CN116761812A - NEUROD1 and DLX2 vectors - Google Patents

NEUROD1 and DLX2 vectors Download PDF

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Publication number
CN116761812A
CN116761812A CN202180080014.7A CN202180080014A CN116761812A CN 116761812 A CN116761812 A CN 116761812A CN 202180080014 A CN202180080014 A CN 202180080014A CN 116761812 A CN116761812 A CN 116761812A
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seq
nucleic acid
sequence
acid sequence
aav
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CN202180080014.7A
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Chinese (zh)
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J·许
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Shenxi Revitalization Treatment Co
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Shenxi Revitalization Treatment Co
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Priority claimed from PCT/US2021/052348 external-priority patent/WO2022072322A1/en
Publication of CN116761812A publication Critical patent/CN116761812A/en
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Abstract

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

Description

NEUROD1 and DLX2 vectors
Cross Reference to Related Applications
The present application claims the benefit of U.S. provisional application No. 63/084,945, filed on 9/29 in 2020, and U.S. provisional application No. 63/247,442, filed on 23 in 2021, both of which are incorporated herein by reference in their entirety.
Incorporation of the sequence Listing
Is contained in the name P34838WO 00/uThe file of sl.txt (28,311 bytes (at MS-Measurement of (r)) and created at 9.27 of 2021 are filed electronically herewith and incorporated by reference in their entirety.
Technical Field
The present disclosure includes methods and compositions for converting glial cells into neurons using AAV vectors comprising nucleic acid sequences encoding human NeuroD1 and Dlx 2.
Background
In subjects suffering from neurological disorders or following injury to the Central Nervous System (CNS) or Peripheral Nervous System (PNS), neurons are often killed or damaged and cannot regenerate.
Glial cells become reactive following CNS or PNS injury (such as brain injury) or neurological disorders.
There is currently no method available for regenerating functional new neurons in human subjects suffering from neurological disorders using adeno-associated virus (AAV).
Disclosure of Invention
In one aspect, the present disclosure provides and includes an adeno-associated virus (AAV) vector comprising: a human neurogenic differentiation factor 1 (hNeuroD 1) sequence, the hNeuroD1 sequence comprising the nucleic acid sequence of SEQ ID NO. 6, and a human distantly related homeobox 2 (hDlx 2) sequence, the hDlx2 sequence comprising the 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 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.
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 (hdld 1) protein, the hdld 1 protein comprising the 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 the amino acid sequence of SEQ ID No. 14, wherein the hdld 1 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.
In one aspect, the present disclosure provides and includes an adeno-associated virus (AAV) vector comprising a NeuroD1 nucleic acid encoding a NeuroD1 (NeuroD 1) protein and a Dlx nucleic acid encoding a distantly related homeobox 2 (Dlx 2) protein, wherein the NeuroD1 encoding sequence and Dlx2 encoding sequence are separated by a linker sequence, wherein the NeuroD1 encoding sequence and Dlx encoding sequence are operably linked to a regulatory element comprising: (a) a Glial Fibrillary Acidic Protein (GFAP) promoter; (b) an enhancer; (c) a chimeric intron; (d) Woodchuck hepatitis virus posttranscriptional regulatory elements (WPREs); and (e) a polyadenylation signal sequence.
In one aspect, the present disclosure provides and includes a composition comprising an adeno-associated virus (AAV) vector for converting human glial cells to functional neurons, wherein the AAV vector comprises: a human neurogenic differentiation factor 1 (hNeuroD 1) sequence, the hNeuroD1 sequence having the nucleic acid sequence of SEQ ID NO. 6, and a human distantly related homeobox 2 (hDlx 2) sequence, the hDlx2 sequence having the 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.
In one aspect, the present disclosure provides and includes a composition comprising an adeno-associated virus (AAV) vector for converting human glial cells to functional neurons, wherein the AAV vector comprises: a nucleic acid coding sequence encoding a human neurogenic differentiation factor 1 (hNeuroD 1) protein, the hNeuroD1 protein comprising the amino acid sequence of SEQ ID No. 10, and a nucleic acid coding sequence encoding a human distantly related homeobox 2 (hDlx 2) 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 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.
