CN116761812A - NEUROD1 and DLX2 vectors - Google Patents

Abstract

The present disclosure relates to AAV vectors, compositions and methods related to converting glial cells into neurons by using NeuroD1 and Dlx2 coding sequences in AAV vectors.

Description

NEUROD1 and DLX2 vectors

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 63/084,945, filed on September 29, 2020, and U.S. Provisional Application No. 63/247,442, filed on September 23, 2021, both of which are incorporated herein by reference in their entirety.

Incorporation of Sequence Listing

Contained in a file named P34838WO00_SL.TXT (28,311 bytes (in MS- The sequence listing created on September 27, 2021 and measured in (measured in)) is submitted electronically herewith and is incorporated by reference in its 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 Dlx2.

Background Art

In subjects with neurological disorders or following injury to the central nervous system (CNS) or peripheral nervous system (PNS), neurons are often killed or damaged and are unable to regenerate.

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

There are currently no methods available for regenerating functional new neurons in human subjects with neurological disorders using adeno-associated viruses (AAV).

Summary of the invention

In one aspect, the present disclosure provides and includes an adeno-associated virus (AAV) vector comprising: a human neurogenic differentiation factor 1 (hNeuroD1) sequence, the hNeuroD1 sequence comprising a nucleic acid sequence of SEQ ID NO:6, and a human distal homeobox-free 2 (hDlx2) sequence, the hDlx2 sequence comprising a nucleic acid sequence of SEQ ID NO:13, wherein the hNeuroD1 sequence and the hDlx2 sequence are separated by: (i) a P2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs:15 and 18, (ii) a T2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs:16 and 19, or (iii) an internal ribosome entry site (IRES) sequence of encephalomyocarditis virus comprising SEQ ID NO:3, wherein the hNeuroD1 sequence and the hDlx2 sequence are operably linked to a regulatory element, the regulatory element comprising: (a) a nucleic acid sequence selected from the group consisting of SEQ ID NOs:15 and 18, (b) a nucleic acid sequence selected from the group consisting of SEQ ID NOs:16 and 19, NO:4, 12 and 26 consisting of a glial fibrillary acid protein (GFAP) promoter; (b) comprising a nucleic acid sequence of SEQ ID NO:2 from the human elongation factor -1 alpha (EF1-alpha) promoter enhancer or comprising a nucleic acid sequence of SEQ ID NO:11 cytomegalovirus (CMV) enhancer; (c) comprising a chimeric intron comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:5 and 27; (d) comprising a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:7 and 29; and (e) comprising a SV40 polyadenylation signal sequence of SEQ ID NO:8, a hGH polyadenylation sequence of SEQ ID NO:17, or a bGH polyadenylation sequence of SEQ ID NO:30.

In one aspect, the present disclosure provides and includes an adeno-associated virus (AAV) vector comprising: a nucleic acid sequence encoding a human neurogenic differentiation factor 1 (hNeuroD1) protein, the hNeuroD1 protein comprising the amino acid coding sequence of SEQ ID NO: 10, and a nucleic acid coding sequence encoding a human distal homeobox-free 2 (hDlx2) protein, the hDlx2 protein comprising the amino acid sequence of SEQ ID NO: 14, wherein the hNeuroD1 coding sequence and the hDlx2 coding sequence are separated by: (i) a P2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 15 and 18, (ii) a T2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16 and 19, or (iii) an internal ribosome entry site (IRES) sequence of encephalomyocarditis virus comprising SEQ ID NO: 3, wherein the hNeuroD1 coding sequence and the hDlx2 coding sequence are operably linked to a regulatory element, the regulatory element comprising: (a) a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 15 and 18, (b) a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16 and 19, or (c) a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16 and 19, NO:4, 12 and 26 consisting of a glial fibrillary acid protein (GFAP) promoter; (b) comprising a nucleic acid sequence of SEQ ID NO:2 from the human elongation factor -1 alpha (EF1-alpha) promoter enhancer or comprising a nucleic acid sequence of SEQ ID NO:11 cytomegalovirus (CMV) enhancer; (c) comprising a chimeric intron comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:5 and 27; (d) comprising a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:7 and 29; and (e) comprising a SV40 polyadenylation signal sequence of SEQ ID NO:8, a hGH polyadenylation sequence of SEQ ID NO:17, or a bGH polyadenylation sequence of SEQ ID NO:30.

In one aspect, the present disclosure provides and includes an adeno-associated virus (AAV) vector comprising a NeuroD1 nucleic acid coding sequence encoding a neurogenic differentiation factor 1 (NeuroD1) protein and a Dlx2 nucleic acid coding sequence encoding a distal homeobox-free 2 (Dlx2) protein, wherein the NeuroD1 coding sequence and the Dlx2 coding sequence are separated by a linker sequence, wherein the NeuroD1 coding sequence and the Dlx2 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) a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE); and (e) a polyadenylation signal sequence.

In one aspect, the present disclosure provides and includes a composition comprising an adeno-associated virus (AAV) vector for converting human glial cells into functional neurons, wherein the AAV vector comprises: a human neurogenic differentiation factor 1 (hNeuroD1) sequence, the hNeuroD1 sequence having a nucleic acid sequence of SEQ ID NO:6, and a human distal homeobox-free 2 (hD1x2) sequence, the hD1x2 sequence having a nucleic acid sequence of SEQ ID NO:13, wherein the hNeuroD1 sequence and the hD1x2 sequence are separated by: (i) a P2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs:15 and 18, (ii) a T2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs:16 and 19, or (iii) an internal ribosome entry site (IRES) sequence of encephalomyocarditis virus comprising SEQ ID NO:3, wherein the hNeuroD1 sequence and the hD1x2 sequence are operably linked to a regulatory element, the regulatory element comprising: (a) a nucleic acid sequence selected from the group consisting of SEQ ID NOs:15 and 18, (b) a nucleic acid sequence selected from the group consisting of SEQ ID NOs:16 and 19, or (c) a nucleic acid sequence selected from the group consisting of SEQ ID NOs:16 and 19, NO:4, 12 and 26 consisting of the human glial fibrillary acid protein (GFAP) promoter; (b) comprising the nucleic acid sequence of SEQ ID NO:2 from the human elongation factor -1 alpha (EF-1 alpha) promoter enhancer or comprising the nucleic acid sequence of SEQ ID NO:11 cytomegalovirus (CMV) enhancer; (c) comprising a chimeric intron of the nucleic acid sequence selected from the group consisting of SEQ ID NO:5 and 27; (d) comprising a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) of the nucleic acid sequence selected from the group consisting of SEQ ID NO:7 and 29; and (e) comprising the SV40 polyadenylation signal sequence of SEQ ID NO:8, the hGH polyadenylation sequence of SEQ ID NO:17, or the bGH polyadenylation sequence of SEQ ID NO:30.

In one aspect, the present disclosure provides and includes a composition comprising an adeno-associated virus (AAV) vector for converting human glial cells into functional neurons, wherein the AAV vector comprises: a nucleic acid coding sequence encoding a human neurogenic differentiation factor 1 (hNeuroD1) protein, the hNeuroD1 protein comprising the amino acid sequence of SEQ ID NO: 10, and a nucleic acid coding sequence encoding a human distal homeobox-free 2 (hDlx2) protein, the hDlx2 protein comprising the amino acid sequence of SEQ ID NO: 14, wherein the hNeuroD1 coding sequence and the hDlx2 coding sequence are separated by: (i) a P2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 15 and 18, (ii) a T2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16 and 19, or (iii) a nucleic acid coding sequence comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 17 and 18. NO:3 of the encephalomyocarditis virus internal ribosome entry site (IRES) sequence, wherein the hNeuroD1 coding sequence and the hD1x2 coding sequence are operably linked to a regulatory element, the regulatory element comprising: (a) a human glial fibrillary acid protein (GFAP) promoter comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:4, 12 and 26; (b) an enhancer from the human elongation factor-1α (EF-1α) 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:11; (c) a chimeric intron comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:5 and 27; (d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:7 and 29; and (e) an SV40 polyadenylation signal sequence comprising the nucleic acid sequence of SEQ ID NO:8, an hGH polyadenylation sequence comprising the nucleic acid sequence of SEQ ID NO:17, or a cytomegalovirus (CMV) enhancer comprising the nucleic acid sequence of SEQ ID NO:11. The bGH polyadenylation sequence of the nucleic acid sequence of NO:30.

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 (NeuroD1) sequence and a distal homeobox-less 2 (Dlx2) sequence, wherein the NeuroD1 sequence and the Dlx2 sequence are separated by a linker sequence, wherein the NeuroD1 sequence and the Dlx2 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) a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE); and (e) a polyadenylation signal.

In one aspect, the present disclosure provides and includes a method for 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, the DNA vector construct comprising: a human neurogenic differentiation factor 1 (hNeuroD1) sequence, the hNeuroD1 sequence comprising a nucleic acid sequence of SEQ ID NO: 6, and a human distal homeobox-free 2 (hD1x2) sequence, the hD1x2 sequence comprising a nucleic acid sequence of SEQ ID NO: 13, wherein the hNeuroD1 sequence and the hD1x2 sequence are separated by: (i) a P2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 15 and 18, (ii) a T2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16 and 19, or (iii) a linker comprising a nucleic acid sequence of SEQ ID NOs: 17 and 18. NO:3 of the encephalomyocarditis virus internal ribosome entry site (IRES) sequence, wherein the hNeuroD1 sequence and the hD1x2 sequence are operably linked to a regulatory element, the regulatory element comprising: (a) a human glial fibrillary acid protein (GFAP) promoter comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4, 12 and 26; (b) an enhancer from a human elongation factor-1α (EF-1α) promoter comprising a nucleic acid sequence of SEQ ID NO: 2 or a cytomegalovirus (CMV) enhancer comprising a nucleic acid sequence of SEQ ID NO: 11; (c) a chimeric intron comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 5 and 27; (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 29; and (e) an SV40 polyadenylation signal sequence comprising a nucleic acid sequence of SEQ ID NO: 8, an hGH polyadenylation sequence comprising a nucleic acid sequence of SEQ ID NO: 17, or a cytomegalovirus (CMV) enhancer comprising a nucleic acid sequence of SEQ ID NO: 11. The bGH polyadenylation sequence of the nucleic acid sequence of NO:30.

In one aspect, the present disclosure provides and includes a method for 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, the DNA vector construct comprising: a nucleic acid coding sequence encoding a human neurogenic differentiation factor 1 (hNeuroD1) protein, the hNeuroD1 protein comprising the amino acid sequence of SEQ ID NO: 10, and a nucleic acid coding sequence encoding a human distal homeobox-free 2 (hDlx2) protein, the hDlx2 protein comprising the amino acid sequence of SEQ ID NO: 14, wherein the hNeuroD1 coding sequence and the hDlx2 coding sequence are separated by: (i) a P2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 15 and 18, (ii) a T2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16 and 19, or (iii) a nucleic acid coding sequence comprising SEQ ID NOs: 1 NO:3 of the encephalomyocarditis virus internal ribosome entry site (IRES) sequence, wherein the hNeuroD1 coding sequence and hD1x2 coding sequence are operably linked to an expression control element, the expression control element comprising: (a) a human glial fibrillary acid protein (GFAP) promoter comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4, 12 and 26; (b) an enhancer from a human elongation factor-1α (EF-1α) promoter comprising a nucleic acid sequence of SEQ ID NO: 2 or a cytomegalovirus (CMV) enhancer comprising a nucleic acid sequence of SEQ ID NO: 11; (c) a chimeric intron comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 5 and 27; (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 29; and (e) an SV40 polyadenylation signal sequence comprising a nucleic acid sequence of SEQ ID NO: 8, a SV40 polyadenylation signal sequence comprising a nucleic acid sequence of SEQ ID NO: 9, a SV40 polyadenylation signal sequence comprising a nucleic acid sequence of SEQ ID NO: 10 The hGH polyadenylation sequence comprises the nucleic acid sequence of SEQ ID NO:17, or the bGH polyadenylation sequence comprises the nucleic acid sequence of SEQ ID NO:30.

In one aspect, the present disclosure provides and includes a method for converting glial cells of a subject in need thereof into neurons, 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 (NeuroD1) sequence and a distal homeobox-less 2 (Dlx2) sequence, wherein the NeuroD1 sequence and the Dlx2 sequence are separated by a linker sequence, wherein the NeuroD1 sequence and the Dlx2 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) a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE); and (e) a polyadenylation signal sequence, wherein the vector is capable of converting at least one glial cell of the subject in need thereof into a neuron.

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 the subject, wherein the AAV administered to the subject in need thereof comprises a DNA vector construct comprising a neurogenic differentiation factor 1 (NeuroD1) sequence and a distal homeobox-less 2 (Dlx2) sequence, wherein the NeuroD1 sequence and the Dlx2 sequence are separated by a linker sequence, wherein the NeuroD1 sequence and the Dlx2 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) a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE); and (e) a polyadenylation signal.

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 (hNeuroD1) sequence, the hNeuroD1 sequence comprising a nucleic acid sequence of SEQ ID NO: 6, and (ii) an adeno-associated virus (AAV) vector comprising a human distal homeobox-free 2 (hD1x2) sequence, the hD1x2 sequence comprising a nucleic acid sequence of SEQ ID NO: 13; wherein the hNeuroD1 sequence is operably linked to a regulatory element, the regulatory element comprising: (a) a glial fibrillary acidic protein (GFAP) promoter comprising a nucleic acid sequence of SEQ ID NO: 26; (b) an enhancer from a human elongation factor-1α (EF1-α) promoter comprising a nucleic acid sequence of SEQ ID NO: 2; (c) a chimeric intron comprising a nucleic acid sequence of SEQ ID NO: 5 or SEQ ID NO: 27; (d) a chimeric intron comprising a nucleic acid sequence of SEQ ID NO: 1 NO:29; and (e) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) comprising the nucleic acid sequence of SEQ ID NO:30.

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 (hNeuroD1) sequence, the hNeuroD1 sequence comprising a nucleic acid sequence of SEQ ID NO: 6, and (ii) an adeno-associated virus (AAV) vector comprising a human distal homeobox-free 2 (hD1x2) sequence, the hD1x2 sequence comprising a nucleic acid sequence of SEQ ID NO: 13; wherein the hNeuroD1 sequence is operably linked to a regulatory element, the regulatory element comprising: (a) a glial fibrillary acidic protein (GFAP) promoter comprising a nucleic acid sequence of SEQ ID NO: 26; (b) a cytomegalovirus (CMV) enhancer comprising a nucleic acid sequence of SEQ ID NO: 11; (c) a chimeric intron comprising a nucleic acid sequence of SEQ ID NO: 5 or SEQ ID NO: 27; (d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) comprising a nucleic acid sequence of SEQ ID NO: 29; and (e) a cytomegalovirus (CMV) enhancer comprising a nucleic acid sequence of SEQ ID NO: 12. The bGH polyadenylation sequence of the nucleic acid sequence of NO:30.

In one aspect, the present disclosure provides and includes a composition comprising: (i) an adeno-associated virus (AAV) vector comprising a nucleic acid sequence encoding a human neurogenic differentiation factor 1 (hNeuroD1) protein, the hNeuroD1 protein comprising the amino acid coding sequence of SEQ ID NO: 10, and (ii) an adeno-associated virus (AAV) vector comprising a nucleic acid coding sequence encoding a human distal homeobox-free 2 (hD1x2) protein, the hD1x2 protein comprising the amino acid sequence of SEQ ID NO: 14; wherein the nucleic acid sequence encoding the hNeuroD1 protein is operably linked to a regulatory element, the regulatory element comprising: (a) a glial fibrillary acidic protein (GFAP) promoter comprising the nucleic acid sequence of SEQ ID NO: 26; (b) an enhancer from a human elongation factor-1 alpha (EF1-α) promoter comprising the nucleic acid sequence of SEQ ID NO: 2; (c) a chimeric intron comprising the nucleic acid sequence of SEQ ID NO: 5 or SEQ ID NO: 27; (d) a chimeric intron comprising the nucleic acid sequence of SEQ ID NO: 1 NO:29; and (e) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) comprising the nucleic acid sequence of SEQ ID NO:30.

In one aspect, the present disclosure provides and includes a composition comprising: (i) an adeno-associated virus (AAV) vector comprising a nucleic acid sequence encoding a human neurogenic differentiation factor 1 (hNeuroD1) protein, the hNeuroD1 protein comprising an amino acid coding sequence of SEQ ID NO: 10, and (ii) an adeno-associated virus (AAV) vector comprising a nucleic acid coding sequence encoding a human distal homeobox-free 2 (hD1x2) protein, the hD1x2 protein comprising an amino acid sequence of SEQ ID NO: 14; wherein the nucleic acid sequence encoding the hNeuroD1 protein is operably linked to a regulatory element, the regulatory element comprising: (a) a glial fibrillary acidic protein (GFAP) promoter comprising a nucleic acid sequence of SEQ ID NO: 26; (b) a cytomegalovirus (CMV) enhancer comprising a nucleic acid sequence of SEQ ID NO: 11; (c) a chimeric intron comprising a nucleic acid sequence of SEQ ID NO: 5 or SEQ ID NO: 27; (d) a chimeric intron comprising a nucleic acid sequence of SEQ ID NO: 1 NO:29; and (e) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) comprising the nucleic acid sequence of SEQ ID NO:30.

In one aspect, the present disclosure provides and includes an adeno-associated virus (AAV) vector comprising: a human neurogenic differentiation factor 1 (hNeuroD1) sequence comprising a nucleic acid sequence of SEQ ID NO:6, and a human distal homeobox-free 2 (hD1x2) sequence comprising a nucleic acid sequence of SEQ ID NO:13, wherein the hNeuroD1 sequence and the hD1x2 sequence are separated by: (i) a P2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs:15 and 18, (ii) a T2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs:16 and 19, or (iii) an internal ribosome entry site (IRES) sequence of encephalomyocarditis virus comprising SEQ ID NO:3, wherein the hNeuroD1 sequence and the hD1x2 sequence are operably linked to a regulatory element comprising: (a) a nucleic acid sequence comprising SEQ ID NO:1 NO:26; (b) an enhancer from the human elongation factor-1α (EF1-α) promoter comprising the nucleic acid sequence of SEQ ID NO:2; (c) a chimeric intron comprising the nucleic acid sequence of SEQ ID NO:5 or SEQ ID NO:27; (d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) comprising the nucleic acid sequence of SEQ ID NO:29; and (e) a bGH polyadenylation sequence comprising the nucleic acid sequence of SEQ ID NO:30.

In one aspect, the present disclosure provides and includes an adeno-associated virus (AAV) vector comprising: a human neurogenic differentiation factor 1 (hNeuroD1) sequence comprising a nucleic acid sequence of SEQ ID NO:6, and a human distal homeobox-free 2 (hD1x2) sequence comprising a nucleic acid sequence of SEQ ID NO:13, wherein the hNeuroD1 sequence and the hD1x2 sequence are separated by: (i) a P2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs:15 and 18, (ii) a T2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs:16 and 19, or (iii) an internal ribosome entry site (IRES) sequence of encephalomyocarditis virus comprising SEQ ID NO:3, wherein the hNeuroD1 sequence and the hD1x2 sequence are operably linked to a regulatory element comprising: (a) a nucleic acid sequence comprising SEQ ID NO:1 NO:26; (b) a glial fibrillary acidic protein (GFAP) promoter comprising the nucleic acid sequence of SEQ ID NO:11; (c) a chimeric intron comprising the nucleic acid sequence of SEQ ID NO:5 or SEQ ID NO:27; (d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) comprising the nucleic acid sequence of SEQ ID NO:29; and (e) a bGH polyadenylation sequence comprising the nucleic acid sequence of SEQ ID NO:30.

In one aspect, the present disclosure provides and includes an adeno-associated virus (AAV) vector comprising: a nucleic acid sequence encoding a human neurogenic differentiation factor 1 (hNeuroD1) protein, the hNeuroD1 protein comprising the amino acid coding sequence of SEQ ID NO: 10, and a nucleic acid coding sequence encoding a human distal homeobox-free 2 (hDlx2) protein, the hDlx2 protein comprising the amino acid sequence of SEQ ID NO: 14, wherein the hNeuroD1 coding sequence and the hDlx2 coding sequence are separated by: (i) a P2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 15 and 18, (ii) a T2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16 and 19, or (iii) an internal ribosome entry site (IRES) sequence of encephalomyocarditis virus comprising SEQ ID NO: 3, wherein the hNeuroD1 coding sequence and the hDlx2 coding sequence are operably linked to a regulatory element, the regulatory element comprising: (a) a nucleic acid sequence comprising SEQ ID NO: 1 NO:26; (b) an enhancer from the human elongation factor-1α (EF1-α) promoter comprising the nucleic acid sequence of SEQ ID NO:2; (c) a chimeric intron comprising the nucleic acid sequence of SEQ ID NO:5 or SEQ ID NO:27; (d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) comprising the nucleic acid sequence of SEQ ID NO:29; and (e) a bGH polyadenylation sequence comprising the nucleic acid sequence of SEQ ID NO:30.

In one aspect, the present disclosure provides and includes an adeno-associated virus (AAV) vector comprising: a nucleic acid sequence encoding a human neurogenic differentiation factor 1 (hNeuroD1) protein, the hNeuroD1 protein comprising the amino acid coding sequence of SEQ ID NO: 10, and a nucleic acid coding sequence encoding a human distal homeobox-free 2 (hDlx2) protein, the hDlx2 protein comprising the amino acid sequence of SEQ ID NO: 14, wherein the hNeuroD1 coding sequence and the hDlx2 coding sequence are separated by: (i) a P2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 15 and 18, (ii) a T2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16 and 19, or (iii) an internal ribosome entry site (IRES) sequence of encephalomyocarditis virus comprising SEQ ID NO: 3, wherein the hNeuroD1 coding sequence and the hDlx2 coding sequence are operably linked to a regulatory element, the regulatory element comprising: (a) a nucleic acid sequence comprising SEQ ID NO: 1 NO:26; (b) a glial fibrillary acidic protein (GFAP) promoter comprising the nucleic acid sequence of SEQ ID NO:11; (c) a chimeric intron comprising the nucleic acid sequence of SEQ ID NO:5 or SEQ ID NO:27; (d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) comprising the nucleic acid sequence of SEQ ID NO:29; and (e) a bGH polyadenylation sequence comprising the nucleic acid sequence of SEQ ID NO:30.

In one aspect, the present disclosure provides and includes an adeno-associated virus (AAV) vector comprising: a human neurogenic differentiation factor 1 (hNeuroD1) sequence comprising a nucleic acid sequence of SEQ ID NO:6, and a human distal homeobox-free 2 (hDlx2) sequence comprising a nucleic acid sequence of SEQ ID NO:13, wherein the hNeuroD1 sequence and the hDlx2 sequence are separated by: a P2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs:15 and 18, or an internal ribosome entry site (IRES) sequence of encephalomyocarditis virus comprising SEQ ID NO:3, wherein the hNeuroD1 sequence and the hDlx2 sequence are operably linked to a regulatory element comprising: (a) a glial fibrillary acidic protein (GFAP) promoter comprising a nucleic acid sequence of SEQ ID NO:26; (b) a cytomegalovirus (CMV) enhancer comprising a nucleic acid sequence of SEQ ID NO:11; (c) a cytomegalovirus (CMV) enhancer comprising a nucleic acid sequence of SEQ ID NO:5 or SEQ ID NO:6. ID NO:27; and (d) a bGH polyadenylation sequence comprising the nucleic acid sequence of SEQ ID NO:30.

In one aspect, the present disclosure provides and includes an adeno-associated virus (AAV) vector comprising: a nucleic acid sequence encoding a human neurogenic differentiation factor 1 (hNeuroD1) protein, the hNeuroD1 protein comprising the amino acid coding sequence of SEQ ID NO: 10, and a nucleic acid coding sequence encoding a human distal homeobox-free 2 (hDlx2) protein, the hDlx2 protein comprising the amino acid sequence of SEQ ID NO: 14, wherein the hNeuroD1 coding sequence and the hDlx2 coding sequence are separated by: a P2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 15 and 18, or an internal ribosome entry site (IRES) sequence of encephalomyocarditis virus comprising SEQ ID NO: 3, wherein the hNeuroD1 coding sequence and the hDlx2 coding sequence are operably linked to a regulatory element, the regulatory element comprising: (a) a glial fibrillary acidic protein (GFAP) promoter comprising the nucleic acid sequence of SEQ ID NO: 26; (b) a promoter comprising the nucleic acid sequence of SEQ ID NO: 30; (c) a chimeric intron comprising the nucleic acid sequence of SEQ ID NO:5 or SEQ ID NO:27; and (d) a bGH polyadenylation sequence comprising the nucleic acid sequence of SEQ ID NO:30.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A depicts a diagram of aCE:Gfa681:NeuroD1:P2A:Dlx2:WPRE:SV40.

FIG. 1B depicts a graph of EF-1α:Gfa681:NeuroD1:P2A:Dlx2:WPRE:SV40.

Figure 1C depicts a graph of CE:Gfa681:NeuroD1:P2A:Dlx2:WPRE:hGH

FIG. 1D depicts a graph of EF-1α:Gfa681:NeuroD1:P2A:Dlx2:WPRE:hGH.

Figure 2A depicts a map of CE:Gfa681 NeuroD1:GSG-P2A:Dlx2:WPRE:SV40.

FIG. 2B depicts a graph of EF-1α:Gfa681 NeuroD1:GSG-P2A:Dlx2:WPRE:SV40.

FIG. 2C depicts a graph of CE:Gfa681 NeuroD1:GSG-P2A:Dlx2:WPRE:hGH.

FIG. 2D depicts a graph of EF-1α:Gfa681 NeuroD1:GSG-P2A:Dlx2:WPRE:hGH.

Figure 3A depicts a map of CE:Gfa681 NeuroD1:T2A:Dlx2:WPRE:SV40

FIG. 3B depicts a graph of EF-1α:Gfa681 NeuroD1:T2A:Dlx2:WPRE:SV40.

FIG. 3C depicts a graph of CE:Gfa681 NeuroD1:T2A:Dlx2:WPRE:hGH.

FIG. 3D depicts a graph of EF-1α:Gfa681 NeuroD1:T2A:Dlx2:WPRE:hGH.

Figure 4A depicts a graph of CE:Gfa681 NeuroD1:GSG-T2A:Dlx2:WPRE:SV40

FIG. 4B depicts a graph of EF-1α:Gfa681 NeuroD1:GSG-T2A:Dlx2:WPRE:SV40.

Figure 4C depicts a graph of CE:Gfa681 NeuroD1:GSG-T2A:Dlx2:WPRE:hGH.

FIG. 4D depicts a graph of EF-1α:Gfa681 NeuroD1:GSG-T2A:Dlx2:WPRE:hGH.

FIG. 5A depicts a graph of U6:shRNA:CE:Gfa681:NeuroD1:P2A:Dlx2:SV40.

FIG. 5B depicts a graph of U6:shRNA:EF-1α:Gfa681:NeuroD1:P2A:Dlx2:SV40.

FIG. 5C depicts a graph of U6:shRNA:CE:Gfa681:NeuroD1:GSG-P2A:Dlx2:SV40.

FIG. 5D depicts a graph of U6:shRNA:EF-1α:Gfa681:NeuroD1:GSG-P2A:Dlx2:SV40.

FIG. 6A depicts a graph of U6:shRNA:CE:Gfa681:NeuroD1:P2A:Dlx2:hGh.

FIG. 6B depicts a graph of U6:shRNA:EF-1α:Gfa681:NeuroD1:P2A:Dlx2:hGh.

FIG. 6C depicts a graph of U6:shRNA:CE:Gfa681:NeuroD1:GSG-P2A:Dlx2:hGh.

FIG. 6D depicts a graph of U6:shRNA:EF-1α:Gfa681:NeuroD1:GSG-P2A:Dlx2:hGh.

FIG. 7A depicts a graph of U6:shRNA:CE:Gfa681:NeuroD1:T2A:Dlx2:SV40.

FIG. 7B depicts a graph of U6:shRNA:EF-1α:Gfa681:NeuroD1:T2A:Dlx2:SV40.

FIG. 7C depicts a graph of U6:shRNA:CE:Gfa681:NeuroD1:GSG-T2A:Dlx2:SV40.

FIG. 7D depicts a graph of EF-1α:Gfa681:NeuroD1:GSG-T2A:Dlx2:SV40.

FIG. 8A depicts a graph of U6:shRNA:CE:Gfa681:NeuroD1:T2A:Dlx2:hGh.

FIG. 8B depicts a graph of U6:shRNA:EF-1α:Gfa681:NeuroD1:T2A:Dlx2:hGh.

FIG. 8C depicts a graph of U6:shRNA:CE:Gfa681:NeuroD1:GSG-T2A:Dlx2:hGh.

FIG. 8D depicts a vector map of U6:shRNA:EF-1α:Gfa681:NeuroD1:GSG-T2A:Dlx2:hGh.

Figure 9 measures AAV virus production from the P31 plasmid. Titer analysis was performed using target gene (GOI) primers, ITR region primers, and reverse packaging primers. Viral titers were calculated as vg/cell.

Figure 10 depicts the establishment of rat astrocyte primary cultures from Sprague-Dawley rat brains at 3 days postnatal. The upper left figure presents images of GFAP stained cells. The upper right figure presents images of SOX9 stained cells. The lower left figure presents images of DAPI stained cells. The lower right figure presents a merged image of GFAP, SOX9 and DAPI stained cells.

Figure 11 depicts a comparison of the NeuroD1 expression efficiency of plasmids. Primary rat astrocytes were transfected with P14 (CE: GfaABC1D: hNeuroD1-P2A-Dlx2: WPRE: SV40), P31 (EF-1αE: GfaABC1D: NeuroD1-P2A-Dlx2: WPRE: SV40) and P63 (CE: GfaABC1D: NeuroD1-GSG P2A-Dlx2: WPRE: SV40). The upper figure shows the NeuroD1 staining of cells, the middle figure shows the Dlx2 staining of cells, and the lower figure shows the merged cell NeuroD1, Dlx2 and DAPI staining.

Figure 12 depicts a comparison of AAV viral particle transduction at different doses using AAV9-P12 (pGfaABC1D: GFP). The upper left graph shows a dose of ^3x1010 vg/well. The upper right graph shows a dose of ^1x1010 vg/well. The lower graph shows a dose of ^2.5x109 vg/well.

Figures 13A and 13B depict quantitative analysis of AAV particle transduction into primary rat astrocytes.Figure 13A presents the percent transduction efficiency of AAV9-P12 (pGfaABC1D:GFP) and AAV5-P7 (pEF-1α:GFP) at MOIs of 5x105 ^vg /cell, ^2x105 vg/cell, and ^5x104 vg/cell. Figure 13B presents the percent transduction of AAV9-P12 (pGfaABC1D:GFP) in cells seeded at a range of densities (2x10 ⁴ cells/well, 1.5x10 ⁴ cells/well, 1x10 ⁴ cells/well, and 5x10 ³ cells/well) and infected with a range of amounts of virus (2 μl, 1 μl, 0.5 μl, 0.25 μl, 0.125 μl of 1x10 ¹³ vg/ml virus in 100 μl medium).

Figure 14 depicts RCA three weeks after transduction with control plasmid AAV9-P21 (CE-pGFA681-CI-GFP-WPRE-SV40pA) at ^2X1010 vg/ml. Cells were immunostained using antibodies against neuronal markers NeuN and MAP2 and DAPI (nuclear dye). GFP fluorescence indicates the presence of cells transduced with the control plasmid.

FIG. 15 depicts RCA immunostained with anti-NeuroD1 (ND1) antibody and DAPI (nuclear stain) 24 hours after transfection with NXL-P134 (CE-pGfa681-CRGI-hND1-oPRE-bGHpA).

FIG. 16 depicts RCA immunostained with anti-ND1 antibody and DAPI (nuclear stain) 6 days after transduction with AAV9-P134 (CE-pGfa681-CRGI-hND1-oPRE-bGHpA) at 2X10 ¹⁰ vg/ml.

Figure 17 depicts RCA immunostained with anti-NeuN and anti-MAP2 antibodies and DAPI (nuclear stain) 3 weeks after transduction with AAV9-P134 (CE-pGfa681-CRGI-hND1-oPRE-bGHpA) at ^2X1010 vg/ml. Neurons (NeuN//MAP2+) were generated from astrocyte cultures using transduction with ND1-containing vectors.

FIG. 18 depicts RCA immunostained with anti-ND1 antibody and DAPI (nuclear stain) 24 hours after transfection with NXL-P138 (EE-pGfa681-CRGI-hND1-oPRE-bGHpA).

FIG. 19 depicts RCA immunostained with anti-ND1 antibody and DAPI (nuclear stain) 6 days after transduction with AAV9-P138 (EE-pGfa681-CRGI-hND1-oPRE-bGHpA) at 2X10 ¹⁰ vg/ml.

Figure 20 depicts RCA immunostained with anti-NeuN and anti-MAP2 antibodies and DAPI (nuclear stain) 3 weeks after transduction with AAV9-P138 (EE-pGfa681-CRGI-hND1-oPRE-bGHpA) at ^2X1010 vg/ml. Neurons (NeuN//MAP2+) were generated from astrocyte cultures using transduction with ND1-containing vectors.

Figure 21 depicts RCA immunostained with anti-ND1 antibody and DAPI (nuclear dye) 6 days after transduction with AAV9-P9 (CE-pGfa681-CI-hND1-p2A-GFP-WPRE-SV40pA) at ^2X1010 vg/ml. GFP fluorescence indicates the presence of transduced cells.

Figure 22 depicts RCA immunostained with anti-NeuN and anti-MAP2 antibodies and DAPI (nuclear dye) 3 weeks after transduction with AAV9-P9 (CE-pGfa681-CI-hND1-p2A-GFP-WPRE-SV40pA) at ^2X1010 vg/ml. Neurons (NeuN//MAP2+) were generated from astrocyte cultures using transduction with ND1-containing vectors.

FIG. 23 depicts RCA immunostained with anti-ND1 antibody and DAPI (nuclear stain) 24 hours after transfection with NXL-P22 (CE-pGfa681-CI-hND1-WPRE-SV40pA).

FIG. 24 depicts RCA immunostained with anti-ND1 antibody and DAPI (nuclear stain) 6 days after transduction with AAV9-P22 (CE-pGfa681-CI-hND1 WPRE-SV40pA) at 2X10 ¹⁰ vg/ml.

Figure 25 depicts RCA immunostained with anti-NeuN and anti-MAP2 antibodies and DAPI (nuclear stain) 3 weeks after transduction with AAV9-P22 (CE-pGfa681-CI-hND1-WPRE-SV40pA) at ^2X1010 vg/ml. Neurons (NeuN//MAP2+) were generated from astrocyte cultures using transduction with ND1-containing vectors.

FIG. 26 depicts RCA immunostained with anti-ND1 antibody and DAPI (nuclear stain) 24 hours after transfection with NXL-P35 (EE-pGfa681-CI-hND1-WPRE-SV40pA).

FIG. 27 depicts RCA immunostained with anti-ND1 antibody and DAPI (nuclear stain) 6 days after transduction with AAV9-P35 (EE-pGfa681-CI-hND1 WPRE-SV40pA) at 2X10 ¹⁰ vg/ml.

Figure 28 depicts RCA immunostained with anti-NeuN antibody and DAPI (nuclear stain) 3 weeks after transduction with AAV9-P35 (EE-pGfa681-CI-hND1-WPRE-SV40pA) at ^2X1010 vg/ml. Transduction with ND1-containing vectors was performed to generate neurons (NeuN+) from astrocyte cultures.

FIG. 29 depicts RCA immunostained with anti-ND1 antibody and DAPI (nuclear stain) 24 hours after transfection with NXL-P107 (CE-pGfa681-CI-hND1-bGHpA).

FIG. 30 depicts RCA immunostained with anti-ND1 antibody and DAPI (nuclear stain) 24 hours after transfection with NXL-P108 (CE-pGfa681-CI-hND1-oPRE-bGHpA).

FIG. 31 depicts RCA immunostained with anti-ND1 antibody and DAPI (nuclear stain) 24 hours after transfection with NXL-P109 (CE-pGfa681-CRGI-hND1-bGHpA).

FIG. 32 depicts cerebral cortical tissues of mice infected with AAV9-P12 (P12 control group), AAV9-P12+AAV9-P134 (P134 group), and AAV9-P12+AAV9-P138 (P138 group) at 10 days post infection (dpi).

FIG. 33 depicts cerebral cortical tissues of mice infected with AAV9-P12+AAV9-P134 (P134 group) and AAV9-P12+AAV9-P138 (P138 group) at 30 days post infection (dpi).

FIG. 34 depicts the cerebral cortical tissues of mice infected with AAV9-P12 (P12 control group) and AAV9-P12+AAV9-P134 (P134 group) (bilateral injury model) at 10 dpi.

