein the SV40 polyadenylation signal comprises a nucleic acid sequence that is at least 80% identical to: SEQ ID NOS.8, 21, 26, or the complements thereof.

127. The method of embodiment 91 or 92, wherein the vector further comprises a nucleic acid sequence encoding an AAV protein sequence.

128. The method of any one of embodiments 89 to 92, wherein the vector comprises an AAV serotype 2 Inverted Terminal Repeat (ITR).

129. The method of any one of embodiments 89 to 92, wherein the vector comprises an AAV serotype 5 Inverted Terminal Repeat (ITR).

130. The method of any one of embodiments 89 to 92, wherein the vector comprises an AAV serotype 9 Inverted Terminal Repeat (ITR).

131. The method of any one of embodiments 89 to 92, wherein the vector comprises at least one ITR nucleic acid sequence at least 80% identical to: SEQ ID NO. 1.

132. The method of any one of embodiments 89 to 92, wherein the vector comprises at least one ITR nucleic acid sequence at least 80% identical to: SEQ ID NO. 9.

133. The method of embodiment 91, wherein the transformation occurs in the Central Nervous System (CNS) or peripheral nervous system.

134. The method of embodiment 91, wherein said transforming occurs in the CNS.

135. The method of embodiment 91 or 92, wherein the subject in need thereof is a mammal. 136. The method of embodiment 135 wherein the mammal is a human.

137. The method of embodiment 135, wherein the mammal is a non-human primate.

138. The method of embodiment 91 or 92, wherein said delivering comprises topical administration.

139. The method of embodiment 91 or 92, wherein said delivering comprises systemic administration.

140. The method of embodiment 91 or 92, wherein said delivering comprises administration selected from the group consisting of: intraperitoneal administration, intramuscular administration, intravenous administration, intrathecal administration, intracerebral administration, intracranial, lateral cerebral compartments of the brain, intracavitary, intravitreal, subretinal, intraparenchymal administration, intranasal administration, and oral administration.

141. The method of embodiment 91 or 92, wherein the injecting comprises an injection selected from the group consisting of: intraperitoneal injection, intramuscular injection, intravenous injection, intrathecal injection, intracerebral injection, intracranial, lateral ventricle of the brain, intracavitary, intravitreal, subretinal, intraparenchymal injection, intranasal injection, and oral injection.

142. The method of embodiment 91 or 92, wherein said delivering comprises injection.

143. The method of any one of embodiments 89, 90 or 142, wherein said injecting is at between 10 ¹⁰ particle/mL and 10 ¹⁴ Concentration between individual particles/mL.

144. The method of embodiment 143, wherein the injecting further comprises a flow rate of between 0.1 uL/min and 5.0 uL/min.

145. The method of embodiment 91, wherein the at least one glial cell is selected from the group consisting of at least one astrocyte and at least one NG2 cell.

146. The method of embodiment 145, wherein the at least one glial cell is at least one astrocyte.

147. The method of embodiment 145 or 146, wherein the at least one astrocyte is a reactive astrocyte.

148. The method of embodiment 91, wherein the neuron is a functional neuron.

149. The method of any one of embodiments 89, 90 or 148, wherein the functional neuron is selected from the group consisting of a glutamatergic neuron, a gabaergic neuron, a dopaminergic neuron, a cholinergic neuron, a serotonergic neuron, an adrenergic neuron, a motor neuron, and a peptidoergic neuron.

150. The method of embodiment 91, wherein the subject exhibits an improvement in at least one symptom of a neurological disorder as compared to the subject prior to the delivering.

151. The method of embodiment 150, wherein the improvement is measured within 1 year of the delivery.

152. The method of any one of embodiments 89, 90 or 142, wherein the method comprises injecting the AAV directly into the brain of the subject.

153. The method of any one of embodiments 89 or 90, wherein the transformation is in the cerebral cortex of the brain.

154. The method of any one of embodiments 89, 90 or 142, wherein the method comprises injecting the AAV directly into the spinal cord of the subject.

155. The method of embodiment 92, wherein the neurological disorder comprises a damage to the Central Nervous System (CNS) or the peripheral nervous system.

156. The method of embodiment 92, wherein the neurological disorder is selected from the group consisting of: alzheimer's disease, parkinson's disease, amyotrophic Lateral Sclerosis (ALS), huntington's disease, epilepsy, physical injury, stroke, cerebral aneurysms, traumatic brain injury, concussion, tumors, inflammation, infection, ataxia, brain atrophy, spinal cord atrophy, multiple sclerosis, traumatic spinal cord injury, ischemic or hemorrhagic myelopathy (myelopathy), global cerebral ischemia, hypoxic ischemic encephalopathy, embolism, fibrocartilage embolic myelopathy, thrombosis, kidney disease, chronic inflammatory diseases, meningitis, and cerebral venous sinus thrombosis.

157. The method of embodiment 92, wherein the neurological disorder is alzheimer's disease.

158. The method of embodiment 92, wherein the neurological disorder is parkinson's disease.

159. The method of embodiment 92, wherein the neurological disorder is ALS.

160. The method of embodiment 92, wherein the neurological disorder is huntington's disease.

161. The method of embodiment 92, wherein the neurological disorder is stroke.

162. The method of embodiment 161, wherein the stroke is an ischemic stroke.

163. The method of embodiment 161, wherein the stroke is a hemorrhagic stroke.

164. The method of embodiment 92, wherein the method is capable of converting at least one glial cell into a neuron.

165. The method of embodiment 164, wherein the glial cell is selected from the group consisting of an astrocyte and a NG2 cell.

166. The method of embodiment 164, wherein the glial cell is an astrocyte.

167. The method of embodiment 166, wherein the astrocytes are reactive astrocytes.

168. The method of embodiment 164, wherein the glial cells are GFAP positive.

169. The method of embodiment 164, wherein the neuron is a functional neuron.

170. The method of embodiment 169, wherein the functional neuron is selected from the group consisting of a glutamatergic neuron, a gabaergic neuron, a dopaminergic neuron, a cholinergic neuron, a serotonergic neuron, an adrenergic neuron, a motor neuron, and a peptidoergic neuron.

171. The method of embodiment 89 or 90, wherein a therapeutically effective dose of the AAV is injected into the subject.

172. The method of embodiment 91 or 92, wherein a therapeutically effective dose of the AAV is delivered into the subject.

173. The method of embodiment 171 or 172, wherein the therapeutically effective dose is administered with a pharmaceutically acceptable carrier.

174. An adeno-associated virus (AAV) vector comprising: a human neurogenic differentiation factor 1 (hNeuroD 1) sequence, the hNeuroD1 sequence comprising a nucleic acid sequence of SEQ ID No. 6, and a second sequence comprising a nucleic acid sequence selected from the group consisting of SEQ ID nos. 11, 13, and 15, wherein the hNeuroD1 sequence and the second sequence are separated by: (i) a P2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID nos. 19 and 22, (ii) a T2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID nos. 20 and 23, or (iii) an Internal Ribosome Entry Site (IRES) sequence of an encephalomyocarditis virus comprising SEQ ID No. 3, wherein the hNeuroD1 sequence and the second sequence are operably linked to a regulatory element comprising:

(a) A Glial Fibrillary Acidic Protein (GFAP) promoter comprising the nucleic acid sequence of SEQ ID No. 27;

(b) A Cytomegalovirus (CMV) enhancer comprising the nucleic acid sequence of SEQ ID No. 17;

(c) A chimeric intron comprising the nucleic acid sequence of SEQ ID NO. 5;

and

(d) A bGH polyadenylation signal comprising the nucleic acid sequence of SEQ ID No. 26.

175. An adeno-associated virus (AAV) vector comprising: a human neurogenic differentiation factor 1 (hNeuroD 1) sequence, the hNeuroD1 sequence comprising a nucleic acid sequence of SEQ ID No. 6, and a second sequence comprising a nucleic acid sequence selected from the group consisting of SEQ ID nos. 11, 13 and 15, wherein the hNeuroD1 sequence and the second sequence are separated by: (i) a P2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID nos. 19 and 22, (ii) a T2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID nos. 20 and 23, or (iii) an Internal Ribosome Entry Site (IRES) sequence of an encephalomyocarditis virus comprising SEQ ID No. 3, wherein the hNeuroD1 sequence and the second sequence are operably linked to a regulatory element comprising:

(c) A chimeric intron comprising the nucleic acid sequence of SEQ ID NO. 28;

And

176. An adeno-associated virus (AAV) vector comprising: a nucleic acid coding sequence encoding a human neurogenic differentiation factor 1 (hNeuroD 1) protein, said hNeuroD1 protein comprising the amino acid sequence of SEQ ID No. 10, and a second nucleic acid coding sequence encoding a second protein having an amino acid selected from the group consisting of SEQ ID nos. 12, 14 and 16, wherein said hNeuroD1 coding sequence and said second coding sequence are separated by: (i) a P2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID nos. 19 and 22, (ii) a T2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID nos. 20 and 23, or (iii) an Internal Ribosome Entry Site (IRES) sequence of an encephalomyocarditis virus comprising SEQ ID No. 3, wherein the hNeuroD1 coding sequence and the second coding sequence are operably linked to a regulatory element comprising:

(c) A chimeric intron comprising the nucleic acid sequence of SEQ ID NO. 5;

And

177. An adeno-associated virus (AAV) vector comprising: a nucleic acid coding sequence encoding a human neurogenic differentiation factor 1 (hNeuroD 1) protein, said hNeuroD1 protein comprising the amino acid sequence of SEQ ID No. 10, and a second nucleic acid coding sequence encoding a second protein having an amino acid selected from the group consisting of SEQ ID nos. 12, 14 and 16, wherein said hNeuroD1 coding sequence and said second coding sequence are separated by: (i) a P2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID nos. 19 and 22, (ii) a T2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID nos. 20 and 23, or (iii) an Internal Ribosome Entry Site (IRES) sequence of an encephalomyocarditis virus comprising SEQ ID No. 3, wherein the hNeuroD1 coding sequence and the second coding sequence are operably linked to a regulatory element comprising:

(c) A chimeric intron comprising the nucleic acid sequence of SEQ ID NO. 28;

And

178. A composition comprising: (i) An adeno-associated virus (AAV) vector comprising a human neurogenic differentiation factor 1 (hNeuroD 1) sequence, the hNeuroD1 sequence comprising a nucleic acid sequence of SEQ ID NO:6, and (ii) an adeno-associated virus (AAV) comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 11, 13, and 15, wherein the hNeuroD1 sequence is operably linked to a regulatory element comprising:

(c) A chimeric intron comprising the nucleic acid sequence of SEQ ID NO. 28;

(d) A woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) comprising the nucleic acid sequence of SEQ ID NO. 30; and

(e) A bGH polyadenylation signal comprising the nucleic acid sequence of SEQ ID No. 26.