In one aspect, the present disclosure provides and includes a composition comprising an adeno-associated virus (AAV) vector for treating a subject in need thereof, wherein the AAV vector comprises a neurogenic differentiation factor 1 (NeuroD 1) sequence and a 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 Dlx sequence are operably linked to an expression control element comprising: (a) a Glial Fibrillary Acidic Protein (GFAP) promoter; (b) an enhancer; (c) a chimeric intron; (d) Woodchuck hepatitis virus posttranscriptional regulatory elements (WPREs); and (e) a polyadenylation signal.
In one aspect, the present disclosure provides and includes a method of converting reactive astrocytes into functional neurons in a living human brain, comprising: injecting an adeno-associated virus (AAV) into a subject in need thereof, wherein the AAV comprises a DNA vector construct comprising: a human neurogenic differentiation factor 1 (hNeuroD 1) sequence, the hNeuroD1 sequence comprising a nucleic acid sequence of SEQ ID NO. 6, and a 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 said hNeuroD1 sequence and said 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.
In one aspect, the present disclosure provides and includes a method of converting reactive astrocytes into functional neurons in a living human brain, comprising: injecting an adeno-associated virus (AAV) into a subject in need thereof, wherein the AAV comprises a DNA vector construct comprising: a nucleic acid coding sequence encoding a human neurogenic differentiation factor 1 (hNeuroD 1) protein, the hNeuroD1 protein comprising the amino acid sequence of SEQ ID No. 10, and a nucleic acid coding sequence encoding a human distantly related homeobox 2 (hDlx 2) 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 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.
In one aspect, the present disclosure provides and includes a method of converting glial cells into neurons in a subject in need thereof, comprising: delivering an adeno-associated virus (AAV) to 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) a Glial Fibrillary Acidic Protein (GFAP) promoter; (b) an enhancer; (c) a chimeric intron; (d) Woodchuck hepatitis virus posttranscriptional regulatory elements (WPREs); and (e) a polyadenylation signal sequence, wherein the vector is capable of converting at least one glial cell to a neuron in the subject in need thereof.
In one aspect, the present disclosure provides and includes a method of treating a neurological disorder in a subject in need thereof, comprising: delivering an adeno-associated virus (AAV) to 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) a Glial Fibrillary Acidic Protein (GFAP) promoter; (b) an enhancer; (c) a chimeric intron; (d) Woodchuck hepatitis virus posttranscriptional regulatory elements (WPREs); and (e) a polyadenylation signal.
In one aspect, the present disclosure provides and includes a composition comprising: (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.
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 (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.
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 (hdld 1) protein, the hdld 1 protein comprising the amino acid sequence of SEQ ID No. 10, and (ii) an adeno-associated virus (AAV) vector comprising a nucleic acid sequence encoding a human distantly related homeobox 2 (hDlx 2) protein, the hDlx2 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 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.
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 (hdld 1) protein, the hdld 1 protein comprising the amino acid sequence of SEQ ID No. 10, and (ii) an adeno-associated virus (AAV) vector comprising a nucleic acid sequence encoding a human distantly related homeobox 2 (hDlx 2) protein, the hDlx2 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 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.
In one aspect, the disclosure provides and includes an adeno-associated virus (AAV) vector comprising: a human neurogenic differentiation factor 1 (hNeuroD 1) sequence, the hNeuroD1 sequence comprising 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 said hNeuroD1 sequence and said 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 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 disclosure provides and includes 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 said hNeuroD1 sequence and said 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 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 disclosure provides and includes 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 the 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 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 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 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 disclosure provides and includes 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 the 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 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 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 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 disclosure provides and includes 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 or SEQ ID No. 27; and (d) a bGH polyadenylation sequence comprising the nucleic acid sequence of SEQ ID No. 30.
In one aspect, the disclosure provides and includes 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 the 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 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 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 or SEQ ID No. 27; and (d) a bGH polyadenylation sequence comprising the nucleic acid sequence of SEQ ID No. 30.
Drawings
FIG. 1A depicts a diagram of aCE: gfa681:NeuroD1:P2A: dlx2:WPRE:SV 40.
FIG. 1B depicts a diagram of EF-1α: gfa681:NeuroD1:P2A: dlx2:WPRE:SV 40.
FIG. 1C depicts a diagram of CE Gfa681:NeuroD1:P2A: dlx2:WPRE:hGH
FIG. 1D depicts a diagram of EF-1α: gfa681:NeuroD1:P2A: dlx 2:WPRE:hGH.