Figure 35 is a measurement graph of AAV virus production of P134, P130, P138 and P21 plasmids. Titer analysis was performed by qPCR using primers that amplify the target gene (GOI) and primers specific to the plasmid. Virus yield was calculated as vg/cell.

Figure 36 depicts the cerebral cortex tissue of mice infected with AAV9-P12 (P12 control group), AAV9-P12+AAV9-P134 (P134 group) and AAV9-P12+AAV9-P138 (P138 group) at 10 days post infection (dpi). Cells were immunostained using antibodies against NeuroD1, GFAP, NeuN and DAPI (nuclear dye). GFP fluorescence indicates the presence of cells infected with the control virus.

Figure 37 depicts the cerebral cortex tissue of mice infected with AAV9-P12 (P12 control group), AAV9-P12+AAV9-P134 (P134 group) and AAV9-P12+AAV9-P138 (P138 group) at 30 days post infection (dpi). Cells were immunostained using antibodies against NeuroD1, GFAP, NeuN and DAPI (nuclear dye). GFP fluorescence indicates the presence of cells infected with the control virus.

Figure 38 depicts the cerebral cortex tissue of mice infected with AAV9-P12 (P12 control group) and AAV9-P12 + AAV9-P134 (P134 group) (bilateral injury model) at 10dpi. Cells were immunostained using antibodies against NeuroD1, GFAP, NeuN and DAPI (nuclear dye). GFP fluorescence indicates the presence of cells infected with the control virus.

Figures 39A and 39B depict cerebral cortical tissue of mice infected with AAV9-P12 (P12 control group) and AAV9-P12+AAV9-P134 (P134 group) (bilateral injury model) at 30 dpi. Figure 39A depicts cells immunostained with antibodies against NeuroD1, GFAP, NeuN and DAPI (nuclear dye). GFP fluorescence indicates the presence of cells infected with the control virus. Figure 39B is a quantification of the conversion rate of glial cells to neurons at 30 dpi.

Figure 40 depicts two general diagrams of ND1 and Dlx2 constructs. Enhancer refers to ef1a enhancer or CMV enhancer. pGfa refers to 2.2 kb or 1.6 kb Gfa promoter. Poly(A) signal refers to SV40, bGH or hGH poly(A) signal.

FIG. 41 depicts RCA immunostained with anti-ND1 antibody, anti-D1x2 antibody and DAPI (nuclear stain) 24 hours after transfection with NXL-P112 (CE-pGfa681-CI-hD1x2-IRES-hND1-bGHpA).

FIG. 42 depicts RCA immunostained with anti-ND1 antibody, anti-D1x2 antibody and DAPI (nuclear stain) 6 days after transduction with AAV9-P112 (CE-pGfa681-CI-hD1x2-IRES-hND1-bGHpA) at 2X10 ¹⁰ vg/ml.

FIG. 43 depicts RCA immunostained with anti-NeuN antibody, anti-MAP2 antibody and DAPI (nuclear stain) 3 weeks after transduction with AAV9-P112 (CE-pGfa681-CI-hD1x2-IRES-hND1-bGHpA) at 2X10 ¹⁰ vg/ml.

FIG. 44 depicts RCA immunostained with anti-ND1 antibody, anti-D1x2 antibody and DAPI (nuclear stain) 24 hours after transfection with NXL-P122 (CE-pGfa681-CI-hD1x2-P2A-hND1-bGHpA).

FIG. 45 depicts RCA immunostained with anti-ND1 antibody, anti-D1x2 antibody and DAPI (nuclear stain) 6 days after transduction with AAV9-P122 (CE-pGfa681-CI-hD1x2-p2A-hND1-bGHpA) at 2X10 ¹⁰ vg/ml.

FIG. 46 depicts RCA immunostained with anti-NeuN antibody, anti-MAP2 antibody and DAPI (nuclear stain) 3 weeks after transduction with AAV9-P122 (CE-pGfa681-CI-hD1x2-P2A-hND1-bGHpA) at 2X10 ¹⁰ vg/ml.

FIG. 47 depicts RCA immunostained with anti-ND1 antibody, anti-D1x2 antibody and DAPI (nuclear stain) 24 hours after transfection with NXL-P124 (CE-pGfa681-CI-hND1-P2A-hD1x2-bGHpA).

FIG. 48 depicts RCA immunostained with anti-ND1 antibody, anti-Dlx2 antibody and DAPI (nuclear stain) 6 days after transduction with AAV9-P124 (CE-pGfa681-CI-hND1-p2A-hD1x2-bGHpA) at 2X10 ¹⁰ vg/ml.

FIG. 49 depicts RCA immunostained with anti-NeuN antibody, anti-MAP2 antibody and DAPI (nuclear stain) 3 weeks after transduction with AAV9-P124 (CE-pGfa681-CI-hND1-P2A-hD1x2-bGHpA) at 2X10 ¹⁰ vg/ml.

FIG. 50 depicts RCA immunostained with anti-ND1 antibody, anti-Dlx2 antibody and DAPI (nuclear stain) 24 hours after transfection with NXL-P20 (CE-pGfa681-CI-hND1-P2A-hDlx2-WPRE-SV40pA).

FIG. 51 depicts RCA immunostained with anti-ND1 antibody, anti-Dlx2 antibody and DAPI (nuclear dye) 6 days after transduction with AAV9-P20 (CE-pGfa681-CI-hDND1-p2A-hD1x2-WPRE-SV40pA) at 2X10 ¹⁰ vg/ml.

FIG. 52 depicts RCA immunostained with anti-NeuN antibody, anti-MAP2 antibody and DAPI (nuclear stain) 3 weeks after transduction with AAV9-P20 (CE-pGfa681-CI-hND1-P2A-hD1x2-bSV40pA) at 2X10 ¹⁰ vg/ml.

FIG. 53 depicts RCA immunostained with anti-ND1 antibody, anti-D1x2 antibody and DAPI (nuclear stain) 24 hours after transfection with NXL-P31 (EE-pGfa681-CI-hND1-P2A-hD1x2-WPRE-SV40pA).

FIG. 54 depicts RCA immunostained with anti-ND1 antibody, anti-D1x2 antibody and DAPI (nuclear stain) 24 hours after transfection with NXL-P123 (EE-pGfa681-CI-hD1x2-p2A-hND1-bGHpA).

FIG. 55 depicts RCA immunostained with anti-ND1 antibody, anti-D1x2 antibody and DAPI (nuclear stain) 24 hours after transfection with NXL-P113 (EE-pGfa681-CI-hD1x2-IRES-hND1-bGHpA).

FIG. 56 depicts RCA immunostained with anti-ND1 antibody, anti-D1x2 antibody and DAPI (nuclear stain) 24 hours after transfection with NXL-P111 (CE-pGfa681-CI-hD1x2-p2A-hND1-SV40pA).

Figures 57A and 57B depict brain striatal tissue of mice infected with AAV9-P12 (virus) at 10 dpi and 30 dpi. Figure 57A shows extensive infection of the mouse striatum by AAV-P12 at 10 dpi as evidenced by GFP fluorescence. Figure 57B shows infection of mice by AAV9-P12 at 30 dpi. Cells were immunostained with antibodies against GFAP and NeuN.

Figure 58 depicts brain striatum tissue of mice co-infected with AAV9-P12 and AAV9-P112 (P112 group) at 10 dpi. GFP fluorescence identified AAV9-P112 infected cells. Cells were immunostained with antibodies against GFAP and NeuN.

Figure 59 depicts brain striatum tissue of mice co-infected with AAV9-P12 and AAV9-P112 (P112 group) at 30dpi. GFP fluorescence identified cells infected with AAV9-P112. Cells were immunostained with antibodies against GFAP and NeuN. White arrows indicate that cells co-infected with AAV9-P12 and AAV9-P112 become NeuN-positive neurons.

Figure 60 depicts brain striatum tissue of mice co-infected with AAV9-P12 and AAV9-P122 (P122 group) at 10 dpi. GFP fluorescence identified AAV9-P112 infected cells. Cells were immunostained with antibodies against GFAP and NeuN.

Figure 61 depicts brain striatum tissue of mice co-infected with AAV9-P12 and AAV9-P122 (P122 group) at 30 dpi. GFP fluorescence identified cells infected with AAV9-P112. Cells were immunostained with antibodies against GFAP and NeuN.

FIG62 depicts rat cortical astrocytes (RCA) immunostained with anti-Dlx2 antibody and DAPI (nuclear stain) 24 hours after transfection with NXL-P104 (CE-pGfa681-CGRI-Dlx2-bGHpA) or NXL-P105 (CE-pGfa681-CI-Dlx2-oPRE-bGHpA).

Figure 63 depicts rat cortical astrocytes (RCA) immunostained with anti-Dlx2 antibody and DAPI (nuclear stain) 24 hours after transfection with NXL-P133 (EE-pGfa681-CGRI-Dlx2-oPRE-bGHpA), NXL-P137 (EE-pGfa681-CGRI-Dlx2-oPRE-bGHpA) or NXL-P131 (EE-pGfa681-CI-Dlx2-oPRE-bGHpA).

FIG. 64 depicts rat cortical astrocytes (RCA) immunostained with anti-Dlx2 antibody and DAPI (nuclear stain) after transduction with AAV9-P133 (CE-pGfa681-CGRI-Dlx2-oPRE-bGHpA).

Sequence Description

A listing of nucleic acid and amino acid sequences is provided in Table 1.

Table 1. Nucleotide sequences

DETAILED DESCRIPTION

Unless otherwise defined, all technical and scientific terms used have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. Where a term is provided in the singular, the inventors of the present invention also contemplate various aspects of the present disclosure described in the plural form of the term. In the event of differences in the terms and definitions used in the references incorporated by reference, the terms used in this 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, such as the American "Dictionary of Science" (Editors of the American Heritage Dictionaries, 2011, Houghton Mifflin Harcourt, Boston and New York), "McGraw-Hill Dictionary of Scientific and Technical Terms" (6th edition, 2002, McGraw-Hill, New York) or "Oxford Dictionary of Biology" (6th edition, 2008, Oxford University Press, Oxford and New York).

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

When a set of alternatives is presented, any and all combinations of the members that make up the set of alternatives are specifically contemplated. For example, if an item is selected from the group consisting of A, B, C, and D, the inventor will specifically contemplate each alternative individually (e.g., A alone, B alone, etc.) and combinations 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 refers to 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, C alone, a combination of A and B, 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 range and any numbers between the defined edges of the range. For example, "between 1 and 10" includes any number between 1 and 10 as well as the number 1 and the number 10.

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

As used herein, "hND1" refers to the human neurogenic differentiation factor (NeuroD1) gene or protein.

As used herein, "CE" refers to the cytomegalovirus (CMV) promoter enhancer sequence.

As used herein, "EE" refers to the Ef1α promoter enhancer sequence.

As used herein, "pGfa681" refers to a truncated sequence of the human glial fibrillary acid protein (GFAP) promoter of 681 bp in size. As used herein, "pGfa681", "Gfa681", "GfaABC1D" and "pGfaABC1D" 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 branch and 3'-acceptor site from an intron of the heavy chain variable region of an immunoglobulin gene.

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

As used herein, "GI" refers to the first intron of human glial fibrillary acidic protein (GFAP).

As used herein, "WPRE" refers to the Woodchuck Hepatitis Virus (WHV) Post-transcriptional Regulatory Element.

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

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

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

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

As used herein, "hDlx2" refers to the human distal homeobox 2 gene or protein.

Any composition or vector provided herein is specifically contemplated for use in any method provided herein.

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

In one aspect, the vector provided herein is a recombinant vector. 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 a laboratory method of genetic recombination (such as, but not limited to, molecular cloning). Gene vectors can be formed by a laboratory method of genetic recombination (such as, but not limited to, molecular cloning). Moreover, those skilled in the art can create recombinant vectors from scratch by synthesizing plasmids from a single nucleotide or by splicing together nucleic acid molecules from different pre-existing vectors, but are not limited thereto.

Adeno-associated viruses (AAV) are replication-defective, non-enveloped viruses of the genus Dependoparvovirus that infect humans and other primate species. AAV are not known to cause disease in any species, but they can induce a mild immune response. AAV can infect both dividing and dormant cells. AAV stably integrates into the human genome at a specific site called the AAVS1 locus on chromosome 19 (nucleotides 7774-11429 of GenBank accession number AC010327.8), although random integration at other loci in the human genome is possible.

AAV contains a linear genome with a single-stranded DNA of approximately 4700 nucleotides in length. The genome of AAV also includes 145 nucleotide-long inverted terminal repeats (ITRs) located at each end of the genome. The ITRs are flanked by two viral genes: rep (for replication, encoding nonstructural proteins) and cap (for capsid, encoding structural proteins). The ITRs contain all the cis-acting elements required for rescue, replication, and packaging of the AAV genome.

When used in gene therapy, the rep and cap genes of the AAV genomic sequence are removed and replaced with the target DNA located between two AAV ITRs. As used herein, "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 an AAV packaged with a DNA vector construct.

As used herein, the term "AAV vector serotype" primarily refers to the variation within the capsid protein of the 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 ITR, AAV serotype 2 ITR, AAV serotype 3 ITR, AAV serotype 4 ITR, AAV serotype 5 ITR, AAV serotype 6 ITR, AAV serotype 7 ITR, AAV serotype 8 ITR, AAV serotype 9 ITR, AAV serotype 10 ITR, AAV serotype 11 ITR and AAV serotype 12 ITR. In one aspect, the AAV vector ITR is an AAV serotype 1 ITR. In one aspect, the AAV vector ITR is an AAV serotype 2 ITR. In one aspect, the AAV vector ITR is an AAV serotype 3 ITR. In one aspect, the AAV vector ITR is an AAV serotype 4 ITR. In one aspect, the AAV vector ITR is an AAV serotype 5 ITR. In one aspect, the AAV vector ITR is an AAV serotype 6 ITR. In one aspect, the AAV vector ITR is an AAV serotype 7 ITR. In one aspect, the AAV vector ITR is an AAV serotype 8 ITR. In one aspect, the AAV vector ITR is an AAV serotype 9 ITR. In one aspect, the AAV vector ITR is an AAV serotype 10 ITR. In one aspect, the AAV vector ITR is an AAV serotype 11 ITR. In one aspect, the AAV vector ITR is an AAV serotype 12 ITR.

In one aspect, at least one AAV vector ITR nucleic acid sequence is selected from the group consisting of: SEQ ID NO: 1 and 9. In one aspect, at least one AAV vector ITR nucleic acid sequence is SEQ ID NO: 1. In one aspect, 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 that is at least 70% identical to SEQ ID NO: 1, or its complement. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence that is at least 75% identical to SEQ ID NO: 1, or its complement. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence that is at least 80% identical to SEQ ID NO: 1, or its complement. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence that is at least 85% identical to SEQ ID NO: 1, or its complement. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence that is at least 90% identical to SEQ ID NO: 1, or its complement. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence that is at least 91% identical to SEQ ID NO: 1, or its complement. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence that is at least 92% identical to SEQ ID NO: 1, or its complement. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence that is at least 93% identical to SEQ ID NO: 1, or its complement. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence that is at least 94% identical to SEQ ID NO: 1, or its complement. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence that is at least 95% identical to SEQ ID NO: 1, or its complement. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence that is at least 96% identical to SEQ ID NO: 1, or its complement. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence that is at least 97% identical to SEQ ID NO: 1, or its complement. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence that is at least 98% identical to SEQ ID NO: 1, or its complement. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence that is at least 99% identical to SEQ ID NO: 1, or its complement. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence that is at least 99.5% identical to SEQ ID NO: 1, or its complement. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence that is at least 99.8% identical to SEQ ID NO: 1, or its complement. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence that is at least 99.9% identical to SEQ ID NO: 1, or a complementary sequence thereof. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence that is 100% identical to SEQ ID NO: 1, or a complementary sequence thereof.

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

As used herein, the term "percent identity" or "identical percent" with respect to two or more nucleotide or amino acid sequences is calculated by (i) comparing two optimally aligned sequences (nucleotides or amino acids) over a comparison window (one or more "comparable" regions), (ii) determining the number of positions at which the same nucleic acid base (for nucleotide sequences) or amino acid residue (for proteins and polypeptides) occurs in the two sequences to produce the 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 the percent identity. If the "percent identity" is calculated relative to a reference sequence without specifying a particular comparison window, the percent identity is determined by dividing the number of matched positions over the comparison region by the total length of the reference sequence. Therefore, for the purposes of this application, when two sequences (query and subject) are optimally aligned (allowing gaps in their alignment), the "percent identity" of the query sequence is equal to the number of identical positions between the two sequences divided by the total number of positions in the query sequence over its length (or comparison window), then multiplied by 100%.

When percent sequence identity is used with reference to amino acids, residue positions that are considered non-identical typically differ by conservative amino acid substitutions, in which an amino acid residue is substituted with another amino acid residue having similar chemical properties (e.g., charge or hydrophobicity) and thus does not alter the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upward to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity."

In order to optimally align sequences to calculate their percent identity, various pairwise or multiple sequence alignment algorithms and programs 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 ^™ ) etc. Although other alignment and comparison methods are known in the art, the alignment and percent identity between two sequences (including the above-mentioned percent identity ranges) can be determined by the ClustalW algorithm, see, e.g., Chenna et al., "Multiple sequence alignment with the Clustal series of programs," Nucleic Acids Research 31:3497-3500 (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," Bioinformatics 23:2947-48 (2007); and Altschul et al., "Basic local alignment search tool." J. Mol. Biol. 215:403-410 (1990), the entire contents and disclosure of which are incorporated herein by reference.

As used herein, the term "complementarity percentage" or "complementarity percentage" with respect to two nucleotide sequences is similar to the concept of identity percentage, but refers to the percentage of nucleotides of the query sequence that best base-pair or hybridize with nucleotides of the subject sequence when the query sequence and the subject sequence are arranged linearly and optimally base-paired without secondary folded structures (such as loops, stems, or hairpins). Such complementarity percentages can be between two DNA chains, two RNA chains, or a DNA chain and an RNA chain. "Complementarity percentage" can 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 structures) over a comparison window, (ii) determining the number of base pair positions between the two sequences over the comparison window to produce the 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 obtain the complementarity percentage of the two sequences. The optimal base pairing of the two sequences can be determined by hydrogen bonding based on known pairings of nucleotide bases (such as G-C, A-T, and A-U). If the "complementarity percentage" is calculated relative to a reference sequence without specifying a particular comparison window, the percent identity is determined by dividing the number of complementary positions between the two linear sequences by the total length of the reference sequence. Thus, for the purposes of this application, when the two sequences (query and subject) are optimally base paired (allowing for mismatches or non-base paired nucleotides), the "complementarity percentage" of the query sequence is equal to the number of base paired positions between the two sequences divided by the total number of positions in the query sequence over its length, 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 appreciate that polynucleotides and nucleic acid molecules can comprise ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogs. The 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 molecules can encode polypeptides or small RNAs.

As used herein, the term "polypeptide" refers to a chain of at least two covalently linked amino acids. A polypeptide can be encoded by a polynucleotide as provided herein. A protein as provided herein can be encoded by a nucleic acid molecule as provided herein. A protein can comprise a polypeptide as provided herein. As used herein, "protein" refers to a chain of amino acid residues that can provide structure or enzymatic activity to a cell. As used herein, "coding sequence" refers to a nucleic acid sequence encoding a protein.

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

As used herein, the term "CpG island" of "CG island" refers to a CpG site that occurs 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 target host cell by replacing at least one codon of a sequence with a codon more frequently or most frequently used in the genes of the host cell while maintaining the original amino acid sequence.

As used herein, the term "enhancer" refers to a DNA sequence region that operates to initiate, assist, influence, cause and/or promote the transcription and expression of a related transcribable DNA sequence or coding sequence at least in certain 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 immunoprecipitation sequencing (ChIP-seq), DNA enzyme I hypersensitivity sequencing (DNase-seq), micrococcal nuclease sequencing (MNase-seq), formaldehyde assisted isolation of regulatory elements sequencing (FAIRE-seq) and chromatin transposase accessibility sequencing assay (ATAC-seq).

As used herein, the term "operably connected" refers to the functional connection between a promoter or other regulatory element and a relevant transcribable DNA sequence or coding sequence of a gene (or transgenic), so that a promoter, etc., operates to start, assist, influence, cause and/or promote transcription and expression of a relevant 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 the expression of an operably connected sequence. "Regulatory element" includes but is not limited to promoters, enhancers, leader sequences, transcription start sites (TSS), joints, 5' and 3' untranslated regions (UTRs), introns, polyadenylation signals and termination regions or sequences, etc., which are suitable, necessary or preferred for regulating or allowing the expression of a gene or transcribable DNA sequence in a cell. Such additional regulatory elements can be optional and used to enhance or optimize the expression of a 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 start site, and/or a TATA box and assists or promotes the transcription and expression of a related transcribable polynucleotide sequence and/or a gene (or transgenic). The promoter can be synthesized from, altered from, or derived from a known or naturally occurring promoter sequence or other promoter sequence. The promoter can also include a chimeric promoter comprising a combination of two or more heterologous sequences. Therefore, the promoter of the present application can include a variant of a promoter sequence that is similar in composition to other promoter sequences known or provided herein, but not identical.

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

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

The expression of mRNA 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), RNA blot (e.g., Northern blot) and RNA sequencing. Expression differences can be described as absolute quantification or relative quantification. See, e.g., Livak and Schmittgen, Methods, 25:402-408 (2001).

As used herein, "genome editing" or "gene editing" refers to targeted mutagenesis, insertion, deletion, inversion, substitution or translocation of a target nucleotide sequence in a genome using targeted editing technology. The target nucleotide sequence can be of any length, for example, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 75, at least 100, at least 250, at least 500, at least 1000, at least 2500, at least 5000, at least 10,000 or at least 25,000 nucleotides. Non-limiting examples of gene editing technology are small interfering RNA (siRNA) technology, small hairpin RNA (shRNA) technology, micro RNA (miRNA) technology, antisense oligonucleotide (ASO) technology or CRISPR/CAS technology.

As used herein, "ASO" or "antisense oligonucleotide" is a small single-stranded nucleic acid that binds to a target RNA sequence within a cell and silences the gene.

As used herein, "coding region", "gene region" or "gene" refers to a polynucleotide that can produce a functional unit. Non-limiting examples include a protein or a non-coding RNA molecule. A "coding region", "gene" or "gene region" may include a promoter, an enhancer sequence, a leader sequence, a transcription start site, a transcription stop site, a polyadenylation site, one or more exons, one or more introns, a 5'-UTR, a 3'-UTR or any combination thereof.

On the one hand, gene editing targets mutant huntingtin protein (Htt) aggregates. In one aspect, gene editing is performed by non-coding RNA molecules. Non-limiting examples of non-coding RNA molecules include microRNA (miRNA), miRNA precursor (pre-miRNA), small interfering RNA (siRNA), small RNA (18-26 nucleotides in length) and precursors encoding them, heterochromatic siRNA (hc-siRNA), Piwi interaction RNA (piRNA), hairpin double-stranded RNA (hairpin dsRNA), trans-acting siRNA (ta-siRNA), naturally occurring antisense siRNA (nat-siRNA), CRISPR RNA (crRNA), tracer RNA (tracrRNA), guide RNA (gRNA) and single guide RNA (sgRNA). In one aspect, shRNA targets Htt gene. In one aspect, siRNA targets Htt gene. In one aspect, ASO targets Htt gene. In one aspect, miRNA targets Htt gene. In one aspect, gRNA targets Htt gene. In one aspect, pre-miRNA targets Htt gene. In one aspect, the small RNA targets the Htt gene. In one aspect, the hc-siRNA targets the Htt gene. In one aspect, the piRNA targets the Htt gene. In one aspect, the hairpin dsRNA targets the Htt gene. In one aspect, the ta-siRNA targets the Htt gene. In one aspect, the nat-siRNA targets the Htt gene. In one aspect, the crRNA targets the Htt gene. In one aspect, the tracrRNA targets the Htt gene. In one aspect, the sgRNA targets the Htt gene. In one aspect, the shRNA comprises a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 23 to 25. In one aspect, the shRNA comprises the nucleic acid sequence SEQ ID NO: 23. In one aspect, the shRNA comprises the nucleic acid sequence SEQ ID NO: 24. In one aspect, the shRNA comprises the nucleic acid sequence SEQ ID NO: 25.

As used herein, "donor molecules" or "donor sequences" are defined as nucleic acid sequences that have been selected for site-directed, targeted insertion into a genome. In one aspect, a donor molecule comprises a "donor sequence". In one aspect, the targeted editing techniques provided herein include the use of one or more, two or more, three or more, four or more, or five or more donor molecules or donor sequences. The donor molecules or donor sequences provided herein can be of any length. For example, the length of the donor molecules or donor sequences provided herein is between 2 and 50,000 nucleotides, between 2 and 10,000 nucleotides, between 2 and 5000 nucleotides, between 2 and 1000 nucleotides, between 2 and 500 nucleotides, between 2 and 250 nucleotides, between 2 and 100 nucleotides, between 2 and 50 nucleotides, between 2 and 30 nucleotides, between 15 and 50 nucleotides, between 15 and 100 nucleotides, between 15 and 5 ... nucleotides, between 15 and 1000 nucleotides, between 15 and 5000 nucleotides, between 18 and 30 nucleotides, between 18 and 26 nucleotides, between 20 and 26 nucleotides, between 20 and 50 nucleotides, between 20 and 100 nucleotides, between 20 and 250 nucleotides, between 20 and 500 nucleotides, between 20 and 1000 nucleotides, between 20 and 5000 nucleotides, or between 20 and 10,000 nucleotides.

As used herein, "HTT" refers to the Htt-specific guide RNA (gRNA) and/or donor sequence.

On the one hand, the present disclosure provides and includes a composition comprising an adeno-associated virus (AAV) vector, wherein the AAV vector comprises a Cas9 nuclease gene, a Htt-specific gRNA, and a donor sequence. On the one hand, the present disclosure provides and includes a composition comprising an adeno-associated virus (AAV) vector, wherein the AAV vector comprises a Cas9 nuclease gene, a Htt-specific gRNA, a donor sequence, and a Dlx2 gene sequence. On the one hand, the present disclosure provides and includes a composition comprising an adeno-associated virus (AAV) vector, wherein the AAV vector comprises a Cas9 nuclease gene, a Htt-specific shRNA, and a donor sequence. On the one hand, the present disclosure provides and includes a composition comprising an adeno-associated virus (AAV) vector, wherein the AAV vector comprises a Cas9 nuclease gene, a Htt-specific shRNA, a donor sequence, and a Dlx2 gene sequence.

Site-specific nucleases provided herein can be used as part of targeted editing techniques. Non-limiting examples of site-specific nucleases include large-range nucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), RNA-guided nucleases (e.g., Cas9 and Cpf1), recombinases (e.g., but not limited to, serine recombinases attached to DNA recognition motifs, tyrosine recombinases attached to DNA recognition motifs), transposases (e.g., but not limited to, DNA transposases attached to DNA binding domains) or any combination thereof.

Site-specific nucleases (such as meganucleases, ZFNs, TALENs, Argonaute proteins (non-limiting examples of Argonaute proteins include Thermus thermophilus Argonaute (TtAgo), Pyrococcus furiosus Argonaute (PfAgo), Natronobacterium gregoryi Argonaute (NgAgo), homologs thereof or modified versions thereof), Cas9 nucleases (non-limiting examples of RNA-guided nucleases include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm 3. Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1, CasX, CasY, homologs thereof, or modified versions thereof)) induce double-stranded DNA breaks at target sites in genomic sequences that are then repaired by natural processes. Sequence modifications then occur at the cleavage sites, which may include inversions, deletions, or insertions that result in gene disruption or integration of nucleic acid sequences. In one aspect, the RNA-guided nuclease provided herein is selected from the group consisting of Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2 , Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, Cpf1, CasX, CasY, homologs thereof or modified versions thereof

In one aspect, the targeted editing techniques described herein comprise the use of RNA-guided nucleases.

Although not limited by any particular scientific theory, CRISPR/CAS nucleases are part of the adaptive immune system of bacteria and archaea, protecting them from invading nucleic acids such as viruses by cleaving target DNA in a sequence-dependent manner. Immunity is acquired by integrating short fragments of invading DNA (called spacers) between CRISPR repeat sequences about 20 nucleotides long at the proximal end of the CRISPR locus (CRISPR array). A well-described Cas protein is the Cas9 nuclease (also known as Csn1), which is part of the type 2 type II CRISPR/Cas system in Streptococcus pyogenes. See Makarova et al. Nature Reviews Microbiology (2015) doi:10.1038/nrmicro3569. Cas9 contains a RuvC-like nuclease domain at its amino terminus and a HNH-like nuclease domain located in the middle of the protein. The Cas9 protein also contains a PAM interaction (PI) domain, a recognition lobe (REC), and a BH domain. The Cpf1 nuclease is another type II system that acts in a similar manner to Cas9, but Cpf1 does not require tracrRNA. See Cong et al. Science (2013) 339:819-823; Zetsche et al., Cell (2015) doi: 10.1016/j.cell.2015.09.038; U.S. Patent Publication No. 2014/0068797; U.S. Patent Publication No. 2014/0273235; U.S. Patent Publication No. 2015/0067922; U.S. Patent No. 8,697,359; U.S. Patent No. 8,771,945; U.S. Patent No. 8,795,965; U.S. Patent No. 8,865,406; U.S. Patent No. 8,871,445; U.S. Patent No. 8,889,356; U.S. Patent No. 8,889,418; U.S. Patent No. 8,895,308; and U.S. Patent No. 8,906,616, each of which is incorporated herein by reference in its entirety.

As used herein, the term "glia" or "glial cell" refers to non-neuronal cells in the CNS or PNS. In one aspect, 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 microglia. In one aspect, at least one glial cell is at least one oligodendrocyte. In one aspect, at least one glial cell is at least one NG2 cell. In one aspect, at least one glial cell is at least one ependymal cell. In one aspect, at least one glial cell is at least one microglia. In one aspect, at least one glial cell is at least one reactive astrocyte. In one aspect, at least one astrocyte is at least one reactive astrocyte.

As used herein, the term "astrocyte" refers to glial cells that are 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 neuro- or glial transmitters, and maintenance of the blood-brain barrier. As used herein, the term "reactive astrocyte" refers to the abnormal state of astrocytes after injury or disease.

As used herein, the term "NG2 cell" or "polydendrocyte" refers to a glial cell that expresses chondroitin sulfate proteoglycan (CSPG4) 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 unipolar neurons, bipolar neurons, pseudo-unipolar neurons, and multipolar neurons. 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 pseudo-unipolar neuron. In one aspect, the neuron is a bipolar neuron. In one aspect, the neuron is selected from the group consisting of sensory neurons, motor neurons, and interneurons. 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, processing and transmission of information and communication via chemical and electrical synapses.

As used herein, the term "glutamatergic neuron" refers to a subclass of neurons that produce glutamate and establish excitatory synapses. As used herein, the term "excitatory synapse" refers to a synapse in which an action potential in a presynaptic neuron increases the probability of an action potential in a postsynaptic cell. As used herein, the term "action potential" or "nerve impulse" refers to an electrical pulse that passes through the axonal membrane. As used herein, the term "axon" or "nerve fiber" refers to a neuron that conducts an action potential. 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 a cell or positively charged potassium ions to flow out of a cell. As used herein, the term "inhibitory synapse" refers to a synapse that keeps the membrane potential of a postsynaptic neuron away from the threshold for generating an action potential. As used herein, the term "dopaminergic neuron" refers to a subset of neurons that produce dopamine. As used herein, the term "dopamine" refers to a neurotransmitter. As used herein, the term "neurotransmitter" refers to an endogenous chemical substance that activates neurotransmission. As used herein, the term "neurotransmission" refers to the process of releasing neurotransmitters by the axon terminals of neurons. As used herein, the term "acetylcholinergic neuron" or "cholinergic neuron" refers to a subset of neurons that secrete acetylcholine. As used herein, the term "acetylcholine" refers to a neurotransmitter. As used herein, the term "serotonergic neuron" refers to a subset of neurons that synthesize serotonin. As used herein, the term "serotonin" refers to a neurotransmitter. As used herein, "adrenergic neuron" refers to a neuron that releases adrenaline 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, brainstem or spinal cord and the axons project to the spinal cord or outside the spinal cord and directly or indirectly control muscles and glands. As used herein, the term peptidergic 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 glutamatergic neurons, GABAergic neurons, dopaminergic neurons, cholinergic neurons, serotonergic neurons, adrenergic neurons, motor neurons and peptidergic neurons. 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 peptidergic neuron.

As used herein, the term "conversion" or "converted" 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 disclosure provides 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 the PNS.

In one aspect, the present disclosure provides and includes an adeno-associated virus (AAV) vector comprising: a human neurogenic differentiation factor 1 (hNeuroD1) sequence, the hNeuroD1 sequence comprising a nucleic acid sequence of SEQ ID NO:6, and a human distal homeobox-free 2 (hDlx2) sequence, the hDlx2 sequence comprising a nucleic acid sequence of SEQ ID NO:13, wherein the hNeuroD1 sequence and the hDlx2 sequence are separated by: (i) a P2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs:15 and 18, (ii) a T2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs:16 and 19, or (iii) an internal ribosome entry site (IRES) sequence of encephalomyocarditis virus comprising SEQ ID NO:3, wherein the hNeuroD1 sequence and the hDlx2 sequence are operably linked to a regulatory element, the regulatory element comprising: (a) a nucleic acid sequence selected from the group consisting of SEQ ID NOs:15 and 18, (b) a nucleic acid sequence selected from the group consisting of SEQ ID NOs:16 and 19, NO:4, 12 and 26 consisting of a glial fibrillary acid protein (GFAP) promoter; (b) comprising a nucleic acid sequence of SEQ ID NO:2 from the human elongation factor -1 alpha (EF1-alpha) promoter enhancer or comprising a nucleic acid sequence of SEQ ID NO:11 cytomegalovirus (CMV) enhancer; (c) comprising a chimeric intron comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:5 and 27; (d) comprising a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:7 and 29; and (e) comprising a SV40 polyadenylation signal sequence of SEQ ID NO:8, a hGH polyadenylation sequence of SEQ ID NO:17, or a bGH polyadenylation sequence of SEQ ID NO:30.

In one aspect, the present disclosure provides and includes an adeno-associated virus (AAV) vector comprising: a nucleic acid sequence encoding a human neurogenic differentiation factor 1 (hNeuroD1) protein, the hNeuroD1 protein comprising the amino acid coding sequence of SEQ ID NO: 10, and a nucleic acid coding sequence encoding a human distal homeobox-free 2 (hDlx2) protein, the hDlx2 protein comprising the amino acid sequence of SEQ ID NO: 14, wherein the hNeuroD1 coding sequence and the hDlx2 coding sequence are separated by: (i) a P2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 15 and 18, (ii) a T2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16 and 19, or (iii) an internal ribosome entry site (IRES) sequence of encephalomyocarditis virus comprising SEQ ID NO: 3, wherein the hNeuroD1 coding sequence and the hDlx2 coding sequence are operably linked to a regulatory element, the regulatory element comprising: (a) a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 15 and 18, (b) a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 15 and 18, (c) a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 15 and 18, (d) a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 15 and 18, (e) a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 15 and 18, (f) a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 15 and 18, (g) a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 15 and 18, (h) a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 15 and 18, (h) a nucleic acid sequence selected from the group consisting of SEQ ID NOs NO:4, 12 and 28 consisting of a glial fibrillary acid protein (GFAP) promoter; (b) comprising a nucleic acid sequence of SEQ ID NO:2 from the human elongation factor -1 alpha (EF1-alpha) promoter enhancer or comprising a nucleic acid sequence of SEQ ID NO:11 cytomegalovirus (CMV) enhancer; (c) comprising a chimeric intron comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:5 and 27; (d) comprising a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:7 and 29; and (e) comprising a SV40 polyadenylation signal sequence of SEQ ID NO:8, a hGH polyadenylation sequence of SEQ ID NO:17, or a bGH polyadenylation sequence of SEQ ID NO:30.

In one aspect, the present disclosure provides and includes an adeno-associated virus (AAV) vector comprising a NeuroD1 nucleic acid coding sequence encoding a neurogenic differentiation factor 1 (NeuroD1) protein and a Dlx2 nucleic acid coding sequence encoding a distal homeobox-free 2 (Dlx2) protein, wherein the NeuroD1 coding sequence and the Dlx2 coding sequence are separated by a linker sequence, wherein the NeuroD1 coding sequence and the Dlx2 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) a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE); and (e) a polyadenylation signal sequence.