179. The composition of embodiment 178, wherein (ii) comprises an AAV comprising a nucleic acid sequence operably linked to regulatory elements, the nucleic acid sequence comprising SEQ ID NO:13, the regulatory element comprising:

(c) A chimeric intron comprising the nucleic acid sequence of SEQ ID NO. 28;

180. The composition of embodiment 178, wherein (ii) comprises an AAV comprising a nucleic acid sequence operably linked to regulatory elements, the nucleic acid sequence comprising SEQ ID NO:11, the regulatory element comprising:

(c) A chimeric intron comprising the nucleic acid sequence of SEQ ID NO. 28;

181. A composition comprising: (i) An adeno-associated virus (AAV) vector comprising a nucleic acid encoding a human neuro-derived differentiation factor 1 (hNeuroD 1) protein, said hNeuroD1 protein comprising the amino acid sequence of SEQ ID NO. 10, and (ii) an adeno-associated virus (AAV) vector comprising a nucleic acid encoding a protein having an amino acid sequence selected from the group consisting of SEQ ID NO. 12, 14 and 16,

Wherein the hNeuroD1 coding sequence is operably linked to a regulatory element comprising:

(c) A chimeric intron comprising the nucleic acid sequence of SEQ ID NO. 28;

182. The composition of embodiment 181, wherein (ii) comprises an AAV vector comprising an operably linked

A nucleic acid coding sequence encoding a protein having an amino acid sequence comprising SEQ ID No. 14, linked to a regulatory element comprising:

(c) A chimeric intron comprising the nucleic acid sequence of SEQ ID NO. 28;

183. The composition of embodiment 181, wherein (ii) comprises an AAV comprising a polypeptide operably linked to

A nucleic acid sequence of a regulatory element comprising SEQ ID NO:12, the regulatory element comprises:

(c) A chimeric intron comprising the nucleic acid sequence of SEQ ID NO. 28;

184. The AAV vector or composition of embodiment 48, wherein the synthetic polyadenylation signal comprises a nucleic acid sequence that is at least 80% identical to: SEQ ID NO. 31, or a complement thereof.

Sequence listing

<110> NEUEXCELL THERAPEUTICS INC

<120> NEUROD1 combination vector

<130> P34840WO00

<140>

<141>

<150> 63/247,439

<151> 2021-09-23

<150> 63/084,971

<151> 2020-09-29

<160> 34

<170> patent in version 3.5

<210> 1

<211> 128

<212> DNA

<213> artificial sequence

<220>

<223> manual sequence description: synthesis of polynucleotides

<400> 1

tgcaggcagc tgcgcgctcg ctcgctcact gaggccgccc gggcgtcggg cgacctttgg 60

tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga gtggccaact ccatcactag 120