FIG. 2A depicts a diagram of CE: gfa681 neuroD1:GSG-P2A: dlx2:WPRE:SV 40.
FIG. 2B depicts a diagram of EF-1α: gfa681 neuroD1:GSG-P2A: dlx2:WPRE:SV 40.
FIG. 2C depicts a diagram of CE: gfa681 neuroD1:GSG-P2A: dlx 2:WPRE:hGH.
FIG. 2D depicts a diagram of EF-1α: gfa681 neuroD1:GSG-P2A: dlx 2:WPRE:hGH.
FIG. 3A depicts a diagram of CE: gfa681 neuroD1:T2A: dlx2:WPRE:SV40
FIG. 3B depicts a diagram of EF-1α: gfa681 NeuroD1:T2A: dlx2:WPRE:SV 40.
FIG. 3C depicts a diagram of CE: gfa681 neuroD1:T2A: dlx 2:WPRE:hGH.
FIG. 3D depicts a diagram of EF-1α: gfa681 NeuroD1:T2A: dlx 2:WPRE:hGH.
FIG. 4A depicts a diagram of CE: gfa681 neuroD1:GSG-T2A: dlx2:WPRE:SV40
FIG. 4B depicts a diagram of EF-1α: gfa681 neuroD1:GSG-T2A: dlx2:WPRE:SV 40.
FIG. 4C depicts a diagram of CE: gfa681 neuroD1:GSG-T2A: dlx 2:WPRE:hGH.
FIG. 4D depicts a diagram of EF-1α: gfa681 neuroD1:GSG-T2A: dlx 2:WPRE:hGH.
FIG. 5A depicts a diagram of U6 shRNA: CE: gfa681: neuroD1: P2A: dlx2: SV 40.
FIG. 5B depicts a diagram of U6. ShRNA: EF-1. Alpha.: gfa681: neuroD1: P2A: dlx. Alpha. SV 40.
FIG. 5C depicts a diagram of U6 shRNA: CE: gfa681: neuroD1: GSG-P2A: dlx2:SV 40.
FIG. 5D depicts a diagram of U6. ShRNA. EF-1. Alpha. Gfa 681. NeuroD 1. GSG-P2A Dlx. SV 40.
FIG. 6A depicts a diagram of U6 shRNA: CE: gfa681: neuroD1: P2A: dlx2: hGh.
FIG. 6B depicts a diagram of U6. ShRNA: EF-1. Alpha.: gfa681: neuroD1: P2A: dlx2: hGh.
FIG. 6C depicts a diagram of U6 shRNA: CE: gfa681: neuroD1: GSG-P2A: dlx2: hGh.
FIG. 6D depicts a diagram of U6. ShRNA: EF-1. Alpha.: gfa681: neuroD1: GSG-P2A: dlx2: hGh.
FIG. 7A depicts a diagram of U6 shRNA: CE: gfa681: neuroD1: T2A: dlx2: SV 40.
FIG. 7B depicts a diagram of U6. ShRNA: EF-1. Alpha.: gfa681: neuroD1: T2A: dlx. Alpha. SV 40.
FIG. 7C depicts a diagram of U6 shRNA: CE: gfa681: neuroD1: GSG-T2A: dlx2:SV 40.
FIG. 7D depicts a diagram of EF-1α: gfa681:NeuroD1:GSG-T2A: dlx2:SV 40.
FIG. 8A depicts a diagram of U6 shRNA: CE: gfa681: neuroD1: T2A: dlx2: hGh.
FIG. 8B depicts a diagram of U6. ShRNA. EF-1. Alpha.: gfa 681. NeuroD 1. T2A Dlx. HGh.
FIG. 8C depicts a diagram of U6 shRNA: CE: gfa681: neuroD1: GSG-T2A: dlx2: hGh.
FIG. 8D depicts a vector diagram of U6 shRNA:EF-1. Alpha.: gfa681:NeuroD1:GSG-T2A: dlx2: hGh.
FIG. 9 measures AAV viral production of the P31 plasmid. Titer analysis was performed using the gene of interest (GOI) primer, ITR region primer and reverse package primer. Viral titer was calculated as vg/cell.