In one aspect, the present disclosure provides and includes a composition comprising an adeno-associated virus (AAV) vector for converting human glial cells into functional neurons, wherein the AAV vector comprises: a human neurogenic differentiation factor 1 (hNeuroD1) sequence, the hNeuroD1 sequence having a nucleic acid sequence of SEQ ID NO:6, and a human distal homeobox-free 2 (hD1x2) sequence, the hD1x2 sequence having a nucleic acid sequence of SEQ ID NO:13, wherein the hNeuroD1 sequence and the hD1x2 sequence are separated by: (i) a P2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs:15 and 18, (ii) a T2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs:16 and 19, or (iii) an internal ribosome entry site (IRES) sequence of encephalomyocarditis virus comprising SEQ ID NO:3, wherein the hNeuroD1 sequence and the hD1x2 sequence are operably linked to a regulatory element, the regulatory element comprising: (a) a nucleic acid sequence selected from the group consisting of SEQ ID NOs:15 and 18, (b) a nucleic acid sequence selected from the group consisting of SEQ ID NOs:16 and 19, or (c) a nucleic acid sequence selected from the group consisting of SEQ ID NOs:16 and 19, NO:4, 12 and 26 consisting of the human glial fibrillary acid protein (GFAP) promoter; (b) comprising the nucleic acid sequence of SEQ ID NO:2 from the human elongation factor -1 alpha (EF-1 alpha) promoter enhancer or comprising the nucleic acid sequence of SEQ ID NO:11 cytomegalovirus (CMV) enhancer; (c) comprising a chimeric intron comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:5 and 27; (d) comprising a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:7 and 29; and (e) comprising the SV40 polyadenylation signal sequence of SEQ ID NO:8, the hGH polyadenylation sequence of SEQ ID NO:17, or the bGH polyadenylation sequence of SEQ ID NO:30.

In one aspect, the present disclosure provides and includes a composition comprising an adeno-associated virus (AAV) vector for converting human glial cells into functional neurons, wherein the AAV vector comprises: a nucleic acid coding sequence encoding a human neurogenic differentiation factor 1 (hNeuroD1) protein, the hNeuroD1 protein comprising the amino acid sequence of SEQ ID NO: 10, and a nucleic acid coding sequence encoding a human distal homeobox-free 2 (hDlx2) protein, the hDlx2 protein comprising the amino acid sequence of SEQ ID NO: 14, wherein the hNeuroD1 coding sequence and the hDlx2 coding sequence are separated by: (i) a P2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 15 and 18, (ii) a T2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16 and 19, or (iii) a nucleic acid coding sequence comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 17 and 18. NO:3 of the encephalomyocarditis virus internal ribosome entry site (IRES) sequence, wherein the hNeuroD1 coding sequence and the hD1x2 coding sequence are operably linked to a regulatory element, the regulatory element comprising: (a) a human glial fibrillary acid protein (GFAP) promoter comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:4, 12 and 26; (b) an enhancer from the human elongation factor-1α (EF-1α) 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:11; (c) a chimeric intron comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:5 and 27; (d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:7 and 29; and (e) an SV40 polyadenylation signal sequence comprising the nucleic acid sequence of SEQ ID NO:8, an hGH polyadenylation sequence comprising the nucleic acid sequence of SEQ ID NO:17, or a cytomegalovirus (CMV) enhancer comprising the nucleic acid sequence of SEQ ID NO:11. The bGH polyadenylation sequence of the nucleic acid sequence of NO:30.

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 (NeuroD1) sequence and a distal homeobox-less 2 (Dlx2) sequence, wherein the NeuroD1 sequence and the Dlx2 sequence are separated by a linker sequence, wherein the NeuroD1 sequence and the Dlx2 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) a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE); and (e) a polyadenylation signal.

In one aspect, the 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.

Neurogenic differentiation factor 1 (NeuroD1; also known as β2) is a basic helix-loop-helix (bHLH) transcription factor that forms heterodimers with other bHLH proteins to activate transcription of genes containing a DNA sequence called an E-box.

In one aspect, the NeuroD1 sequence is a human NeuroD1 (hNeuroD1) sequence. In one aspect, the NeuroD1 sequence is selected from the group consisting of a chimpanzee NeuroD1 sequence, a bonobo NeuroD1 sequence, an orangutan NeuroD1 sequence, a gorilla NeuroD1 sequence, a macaque NeuroD1 sequence, a marmoset NeuroD1 sequence, a capuchin NeuroD1 sequence, a baboon NeuroD1 sequence, a gibbon NeuroD1 sequence, and a lemur NeuroD1 sequence. In one aspect, the NeuroD1 sequence is a chimpanzee NeuroD1 sequence. In one aspect, the NeuroD1 sequence is a bonobo NeuroD1 sequence. In one aspect, the NeuroD1 sequence is an orangutan NeuroD1 sequence. In one aspect, the NeuroD1 sequence is a gorilla NeuroD1 sequence. In one aspect, the NeuroD1 sequence is a macaque NeuroD1 sequence. In one aspect, the NeuroD1 sequence is a marmoset NeuroD1 sequence. In one aspect, the NeuroD1 sequence is a capuchin 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 lemur NeuroD1 sequence.

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

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

Distal-less homeobox 2 (Dlx2; also known as TES1) is a member of the Dlx gene family and is a homeobox containing genes that function in forebrain and craniofacial development.

In one aspect, the Dlx2 sequence is a human Dlx2 (hDlx2) sequence. In one aspect, the Dlx2 sequence is selected from the group consisting of a chimpanzee Dlx2 sequence, a bonobo Dlx2 sequence, an orangutan Dlx2 sequence, a gorilla Dlx2 sequence, a macaque Dlx2 sequence, a marmoset Dlx2 sequence, a capuchin Dlx2 sequence, a baboon Dlx2 sequence, a gibbon Dlx2 sequence, and a lemur Dlx2 sequence. In one aspect, the Dlx2 sequence is a chimpanzee Dlx2 sequence. In one aspect, the Dlx2 sequence is a bonobo Dlx2 sequence. In one aspect, the Dlx2 sequence is an orangutan Dlx2 sequence. In one aspect, the Dlx2 sequence is a gorilla Dlx2 sequence. In one aspect, the Dlx2 sequence is a macaque Dlx2 sequence. In one aspect, the Dlx2 sequence is a marmoset Dlx2 sequence. In one aspect, the Dlx2 sequence is a capuchin Dlx2 sequence. In one aspect, the Dlx2 sequence is a baboon Dlx2 sequence. In one aspect, the Dlx2 sequence is a gibbon Dlx2 sequence. In one aspect, the Dlx2 sequence is a lemur Dlx2 sequence.

In one aspect, the Dlx2 nucleic acid sequence comprises a sequence that is at least 70% identical to SEQ ID NO: 13, or its complement. In one aspect, the Dlx2 nucleic acid sequence comprises a sequence that is at least 75% identical to SEQ ID NO: 13, or its complement. In one aspect, the Dlx2 nucleic acid sequence comprises a sequence that is at least 80% identical to SEQ ID NO: 13, or its complement. In one aspect, the Dlx2 nucleic acid sequence comprises a sequence that is at least 85% identical to SEQ ID NO: 13, or its complement. In one aspect, the Dlx2 nucleic acid sequence comprises a sequence that is at least 90% identical to SEQ ID NO: 13, or its complement. In one aspect, the Dlx2 nucleic acid sequence comprises a sequence that is at least 91% identical to SEQ ID NO: 13, or its complement. In one aspect, the Dlx2 nucleic acid sequence comprises a sequence that is at least 92% identical to SEQ ID NO: 13, or its complement. In one aspect, the Dlx2 nucleic acid sequence comprises a sequence that is at least 93% identical to SEQ ID NO: 13, or its complement. In one aspect, the Dlx2 nucleic acid sequence comprises a sequence that is at least 94% identical to SEQ ID NO: 13, or its complement. In one aspect, the Dlx2 nucleic acid sequence comprises a sequence that is at least 95% identical to SEQ ID NO: 13, or its complement. In one aspect, the Dlx2 nucleic acid sequence comprises a sequence that is at least 913% identical to SEQ ID NO: 13, or its complement. In one aspect, the Dlx2 nucleic acid sequence comprises a sequence that is at least 97% identical to SEQ ID NO: 13, or its complement. In one aspect, the Dlx2 nucleic acid sequence comprises a sequence that is at least 98% identical to SEQ ID NO: 13, or its complement. In one aspect, the Dlx2 nucleic acid sequence comprises a sequence that is at least 99% identical to SEQ ID NO: 13, or its complement. In one aspect, the Dlx2 nucleic acid sequence comprises a sequence that is at least 99.5% identical to SEQ ID NO: 13, or its complement. In one aspect, the Dlx2 nucleic acid sequence comprises a sequence that is at least 99.8% identical to SEQ ID NO: 13, or its complement. In one aspect, the Dlx2 nucleic acid sequence comprises a sequence that is at least 99.9% identical to SEQ ID NO: 13, or the complement thereof. In one aspect, the Dlx2 nucleic acid sequence comprises a sequence that is 100% identical to SEQ ID NO: 13, or the complement thereof.

In one aspect, the nucleic acid coding sequence encodes a Dlx2 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 a Dlx2 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 a Dlx2 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 a Dlx2 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 a Dlx2 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 a Dlx2 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 a Dlx2 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 a Dlx2 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 a Dlx2 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 a Dlx2 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 a Dlx2 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 a Dlx2 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 a Dlx2 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 a Dlx2 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 a Dlx2 protein comprising an amino acid sequence at least 99.5% identical or similar to: SEQ ID NO: 14. In one aspect, the nucleic acid coding sequence encodes a Dlx2 protein comprising an amino acid sequence at least 99.8% identical or similar to: SEQ ID NO: 14. In one aspect, the nucleic acid coding sequence encodes a Dlx2 protein comprising an amino acid sequence at least 99.9% identical or similar to: SEQ ID NO: 14. In one aspect, the nucleic acid coding sequence encodes a Dlx2 protein comprising an amino acid sequence 100% identical or similar to: SEQ ID NO: 14.

In one aspect, the AAV vector comprises a NeuroD1 sequence and a Dlx2 sequence. In one aspect, the AAV vector comprises a NeuroD1 sequence. In one aspect, the AAV comprises a Dlx2 sequence.

As used herein, a "linker" or "spacer" is a short sequence that separates multiple protein and coding domains. Linkers can be cleavable or non-cleavable and facilitate co-expression of multiple genes in a single vector.

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

As used herein, "P2A linker," "p2A," or "P2A" refers to the porcine teschovirus-1 (P2A) linker, which is a member of the 2A self-cleaving peptides.

In one aspect, the P2A linker has a nucleic acid sequence selected from the group consisting of: SEQ ID NOs: 15 and 18. In one aspect, the P2A linker has a nucleic acid sequence selected from: SEQ ID NOs: 15 and 18. In one aspect, the P2A linker has a nucleic acid sequence selected from: SEQ ID NOs: 15. In one aspect, the P2A linker has a nucleic acid sequence selected from: SEQ ID NOs: 18. In one aspect, P2A is SEQ ID NO: 15. In one aspect, GSG-P2A is SEQ ID NO: 18. In one aspect, the P2A linker has a nucleic acid sequence selected from: SEQ ID NOs: 18. In one aspect, the P2A linker protein has a nucleic acid encoding sequence selected from: SEQ ID NOs: 20.

As used herein, "T2A linker" refers to the thosea asigna virus 2A (T2A) linker, which is a member of the 2A self-cleaving peptides.

In one aspect, the T2A linker has a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 16 and 19. In one aspect, the T2A linker has a nucleic acid sequence of: SEQ ID NO: 16. In one aspect, the T2A linker has a nucleic acid sequence of: SEQ ID NO: 19. In one aspect, the T2A linker protein has a nucleic acid encoding sequence of: SEQ ID NO: 21

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

As used herein, "F2A linker" refers to the 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 a 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 a F2A linker.

In one aspect, the linker sequence comprises 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: 15, or its complement. In one aspect, the P2A linker nucleic acid sequence comprises a sequence that is at least 75% identical to SEQ ID NO: 15, or its complement. In one aspect, the P2A linker nucleic acid sequence comprises a sequence that is at least 80% identical to SEQ ID NO: 15, or its complement. In one aspect, the P2A linker nucleic acid sequence comprises a sequence that is at least 85% identical to SEQ ID NO: 15, or its complement. In one aspect, the P2A linker nucleic acid sequence comprises a sequence that is at least 90% identical to SEQ ID NO: 15, or its complement. In one aspect, the P2A linker nucleic acid sequence comprises a sequence that is at least 91% identical to SEQ ID NO: 15, or its complement. In one aspect, the P2A linker nucleic acid sequence comprises a sequence that is at least 92% identical to SEQ ID NO: 15, or its complement. In one aspect, the P2A linker nucleic acid sequence comprises a sequence that is at least 93% identical to SEQ ID NO: 15, or its complement. In one aspect, the P2A linker nucleic acid sequence comprises a sequence that is at least 94% identical to SEQ ID NO: 15, or its complement. In one aspect, the P2A linker nucleic acid sequence comprises a sequence that is at least 95% identical to SEQ ID NO: 15, or its complement. In one aspect, the P2A linker nucleic acid sequence comprises a sequence that is at least 96% identical to SEQ ID NO: 15, or its complement. In one aspect, the P2A linker nucleic acid sequence comprises a sequence that is at least 97% identical to SEQ ID NO: 15, or its complement. In one aspect, the P2A linker nucleic acid sequence comprises a sequence that is at least 98% identical to SEQ ID NO: 15, or its complement. In one aspect, the P2A linker nucleic acid sequence comprises a sequence that is at least 99% identical to SEQ ID NO: 15, or its complement. In one aspect, the P2A linker nucleic acid sequence comprises a sequence that is at least 99.5% identical to SEQ ID NO: 15, or its complement. In one aspect, the P2A linker nucleic acid sequence comprises a sequence that is at least 99.8% identical to SEQ ID NO: 15, or its complement. In one aspect, the P2A linker nucleic acid sequence comprises a sequence that is at least 99.9% identical to SEQ ID NO: 15, or its complement. In one aspect, the P2A linker nucleic acid sequence comprises a sequence that is 100% identical to SEQ ID NO: 15, or its complement.

In one aspect, the linker sequence comprises 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: 18, or its complement. In one aspect, the P2A linker nucleic acid sequence comprises a sequence that is at least 75% identical to SEQ ID NO: 18, or its complement. In one aspect, the P2A linker nucleic acid sequence comprises a sequence that is at least 80% identical to SEQ ID NO: 18, or its complement. In one aspect, the P2A linker nucleic acid sequence comprises a sequence that is at least 85% identical to SEQ ID NO: 18, or its complement. In one aspect, the P2A linker nucleic acid sequence comprises a sequence that is at least 90% identical to SEQ ID NO: 18, or its complement. In one aspect, the P2A linker nucleic acid sequence comprises a sequence that is at least 91% identical to SEQ ID NO: 18, or its complement. In one aspect, the P2A linker nucleic acid sequence comprises a sequence that is at least 92% identical to SEQ ID NO: 18, or its complement. In one aspect, the P2A linker nucleic acid sequence comprises a sequence that is at least 93% identical to SEQ ID NO: 18, or its complement. In one aspect, the P2A linker nucleic acid sequence comprises a sequence that is at least 94% identical to SEQ ID NO: 18, or its complement. In one aspect, the P2A linker nucleic acid sequence comprises a sequence that is at least 95% identical to SEQ ID NO: 18, or its complement. In one aspect, the P2A linker nucleic acid sequence comprises a sequence that is at least 96% identical to SEQ ID NO: 18, or its complement. In one aspect, the P2A linker nucleic acid sequence comprises a sequence that is at least 97% identical to SEQ ID NO: 18, or its complement. In one aspect, the P2A linker nucleic acid sequence comprises a sequence that is at least 98% identical to SEQ ID NO: 18, or its complement. In one aspect, the P2A linker nucleic acid sequence comprises a sequence that is at least 99% identical to SEQ ID NO: 18, or its complement. In one aspect, the P2A linker nucleic acid sequence comprises a sequence that is at least 99.5% identical to SEQ ID NO: 18, or its complement. In one aspect, the P2A linker nucleic acid sequence comprises a sequence at least 99.8% identical to SEQ ID NO: 18, or its complement. In one aspect, the P2A linker nucleic acid sequence comprises a sequence at least 99.9% identical to SEQ ID NO: 18, or its complement. In one aspect, the P2A linker nucleic acid sequence comprises a sequence 100% identical to SEQ ID NO: 18, or its complement.

In one aspect, the nucleic acid coding sequence encodes a P2A protein comprising an amino acid sequence that is at least 70% identical or similar to SEQ ID NO: 20. In one aspect, the nucleic acid coding sequence encodes a P2A protein comprising an amino acid sequence that is at least 75% identical or similar to SEQ ID NO: 20. In one aspect, the nucleic acid coding sequence encodes a P2A protein comprising an amino acid sequence that is at least 80% identical or similar to SEQ ID NO: 20. In one aspect, the nucleic acid coding sequence encodes a P2A protein comprising an amino acid sequence that is at least 85% identical or similar to SEQ ID NO: 20. In one aspect, the nucleic acid coding sequence encodes a P2A protein comprising an amino acid sequence that is at least 90% identical or similar to SEQ ID NO: 20. In one aspect, the nucleic acid coding sequence encodes a P2A protein comprising an amino acid sequence that is at least 91% identical or similar to SEQ ID NO: 20. In one aspect, the nucleic acid coding sequence encodes a P2A protein comprising an amino acid sequence that is at least 92% identical or similar to SEQ ID NO: 20. In one aspect, the nucleic acid coding sequence encodes a P2A protein comprising an amino acid sequence that is at least 93% identical or similar to SEQ ID NO: 20. In one aspect, the nucleic acid coding sequence encodes a P2A protein comprising an amino acid sequence that is at least 94% identical or similar to SEQ ID NO: 20. In one aspect, the nucleic acid coding sequence encodes a P2A protein comprising an amino acid sequence that is at least 95% identical or similar to SEQ ID NO: 20. In one aspect, the nucleic acid coding sequence encodes a P2A protein comprising an amino acid sequence that is at least 96% identical or similar to SEQ ID NO: 20. In one aspect, the nucleic acid coding sequence encodes a P2A protein comprising an amino acid sequence that is at least 97% identical or similar to SEQ ID NO: 20. In one aspect, the nucleic acid coding sequence encodes a P2A protein comprising an amino acid sequence that is at least 98% identical or similar to SEQ ID NO: 20. In one aspect, the nucleic acid coding sequence encodes a P2A protein comprising an amino acid sequence that is at least 99% identical or similar to SEQ ID NO: 20. In one aspect, the nucleic acid coding sequence encodes a P2A protein comprising an amino acid sequence at least 99.5% identical or similar to: SEQ ID NO: 20. In one aspect, the nucleic acid coding sequence encodes a P2A protein comprising an amino acid sequence at least 99.8% identical or similar to: SEQ ID NO: 20. In one aspect, the nucleic acid coding sequence encodes a P2A protein comprising an amino acid sequence at least 99.9% identical or similar to: SEQ ID NO: 20. In one aspect, the nucleic acid coding sequence encodes a P2A protein comprising an amino acid sequence 100% identical or similar to: SEQ ID NO: 20.

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: 16, or its complement. In one aspect, the T2A linker nucleic acid sequence comprises a sequence that is at least 75% identical to SEQ ID NO: 16, or its complement. In one aspect, the T2A linker nucleic acid sequence comprises a sequence that is at least 80% identical to SEQ ID NO: 16, or its complement. In one aspect, the T2A linker nucleic acid sequence comprises a sequence that is at least 85% identical to SEQ ID NO: 16, or its complement. In one aspect, the T2A linker nucleic acid sequence comprises a sequence that is at least 90% identical to SEQ ID NO: 16, or its complement. In one aspect, the T2A linker nucleic acid sequence comprises a sequence that is at least 91% identical to SEQ ID NO: 16, or its complement. In one aspect, the T2A linker nucleic acid sequence comprises a sequence that is at least 92% identical to SEQ ID NO: 16, or its complement. In one aspect, the T2A linker nucleic acid sequence comprises a sequence that is at least 93% identical to SEQ ID NO: 16, or its complement. In one aspect, the T2A linker nucleic acid sequence comprises a sequence that is at least 94% identical to SEQ ID NO: 16, or its complement. In one aspect, the T2A linker nucleic acid sequence comprises a sequence that is at least 95% identical to SEQ ID NO: 16, or its complement. In one aspect, the T2A linker nucleic acid sequence comprises a sequence that is at least 96% identical to SEQ ID NO: 16, or its complement. In one aspect, the T2A linker nucleic acid sequence comprises a sequence that is at least 97% identical to SEQ ID NO: 16, or its complement. In one aspect, the T2A linker nucleic acid sequence comprises a sequence that is at least 98% identical to SEQ ID NO: 16, or its complement. In one aspect, the T2A linker nucleic acid sequence comprises a sequence that is at least 99% identical to SEQ ID NO: 16, or its complement. In one aspect, the T2A linker nucleic acid sequence comprises a sequence that is at least 99.5% identical to SEQ ID NO: 16, or its complement. In one aspect, the T2A linker nucleic acid sequence comprises a sequence that is at least 99.8% identical to SEQ ID NO: 16, or its complement. In one aspect, the T2A linker nucleic acid sequence comprises a sequence that is at least 99.9% identical to SEQ ID NO: 16, or its complement. In one aspect, the T2A linker nucleic acid sequence comprises a sequence that is 100% identical to SEQ ID NO: 16, or its complement.

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: 19, or its complement. In one aspect, the T2A linker nucleic acid sequence comprises a sequence that is at least 75% identical to SEQ ID NO: 19, or its complement. In one aspect, the T2A linker nucleic acid sequence comprises a sequence that is at least 80% identical to SEQ ID NO: 19, or its complement. In one aspect, the T2A linker nucleic acid sequence comprises a sequence that is at least 85% identical to SEQ ID NO: 19, or its complement. In one aspect, the T2A linker nucleic acid sequence comprises a sequence that is at least 90% identical to SEQ ID NO: 19, or its complement. In one aspect, the T2A linker nucleic acid sequence comprises a sequence that is at least 91% identical to SEQ ID NO: 19, or its complement. In one aspect, the T2A linker nucleic acid sequence comprises a sequence that is at least 92% identical to SEQ ID NO: 19, or its complement. In one aspect, the T2A linker nucleic acid sequence comprises a sequence that is at least 93% identical to SEQ ID NO: 19, or its complement. In one aspect, the T2A linker nucleic acid sequence comprises a sequence that is at least 94% identical to SEQ ID NO: 19, or its complement. In one aspect, the T2A linker nucleic acid sequence comprises a sequence that is at least 95% identical to SEQ ID NO: 19, or its complement. In one aspect, the T2A linker nucleic acid sequence comprises a sequence that is at least 96% identical to SEQ ID NO: 19, or its complement. In one aspect, the T2A linker nucleic acid sequence comprises a sequence that is at least 97% identical to SEQ ID NO: 19, or its complement. In one aspect, the T2A linker nucleic acid sequence comprises a sequence that is at least 98% identical to SEQ ID NO: 19, or its complement. In one aspect, the T2A linker nucleic acid sequence comprises a sequence that is at least 99% identical to SEQ ID NO: 19, or its complement. In one aspect, the T2A linker nucleic acid sequence comprises a sequence that is at least 99.5% identical to SEQ ID NO: 19, or its complement. In one aspect, the T2A linker nucleic acid sequence comprises a sequence that is at least 99.8% identical to SEQ ID NO: 19, or its complement. In one aspect, the T2A linker nucleic acid sequence comprises a sequence that is at least 99.9% identical to SEQ ID NO: 19, or its complement. In one aspect, the T2A linker nucleic acid sequence comprises a sequence that is 100% identical to SEQ ID NO: 19, or its complement.

In one aspect, the nucleic acid coding sequence encodes a T2A protein comprising an amino acid sequence that is at least 70% identical or similar to SEQ ID NO: 21. In one aspect, the nucleic acid coding sequence encodes a T2A protein comprising an amino acid sequence that is at least 75% identical or similar to SEQ ID NO: 21. In one aspect, the nucleic acid coding sequence encodes a T2A protein comprising an amino acid sequence that is at least 80% identical or similar to SEQ ID NO: 21. In one aspect, the nucleic acid coding sequence encodes a T2A protein comprising an amino acid sequence that is at least 85% identical or similar to SEQ ID NO: 21. In one aspect, the nucleic acid coding sequence encodes a T2A protein comprising an amino acid sequence that is at least 90% identical or similar to SEQ ID NO: 21. In one aspect, the nucleic acid coding sequence encodes a T2A protein comprising an amino acid sequence that is at least 91% identical or similar to SEQ ID NO: 21. In one aspect, the nucleic acid coding sequence encodes a T2A protein comprising an amino acid sequence that is at least 92% identical or similar to SEQ ID NO: 21. In one aspect, the nucleic acid coding sequence encodes a T2A protein comprising an amino acid sequence that is at least 93% identical or similar to SEQ ID NO: 21. In one aspect, the nucleic acid coding sequence encodes a T2A protein comprising an amino acid sequence that is at least 94% identical or similar to SEQ ID NO: 21. In one aspect, the nucleic acid coding sequence encodes a T2A protein comprising an amino acid sequence that is at least 95% identical or similar to SEQ ID NO: 21. In one aspect, the nucleic acid coding sequence encodes a T2A protein comprising an amino acid sequence that is at least 96% identical or similar to SEQ ID NO: 21. In one aspect, the nucleic acid coding sequence encodes a T2A protein comprising an amino acid sequence that is at least 97% identical or similar to SEQ ID NO: 21. In one aspect, the nucleic acid coding sequence encodes a T2A protein comprising an amino acid sequence that is at least 98% identical or similar to SEQ ID NO: 21. In one aspect, the nucleic acid coding sequence encodes a T2A protein comprising an amino acid sequence that is at least 99% identical or similar to SEQ ID NO: 21. In one aspect, the nucleic acid coding sequence encodes a T2A protein comprising an amino acid sequence at least 99.5% identical or similar to SEQ ID NO: 21. In one aspect, the nucleic acid coding sequence encodes a T2A protein comprising an amino acid sequence at least 99.8% identical or similar to SEQ ID NO: 21. In one aspect, the nucleic acid coding sequence encodes a T2A protein comprising an amino acid sequence at least 99.9% identical or similar to SEQ ID NO: 21. In one aspect, the nucleic acid coding sequence encodes a T2A protein comprising an amino acid sequence 100% identical or similar to SEQ ID NO: 21.

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

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

Glial fibrillary acid protein (GFAP); also known as glial fibrillary acidic protein, is a member of the type III intermediate filament protein family that 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 (Lcn2) promoter, glutamine synthetase promoter, aquaporin-4 (AQP4) promoter, oligodendrocyte transcription factor (Olig2) promoter and synaptophysin promoter, NG2 promoter, ionized calcium binding adapter molecule 1 (Iba1) promoter, cluster of differentiation 86 (CD86) promoter, platelet-derived growth factor receptor alpha (PDGFRA) promoter, platelet-derived growth factor receptor beta (PDGFRB) promoter, elongation factor 1-alpha (EF1a) promoter, CAG promoter, cytomegalovirus (CMV) promoter, ubiquitin promoter. In one aspect, the promoter is GFAP promoter. In one aspect, the promoter is a truncated GFAP promoter. In one aspect, the promoter is Sox9 promoter. In one aspect, the promoter is S100b promoter. In one aspect, the promoter is the Aldh1l1 promoter. In one aspect, the promoter is the Lcn2 promoter. In one aspect, the promoter is the glutamine synthetase promoter. In one aspect, the promoter is the AQP4 promoter. In one aspect, the promoter is the Olig2 promoter. In one aspect, the promoter is the synapsin promoter. In one aspect, the promoter is the Iba1 promoter. In one aspect, the promoter is the CD86 promoter. In one aspect, the promoter is the PDGFRA promoter. In one aspect, the promoter is the PDGFRB promoter. In one aspect, the promoter is the EF1a promoter. In one aspect, the promoter is the CAG promoter. In one aspect, the promoter is the CMV promoter. In one aspect, the promoter is the ubiquitin promoter.

In one aspect, the ubiquitin promoter is selected from the group consisting of U6, H1, 7SK and U1. In one aspect, the ubiquitin promoter is U6. In one aspect, the ubiquitin promoter is H1. In one aspect, the ubiquitin promoter is H1. In one aspect, the ubiquitin promoter is 7SK. In one aspect, the ubiquitin promoter is U1. In one aspect, U6 comprises the following nucleic acid sequence: SEQ ID NO: 22.

In one aspect, the GFAP promoter is a promoter that directs astrocyte-specific expression of a protein called glial fibrillary acid protein (GFAP) in cells. 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 known as "pGfa681"), Gfa1.6, and hGFA2.2. In one aspect, the GFAP promoter is GfaABC1D (also known as "pGfa681"). 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: 26. In one aspect, GFAP Gfa1.6 is SEQ ID NO: 4. In one aspect, hGFa2.2 is SEQ ID NO: 12. In one aspect, the GFAP promoter is selected from the group consisting of: SEQ ID NO: 4, 12, and 26. In one aspect, the GFAP promoter is SEQ ID NO: 4. In one aspect, the GFAP promoter is SEQ ID NO: 12. In one aspect, the GFAP promoter is SEQ ID NO:26.

In one aspect, the GFAP promoter sequence is selected from the group consisting of a chimpanzee GFAP promoter sequence, a bonobo GFAP promoter sequence, an orangutan GFAP promoter sequence, a gorilla GFAP promoter sequence, a macaque GFAP promoter sequence, a marmoset GFAP promoter sequence, a capuchin GFAP promoter sequence, a baboon GFAP promoter sequence, a gibbon GFAP promoter sequence, and a lemur 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 an orangutan 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 capuchin 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 lemur 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 1400 nucleotides.

It is understood in the art that a fragment of a promoter sequence can 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 able to drive transcription, then 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 140 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 that is at least 70% identical to a sequence selected from the group consisting of: SEQ ID NO: 4, 12, 26 and functional fragments thereof. In one aspect, the GFAP promoter nucleic acid sequence comprises a sequence that is at least 75% identical to a sequence selected from the group consisting of: SEQ ID NO: 4, 12, 26 and functional fragments thereof. In one aspect, the GFAP promoter nucleic acid sequence comprises a sequence that is at least 80% identical to a sequence selected from the group consisting of: SEQ ID NO: 4, 12, 26 and functional fragments thereof. In one aspect, the GFAP promoter nucleic acid sequence comprises a sequence that is at least 85% identical to a sequence selected from the group consisting of: SEQ ID NO: 4, 12, 26 and functional fragments thereof. In one aspect, the GFAP promoter nucleic acid sequence comprises a sequence that is at least 90% identical to a sequence selected from the group consisting of: SEQ ID NO: 4, 12, 26 and functional fragments thereof. In one aspect, the GFAP promoter nucleic acid sequence comprises a sequence that is at least 91% identical to a sequence selected from the group consisting of: SEQ ID NO: 4, 12, 26 and functional fragments thereof. In one aspect, the GFAP promoter nucleic acid sequence comprises a sequence that is at least 92% identical to a sequence selected from the group consisting of: SEQ ID NO: 4, 12, 26 and functional fragments thereof. In one aspect, the GFAP promoter nucleic acid sequence comprises a sequence that is at least 93% identical to a sequence selected from the group consisting of: SEQ ID NO: 4, 12, 26 and functional fragments thereof. In one aspect, the GFAP promoter nucleic acid sequence comprises a sequence that is at least 94% identical to a sequence selected from the group consisting of: SEQ ID NO: 4, 12, 26 and functional fragments thereof. In one aspect, the GFAP promoter nucleic acid sequence comprises a sequence that is at least 95% identical to a sequence selected from the group consisting of: SEQ ID NO: 4, 12, 26 and functional fragments thereof. In one aspect, the GFAP promoter nucleic acid sequence comprises a sequence that is at least 96% identical to a sequence selected from the group consisting of: SEQ ID NO: 4, 12, 26 and functional fragments thereof. In one aspect, the GFAP promoter nucleic acid sequence comprises a sequence that is at least 97% identical to a sequence selected from the group consisting of: SEQ ID NO: 4, 12, 26 and functional fragments thereof. In one aspect, the GFAP promoter nucleic acid sequence comprises a sequence at least 98% identical to a sequence selected from the group consisting of: SEQ ID NO: 4, 12, 26 and functional fragments thereof. In one aspect, the GFAP promoter nucleic acid sequence comprises a sequence at least 99% identical to a sequence selected from the group consisting of: SEQ ID NO: 4, 12, 26 and functional fragments thereof. In one aspect, the GFAP promoter nucleic acid sequence comprises a sequence at least 99.5% identical to a sequence selected from the group consisting of: SEQ ID NO: 4, 12, 26 and functional fragments thereof. In one aspect, the GFAP promoter nucleic acid sequence comprises a sequence at least 99.8% identical to a sequence selected from the group consisting of: SEQ ID NO: 4, 12, 26 and functional fragments thereof. In one aspect, the GFAP promoter nucleic acid sequence comprises a sequence at least 99.9% identical to a sequence selected from the group consisting of: SEQ ID NO: 4, 12, 26 and functional fragments thereof. In one aspect, the GFAP promoter nucleic acid sequence comprises a sequence that is 100% identical to a sequence selected from the group consisting of SEQ ID NO: 4, 12, 26, and functional fragments thereof.

In one aspect, the nucleic acid sequences provided herein are codon optimized.

In one aspect, the nucleic acid sequences provided herein are depleted of CpG sites.

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

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

As used herein, the term "striatum" or "corpus striatum" refers to the clusters of neurons in the basal ganglia beneath the forebrain 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 beneath the midbrain cortex and includes the pars compacta and pars reticularis.

As used herein, the term "forebrain" refers to the front-most part of the brain.

As used herein, the term "putamen" refers to a rounded structure at the base of the forebrain and is a component of the dorsal striatum.

As used herein, the term "caudate nucleus" refers to a structure in the base of the forebrain and is a component of the dorsal striatum.

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

As used herein, the term "spinal cord" refers to the structure that plays a role in transmitting nerve signals from the motor cortex to the body.

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

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

Elongation factor 1 alpha (EF-1α; also known as eEF1a1) is an isoform of the alpha subunit of the elongation factor 1 complex. The complex is involved in the enzymatic delivery of aminoacyl tRNAs to the ribosome. EF-1α isoforms are expressed in the brain, placenta, lung, liver, kidney, and pancreas.

In one aspect, the enhancer sequence from the EF-1α promoter is a human enhancer sequence from the EF-1α promoter. In one aspect, the enhancer sequence from the EF-1α promoter is selected from the group consisting of the following items: a chimpanzee enhancer sequence from the EF-1α promoter, a bonobo enhancer sequence from the EF-1α promoter, an orangutan enhancer sequence from the EF-1α promoter, a gorilla enhancer sequence from the EF-1α promoter, a macaque enhancer sequence from the EF-1α promoter, a marmoset enhancer sequence from the EF-1α promoter, a capuchin enhancer sequence from the EF-1α promoter, a baboon enhancer sequence from the EF-1α promoter, a gibbon enhancer sequence from the EF-1α promoter, and a lemur enhancer sequence from the EF-1α promoter. In one aspect, the enhancer sequence from the EF-1α promoter is a chimpanzee enhancer sequence from the EF-1α promoter. In one aspect, the enhancer sequence from the EF-1α promoter is a bonobo enhancer sequence from the EF-1α promoter. In one aspect, the enhancer sequence from the EF-1α promoter is an orangutan enhancer sequence from the EF-1α promoter. In one aspect, the enhancer sequence from the EF-1α promoter is a gorilla enhancer sequence from the EF-1α promoter. In one aspect, the enhancer sequence from the EF-1α promoter is a macaque enhancer sequence from the EF-1α promoter. In one aspect, the enhancer sequence from the EF-1α promoter is a marmoset enhancer sequence from the EF-1α promoter. In one aspect, the enhancer sequence from the EF-1α promoter is a capuchin enhancer sequence from the EF-1α promoter. In one aspect, the enhancer sequence from the EF-1α promoter is a baboon enhancer sequence from the EF-1α promoter. In one aspect, the enhancer sequence from the EF-1α promoter is a gibbon enhancer sequence from the EF-1α promoter. In one aspect, the enhancer sequence from the EF-1α promoter is a lemur enhancer sequence from the EF-1α promoter.