gggttcct 128

<210> 2

<211> 153

<212> DNA

<213> artificial sequence

<220>

<223> manual sequence description: synthesis of polynucleotides

<400> 2

tgcaaagatg gataaagttt taaacagaga ggaatctttg cagctaatgg accttctagg 60

tcttgaaagg agtgggaatt ggctccggtg cccgtcagtg ggcagagcgc acatcgccca 120

cagtccccga gaagttgggg ggaggggtcg gca 153

<210> 3

<211> 553

<212> DNA

<213> artificial sequence

<220>

<223> manual sequence description: synthesis of polynucleotides

<400> 3

acgttactgg ccgaagccgc ttggaataag gccggtgtgc gtttgtctat atgttatttt 60

ccaccatatt gccgtctttt ggcaatgtga gggcccggaa acctggccct gtcttcttga 120

cgagcattcc taggggtctt tcccctctcg ccaaaggaat gcaaggtctg ttgaatgtcg 180

tgaaggaagc agttcctctg gaagcttctt gaagacaaac aacgtctgta gcgacccttt 240

gcaggcagcg gaacccccca cctggcgaca ggtgcctctg cggccaaaag ccacgtgtat 300

aagatacacc tgcaaaggcg gcacaacccc agtgccacgt tgtgagttgg atagttgtgg 360

aaagagtcaa atggctctcc tcaagcgtat tcaacaaggg gctgaaggat gcccagaagg 420

taccccattg tatgggatct gatctggggc ctcggtgcac atgctttaca tgtgtttagt 480

cgaggttaaa aaaacgtcta ggccccccga accacgggga cgtggttttc ctttgaaaaa 540

cacgatgata ata 553

<210> 4

<211> 1667

<212> DNA

<213> artificial sequence

<220>

<223> manual sequence description: synthesis of polynucleotides

<400> 4

ctgcaagcag acctggcagc attgggctgg ccgcccccca gggcctcctc ttcatgccca 60

gtgaatgact caccttggca cagacacaat gttcggggtg ggcacagtgc ctgcttcccg 120

ccgcacccca gcccccctca aatgccttcc gagaagccca ttgagtaggg ggcttgcatt 180

gcaccccagc ctgacagcct ggcatcttgg gataaaagca gcacagcccc ctaggggctg 240

cccttgctgt gtggcgccac cggcggtgga gaacaaggct ctattcagcc tgtgcccagg 300

aaaggggatc aggggatgcc caggcatgga cagtgggtgg caggggggga gaggagggct 360

gtctgcttcc cagaagtcca aggacacaaa tgggtgaggg gactgggcag ggttctgacc 420

ctgtgggacc agagtggagg gcgtagatgg acctgaagtc tccagggaca acagggccca 480

ggtctcaggc tcctagttgg gcccagtggc tccagcgttt ccaaacccat ccatccccag 540

aggttcttcc catctctcca ggctgatgtg tgggaactcg aggaaataaa tctccagtgg 600

gagacggagg ggtggccagg gaaacggggc gctgcaggaa taaagacgag ccagcacagc 660

cagctcatgc gtaacggctt tgtggagctg tcaaggcctg gtctctggga gagaggcaca 720

gggaggccag acaaggaagg ggtgacctgg agggacagat ccaggggcta aagtcctgat 780

aaggcaagag agtgccggcc ccctcttgcc ctatcaggac ctccactgcc acatagaggc 840

catgattgac ccttagacaa agggctggtg tccaatccca gcccccagcc ccagaactcc 900

agggaatgaa tgggcagaga gcaggaatgt gggacatctg tgttcaaggg aaggactcca 960

ggagtctgct gggaatgagg cctagtagga aatgaggtgg cccttgaggg tacagaacag 1020

gttcattctt cgccaaattc ccagcacctt gcaggcactt acagctgagt gagataatgc 1080

ctgggttatg aaatcaaaaa gttggaaagc aggtcagagg tcatctggta cagcccttcc 1140

ttcccttttt tttttttttt ttttgtgaga caaggtctct ctctgttgcc caggctggag 1200

tggcgcaaac acagctcact gcagcctcaa cctactgggc tcaagcaatc ctccagcctc 1260

agcctcccaa agtgctggga ttacaagcat gagccacccc actcagccct ttccttcctt 1320

tttaattgat gcataataat tgtaagtatt catcatggtc caaccaaccc tttcttgacc 1380

caccttccta gagagagggt cctcttgatt cagcggtcag ggccccagac ccatggtctg 1440

gctccaggta ccacctgcct catgcaggag ttggcgtgcc caggaagctc tgcctctggg 1500

cacagtgacc tcagtggggt gaggggagct ctccccatag ctgggctgcg gcccaacccc 1560

accccctcag gctatgccag ggggtgttgc caggggcacc cgggcatcgc cagtctagcc 1620

cactccttca taaagccctc gcatcccagg agcgagcaga gccagag 1667

<210> 5

<211> 133

<212> DNA

<213> artificial sequence

<220>

<223> manual sequence description: synthesis of polynucleotides

<400> 5

gtaagtatca aggttacaag acaggtttaa ggagaccaat agaaactggg cttgtcgaga 60

cagagaagac tcttgcgttt ctgataggca cctattggtc ttactgacat ccactttgcc 120

tttctctcca cag 133

<210> 6

<211> 1068

<212> DNA

<213> Chile person

<400> 6

atgaccaaat cgtacagcga gagtgggctg atgggcgagc ctcagcccca aggtcctcca 60

agctggacag acgagtgtct cagttctcag gacgaggagc acgaggcaga caagaaggag 120

gacgacctcg aagccatgaa cgcagaggag gactcactga ggaacggggg agaggaggag 180

gacgaagatg aggacctgga agaggaggaa gaagaggaag aggaggatga cgatcaaaag 240

cccaagagac gcggccccaa aaagaagaag atgactaagg ctcgcctgga gcgttttaaa 300

ttgagacgca tgaaggctaa cgcccgggag cggaaccgca tgcacggact gaacgcggcg 360

ctagacaacc tgcgcaaggt ggtgccttgc tattctaaga cgcagaagct gtccaaaatc 420

gagactctgc gcttggccaa gaactacatc tgggctctgt cggagatcct gcgctcaggc 480

aaaagcccag acctggtctc cttcgttcag acgctttgca agggcttatc ccaacccacc 540

accaacctgg ttgcgggctg cctgcaactc aatcctcgga cttttctgcc tgagcagaac 600

caggacatgc ccccccacct gccgacggcc agcgcttcct tccctgtaca cccctactcc 660

taccagtcgc ctgggctgcc cagtccgcct tacggtacca tggacagctc ccatgtcttc 720

cacgttaagc ctccgccgca cgcctacagc gcagcgctgg agcccttctt tgaaagccct 780

ctgactgatt gcaccagccc ttcctttgat ggacccctca gcccgccgct cagcatcaat 840

ggcaacttct ctttcaaaca cgaaccgtcc gccgagtttg agaaaaatta tgcctttacc 900

atgcactatc ctgcagcgac actggcaggg gcccaaagcc acggatcaat cttctcaggc 960

accgctgccc ctcgctgcga gatccccata gacaatatta tgtccttcga tagccattca 1020

catcatgagc gagtcatgag tgcccagctc aatgccatat ttcatgat 1068

<210> 7

<211> 589

<212> DNA

<213> artificial sequence

<220>

<223> manual sequence description: synthesis of polynucleotides

<400> 7

aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct 60

ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt 120

atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg 180

tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact 240

ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct 300

attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg 360

ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc 420

gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc 480

aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt 540

cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgc 589

<210> 8

<211> 251

<212> DNA

<213> artificial sequence

<220>

<223> manual sequence description: synthesis of polynucleotides

<400> 8

cgatccaccg gatctagata actgatcata atcagccata ccacatttgt agaggtttta 60

cttgctttaa aaaacctccc acacctcccc ctgaacctga aacataaaat gaatgcaatt 120

gttgttgtta acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca 180

aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc 240

aatgtatctt a 251

<210> 9

<211> 139

<212> DNA

<213> artificial sequence

<220>

<223> manual sequence description: synthesis of polynucleotides

<400> 9

aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 60

ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc 120

gagcgcgcag ctgcctgca 139

<210> 10

<211> 356

<212> PRT

<213> Chile person

<400> 10

Met Thr Lys Ser Tyr Ser Glu Ser Gly Leu Met Gly Glu Pro Gln Pro

1 5 10 15

Gln Gly Pro Pro Ser Trp Thr Asp Glu Cys Leu Ser Ser Gln Asp Glu

20 25 30

Glu His Glu Ala Asp Lys Lys Glu Asp Asp Leu Glu Ala Met Asn Ala

35 40 45

Glu Glu Asp Ser Leu Arg Asn Gly Gly Glu Glu Glu Asp Glu Asp Glu

50 55 60

Asp Leu Glu Glu Glu Glu Glu Glu Glu Glu Glu Asp Asp Asp Gln Lys

65 70 75 80

Pro Lys Arg Arg Gly Pro Lys Lys Lys Lys Met Thr Lys Ala Arg Leu

85 90 95

Glu Arg Phe Lys Leu Arg Arg Met Lys Ala Asn Ala Arg Glu Arg Asn

100 105 110

Arg Met His Gly Leu Asn Ala Ala Leu Asp Asn Leu Arg Lys Val Val

115 120 125

Pro Cys Tyr Ser Lys Thr Gln Lys Leu Ser Lys Ile Glu Thr Leu Arg

130 135 140

Leu Ala Lys Asn Tyr Ile Trp Ala Leu Ser Glu Ile Leu Arg Ser Gly

145 150 155 160

Lys Ser Pro Asp Leu Val Ser Phe Val Gln Thr Leu Cys Lys Gly Leu

165 170 175

Ser Gln Pro Thr Thr Asn Leu Val Ala Gly Cys Leu Gln Leu Asn Pro

180 185 190

Arg Thr Phe Leu Pro Glu Gln Asn Gln Asp Met Pro Pro His Leu Pro

195 200 205

Thr Ala Ser Ala Ser Phe Pro Val His Pro Tyr Ser Tyr Gln Ser Pro

210 215 220

Gly Leu Pro Ser Pro Pro Tyr Gly Thr Met Asp Ser Ser His Val Phe

225 230 235 240

His Val Lys Pro Pro Pro His Ala Tyr Ser Ala Ala Leu Glu Pro Phe

245 250 255

Phe Glu Ser Pro Leu Thr Asp Cys Thr Ser Pro Ser Phe Asp Gly Pro

260 265 270

Leu Ser Pro Pro Leu Ser Ile Asn Gly Asn Phe Ser Phe Lys His Glu

275 280 285

Pro Ser Ala Glu Phe Glu Lys Asn Tyr Ala Phe Thr Met His Tyr Pro

290 295 300

Ala Ala Thr Leu Ala Gly Ala Gln Ser His Gly Ser Ile Phe Ser Gly

305 310 315 320

Thr Ala Ala Pro Arg Cys Glu Ile Pro Ile Asp Asn Ile Met Ser Phe

325 330 335

Asp Ser His Ser His His Glu Arg Val Met Ser Ala Gln Leu Asn Ala

340 345 350

Ile Phe His Asp

355

<210> 11

<211> 711

<212> DNA

<213> Chile person

<400> 11

atggaaagct ctgccaagat ggagagcggc ggcgccggcc agcagcccca gccgcagccc 60

cagcagccct tcctgccgcc cgcagcctgt ttctttgcca cggccgcagc cgcggcggcc 120

gcagccgccg cagcggcagc gcagagcgcg cagcagcagc agcagcagca gcagcagcag 180

cagcaggcgc cgcagctgag accggcggcc gacggccagc cctcaggggg cggtcacaag 240

tcagcgccca agcaagtcaa gcgacagcgc tcgtcttcgc ccgaactgat gcgctgcaaa 300

cgccggctca acttcagcgg ctttggctac agcctgccgc agcagcagcc ggccgccgtg 360

gcgcgccgca acgagcgcga gcgcaaccgc gtcaagttgg tcaacctggg ctttgccacc 420

cttcgggagc acgtccccaa cggcgcggcc aacaagaaga tgagtaaggt ggagacactg 480

cgctcggcgg tcgagtacat ccgcgcgctg cagcagctgc tggacgagca tgacgcggtg 540

agcgccgcct tccaggcagg cgtcctgtcg cccaccatct cccccaacta ctccaacgac 600

ttgaactcca tggccggctc gccggtctca tcctactcgt cggacgaggg ctcttacgac 660

ccgctcagcc ccgaggagca ggagcttctc gacttcacca actggttctg a 711

<210> 12

<211> 236

<212> PRT

<213> Chile person

<400> 12

Met Glu Ser Ser Ala Lys Met Glu Ser Gly Gly Ala Gly Gln Gln Pro

1 5 10 15

Gln Pro Gln Pro Gln Gln Pro Phe Leu Pro Pro Ala Ala Cys Phe Phe

20 25 30

Ala Thr Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Gln

35 40 45

Ser Ala Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Ala Pro

50 55 60

Gln Leu Arg Pro Ala Ala Asp Gly Gln Pro Ser Gly Gly Gly His Lys

65 70 75 80

Ser Ala Pro Lys Gln Val Lys Arg Gln Arg Ser Ser Ser Pro Glu Leu

85 90 95

Met Arg Cys Lys Arg Arg Leu Asn Phe Ser Gly Phe Gly Tyr Ser Leu

100 105 110