FIG. 10 depicts the establishment of a primary culture of rat astrocytes from Sprague-Dawley rat brain 3 days after birth. The upper left panel presents images of GFAP stained cells. The upper right panel presents an image of SOX9 stained cells. The lower left panel presents images of DAPI stained cells. The bottom right panel presents a combined image of GFAP, SOX9 and DAPI stained cells.
FIG. 11 depicts a comparison of the expression efficiency of neuroD1 of plasmids. Primary rat astrocytes were transfected with P14 (CE: gfaABC1D: hNeuroD1-P2A-Dlx2: WPRE: SV 40), P31 (EF-1αE: gfaABC1D: neuroD1-P2A-Dlx2: WPRE: SV 40) and P63 (CE: gfaABC1D: neuroD1-GSG P2A-Dlx2: WPRE: SV 40). The upper panel shows the NeuroD1 staining of cells, the middle panel shows the Dlx staining of cells, and the lower panel shows the pooled NeuroD1, dlx, and DAPI staining of cells.
FIG. 12 depicts a comparison of AAV viral particle transduction at different doses using AAV9-P12 (pGfaABC 1D: GFP). The upper left panel shows 3x10 10 Dose of vg per well. The upper right panel shows 1x10 10 Dose of vg per well. The lower graph shows 2.5x10 9 Dose of vg per well.
Fig. 13A and 13B depict quantitative analysis of AAV particle transduction into primary rat astrocytes. FIG. 13A shows AAV9-P12 (pGfaABC 1D: GFP) and AAV5-P7 (pEF-1. Alpha.: GFP) at an MOI of 5X10 5 vg/cell, 2X10 5 vg/cell and 5x10 4 Percent transduction at vg/cell. FIG. 13B shows AAV9-P12 (pGfaABC 1D: GFP) at a range of densities (2X 10) 4 Individual cells/well, 1.5x10 4 Individual cells/well, 1x10 4 Individual cells/well and 5x10 3 Individual cells/well) and inoculated with a series of amounts of virus (1 x10 in 100 μl of medium 13 2. Mu.l, 1. Mu.l, 0.5. Mu.l, 0.25. Mu.l, 0.125. Mu.l) of vg/ml virus.
FIG. 14 depicts AAV9-P21 (CE-pGFA 681-CI-GFP-WPRE-SV40 pA) using a control plasmid2X10 10 RCA three weeks after vg/ml transduction. Cells were immunostained with antibodies to the neuronal markers NeuN and MAP2, DAPI (nuclear dye). GFP fluorescence indicates the presence of cells transduced with the control plasmid.
FIG. 15 depicts RCA immunostained with anti-NeuroD 1 (ND 1) antibody and DAPI (nuclear dye) 24 hours after transfection with NXL-P134 (CE-pGfa 681-CRGI-hND 1-oPRE-bGHPA).
FIG. 16 depicts AAV9-P134 (CE-pGfa 681-CRGI-hND 1-oPRE-bGHPA) at 2X10 10 RCA immunostained with anti-ND 1 antibody and DAPI (nuclear dye) 6 days after vg/ml transduction.
FIG. 17 depicts AAV9-P134 (CE-pGfa 681-CRGI-hND 1-oPRE-bGHPA) at 2X10 10 RCA immunostained with anti-NeuN and anti-MAP 2 antibodies and DAPI (nuclear dye) 3 weeks after vg/ml transduction. Transduction was performed using ND 1-containing vectors to generate neurons (NeuN// MAP 2+) from astrocyte cultures.
FIG. 18 depicts RCA immunostained with anti-ND 1 antibody and DAPI (nuclear dye) 24 hours after transfection with NXL-P138 (EE-pGfa 681-CRGI-hND 1-oPRE-bGHPA).
FIG. 19 depicts the use of AAV9-P138 (EE-pGfa 681-CRGI-hND 1-oPRE-bGHPA) at 2X10 10 RCA immunostained with anti-ND 1 antibody and DAPI (nuclear dye) 6 days after vg/ml transduction.
FIG. 20 depicts the use of AAV9-P138 (EE-pGfa 681-CRGI-hND 1-oPRE-bGHPA) at 2X10 10 RCA immunostained with anti-NeuN and anti-MAP 2 antibodies and DAPI (nuclear dye) 3 weeks after vg/ml transduction. Transduction was performed using ND 1-containing vectors to generate neurons (NeuN// MAP 2+) from astrocyte cultures.