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

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

In one aspect, the enhancer sequence from CMV is the human enhancer sequence from CMV. In one aspect, the enhancer sequence from CMV is selected from the group consisting of the following items: the chimpanzee enhancer sequence from CMV, the bonobo enhancer sequence from CMV, the orangutan enhancer sequence from CMV, the gorilla enhancer sequence from CMV, the macaque enhancer sequence from CMV, the marmoset enhancer sequence from CMV, the capuchin enhancer sequence from CMV, the baboon enhancer sequence from CMV, the gibbon enhancer sequence from CMV and the lemur enhancer sequence from CMV. In one aspect, the enhancer sequence from CMV is the chimpanzee enhancer sequence from CMV. In one aspect, the enhancer sequence from CMV is the bonobo enhancer sequence from CMV. In one aspect, the enhancer sequence from CMV is the orangutan enhancer sequence from CMV. In one aspect, the enhancer sequence from CMV is the gorilla enhancer sequence from CMV. In one aspect, the enhancer sequence from CMV is a macaque 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 capuchin 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 lemur enhancer sequence from CMV.

In one aspect, the enhancer from the CMV nucleic acid sequence comprises a sequence that is at least 70% identical to SEQ ID NO: 11, or its complement. In one aspect, the enhancer from the CMV nucleic acid sequence comprises a sequence that is at least 75% identical to SEQ ID NO: 11, or its complement. In one aspect, the enhancer from the CMV nucleic acid sequence comprises a sequence that is at least 80% identical to SEQ ID NO: 11, or its complement. In one aspect, the enhancer from the CMV nucleic acid sequence comprises a sequence that is at least 85% identical to SEQ ID NO: 11, or its complement. In one aspect, the enhancer from the CMV nucleic acid sequence comprises a sequence that is at least 90% identical to SEQ ID NO: 11, or its complement. In one aspect, the enhancer from the CMV nucleic acid sequence comprises a sequence that is at least 91% identical to SEQ ID NO: 11, or its complement. In one aspect, the enhancer from the CMV nucleic acid sequence comprises a sequence that is at least 911% identical to SEQ ID NO: 11, or its complement. In one aspect, the enhancer from the CMV nucleic acid sequence comprises a sequence that is at least 93% identical to SEQ ID NO: 11, or its complement. In one aspect, the enhancer from the CMV nucleic acid sequence comprises a sequence that is at least 94% identical to SEQ ID NO: 11, or its complement. In one aspect, the enhancer from the CMV nucleic acid sequence comprises a sequence that is at least 95% identical to SEQ ID NO: 11, or its complement. In one aspect, the enhancer from the CMV nucleic acid sequence comprises a sequence that is at least 96% identical to SEQ ID NO: 11, or its complement. In one aspect, the enhancer from the CMV nucleic acid sequence comprises a sequence that is at least 97% identical to SEQ ID NO: 11, or its complement. In one aspect, the enhancer from the CMV nucleic acid sequence comprises a sequence that is at least 98% identical to SEQ ID NO: 11, or its complement. In one aspect, the enhancer from the CMV nucleic acid sequence comprises a sequence that is at least 99% identical to SEQ ID NO: 11, or its complement. In one aspect, the enhancer from the CMV nucleic acid sequence comprises a sequence that is at least 99.5% identical to SEQ ID NO: 11, or its complement. In one aspect, the enhancer from the CMV nucleic acid sequence comprises a sequence that is at least 99.8% identical to SEQ ID NO: 11, or its complement. In one aspect, the enhancer from the CMV nucleic acid sequence comprises a sequence that is at least 99.9% identical to SEQ ID NO: 11, or its complement. In one aspect, the enhancer from the CMV nucleic acid sequence comprises a sequence that is 100% identical to SEQ ID NO: 11, or its complement.

In one aspect, the enhancer is selected from the group consisting of an enhancer from the EF1-alpha promoter and a CMV enhancer. In one aspect, the enhancer is from the EF1-alpha promoter. In one aspect, the enhancer is a CMV enhancer.

In one aspect, the vector of the present disclosure comprises a chimeric intron. In one aspect, the chimeric intron is composed of a 5'-donor site from the first intron of the human β-globulin gene and a branch and a 3'-acceptor site from an intron of the heavy chain variable region of an immunoglobulin gene. In one aspect, the chimeric intron is a chimeric intron of rabbit β-globulin and chicken β-actin similar in the CAG promoter. In one aspect, the vector of the present disclosure comprises a glial fibrillary acidic protein (GFAP) intron. In one aspect, the vector of the present disclosure comprises a glial fibrillary acidic protein (GFAP) first intron.

Introns can be divided into at least five categories, including: spliceosomal introns; transfer RNA introns; group I introns; group II introns; and group III introns. Introns can be synthesized, altered, or derived from known or naturally occurring intron sequences or other intron sequences. Introns can also include chimeric introns comprising a combination of two or more heterologous sequences. Therefore, the introns of the present application can include variants of intron sequences that are similar but not identical to other intron sequences known or provided herein in composition. 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 140 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, an intron comprises between 10 nucleotides and 500 nucleotides.

In one aspect, the chimeric intronic nucleic acid sequence is selected from the group consisting of SEQ ID NO: 5 and 27. In one aspect, the chimeric intronic nucleic acid sequence is SEQ ID NO: 5. In one aspect, the chimeric intronic nucleic acid sequence is SEQ ID NO: 27.

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

In one aspect, the chimeric intron nucleic acid sequence comprises a sequence that is at least 70% identical to SEQ ID NO: 27, or its complement. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence that is at least 75% identical to SEQ ID NO: 27, or its complement. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence that is at least 80% identical to SEQ ID NO: 27, or its complement. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence that is at least 85% identical to SEQ ID NO: 27, or its complement. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence that is at least 90% identical to SEQ ID NO: 27, or its complement. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence that is at least 91% identical to SEQ ID NO: 27, or its complement. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence that is at least 92% identical to SEQ ID NO: 27, or its complement. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence that is at least 93% identical to SEQ ID NO: 27, or its complement. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence that is at least 94% identical to SEQ ID NO: 27, or its complement. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence that is at least 95% identical to SEQ ID NO: 27, or its complement. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence that is at least 96% identical to SEQ ID NO: 27, or its complement. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence that is at least 97% identical to SEQ ID NO: 27, or its complement. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence that is at least 98% identical to SEQ ID NO: 27, or its complement. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence that is at least 99% identical to SEQ ID NO: 27, or its complement. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence that is at least 99.5% identical to SEQ ID NO: 27, or a sequence complementary thereto. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence that is at least 99.8% identical to SEQ ID NO: 27, or a sequence complementary thereto. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence that is at least 99.9% identical to SEQ ID NO: 27, or a sequence complementary thereto. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence that is 100% identical to SEQ ID NO: 27, or a sequence complementary thereto.

The woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) is a DNA sequence that creates a tertiary structure that enhances the expression of genes delivered in viral vectors.

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 NO: 7 and 29. 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: 29.

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

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

The SV40 polyadenylation signal sequence (also known as SV40 PolyA; Simian virus 40 PolyA; and PolyA) is a DNA sequence that terminates transcription and adds a PolyA tail to the 3' end of messenger RNA (mRNA).

The hGH polyadenylation signal sequence (also known as hGH PolyA) is a DNA sequence that terminates transcription and adds a PolyA tail to the 3' end of the messenger RNA (mRNA).

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

As used herein, a "PolyA tail" refers to a segment of RNA containing only the nucleobase adenine. In one aspect, an RNA molecule transcribed from an AAV vector construct provided herein comprises a PolyA tail. In one aspect, the PolyA tail comprises at least two adenines. In one aspect, the PolyA tail comprises at least ten adenines. In one aspect, the PolyA tail comprises at least 50 adenines. In one aspect, the PolyA tail comprises at least 100 adenines. In one aspect, the PolyA tail comprises at least 140 adenines. In one aspect, the PolyA tail comprises at least 200 adenines. In one aspect, the PolyA tail comprises at least 250 adenines. In one aspect, the PolyA tail comprises between 50 and 300 adenines.

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

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

In one aspect, the bGH polyadenylation signal nucleic acid sequence comprises a sequence that is at least 70% identical to SEQ ID NO: 30, or its complement. In one aspect, the bGH polyadenylation signal nucleic acid sequence comprises a sequence that is at least 75% identical to SEQ ID NO: 30, or its complement. In one aspect, the bGH polyadenylation signal nucleic acid sequence comprises a sequence that is at least 80% identical to SEQ ID NO: 30, or its complement. In one aspect, the bGH polyadenylation signal nucleic acid sequence comprises a sequence that is at least 85% identical to SEQ ID NO: 30, or its complement. In one aspect, the bGH polyadenylation signal nucleic acid sequence comprises a sequence that is at least 90% identical to SEQ ID NO: 30, or its complement. In one aspect, the bGH polyadenylation signal nucleic acid sequence comprises a sequence that is at least 91% identical to SEQ ID NO: 30, or its complement. In one aspect, the bGH polyadenylation signal nucleic acid sequence comprises a sequence that is at least 92% identical to SEQ ID NO: 30, or its complement. In one aspect, the bGH polyadenylation signal nucleic acid sequence comprises a sequence that is at least 93% identical to SEQ ID NO:30, or its complement. In one aspect, the bGH polyadenylation signal nucleic acid sequence comprises a sequence that is at least 94% identical to SEQ ID NO:30, or its complement. In one aspect, the bGH polyadenylation signal nucleic acid sequence comprises a sequence that is at least 95% identical to SEQ ID NO:30, or its complement. In one aspect, the bGH polyadenylation signal nucleic acid sequence comprises a sequence that is at least 96% identical to SEQ ID NO:30, or its complement. In one aspect, the bGH polyadenylation signal nucleic acid sequence comprises a sequence that is at least 97% identical to SEQ ID NO:30, or its complement. In one aspect, the bGH polyadenylation signal nucleic acid sequence comprises a sequence that is at least 917% identical to SEQ ID NO:30, or its complement. In one aspect, the bGH polyadenylation signal nucleic acid sequence comprises a sequence that is at least 99% identical to SEQ ID NO:30, or its complement. In one aspect, the bGH polyadenylation signal nucleic acid sequence comprises a sequence that is at least 99.5% identical to SEQ ID NO:30, or its complement. In one aspect, the bGH polyadenylation signal nucleic acid sequence comprises a sequence that is at least 99.17% identical to SEQ ID NO:30, or its complement. In one aspect, the bGH polyadenylation signal nucleic acid sequence comprises a sequence that is at least 99.9% identical to SEQ ID NO:30, or its complement. In one aspect, the bGH polyadenylation signal nucleic acid sequence comprises a sequence that is 100% identical to SEQ ID NO:30, or its complement.

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

As used herein, the term "peripheral nervous system" or "PNS" refers to the nerves and ganglia outside the brain and spinal cord, excluding the retina, optic nerves, olfactory nerves, and olfactory epithelium. 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 the PNS associated with voluntary control of body movement.

As used herein, the term "autonomic nervous system" refers to the part of the PNS that regulates the function of internal organs.

As used herein, the term "GFAP positive" refers to cells with accumulation of human glial fibrillary acid protein (GFAP) protein detectable using standard techniques in the art or accumulation of GFAP mRNA expression detectable using standard techniques in the art. In one aspect, the glial cells are GFAP positive.

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

Antibodies can be used to identify protein accumulation. Non-limiting examples of measuring protein accumulation include Western blots, enzyme-linked immunosorbent assays (ELISA), immunoprecipitation, and immunofluorescence. The antibodies provided herein can be polyclonal antibodies or monoclonal antibodies. Methods well known in the art can be used to generate antibodies provided herein that have specific binding affinity to proteins. Methods known in the art can be used to connect the antibodies provided herein to solid supports (such as microtiter plates).

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 the infectious form of a virus outside of a host cell.

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

As used herein, the term "injury" refers to damage to the central nervous system or the 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 aneurysm, traumatic brain injury, concussion, tumor, 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, fibrocartilaginous embolic myelopathy, thrombosis, nephropathy, chronic inflammatory disease, 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 disorder is physical injury. In one aspect, the neurological disorder is stroke. In one aspect, the neurological disorder is ischemic stroke. In one aspect, the nervous system disorder is hemorrhagic stroke. In one aspect, the nervous system disorder is cerebral aneurysm. In one aspect, the nervous system disorder is traumatic brain injury. In one aspect, the nervous system disorder is concussion. In one aspect, the nervous system disorder is a tumor. In one aspect, the nervous system disorder is inflammation. In one aspect, the nervous system disorder is infection. In one aspect, the nervous system disorder is ataxia. In one aspect, the nervous system disorder is brain atrophy. In one aspect, the nervous system disorder is spinal cord atrophy. In one aspect, the nervous system disorder is multiple sclerosis. In one aspect, the nervous system disorder is traumatic spinal cord injury. In one aspect, the nervous system disorder is ischemic or hemorrhagic myelopathy (myelopathy). In one aspect, the nervous system disorder is global cerebral ischemia. In one aspect, the nervous system disorder is hypoxic-ischemic encephalopathy. In one aspect, the nervous system disorder is embolism. In one aspect, the nervous system disorder is fibrocartilaginous embolic myelopathy. In one aspect, the nervous system disorder is thrombosis. In one aspect, the nervous system disorder is nephropathy. In one aspect, the nervous system disorder is a chronic inflammatory disease. In one aspect, the neurological disorder is meningitis. In one aspect, the neurological disorder is cerebral venous sinus thrombosis.

In one aspect, a nervous system disorder comprises damage to the CNS or PNS. In one aspect, a nervous system disorder comprises damage to the CNS. In one aspect, a nervous system disorder comprises damage to the PNS.

In one aspect, the present disclosure provides and includes a method for 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, the DNA vector construct comprising: a human neurogenic differentiation factor 1 (hNeuroD1) sequence, the hNeuroD1 sequence comprising a nucleic acid sequence of SEQ ID NO: 6, and a human distal homeobox-free 2 (hD1x2) sequence, the hD1x2 sequence comprising a nucleic acid sequence of SEQ ID NO: 13, wherein the hNeuroD1 sequence and the hD1x2 sequence are separated by: (i) a P2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 15 and 18, (ii) a T2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16 and 19, or (iii) a linker comprising a nucleic acid sequence of SEQ ID NOs: 17 and 18. NO:3 of the encephalomyocarditis virus internal ribosome entry site (IRES) sequence, wherein the hNeuroD1 sequence and the hD1x2 sequence are operably linked to a regulatory element, the regulatory element comprising: (a) a human glial fibrillary acid protein (GFAP) promoter comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4, 12 and 26; (b) an enhancer from a human elongation factor-1α (EF-1α) promoter comprising a nucleic acid sequence of SEQ ID NO: 2 or a cytomegalovirus (CMV) enhancer comprising a nucleic acid sequence of SEQ ID NO: 11; (c) a chimeric intron comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 5 and 27; (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 29; and (e) an SV40 polyadenylation signal sequence comprising a nucleic acid sequence of SEQ ID NO: 8, an hGH polyadenylation sequence comprising a nucleic acid sequence of SEQ ID NO: 17, or a cytomegalovirus (CMV) enhancer comprising a nucleic acid sequence of SEQ ID NO: 11. The bGH polyadenylation sequence of the nucleic acid sequence of NO:30.

In one aspect, the present disclosure provides and includes a method for 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, the DNA vector construct comprising: a nucleic acid coding sequence encoding a human neurogenic differentiation factor 1 (hNeuroD1) protein, the hNeuroD1 protein comprising the amino acid sequence of SEQ ID NO: 10, and a nucleic acid coding sequence encoding a human distal homeobox-free 2 (hDlx2) protein, the hDlx2 protein comprising the amino acid sequence of SEQ ID NO: 14, wherein the hNeuroD1 coding sequence and the hDlx2 coding sequence are separated by: (i) a P2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 15 and 18, (ii) a T2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16 and 19, or (iii) a nucleic acid coding sequence comprising SEQ ID NOs: 1 NO:3 of the encephalomyocarditis virus internal ribosome entry site (IRES) sequence, wherein the hNeuroD1 coding sequence and hD1x2 coding sequence are operably linked to an expression control element, the expression control element comprising: (a) a human glial fibrillary acid protein (GFAP) promoter comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4, 12 and 26; (b) an enhancer from a human elongation factor-1α (EF-1α) promoter comprising a nucleic acid sequence of SEQ ID NO: 2 or a cytomegalovirus (CMV) enhancer comprising a nucleic acid sequence of SEQ ID NO: 11; (c) a chimeric intron comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 5 and 27; (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 29; and (e) an SV40 polyadenylation signal sequence comprising a nucleic acid sequence of SEQ ID NO: 8, a SV40 polyadenylation signal sequence comprising a nucleic acid sequence of SEQ ID NO: 9, a SV40 polyadenylation signal sequence comprising a nucleic acid sequence of SEQ ID NO: 10 The hGH polyadenylation sequence comprises the nucleic acid sequence of SEQ ID NO:17, or the bGH polyadenylation sequence comprises the nucleic acid sequence of SEQ ID NO:30.

In one aspect, the present disclosure provides and includes a method for converting glial cells of a subject in need thereof into neurons, 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 (NeuroD1) sequence and a distal homeobox-less 2 (Dlx2) sequence, wherein the NeuroD1 sequence and the Dlx2 sequence are separated by a linker sequence, wherein the NeuroD1 sequence and the Dlx2 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) a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE); and (e) a polyadenylation signal sequence, wherein the AAV vector is capable of converting at least one glial cell of the subject in need thereof into a neuron.

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 the subject, wherein the AAV administered to the subject in need thereof comprises a DNA vector comprising a neurogenic differentiation factor 1 (NeuroD1) sequence and a distal homeobox-less 2 (Dlx2) sequence, wherein the NeuroD1 sequence and the Dlx2 sequence are separated by a linker sequence, wherein the NeuroD1 sequence and the Dlx2 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) a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE); and (e) a polyadenylation signal.

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 are capable of converting at least one glial cell into a neuron.

Achaete-scute family BHLH transcription factor 1 (Ascl1; also known as ASH1, HASH1, MASH-1, and bHLHa46) encodes a member of the basic helix-loop-helix family of transcription factors and is a gene that plays a role in neuronal commitment and differentiation.

Insulin gene enhancer protein (ISL1; also known as ISL LIM homeobox 1 and ISLET1) is a gene encoding a transcription factor that contains two N-terminal LIM domains and a C-terminal homeodomain. The encoded protein plays a role in embryogenesis of the pancreatic islets of Langerhans.

The LIM-homeobox 3 (LHX3; also known as LIM3 and CPHD3) gene encodes a protein from a family of proteins with a unique cysteine-rich zinc-binding domain (LIM domain).

The Huntington's disease gene (Htt) gene encodes the Huntington protein. The wild type contains 6-35 glutamine residues, and the mutant Htt contains more than 36 glutamine residues.

In one aspect, the methods provided herein use an AAV vector comprising a NeuroD1 coding sequence and a Dlx2 coding sequence according to the present disclosure. In one aspect, the methods provided herein use a combination of an AAV vector comprising a NeuroD1 coding sequence and a second AAV vector comprising a Dlx2 coding sequence. In one aspect, the methods provided herein use a combination of an AAV vector comprising a NeuroD1 or Dlx2 coding sequence and a second AAV vector comprising a third transcription factor coding sequence. In one aspect, the third transcription factor is selected from the group consisting of Ascl1, ISL1, and LHX3. In one aspect, the third transcription factor is Ascl1. In one aspect, the second transcription factor is ISL1. In one aspect, the third transcription factor is LHX3. In one aspect, the methods provided herein use an AAV vector comprising a NeuroD1 coding sequence, a Dlx2 coding sequence, a second NeuroD1 coding sequence, and a second Dlx2 coding sequence. In one aspect, the methods provided herein use a combination of an AAV vector comprising NeuroD1 and Dlx2 coding sequences and a second AAV vector comprising NeuroD1 and Dlx2 coding sequences.

In one aspect, the method provided herein uses a combination of an AAV vector comprising NeuroD1 and Dlx2 coding sequences and an AAV vector comprising an shRNA sequence targeting Htt. In one aspect, the method provided herein uses a combination of an AAV vector comprising NeuroD1 and Dlx2 coding sequences and an AAV vector comprising an shRNA sequence targeting Htt and a second shRNA sequence targeting Htt. In one aspect, the method provided herein uses an AAV vector comprising NeuroD1 and Dlx2 coding sequences and an AAV vector comprising an shRNA sequence targeting Htt, a second shRNA sequence targeting Htt, and a third shRNA targeting Htt. In one aspect, the method provided herein uses an AAV vector comprising NeuroD1 and Dlx2 coding sequences and an shRNA sequence targeting Htt. In one aspect, the method provided herein uses an AAV vector comprising NeuroD1 and Dlx2 coding sequences and an shRNA sequence targeting Htt and an AAV vector comprising a second shRNA sequence targeting Htt. In one aspect, the methods provided herein use an AAV vector comprising NeuroD1 and Dlx2 coding sequences and a shRNA sequence targeting Htt, a second shRNA sequence targeting Htt, and a third shRNA targeting Htt.

In one aspect, the methods provided herein use an AAV vector comprising a combination of NeuroD1 and Dlx2 coding sequences and an ASO sequence targeting Htt. In one aspect, the methods provided herein use an AAV vector comprising a combination of NeuroD1 and Dlx2 coding sequences and an ASO sequence targeting Htt and a second ASO sequence targeting Htt. In one aspect, the methods provided herein use an AAV vector comprising a combination of NeuroD1 and Dlx2 coding sequences and an ASO sequence targeting Htt, a second ASO sequence targeting Htt, and a third ASO targeting Htt.

In one aspect, the methods provided herein use an AAV vector comprising a combination of NeuroD1 and Dlx2 coding sequences and a siRNA sequence targeting Htt. In one aspect, the methods provided herein use an AAV vector comprising a combination of NeuroD1 and Dlx2 coding sequences and a siRNA sequence targeting Htt and a second siRNA sequence targeting Htt. In one aspect, the methods provided herein use an AAV vector comprising a combination of NeuroD1 and Dlx2 coding sequences and a siRNA sequence targeting Htt, a second siRNA sequence targeting Htt, and a third siRNA targeting Htt.

In one aspect, the method provided herein uses a combination of an AAV vector comprising NeuroD1 and Dlx2 coding sequences and an AAV vector comprising a miRNA sequence targeting Htt. In one aspect, the method provided herein uses a combination of an AAV vector comprising NeuroD1 and Dlx2 coding sequences and an AAV vector comprising a miRNA sequence targeting Htt and a second miRNA sequence targeting Htt. In one aspect, the method provided herein uses a combination of an AAV vector comprising NeuroD1 and Dlx2 coding sequences and an AAV vector comprising a miRNA sequence targeting Htt, a second miRNA sequence targeting Htt, and a third miRNA targeting Htt. In one aspect, the method provided herein uses an AAV vector comprising NeuroD1 and Dlx2 coding sequences and a miRNA sequence targeting Htt. In one aspect, the method provided herein uses an AAV vector comprising NeuroD1 and Dlx2 coding sequences and a miRNA sequence targeting Htt and a second miRNA sequence targeting Htt. In one aspect, the methods provided herein use an AAV vector comprising NeuroD1 and Dlx2 coding sequences and a miRNA sequence targeting Htt, a second miRNA sequence targeting Htt, and a third miRNA targeting Htt.

In one aspect, the methods provided herein use a combination of an AAV vector comprising NeuroD1 and Dlx2 coding sequences and an AAV vector comprising a gRNA sequence targeting Htt and CAS nuclease. In one aspect, the methods provided herein use an AAV vector comprising NeuroD1 and Dlx2 coding sequences and a gRNA sequence targeting Htt and CAS nuclease.

In one aspect, the functionality of the AAV vectors provided herein is measured by assessing the transcript and protein levels of NeuN, doublecortin (DCX), β3-tubulin, (neurofilament 200) NF-200, (microtubule-associated protein 2) MAP2, and ionized calcium binding adaptor molecule (Iba1).

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 the sex determination gene in Caenorhabditis elegans and is a neuronal nuclear antigen.

As used herein, the term "DCX" or "doubling" or "lissencephalin-X" refers to a microtubule-associated protein 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 microtubule element of the tubulin family found in neurons.

As used herein, the term "NF-200" refers to a class of proteins that are type IV intermediate filament proteins 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 plays a role in determining and stabilizing neuronal morphology during neuronal development.

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

In one aspect, the method provided herein combines gene editing technology to convert glial cells into neurons. In one aspect, gene editing technology targets mutant Htt. In one aspect, gene editing technology is selected from the group consisting of siRNA, miRNA, ASO and CRISPR/CAS. In one aspect, gene editing technology is siRNA. In one aspect, gene editing technology is miRNA. In one aspect, gene editing technology is ASO. In one aspect, gene editing technology is CRISPR/CAS.

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 are capable of converting at least one glial cell into a neuron.

As used herein, the term "mammal" refers to any species classified into the class Mammalia.

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 person" refers to a person with 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, bonobos, orangutans, gorillas, macaques, marmosets, capuchins, baboons, gibbons, and lemurs.

As used herein, the term "delivering" or "delivery" refers to treating a mammal with an AAV vector or composition as provided herein. In one aspect, the AAV vector or composition provided herein is delivered to a subject in need thereof. In one aspect, the AAV vector or composition provided herein is formulated to be delivered to a subject in need thereof. In one aspect, delivery includes local delivery. In one aspect, the AAV vector or composition provided herein is formulated for local delivery. In one aspect, delivery includes systemic delivery. In one aspect, the AAV vector or composition provided herein is formulated for systemic delivery. In one aspect, delivery includes injecting the AAV vector or composition provided herein into a subject in need thereof. In one aspect, delivery is selected from the group consisting of: intraperitoneal, intramuscular, intravenous, intrathecal, intracerebral, intracranial, intraventricular of the brain, intracerebellar medullary cisterna, intravitreal, subretinal, intraparenchymal, intranasal or oral administration. In one aspect, delivery includes intraperitoneal delivery. In one aspect, delivery includes intramuscular delivery. In one aspect, delivery includes intravenous delivery. In one aspect, delivery includes intrathecal delivery. In one aspect, delivery comprises intracerebral delivery. In one aspect, delivery comprises intracranial delivery. In one aspect, delivery comprises intracerebroventricular delivery of the brain. In one aspect, delivery comprises intracisterna magna delivery. In one aspect, delivery comprises intravitreal delivery. In one aspect, delivery comprises intraretinal delivery. In one aspect, delivery comprises intraparenchymal delivery. In one aspect, delivery comprises intranasal delivery. In one aspect, delivery comprises oral administration.

As used herein, the term "injection" refers to delivering an AAV vector or composition provided herein under pressure and force. As a non-limiting example, injection can include the use of a syringe and a needle.

In one aspect, the 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, the AAV vector or composition provided herein is injected into the striatum of a subject. In one aspect, the AAV vector or composition provided herein is injected into the spinal cord or a subject. In one aspect, the AAV vector or composition is injected into the striatum of a 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, the AAV vectors or compositions provided herein have spread between about 1% and about 100% in the brain. In one aspect, the 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 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 70% and about 100%, between about 80% and about 90%, between about 80% and about 100%, or between about 90% and about 100%.

In one aspect, the AAV vectors or compositions provided herein have spread between about 1% and about 100% in the cerebral cortex. In one aspect, the 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%. In some embodiments, the present invention relates to an amount of at least one molecule of the present invention that is at least about 50% of the present invention and about 80% of the present invention. In some embodiments, the present invention relates to an amount of at least one molecule of the present invention that is at least about 50% of the present invention and about 80% of the present invention.

In one aspect, the AAV vectors or compositions provided herein have spread between about 1% and about 100% in the spinal cord. In one aspect, the 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 60% and about 70%, between about 70% and about 80%, between about 80% and about 90%, between about 90% and about 100%, between about 10 ... In some embodiments, the present invention relates to an amount of at least one molecule of the present invention that is ...

In one aspect, the AAV vectors or compositions provided herein have spread between about 1% and about 100% in the striatum. In one aspect, the 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 60% and about 70%, between about 70% and about 80%, between about 80% and about 90%, between about 90% and about 100%, between about 10 ... In some embodiments, the present invention relates to an amount of at least one molecule of the present invention that is ...

In one aspect, the AAV vectors or compositions provided herein have spread between about 1% and about 100% in the dorsal striatum. In one aspect, the 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%, In some embodiments, the present invention relates to an amount of at least one molecule of the present invention that is at least about 50% of the present invention and about 80% of the present invention. In some embodiments, the present invention relates to an amount of at least one molecule of the present invention that is at least about 50% of the present invention and about 80% of the present invention.

In one aspect, the AAV vectors or compositions provided herein have diffused between about 1% and about 100% in the putamen. In one aspect, the 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 60% and about 70%, between about 70% and about 80%, between about 8 ... In some embodiments, the present invention relates to an amount of at least one molecule of the present invention that is ...

In one aspect, the AAV vectors or compositions provided herein have spread between about 1% and about 100% in the caudate nucleus. In one aspect, the 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 60% and about 70%, between about 70% and about 80%, between about 8 ... In some embodiments, the present invention relates to an amount of at least one molecule of the present invention that is ...

In one aspect, the AAV vector or composition provided herein has diffused between about 1% and about 100% from the injection site. In one aspect, the 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%, In some embodiments, the present invention relates to an amount of at least one molecule of the present invention that is ...

In one aspect, the AAV vectors or compositions provided herein have diffused between about 1% and about 100% in the substantia nigra. In one aspect, the 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 60% and about 70%, between about 70% and about 80%, between about 80% and about 90%, between about 90% and about 100%, between about 10 ... In some embodiments, the present invention relates to an amount of at least one molecule of the present invention that is ...

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

In one aspect, a composition comprising AAV particles encoded by an AAV vector provided herein is injected at a concentration between 10 ¹⁰ AAV particles/mL and 10 ¹⁴ AAV particles/mL. In one aspect, a composition comprising AAV particles encoded by an AAV vector provided herein is prepared at between 10 ¹⁰ AAV particles/mL and 10 ¹¹ AAV particles/mL, between 10 ¹⁰ AAV particles/mL and 10 ¹² AAV particles/mL, between 10 ¹⁰ AAV particles/mL and 10 ¹³ AAV particles/mL, between 10 ¹¹ AAV particles/mL and 10 ¹² AAV particles/mL, between 10 ¹¹ AAV particles/mL and 10 ¹³ AAV particles/mL, between 10 ¹¹ AAV particles/mL and 10 ¹⁴ AAV particles/mL, between 10 ¹² AAV particles/mL and 10 ¹³ AAV particles/mL, between 10 ¹² AAV particles/mL and 10 ¹⁴ AAV particles/mL, or between 10 ¹³ AAV particles/mL and 10 14 AAV particles/mL. Concentrations between ¹⁴ AAV particles/mL were injected.

In one aspect, the composition comprising the AAV particles encoded by the AAV vectors provided herein is injected in a volume between 10 μL and 1000 μL. In one aspect, the composition comprising the AAV particles encoded by the AAV vectors provided herein is injected in 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 800 μL, between 600 μL and 700 μL, between 600 μL and 800 μL, between 600 μL and 900 μL, between 700 μL and 800 μL, between 700 μL and 900 μL, between 700 μL and 1000 μL, between 800 μL and 900 μL, between 800 μL and 1000 μL, or between 900 μL and 1000 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), livestock (e.g., cattle, sheep, goats, pigs, turkeys, chickens), and household 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 suffers from 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 aneurysm, traumatic brain injury, concussion, tumor, 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, fibrocartilaginous embolic myelopathy, thrombosis, nephropathy, chronic inflammatory disease, 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 suffers from 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 suffers from ischemic stroke. In one aspect, a subject in need thereof suffers from hemorrhagic stroke. In one aspect, a subject in need thereof suffers from 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 suffers from a tumor. In one aspect, a subject in need thereof suffers from inflammation. In one aspect, a subject in need thereof suffers from 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 cord atrophy. In one aspect, a subject in need thereof suffers from multiple sclerosis. In one aspect, a subject in need thereof suffers from 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 has hypoxic-ischemic encephalopathy. In one aspect, a subject in need thereof has embolism. In one aspect, a subject in need thereof has fibrocartilaginous embolic myelopathy. In one aspect, a subject in need thereof has thrombosis. In one aspect, a subject in need thereof has nephropathy. In one aspect, a subject in need thereof has a chronic inflammatory disease. In one aspect, a subject in need thereof has meningitis. In one aspect, a subject in need thereof has cerebral venous sinus thrombosis.

In one aspect, the subject in need is a mammal. In one aspect, the subject in need is a human. In one aspect, the subject in need is a non-human primate. In one aspect, the subject in need is selected from the group consisting of chimpanzees, bonobos, orangutans, gorillas, macaques, marmosets, capuchins, baboons, gibbons, and lemurs. In one aspect, the subject in need is a chimpanzee. In one aspect, the subject in need is a bonobo. In one aspect, the subject in need is an orangutan. In one aspect, the subject in need is a gorilla. In one aspect, the subject in need is a macaque. In one aspect, the subject in need is a marmoset. In one aspect, the subject in need is a capuchin. In one aspect, the subject in need is a baboon. In one aspect, the subject in need is a gibbon. In one aspect, the subject in need is a lemur.

In one aspect, the subject in need is male. In one aspect, the subject in need is female. In one aspect, the subject in need is neuter. In one aspect, the subject in need is premature infant. In one aspect, premature infant is born before 36 weeks of gestation. In one aspect, the subject in need is full-term infant. In one aspect, full-term infant is less than about 2 months old. In one aspect, the subject in need is a newborn. In one aspect, a newborn is less than about 1 month old. In one aspect, the subject in need is an infant. In one aspect, the infant is between 2 months old and 24 months old. In one aspect, the infant is between 2 and 3 months old, between 2 and 4 months old, between 2 and 5 months old, between 3 and 4 months old, between 3 and 5 months old, between 3 and 6 months old, between 4 and 5 months old, between 4 and 6 months old, between 4 and 7 months old, between 5 and 6 months old, between 5 and 7 months old, between 5 and 8 months old, between 6 and 7 months old, between 6 and 8 months old, between 6 and 9 months old, between 7 and 8 months old, between 7 and 9 months old, Between 7 and 10 months, Between 8 and 9 months, Between 8 and 10 months, Between 8 and 11 months, Between 9 and 10 months, Between 9 and 11 months, Between 9 and 12 months, Between 10 and 11 months, Between 10 and 12 months, Between 10 and 13 months, Between 11 and 12 months, Between 11 and 13 months, Between 11 and 14 months, Between 12 and 13 months, Between 12 and 14 months, Between 12 and 14 months between 13 and 14 months, between 13 and 14 months, between 13 and 16 months, between 14 and 14 months, between 14 and 16 months, between 14 and 17 months, between 14 and 16 months, between 14 and 17 months, between 14 and 18 months, between 16 and 17 months, between 16 and 18 months, between 16 and 19 months, between 17 and 18 months, between 17 and 19 months, between 17 and 20 months In one aspect, the experimenter in need is a child. In one aspect, the child is between 1 year old and 4 years old. In one aspect, the toddler 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 is a child. In one aspect, the child is between 2 and 5 years old. 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 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 a teenager. In one aspect, the teenager is between 13 and 19 years old. In one aspect, the adolescent is between 13 and 14 years old, between 13 and 14 years old, between 13 and 16 years old, between 14 and 14 years old, between 14 and 16 years old, between 14 and 17 years old, between 14 and 16 years old, between 14 and 17 years old, between 14 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 old and 18 years old. In one aspect, the pediatric subject is 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 years old and 5 years old, between 4 years old and 6 years old, between 4 years old and 7 years old, between 5 years old and 6 years old, between 5 years old and 7 years old, between 5 years old and 8 years old, between 6 years old and 7 years old, between 6 years old and 8 years old, between 6 years old and 9 years old, between 7 years old and 8 years old, between 7 years old and 9 years old, between 7 years old and 10 years old, between 8 years old and 9 years old, between 8 years old and 10 years old, between 8 years old 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, between 10 and 13 years old, between 11 and 12 years old, between 11 and 13 years old, between 11 and 14 years old, between 12 and 13 years old, between 12 and 14 years old, between 12 and 14 years old, between 13 and 14 years old, between 13 and 14 years old, between 13 and 16 years old, between 14 and 14 years old, between 14 and 16 years old, between 14 and 17 years old, between 14 and 16 years old, between 14 and 17 years old, between 14 and 18 years old, between 16 and 17 years old, between 16 and 18 years old, and between 17 and 18 years old. In one aspect, the subject in need is an elderly subject. In one aspect, the elderly subject is between 65 and 95 years old or higher. In one aspect, the 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 is an adult subject. In one aspect, the adult subject is between 20 and 95 years old or higher. In one aspect, the 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 years, between 55 and 60 years, between 55 and 65 years, between 55 and 70 years, between 60 and 65 years, between 60 and 70 years, between 60 and 75 years, between 65 and 70 years, between 65 and 75 years, between 65 and 80 years, between 70 and 75 years, between 70 and 80 years, between 70 and 85 years, between 75 and 80 years, between 75 and 85 years, between 75 and 90 years, between 80 and 85 years, between 80 and 90 years, between 80 and 95 years, between 85 and 90 years and between 85 and 95 years. In one aspect, the subject in need is between 1 year old and 5 years old, between 2 years old and 10 years old, between 3 years old and 18 years old, between 21 years old and 50 years old, between 21 years old and 40 years old, between 21 years old and 30 years old, between 50 years old and 90 years old, between 60 years old and 90 years old, between 70 years old and 90 years old, between 60 years old and 80 years old, or between 65 years old and 75 years old. In one aspect, the subject in need is young elderly subject (65 to 74 years old). In one aspect, the subject in need is middle elderly subject (75 to 84 years old). In one aspect, the subject in need is elderly subject (>85 years old).