Pro Gln Gln Gln Pro Ala Ala Val Ala Arg Arg Asn Glu Arg Glu Arg

115 120 125

Asn Arg Val Lys Leu Val Asn Leu Gly Phe Ala Thr Leu Arg Glu His

130 135 140

Val Pro Asn Gly Ala Ala Asn Lys Lys Met Ser Lys Val Glu Thr Leu

145 150 155 160

Arg Ser Ala Val Glu Tyr Ile Arg Ala Leu Gln Gln Leu Leu Asp Glu

165 170 175

His Asp Ala Val Ser Ala Ala Phe Gln Ala Gly Val Leu Ser Pro Thr

180 185 190

Ile Ser Pro Asn Tyr Ser Asn Asp Leu Asn Ser Met Ala Gly Ser Pro

195 200 205

Val Ser Ser Tyr Ser Ser Asp Glu Gly Ser Tyr Asp Pro Leu Ser Pro

210 215 220

Glu Glu Gln Glu Leu Leu Asp Phe Thr Asn Trp Phe

225 230 235

<210> 13

<211> 1050

<212> DNA

<213> Chile person

<400> 13

atgggagaca tgggagatcc accaaaaaaa aaacgtctga tttccctatg tgttggttgc 60

ggcaatcaga ttcacgatca gtatattctg agggtttctc cggatttgga atggcatgcg 120

gcatgtttga aatgtgcgga gtgtaatcag tatttggacg agagctgtac atgctttgtt 180

agggatggga aaacctactg taaaagagat tatatcaggt tgtacgggat caaatgcgcc 240

aagtgcagca tcggcttcag caagaacgac ttcgtgatgc gtgcccgctc caaggtgtat 300

cacatcgagt gtttccgctg tgtggcctgc agccgccagc tcatccctgg ggacgaattt 360

gcgcttcggg aggacggtct cttctgccga gcagaccacg atgtggtgga gagggccagt 420

ctaggcgctg gcgacccgct cagtcccctg catccagcgc ggccactgca aatggcagcg 480

gagcccatct ccgccaggca gccggccctg cggccccacg tccacaagca gccggagaag 540

accacccgcg tgcggactgt gctgaacgag aagcagctgc acaccttgcg gacctgctac 600

gccgcaaacc cgcggccaga tgcgctcatg aaggagcaac tggtagagat gacgggcctc 660

agtccccgtg tgatccgggt ctggtttcaa aacaagcggt gcaaggacaa gaagcgaagc 720

atcatgatga agcaactcca gcagcagcag cccaatgaca aaactaatat ccaggggatg 780

acaggaactc ccatggtggc tgccagtcca gagagacacg acggtggctt acaggctaac 840

ccagtggaag tacaaagtta ccagccacct tggaaagtac tgagcgactt cgccttgcag 900

agtgacatag atcagcctgc ttttcagcaa ctggtcaatt tttcagaagg aggaccgggc 960

tctaattcca ctggcagtga agtagcatca atgtcctctc aacttccaga tacacctaac 1020

agcatggtag ccagtcctat tgaggcatga 1050

<210> 14

<211> 349

<212> PRT

<213> Chile person

<400> 14

Met Gly Asp Met Gly Asp Pro Pro Lys Lys Lys Arg Leu Ile Ser Leu

1 5 10 15

Cys Val Gly Cys Gly Asn Gln Ile His Asp Gln Tyr Ile Leu Arg Val

20 25 30

Ser Pro Asp Leu Glu Trp His Ala Ala Cys Leu Lys Cys Ala Glu Cys

35 40 45

Asn Gln Tyr Leu Asp Glu Ser Cys Thr Cys Phe Val Arg Asp Gly Lys

50 55 60

Thr Tyr Cys Lys Arg Asp Tyr Ile Arg Leu Tyr Gly Ile Lys Cys Ala

65 70 75 80

Lys Cys Ser Ile Gly Phe Ser Lys Asn Asp Phe Val Met Arg Ala Arg

85 90 95

Ser Lys Val Tyr His Ile Glu Cys Phe Arg Cys Val Ala Cys Ser Arg

100 105 110

Gln Leu Ile Pro Gly Asp Glu Phe Ala Leu Arg Glu Asp Gly Leu Phe

115 120 125

Cys Arg Ala Asp His Asp Val Val Glu Arg Ala Ser Leu Gly Ala Gly

130 135 140

Asp Pro Leu Ser Pro Leu His Pro Ala Arg Pro Leu Gln Met Ala Ala

145 150 155 160

Glu Pro Ile Ser Ala Arg Gln Pro Ala Leu Arg Pro His Val His Lys

165 170 175

Gln Pro Glu Lys Thr Thr Arg Val Arg Thr Val Leu Asn Glu Lys Gln

180 185 190

Leu His Thr Leu Arg Thr Cys Tyr Ala Ala Asn Pro Arg Pro Asp Ala

195 200 205

Leu Met Lys Glu Gln Leu Val Glu Met Thr Gly Leu Ser Pro Arg Val

210 215 220

Ile Arg Val Trp Phe Gln Asn Lys Arg Cys Lys Asp Lys Lys Arg Ser

225 230 235 240

Ile Met Met Lys Gln Leu Gln Gln Gln Gln Pro Asn Asp Lys Thr Asn

245 250 255

Ile Gln Gly Met Thr Gly Thr Pro Met Val Ala Ala Ser Pro Glu Arg

260 265 270

His Asp Gly Gly Leu Gln Ala Asn Pro Val Glu Val Gln Ser Tyr Gln

275 280 285

Pro Pro Trp Lys Val Leu Ser Asp Phe Ala Leu Gln Ser Asp Ile Asp

290 295 300

Gln Pro Ala Phe Gln Gln Leu Val Asn Phe Ser Glu Gly Gly Pro Gly

305 310 315 320

Ser Asn Ser Thr Gly Ser Glu Val Ala Ser Met Ser Ser Gln Leu Pro

325 330 335

Asp Thr Pro Asn Ser Met Val Ala Ser Pro Ile Glu Ala

340 345

<210> 15

<211> 1209

<212> DNA

<213> Chile person

<400> 15

atggaggcgc gcggggagct gggcccggcc cgggagtcgg cgggaggcga cctgctgcta 60

gcactgctgg cgcggagggc ggacctgcgc cgagagatcc cgctgtgcgc tggctgtgac 120

cagcacatcc tggaccgctt catcctcaag gctctggacc gccactggca cagcaagtgt 180

ctcaagtgca gcgactgcca cacgccactg gccgagcgct gcttcagccg aggggagagc 240

gtttactgca aggacgactt tttcaagcgc ttcgggacca agtgcgccgc gtgccagctg 300

ggcatcccgc ccacgcaggt ggtgcgccgc gcccaggact tcgtgtacca cctgcactgc 360

tttgcctgcg tcgtgtgcaa gcggcagctg gccacgggcg acgagttcta cctcatggag 420

gacagccggc tcgtgtgcaa ggcggactac gaaaccgcca agcagcgaga ggccgaggcc 480

acggccaagc ggccgcgcac gaccatcacc gccaagcagc tggagacgct gaagagcgct 540

tacaacacct cgcccaagcc ggcgcgccac gtgcgcgagc agctctcgtc cgagacgggc 600

ctggacatgc gcgtggtgca ggtttggttc cagaaccgcc gggccaagga gaagaggctg 660

aagaaggacg ccggccggca gcgctggggg cagtatttcc gcaacatgaa gcgctcccgc 720

ggcggctcca agtcggacaa ggacagcgtt caggaggggc aggacagcga cgctgaggtc 780

tccttccccg atgagccttc cttggcggaa atgggcccgg ccaatggcct ctacgggagc 840

ttgggggaac ccacccaggc cttgggccgg ccctcgggag ccctgggcaa cttctccctg 900

gagcatggag gcctggcagg cccagagcag taccgagagc tgcgtcccgg cagcccctac 960

ggtgtccccc catcccccgc cgccccgcag agcctccctg gcccccagcc cctcctctcc 1020

agcctggtgt acccagacac cagcttgggc cttgtgccct cgggagcccc cggcgggccc 1080

ccacccatga gggtgctggc agggaacgga cccagttctg acctatccac ggggagcagc 1140

gggggttacc ccgacttccc tgccagcccc gcctcctggc tggatgaggt agaccacgct 1200

cagttctga 1209

<210> 16

<211> 402

<212> PRT

<213> Chile person

<400> 16

Met Glu Ala Arg Gly Glu Leu Gly Pro Ala Arg Glu Ser Ala Gly Gly

1 5 10 15

Asp Leu Leu Leu Ala Leu Leu Ala Arg Arg Ala Asp Leu Arg Arg Glu

20 25 30

Ile Pro Leu Cys Ala Gly Cys Asp Gln His Ile Leu Asp Arg Phe Ile

35 40 45

Leu Lys Ala Leu Asp Arg His Trp His Ser Lys Cys Leu Lys Cys Ser

50 55 60

Asp Cys His Thr Pro Leu Ala Glu Arg Cys Phe Ser Arg Gly Glu Ser

65 70 75 80

Val Tyr Cys Lys Asp Asp Phe Phe Lys Arg Phe Gly Thr Lys Cys Ala

85 90 95

Ala Cys Gln Leu Gly Ile Pro Pro Thr Gln Val Val Arg Arg Ala Gln

100 105 110

Asp Phe Val Tyr His Leu His Cys Phe Ala Cys Val Val Cys Lys Arg

115 120 125

Gln Leu Ala Thr Gly Asp Glu Phe Tyr Leu Met Glu Asp Ser Arg Leu

130 135 140

Val Cys Lys Ala Asp Tyr Glu Thr Ala Lys Gln Arg Glu Ala Glu Ala

145 150 155 160

Thr Ala Lys Arg Pro Arg Thr Thr Ile Thr Ala Lys Gln Leu Glu Thr

165 170 175

Leu Lys Ser Ala Tyr Asn Thr Ser Pro Lys Pro Ala Arg His Val Arg

180 185 190

Glu Gln Leu Ser Ser Glu Thr Gly Leu Asp Met Arg Val Val Gln Val

195 200 205

Trp Phe Gln Asn Arg Arg Ala Lys Glu Lys Arg Leu Lys Lys Asp Ala

210 215 220

Gly Arg Gln Arg Trp Gly Gln Tyr Phe Arg Asn Met Lys Arg Ser Arg

225 230 235 240

Gly Gly Ser Lys Ser Asp Lys Asp Ser Val Gln Glu Gly Gln Asp Ser

245 250 255

Asp Ala Glu Val Ser Phe Pro Asp Glu Pro Ser Leu Ala Glu Met Gly

260 265 270

Pro Ala Asn Gly Leu Tyr Gly Ser Leu Gly Glu Pro Thr Gln Ala Leu

275 280 285

Gly Arg Pro Ser Gly Ala Leu Gly Asn Phe Ser Leu Glu His Gly Gly

290 295 300

Leu Ala Gly Pro Glu Gln Tyr Arg Glu Leu Arg Pro Gly Ser Pro Tyr

305 310 315 320

Gly Val Pro Pro Ser Pro Ala Ala Pro Gln Ser Leu Pro Gly Pro Gln

325 330 335

Pro Leu Leu Ser Ser Leu Val Tyr Pro Asp Thr Ser Leu Gly Leu Val

340 345 350

Pro Ser Gly Ala Pro Gly Gly Pro Pro Pro Met Arg Val Leu Ala Gly

355 360 365

Asn Gly Pro Ser Ser Asp Leu Ser Thr Gly Ser Ser Gly Gly Tyr Pro

370 375 380

Asp Phe Pro Ala Ser Pro Ala Ser Trp Leu Asp Glu Val Asp His Ala

385 390 395 400

Gln Phe

<210> 17

<211> 380

<212> DNA

<213> artificial sequence

<220>

<223> manual sequence description: synthesis of polynucleotides

<400> 17

gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc 60

catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca 120

acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga 180

ctttccattg acgtcaatgg gtggactatt tacggtaaac tgcccacttg gcagtacatc 240

aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 300

ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 360

tagtcatcgc tattaccatg 380

<210> 18

<211> 2214

<212> DNA

<213> Chile person

<400> 18

cgcgtcccac ctccctctct gtgctgggac tcacagaggg agacctcagg aggcagtctg 60

tccatcacat gtccaaatgc agagcatacc ctgggctggg cgcagtggcg cacaactgta 120

attccagcac tttgggaggc tgatgtggaa ggatcacttg agcccagaag ttctagacca 180

gcctgggcaa catggcaaga ccctatctct acaaaaaaag ttaaaaaatc agccacgtgt 240

ggtgacacac acctgtagtc ccagctattc aggaggctga ggtgagggga tcacttaagg 300

ctgggaggtt gaggctgcag tgagtcgtgg ttgcgccact gcactccagc ctgggcaaca 360

gtgagaccct gtctcaaaag acaaaaaaaa aaaaaaaaaa aaaaagaaca tatcctggtg 420

tggagtaggg gacgctgctc tgacagaggc tcgggggcct gagctggctc tgtgagctgg 480

ggaggaggca gacagccagg ccttgtctgc aagcagacct ggcagcattg ggctggccgc 540

cccccagggc ctcctcttca tgcccagtga atgactcacc ttggcacaga cacaatgttc 600

ggggtgggca cagtgcctgc ttcccgccgc accccagccc ccctcaaatg ccttccgaga 660

agcccattga gcagggggct tgcattgcac cccagcctga cagcctggca tcttgggata 720

aaagcagcac agccccctag gggctgccct tgctgtgtgg cgccaccggc ggtggagaac 780

aaggctctat tcagcctgtg cccaggaaag gggatcaggg gatgcccagg catggacagt 840

gggtggcagg gggggagagg agggctgtct gcttcccaga agtccaagga cacaaatggg 900

tgaggggact gggcagggtt ctgaccctgt gggaccagag tggagggcgt agatggacct 960

gaagtctcca gggacaacag ggcccaggtc tcaggctcct agttgggccc agtggctcca 1020

gcgtttccaa acccatccat ccccagaggt tcttcccatc tctccaggct gatgtgtggg 1080

aactcgagga aataaatctc cagtgggaga cggaggggtg gccagggaaa cggggcgctg 1140

caggaataaa gacgagccag cacagccagc tcatgtgtaa cggctttgtg gagctgtcaa 1200

ggcctggtct ctgggagaga ggcacaggga ggccagacaa ggaaggggtg acctggaggg 1260

acagatccag gggctaaagt cctgataagg caagagagtg ccggccccct cttgccctat 1320

caggacctcc actgccacat agaggccatg attgaccctt agacaaaggg ctggtgtcca 1380

atcccagccc ccagccccag aactccaggg aatgaatggg cagagagcag gaatgtggga 1440

catctgtgtt caagggaagg actccaggag tctgctggga atgaggccta gtaggaaatg 1500

aggtggccct tgagggtaca gaacaggttc attcttcgcc aaattcccag caccttgcag 1560