FIG. 21 depicts AAV9-P9 (CE-pGfa 681-CI-hND1-P2A-GFP-WPRE-SV40 pA) at 2X10 10 RCA immunostained with anti-ND 1 antibody and DAPI (nuclear dye) 6 days after vg/ml transduction. GFP fluorescence indicates the presence of transduced cells.
FIG. 22 depicts AAV9-P9 (CE-pGfa 681-CI-hND1-P2A-GFP-WPRE-SV40 pA) at 2X10 10 RCA immunostained with anti-NeuN and anti-MAP 2 antibodies and DAPI (nuclear dye) 3 weeks after vg/ml transduction. Transduction using ND 1-containing vectors for production from astrocyte culturesNeurogenesis (NeuN// MAP 2+).
FIG. 23 depicts RCA immunostained with anti-ND 1 antibody and DAPI (nuclear dye) 24 hours after transfection with NXL-P22 (CE-pGfa 681-CI-hND1-WPRE-SV40 pA).
FIG. 24 depicts AAV9-P22 (CE-pGfa 681-CI-hND1 WPRE-SV40 pA) at 2X10 10 RCA immunostained with anti-ND 1 antibody and DAPI (nuclear dye) 6 days after vg/ml transduction.
FIG. 25 depicts AAV9-P22 (CE-pGfa 681-CI-hND1-WPRE-SV40 pA) at 2X10 10 RCA immunostained with anti-NeuN and anti-MAP 2 antibodies and DAPI (nuclear dye) 3 weeks after vg/ml transduction. Transduction was performed using ND 1-containing vectors to generate neurons (NeuN// MAP 2+) from astrocyte cultures.
FIG. 26 depicts RCA immunostained with anti-ND 1 antibody and DAPI (nuclear dye) 24 hours after transfection with NXL-P35 (EE-pGfa 681-CI-hND1-WPRE-SV40 pA).
FIG. 27 depicts AAV9-P35 (EE-pGfa 681-CI-hND1 WPRE-SV40 pA) at 2X10 10 RCA immunostained with anti-ND 1 antibody and DAPI (nuclear dye) 6 days after vg/ml transduction.
FIG. 28 depicts AAV9-P35 (EE-pGfa 681-CI-hND1-WPRE-SV40 pA) at 2X10 10 RCA immunostained with anti-NeuN antibody and DAPI (nuclear dye) 3 weeks after vg/ml transduction. Transduction with ND 1-containing vectors resulted in the production of neurons (NeuN+) from astrocyte cultures.
FIG. 29 depicts RCA immunostained with anti-ND 1 antibody and DAPI (nuclear dye) 24 hours after transfection with NXL-P107 (CE-pGfa 681-CI-hND 1-bGHPA).
FIG. 30 depicts RCA immunostained with anti-ND 1 antibody and DAPI (nuclear dye) 24 hours after transfection with NXL-P108 (CE-pGfa 681-CI-hND 1-oPRE-bGHPA).
FIG. 31 depicts RCA immunostained with anti-ND 1 antibody and DAPI (nuclear dye) 24 hours after transfection with NXL-P109 (CE-pGfa 681-CRGI-hND 1-bGHPA).
FIG. 32 depicts cortical tissue 10 days post infection (dpi) in mice infected with AAV9-P12 (P12 control group), AAV9-P12+AAV9-P134 (P134 group), and AAV9-P12+AAV9-P138 (P138 group).
FIG. 33 depicts cortical tissue 30 days post infection (dpi) in mice infected with AAV9-P12+ AAV9-P134 (group P134) and AAV9-P12+ AAV9-P138 (group P138).
FIG. 34 depicts cortical tissue at 10dpi in mice infected with AAV9-P12 (P12 control group) and AAV9-P12+ AAV9-P134 (P134 group) (bilateral injury model).
FIG. 35 is a graph of measurements of AAV viral production from the P134, P130, P138 and P21 plasmids. Titer analysis was performed by qPCR using primers that amplify the gene of interest (GOI) and primers specific for the plasmid. The viral yield was calculated as vg/cell.
FIG. 36 depicts cortical tissue 10 days post infection (dpi) in mice infected with AAV9-P12 (P12 control group), AAV9-P12+AAV9-P134 (P134 group), and AAV9-P12+AAV9-P138 (P138 group). Cells were immunostained with antibodies to NeuroD1, GFAP, neuN and DAPI (nuclear stain). GFP fluorescence indicates the presence of cells infected with the control virus.