As used herein, the term "flow rate" refers to the delivery rate 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 μL/min and 0.2 μL/min, between 0.1 μL/min and 0.3 μL/min, between 0.1 μL/min and 0.4 μL/min, between 0.2 μL/min and 0.3 μL/min, between 0.2 μL/min and 0.4 μL/min, between 0.2 μL/min and 0.5 μL/min, between 0.3 μL/min and 0.4 μL/min, between 0.3 μL/min and 0.5 μL/min, between 0.3 μL/min and 0.6 μL/min, between 0.4μL/min and 0.5μL/min, between 0.4μL/min and 0.6μL/min, between 0.4μL/min and 0.7μL/min, between 0.5μL/min and 0.6μL/min, between 0.5μL/min and 0.7μL/min, between 0.5μL/min and 0.8μL/min, between 0.6μL/min and 0.7μL/min, between 0.6μL/min and 0.8μL/min, between 0.6μL/min and 0.9μ L/min, between 0.7μL/min and 0.8μL/min, between 0.7μL/min and 0.9μL/min, between 0.7μL/min and 1.0μL/min, between 0.8μL/min and 0.9μL/min, between 0.8μL/min and 1.0μL/min, between 0.8μL/min and 1.1μL/min, between 0.9μL/min and 1.0μL/min, between 0.9μL/min and 1.1μL/min, between 0.9μL/min and 1.2μ L/min, between 1.0μL/min and 1.1μL/min, between 1.0μL/min and 1.2μL/min, between 1.0μL/min and 1.3μL/min, between 1.1μL/min and 1.2μL/min, between 1.1μL/min and 1.3μL/min, between 1.1μL/min and 1.4μL/min, between 1.2μL/min and 1.3μL/min, between 1.2μL/min and 1.4μL/min, between 1.2μL/min and 1.5μL /min, between 1.3μL/min and 1.4μL/min, between 1.3μL/min and 1.5μL/min, between 1.3μL/min and 1.6μL/min, between 1.4μL/min and 1.5μL/min, between 1.4μL/min and 1.6μL/min, between 1.4μL/min and 1.7μL/min, between 1.5μL/min and 1.6μL/min, between 1.5μL/min and 1.7μL/min, between 1.5μL/min and 1.8μL /min, between 1.6μL/min and 1.7μL/min, between 1.6μL/min and 1.8μL/min, between 1.6μL/min and 1.9μL/min, between 1.7μL/min and 1.8μL/min, between 1.7μL/min and 1.9μL/min, between 1.7μL/min and 2.0μL/min, between 1.8μL/min and 1.9μL/min, between 1.8μL/min and 2.0μL/min, between 1.8μL/min and 2.1μL/min min, between 1.9 μL/min and 2.0 μL/min, between 1.9 μL/min and 2.1 μL/min, between 1.9 μL/min and 2.2 μL/min, between 2.0 μL/min and 2.1 μL/min, between 2.0 μL/min and 2.2 μL/min, between 2.0 μL/min and 2.3 μL/min, between 2.1 μL/min and 2.2 μL/min, between 2.1 μL/min and 2.3 μL/min, between 2.1 μL/min and 2.4 μL/min min, between 2.2 μL/min and 2.3 μL/min, between 2.2 μL/min and 2.4 μL/min, between 2.2 μL/min and 2.5 μL/min, between 2.3 μL/min and 2.4 μL/min, between 2.3 μL/min and 2.5 μL/min, between 2.3 μL/min and 2.6 μL/min, between 2.4 μL/min and 2.5 μL/min, between 2.4 μL/min and 2.6 μL/min, between 2.4 μL/min and 2.7 μL/min between 2.5 μL/min and 2.6 μL/min, between 2.5 μL/min and 2.7 μL/min, between 2.5 μL/min and 2.8 μL/min, between 2.6 μL/min and 2.7 μL/min, between 2.6 μL/min and 2.8 μL/min, between 2.6 μL/min and 2.9 μL/min, between 2.7 μL/min and 2.8 μL/min, between 2.7 μL/min and 2.9 μL/min, between 2.7 μL/min 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, Between 3.1μL/min and 3.2μL/min, Between 3.1μL/min and 3.3μL/min, Between 3.1μL/min and 3.4μL/min, Between 3.2μL/min and 3.3μL/min, Between 3.2μL/min and 3.4μL/min, Between 3.2μL/min and 3.5μL/min, Between 3.3μL/min and 3.4μL/min, Between 3.3μL/min and 3.5μL/min, Between 3.3μL/min and 3.6μL/min between 3.4μL/min and 3.5μL/min, between 3.4μL/min and 3.6μL/min, between 3.4μL/min and 3.7μL/min, between 3.5μL/min and 3.6μL/min, between 3.5μL/min and 3.7μL/min, between 3.5μL/min and 3.8μL/min, between 3.6μL/min and 3.7μL/min, between 3.6μL/min and 3.8μL/min, between 3.6μL/min and 3.9μL/min between 3.7μL/min and 3.8μL/min, between 3.7μL/min and 3.9μL/min, between 3.7μL/min and 4.0μL/min, between 3.8μL/min and 3.9μL/min, between 3.8μL/min and 4.0μL/min, between 3.8μL/min and 4.1μL/min, between 3.9μL/min and 4.0μL/min, between 3.9μL/min and 4.1μL/min, between 3.9μL/min and 4.2μL/min , between 4.0 μL/min and 4.1 μL/min, between 4.0 μL/min and 4.2 μL/min, between 4.0 μL/min and 4.3 μL/min, between 4.1 μL/min and 4.2 μL/min, between 4.1 μL/min and 4.3 μL/min, between 4.1 μL/min and 4.4 μL/min, between 4.2 μL/min and 4.3 μL/min, between 4.2 μL/min and 4.4 μL/min, between 4.2 μL/min and 4.5 μL/min , between 4.3μL/min and 4.4μL/min, between 4.3μL/min and 4.5μL/min, between 4.3μL/min and 4.6μL/min, between 4.4μL/min and 4.5μL/min, between 4.4μL/min and 4.6μL/min, between 4.4μL/min and 4.7μL/min, between 4.5μL/min and 4.6μL/min, between 4.5μL/min and 4.7μL/min, between 4.5μL/min and 4.8μL/min , between 4.6 μL/min and 4.7 μL/min, between 4.6 μL/min and 4.8 μL/min, between 4.6 μL/min and 4.9 μL/min, between 4.7 μL/min and 4.8 μL/min, between 4.7 μL/min and 4.9 μL/min, between 4.7 μL/min and 5.0 μL/min, between 4.8 μL/min and 4.9 μL/min, between 4.8 μL/min and 5.0 μL/min, or between 4.9 μL/min and 5.0 μL/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, an AAV particle or composition provided herein can be provided together with a pharmaceutically acceptable carrier. As used herein, "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 encapsulated components in liquid (e.g., saline), gels, nanoparticles, exosomes, lipid vesicles or solid forms. Non-limiting examples of suitable diluents and excipients include pharmaceutical grade physiological saline, glucose, glycerol, mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc., and combinations thereof. On the one hand, the therapeutically effective dose contains auxiliary substances, such as wetting agents or emulsifiers, stabilizers or pH buffers. In one aspect, the AAV particles or compositions provided herein for the treatment of an effective dose are injected into a subject. In one aspect, the AAV particles or compositions provided herein for the treatment of an effective dose are delivered to a subject. In one aspect, the therapeutically effective dose is administered together 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 14%, between about 14% 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 14% and about 95%, 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 14% and about 95%, between about 2 Between about 0% 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 65% and about 95%, between about 70% and about 95%, between about 45% and about 95%, between about 80% and about 95%, or between about 85% and about 95% of the AAV particles or compositions provided herein.

In one aspect, at least once a day or at least once a week, a therapeutically effective dose is delivered to a subject in need thereof for at least two consecutive days or weeks. In one aspect, at least once a day or at least once a week, a therapeutically effective dose is delivered to a subject in need thereof for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 14 consecutive days or weeks. In one aspect, at least once a day or at least once a week, a therapeutically effective dose is delivered to a subject in need thereof for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 consecutive weeks. In one aspect, at least once a day or at least once a week, a therapeutically effective dose is delivered to a subject in need thereof for up to 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 14, 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 a day or at least once a week 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, at least once, for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 consecutive months or years, for a long period of time for the entire life cycle of the subject, or for an indefinite period of time. In one aspect, a therapeutically effective dose is delivered to a subject in need thereof once a year for 2 consecutive years, 3 consecutive years or 5 consecutive years. In one aspect, a therapeutically effective dose is delivered to a subject in need thereof once a year for 2 consecutive years. In one aspect, a therapeutically effective dose is delivered to a subject in need thereof once a year for 3 consecutive years. In one aspect, a therapeutically effective dose is delivered to a subject in need thereof once a year for 5 consecutive years.

As used herein, the terms "remission," "cure," or "remission rate" refer to the percentage of subjects in need thereof who are cured of a neurological disorder or who achieve remission or complete regression of a neurological disorder in response to a therapeutically effective dose.

As used herein, the term "response rate" refers to the percentage of subjects in need thereof who respond positively (eg, decrease in severity or frequency of one or more symptoms) to a therapeutically effective dose.

In one aspect, the therapeutically effective dose achieves at least about 50% of the alleviation, cure, response rate or regression rate of the nervous system disorder. In one aspect, the therapeutically effective dose eliminates, reduces, slows down or delays one or more nervous system disorder symptoms. The non-limiting examples of nervous system disorder symptoms include tremor, slow movement (bradykinesia), muscle stiffness, posture and impaired balance, automatic movement loss, uncoordinated movement, uncontrolled movement, spontaneous impact movement, speech changes, numbness, writing changes, involuntary movement (such as chorea), uncontrolled posture, mood changes and sleep disorders. In one aspect, nervous system disorder symptoms are motor symptoms. The non-limiting examples of motor symptoms include involuntary movement disorders or autonomous movement disorders. In one aspect, nervous system disorder symptoms are cognitive symptoms. Non-limiting examples of cognitive symptoms include fine motor skills, tremors, seizures, chorea, dystonia, dyskinesia, slow or abnormal eye movements, impaired gait, impaired posture, impaired balance, difficulty speaking, difficulty swallowing, difficulty organizing, difficulty prioritizing, difficulty focusing on tasks, lack of flexibility, lack of impulse control, outbursts, lack of awareness of one's own behavior and/or abilities, slow processing of thoughts, difficulty learning new information, difficulty remembering things, difficulty communicating, difficulty following commands, difficulty performing tasks.

In one aspect, the neurological disorder symptom is a psychiatric symptom. Non-limiting examples of psychiatric symptoms include depression, irritability, sadness or apathy, social withdrawal, insomnia, fatigue, lack of energy, obsessive-compulsive disorder, mania, bipolar disorder, and weight loss. In one aspect, the neurological disorder symptom is at least one damaged blood vessel. In one aspect, the neurological disorder symptom is an impaired blood-brain barrier. In one aspect, the neurological disorder symptom is impaired blood flow. Non-limiting examples of tests used to evaluate the elimination, reduction, slowing or delay of symptoms of a neurological condition 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 Rating Scale (HAM-D), the Columbia-Suicide Severity Rating Scale (C-SSRS), the Montreal Cognitive Assessment (MoCA), the Modified Rankin Scale (mRS), the National Institutes of Health Stroke Scale (NIHSS) and the Barthel Index (BI), the Timed Up and Go Test (TUG), the Chedoke Hand and Arm Activity Inventory (CAHAI), the Symbol Digit Modality Test, Controlled Oral Word Association tasks, magnetic resonance imaging (MRI), functional magnetic resonance imaging (fMRI), and positron emission tomography (PET) scans.

In one aspect, the therapeutically effective dose achieves a relief, cure, response rate, or regression rate of a neurological disorder between about 10% and about 99% or more. In one aspect, the therapeutically effective dose achieves a relief, cure, response rate, or regression rate of a neurological disorder between 10% and 100%, such as between 10% and 14%, between 10% and 20%, between 10% and 25%, between 14% and 20%, between 14% and 25%, between 14% and 30%, between 20% and 25%, between 20% and 30%, between 20% and 35%, between 10% and 100%, between 10% and 140 ...5%, between 14% and 30%, between 14% and 30%, between 14% and 30%, between 14% and 30%, between 14% and 35%, between 14% and 30%, between 14% and 30%, between 14% and 30%, between 14% and 30%, between 14% and 30%, between 14% and 30%, between 14% and 30%, between 14% and 30%, between 14% and 30%, between 14% and 30%, between 14% and 30%, between 14% and 30%, between 14% and 30%, between 14% 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 Between 55%, 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 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 90% and 100%, or between 95% and 100%.

In one aspect, a therapeutically effective dose eliminates, reduces, slows or delays one or more symptoms of a nervous system disorder by between 10% and 100%, such as between 10% and about 14%, between 10% and 20%, between 10% and 25%, between 14% and 20%, between 14% and 25%, between 14% and 30%, between 20% and 25%, between 20% and 30%, between 20% and 35 ...14% and 20%, between 14% and 20%, between 14% and 25%, between 14% and 25%, between 20% and 30%, between 20% and 35%, between 20% and 35%, between 20% and 35%, between 20% and 35%, between 20% and 35%, between 20% and 35%, between 14% and 20%, between 14% and 20%, between 14% and 25%, between 14% and 30%, between 20% and 35%, between 20% and 35%, between 20% and 35%, between 20% and 35%, between 20% and 35%, between 14% and 2 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% 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 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 90% and 100%, or between 95% and 100%.

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

On the one hand, nervous system disorder symptoms are assessed between 1 day after treatment and 7 days after treatment.In one aspect, symptom can be assessed between 1 day after treatment and 2 days after treatment, between 1 day after treatment and 3 days after treatment, between 1 day after treatment and 4 days after treatment, between 2 days after treatment and 3 days after treatment, between 2 days after treatment and 4 days after treatment, between 2 days after treatment and 5 days after treatment, between 3 days after treatment and 4 days after treatment, between 3 days after treatment and 5 days after treatment, between 3 days after treatment and 6 days after treatment, between 4 days after treatment and 5 days after treatment, between 4 days after treatment and 6 days after treatment, between 4 days after treatment and 7 days after treatment, between 5 days after treatment and 6 days after treatment, between 5 days after treatment and 7 days after treatment, or between 6 days after treatment and 7 days after treatment.In one aspect, symptom can be assessed between 1 week after treatment and 4 weeks after treatment. In one aspect, symptoms can be assessed between 1 week after treatment and 2 weeks after treatment, between 1 week after treatment and 3 weeks after treatment, between 1 week after treatment and 4 weeks after treatment, between 2 weeks after treatment and 3 weeks after treatment, between 2 weeks after treatment and 4 weeks after treatment, or between 3 weeks after treatment 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, the symptoms may be between 1 month after treatment and 2 months after treatment, between 1 month after treatment and 3 months after treatment, between 1 month after treatment and 4 months after treatment, between 2 months after treatment and 3 months after treatment, between 2 months after treatment and 4 months after treatment, between 2 months after treatment and 5 months after treatment, between 3 months after treatment and 4 months after treatment, between 3 months after treatment and 5 months after treatment, between 3 months after treatment and 6 months after treatment, between 4 months after treatment and 5 months after treatment, between 4 months after treatment and 6 months after treatment, between 4 months after treatment and 7 months after treatment, between 5 months after treatment and 6 months after treatment, between 5 months after treatment and 7 months after treatment, between 5 months after treatment and 8 months after treatment, or between 1 month after treatment and 2 months after treatment. Between 6 months after treatment and 7 months after treatment, between 6 months after treatment and 8 months after treatment, between 6 months after treatment and 9 months after treatment, between 7 months after treatment and 8 months after treatment, between 7 months after treatment and 9 months after treatment, between 7 months after treatment and 10 months after treatment, between 8 months after treatment and 9 months after treatment, between 8 months after treatment and 10 months after treatment, between 8 months after treatment and 11 months after treatment, between 9 months after treatment and 10 months after treatment, between 9 months after treatment and 11 months after treatment, between 9 months after treatment and 12 months after treatment, between 10 months after treatment and 11 months after treatment, between 10 months after treatment and 12 months after treatment, or between 11 months after treatment and 12 months after treatment. In one aspect, the symptom can be assessed between 1 year after treatment and about 20 years after treatment. In one aspect, symptoms can be assessed between 1 year after treatment and 5 years after treatment, between 1 year after treatment and 10 years after treatment, between 1 year after treatment and 14 years after treatment, between 5 years after treatment and 10 years after treatment, between 5 years after treatment and 14 years after treatment, between 5 years after treatment and 20 years after treatment, between 10 years after treatment and 14 years after treatment, between 10 years after treatment and 20 years after treatment, or between 14 years after treatment and 20 years after treatment.

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

In one aspect, a therapeutically effective dose achieves an increase in survival of between about 10% and 99% or more. In one aspect, a therapeutically effective dose achieves an increase in survival of between 10% and 100%, such as between 10% and 14%, between 10% and 20%, between 10% and 25%, between 14% and 20%, between 14% and 25%, between 14% and 30%, between 20% and 25%, between 20% and 30%, between 20% and 35%, between 25 ... 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 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 90% and 100%, or between 95% and 100%.

As used herein, the term "life expectancy" refers to the period of time that a subject is expected to live.

In one aspect, a therapeutically effective dose increases life expectancy by between about 10% and 99% or more. In one aspect, a therapeutically effective dose increases life expectancy by between 10% and 100%, such as between 10% and 14%, between 10% and 20%, between 10% and 25%, between 14% and 20%, between 14% and 25%, between 14% and 30%, between 20% and 25%, between 20% and 30%, between 20% and 35%, between 25% and 30%, between 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 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 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 14%, between 10% and 20%, between 10% and 25%, between 14% and 20%, between 14% and 25%, between 14% and 30%, between 20% and 25%, between 20% and 30%, between 20% and 35%, between 25% and 30%. Between 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 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 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 on the day of treatment, 1 day after treatment, 3 months after treatment, 6 months after treatment, 1 year after treatment, and annually thereafter.

On the one hand, the amount of atrophy in the subject's brain in need is assessed between 1 day after treatment and 7 days after treatment. In one aspect, symptoms can be assessed between 1 day after treatment and 2 days after treatment, between 1 day after treatment and 3 days after treatment, between 1 day after treatment and 4 days after treatment, between 2 days after treatment and 3 days after treatment, between 2 days after treatment and 4 days after treatment, between 2 days after treatment and 5 days after treatment, between 3 days after treatment and 4 days after treatment, between 3 days after treatment and 5 days after treatment, between 3 days after treatment and 6 days after treatment, between 4 days after treatment and 5 days after treatment, between 4 days after treatment and 6 days after treatment, between 4 days after treatment and 7 days after treatment, between 5 days after treatment and 6 days after treatment, between 5 days after treatment and 7 days after treatment, or between 6 days after treatment and 7 days after 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 after treatment and 2 weeks after treatment, between 1 week after treatment and 3 weeks after treatment, between 1 week after treatment and 4 weeks after treatment, between 2 weeks after treatment and 3 weeks after treatment, between 2 weeks after treatment and 4 weeks after treatment, or between 3 weeks after treatment 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, the symptoms may be between 1 month after treatment and 2 months after treatment, between 1 month after treatment and 3 months after treatment, between 1 month after treatment and 4 months after treatment, between 2 months after treatment and 3 months after treatment, between 2 months after treatment and 4 months after treatment, between 2 months after treatment and 5 months after treatment, between 3 months after treatment and 4 months after treatment, between 3 months after treatment and 5 months after treatment, between 3 months after treatment and 6 months after treatment, between 4 months after treatment and 5 months after treatment, between 4 months after treatment and 6 months after treatment, between 4 months after treatment and 7 months after treatment, between 5 months after treatment and 6 months after treatment, between 5 months after treatment and 7 months after treatment, between 5 months after treatment and 8 months after treatment, or between 1 month after treatment and 2 months after treatment. Between 6 months after treatment and 7 months after treatment, between 6 months after treatment and 8 months after treatment, between 6 months after treatment and 9 months after treatment, between 7 months after treatment and 8 months after treatment, between 7 months after treatment and 9 months after treatment, between 7 months after treatment and 10 months after treatment, between 8 months after treatment and 9 months after treatment, between 8 months after treatment and 10 months after treatment, between 8 months after treatment and 11 months after treatment, between 9 months after treatment and 10 months after treatment, between 9 months after treatment and 11 months after treatment, between 9 months after treatment and 12 months after treatment, between 10 months after treatment and 11 months after treatment, between 10 months after treatment and 12 months after treatment, or between 11 months after treatment 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 can be assessed between 1 year after treatment and 5 years after treatment, between 1 year after treatment and 10 years after treatment, between 1 year after treatment and 14 years after treatment, between 5 years after treatment and 10 years after treatment, between 5 years after treatment and 14 years after treatment, between 5 years after treatment and 20 years after treatment, between 10 years after treatment and 14 years after treatment, between 10 years after treatment and 20 years after treatment, or between 14 years after treatment and 20 years after 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 imaging (fMRI), and PET scans.

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

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

Examples

Example 1. AAV vector constructs

Forty-eight AAV vector constructs were constructed:

EF-1α:GfaABC1D:NeuroD1:P2A:Dlx2:WPRE:SV40 ( Figure 1B );

EF-1α:Gfa1.6:NeuroD1:P2A:Dlx2:WPRE:SV40;

EF-1α:GFA2.2:NeuroD1:P2A:Dlx2:WPRE:SV40;

EF-1α:GfaABC1D:NeuroD1:P2A:Dlx2:WPRE:hGH;

EF-1α:Gfa1.6:NeuroD1:P2A:Dlx2:WPRE:hGH;

EF-1α:GFA2.2:NeuroD1:P2A:Dlx2:WPRE:hGH;

CE:GfaABC1D:NeuroD1:P2A:Dlx2:WPRE:SV40(P31) (Figure 1A);

CE:Gfa1.6:NeuroD1:P2A:Dlx2:WPRE:SV40;

CE:GFA2.2:NeuroD1:P2A:Dlx2:WPRE:SV40;

CE:GfaABC1D:NeuroD1:P2A:Dlx2:WPRE:hGH;

CE:Gfa1.6:NeuroD1:P2A:Dlx2:WPRE:hGH;

CE:GFA2.2:NeuroD1:P2A:Dlx2:WPRE:hGH;

EF-1α:GfaABC1D:NeuroD1:T2A:Dlx2:WPRE:SV40 ( Figure 3B );

EF-1α:Gfa1.6:NeuroD1:T2A:Dlx2:WPRE:SV40;

EF-1α:GFA2.2:NeuroD1:T2A:Dlx2:WPRE:SV40;

EF-1α:GfaABC1D:NeuroD1:T2A:Dlx2:WPRE:hGH ( Fig. 3D );

EF-1α:Gfa1.6:NeuroD1:T2A:Dlx2:WPRE:hGH;

EF-1α:GFA2.2:NeuroD1:T2A:Dlx2:WPRE:hGH;

CE:GfaABC1D:NeuroD1:T2A:Dlx2:WPRE:SV40 ( Figure 3A );

CE:Gfa1.6:NeuroD1:T2A:Dlx2:WPRE:SV40;

CE:GFA2.2:NeuroD1:T2A:Dlx2:WPRE:SV40;

CE:GfaABC1D:NeuroD1:T2A:Dlx2:WPRE:hGH ( Figure 3C );

CE:Gfa1.6:NeuroD1:T2A:Dlx2:WPRE:hGH;

CE:GFA2.2:NeuroD1:T2A:Dlx2:WPRE:hGH;

EF-1α:GfaABC1D:NeuroD1:GSG-P2A:Dlx2:WPRE:SV40 ( Figure 2B );

EF-1α:Gfa1.6:NeuroD1:GSG-P2A:Dlx2:WPRE:SV40;

EF-1α:GFA2.2:NeuroD1:GSG-P2A:Dlx2:WPRE:SV40;

EF-1α:GfaABC1D:NeuroD1:GSG-P2A:Dlx2:WPRE:hGH (Figure 2D);

EF-1α:Gfa1.6:NeuroD1:GSG-P2A:Dlx2:WPRE:hGH;

EF-1α:GFA2.2:NeuroD1:GSG-P2A:Dlx2:WPRE:hGH;

CE:GfaABC1D:NeuroD1:GSG-P2A:Dlx2:WPRE:SV40 (Figure 2A);

CE:Gfa1.6:NeuroD1:GSG-P2A:Dlx2:WPRE:SV40;

CE:GFA2.2:NeuroD1:GSG-P2A:Dlx2:WPRE:SV40; CE:GfaABC1D:NeuroD1:GSG-P2A:Dlx2:WPRE:hGH (Figure 2C);

CE:Gfa1.6:NeuroD1:GSG-P2A:Dlx2:WPRE:hGH;

CE:GFA2.2:NeuroD1:GSG-P2A:Dlx2:WPRE:hGH;

EF-1α:GfaABC1D:NeuroD1:GSG-T2A:Dlx2:WPRE:SV40 ( Figure 4B );

EF-1α:Gfa1.6:NeuroD1:GSG-T2A:Dlx2:WPRE:SV40;

EF-1α:GFA2.2:NeuroD1:GSG-T2A:Dlx2:WPRE:SV40;

EF-1α:GfaABC1D:NeuroD1:GSG-T2A:Dlx2:WPRE:hGH (Figure 4D);

EF-1α:Gfa1.6:NeuroD1:GSG-T2A:Dlx2:WPRE:hGH;

EF-1α:GFA2.2:NeuroD1:GSG-T2A:Dlx2:WPRE:hGH;

CE:GfaABC1D:NeuroD1:GSG-T2A:Dlx2:WPRE:SV40 ( Figure 4A );

CE:Gfa1.6:NeuroD1:GSG-T2A:Dlx2:WPRE:SV40;

CE:GFA2.2:NeuroD1:GSG-T2A:Dlx2:WPRE:SV40; CE:GfaABC1D:NeuroD1:GSG-

T2A:Dlx2:WPRE:hGH ( Fig. 4C );

CE:Gfa1.6:NeuroD1:GSG-T2A:Dlx2:WPRE:hGH; and CE:GFA2.2:NeuroD1:GSG-

T2A:Dlx2:WPRE:hGH.

All 48 vector constructs utilized PHSG-299 (Takara, Mountain View, CA), a pUC-based vector construct containing an origin of replication, a kanamycin resistance gene, and a multiple cloning site (MSC) with the lacZ gene as a 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 a cytomegalovirus enhancer (CMV enhancer; SEQ ID NO:11), which is placed 5' to the following promoters: the 758 nucleotide GFAP promoter (GfaABC1D; SEQ ID NO:26), the 1667 nucleotide GFAP promoter (Gfa1.6; SEQ ID NO:4), or the 2214 nucleotide GFAP promoter (GFA2.2 SEQ ID NO:12).

After the enhancer/GFAP promoter (e.g., 3' to the enhancer/GFAP promoter), several additional sequences were introduced into the expression cassette in a 5' to 3' direction, including: a chimeric intron (SEQ ID NO: 5); a human NeuroD1 coding sequence (hNeuroD1; SEQ ID NO: 6); a human Dlx2 coding sequence (hDlx2; SEQ ID NO: 13); a linker sequence (P2A; SEQ ID NO: 15), (GSG-P2A; SEQ ID NO: 18), (T2A; SEQ ID NO: 16) or (GSG-T2A; SEQ ID NO: 19); and a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; SEQ ID NO: 7). These sequences are all operably linked to an SV40 poly (A) signal (SEQ ID NO: 8) or an hGH poly (A) signal (SEQ ID NO: 17) or a bGH poly (A) signal (SEQ ID NO: 30). The enhancer, GFAP promoter, chimeric intron, hNeuroD1 coding sequence, hD1x2 coding sequence, linker, WPRE and SV40 poly(A) signal are flanked by two AAVITR sequences.

Example 2. AAV virus production

Each of the 24 plasmids was co-transfected into 293AAV cells using polyethyleneimine together with the Rep-Cap plasmid (a plasmid containing promoters driving the expression of the AAV rep and cap genes) and a helper plasmid (a plasmid containing promoters driving the expression of the adenoviral E2A, E4, and VA RNAs) to produce recombinant AAV viral particles.

72 hours after transfection, the transfected cells were scraped and centrifuged. The cell pellet was frozen and thawed, i.e., placed in a dry ice/ethanol mixture and then placed in a 37°C water bath. The freeze/thaw cycle was repeated three more times. The AAV lysate was purified (e.g., to remove cell debris) 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. The viral titer was then determined by qPCR using primers that amplified the ITR region or gene/expression cassette-specific sequences.

Example 3. Astrocyte Culture

Human cortical astrocytes (HA1800; ScienCell Research Laboratories, Inc., Carlsbad, California) were subcultured at a confluence of more than 90%. For subculture, cells were trypsinized using TrypLE ^™ Select (Invitrogen, Carlsbad, California), centrifuged at 200×g for 5 minutes, and then resuspended and plated in a medium containing DMEM/F12 (Gibco); 10% fetal bovine serum (Gibco); penicillin/streptomycin (Gibco); 3.5 mM glucose (Sigma-Aldrich); B27 (Gibco); 10 ng/mL epidermal growth factor (Invitrogen); and 10 ng/mL fibroblast growth factor 2 (Invitrogen). Astrocytes were cultured on poly-D-lysine (Sigma-Aldrich) coated coverslips (12 mm) in 24-well plates (BD Biosciences) at a density of approximately 20,000 cells per coverslip.

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

All cells were maintained at 37°C in a humidified atmosphere containing 5% carbon dioxide.

Example 4. Testing of AAV vectors in astrocyte cultures (in vitro)

The recombinant AAV obtained from the method of Example 2 was used to infect the human cortical astrocytes and rat primary astrocytes of Example 3 at a concentration range of 10 ¹⁰ particles/mL and 10 ¹⁴ particles/ml. Twenty-four hours after the cells were infected, the culture medium was replaced with a differentiation medium containing DMEM/F12 (Gibco); N2 supplement (Gibco); and 20 ng/mL 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).

Filling of gaps in cell culture with additional human astrocytes to support functional development of converted neurons when astrocytes or rat primary astrocytes are converted into neurons.

Example 5. Testing of AAV vector efficacy

The recombinant AAV obtained by the method of Example 2 was used to infect the human cortical astrocytes of Example 3 and rat primary astrocytes (or astrocytes of other brain regions or spinal cord) at passages 4 ^to 7 at a concentration range of 10 ¹⁰ particles/mL and 10 14 particles/mL. qPCR, enzyme-linked immunosorbent assay (ELISA) and western blotting were performed to determine the expression of NeuroD1 transcripts and protein levels.

The expression of NeuN, doublecortin (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. Testing of AAV vector titer and infection rate

The purified AAV vector was treated with DNase I to eliminate residual plasmid contamination. AAV vector serial dilution was performed at 100 times, 500 times, 2500 times and 12500 times. AAV plasmid was diluted to generate a standard curve by serial dilution. Plasmid dilution was 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ and 10 ⁸ molecules/ul. qPCR was performed on the diluted AAV vector and diluted AAV plasmid. The primers used were for the ITR region (forward ITR primer 5'-GGAACCCCTAGTGATGGAGTT, reverse ITR primer 5'-CGGCCTCAGTGAGCGA). The qPCR mixture contained 10uL of Universal SYBR Master Mix 2X, 2uL of 5uM forward ITR primer, 2uL of 5uM reverse ITR primer, 5uL of test sample or dilution standard and 1uL of H ₂ O. The qPCR program was 95°C for 10 min, followed by 40 cycles of 95°C for 15 s, 60°C for 30 s, and then a melting curve. The data were analyzed using the qPCR cycler software. The physical titer (viral genome (vg)/ml) of the AAV samples was calculated based on the standard curve.

AAV vector infectivity was tested by 50% tissue culture infectious dose (TCID50) assay using the standard protocol of the American Type Culture Collection (ATCC; Manassas, VA).

Example 7. Testing of AAV Dose Range (In Vivo)

The 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 was injected with a volume of 1uL at a dosage of 1x10 ¹¹ , 3x10 ¹¹ , 1x10 ¹² , 3x10 ¹² , 1x10 ¹² , 3x10 ¹² , 1x10 ¹³ viral genomes/mL. Each dosage was evaluated 4 days, 20 days and 60 days after injection to determine the optimal effective dose (OED), maximum tolerated dose (MTD) and minimum effective dose (MED) at cell and tissue levels. Three mice at each time point. OED, MTD and MED were determined by evaluating the conversion efficiency and potential toxicity of astrocytes to neurons via immunostaining of NeuroD1, GFAP, NeuN and Iba1. If the first dose range was insufficient to determine the OED, MTD, and MED, a second dose range was performed at ^1x1010 viral genomes/mL to ^1x1014 GC/mL in 1 uL volume.

Example 8. Comparison of neuronal conversion rates of recombinant AAV obtained from various AAV vector constructs in human cell culture (in vitro)

AAV vector constructs were designed as described in Example 1 to express NeuroD1 alone or Dlx2 alone. The following recombinant AAVs were obtained as described in Example 2: (1) an AAV vector construct expressing NeuroD1 alone; (2) an AAV vector construct expressing Dlx2 alone; (3) a combination of AAV vector constructs (1) and (2); and (4) an AAV vector construct expressing NeuroD1 and a linker and Dlx2. The resulting recombinant AAV was used to infect human cortical astrocytes and rat primary astrocytes of Example 3. Twenty-four hours after infecting the cells, the culture medium was replaced with differentiation medium containing DMEM/F12 (Gibco); N2 supplement (Gibco); and 20 ng/mL 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). Additional human astrocytes were used to fill the gaps in the cell culture to support the functional development of converted neurons when astrocytes or microglia were converted to neurons. The level of neuronal conversion was measured and compared for each treatment.

Example 9. Testing of AAV Vectors in Human Subjects (In Vivo)

The recombinant AAV obtained from the method of Example 2 is used to infect human brain or spinal cord astrocytes in vivo. The recombinant AAV is injected into the brain or spinal cord of a human subject with a neurological disorder at a concentration range of 10 ¹⁰ particles/mL and 10 ¹⁴ particles/mL in a volume range of 10 μL to 1 mL. Symptoms of neurological disorders, brain imaging (including MRI, PET scans, or a combination of MRI and PET), and behavioral indicators of human subjects are observed before, during, and after the injection. Post-injection observations are performed once a week until the first month after injection. After the first month after injection, observations are performed once a month for the next 11 months and can be extended to 2 years after viral injection.

Example 10. Dose scaling in non-human primates

The volume of brain tissue expressing NeuroD1 from Example 7 divided by the number of vector genomes (mm ³ /vector genome) was used to determine the viral infection rate of brain tissue. The volume of the specific brain region to be treated in non-human primates was calculated (mm ³ ), and the dose range of vector genomes was determined proportionally according to the infection rate obtained in Example 7. A dose ranging study was performed as in Example 7 and the OED, MTD and MED were determined by evaluating the conversion efficiency of astrocytes to neurons and potential toxicity via immunostaining of NeuroD1, GFAP, NeuN and Iba1.

Example 11. Treatment of a subject in need thereof with Huntington's disease (in vivo)

The recombinant AAV obtained from the method of Example 2 is used to treat subjects with Huntington's disease. The neurological symptoms of the subject include involuntary movements (such as chorea), uncontrolled postures, mood changes, sleep disorders, language changes, dysphagia, and impaired cognitive function (such as learning and memory defects). The recombinant AAV is injected into the striatum (nucleus shell and caudate nucleus) of human subjects with neurological disorders in a volume range of 10 μL to 1000 μL at a concentration range of 10 ¹⁰ particles/mL and 10 ¹⁴ particles/mL. Before, during and after the injection, the symptoms of neurological disorders, brain imaging (including MRI, PET scans or a combination of MRI and PET) and behavioral indicators of human subjects are observed. Post-injection observations are performed once a week until the first month after injection. After the first month after injection, observations are performed once a month for the next 11 months and can be extended to 2 years after viral injection.