gcacttacag ctgagtgaga taatgcctgg gttatgaaat caaaaagttg gaaagcaggt 1620

cagaggtcat ctggtacagc ccttccttcc cttttttttt tttttttttt gtgagacaag 1680

gtctctctct gttgcccagg ctggagtggc gcaaacacag ctcactgcag cctcaaccta 1740

ctgggctcaa gcaatcctcc agcctcagcc tcccaaagtg ctgggattac aagcatgagc 1800

caccccactc agccctttcc ttccttttta attgatgcat aataattgta agtattcatc 1860

atggtccaac caaccctttc ttgacccacc ttcctagaga gagggtcctc ttgcttcagc 1920

ggtcagggcc ccagacccat ggtctggctc caggtaccac ctgcctcatg caggagttgg 1980

cgtgcccagg aagctctgcc tctgggcaca gtgacctcag tggggtgagg ggagctctcc 2040

ccatagctgg gctgcggccc aaccccaccc cctcaggcta tgccaggggg tgttgccagg 2100

ggcacccggg catcgccagt ctagcccact ccttcataaa gccctcgcat cccaggagcg 2160

agcagagcca gagcaggttg gagaggagac gcatcacctc cgctgctcgc cggg 2214

<210> 19

<211> 57

<212> DNA

<213> artificial sequence

<220>

<223> manual sequence description: synthetic oligonucleotides

<400> 19

gctactaact tcagcctgct gaagcaggct ggagacgtgg aggagaaccc tggacct 57

<210> 20

<211> 54

<212> DNA

<213> artificial sequence

<220>

<223> manual sequence description: synthetic oligonucleotides

<400> 20

gaggggagag gaagtcttct gacctgcgga gacgtcgaag agaatcctgg accc 54

<210> 21

<211> 477

<212> DNA

<213> Chile person

<400> 21

gggtggcatc cctgtgaccc ctccccagtg cctctcctgg ccctggaagt tgccactcca 60

gtgcccacca gccttgtcct aataaaatta agttgcatca ttttgtctga ctaggtgtcc 120

ttctataata ttatggggtg gaggggggtg gtatggagca aggggcaagt tgggaagaca 180

acctgtaggg cctgcggggt ctattgggaa ccaagctgga gtgcagtggc acaatcttgg 240

ctcactgcaa tctccgcctc ctgggttcaa gcgattctcc tgcctcagcc tcccgagttg 300

ttgggattcc aggcatgcat gaccaggctc agctaatttt tgtttttttg gtagagacgg 360

ggtttcacca tattggccag gctggtctcc aactcctaat ctcaggtgat ctacccacct 420

tggcctccca aattgctggg attacaggcg tgaaccactg ctcccttccc tgtcctt 477

<210> 22

<211> 66

<212> DNA

<213> artificial sequence

<220>

<223> manual sequence description: synthetic oligonucleotides

<400> 22

ggaagcggag ctactaactt cagcctgctg aagcaggctg gagacgtgga ggagaaccct 60

ggacct 66

<210> 23

<211> 66

<212> DNA

<213> artificial sequence

<220>

<223> manual sequence description: synthetic oligonucleotides

<400> 23

ggaagcggag ctactaactt cagcctgctg aagcaggctg gagacgtgga ggagaaccct 60

ggacct 66

<210> 24

<211> 22

<212> PRT

<213> artificial sequence

<220>

<223> manual sequence description: synthetic peptides

<400> 24

Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val

1 5 10 15

Glu Glu Asn Pro Gly Pro

20

<210> 25

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> manual sequence description: synthetic peptides

<400> 25

Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu

1 5 10 15

Glu Asn Pro Gly Pro

20

<210> 26

<211> 208

<212> DNA

<213> artificial sequence

<220>

<223> manual sequence description: synthesis of polynucleotides

<400> 26

ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc 60

tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc 120

tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt 180

gggaagagaa tagcaggcat gctgggga 208

<210> 27

<211> 681

<212> DNA

<213> artificial sequence

<220>

<223> manual sequence description: synthesis of polynucleotides

<400> 27

aacatatcct ggtgtggagt aggggacgct gctctgacag aggctcgggg gcctgagctg 60

gctctgtgag ctggggagga ggcagacagc caggccttgt ctgcaagcag acctggcagc 120

attgggctgg ccgcccccca gggcctcctc ttcatgccca gtgaatgact caccttggca 180

cagacacaat gttcggggtg ggcacagtgc ctgcttcccg ccgcacccca gcccccctca 240

aatgccttcc gagaagccca ttgagcaggg ggcttgcatt gcaccccagc ctgacagcct 300

ggcatcttgg gataaaagca gcacagcccc ctaggggctg cccttgctgt gtggcgccac 360

cggcggtgga gaacaaggct ctattcagcc tgtgcccagg aaaggggatc aggggatgcc 420

caggcatgga cagtgggtgg caggggggga gaggagggct gtctgcttcc cagaagtcca 480

aggacacaaa tgggtgaggg gagagctctc cccatagctg ggctgcggcc caaccccacc 540

ccctcaggct atgccagggg gtgttgccag gggcacccgg gcatcgccag tctagcccac 600

tccttcataa agccctcgca tcccaggagc gagcagagcc agagcaggtt ggagaggaga 660

cgcatcacct ccgctgctcg c 681

<210> 28

<211> 1062

<212> DNA

<213> artificial sequence

<220>

<223> manual sequence description: synthesis of polynucleotides

<400> 28

ggagtcgctg cgttgccttc gccccgtgcc ccgctccgcg ccgcctcgcg ccgcccgccc 60

cggctctgac tgaccgcgtt actcccacag gtgagcgggc gggacggccc ttctccctcc 120

gggctgtaat tagcgcttgg tttaatgacg gctcgtttct tttctgtggc tgcgtgaaag 180

ccttaaaggg ctccgggagg gcctttgtgc gggggggagc ggctcggggg gtgcgtgcgt 240

gtgtgtgtgc gtggggagcg ccgcgtgcgg cccgcgctgc ccggcggctg tgagcgctgc 300

gggcgcggcg cggggctttg tgcgctccgc gtgtgcgcga ggggagcgcg ggccgggggc 360

ggtgccccgc ggtgcggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg 420

tgggggggtg agcagggggt gtgggcgcgg cggtcgggct gtaacccccc cctggcaccc 480

ccctccccga gttgctgagc acggcccggc ttcgggtgcg gggctccgtg cggggcgtgg 540

cgcggggctc gccgtgccgg gcggggggtg gcggcaggtg ggggtgccgg gcggggcggg 600

gccgcctcgg gccggggagg gctcggggga ggggcgcggc ggccccggag cgccggcggc 660

tgtcgaggcg cggcgagccg cagccattgc cttttatggt aatcgtgcga gagggcgcag 720

ggacttcctt tgtcccaaat ctggcggagc cgaaatctgg gaggcgccgc cgcaccccct 780

ctagcgggcg cgggcgaagc ggtgcggcgc cggcaggaag gaaatgggcg gggagggcct 840

tcgtgcgtcg ccgcgccgcc gtccccttct ccatctccag cctcggggct gccgcagggg 900

gacggctgcc ttcggggggg acggggcagg gcggggttcg gcttctggcg tgtgaccggc 960

ggctttagag cctctgctaa ccatgttcat gccttcttct ttttcctaca gctcctgggc 1020

aacgtgctgg ttgttgtgct gtctcatcat tttggcaaag at 1062

<210> 29

<211> 978

<212> DNA

<213> artificial sequence

<220>

<223> manual sequence description: synthesis of polynucleotides

<400> 29

ggccactgtg aggcagaagt gaggagggga tggggaaggg gggccttgtg agcagaaggg 60

gctgaatccc caagaaggag tgcccgagaa gtctcaggga ggggccgaac ctccctgctc 120

cctgggcctc cctacctctt gatggggcac tatccttgcc ccccaacatg atgggaggga 180

ccagaaacag gcccagggcc ccggggatct gatgcccgca tgccttctgc caggagtcca 240

gggtcccctc agcacctccc tactggggaa agcagtgcag gagcagcggg gcccctgtgt 300

ttcattcatg gctgggcttt gtgactgtgg gcagcgagct cacctattct gagcctgtgt 360

ccatataaag gaggagttgg aagcggagaa ggttgatgtc catgagggag attggattct 420

ggggtgaaga aagtgaggga aagagcaggc aggtctgggc gcaaagcaca ggtgactgcc 480

tgccaccagc ttgtgacccc catcaagtta ctttgacttg cacagctgtg aagcggtggt 540

cataataaaa ttcatttcaa aaggtggtta cctgggatca gaggaatccc caggggcatg 600

gcgcttcact gagctgacag gacatgcatg tgtgccttca agtgcaggag gacatgtgcg 660

tgtgtgtgtg tgtgtgtgca acagtgagtg tatgcttgtg gatgcgcctg tgtgagcaga 720

agcaggtgca ccaaccctga taaggcacct tagtaatgag ttaaggcaaa agcccacatc 780

tgctcatcct ccagacaagt cctctgtcta aggcccccca acccttaatc ctcctgctgc 840

tctactggtc ctgggtgggg gtggtctctg tgacagctgc ctcaagggag actgaggcag 900

gtattcaagt gtcctcagaa gagcctggac ccaggaatgt gtccccccac tccaggctcc 960

aggatgaaac caacctga 978

<210> 30

<211> 581

<212> DNA

<213> artificial sequence

<220>

<223> manual sequence description: synthesis of polynucleotides

<400> 30

gagcatctta ccgccattta tacccatatt tgttctgttt ttcttgattt gggtatacat 60

ttaaatgtta ataaaacaaa atggtggggc aatcatttac atttttaggg atatgtaatt 120

actagttcag gtgtattgcc acaagacaaa catgttaaga aactttcccg ttatttacgc 180

tctgttcctg ttaatcaacc tctggattac aaaatttgtg aaagattgac tgatattctt 240

aactatgttg ctccttttac gctgtgtgga tatgctgctt tatagcctct gtatctagct 300

attgcttccc gtacggcttt cgttttctcc tccttgtata aatcctggtt gctgtctctt 360

ttagaggagt tgtggcccgt tgtccgtcaa cgtggcgtgg tgtgctctgt gtttgctgac 420

gcaaccccca ctggctgggg cattgccacc acctgtcaac tcctttctgg gactttcgct 480

ttccccctcc cgatcgccac ggcagaactc atcgccgcct gccttgcccg ctgctggaca 540

ggggctaggt tgctgggcac tgataattcc gtggtgttgt c 581

<210> 31

<211> 49

<212> DNA

<213> artificial sequence

<220>

<223> manual sequence description: synthetic oligonucleotides

<400> 31

cacacaaaaa accaacacac atccatcttc gatggatagc gattttatt 49

<210> 32

<211> 300

<212> RNA

<213> artificial sequence

<220>

<223> manual sequence description: synthesis of polynucleotides

<220>

<221> misc_feature

<222> (1)..(300)

<223> the sequence may cover 50-300 nucleotides

<220>

<223> please refer to the submitted description for a detailed description of the alternative and preferred embodiments

<400> 32

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 60

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 120

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 180

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 240

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 300

<210> 33

<211> 21

<212> DNA

<213> artificial sequence

<220>

<223> manual sequence description: synthetic primers

<400> 33

ggaaccccta gtgatggagt t 21

<210> 34

<211> 16

<212> DNA

<213> artificial sequence

<220>

<223> manual sequence description: synthetic primers

<400> 34

cggcctcagt gagcga 16

Claims

2. An adeno-associated virus (AAV) vector comprising: a nucleic acid coding sequence encoding a human neurogenic differentiation factor 1 (hNeuroD 1) protein, the hNeuroD1 protein comprising the amino acid sequence of SEQ ID NO:10, and a second nucleic acid coding sequence encoding a second protein having amino acids selected from the group consisting of SEQ ID NOs: 12, 14 and 16, wherein the hNeuroD1 coding sequence and the second coding sequence are separated by: (i) a P2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID nos. 19 and 22, (ii) a T2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID nos. 20 and 23, or (iii) an Internal Ribosome Entry Site (IRES) sequence of an encephalomyocarditis virus comprising SEQ ID No. 3, wherein the hNeuroD1 coding sequence and the second coding sequence are operably linked to a regulatory element comprising:

(a) Glial Fibrillary Acidic Protein (GFAP) promoter;

(b) An enhancer;

(c) Chimeric introns;

(e) Polyadenylation signals.

5. A composition comprising an adeno-associated virus (AAV) vector for converting human glial cells to functional neurons, wherein the AAV vector comprises: a nucleic acid coding sequence encoding a human neurogenic differentiation factor 1 (hNeuroD 1) protein, the hNeuroD1 protein comprising the amino acid sequence of SEQ ID NO:10, and a second nucleic acid coding sequence encoding a second protein having amino acids selected from the group consisting of SEQ ID NOs: 12, 14 and 16, wherein the hNeuroD1 coding sequence and the second coding sequence are separated by: (i) a P2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID nos. 19 and 22, (ii) a T2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID nos. 20 and 23, or (iii) an Internal Ribosome Entry Site (IRES) sequence of an encephalomyocarditis virus comprising SEQ ID No. 3, wherein the hNeuroD1 coding sequence and the second coding sequence are operably linked to a regulatory element comprising:

6. A composition comprising an adeno-associated virus (AAV) vector, wherein the AAV vector comprises a neurogenic differentiation factor 1 (NeuroD 1) sequence and a second protein sequence, wherein the NeuroD1 sequence and the second protein sequence are separated by a linker sequence, wherein the NeuroD1 sequence and the second sequence are operably linked to an expression control element, the expression control element comprising:

(a) Glial Fibrillary Acidic Protein (GFAP) promoter;

(b) An enhancer;

(c) Chimeric introns;

(e) Polyadenylation signals.

7. The AAV vector of any one of claims 1-3, or composition of any one of claims 4-6, wherein the AAV vector is selected from the group consisting of AAV serotype 2, AAV serotype 5, and AAV serotype 9.

8. The AAV vector or composition of claim 7, wherein the AAV vector is AAV serotype 2.

9. The AAV vector or composition of claim 7, wherein the AAV vector is AAV serotype 5.

10. The AAV vector or composition of claim 7, wherein the AAV vector is AAV serotype 9.

11. The composition of claim 4 or 5, wherein the glial cell is a reactive astrocyte.

12. The composition of claim 4 or 5, wherein the functional neuron is selected from the group consisting of a glutamatergic neuron, a gabaergic neuron, a dopaminergic neuron, a cholinergic neuron, a serotonergic neuron, an adrenergic neuron, a motor neuron, and a peptidoergic neuron.

13. The composition of claim 4 or 5, wherein the human suffers from a neurological disorder.

14. The AAV vector of claim 3 or composition of claim 6, wherein the NeuroD1 is human NeuroD1 (hNeuroD 1).

15. The AAV vector of claim 3 or composition of claim 6, wherein the second protein is selected from the group consisting of acetate-scale family BHLH transcription factor 1 (Ascl 1), insulin gene enhancer protein (ISL 1), and LIM-homeobox 3 (LHX 2).

16. The AAV vector or composition of claim 15, wherein the second protein is Ascl1.

17. The AAV vector or composition of claim 16, wherein the Ascl1 is human Ascl1 (hAscl 1).

18. The AAV vector or composition of claim 15, wherein the second protein is ISL1.

19. The AAV vector or composition of claim 18, wherein the ISL1 is human ISL1 (hsl 1).

20. The AAV vector or composition of claim 15, wherein the second protein is LHX3.

21. The AAV vector or composition of claim 20, wherein the LHX3 is human LHX3 (hLHX 3).

22. The AAV vector of claim 3 or composition of claim 6, wherein the NeuroD1 is selected from the group consisting of chimpanzee NeuroD1, bonobo NeuroD1, red-chimpanzee NeuroD1, gorilla NeuroD1, macaque NeuroD1, marmoset NeuroD1, pigtail NeuroD1, baboon NeuroD1, gibbon NeuroD1, and marmoset NeuroD 1.

23. The AAV vector or composition of claim 14, wherein the hNeuroD1 comprises a nucleic acid encoding sequence encoding an amino acid sequence at least 80% identical or similar to: SEQ ID NO. 10.

24. The AAV vector or composition of claim 17, wherein the hAscl1 comprises a nucleic acid encoding sequence encoding an amino acid sequence at least 80% identical or similar to: SEQ ID NO. 12.

25. The AAV vector or composition of claim 19, wherein the hsl 1 comprises a nucleic acid coding sequence encoding an amino acid sequence at least 80% identical or similar to: SEQ ID NO. 14.

26. The AAV vector or composition of claim 21, wherein the hLHX3 comprises a nucleic acid coding sequence encoding an amino acid sequence that is at least 80% identical or similar to: SEQ ID NO. 16.

27. The AAV vector or composition of claim 14, wherein the hNeuroD1 sequence comprises a nucleic acid sequence that is at least 80% identical to: SEQ ID NO. 6, or a complement thereof.

28. The AAV vector or composition of claim 17, wherein the hAscl1 sequence comprises a nucleic acid sequence that is at least 80% identical to: SEQ ID NO. 11, or a complement thereof.

29. The AAV vector or composition of claim 19, wherein the hsl 1 sequence comprises a nucleic acid sequence that is at least 80% identical to: SEQ ID NO. 13, or a complement thereof.

30. The AAV vector or composition of claim 21, wherein the hLHX3 sequence comprises a nucleic acid sequence that is at least 80% identical to: SEQ ID NO. 15, or a complement thereof.

31. The AAV vector of claim 3 or composition of claim 6, wherein the linker is selected from the group consisting of P2A and T2A.

32. The AAV vector or composition of claim 31, wherein the linker is the P2A.

33. The AAV vector or composition of claim 31, wherein the linker is the T2A.

34. The AAV vector or composition of claim 31, wherein the P2A linker comprises a nucleic acid sequence that is at least 80% identical to a sequence selected from the group consisting of: SEQ ID NOS 19 and 22, or the complements thereof.

35. The AAV vector or composition of claim 31, wherein the T2A linker comprises a nucleic acid sequence that is at least 80% identical to a sequence selected from the group consisting of: SEQ ID NOS 20 and 23, or the complements thereof.

36. The AAV vector of claim 3 or composition of claim 6, wherein the GFAP promoter is a human GFAP (hGFAP) promoter.

37. The AAV vector of claim 3 or composition of claim 6, wherein the GFAP promoter is selected from the group consisting of a chimpanzee GFAP promoter, a bonoban GFAP promoter, a chimpanzee GFAP promoter, a macaque GFAP promoter, a marmoset GFAP promoter, a pigtail GFAP promoter, a baboon GFAP promoter, a gibbon GFAP promoter, and a lemur GFAP promoter.

38. The AAV vector or composition of any one of the preceding claims, wherein the IRES sequence comprises a nucleic acid sequence at least 80% identical to: SEQ ID NO. 3, or a complement thereof.

39. The AAV vector or composition of claim 36, wherein the hGFAP promoter comprises a nucleic acid sequence that is at least 80% identical to: SEQ ID NO. 4, or a complement thereof.

40. The AAV vector or composition of claim 36, wherein the hGFAP promoter comprises a nucleic acid sequence that is at least 80% identical to: 18, or the complement thereof.

41. The AAV vector or composition of claim 36, wherein the hGFAP promoter comprises a nucleic acid sequence that is at least 80% identical to: SEQ ID NO. 27, or a complement thereof.

42. The AAV vector of claim 3 or composition of claim 6, wherein the enhancer is selected from the group consisting of an enhancer from the human elongation factor-1 a (EF 1-a) promoter and a Cytomegalovirus (CMV) enhancer.

43. The AAV vector or composition of claim 42, wherein the EF 1-a comprises a nucleic acid sequence that is at least 80% identical to: SEQ ID NO. 2, or a complement thereof.

44. The AAV vector or composition of claim 42, wherein the CMV enhancer comprises a nucleic acid sequence at least 80% identical to: SEQ ID NO. 17, or a complement thereof.

45. The AAV vector of claim 3 or composition of claim 6, wherein the chimeric intron comprises a nucleic acid sequence at least 80% identical to: SEQ ID NO. 5, or a complement thereof.

46. The AAV vector of claim 3 or composition of claim 6, wherein the chimeric intron comprises a nucleic acid sequence at least 80% identical to: SEQ ID NO. 28, or a complement thereof.

47. The AAV vector of claim 3 or composition of claim 6, wherein the WPRE comprises a nucleic acid sequence at least 80% identical to a nucleic acid sequence selected from the group consisting of: SEQ ID NOS.7 and 30, or the complements thereof.

48. The AAV vector of claim 3 or composition of claim 6, wherein the polyadenylation signal is selected from the group consisting of an SV40 polyadenylation signal, an hGH polyadenylation signal, a synthetic polyadenylation signal, and a bGH polyadenylation signal.

49. The AAV vector or composition of claim 48, wherein the SV40 polyadenylation signal comprises a nucleic acid sequence that is at least 80% identical to: SEQ ID NO. 8, or a complement thereof.

50. The AAV vector or composition of claim 48, wherein the hGH polyadenylation signal comprises a nucleic acid sequence that is at least 80% identical to: SEQ ID NO. 21, or a complement thereof.

51. The AAV vector or composition of claim 48, wherein the hGH polyadenylation signal comprises a nucleic acid sequence that is at least 80% identical to: SEQ ID NO. 26, or a complement thereof.

52. The AAV vector of claim 3, or composition of claim 6, wherein the AAV vector further comprises a nucleic acid sequence encoding an AAV protein sequence.

53. The AAV vector of any one of claims 1-3, or composition of any one of claims 4-6, wherein the AAV vector comprises an AAV serotype 2 Inverted Terminal Repeat (ITR).

54. The AAV vector of any one of claims 1-3, or composition of any one of claims 4-6, wherein the AAV vector comprises an AAV serotype 5 Inverted Terminal Repeat (ITR).

55. The AAV vector of any one of claims 1-3, or composition of any one of claims 4-6, wherein the AAV vector comprises an AAV serotype 9 Inverted Terminal Repeat (ITR).