Example 12. Combinatorial method for directly converting glial cells into neurons in combination with shRNA for knocking down Htt gene expression

Identify the target sequence complementary to the Htt gene. shRNA is designed to target the Htt gene. NeuroD1, Dlx2 and target shRNA are packaged into AAV vectors (hU6::Htt shRNA-hGFAP::hNeuroD1-P2A-hDlx2) (Figures 5A-8D), and recombinant AAV is produced as described in Example 2. Recombinant AAV is injected into the striatum of mice with mutant Htt gene. The behavioral indicators of the mice treated are tested, such as cat walk, open field test, clenching, mouse weight and grip strength, and brain imaging (including MRI, PET scans or a combination of MRI and PET). The behavioral test results and brain imaging of the following groups are compared: (i) not treated, (ii) receiving recombinant AAV from Example 2, and (iii) receiving recombinant AAV (hU6::Htt shRNA-hGFAP::hNeuroD1-P2A-hDlx2) (Figures 5A-8D).

Alternatively, the target shRNA is packaged into an AAV vector (hU6::hHtt shRNA) and another recombinant AAV is produced as described in Example 2. The two recombinant AAVs are injected into the striatum of mice with mutant Htt. The treated mice are tested for behavioral indicators such as catwalk, open field test, clenching, mouse weight and grip strength, and brain imaging (including MRI, PET scans, or a combination of MRI and PET). The behavioral test results and brain imaging of the following groups are compared: (i) no treatment, (ii) only receiving the recombinant AAV from Example 2, and (iii) receiving a combination of recombinant AAV (hU6::hHtt shRNA) and the recombinant AAV from Example 2.

Example 13. Combination method for directly converting glial cells into neurons combined with CRISPR/CAS gene editing of the Htt gene

Identify a target sequence complementary to the Htt gene. The guide RNA (gRNA) sequence is designed to target the Htt gene. The donor sequence is designed to modify the number of CAG repeats of the Htt gene to less than 36. The Cas9 nuclease, Htt-specific gRNA, and donor sequence are packaged into an AAV vector (AAV-Cas9-HTT). Recombinant AAV is produced as described in Example 2.

Recombinant AAV (AAV-Cas9-HTT) was injected into the striatum of mice with mutant Htt simultaneously with the recombinant AAV from Example 2. Behavioral indicators such as catwalk, open field test, clenching, mouse weight and grip strength, and brain imaging (including MRI, PET scan, or a combination of MRI and PET) of the treated mice were tested. The behavioral test results and brain imaging of the following groups were compared: (i) not receiving treatment, (ii) receiving recombinant AAV from Example 2, and (iii) receiving recombinant AAV-Cas9-HTT and recombinant AAV from Example 2 to identify the synergistic effect between mutant Htt gene editing and glial to neuronal conversion. Recombinant AAV-Cas9-HTT and recombinant AAV from Example 2 can be injected simultaneously or at different times.

Alternatively, NeuroD1, a linker (P2A), Dlx2, a second linker (P2A), Cas9 nuclease, Htt-specific gRNA, and a donor sequence are packaged into an AAV vector (AAV-hNeuroD1-P2A-hDlx2-P2A-Cas9-HTT). Recombinant AAV is produced as described in Example 2. Recombinant AAV (AAV-hNeuroD1-P2A-hDlx2-P2A-Cas9-HTT) is injected into the striatum of mice with mutant Htt simultaneously with the recombinant AAV from Example 2. The treated mice are tested for behavioral indicators such as catwalk, open field test, clenching, mouse weight and grip strength, and brain imaging (including MRI, PET scan, or a combination of MRI and PET). The behavioral test results and brain imaging of the following groups were compared: (i) receiving no treatment, (ii) receiving recombinant AAV from Example 2, and (iii) receiving recombinant AAV-hNeuroD1-P2A-hDlx2-P2A-Cas9-HTT and recombinant AAV from Example 2 to identify the synergistic effect between mutant Htt gene editing and glial to neuronal conversion.

Example 14. Combinatorial method for directly converting glial cells into neurons in combination with antisense oligonucleotides (ASOs) that knock down Htt gene expression

Identify target sequences complementary to the Htt gene. Design and synthesize ASOs that knock down Htt gene expression. The recombinant AAV from Example 2 is injected into the striatum of mice with mutant Htt together with the Htt ASO. The behavioral indicators of the treated mice are tested, such as catwalk, open field test, clenching, mouse weight and grip strength, and brain imaging (including MRI, PET scans, or a combination of MRI and PET). Compare the behavioral test results and brain imaging of the following groups: (i) not receiving treatment, (ii) receiving the recombinant AAV from Example 2, and (iii) receiving the recombinant AAV and Htt ASO from Example 2.

Example 15. Combinatorial method for directly converting glial cells into neurons combined with siRNA that knocks down Htt gene expression

Identify target sequences complementary to the Htt gene. Design and synthesize siRNAs that knock down Htt gene expression. The recombinant AAV from Example 2 is injected into the striatum of mice with mutant Htt together with Htt siRNA. The behavioral indicators of the treated mice are tested, such as catwalk, open field test, clenching, mouse weight and grip strength, and brain imaging (including MRI, PET scans, or a combination of MRI and PET). Compare the behavioral test results and brain imaging of the following groups: (i) not receiving treatment, (ii) receiving recombinant AAV from Example 2, and (iii) receiving recombinant AAV and Htt siRNA from Example 2.

Example 16. Combinatorial approach to directly convert glial cells into neurons in combination with miRNA that knocks down Htt gene expression

Identify miRNAs that regulate Htt gene expression. NeuroD1, Dlx2 and miRNA are packaged into AAV vectors (CAG::Htt miRNA-hGFAP::hNeuroD1-P2A-hDlx2), and recombinant AAV is produced as described in Example 2. Recombinant AAV is injected into the striatum of mice with mutant Htt. Behavioral indicators of treated mice are tested, such as catwalk, open field test, clenching, mouse weight and grip strength, and brain imaging (including MRI, PET scans or a combination of MRI and PET). Compare the behavioral test results and brain imaging of the following groups: (i) not receiving treatment, (ii) receiving recombinant AAV from Example 2, and (iii) receiving recombinant AAV (CAG::Htt miRNA-hGFAP::hNeuroD1-P2A-hDlx2).

Alternatively, the target miRNA is packaged into an AAV vector (CAG::hHtt miRNA) and recombinant AAV is produced as described in Example 2. The recombinant AAV is injected into the striatum of mice with mutant Htt. The treated mice are tested for behavioral indicators such as catwalk, open field test, clenching, mouse weight and grip strength, and brain imaging (including MRI, PET scans, or a combination of MRI and PET). The behavioral test results and brain imaging of the following groups are compared: (i) not receiving treatment, (ii) receiving only the recombinant AAV from Example 2, and (iii) receiving a combination of recombinant AAV (CAG::hHtt miRNA) and the recombinant AAV from Example 2.

Example 17. AAV virus production of P31

Recombinant AAV was obtained as described in Example 2. The P31 plasmid was co-transfected into AAV293 cells with a Rep-Cap plasmid expressing serotype 5 capsid protein and a helper plasmid P40Helper (P40H) or pALD-X80 (X80) to produce recombinant AAV virus particles (P31-P40H or P31-X80). Viral titers were determined by qPCR using primers that amplify the target gene (GOI), primers specific to the P31 plasmid and ITR regions. Reverse packaging primers were used to evaluate nonspecific packaging. Compared with the P40H helper plasmid, an increase in viral yield was observed with the X80 helper plasmid (Figure 9).

Example 18. Successful establishment of primary culture of rat astrocytes

Cortical and striatal tissues were isolated from the brains of Sprague Dawley rats at 3 days postnatal. The tissues were treated with papain to generate single cell suspensions and seeded in poly-D-lysine coated flasks. The cells were immunostained with GFAP antibodies and SOX9 antibodies. The cells were counterstained with DAPI antibodies. More than 95% of the cells were astrocytes identified by GFAP and SOX9 staining (Figure 10). The leftmost figure presents images of GFAP stained cells. The middle left figure presents images of SOX9 stained cells. The middle right figure presents images of DAPI stained cells. The rightmost figure presents a merged image of GFAP, SOX9 and DAPI stained cells.

Example 19. Comparison of plasmid transfection

Primary rat astrocytes were plated and transfected with expression vectors P14 (CE: GfaABC1D: hNeuroD1-P2A-Dlx2: WPRE: SV40), P31 (EF-1α: GfaABC1D: NeuroD1-P2A-Dlx2: WPRE: SV40) and P63 (CE: GfaABC1D: NeuroD1-GSG P2A-Dlx2: WPRE: SV40) as described in Example 18 to test the expression efficiency of NeuroD1 and Dlx2 in transfected cells. P14 resulted in the expression of NeuroD1 and Dlx2 as shown by NeuroD1 staining and Dlx2 staining of cells ( FIG. 11 ; the upper panel shows NeuroD1 staining of cells, the middle panel shows Dlx2 staining of cells, and the lower panel shows merged NeuroD1, Dlx2 and DAPI staining of cells).

Example 20. Successful transduction of AAV viral particles into primary rat astrocytes

AAV9-P12 (pGfaABC1D: GFP) was used to transduce the recombinant AAV obtained from the method of Example 2 into primary rat astrocytes inoculated in 96-well plates at different doses of 3x10 ¹⁰ vg/well, 1x10 ¹⁰ vg/well and 2.5x10 ⁹ vg/well. 24 hours before transduction, RCA of the 5th-7th generation was inoculated on a glass coverslip coated with poly-D-lysine (PDL) in a 96-well plate, and the confluence was 50%. Cells were transduced with viruses of a specified titer in fresh astrocyte culture medium. The culture medium was updated the next day and updated every 3-4 days. Images obtained six days after GFP-positive cell transduction showed that the transduction rate was higher when the virus titer was higher (Figure 12).

Example 21. 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 inoculated in 24-well plates or 96-well plates with viral particles AAV9-P12 (pGfaABC1D: GFP) and AAV5-P7 (pEF-1α: GFP). Cells were harvested by trypsinization 7 days after infection. The cells were fixed, washed and suspended in PBS. Flow cytometry was used to analyze the viral transduction rate to calculate the GFP-positive cells compared to all cells (Figures 13A and 13B). Figure 13A shows the percentage of transduction rate at different MOIs. Cells inoculated in 24-well plates at 1x10 ⁵ cells/well were infected with an MOI of 5x10 ⁵ / cell, 2x10 ⁵ / cell and 5x10 ⁴ / cell. The viral transduction rate decreased with the decrease of MOI. Figure 13B shows the transduction rate of AAV viral particles in cells seeded in 96-well plates at a range of densities (2x10 ⁴ cells/well, 1.5x10 ⁴ cells/well, 1x10 ⁴ cells/well, and 5x10 ³ cells/well) and infected with a range of amounts of virus (2μl, 1μl, 0.5μl, 0.25μl, 0.125μl of 1x10 ¹³ /ml virus in 100μl medium). This is equivalent to 2x10 ¹⁰ vg, 1x10 ¹⁰ vg, 5x10 ⁹ vg, 2.5x10 ⁹ vg, and 1.25x10 ⁹ vg per well, respectively. As the number of cells per well decreases, the viral transduction rate remains unchanged.

Example 22. NeuroD1 vector-induced transgene expression and astrocyte-to-neuron conversion in vitro.

Materials and methods

Primary rat astrocyte cultures: Rat cortical astrocytes (RCA) were isolated from the cortical tissue of Sprague Dawley rats at 3 days postnatal. The cells were maintained in astrocyte medium (AM), which consisted of DMEM supplemented with 10% FBS, 2.5mM glutamine, 3.5mM glucose, and penicillin/streptomycin. The first two generations of cells were subcultured at a ratio of 1:3-1:4 at low cell density to promote the differentiation of residual progenitor cells. When 90%-100% confluence was reached, the ratio of subsequent subculture was 1:2 or 1:3. The 5th-7th generation cells were used for transfection and transduction. Immunostaining with GFAP antibody showed that >90% of the cells were GFAP-positive astrocytes. The 6th generation astrocyte cultures were immunostained with astrocyte markers GFAP and Sox9 (Figure 14).

Vectors : AAVs were generated with 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 used for in vitro studies were produced using adherent AAV293 cells by triple transfection (GOI, helper and Rep/Cap plasmids) using polyethyleneimine (PEI). Virus recovery and purification were achieved by ultracentrifugation or using commercial purification kits.

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

Virus titers were determined by real-time quantitative PCR using primer pairs in the ITR region, primers amplifying the target gene (GOI), or vector-specific primers. Plasmid DNA was used as a standard. Production yields were approximately 10 ³ -10 ⁴ vg/cell levels. FIG. 35 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 the helper plasmid pALD-X80 (X80) produced recombinant AAV virus particles as measured by qPCR.

Transfection and immunofluorescence: 24-48 hours before transfection, rat cortical astrocytes (RCA) of passage 5-7 were seeded on glass coverslips coated with poly-D-lysine (PDL) in 24-well plates at a confluence of 30%-50%. Cells were transfected with 300ng of 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, then washed and immunostained with anti-NeuroD1 (anti-ND1) antibody (Abcam Cat#ab60704), followed by immunostaining with secondary antibodies conjugated to fluorescent dyes (Invitrogen, AlexaFluor). Images were taken under a fluorescence microscope (Zeiss Axiovert A1, Zen Blue). Gene expression levels were assessed by comparing fluorescence intensity.

Transduction and immunofluorescence: 24-48 hours before transduction, RCAs of passages 5-7 were seeded on glass coverslips coated with poly-D-lysine (PDL) in 24-well plates at a confluence of 30%-50%. Cells were transduced with 2-6X10 ¹⁰ viral genomes (vg)/ml of AAV in fresh astrocyte culture medium. The culture medium was updated the next day and every 3-4 days. Three to six days after transduction, cells were fixed with 4% paraformaldehyde in PBS, then washed and immunostained with anti-ND1 antibody (Abcam Cat#ab60704), and then immunostained with secondary antibodies conjugated to fluorescent dyes (Invitrogen, Alexafluor) for observation and image capture under a fluorescence microscope (Zeiss Axiovert A1, Zen Blue). Gene expression levels were assessed by comparing fluorescence intensity.

Astrocyte to neuron conversion assessment 24-48 hours before transduction, the 5-7th generation RCA was inoculated on a glass coverslip coated with poly-D-lysine (PDL) in a 24-well plate, and the confluence was 30%-50%. 2-6X10 10 vg/ml of virus transduced cells were used in 500ul fresh astrocyte culture medium (supplemented with 10% FBS, 2.5mM glutamine, 3.5mM glucose, penicillin/streptomycin DMEM). 48 hours after transduction, the culture medium was replaced with 5% FBS astrocyte culture medium. Subsequently, 100ul conversion culture medium (DMEM/F12+1% FBS+B27+N2 and ^1uM Rock inhibitor and 10ng/ml BDNF) was added every day for 4 days. After 4 days, the culture medium was completely replaced with conversion culture medium.

At different desired time points (three days, one to five weeks after transduction), cells were fixed with 4% paraformaldehyde in PBS, then washed and immunostained with antibodies against ND1 (Abcam Cat#ab60704), NeuN (Millipore, Cat#ABN78), Map2 (Invitrogen, Cat#PA5-17646), and then immunostained with secondary antibodies conjugated with fluorescent dyes (Invitrogen, Alexafluor) for visualization and imaging under a fluorescence microscope (Zeiss Axiovert A1, Zen Blue).

In vitro study results:

All tested NeuroD1 (ND1) plasmids effectively drove the expression of NeuroD1 (Figures 16-31). The expression level of NeuroD1 was affected by the elements in the vector. Among the three versions of the GFA promoter, the 681bp promoter showed the highest NeuroD1 expression level, and the 1.6kb promoter showed the weakest NeuroD1 expression level. Promoter enhancer elements significantly affected the expression level of NeuroD1. The CMV enhancer increased the expression level of NeuroD1 more than the ef1α enhancer. Chimeric introns and WPRE also increased the expression level of NeuroD1.

All tested AAVs containing ND1 efficiently drove expression of ND1 and induced astrocyte-to-neuron conversion in cultured rat astrocytes, as indicated by positive staining for NeuN and/or MAP2 (Figures 17, 30, 22, 25, and 28). Conversion was higher when astrocytes were transduced with vectors driving higher ND1 expression. Vectors NXL-P134 and NXL-P138, as well as viruses generated using these vectors (i.e., AAV9-P134 and AAV9-P138, respectively) were the most efficient in driving ND1 expression and inducing astrocyte-to-neuron conversion, with AAV-P134 being the most potent (Figures 15-20). Plasmid AAV9-P21 (CE-pGFA681-CI-GFP-WPRE-SV40pA) without the ND1 sequence was used as a control and did not induce astrocyte-to-neuron conversion, as shown by the lack of positive staining for NeuN and/or Map2 ( FIG. 14 ).

NeuN/RBFOX3 (neuronal nuclear protein) is a neuronal differentiation marker that stains the nuclei and perinuclear cytoplasm of neurons. MAP2 (microtubule-associated protein 2) is another neuronal marker that stains cytoplasmic 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 23. NeuroD1 viral vector induced transgene expression and astrocyte-to-neuron conversion in vivo.

AAV9-P134 and AAV9-P138 viruses were used for in vivo studies. AAV9-P12 driving GFP expression alone (without ND1) under the GFAP promoter was used for control and identification of GFAP-expressing cells (astrocytes).

The single-stranded adeno-associated virus (ssAAV, abbreviated AAV) vectors NXL-P12, NXL-P134, and NXL-P138 were packaged into AAV serotype 9 (AAV9) and then subjected to subsequent iodixanol gradient ultracentrifugation and concentration. The purified AAV viruses were titrated using a quantitative PCR-based method. All AAVs 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/6 mice older than 8 weeks were injected with AAV9-P134, AAV-P138, and AAV9-P12 viruses as follows:

P12 control group: AAV9-P12 5x10 ¹¹ GC/ml, 1 μL, once injected into the cortex (unilaterally) (n=6)

P134 group: AAV9-P12 2.5x10 ¹¹ GC/ml+AAV9-P134 2.5x10 ¹¹ GC/ml, 1 μL, cortical injection once (unilateral) (n=6)

P138 group: AAV9-P12 2.5x10 ¹¹ GC/ml+AAV9-P138 2.5x10 ¹¹ GC/ml, 1 μL, cortical injection once (unilateral) (n=6)

Mice were sacrificed at 10 days post infection (dpi) and 30 dpi and cerebral cortical tissue was analyzed. Animals were anesthetized with 1.25% Avertin and then perfused intracardially with saline (0.9% NaCl) followed by 4% paraformaldehyde (PFA). Brains were collected and fixed overnight in 4% PFA and then placed in 20% and 30% sucrose at 4°C until the tissue sank. Dehydrated brains were embedded in optimal cutting temperature (Tissue- OCT compounds, Finetek, Torrance, CA, USA), and then serially sectioned in the coronal plane at 30 μm thickness on a cryostat (Thermo Scientific, Shanghai, China). For immunofluorescence, free-floating brain sections were first washed with PBS and blocked in 5% normal donkey serum, 3% bovine serum albumin, and 0.3% TritonX-100 prepared in PBS for 1 h at room temperature (RT), and then incubated overnight at 4°C with primary antibodies diluted in blocking solution. After additional washing with 0.2% PBST (0.2% tween-20 in PBS), the samples were incubated with 0.5 μg/μL of 4′,6-diamidino-2-phenylindole (DAPI; F. Hoffmann-La Roche, Natley, NJ, USA) and appropriate secondary antibodies conjugated to Alexa Fluor 555, goat anti-chicken conjugated to Alexa Fluor 488 (1:1000, Life technologies, Carlsbad, CA, USA), and goat anti-rat (Life technologies)/guinea pig (Jackson immune research) conjugated to Alexa Fluor 647 (1:500) for 2 h at room temperature and then washed extensively with PBS. The samples were finally incubated with The slides were mounted with mounting medium (VECTOR Laboratories, Burlingame, CA, USA) and sealed with nail polish. Representative images were taken with a confocal microscope (LSM880, 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-NeuroD1 (1:500, Cat#ab60704, Abcam) and chicken anti-GFP (1:1000, Cat#ab13970, Abcam). The images were acquired by a Zeiss Axioplan fluorescence microscope (Axio Imager Z2, Zeiss, Representative images were taken with a confocal microscope (LSM880, Zeiss, Jena, Germany) or a confocal microscope (LSM880, Zeiss, Jena, Germany). Quantitative analysis was performed based on 4 randomly selected fields of view (212 μm x 212 μm, obtained from a LSM880 confocal microscope at 400 times magnification) from 3 brain sections of each mouse (3 mice per group). Data are shown as mean ± SEM.

First, the control virus P12 expressing a single GFP reporter gene was compared with the viruses P134 and P138 expressing NeuroD1 (all P12 was added together to track the neurons converted). When the control virus P12 was injected into the uninjured mouse cortex, the infected cells were mainly astrocytes without NeuroD1 expression at 10dpi (days after injection) (Figure 36). On the contrary, NeuroD1 expression was clearly detected in P134 and P138 groups. Although most of the cells expressing NeuroD1 in the P138 group at 10dpi were still astrocytes, a part of the cells expressing NeuroD1 in the P134 group was already NeuN+ neurons (Figure 36), indicating that P134 may have a better conversion ability than P138. In addition, at 10dpi, the analysis of the cortical brain tissue of the P134 group mice showed that the conversion level of astrocytes to neurons was high, and the morphological changes (such as the presence of long protrusions) of the GFP positive cells proved this point (Figure 32). The P138 group showed a lower level of conversion.

30 days after virus injection, the infected cells in the control group (P12) were still astrocytes, but most of the GFP-positive cells in the P134 group were neurons expressing NeuN (Figure 37). However, the conversion rate in the P138 group was lower than that in the P134 group. At this stage, most of the infected cells in the P138 group were still astrocytes, and the GFP signal in the converted neurons was weak (Figure 37). In addition, at 30dpi, analysis of the cortical brain tissue of mice in the P134 group showed that the level of conversion of astrocytes to neurons was even higher, as evidenced by the presence of long processes in GFP-positive cells (Figure 33)

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

P12 group: AAV9-P12 5x10 ¹¹ GC/ml, 1μL, once per cortex (bilaterally)

P14 group: AAV9-P12 2.5x10 ¹¹ GC/ml+AAV9-P134 2.5x10 ¹¹ GC/ml, 1 μL, once per cortex (bilaterally)

Mice were put to death 10 days (dpi) after virus injection, and cerebral cortex tissue was analyzed. When control virus P12 was injected into ET-1 damaged mouse cortex, the infected cells were mainly astrocytes (Figure 38) without NeuroD1 expression at 10dpi (days after injection). On the contrary, NeuroD1 expression (Figure 38) was detected in P134 group. Mice were put to death 10 days (dpi) after virus injection, and cerebral cortex tissue was analyzed. At 10dpi, the analysis of P134 group mouse cortical brain tissue showed that astrocytes had a high level of conversion to neurons, and the morphological changes (such as the presence of long protrusions) observed in GFP positive cells proved this point (Figure 34).

Example 24. NeuroD1 and Dlx2 expression induced in vitro transgene expression and astrocyte to neuron conversion

Materials and methods

Vectors : Vectors were tested by transfection of rat cortical astrocytes (RCA). In addition, AAVs were produced from selected vectors and tested in vitro by transduction using rat astrocytes:

·NXL-P20(CE-pGfa681-CI-hND1-p2A-hDLX2-WPRE-SV40pA)

·NXL-P31(EE-pGfa681-CI-hND1-p2A-hDlx2-WPRE-SV40pA)

·NXL-P111(CE-pGfa1681-CI-hDlx2-IRES-hND1-SV40pA)

·NXL-P112(CE-pGfa1681-CI-hDlx2-IRES-hND1-bGHpA)

·NXL-P113(EE-pGfa1681-CI-hDlx2-IRES-hND1-bGHpA)

·NXL-P122(CE-pGfa681-CI-hDlx2-P2A-hND1-bGHpA)

·NXL-P123(EE-pGfa681-CI-hDlx2-P2A-hND1-bGHpA)

·NXL-P124(CE-pGfa681-CI-hND1-P2A-hDlx2-bGHpA)

Figure 40 depicts two general diagrams of the constructs.

Cell culture: 24-48 hours before transduction, rat cortical astrocytes (RCA) at passage 5-7 were plated on glass coverslips coated with poly-D-lysine (PDL) in 24-well plates at a confluence of 30%-50%.

Transduction and immunofluorescence: Transduce cells with 2-6X10 ¹⁰ vg/ml of virus in 500ul fresh astrocyte culture medium (DMEM supplemented with 10% FBS, 2.5mM glutamine, 3.5mM glucose, penicillin/streptomycin). 48 hours after transduction, replace the culture medium with 5% FBS astrocyte culture medium. For the next 4 days, add 100ul transformation medium (DMEM/F12+1% FBS+B27+N2 and 1uM Rock inhibitor and 10ng/ml BDNF) every day. Then completely replace the culture medium with transformation medium.

Cells were fixed with 4% paraformaldehyde in PBS at different desired time points (e.g., three days, one to five weeks after transduction), then washed and immunostained with antibodies to ND1 (Abcam Cat#ab60704), antibodies to Dlx2 (Millipore Cat#AB5726), antibodies to NeuN (Millipore, Cat#ABN78), antibodies to Map2 (Invitrogen, Cat#PA5-17646), and then immunostained with secondary antibodies conjugated to fluorescent dyes (Invitrogen, Alexafluor). Images were taken under a fluorescence microscope (Zeiss Axiovert A1, Zen Blue). Gene expression levels were assessed by comparing fluorescence intensity.

In vitro study results:

The tested ND1-Dlx2 constructs effectively drove the expression of ND1 and Dlx2 by transfection and/or transduction of cultured rat astrocytes, as evidenced by positive staining of ND1 and Dlx2 in these cells (Figures 41-56). In addition, as shown by positive staining of the neuronal markers NeuN and/or MAP2 (Figures 43, 46, 49 and 52), AAVs containing ND1/Dlx2 (AAV9-P122, AAV-P122, AAV-P124 and AAV-P20) effectively drove the expression of ND1, Dlx2 in cultured rat astrocytes and induced the conversion of astrocytes to neurons. Some NeuN/MAP2 positive cells showed typical neuronal morphology.

Example 25. In vivo transgene expression and astrocyte-to-neuron conversion induced by NeuroD1 and Dlx2 expression

AAV9-P112 and AAV9-P122 viruses were used for in vivo studies. AAV9-P12 driving GFP expression alone (without ND1 and Dlx2) under the GFAP promoter was used for control and identification of GFAP-expressing cells (astrocytes).

Normal C57BL/6 mice older than 8 weeks were injected with AAV-P12, AAV9-P112, and AAV9-P122 viruses as follows:

P12 control group: AAV9-P12 2.5x10 ¹¹ GC/ml, 2 μL, injected into striatum once (n=6)

P112 group: AAV9-P12 2.5x10 ¹¹ GC/ml+AAV9-P112 2.5x10 ¹¹ GC/ml, 2 μL, once injected into striatum (n=6)

P122 group: AAV9-P12 2.5x10 ¹¹ GC/ml+AAV9-P122 2.5x ¹¹ GC/ml, 2 μL, once injected into striatum (n=6)

Mice were killed at 10 days (dpi) and 30dpi after infection and cerebral cortical tissue was analyzed. The infection of AAV9-P12 to mouse striatum was confirmed by the presence of GFP fluorescence at 10dpi. At 30dpi, the cells infected by AAV9-P12 were still GFAP-positive astrocytes, indicating that AAV9-P12 is specific in infecting astrocytes (Figure 57A-B). In the P112 group, at 10dpi, most of the cells co-infected by AAV9-P12 and AAV9-P112 were GFAP-positive stained astrocytes (Figure 58). By 30 days after viral infection, many cells co-infected by AAV9-P12 and AAV9-P112 became NeuN-positive neurons (Figure 59, white arrows). In the P122 group, at 10 dpi, most of the cells co-infected with AAV9-P12 and AAV9-P122 were GFAP-positive astrocytes (Figure 60). By 30 days after viral infection, many cells co-infected with AAV9-P12 and AAV9-P122 became NeuN-positive neurons (Figure 61, white arrows).

Example 19. In vitro transgenic expression of Dlx2

Vectors : Vectors were tested by transfection of rat cortical astrocytes (RACs). In addition, AAV was produced with the selected vectors and tested in vitro by transduction: NXL-P44:EE-pGfa681-CI-Dlx2-WPRE-SV40pA

·NXL-P60:EE-pGfa681-Dlx2-WPRE-SV40pA

·NXL-P75:CE-pGfa681-CI-Dlx2-WPRE-SV40pA

·NXL-P104:CE-pGfa681-CGRI-Dlx2-bGHpA

·NXL-P105:CE-pGfa681-CI-Dlx2-oPRE-bGHpA

·NXL-P131:EE-pGfa681-CI-Dlx2-oPRE-bGHpA

·NXL-P133:CE-pGfa681-CGRI-Dlx2-oPRE-bGHpA

·NXL-P137:EE-pGfa681-CGRI-Dlx2-oPRE-bGHpA

24 hours after transfection of cultured RACs, NXL-P104 and NXL-P105 constructs effectively drove the expression of Dlx2 as evidenced by positive Dlx2 staining in these cells (Figure 62). 24 hours after transfection of cultured RACs, NXL-P133, NXL-P137 and NXL-P131 constructs effectively drove the expression of Dlx2 as evidenced by positive Dlx2 staining in these cells (Figure 63). After transduction of cultured RACs with AAV9-P133 (AAV generated with NLX-P133), the virus effectively drove the expression of Dlx2 as evidenced by positive Dlx2 staining in these cells (Figure 64).

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

1. An adeno-associated virus (AAV) vector comprising: a human neurogenic differentiation factor 1 (hNeuroD1) sequence, the hNeuroD1 sequence comprising the nucleic acid sequence of SEQ ID NO: 6, and a human distal homeobox-free 2 (hD1x2) sequence, the hD1x2 sequence comprising the nucleic acid sequence of SEQ ID NO: 13, wherein the hNeuroD1 sequence and the hD1x2 sequence are separated by: (i) a P2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 15 and 18, (ii) a T2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16 and 19, or (iii) an internal ribosome entry site (IRES) sequence of encephalomyocarditis virus comprising SEQ ID NO: 3, wherein the hNeuroD1 sequence and the hD1x2 sequence are operably linked to a regulatory element, the 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, 12 and 26;

(b) an enhancer from the human elongation factor-1α (EF1-α) 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: 11;

(c) a chimeric intron comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 5 and 27;

(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 29; and

(e) an SV40 polyadenylation signal sequence comprising the nucleic acid sequence of SEQ ID NO:8, an hGH polyadenylation sequence comprising the nucleic acid sequence of SEQ ID NO:17, or a bGH polyadenylation sequence comprising the nucleic acid sequence of SEQ ID NO:30.

2. An adeno-associated virus (AAV) vector comprising: a nucleic acid sequence encoding a human neurogenic differentiation factor 1 (hNeuroD1) protein, the hNeuroD1 protein comprising the amino acid coding sequence of SEQ ID NO: 10, and a nucleic acid coding sequence encoding a human distal homeobox-free 2 (hD1x2) protein, the hD1x2 protein comprising the amino acid sequence of SEQ ID NO: 14, wherein the hNeuroD1 coding sequence and the hD1x2 coding sequence are separated by: (i) a P2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 15 and 18, (ii) a T2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16 and 19, or (iii) an internal ribosome entry site (IRES) sequence of encephalomyocarditis virus comprising SEQ ID NO: 3, wherein the hNeuroD1 coding sequence and the hD1x2 coding sequence are operably linked to a regulatory region.

The control element comprises:

(a) a glial fibrillary acidic protein (GFAP) promoter comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4, 12 and 26;

(b) an enhancer from the human elongation factor-1α (EF1-α) 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: 11;

(c) a chimeric intron comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 5 and 27;

(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 29; and

(e) an SV40 polyadenylation signal sequence comprising the nucleic acid sequence of SEQ ID NO:8, an hGH polyadenylation sequence comprising the nucleic acid sequence of SEQ ID NO:17, or a bGH polyadenylation sequence comprising the nucleic acid sequence of SEQ ID NO:30.

3. An adeno-associated virus (AAV) vector comprising a NeuroD1 nucleic acid coding sequence encoding a neurogenic differentiation factor 1 (NeuroD1) protein and a Dlx2 nucleic acid coding sequence encoding a distal homeobox-free 2 (Dlx2) protein, wherein the NeuroD1 coding sequence and the Dlx2 coding sequence are separated by a linker sequence, wherein the NeuroD1 coding sequence and the Dlx2 coding sequence are operably linked to a regulatory element, wherein the regulatory element comprises:

(a) Glial fibrillary acidic protein (GFAP) promoter;

(b) enhancers;

(c) chimeric introns;

(d) woodchuck hepatitis virus posttranscriptional regulatory element (WPRE); and

(e) Polyadenylation signal sequence.

4. A composition comprising an adeno-associated virus (AAV) vector for converting human glial cells into functional neurons, wherein the AAV vector comprises: a human neurogenic differentiation factor 1 (hNeuroD1) sequence, the hNeuroD1 sequence having a nucleic acid sequence of SEQ ID NO: 6, and a human distal homeobox-free 2 (hD1x2) sequence, the hD1x2 sequence having a nucleic acid sequence of SEQ ID NO: 13, wherein the hNeuroD1 sequence and the hD1x2 sequence are separated by: (i) a P2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 15 and 18, (ii) a T2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16 and 19, or (iii) an internal ribosome entry site (IRES) sequence of encephalomyocarditis virus of SEQ ID NO: 3, wherein the hNeuroD1 sequence and the hD1x2 sequence can be

is operably linked to a regulatory element, the 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, 12 and 26;

(b) an enhancer from the human elongation factor-1α (EF-1α) 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: 11;

(c) a chimeric intron comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 5 and 27;

(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 29; and

(e) an SV40 polyadenylation signal sequence comprising the nucleic acid sequence of SEQ ID NO:8, an hGH polyadenylation sequence comprising the nucleic acid sequence of SEQ ID NO:17, or a bGH polyadenylation sequence comprising the nucleic acid sequence of SEQ ID NO:30.

5. A composition comprising an adeno-associated virus (AAV) vector for converting human glial cells into functional neurons, wherein the AAV vector comprises: a nucleic acid coding sequence encoding a human neurogenic differentiation factor 1 (hNeuroD1) protein, the hNeuroD1 protein comprising the amino acid sequence of SEQ ID NO: 10, and a nucleic acid coding sequence encoding a human distal homeobox-free 2 (hD1x2) protein, the hD1x2 protein comprising the amino acid sequence of SEQ ID NO: 14, wherein the hNeuroD1 coding sequence and the hD1x2 coding sequence are separated by: (i) a P2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 15 and 18, (ii) a T2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16 and 19, or (iii) an internal ribosome entry site (IRES) sequence of encephalomyocarditis virus comprising SEQ ID NO: 3, wherein the hNeuroD1 coding sequence and the hD1x2 coding sequence are operably linked to a regulatory element, the 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, 12 and 26;

(b) an enhancer from the human elongation factor-1α (EF-1α) 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: 11;

(c) a chimeric intron comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 5 and 27;

(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 29; and

(e) an SV40 polyadenylation signal sequence comprising the nucleic acid sequence of SEQ ID NO:8, an hGH polyadenylation sequence comprising the nucleic acid sequence of SEQ ID NO:17, or a bGH polyadenylation sequence comprising the nucleic acid sequence of SEQ ID NO:30.

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 (NeuroD1) sequence and a distal homeobox-free 2 (Dlx2) sequence, wherein the NeuroD1 sequence and the Dlx2 sequence are separated by a linker sequence, wherein the NeuroD1 sequence and the Dlx2 sequence are operably linked to an expression control element, the expression control element comprising:

(a) Glial fibrillary acidic protein (GFAP) promoter;

(b) enhancers;

(c) chimeric introns;

(d) woodchuck hepatitis virus posttranscriptional regulatory element (WPRE); and

(e) Polyadenylation signal.

7. The AAV vector of any one of embodiments 1 to 3 or the composition of any one of embodiments 4 to 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 Example 7, wherein the AAV vector is AAV serotype 2.

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

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

11. The composition of embodiment 4 or 5, wherein the glial cells are reactive astrocytes.

12. The composition of embodiment 4 or 5, wherein the functional neurons are selected from the group consisting of glutamatergic neurons, GABAergic neurons, dopaminergic neurons, cholinergic neurons, serotonergic neurons, adrenergic neurons, motor neurons, and peptidergic neurons.

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

14. The AAV vector of embodiment 3 or the composition of embodiment 6, wherein the NeuroD1 is human NeuroD1 (hNeuroD1).

15. The AAV vector of embodiment 3 or the composition of embodiment 6, wherein the Dlx2 is human Dlx2 (hDlx2).

16. The AAV vector of embodiment 3 or the composition of embodiment 6, wherein the NeuroD1 is selected from the group consisting of chimpanzee NeuroD1, bonobo NeuroD1, orangutan NeuroD1, gorilla NeuroD1, macaque NeuroD1, marmoset NeuroD1, capuchin NeuroD1, baboon NeuroD1, gibbon NeuroD1 and lemur NeuroD1.

17. The AAV vector of embodiment 3 or the composition of embodiment 6, wherein the Dlx2 is selected from the group consisting of chimpanzee Dlx2, bonobo Dlx2, orangutan Dlx2, gorilla Dlx2, macaque Dlx2, marmoset Dlx2, capuchin Dlx2, baboon Dlx2, gibbon Dlx2 and lemur Dlx2.

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

19. The AAV vector or composition of embodiment 15, wherein the hDlx2 comprises a nucleic acid sequence encoding an amino acid sequence that is at least 80% identical or similar to SEQ ID NO:14.

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

21. The AAV vector or composition of embodiment 15, wherein the hDlx2 coding sequence comprises a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 13, or a complementary sequence thereof.

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

23. An AAV vector or composition according to embodiment 22, wherein the linker is the P2A.

24. The AAV vector or composition of embodiment 22, wherein the linker is the T2A.

25. The AAV vector or composition of embodiment 22, 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: 15 and 18, or a complementary sequence thereof.

26. The AAV vector or composition of embodiment 22, 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: 16 and 19, or a complementary sequence thereof.

27. The AAV vector of Example 3 or the composition of Example 6, wherein the GFAP promoter is the human GFAP (hGFAP) promoter.

28. The AAV vector of embodiment 3 or the composition of embodiment 6, wherein the GFAP promoter is selected from the group consisting of a chimpanzee GFAP promoter, a bonobo GFAP promoter, an orangutan GFAP promoter, a gorilla GFAP promoter, a macaque GFAP promoter, a marmoset GFAP promoter, a capuchin GFAP promoter, a baboon GFAP promoter, a gibbon GFAP promoter, and a lemur GFAP promoter.

29. The AAV vector or composition of any preceding embodiment, wherein the IRES sequence comprises a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 3, or a complementary sequence thereof.

30. The AAV vector or composition of embodiment 27, wherein the hGFAP promoter comprises a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 4, or a complementary sequence thereof.

31. The AAV vector or composition of embodiment 27, wherein the hGFAP promoter comprises a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 12, or a complementary sequence thereof.

32. The AAV vector or composition of embodiment 27, wherein the hGFAP promoter comprises a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 26, or a complementary sequence thereof.

33. The AAV vector according to embodiment 3 or the composition according to embodiment 6, wherein the enhancer is selected from the group consisting of an enhancer from the human elongation factor-1α (EF1-α) promoter and a cytomegalovirus (CMV) enhancer.

34. The AAV vector or composition of embodiment 33, wherein the EF1-α comprises a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 2, or a complementary sequence thereof.

35. The AAV vector or composition of embodiment 33, wherein the CMV enhancer comprises a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 11, or a complementary sequence thereof.

36. The AAV vector of embodiment 3 or the composition of embodiment 6, wherein the chimeric intron comprises a nucleic acid sequence that is at least 80% identical to a nucleic acid selected from the group consisting of SEQ ID NOs: 5 and 27, or a complementary sequence thereof.

37. The AAV vector of embodiment 3 or the composition of embodiment 6, wherein the WPRE comprises a nucleic acid sequence that is at least 80% identical to a nucleic acid selected from the group consisting of SEQ ID NOs: 7 and 29, or a complementary sequence thereof.

38. The AAV vector of embodiment 3 or the composition of embodiment 6, wherein the polyadenylation signal is selected from the group consisting of an SV40 polyadenylation signal, an hGH polyadenylation signal, and a bGH polyadenylation signal.

39. The AAV vector or composition of embodiment 38, wherein the SV40 polyadenylation signal comprises a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 8, or a complementary sequence thereof.

40. The AAV vector or composition of embodiment 38, wherein the hGH polyadenylation signal comprises a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 17, or a complementary sequence thereof.

41. The AAV vector or composition of embodiment 38, wherein the bGH polyadenylation signal comprises a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 30, or a complementary sequence thereof.

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

43. The AAV vector of any one of embodiments 1 to 3 or the composition of any one of embodiments 4 to 6, wherein the AAV vector comprises an AAV serotype 2 inverted terminal repeat (ITR).

44. The AAV vector of any one of embodiments 1 to 3 or the composition of any one of embodiments 4 to 6, wherein the AAV vector comprises an AAV serotype 5 inverted terminal repeat (ITR).

45. The AAV vector of any one of embodiments 1 to 3 or the composition of any one of embodiments 4 to 6, wherein the AAV vector comprises an AAV serotype 9 inverted terminal repeat (ITR).

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

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

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

49. The composition of embodiment 48, wherein the mammal is a human.

50. The composition of embodiment 48, wherein the mammal is a non-human primate.

51. The composition of embodiment 6, wherein the subject in need thereof suffers from a neurological disorder.

52. The composition of embodiment 13 or 51, wherein the neurological disorder comprises damage to the central nervous system (CNS) or peripheral nervous system.

53. The composition of embodiment 13 or 51, wherein the neurological disorder comprises damage to the CNS.

54. The composition of embodiment 13 or 51, wherein the nervous system 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 aneurysm, traumatic brain injury, concussion, tumor, 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, fibrocartilaginous embolic myelopathy, thrombosis, kidney disease, chronic inflammatory disease, meningitis, and cerebral venous sinus thrombosis.

55. The composition of embodiment 13 or 51, wherein the neurological disorder is Alzheimer's disease.

56. The composition of embodiment 13 or 51, wherein the neurological disorder is Parkinson's disease.

57. The composition of embodiment 13 or 51, wherein the neurological disorder is ALS.

58. The composition of embodiment 13 or 51, wherein the neurological disorder is Huntington's disease.

59. The composition of embodiment 13 or 51, wherein the neurological disorder is stroke.

60. The composition of embodiment 59, wherein the stroke is ischemic stroke.

61. The composition of embodiment 59, wherein the stroke is a hemorrhagic stroke.

62. The composition of embodiment 51, wherein the composition is capable of converting at least one glial cell into a neuron.

63. The composition of embodiment 62, wherein the glial cells are selected from the group consisting of astrocytes and NG2 cells.

64. The composition of embodiment 62, wherein the glial cells are astrocytes.

65. The composition of embodiment 62, wherein the astrocytes are reactive astrocytes.

66. A composition according to embodiment 62, wherein the glial cells are GFAP positive.

67. The composition of embodiment 62, wherein the neurons are functional neurons.

68. A composition according to embodiment 62, wherein the functional neurons are selected from the group consisting of glutamatergic neurons, GABAergic neurons, dopaminergic neurons, cholinergic neurons, serotonergic neurons, adrenergic neurons, motor neurons and peptidergic neurons.

69. The composition of embodiment 68, wherein the functional neurons are glutamatergic neurons.

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

71. The composition of embodiment 70, wherein the composition is formulated for topical delivery.

72. The composition of embodiment 70, wherein the composition is formulated for systemic delivery.

73. The composition of any one of embodiments 70 to 72, wherein the composition is formulated for delivery via intraperitoneal, intramuscular, intravenous, intrathecal, intracerebral, intracranial, intracerebroventricular, intracisternal, intravitreal, subretinal, intraparenchymal, intranasal, or oral administration.

74. A method comprising delivering the composition of embodiment 6 to a subject in need thereof.

75. The method of embodiment 74, wherein the composition is formulated for delivery to a subject in need thereof.

76. The method of embodiment 74, wherein said delivering comprises topical administration.

77. The method of embodiment 74, wherein said delivering comprises systemic administration.

78. The method of any one of embodiments 74 to 77, wherein the delivery comprises intraperitoneal, intramuscular, intravenous, intrathecal, intracerebral, intracranial, intracerebroventricular, intracisternal, intravitreal, subretinal, intraparenchymal, intranasal, or oral administration.

79. A method for 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, the DNA vector construct comprising: a human neurogenic differentiation factor 1 (hNeuroD1) sequence, the hNeuroD1 sequence comprising a nucleic acid sequence of SEQ ID NO: 6, and a human distal homeobox-free 2 (hD1x2) sequence, the hD1x2 sequence comprising a nucleic acid sequence of SEQ ID NO: 13, wherein the hNeuroD1 sequence and the hD1x2 sequence are separated by: (i) a P2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 15 and 18, (ii) a T2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16 and 19, or (iii) a linker comprising a nucleic acid sequence of SEQ ID NOs: 1 NO:3 the internal ribosome entry site (IRES) sequence of the encephalomyocarditis virus, wherein the hNeuroD1 sequence and the hD1x2 sequence are operably linked to a regulatory element, the 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, 12 and 26;

(b) an enhancer from the human elongation factor-1α (EF-1α) 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: 11;

(c) a chimeric intron comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 5 and 27;

(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 29; and

(e) an SV40 polyadenylation signal sequence comprising the nucleic acid sequence of SEQ ID NO:8, an hGH polyadenylation sequence comprising the nucleic acid sequence of SEQ ID NO:17, or a bGH polyadenylation sequence comprising the nucleic acid sequence of SEQ ID NO:30.

80. A method for 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, the DNA vector construct comprising: a nucleic acid coding sequence encoding a human neurogenic differentiation factor 1 (hNeuroD1) protein, the hNeuroD1 protein comprising the amino acid sequence of SEQ ID NO: 10, and a nucleic acid coding sequence encoding a human distal homeobox-free 2 (hD1x2) protein, the hD1x2 protein comprising the amino acid sequence of SEQ ID NO: 14, wherein the hNeuroD1 coding sequence and the hD1x2 coding sequence are separated by: (i) a P2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 15 and 18, (ii) a T2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16 and 19, or (iii) a nucleic acid linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 17 and 18. NO:3 the internal ribosome entry site (IRES) sequence of the encephalomyocarditis virus, wherein the hNeuroD1 coding sequence and the hD1x2 coding sequence are operably linked to an expression control element, the 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, 12 and 26;

(b) an enhancer from the human elongation factor-1α (EF-1α) 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: 11;

(c) a chimeric intron comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 5 and 27;

(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 29; and

(e) an SV40 polyadenylation signal sequence comprising the nucleic acid sequence of SEQ ID NO:8, an hGH polyadenylation sequence comprising the nucleic acid sequence of SEQ ID NO:17, or a bGH polyadenylation sequence comprising the nucleic acid sequence of SEQ ID NO:30.

81. A method for converting glial cells of a subject in need thereof into neurons, comprising: delivering an adeno-associated virus (AAV) to the subject in need thereof, wherein the AAV comprises a DNA vector construct, the DNA vector construct comprising a neurogenic differentiation factor 1 (NeuroD1) sequence and a distal homeobox-free 2 (Dlx2) sequence, wherein the NeuroD1 sequence and the Dlx2 sequence are separated by a linker sequence, wherein the NeuroD1 sequence and the Dlx2 sequence are operably linked to an expression control element, the expression control element comprising:

(a) Glial fibrillary acidic protein (GFAP) promoter;

(b) enhancers;

(c) chimeric introns;

(d) woodchuck hepatitis virus posttranscriptional regulatory element (WPRE); and

(e) and a polyadenylation signal sequence,

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

82. 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 (NeuroD1) sequence and a distal homeobox-free 2 (Dlx2) sequence, wherein the NeuroD1 sequence and the Dlx2 sequence are separated by a linker sequence, wherein the NeuroD1 sequence and the Dlx2 sequence are operably linked to an expression control element, the expression control element comprising:

(a) Glial fibrillary acidic protein (GFAP) promoter;

(b) enhancers;

(c) chimeric introns;

(d) woodchuck hepatitis virus posttranscriptional regulatory element (WPRE); and

(e) Polyadenylation signal.

83. The method of any one of embodiments 79 to 82, wherein the AAV is selected from the group consisting of AAV serotype 2, AAV serotype 5, and AAV serotype 9.

84. The method of embodiment 83, wherein the AAV is AAV serotype 2.

85. The method of embodiment 83, wherein the AAV is AAV serotype 5.

86. The method of embodiment 83, wherein the AAV is AAV serotype 9.

87. A method according to embodiment 79 or 80, wherein the functional neurons are glutamatergic neurons, GABAergic neurons, dopaminergic neurons, cholinergic neurons, serotonergic neurons, adrenergic neurons, motor neurons and peptidergic neurons.

88. The method of embodiment 81 or 82, wherein the NeuroD1 is human NeuroD1 (hNeuroD1).

89. The method of embodiment 81 or 82, wherein the Dlx2 is human Dlx2 (hDlx2).

90. The method according to embodiment 81 or 82, wherein the NeuroD1 is selected from the group consisting of chimpanzee NeuroD1, bonobo NeuroD1, orangutan NeuroD1, gorilla NeuroD1, macaque NeuroD1, marmoset NeuroD1, capuchin NeuroD1, baboon NeuroD1, gibbon NeuroD1 and lemur NeuroD1.

91. The method of embodiment 75 or 76, wherein the Dlx2 is selected from the group consisting of chimpanzee Dlx2, bonobo Dlx2, orangutan Dlx2, gorilla Dlx2, macaque Dlx2, marmoset Dlx2, capuchin Dlx2, baboon Dlx2, gibbon Dlx2 and lemur Dlx2.

92. The method of embodiment 88, wherein the hNeuroD1 comprises an amino acid sequence encoding an amino acid coding sequence that is at least 80% identical or similar to SEQ ID NO:10.

93. The method of embodiment 89, wherein said hDlx2 comprises an amino acid sequence encoding an amino acid sequence that is at least 80% identical or similar to SEQ ID NO:14.

94. The method of embodiment 88, wherein the hNeuroD1 coding sequence comprises a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 6, or a complementary sequence thereof.

95. The method of embodiment 89, wherein the hDlx2 encoding sequence comprises a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 13, or a complementary sequence thereof.

96. A method according to embodiment 81 or 82, wherein the GFAP promoter is human GFAP (hGFAP) promoter.

97. A method according to embodiment 81 or 82, wherein the GFAP promoter is selected from the group consisting of chimpanzee GFAP promoter, bonobo GFAP promoter, orangutan GFAP promoter, gorilla GFAP promoter, macaque GFAP promoter, marmoset GFAP promoter, capuchin GFAP promoter, baboon GFAP promoter, gibbon GFAP promoter and lemur GFAP promoter.

98. The method of any one of embodiments 79 to 97, wherein the IRES sequence comprises a nucleic acid sequence at least 80% identical to SEQ ID NO: 3, or a complementary sequence thereof.

99. The method of embodiment 96, wherein the hGFAP promoter comprises a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 4, or a complementary sequence thereof.

100. The method of embodiment 96, wherein the hGFAP promoter comprises a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 12, or a complementary sequence thereof.

101. The method of embodiment 96, wherein the hGFAP promoter comprises a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 26, or a complementary sequence thereof.

102. The method of embodiment 81 or 82, wherein the linker is selected from the group consisting of P2A and T2A.

103. The method of embodiment 102, wherein the P2A linker comprises a nucleic acid sequence at least 80% identical to a sequence selected from the group consisting of SEQ ID NOs: 15 and 18, or a complementary sequence thereof.

104. The method of embodiment 102, wherein the T2A linker comprises a nucleic acid sequence at least 80% identical to a sequence selected from the group consisting of SEQ ID NOs: 16 and 19, or a complementary sequence thereof.

105. The method of embodiment 81 or 82, wherein the enhancer is selected from the group consisting of an enhancer from the human elongation factor-1 alpha (EF1-α) promoter and a cytomegalovirus (CMV) enhancer.

106. The method of embodiment 105, wherein said EF1-α comprises a nucleic acid sequence at least 80% identical to SEQ ID NO: 2, or a complementary sequence thereof.

107. The method of embodiment 105, wherein the CMV enhancer comprises a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 11, or a complementary sequence thereof.

108. The method of embodiment 81 or 82, wherein the chimeric intron comprises a nucleic acid sequence that is at least 80% identical to a nucleic acid selected from the group consisting of SEQ ID NO: 5 and 27, or a complementary sequence thereof.

109. The method of embodiment 81 or 82, wherein the WPRE comprises a nucleic acid sequence at least 80% identical to a nucleic acid selected from the group consisting of SEQ ID NO: 7 and 29, or a complementary sequence thereof.

110. The method of embodiment 81 or 82, wherein the polyadenylation signal is selected from the group consisting of an SV40 polyadenylation signal and an hGH polyadenylation signal.

111. The method of embodiment 110, wherein the SV40 polyadenylation signal comprises a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 8, or a complementary sequence thereof.

112. The method of embodiment 110, wherein the hGH polyadenylation signal comprises a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 13, or a complementary sequence thereof.

113. A method according to embodiment 81 or 82, wherein the vector further comprises a nucleic acid sequence encoding an AAV protein sequence.

114. The method of any one of embodiments 79 to 82, wherein the vector comprises AAV serotype 2 inverted terminal repeats (ITRs).

115. The method of any one of embodiments 79 to 82, wherein the vector comprises an AAV serotype 5 inverted terminal repeat (ITR).

116. The method of any one of embodiments 79 to 82, wherein the vector comprises an AAV serotype 9 inverted terminal repeat (ITR).

117. The method of any one of embodiments 79 to 82, wherein said vector comprises at least one ITR nucleic acid sequence that is at least 80% identical to SEQ ID NO:1.

118. The method of any one of embodiments 79 to 82, wherein said vector comprises at least one ITR nucleic acid sequence that is at least 80% identical to SEQ ID NO:9.

119. The method of embodiment 81, wherein the conversion occurs in the central nervous system (CNS) or the peripheral nervous system.

120. The method of embodiment 81, wherein said conversion occurs in the CNS.

121. The method of embodiment 81 or 82, wherein the subject in need thereof is a mammal.

122. The method of embodiment 121, wherein the mammal is a human.

123. The method of embodiment 121, wherein the mammal is a non-human primate.

124. The method of embodiment 81 or 82, wherein said delivering comprises topical administration.

125. The method of embodiment 81 or 82, wherein the delivering comprises systemic administration.

126. The method of embodiment 81 or 82, wherein the delivery comprises administration selected from the group consisting of intraperitoneal administration, intramuscular administration, intravenous administration, intrathecal administration, intracerebral administration, intracranial administration, intracerebroventricular administration, intracerebellar cisterna magna, intravitreal administration, subretinal administration, intraparenchymal administration, intranasal administration, and oral administration.

127. The method of embodiment 79 or 80, wherein the injection comprises an injection selected from the group consisting of intraperitoneal injection, intramuscular injection, intravenous injection, intrathecal injection, intracerebral injection, intracranial injection, intracerebroventricular injection, intracerebellar cisterna magna, intravitreal injection, subretinal injection, intraparenchymal injection, intranasal injection, and oral injection.

128. The method of embodiment 81 or 82, wherein the delivering comprises injection.

129. The method of any one of embodiments 79, 80 or 128, wherein the injection is performed at a concentration between 10 ¹⁰ particles/mL and 10 ¹⁴ particles/mL.

130. The method of embodiment 129, wherein the injection further comprises a flow rate between 0.1 uL/min and 5.0 uL/min.

131. The method of embodiment 81, wherein the at least one glial cell is selected from the group consisting of at least one astrocyte and at least one NG2 cell.

132. A method according to embodiment 131, wherein the at least one glial cell is at least one astrocyte.

133. A method according to embodiment 131 or 132, wherein at least one astrocyte is a reactive astrocyte.

134. The method of embodiment 81, wherein the neurons are functional neurons.

135. A method according to any one of embodiments 79, 80 and 134, wherein the functional neurons are selected from the group consisting of glutamatergic neurons, GABAergic neurons, dopaminergic neurons, cholinergic neurons, serotonergic neurons, adrenergic neurons, motor neurons and peptidergic neurons.

136. The method of embodiment 81, wherein said subject exhibits an improvement in at least one symptom of a neurological disorder compared to said subject prior to said delivering.

137. The method of embodiment 136, wherein said improvement is measured within 1 year of said delivery.

138. The method of any one of embodiments 79, 80, or 128, wherein the method comprises injecting the AAV directly into the brain of the subject.

139. The method of any one of embodiments 79 or 80, wherein said transformation is in the cerebral cortex of said brain.

140. The method of any one of embodiments 79, 80, or 128, wherein the method comprises injecting the AAV directly into the spinal cord of the subject.

141. The method of embodiment 82, wherein the neurological disorder comprises damage to the central nervous system (CNS) or the peripheral nervous system.

142. The method of embodiment 82, 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 aneurysm, traumatic brain injury, concussion, tumor, 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, fibrocartilaginous embolic myelopathy, thrombosis, kidney disease, chronic inflammatory disease, meningitis, and cerebral venous sinus thrombosis.

143. The method of embodiment 82, wherein the neurological disorder is Alzheimer's disease.

144. The method of embodiment 82, wherein the neurological disorder is Parkinson's disease.

145. The method of embodiment 82, wherein the neurological disorder is ALS.

146. The method of embodiment 82, wherein the neurological disorder is Huntington's disease.

147. The method of embodiment 82, wherein the neurological disorder is stroke.

148. The method of embodiment 147, wherein the stroke is an ischemic stroke.

149. The method of embodiment 147, wherein the stroke is a hemorrhagic stroke.

150. The method of embodiment 82, wherein the method is capable of converting at least one glial cell into a neuron.

151. The method of embodiment 150, wherein the glial cells are selected from the group consisting of astrocytes and NG2 cells.

152. The method of embodiment 150, wherein the glial cells are astrocytes.

153. A method according to embodiment 152, wherein the astrocytes are reactive astrocytes.

154. A method according to embodiment 150, wherein the glial cells are GFAP positive.

155. The method of embodiment 150, wherein the neurons are functional neurons.

156. A method according to embodiment 145, wherein the functional neurons are selected from the group consisting of glutamatergic neurons, GABAergic neurons, dopaminergic neurons, cholinergic neurons, serotonergic neurons, adrenergic neurons, motor neurons and peptidergic neurons.

157. The method of embodiment 79 or 80, wherein a therapeutically effective dose of the AAV is injected into the subject.

158. The method of embodiment 81 or 82, wherein a therapeutically effective dose of the AAV is delivered to the subject.

159. The method of embodiment 147 or 148, wherein the therapeutically effective dose is administered with a pharmaceutically acceptable carrier.

160. A composition comprising: (i) an adeno-associated virus (AAV) vector comprising a human neurogenic differentiation factor 1 (hNeuroD1) sequence, the hNeuroD1 sequence comprising the nucleic acid sequence of SEQ ID NO: 6, and (ii) an adeno-associated virus (AAV) vector comprising a human distal homeobox-free 2 (hD1x2) sequence, the hD1x2 sequence comprising the nucleic acid sequence of SEQ ID NO: 13;

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

(a) a glial fibrillary acidic protein (GFAP) promoter comprising the nucleic acid sequence of SEQ ID NO: 26;

(b) an enhancer from human elongation factor-1α (EF1-α) promoter comprising the nucleic acid sequence of SEQ ID NO: 2;

(c) a chimeric intron comprising the nucleic acid sequence of SEQ ID NO: 5 or SEQ ID NO: 27;

(d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) comprising the nucleic acid sequence of SEQ ID NO: 29; and

(e) a bGH polyadenylation sequence comprising the nucleic acid sequence of SEQ ID NO:30.

161. A composition comprising: (i) an adeno-associated virus (AAV) vector comprising a human neurogenic differentiation factor 1 (hNeuroD1) sequence, the hNeuroD1 sequence comprising the nucleic acid sequence of SEQ ID NO: 6, and (ii) an adeno-associated virus (AAV) vector comprising a human distal homeobox-free 2 (hD1x2) sequence, the hD1x2 sequence comprising the nucleic acid sequence of SEQ ID NO: 13;

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

(a) a glial fibrillary acidic protein (GFAP) promoter comprising the nucleic acid sequence of SEQ ID NO: 26;

(b) a cytomegalovirus (CMV) enhancer comprising the nucleic acid sequence of SEQ ID NO: 11;

(c) a chimeric intron comprising the nucleic acid sequence of SEQ ID NO: 5 or SEQ ID NO: 27;

(d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) comprising the nucleic acid sequence of SEQ ID NO: 29; and

(e) a bGH polyadenylation sequence comprising the nucleic acid sequence of SEQ ID NO:30.

162. The composition of embodiment 160 or 161, wherein (ii) comprises an AAV vector comprising an hD1x2 sequence operably linked to a regulatory element, the hD1x2 sequence comprising the nucleic acid sequence of 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: 26;

(b) an enhancer from the human elongation factor-1α (EF1-α) 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: 11;

(c) a chimeric intron comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 5 and 27;

(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 29; and

(e) an SV40 polyadenylation signal sequence comprising the nucleic acid sequence of SEQ ID NO:8, an hGH polyadenylation sequence comprising the nucleic acid sequence of SEQ ID NO:17, or a bGH polyadenylation sequence comprising the nucleic acid sequence of SEQ ID NO:30.

163. A composition comprising: (i) an adeno-associated virus (AAV) vector comprising a nucleic acid sequence encoding a human neurogenic differentiation factor 1 (hNeuroD1) protein, the hNeuroD1 protein comprising the amino acid coding sequence of SEQ ID NO: 10, and (ii) an adeno-associated virus (AAV) vector comprising a nucleic acid coding sequence encoding a human distal homeobox-free 2 (hD1x2) protein, the hD1x2 protein comprising the amino acid sequence of SEQ ID NO: 14;

The nucleic acid sequence encoding the hNeuroD1 protein is operably linked to a regulatory element, and the regulatory element comprises:

(a) a glial fibrillary acidic protein (GFAP) promoter comprising the nucleic acid sequence of SEQ ID NO: 26;

(b) an enhancer from human elongation factor-1α (EF1-α) promoter comprising the nucleic acid sequence of SEQ ID NO: 2;

(c) a chimeric intron comprising the nucleic acid sequence of SEQ ID NO: 5 or SEQ ID NO: 27;

(d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) comprising the nucleic acid sequence of SEQ ID NO: 29; and

(e) a bGH polyadenylation sequence comprising the nucleic acid sequence of SEQ ID NO:30.

164. A composition comprising: (i) an adeno-associated virus (AAV) vector comprising a nucleic acid sequence encoding a human neurogenic differentiation factor 1 (hNeuroD1) protein, the hNeuroD1 protein comprising the amino acid coding sequence of SEQ ID NO: 10, and (ii) an adeno-associated virus (AAV) vector comprising a nucleic acid coding sequence encoding a human distal homeobox-free 2 (hDlx2) protein, the hDlx2 protein comprising the amino acid sequence of SEQ ID NO: 14;

The nucleic acid sequence encoding the hNeuroD1 protein is operably linked to a regulatory element, and the regulatory element comprises:

(a) a glial fibrillary acidic protein (GFAP) promoter comprising the nucleic acid sequence of SEQ ID NO: 26;

(b) a cytomegalovirus (CMV) enhancer comprising the nucleic acid sequence of SEQ ID NO: 11;

(c) a chimeric intron comprising the nucleic acid sequence of SEQ ID NO: 5 or SEQ ID NO: 27;

(d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) comprising the nucleic acid sequence of SEQ ID NO: 29; and

(e) a bGH polyadenylation sequence comprising the nucleic acid sequence of SEQ ID NO: 30

165. The composition of embodiment 163 or 164, wherein (ii) comprises an AAV vector comprising a nucleic acid coding sequence encoding an hD1x2 protein, wherein the nucleic acid 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: 26;

(b) an enhancer from the human elongation factor-1α (EF1-α) 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: 11;

(c) a chimeric intron comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 5 and 27;

(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 29; and

(e) an SV40 polyadenylation signal sequence comprising the nucleic acid sequence of SEQ ID NO:8, an hGH polyadenylation sequence comprising the nucleic acid sequence of SEQ ID NO:17, or a bGH polyadenylation sequence comprising the nucleic acid sequence of SEQ ID NO:30.

166. An adeno-associated virus (AAV) vector comprising: a human neurogenic differentiation factor 1 (hNeuroD1) sequence, the hNeuroD1 sequence comprising the nucleic acid sequence of SEQ ID NO:6, and a human distal homeobox-free 2 (hD1x2) sequence, the hD1x2 sequence comprising the nucleic acid sequence of SEQ ID NO:13, wherein the hNeuroD1 sequence and the hD1x2 sequence are separated by: (i) a P2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs:15 and 18, (ii) a T2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs:16 and 19, or (iii) an internal ribosome entry site (IRES) sequence of encephalomyocarditis virus comprising SEQ ID NO:3, wherein the hNeuroD1 sequence and the hD1x2 sequence are operably linked to a regulatory element, the regulatory element comprising:

(a) a glial fibrillary acidic protein (GFAP) promoter comprising the nucleic acid sequence of SEQ ID NO: 26;

(b) an enhancer from human elongation factor-1α (EF1-α) promoter comprising the nucleic acid sequence of SEQ ID NO: 2;

(c) comprising SEQ ID NO:5 or a chimeric intron of the nucleic acid sequence of SEQ ID NO:5;

(d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) comprising the nucleic acid sequence of SEQ ID NO: 29; and

(e) a bGH polyadenylation sequence comprising the nucleic acid sequence of SEQ ID NO:30.

167. An adeno-associated virus (AAV) vector comprising: a human neurogenic differentiation factor 1 (hNeuroD1) sequence comprising the nucleic acid sequence of SEQ ID NO:6, and a human distal homeobox-free 2 (hD1x2) sequence comprising the nucleic acid sequence of SEQ ID NO:13, wherein the hNeuroD1 sequence and the hD1x2 sequence are separated by: (i) a P2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs:15 and 18, (ii) a T2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs:16 and 19, or (iii) an internal ribosome entry site (IRES) sequence of encephalomyocarditis virus comprising SEQ ID NO:3, wherein the hNeuroD1 sequence and the hD1x2 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: 26;

(b) a cytomegalovirus (CMV) enhancer comprising the nucleic acid sequence of SEQ ID NO: 11;

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

(d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) comprising the nucleic acid sequence of SEQ ID NO: 29; and

(e) a bGH polyadenylation sequence comprising the nucleic acid sequence of SEQ ID NO:30.

168. An adeno-associated virus (AAV) vector comprising: a nucleic acid sequence encoding a human neurogenic differentiation factor 1 (hNeuroD1) protein, the hNeuroD1 protein comprising the amino acid coding sequence of SEQ ID NO: 10, and a nucleic acid coding sequence encoding a human distal homeobox-free 2 (hD1x2) protein, the hD1x2 protein comprising the amino acid sequence of SEQ ID NO: 14, wherein the hNeuroD1 coding sequence and the hD1x2 coding sequence are separated by: (i) a P2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 15 and 18, (ii) a T2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16 and 19, or (iii) an internal ribosome entry site (IRES) sequence of encephalomyocarditis virus comprising SEQ ID NO: 3, wherein the hNeuroD1 coding sequence and the hD1x2 coding sequence are operably linked to a regulatory element, the regulatory element comprising:

(a) a glial fibrillary acidic protein (GFAP) promoter comprising the nucleic acid sequence of SEQ ID NO: 26;

(b) an enhancer from human elongation factor-1α (EF1-α) promoter comprising the nucleic acid sequence of SEQ ID NO: 2;

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

(d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) comprising the nucleic acid sequence of SEQ ID NO: 29; and

(e) a bGH polyadenylation sequence comprising the nucleic acid sequence of SEQ ID NO:30.

169. An adeno-associated virus (AAV) vector comprising: a nucleic acid sequence encoding a human neurogenic differentiation factor 1 (hNeuroD1) protein, the hNeuroD1 protein comprising the amino acid coding sequence of SEQ ID NO: 10, and a nucleic acid coding sequence encoding a human distal homeobox-free 2 (hD1x2) protein, the hD1x2 protein comprising the amino acid sequence of SEQ ID NO: 14, wherein the hNeuroD1 coding sequence and the hD1x2 coding sequence are separated by: (i) a P2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 15 and 18, (ii) a T2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16 and 19, or (iii) an internal ribosome entry site (IRES) sequence of encephalomyocarditis virus comprising SEQ ID NO: 3, wherein the hNeuroD1 coding sequence and the hD1x2 coding sequence are operably linked to a regulatory element, the regulatory element comprising:

(a) a glial fibrillary acidic protein (GFAP) promoter comprising the nucleic acid sequence of SEQ ID NO: 26;

(b) a cytomegalovirus (CMV) enhancer comprising the nucleic acid sequence of SEQ ID NO: 11;

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

(d) a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) comprising the nucleic acid sequence of SEQ ID NO: 29; and

(e) a bGH polyadenylation sequence comprising the nucleic acid sequence of SEQ ID NO:30.

170. An adeno-associated virus (AAV) vector comprising: a human neurogenic differentiation factor 1 (hNeuroD1) sequence comprising the nucleic acid sequence of SEQ ID NO: 6, and a human distal homeobox-free 2 (hD1x2) sequence comprising the nucleic acid sequence of SEQ ID NO: 13, wherein the hNeuroD1 sequence and the hD1x2 sequence are separated by: a P2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 15 and 18, or an internal ribosome entry site (IRES) sequence of encephalomyocarditis virus comprising SEQ ID NO: 3, wherein the hNeuroD1 sequence and the hD1x2 sequence are operably linked to a regulatory element, the regulatory element comprising:

(a) a glial fibrillary acidic protein (GFAP) promoter comprising the nucleic acid sequence of SEQ ID NO: 26;

(b) a cytomegalovirus (CMV) enhancer comprising the nucleic acid sequence of SEQ ID NO: 11;

(c) a chimeric intron comprising the nucleic acid sequence of SEQ ID NO: 5; and

(d) a bGH polyadenylation sequence comprising the nucleic acid sequence of SEQ ID NO:30.

171. An adeno-associated virus (AAV) vector comprising: a nucleic acid sequence encoding a human neurogenic differentiation factor 1 (hNeuroD1) protein, the hNeuroD1 protein comprising the amino acid coding sequence of SEQ ID NO: 10, and a nucleic acid coding sequence encoding a human distal homeobox-free 2 (hD1x2) protein, the hD1x2 protein comprising the amino acid sequence of SEQ ID NO: 14, wherein the hNeuroD1 coding sequence and the hD1x2 coding sequence are separated by: a P2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 15 and 18, or an internal ribosome entry site (IRES) sequence of encephalomyocarditis virus comprising SEQ ID NO: 3, wherein the hNeuroD1 coding sequence and the hD1x2 coding sequence are operably linked to a regulatory element, the regulatory element comprising:

(a) a glial fibrillary acidic protein (GFAP) promoter comprising the nucleic acid sequence of SEQ ID NO: 26;

(b) a cytomegalovirus (CMV) enhancer comprising the nucleic acid sequence of SEQ ID NO: 11;

(c) a chimeric intron comprising the nucleic acid sequence of SEQ ID NO: 5; and

(d) a bGH polyadenylation sequence comprising the nucleic acid sequence of SEQ ID NO:30.