56. The AAV vector of any one of claims 1-3, or composition of any one of claims 4-6, wherein the AAV vector comprises at least one ITR nucleic acid sequence at least 80% identical to: SEQ ID NO. 1.

57. The AAV vector of any one of claims 1-3, or composition of any one of claims 4-6, wherein the AAV vector comprises at least one ITR nucleic acid sequence at least 80% identical to: SEQ ID NO. 9.

58. The composition of claim 6, wherein the subject in need thereof is a mammal.

59. The composition of claim 58, wherein the mammal is a human.

60. The composition of claim 58, wherein the mammal is a non-human primate.

61. The composition of claim 6, wherein the subject in need thereof has a neurological disorder.

62. The composition of claim 13 or 61, wherein the neurological disorder comprises an injury to the Central Nervous System (CNS) or peripheral nervous system.

63. The composition of claim 13 or 61, wherein the neurological condition comprises injury to the CNS.

64. The composition of claim 13 or 61, wherein the neurological disorder is selected from the group consisting of: alzheimer's disease, parkinson's disease, amyotrophic Lateral Sclerosis (ALS), huntington's disease, epilepsy, physical injury, stroke, cerebral aneurysms, traumatic brain injury, concussion, tumors, inflammation, infection, ataxia, brain atrophy, spinal cord atrophy, multiple sclerosis, traumatic spinal cord injury, ischemic or hemorrhagic myelopathy (myelopathy), global cerebral ischemia, hypoxic ischemic encephalopathy, embolism, fibrocartilage embolic myelopathy, thrombosis, kidney disease, chronic inflammatory diseases, meningitis, and cerebral venous sinus thrombosis.

65. The composition of claim 13 or 61, wherein the neurological disorder is alzheimer's disease.

66. The composition of claim 13 or 61, wherein the neurological disorder is parkinson's disease.

67. The composition of claim 13 or 61, wherein the neurological disorder is ALS.

68. The composition of claim 13 or 61, wherein the neurological disorder is huntington's disease.

69. The composition of claim 13 or 61, wherein the neurological disorder is stroke.

70. The composition of claim 69, wherein the stroke is ischemic stroke.

71. The composition of claim 69, wherein the stroke is hemorrhagic stroke.

72. The composition of claim 61, wherein the composition is capable of converting at least one glial cell into a neuron.

73. The composition of claim 72, wherein said glial cell is selected from the group consisting of an astrocyte and a NG2 cell.

74. The composition of claim 72, wherein the glial cell is an astrocyte.

75. The composition of claim 74, wherein said astrocytes are reactive astrocytes.

76. The composition of claim 72, wherein the glial cells are GFAP positive.

77. The composition of claim 72, wherein the neuron is a functional neuron.

78. The composition of claim 72, wherein the functional neuron is selected from the group consisting of a glutamatergic neuron, a gabaergic neuron, a dopaminergic neuron, a cholinergic neuron, a serotonergic neuron, an adrenergic neuron, a motor neuron, and a peptidoergic neuron.

79. The composition of claim 78, wherein the functional neuron is a glutamatergic neuron.

80. The composition of claim 6, wherein the composition is formulated for delivery to a subject in need thereof.

81. The composition of claim 80, wherein the composition is formulated for topical delivery.

82. The composition of claim 80, wherein the composition is formulated for systemic delivery.

83. The composition of any one of claims 80-82, wherein the composition is formulated for delivery via intraperitoneal, intramuscular, intravenous, intrathecal, intracerebral, intracranial, intraventricular of the brain, intracameral of the cerebellum, intravitreal, subretinal, intraparenchymal, intranasal, or oral administration.

84. A method comprising delivering the composition of claim 6 to the subject in need thereof.

85. The method of claim 84, wherein the composition is formulated for delivery to a subject in need thereof.

86. The method of claim 84, wherein the delivering comprises topical administration.

87. The method of claim 84, wherein the delivering comprises systemic administration.

88. The method of any one of claims 84-87, wherein said delivering comprises intraperitoneal, intramuscular, intravenous, intrathecal, intracerebral, intracranial, lateral intracerebroventricular, intravitreal, intraretinal, intraparenchymal, intranasal, or oral administration.

90. A method of converting reactive astrocytes in a living human brain into functional neurons, comprising: injecting an adeno-associated virus (AAV) into a subject in need thereof, wherein the AAV comprises a DNA vector construct comprising: a nucleic acid coding sequence encoding a human neurogenic differentiation factor 1 (hNeuroD 1) protein, the hNeuroD1 protein comprising the amino acid sequence of SEQ ID NO:10, and a second nucleic acid coding sequence encoding a second protein having amino acids selected from the group consisting of SEQ ID NOs: 12, 14 and 16, wherein the hNeuroD1 coding sequence and the second coding sequence are separated by: (i) a P2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID nos. 19 and 22, (ii) a T2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID nos. 20 and 23, or (iii) an Internal Ribosome Entry Site (IRES) sequence of an encephalomyocarditis virus comprising SEQ ID No. 3, wherein the hNeuroD1 coding sequence and the second nucleic acid coding sequence are operably linked to an expression control element comprising:

(a) Glial Fibrillary Acidic Protein (GFAP) promoter;

(b) An enhancer;

(c) Chimeric introns;

(e) And a polyadenylation signal, which is a polyadenylation signal,

92. A method of treating a neurological disorder in a subject in need thereof, comprising: delivering an adeno-associated virus (AAV) to the subject, wherein the AAV administered to the subject in need thereof comprises a DNA vector construct comprising a neurogenic differentiation factor 1 (NeuroD 1) sequence and a second sequence, wherein the NeuroD1 sequence and the second protein sequence are separated by a linker, wherein the NeuroD1 sequence and the second protein sequence are operably linked to an expression control element comprising:

(a) Glial Fibrillary Acidic Protein (GFAP) promoter;

(b) An enhancer from the human elongation factor-1 alpha (EF-1 alpha) promoter;

(c) Chimeric introns;

(e) SV40 polyadenylation signal.

93. The method of any one of claims 89-92, wherein the AAV vector is selected from the group consisting of AAV serotype 2, AAV serotype 5, and AAV serotype 9.

94. The method of claim 93, wherein the AAV is AAV serotype 2.

95. The method of claim 93, wherein the AAV is AAV serotype 5.

96. The method of claim 93, wherein the AAV is AAV serotype 9.

97. The method of claim 89 or 90, wherein the functional neurons are glutamatergic neurons, gabaergic neurons, dopaminergic neurons, cholinergic neurons, serotonergic neurons, adrenergic neurons, motor neurons, and peptidoergic neurons.

98. The method of claim 91 or 92, wherein the NeuroD1 is human NeuroD1 (hNeuroD 1).

99. The method of claim 91 or 92, wherein said second protein is selected from the group consisting of actete-scale family BHLH transcription factor 1 (Ascl 1), insulin gene enhancer protein (ISL 1), and LIM-homeobox 3 (LHX 2).

100. The method of claim 99, wherein the second protein is Ascl1.

101. The method of claim 100, wherein the Ascl1 is human Ascl1 (hAscl 1).

102. The method of claim 99, wherein the second protein is ISL1.

103. The method of claim 102, wherein the ISL1 is human ISL1 (hsl 1)

104. The method of claim 99, wherein the second protein is LHX3.

105. The method of claim 104 wherein the LHX3 is human LHX3 (hLHX 3).

106. The method of claim 91 or 92, wherein the NeuroD1 is selected from the group consisting of chimpanzee NeuroD1, bonobo NeuroD1, chimpanzee NeuroD1, gorilla NeuroD1, macaque NeuroD1, marmoset NeuroD1, pigtail NeuroD1, baboon NeuroD1, gibbon NeuroD1, and marmoset NeuroD 1.

107. The method of claim 98, wherein the hNeuroD1 comprises an amino acid coding sequence encoding an amino acid sequence at least 80% identical or similar to: SEQ ID NO. 10.

108. The method of claim 98, wherein the hAscl1 comprises an amino acid coding sequence that encodes an amino acid sequence that is at least 80% identical or similar to: SEQ ID NO. 12.

109. The method of claim 103, wherein the hsl 1 comprises an amino acid coding sequence encoding an amino acid sequence at least 80% identical or similar to: SEQ ID NO. 14.

110. The method of claim 105, wherein the hLHX3 comprises an amino acid coding sequence that encodes an amino acid sequence that is at least 80% identical or similar to: SEQ ID NO. 16. The method of claim 98, wherein the hNeuroD1 coding sequence comprises a nucleic acid sequence that is at least 80% identical to: SEQ ID NO. 6, or a complement thereof.

111. The method of claim 98, wherein the fscl 1 coding sequence comprises a nucleic acid sequence that is at least 80% identical to: SEQ ID NO. 11, or a complement thereof.

112. The method of claim 103, wherein the hsl 1 coding sequence comprises a nucleic acid sequence that is at least 80% identical to: SEQ ID NO. 13, or a complement thereof.

113. The method of claim 105, wherein the hLHX3 coding sequence comprises a nucleic acid sequence that is at least 80% identical to: SEQ ID NO. 15, or a complement thereof.

114. The method of claim 91 or 92, wherein the GFAP promoter is a human GFAP (hGFAP) promoter.

115. The method of claim 91 or 92, wherein the GFAP promoter is selected from the group consisting of a chimpanzee GFAP promoter, a bonobo GFAP promoter, a chimpanzee GFAP promoter, a gorilla GFAP promoter, a macaque GFAP promoter, a marmoset GFAP promoter, a pigtail monkey GFAP promoter, a baboon GFAP promoter, a gibbon ape GFAP promoter, and a marmoset GFAP promoter.

116. The method of any one of claims 89-115, wherein the IRES sequence comprises a nucleic acid sequence that is at least 80% identical to: SEQ ID NO. 3, or a complement thereof.

117. The method of claim 114, wherein the hGFAP promoter comprises a nucleic acid sequence that is at least 80% identical to: SEQ ID NO. 4, or a complement thereof.

118. The method of claim 114, wherein the hGFAP promoter comprises a nucleic acid sequence that is at least 80% identical to: 18, or the complement thereof.

119. The method of claim 114, wherein the hGFAP promoter comprises a nucleic acid sequence that is at least 80% identical to: SEQ ID NO. 27, or a complement thereof.

120. The method of claim 91 or 92, wherein the linker is selected from the group consisting of P2A or T2A.

121. The method of claim 120, wherein the P2A linker comprises a nucleic acid sequence that is at least 80% identical to a sequence selected from the group consisting of: SEQ ID NOS 19 and 22, or the complements thereof.

122. The method of claim 120, wherein the T2A linker comprises a nucleic acid sequence that is at least 80% identical to a sequence selected from the group consisting of: SEQ ID NOS 20 and 23, or the complements thereof.