<110> NEUEXCELL THERAPEUTICS INC

<120> NEUROD1 and DLX2 vectors

<130> P34838WO00

<140>

<141>

<150> 63/247,442

<151> 2021-09-23

<150> 63/084,945

<151> 2020-09-29

<160> 30

<170> PatentIn version 3.5

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atgaccaaat cgtacagcga gagtgggctg atgggcgagc ctcagcccca aggtcctcca 60

agctggacag acgagtgtct cagttctcag gacgaggagc acgaggcaga caagaaggag 120

gacgacctcg aagccatgaa cgcagaggag gactcactga ggaacggggg agaggagggag 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 agggctttatc 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> Artificial sequence description: synthetic polynucleotide

<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 ctttcgctttccccctccct 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> Artificial sequence description: synthetic polynucleotide

<400> 8

cgatccaccg gatctagata actgatcata atcagccata ccacatttgt agaggtttta 60

cttgctttaa aaaacctccc acacctcccc ctgaacctga aacataaaat gaatgcaatt 120

gttgttgtta acttgtttat tgcagctttat aatggttaca aataaagcaa tagcatcaca 180

aatttcacaa ataaagcatttttttcactg cattctagtt gtggtttgtc caaactcatc 240

aatgtatctt a 251

<210> 9

<211> 139

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic polynucleotide

<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> Homo sapiens

<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 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> 380

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic polynucleotide

<400> 11

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

tagtcatcgctattaccatg 380

<210> 12

<211> 2214

<212> DNA

<213> Homo sapiens

<400> 12

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 tcttggggata 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 ctggggagaga 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> 13

<211> 987

<212> DNA

<213> Homo sapiens

<400> 13

atgactggag tctttgacag tctagtggct gatatgcact cgacccagat cgccgcctcc 60

agcacgtacc accagcacca gcagcccccg agcggcggcg gcgccggccc gggtggcaac 120

agcagcagca gcagcagcct ccacaagccc caggagtcgc ccacccttcc ggtgtccacc 180

gccaccgaca gcagctacta caccaaccag cagcacccgg cgggcggcgg cggcggcggg 240

ggctcgccct acgcgcacat gggttcctac cagtaccaag ccagcggcct caacaacgtc 300

ccttactccg ccaagagcag ctatgacctg ggctacaccg ccgcctacac ctcctacgct 360

ccctatggaa ccagttcgtc cccagccaac aacgagcctg agaaggagga ccttgagcct 420

gaaattcgga tagtgaacgg gaagccaaag aaagtccgga aaccccgcac catctactcc 480

agtttccagc tggcggctct tcagcggcgt ttccaaaaga ctcaatactt ggccttgccg 540

gagcgagccg agctggcggc ctctctgggc ctcacccaga ctcaggtcaa aatctggttc 600

cagaaccgcc ggtccaagtt caagaagatg tggaaaagtg gtgagatccc ctcggagcag 660

caccctgggg ccagcgcttc tccaccttgt gcttcgccgc cagtctcagc gccggcctcc 720

tgggactttg gtgtgccgca gcggatggcg ggcggcggtg gtccgggcag tggcggcagc 780

ggcgccggca gctcgggctc cagcccgagc agcgcggcct cggcttttct gggcaactac 840

ccctggtacc accagacctc gggatccgcc tcacacctgc aggccacggc gccgctgctg 900

caccccactc agaccccgca gccgcatcac caccaccacc atcacggcgg cgggggcgcc 960

ccggtgagcg cggggacgattttctga 987

<210> 14

<211> 328

<212> PRT

<213> Homo sapiens

<400> 14

Met Thr Gly Val Phe Asp Ser Leu Val Ala Asp Met His Ser Thr Gln

1 5 10 15

Ile Ala Ala Ser Ser Thr Tyr His Gln His Gln Gln Pro Pro Ser Gly

20 25 30

Gly Gly Ala Gly Pro Gly Gly Asn Ser Ser Ser Ser Ser Ser Leu His

35 40 45

Lys Pro Gln Glu Ser Pro Thr Leu Pro Val Ser Thr Ala Thr Asp Ser

50 55 60

Ser Tyr Tyr Thr Asn Gln Gln His Pro Ala Gly Gly Gly Gly Gly Gly

65 70 75 80

Gly Ser Pro Tyr Ala His Met Gly Ser Tyr Gln Tyr Gln Ala Ser Gly

85 90 95

Leu Asn Asn Val Pro Tyr Ser Ala Lys Ser Ser Ser Tyr Asp Leu Gly Tyr

100 105 110

Thr Ala Ala Tyr Thr Ser Tyr Ala Pro Tyr Gly Thr Ser Ser Ser Pro

115 120 125

Ala Asn Asn Glu Pro Glu Lys Glu Asp Leu Glu Pro Glu Ile Arg Ile

130 135 140

Val Asn Gly Lys Pro Lys Lys Val Arg Lys Pro Arg Thr Ile Tyr Ser

145 150 155 160

Ser Phe Gln Leu Ala Ala Leu Gln Arg Arg Phe Gln Lys Thr Gln Tyr

165 170 175

Leu Ala Leu Pro Glu Arg Ala Glu Leu Ala Ala Ser Leu Gly Leu Thr

180 185 190

Gln Thr Gln Val Lys Ile Trp Phe Gln Asn Arg Arg Ser Lys Phe Lys

195 200 205

Lys Met Trp Lys Ser Gly Glu Ile Pro Ser Glu Gln His Pro Gly Ala

210 215 220

Ser Ala Ser Pro Pro Cys Ala Ser Pro Pro Val Ser Ala Pro Ala Ser

225 230 235 240

Trp Asp Phe Gly Val Pro Gln Arg Met Ala Gly Gly Gly Gly Pro Gly

245 250 255

Ser Gly Gly Ser Gly Ala Gly Ser Ser Ser Gly Ser Ser Pro Ser Ser Ala

260 265 270

Ala Ser Ala Phe Leu Gly Asn Tyr Pro Trp Tyr His Gln Thr Ser Gly

275 280 285

Ser Ala Ser His Leu Gln Ala Thr Ala Pro Leu Leu His Pro Thr Gln

290 295 300

Thr Pro Gln Pro His His His His His His Gly Gly Gly Gly Ala

305 310 315 320

Pro Val Ser Ala Gly Thr Ile Phe

325

<210> 15

<211> 57

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic oligonucleotide

<400> 15

gctactaact tcagcctgct gaagcaggct ggagacgtgg aggagaaccc tggacct 57

<210> 16

<211> 54

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic oligonucleotide

<400> 16

gaggggagag gaagtcttct gacctgcgga gacgtcgaag agaatcctgg accc 54

<210> 17

<211> 477

<212> DNA

<213> Homo sapiens

<400> 17

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> 18

<211> 66

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic oligonucleotide

<400> 18

ggaagcggag ctactaactt cagcctgctg aagcaggctg gagacgtgga ggagaaccct 60

ggacct 66

<210> 19

<211> 66

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic oligonucleotide

<400> 19

ggaagcggag ctactaactt cagcctgctg aagcaggctg gagacgtgga ggagaaccct 60

ggacct 66

<210> 20

<211> 22

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic peptide

<400> 20

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> 21

<211> 21

<212> PRT

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic peptide

<400> 21

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> 22

<211> 241

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic polynucleotide

<400> 22

gtcctttcca caagatatat aaacccaaga aatcgaaata ctttcaagtt acggtaagca 60

tatgatagtc cattttaaaa cataatttta aaactgcaaa ctacccaaga aattattact 120

ttctacgtca cgtattttgt actaatatct ttgtgtttac agtcaaatta attctaatta 180

tctctctaac agccttgtat cgtatatgca aatatgaagg aatcatggga aataggccct 240

c 241

<210> 23

<211> 58

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic oligonucleotide

<400> 23

ccggtggttc agttacgggt taattctcga gaattaaccc gtaactgaac catttttg 58

<210> 24

<211> 60

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic oligonucleotide

<400> 24

ccggcagtta cgggttaatt aatacctgac ccatattaat taacccgtaa ctgctttttg 60

<210> 25

<211> 57

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic oligonucleotide

<400> 25

ccggtgttgc cgcagcatca ctaatctcga gattagtgat gctgcggcaa cattttg 57

<210> 26

<211> 681

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic polynucleotide

<400> 26

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> 27

<211> 1062

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic polynucleotide

<400> 27

ggagtcgctg cgttgccttc gccccgtgcc ccgctccgcg ccgcctcgcg ccgcccgccc 60

cggctctgac tgaccgcgtt actcccacag gtgagcgggc gggacggccc ttctccctcc 120

gggctgtaat tagcgcttgg tttaatgacg gctcgtttct tttctgtggc tgcgtgaaag 180

ccttaaaggg ctccgggggg 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

tggggggggtg agcagggggt gtgggcgcgg cggtcgggct gtaaccccccc 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> 28

<211> 978

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic polynucleotide

<400> 28

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 catgagggg attggattct 420

ggggtgaaga aagtgaggga aagagcaggc aggtctgggc gcaaagcaca ggtgactgcc 480

tgccaccagc ttgtgacccc catcaagtta ctttgacttg cacagctgtg aagcggtggt 540

cataataaaa ttcatttcaa aaggtggtta cctggggatca 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> 29

<211> 581

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic polynucleotide

<400> 29

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> 30

<211> 208

<212> DNA

<213> Artificial sequence

<220>

<223> Artificial sequence description: synthetic polynucleotide

<400> 30

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

Claims (171)

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 human distantly related homeobox 2 (hDlx 2) sequence, the hDlx2 sequence comprising a nucleic acid sequence of SEQ ID NO. 13, wherein the hNeuroD1 sequence and the hDlx2 sequence are separated by: (i) a P2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs 15 and 18, (ii) a T2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs 16 and 19, or (iii) an Internal Ribosome Entry Site (IRES) sequence of an encephalomyocarditis virus comprising SEQ ID NO 3, wherein the hNeuroD1 sequence and the hDlx2 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, 12 and 26;

(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. 11;

(c) A chimeric intron comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs 5 and 27;

(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 29; and

(e) An SV40 polyadenylation signal sequence comprising the nucleic acid sequence of SEQ ID NO. 8, an hGH polyadenylation sequence comprising the nucleic acid sequence of SEQ ID NO. 17, or a bGH polyadenylation sequence comprising the nucleic acid sequence of SEQ ID NO. 30.

2. An adeno-associated virus (AAV) vector comprising: a nucleic acid sequence encoding a human neurogenic differentiation factor 1 (hNeuroD 1) protein, the hNeuroD1 protein comprising an amino acid coding sequence of SEQ ID No. 10, and a nucleic acid coding sequence encoding a human distantly related homeobox 2 (hDlx 2) protein, the hDlx2 protein comprising an amino acid sequence of SEQ ID No. 14, wherein the hNeuroD1 coding sequence and the hDlx2 coding sequence are separated by: (i) a P2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs 15 and 18, (ii) a T2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs 16 and 19, 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 hDlx2 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, 12 and 26;

(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. 11;

(c) A chimeric intron comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs 5 and 27;

(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 29; and

(e) An SV40 polyadenylation signal sequence comprising the nucleic acid sequence of SEQ ID NO. 8, an hGH polyadenylation sequence comprising the nucleic acid sequence of SEQ ID NO. 17, or a bGH polyadenylation sequence comprising the nucleic acid sequence of SEQ ID NO. 30.

3. An adeno-associated virus (AAV) vector comprising a NeuroD1 nucleic acid coding sequence encoding a NeuroD1 (NeuroD 1) protein and a Dlx nucleic acid coding sequence encoding a distantly related homeobox 2 (Dlx 2) protein, wherein the NeuroD1 coding sequence and the Dlx2 coding sequence are separated by a linker sequence, wherein the NeuroD1 coding sequence and the Dlx2 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 signal sequences.

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 a nucleic acid sequence of SEQ ID NO. 6, and a human distantly related homeobox 2 (hDlx 2) sequence, the hDlx2 sequence having a nucleic acid sequence of SEQ ID NO. 13, wherein the hNeuroD1 sequence and the hDlx2 sequence are separated by: (i) a P2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs 15 and 18, (ii) a T2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs 16 and 19, or (iii) an Internal Ribosome Entry Site (IRES) sequence of an encephalomyocarditis virus comprising SEQ ID NO 3, wherein the hNeuroD1 sequence and hDlx2 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, 12 and 26;

(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. 11;

(c) A chimeric intron comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs 5 and 27;

(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 29; and

(e) An SV40 polyadenylation signal sequence comprising the nucleic acid sequence of SEQ ID NO. 8, an hGH polyadenylation sequence comprising the nucleic acid sequence of SEQ ID NO. 17, or a bGH polyadenylation sequence comprising the nucleic acid sequence of SEQ ID NO. 30.

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 (hDlx 1) protein, said hdbosses 1 protein comprising the amino acid sequence of SEQ ID No. 10, and a nucleic acid coding sequence encoding a human distantly related homeobox 2 (hDlx 2) protein, said hDlx2 protein comprising the amino acid sequence of SEQ ID No. 14, wherein said hdbosses 1 coding sequence and said hDlx2 coding sequence are separated by: (i) a P2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs 15 and 18, (ii) a T2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs 16 and 19, 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 hDlx2 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, 12 and 26;

(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. 11;

(c) A chimeric intron comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs 5 and 27;

(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 29; and

(e) An SV40 polyadenylation signal sequence comprising the nucleic acid sequence of SEQ ID NO. 8, an hGH polyadenylation sequence comprising the nucleic acid sequence of SEQ ID NO. 17, or a bGH polyadenylation sequence comprising the nucleic acid sequence of SEQ ID NO. 30.

6. A composition comprising an adeno-associated virus (AAV) vector, wherein the AAV vector comprises a neuro-derived differentiation factor 1 (NeuroD 1) sequence and a distantly-free homeobox 2 (Dlx 2) sequence, wherein the NeuroD1 sequence and the Dlx2 sequence are separated by a linker sequence, wherein the NeuroD1 sequence and Dlx2 sequence are operably linked to an expression control 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.

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 Dlx2 is human Dlx2 (hDlx 2).

16. 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.

17. The AAV vector of claim 3 or composition of claim 6, wherein the Dlx2 is selected from the group consisting of chimpanzee Dlx2, bonobo Dlx2, chimpanzee Dlx2, gorilla Dlx2, macaque Dlx2, marmoset Dlx2, beginner Dlx2, baboon Dlx2, gibbon Dlx2, and lemma Dlx 2.

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

19. The AAV vector or composition of claim 15, wherein the hDlx2 comprises a nucleic acid sequence encoding an amino acid sequence at least 80% identical or similar to: SEQ ID NO. 14.

20. The AAV vector or composition of claim 14, 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.

21. The AAV vector or composition of claim 15, the hDlx2 coding sequence comprising a nucleic acid sequence that is at least 80% identical to: SEQ ID NO. 13, or a complement thereof.

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

23. The AAV vector or composition of claim 22, wherein the linker is the P2A.

24. The AAV vector or composition of claim 22, wherein the linker is the T2A

25. The AAV vector or composition of claim 22, 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 15 and 18, or the complements thereof.

26. The AAV vector or composition of claim 22, 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 16 and 19, or the complements thereof.

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

28. 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.

29. 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.

30. The AAV vector or composition of claim 27, wherein the hGFAP promoter comprises a nucleic acid sequence that is at least 80% identical to: SEQ ID NO. 4, or a complement thereof.

31. The AAV vector or composition of claim 27, wherein the hGFAP promoter comprises a nucleic acid sequence that is at least 80% identical to: SEQ ID NO. 12, or a complement thereof.

32. The AAV vector or composition of claim 27, wherein the hGFAP promoter comprises a nucleic acid sequence that is at least 80% identical to: SEQ ID NO. 26, or a complement thereof.

33. 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

34. The AAV vector or composition of claim 33, 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.

35. The AAV vector or composition of claim 33, wherein the CMV enhancer comprises a nucleic acid sequence at least 80% identical to: SEQ ID NO. 11, or a complement thereof.

36. 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 a nucleic acid selected from the group consisting of: SEQ ID NOS 5 and 27, or the complements thereof.

37. 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 selected from the group consisting of: SEQ ID NOS.7 and 29, or the complements thereof.

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

39. The AAV vector or composition of claim 38, 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.

40. The AAV vector or composition of claim 38, wherein the hGH polyadenylation signal comprises a nucleic acid sequence that is at least 80% identical to: SEQ ID NO. 17, or a complement thereof.

41. The AAV vector or composition of claim 38, wherein the bGH polyadenylation signal comprises a nucleic acid sequence that is at least 80% identical to: SEQ ID NO. 30, or a complement thereof.

42. 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.

43. 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).

44. 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).

45. 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).

46. 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.

47. 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.

48. The composition of claim 6, wherein the subject in need thereof is a mammal.

49. The composition of claim 48, wherein said mammal is a human.

50. The composition according to claim 48, wherein said mammal is a non-human primate.

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

52. The composition of claim 13 or 51, wherein the neurological condition comprises an injury to the Central Nervous System (CNS) or peripheral nervous system.

53. The composition of claim 13 or 51, wherein the neurological condition comprises injury to the CNS.

54. The composition of claim 13 or 51, 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.

55. The composition of claim 13 or 51, wherein the neurological disorder is alzheimer's disease.

56. The composition of claim 13 or 51, wherein the neurological disorder is parkinson's disease.

57. The composition of claim 13 or 51, wherein the neurological disorder is ALS.

58. The composition of claim 13 or 51, wherein the neurological disorder is huntington's disease.

59. The composition of claim 13 or 51, wherein the neurological disorder is stroke.

60. The composition of claim 59, wherein the stroke is ischemic stroke.

61. The composition of claim 59, wherein the stroke is hemorrhagic stroke.

62. The composition of claim 51, wherein the composition is capable of converting at least one glial cell into neurons.

63. The composition of claim 62, wherein said glial cell is selected from the group consisting of an astrocyte and a NG2 cell.

64. The composition of claim 62, wherein the glial cell is an astrocyte.

65. The composition of claim 62, wherein said astrocytes are reactive astrocytes.

66. The composition of claim 62, wherein the glial cells are GFAP positive.

67. The composition of claim 62, wherein the neuron is a functional neuron.

68. The composition of claim 62, 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.

69. The composition of claim 68, wherein the functional neuron is a glutamatergic neuron.

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

71. The composition of claim 70, wherein the composition is formulated for topical delivery.

72. The composition of claim 70, wherein the composition is formulated for systemic delivery.

73. The composition of any one of claims 70-72, 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.

74. A method comprising delivering the composition of claim 6 to the subject in need thereof.

75. The method of claim 74, wherein the composition is formulated for delivery to a subject in need thereof.

76. The method of claim 74, wherein the delivering comprises topical administration.

77. The method of claim 74, wherein the delivering comprises systemic administration.

78. The method of any one of claims 74-77, wherein said delivering comprises intraperitoneal, intramuscular, intravenous, intrathecal, intracerebral, intracranial, lateral intracerebroventricular, intravitreal, intraretinal, intraparenchymal, intranasal, or oral administration.

79. 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 human distantly related homeobox 2 (hDlx 2) sequence, the hDlx2 sequence comprising a nucleic acid sequence of SEQ ID NO. 13, wherein the hNeuroD1 sequence and the hDlx2 sequence are separated by: (i) a P2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs 15 and 18, (ii) a T2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs 16 and 19, or (iii) an Internal Ribosome Entry Site (IRES) sequence of an encephalomyocarditis virus comprising SEQ ID NO 3, wherein the hNeuroD1 sequence and the hDlx2 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, 12 and 26;

(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. 11;

(c) A chimeric intron comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs 5 and 27;

(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 29; and

(e) An SV40 polyadenylation signal sequence comprising the nucleic acid sequence of SEQ ID NO. 8, an hGH polyadenylation sequence comprising the nucleic acid sequence of SEQ ID NO. 17, or a bGH polyadenylation sequence comprising the nucleic acid sequence of SEQ ID NO. 30.

80. 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 (hDlx 1) protein, said hdbosses 1 protein comprising the amino acid sequence of SEQ ID No. 10, and a nucleic acid coding sequence encoding a human distantly related homeobox 2 (hDlx 2) protein, said hDlx2 protein comprising the amino acid sequence of SEQ ID No. 14, wherein said hdbosses 1 coding sequence and said hDlx2 coding sequence are separated by: (i) a P2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs 15 and 18, (ii) a T2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs 16 and 19, or (iii) an Internal Ribosome Entry Site (IRES) sequence of an encephalomyocarditis virus comprising SEQ ID NO 3, wherein the hNeuroD1 coding sequence and hDlx2 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, 12 and 26;

(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. 11;

(c) A chimeric intron comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs 5 and 27;

(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 29; and

(e) An SV40 polyadenylation signal sequence comprising the nucleic acid sequence of SEQ ID NO. 8, an hGH polyadenylation sequence comprising the nucleic acid sequence of SEQ ID NO. 17, or a bGH polyadenylation sequence comprising the nucleic acid sequence of SEQ ID NO. 30.

81. 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 distantly related homeobox 2 (Dlx 2) sequence, wherein the NeuroD1 sequence and Dlx2 sequence are separated by a linker sequence, wherein the NeuroD1 sequence and Dlx2 sequence are operably linked to an expression control 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) And a polyadenylation signal sequence,

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

82. 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 distantly homologous box 2 (Dlx 2) sequence, wherein the NeuroD1 sequence and Dlx2 sequence are separated by a linker sequence, wherein the NeuroD1 sequence and the Dlx2 sequence are operably linked to an expression control 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.

83. The method of any one of claims 79 to 82, wherein the AAV is selected from the group consisting of AAV serotype 2, AAV serotype 5, and AAV serotype 9.

84. The method of claim 83, wherein the AAV is AAV serotype 2.

85. The method of claim 83, wherein the AAV is AAV serotype 5.

86. The method of claim 83, wherein the AAV is AAV serotype 9.

87. The method of claim 79 or 80, wherein the functional neurons are glutamatergic neurons, gabaergic neurons, dopaminergic neurons, cholinergic neurons, serotonergic neurons, adrenergic neurons, motor neurons, and peptidoergic neurons.

88. The method of claim 81 or 82, wherein the NeuroD1 is human NeuroD1 (hNeuroD 1).

89. The method of claim 81 or 82, wherein the Dlx2 is human Dlx2 (hDlx 2).

90. The method of claim 81 or 82, 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.

91. The method of claim 75 or 76, wherein the Dlx2 is selected from the group consisting of chimpanzee Dlx2, bonobo Dlx2, chimpanzee Dlx2, gorilla Dlx2, macaque Dlx2, marmoset Dlx2, pigtail Dlx2, baboon Dlx2, gibbon Dlx2, and marmoset Dlx 2.

92. The method of claim 88, wherein the hNeuroD1 comprises an amino acid sequence encoding an amino acid coding sequence that is at least 80% identical or similar to: SEQ ID NO. 10.

93. The method of claim 89, the hDlx2 comprising an amino acid sequence encoding an amino acid sequence that is at least 80% identical or similar to: SEQ ID NO. 14.

94. The method of claim 88, 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.

95. The method of claim 89, the hDlx2 coding sequence comprising a nucleic acid sequence that is at least 80% identical to: SEQ ID NO. 13, or a complement thereof.

96. The method of claim 81 or 82, wherein the GFAP promoter is a human GFAP (hGFAP) promoter.

97. The method of claim 81 or 82, 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.

98. The method of any one of claims 79 to 97, wherein the IRES sequence comprises a nucleic acid sequence that is at least 80% identical to: SEQ ID NO. 3, or a complement thereof.

99. The method of claim 96, wherein the hGFAP promoter comprises a nucleic acid sequence at least 80% identical to: SEQ ID NO. 4, or a complement thereof.

100. The method of claim 96, wherein the hGFAP promoter comprises a nucleic acid sequence at least 80% identical to: SEQ ID NO. 12, or a complement thereof.

101. The method of claim 96, wherein the hGFAP promoter comprises a nucleic acid sequence at least 80% identical to: SEQ ID NO. 26, or a complement thereof.

102. The method of claim 81 or 82, wherein the linker is selected from the group consisting of P2A and T2A.

103. The method of claim 102, 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 15 and 18, or the complements thereof.

104. The method of claim 102, 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 16 and 19, or the complements thereof.

105. The method of claim 81 or 82, 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.

106. The method of claim 105, 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.

107. The method of claim 105, wherein the CMV enhancer comprises a nucleic acid sequence at least 80% identical to: SEQ ID NO. 11, or a complement thereof.

108. The method of claim 81 or 82, wherein the chimeric intron comprises a nucleic acid sequence at least 80% identical to a nucleic acid selected from the group consisting of: SEQ ID NOS 5 and 27, or the complements thereof.

109. The method of claim 81 or 82 wherein the WPRE comprises a nucleic acid sequence at least 80% identical to a nucleic acid selected from the group consisting of: SEQ ID NOS.7 and 29, or the complements thereof.

110. The method of claim 81 or 82, wherein the polyadenylation signal is selected from the group consisting of an SV40 polyadenylation signal and an hGH polyadenylation signal

111. The method of claim 110, 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.

112. The method of claim 110, wherein the hGH polyadenylation signal 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 81 or 82, wherein the vector further comprises a nucleic acid sequence encoding an AAV protein sequence.

114. The method of any one of claims 79 to 82, wherein the vector comprises an AAV serotype 2 Inverted Terminal Repeat (ITR).

115. The method of any one of claims 79 to 82, wherein the vector comprises an AAV serotype 5 Inverted Terminal Repeat (ITR).

116. The method of any one of claims 79 to 82, wherein the vector comprises an AAV serotype 9 Inverted Terminal Repeat (ITR).

117. The method of any one of claims 79 to 82, wherein the vector comprises at least one ITR nucleic acid sequence at least 80% identical to: SEQ ID NO. 1.

118. The method of any one of claims 79 to 82, wherein the vector comprises at least one ITR nucleic acid sequence at least 80% identical to: SEQ ID NO. 9.

119. The method of claim 81, wherein the transformation occurs in the Central Nervous System (CNS) or peripheral nervous system.

120. The method of claim 81, wherein the transformation occurs in the CNS.

121. The method of claim 81 or 82, wherein the subject in need thereof is a mammal.

122. The method of claim 121, wherein the mammal is a human.

123. The method of claim 121, wherein the mammal is a non-human primate.

124. The method of claim 81 or 82, wherein the delivering comprises topical administration.

125. The method of claim 81 or 82, wherein the delivering comprises systemic administration.

126. The method of claim 81 or 82, 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.

127. The method of claim 79 or 80, 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.

128. The method of claim 81 or 82, wherein the delivering comprises injection.

129. The method of any one of claims 79, 80, or 128, wherein the injecting is at between 10 ¹⁰ particle/mL and 10 ¹⁴ Concentration between individual particles/mL.

130. The method of claim 129, wherein the injecting further comprises a flow rate of between 0.1 μl/min and 5.0 μl/min.

131. The method of claim 81, wherein the at least one glial cell is selected from the group consisting of at least one astrocyte and at least one NG2 cell.

132. The method of claim 131, wherein the at least one glial cell is at least one astrocyte.

133. The method of claim 131 or 132, wherein the at least one astrocyte is a reactive astrocyte.

134. The method of claim 81, wherein the neuron is a functional neuron.

135. The method of any one of claims 79, 80 and 134, 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.

136. The method of claim 81, wherein the subject exhibits an improvement in at least one symptom of a neurological disorder compared to the subject prior to the delivering.

137. The method of claim 136, wherein the improvement is measured within 1 year of the delivery.

138. The method of any one of claims 79, 80, or 128, wherein the method comprises injecting the AAV directly into the brain of the subject.

139. The method of any one of claims 79 or 80, wherein the transformation is in the cerebral cortex of the brain.

140. The method of any one of claims 79, 80, or 128, wherein the method comprises injecting the AAV directly into the spinal cord of the subject.

141. The method of claim 82, wherein the neurological disorder comprises an injury to the Central Nervous System (CNS) or peripheral nervous system.

142. The method of claim 82, 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.

143. The method of claim 82, wherein the neurological disorder is alzheimer's disease.

144. The method of claim 82, wherein the neurological disorder is parkinson's disease.

145. The method of claim 82, wherein the neurological disorder is ALS.

146. The method of claim 82, wherein the neurological disorder is huntington's disease.

147. The method of claim 82, wherein the neurological disorder is stroke.

148. The method of claim 147, wherein the stroke is an ischemic stroke.

149. The method of claim 147, wherein the stroke is a hemorrhagic stroke.

150. The method of claim 82, wherein the method is capable of converting at least one glial cell into a neuron.

151. The method of claim 150, wherein the glial cell is selected from the group consisting of an astrocyte and a NG2 cell.

152. The method of claim 150, wherein the glial cell is an astrocyte.

153. The method of claim 152, wherein the astrocytes are reactive astrocytes.

154. The method of claim 150, wherein the glial cell is GFAP positive.

155. The method of claim 150, wherein the neuron is a functional neuron.

156. The method of claim 145, 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.

157. The method of claim 79 or 80, wherein a therapeutically effective dose of the AAV is injected into the subject.

158. The method of claim 81 or 82, wherein a therapeutically effective dose of the AAV is delivered to the subject.

159. The method of claim 147 or 148 wherein the therapeutically effective dose is administered with a pharmaceutically acceptable carrier.

160. A composition comprising: (i) An adeno-associated virus (AAV) vector comprising a human neurogenic differentiation factor 1 (hdld 1) sequence, the hdld 1 sequence comprising a nucleic acid sequence of SEQ ID No. 6, and (ii) an adeno-associated virus (AAV) vector comprising a human distantly related homeobox 2 (hDlx 2) sequence, the hDlx2 sequence comprising a nucleic acid sequence of SEQ ID No. 13;

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. 26;

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

(c) A chimeric intron comprising the nucleic acid sequence of SEQ ID No. 5 or SEQ ID No. 27;

(d) A woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) comprising the nucleic acid sequence of SEQ ID NO. 29; and

(e) A bGH polyadenylation sequence comprising the nucleic acid sequence of SEQ ID No. 30.

161. A composition comprising: (i) An adeno-associated virus (AAV) vector comprising a human neurogenic differentiation factor 1 (hdld 1) sequence, the hdld 1 sequence comprising a nucleic acid sequence of SEQ ID No. 6, and (ii) an adeno-associated virus (AAV) vector comprising a human distantly related homeobox 2 (hDlx 2) sequence, the hDlx2 sequence comprising a nucleic acid sequence of SEQ ID No. 13;

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. 26;

(b) A Cytomegalovirus (CMV) enhancer comprising the nucleic acid sequence of SEQ ID No. 11;

(c) A chimeric intron comprising the nucleic acid sequence of SEQ ID No. 5 or SEQ ID No. 27;

(d) A woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) comprising the nucleic acid sequence of SEQ ID NO. 29; and

(e) A bGH polyadenylation sequence comprising the nucleic acid sequence of SEQ ID No. 30.

162. The composition of claim 160 or 161, wherein (ii) comprises an AAV vector comprising an hDlx2 sequence operably linked to a regulatory element, the hDlx2 sequence comprising a nucleic acid sequence of 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. 26;

(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. 11;

(c) A chimeric intron comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs 5 and 27;

(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 29; and

(e) An SV40 polyadenylation signal sequence comprising the nucleic acid sequence of SEQ ID NO. 8, an hGH polyadenylation sequence comprising the nucleic acid sequence of SEQ ID NO. 17, or a bGH polyadenylation sequence comprising the nucleic acid sequence of SEQ ID NO. 30.

163. A composition comprising: (i) An adeno-associated virus (AAV) vector comprising a nucleic acid sequence encoding a human neurogenic differentiation factor 1 (hDlx 1) protein, said hdlex 1 protein comprising the amino acid coding sequence of SEQ ID No. 10, and (ii) an adeno-associated virus (AAV) vector comprising a nucleic acid coding sequence encoding a human distantly related homeobox 2 (hDlx 2) protein, said hDlx2 protein comprising the amino acid sequence of SEQ ID No. 14;

wherein the nucleic acid sequence encoding a hNeuroD1 protein 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. 26;

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

(c) A chimeric intron comprising the nucleic acid sequence of SEQ ID No. 5 or SEQ ID No. 27;

(d) A woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) comprising the nucleic acid sequence of SEQ ID NO. 29; and

(e) A bGH polyadenylation sequence comprising the nucleic acid sequence of SEQ ID No. 30.

164. A composition comprising: (i) An adeno-associated virus (AAV) vector comprising a nucleic acid sequence encoding a human neurogenic differentiation factor 1 (hDlx 1) protein, said hdlex 1 protein comprising the amino acid coding sequence of SEQ ID No. 10, and (ii) an adeno-associated virus (AAV) vector comprising a nucleic acid coding sequence encoding a human distantly related homeobox 2 (hDlx 2) protein, said hDlx2 protein comprising the amino acid sequence of SEQ ID No. 14;

Wherein the nucleic acid sequence encoding a hNeuroD1 protein 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. 26;

(b) A Cytomegalovirus (CMV) enhancer comprising the nucleic acid sequence of SEQ ID No. 11;

(c) A chimeric intron comprising the nucleic acid sequence of SEQ ID No. 5 or SEQ ID No. 27;

(d) A woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) comprising the nucleic acid sequence of SEQ ID NO. 29; and

(e) bGH polyadenylation sequence comprising the nucleic acid sequence of SEQ ID NO. 30

165. The composition of claim 163 or 164, wherein (ii) comprises an AAV vector comprising a nucleic acid coding sequence encoding an hDlx2 protein, wherein the nucleic acid 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. 26;

(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. 11;

(c) A chimeric intron comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs 5 and 27;

(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 29; and

(e) An SV40 polyadenylation signal sequence comprising the nucleic acid sequence of SEQ ID NO. 8, an hGH polyadenylation sequence comprising the nucleic acid sequence of SEQ ID NO. 17, or a bGH polyadenylation sequence comprising the nucleic acid sequence of SEQ ID NO. 30.

166. 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 human distantly related homeobox 2 (hDlx 2) sequence, the hDlx2 sequence comprising a nucleic acid sequence of SEQ ID NO. 13, wherein the hNeuroD1 sequence and the hDlx2 sequence are separated by: (i) a P2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs 15 and 18, (ii) a T2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs 16 and 19, or (iii) an Internal Ribosome Entry Site (IRES) sequence of an encephalomyocarditis virus comprising SEQ ID NO 3, wherein the hNeuroD1 sequence and the hDlx2 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. 26;

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

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

(d) A woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) comprising the nucleic acid sequence of SEQ ID NO. 29; and

(e) A bGH polyadenylation sequence comprising the nucleic acid sequence of SEQ ID No. 30.

167. 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 human distantly related homeobox 2 (hDlx 2) sequence, the hDlx2 sequence comprising a nucleic acid sequence of SEQ ID NO. 13, wherein the hNeuroD1 sequence and the hDlx2 sequence are separated by: (i) a P2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs 15 and 18, (ii) a T2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs 16 and 19, or (iii) an Internal Ribosome Entry Site (IRES) sequence of an encephalomyocarditis virus comprising SEQ ID NO 3, wherein the hNeuroD1 sequence and the hDlx2 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. 26;

(b) A Cytomegalovirus (CMV) enhancer comprising the nucleic acid sequence of SEQ ID No. 11;

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

(d) A woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) comprising the nucleic acid sequence of SEQ ID NO. 29; and

(e) A bGH polyadenylation sequence comprising the nucleic acid sequence of SEQ ID No. 30.

168. An adeno-associated virus (AAV) vector comprising: a nucleic acid sequence encoding a human neurogenic differentiation factor 1 (hNeuroD 1) protein, the hNeuroD1 protein comprising an amino acid coding sequence of SEQ ID No. 10, and a nucleic acid coding sequence encoding a human distantly related homeobox 2 (hDlx 2) protein, the hDlx2 protein comprising an amino acid sequence of SEQ ID No. 14, wherein the hNeuroD1 coding sequence and the hDlx2 coding sequence are separated by: (i) a P2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs 15 and 18, (ii) a T2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs 16 and 19, 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 hDlx2 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. 26;

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

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

(d) A woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) comprising the nucleic acid sequence of SEQ ID NO. 29; and

(e) A bGH polyadenylation sequence comprising the nucleic acid sequence of SEQ ID No. 30.

169. An adeno-associated virus (AAV) vector comprising: a nucleic acid sequence encoding a human neurogenic differentiation factor 1 (hNeuroD 1) protein, the hNeuroD1 protein comprising an amino acid coding sequence of SEQ ID No. 10, and a nucleic acid coding sequence encoding a human distantly related homeobox 2 (hDlx 2) protein, the hDlx2 protein comprising an amino acid sequence of SEQ ID No. 14, wherein the hNeuroD1 coding sequence and the hDlx2 coding sequence are separated by: (i) a P2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs 15 and 18, (ii) a T2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs 16 and 19, 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 hDlx2 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. 26;

(b) A Cytomegalovirus (CMV) enhancer comprising the nucleic acid sequence of SEQ ID No. 11;

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

(d) A woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) comprising the nucleic acid sequence of SEQ ID NO. 29; and

(e) A bGH polyadenylation sequence comprising the nucleic acid sequence of SEQ ID No. 30.

170. 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 human distantly related homeobox 2 (hDlx 2) sequence, the hDlx2 sequence comprising a nucleic acid sequence of SEQ ID NO. 13, wherein the hNeuroD1 sequence and the hDlx2 sequence are separated by: a P2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs 15 and 18, or an Internal Ribosome Entry Site (IRES) sequence of an encephalomyocarditis virus comprising SEQ ID NO 3, wherein the hNeuroD1 sequence and the hDlx2 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. 26;

(b) A Cytomegalovirus (CMV) enhancer comprising the nucleic acid sequence of SEQ ID No. 11;

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

(d) A bGH polyadenylation sequence comprising the nucleic acid sequence of SEQ ID No. 30.

171. An adeno-associated virus (AAV) vector comprising: a nucleic acid sequence encoding a human neurogenic differentiation factor 1 (hNeuroD 1) protein, the hNeuroD1 protein comprising an amino acid coding sequence of SEQ ID No. 10, and a nucleic acid coding sequence encoding a human distantly related homeobox 2 (hDlx 2) protein, the hDlx2 protein comprising an amino acid sequence of SEQ ID No. 14, wherein the hNeuroD1 coding sequence and the hDlx2 coding sequence are separated by: a P2A linker comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs 15 and 18, or an Internal Ribosome Entry Site (IRES) sequence of an encephalomyocarditis virus comprising SEQ ID NO 3, wherein the hNeuroD1 coding sequence and the hDlx2 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. 26;

(b) A Cytomegalovirus (CMV) enhancer comprising the nucleic acid sequence of SEQ ID No. 11;

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

(d) A bGH polyadenylation sequence comprising the nucleic acid sequence of SEQ ID No. 30.