123. The method of claim 91 or 92, wherein the enhancer comprises a nucleic acid sequence that is at least 80% identical to: SEQ ID NO. 2, or a complement thereof.

124. The method of claim 91 or 92, wherein the chimeric intron comprises a nucleic acid sequence that is at least 80% identical to: SEQ ID NO. 5 or 28, or a complement thereof.

125. The method of claim 91 or 92, wherein the WPRE comprises a nucleic acid sequence at least 80% identical to a nucleic acid sequence selected from the group consisting of: SEQ ID NOS.7 and 30, or the complements thereof.

126. The method of claim 91 or 92, wherein the SV40 polyadenylation signal comprises a nucleic acid sequence that is at least 80% identical to: SEQ ID NOS.8, 21, 26, or the complements thereof.

127. The method of claim 91 or 92, wherein the vector further comprises a nucleic acid sequence encoding an AAV protein sequence.

128. The method of any one of claims 89-92, wherein the vector comprises an AAV serotype 2 Inverted Terminal Repeat (ITR).

129. The method of any one of claims 89-92, wherein the vector comprises an AAV serotype 5 Inverted Terminal Repeat (ITR).

130. The method of any one of claims 89-92, wherein the vector comprises an AAV serotype 9 Inverted Terminal Repeat (ITR).

131. The method of any one of claims 89-92, wherein the vector comprises at least one ITR nucleic acid sequence at least 80% identical to: SEQ ID NO. 1.

132. The method of any one of claims 89-92, wherein the vector comprises at least one ITR nucleic acid sequence at least 80% identical to: SEQ ID NO. 9.

133. The method of claim 91, wherein the transformation occurs in the Central Nervous System (CNS) or peripheral nervous system.

134. The method of claim 91, wherein the transformation occurs in the CNS.

135. The method of claim 91 or 92, wherein the subject in need thereof is a mammal.

136. The method of claim 135, wherein the mammal is a human.

137. The method of claim 135, wherein the mammal is a non-human primate.

138. The method of claim 91 or 92, wherein said delivering comprises topical administration.

139. The method of claim 91 or 92, wherein said delivering comprises systemic administration.

140. The method of claim 91 or 92, wherein the delivering comprises administration selected from the group consisting of: intraperitoneal administration, intramuscular administration, intravenous administration, intrathecal administration, intracerebral administration, intracranial, lateral ventricle of the brain, intracavitary, intravitreal, subretinal, intraparenchymal administration, intranasal administration, and oral administration.

141. The method of claim 91 or 92, wherein the injecting comprises injecting selected from the group consisting of: intraperitoneal injection, intramuscular injection, intravenous injection, intrathecal injection, intracerebral injection, intracranial, lateral ventricle of the brain, intracavitary, intravitreal, subretinal, intraparenchymal injection, intranasal injection, and oral injection.

142. The method of claim 91 or 92, wherein the delivering comprises injection.

143. The method of any one of claims 89, 90, or 142, wherein the injecting is at between 10 ¹⁰ particle/mL and 10 ¹⁴ Concentration between individual particles/mL.

144. The method of claim 143, wherein the injecting further comprises a flow rate of between 0.1 μl/min and 5.0 μl/min.

145. The method of claim 91, wherein the at least one glial cell is selected from the group consisting of at least one astrocyte and at least one NG2 cell.

146. The method of claim 145, wherein the at least one glial cell is at least one astrocyte.

147. The method of claim 145 or 146, wherein the at least one astrocyte is a reactive astrocyte.

148. The method of claim 91, wherein the neuron is a functional neuron.

149. The method of any one of claims 89, 90 or 148, wherein the functional neuron is selected from the group consisting of a glutamatergic neuron, a gabaergic neuron, a dopaminergic neuron, a cholinergic neuron, a serotonergic neuron, an adrenergic neuron, a motor neuron, and a peptidoergic neuron.

150. The method of claim 91, wherein the subject exhibits an improvement in at least one symptom of a neurological disorder compared to the subject prior to the delivering.

151. The method of claim 150, wherein the improvement is measured within 1 year of the delivery.

152. The method of any one of claims 89, 90, or 142, wherein the method comprises injecting the AAV directly into the brain of the subject.

153. The method of any one of claims 89 or 90, wherein the transformation is in the cerebral cortex of the brain.

154. The method of any one of claims 89, 90, or 142, wherein the method comprises injecting the AAV directly into the spinal cord of the subject.

155. The method of claim 92, wherein the neurological disorder comprises an injury to the Central Nervous System (CNS) or peripheral nervous system.

156. The method of claim 92, wherein the neurological disorder is selected from the group consisting of: alzheimer's disease, parkinson's disease, amyotrophic Lateral Sclerosis (ALS), huntington's disease, epilepsy, physical injury, stroke, cerebral aneurysms, traumatic brain injury, concussion, tumors, inflammation, infection, ataxia, brain atrophy, spinal cord atrophy, multiple sclerosis, traumatic spinal cord injury, ischemic or hemorrhagic myelopathy (myelopathy), global cerebral ischemia, hypoxic ischemic encephalopathy, embolism, fibrocartilage embolic myelopathy, thrombosis, kidney disease, chronic inflammatory diseases, meningitis, and cerebral venous sinus thrombosis.

157. The method of claim 92, wherein the neurological disorder is alzheimer's disease.

158. The method of claim 92, wherein the neurological disorder is parkinson's disease.

159. The method of claim 92, wherein the neurological disorder is ALS.

160. The method of claim 92, wherein the neurological disorder is huntington's disease.

161. The method of claim 92, wherein the neurological disorder is stroke.

162. The method of claim 161, wherein the stroke is ischemic stroke.

163. The method of claim 161, wherein the stroke is a hemorrhagic stroke.

164. The method of claim 92, wherein the method is capable of converting at least one glial cell into a neuron.

165. The method of claim 164, wherein the glial cell is selected from the group consisting of an astrocyte and a NG2 cell.

166. The method of claim 164, wherein the glial cell is an astrocyte.

167. The method of claim 166, wherein the astrocytes are reactive astrocytes.

168. The method of claim 164, wherein the glial cell is GFAP positive.

169. The method of claim 164, wherein the neuron is a functional neuron.

170. The method of claim 169, wherein the functional neuron is selected from the group consisting of a glutamatergic neuron, a gabaergic neuron, a dopaminergic neuron, a cholinergic neuron, a serotonergic neuron, an adrenergic neuron, a motor neuron, and a peptidoergic neuron.

171. The method of claim 89 or 90, wherein a therapeutically effective dose of the AAV is injected into the subject.

172. The method of claim 91 or 92, wherein a therapeutically effective dose of the AAV is delivered to the subject.

173. The method of claim 171 or 172, wherein the therapeutically effective dose is administered with a pharmaceutically acceptable carrier.

(c) A chimeric intron comprising the nucleic acid sequence of SEQ ID NO. 5;

and

175. An adeno-associated virus (AAV) vector comprising: a human neurogenic differentiation factor 1 (hNeuroD 1) sequence, the hNeuroD1 sequence comprising a nucleic acid sequence of SEQ ID No. 6, and a second sequence comprising a nucleic acid sequence selected from the group consisting of SEQ ID nos. 11, 13, and 15, wherein the hNeuroD1 sequence and the second sequence are separated by: (i) a P2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID nos. 19 and 22, (ii) a T2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID nos. 20 and 23, or (iii) an Internal Ribosome Entry Site (IRES) sequence of an encephalomyocarditis virus comprising SEQ ID No. 3, wherein the hNeuroD1 sequence and the second sequence are operably linked to a regulatory element comprising:

(c) A chimeric intron comprising the nucleic acid sequence of SEQ ID NO. 28;

And

176. An adeno-associated virus (AAV) vector comprising: a nucleic acid coding sequence encoding a human neurogenic differentiation factor 1 (hNeuroD 1) protein, the hNeuroD1 protein comprising the amino acid sequence of SEQ ID NO:10, and a second nucleic acid coding sequence encoding a second protein having amino acids selected from the group consisting of SEQ ID NOs: 12, 14 and 16, wherein the hNeuroD1 coding sequence and the second coding sequence are separated by: (i) a P2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID nos. 19 and 22, (ii) a T2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID nos. 20 and 23, or (iii) an Internal Ribosome Entry Site (IRES) sequence of an encephalomyocarditis virus comprising SEQ ID No. 3, wherein the hNeuroD1 coding sequence and the second coding sequence are operably linked to a regulatory element comprising:

(c) A chimeric intron comprising the nucleic acid sequence of SEQ ID NO. 5;

And

177. An adeno-associated virus (AAV) vector comprising: a nucleic acid coding sequence encoding a human neurogenic differentiation factor 1 (hNeuroD 1) protein, the hNeuroD1 protein comprising the amino acid sequence of SEQ ID NO:10, and a second nucleic acid coding sequence encoding a second protein having amino acids selected from the group consisting of SEQ ID NOs: 12, 14 and 16, wherein the hNeuroD1 coding sequence and the second coding sequence are separated by: (i) a P2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID nos. 19 and 22, (ii) a T2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID nos. 20 and 23, or (iii) an Internal Ribosome Entry Site (IRES) sequence of an encephalomyocarditis virus comprising SEQ ID No. 3, wherein the hNeuroD1 coding sequence and the second coding sequence are operably linked to a regulatory element comprising:

(c) A chimeric intron comprising the nucleic acid sequence of SEQ ID NO. 28;

And

(c) A chimeric intron comprising the nucleic acid sequence of SEQ ID NO. 28;

179. The composition of claim 178, wherein (ii) comprises an AAV comprising a nucleic acid sequence operably linked to a regulatory element comprising SEQ ID No. 13, the regulatory element comprising:

(c) A chimeric intron comprising the nucleic acid sequence of SEQ ID NO. 28;

180. The composition of claim 178, wherein (ii) comprises an AAV comprising a nucleic acid sequence comprising SEQ ID No. 11 operably linked to a regulatory element comprising:

(c) A chimeric intron comprising the nucleic acid sequence of SEQ ID NO. 28;

182. The composition of claim 181, wherein (ii) comprises an AAV vector comprising a nucleic acid coding sequence encoding a protein having an amino acid sequence comprising SEQ ID No. 14 operably linked to a regulatory element comprising:

(c) A chimeric intron comprising the nucleic acid sequence of SEQ ID NO. 28;

183. The composition of claim 181, wherein (ii) comprises an AAV comprising a nucleic acid sequence comprising SEQ ID No. 12 operably linked to a regulatory element comprising:

(c) A chimeric intron comprising the nucleic acid sequence of SEQ ID NO. 28;

184. The AAV vector or composition of claim 48, wherein the synthetic polyadenylation signal comprises a nucleic acid sequence that is at least 80% identical to: SEQ ID NO. 31, or a complement thereof.