FIG. 37 depicts cortical tissue 30 days post infection (dpi) in mice infected with AAV9-P12 (P12 control group), AAV9-P12+AAV9-P134 (P134 group), and AAV9-P12+AAV9-P138 (P138 group). Cells were immunostained with antibodies to NeuroD1, GFAP, neuN and DAPI (nuclear stain). GFP fluorescence indicates the presence of cells infected with the control virus.
FIG. 38 depicts cortical tissue at 10dpi in mice infected with AAV9-P12 (P12 control group) and AAV9-P12+ AAV9-P134 (P134 group) (bilateral injury model). Cells were immunostained with antibodies to NeuroD1, GFAP, neuN and DAPI (nuclear stain). GFP fluorescence indicates the presence of cells infected with the control virus.
FIGS. 39A and 39B depict cortical tissue at 30dpi in mice (bilateral injury models) infected with AAV9-P12 (P12 control group) and AAV9-P12+ AAV9-P134 (P134 group). Fig. 39A depicts cells immunostained with antibodies to NeuroD1, GFAP, neuN and DAPI (nuclear dye). GFP fluorescence indicates the presence of cells infected with the control virus. FIG. 39B is a quantification of glial cell to neuron conversion at 30 dpi.
Fig. 40 depicts two general diagrams of ND1 and Dlx2 constructs. Enhancers refer to either the ef1a enhancer or the CMV enhancer. pGfa refers to the 2.2kb or 1.6kb Gfa promoter. poly (a) signal refers to SV40, bGH or hGH poly (a) signal.
FIG. 41 depicts RCA immunostained with anti-ND 1 antibody, anti-Dlx antibody and DAPI (nuclear dye) 24 hours after transfection with NXL-P112 (CE-pGfa 681-CI-hDlx2-IRES-hND 1-bGHPA).
FIG. 42 depicts AAV9-P112 (CE-pGfa 681-CI-hDlx2-IRES-hND 1-bGHPA) at 2X10 10 RCA immunostained with anti-ND 1 antibody, anti-Dlx antibody and DAPI (nuclear dye) 6 days after vg/ml transduction.
FIG. 43 depicts AAV9-P112 (CE-pGfa 681-CI-hDlx2-IRES-hND 1-bGHPA) at 2X10 10 RCA immunostained with anti-NeuN antibodies, anti-MAP 2 antibodies and DAPI (nuclear dye) 3 weeks after vg/ml transduction.
FIG. 44 depicts RCA immunostained with anti-ND 1 antibody, anti-Dlx antibody and DAPI (nuclear dye) 24 hours after transfection with NXL-P122 (CE-pGfa 681-CI-hDlx2-P2A-hND 1-bGHPA).
FIG. 45 depicts AAV9-P122 (CE-pGfa 681-CI-hDlx2-P2A-hND 1-bGHPA) at 2X10 10 RCA immunostained with anti-ND 1 antibody, anti-Dlx antibody and DAPI (nuclear dye) 6 days after vg/ml transduction.
FIG. 46 depicts AAV9-P122 (CE-pGfa 681-CI-hDlx2-P2A-hND 1-bGHPA) at 2X10 10 RCA immunostained with anti-NeuN antibodies, anti-MAP 2 antibodies and DAPI (nuclear dye) 3 weeks after vg/ml transduction.
FIG. 47 depicts RCA immunostained with anti-ND 1 antibody, anti-Dlx antibody and DAPI (nuclear dye) 24 hours after transfection with NXL-P124 (CE-pGfa 681-CI-hND1-P2A-hDlx 2-bGHPA).
FIG. 48 depicts AAV9-P124 (CE-pGfa 681-CI-hND1-P2A-hDlx 2-bGHPA) at 2X10 10 RCA immunostained with anti-ND 1 antibody, anti-Dlx antibody and DAPI (nuclear dye) 6 days after vg/ml transduction.
FIG. 49 depicts AAV9-P124 (CE-pGfa 681-CI-hND1-P2A-hDlx 2-bGHPA) at 2X10 10 RCA immunostained with anti-NeuN antibodies, anti-MAP 2 antibodies and DAPI (nuclear dye) 3 weeks after vg/ml transduction.
FIG. 50 depicts RCA immunostained with anti-ND 1 antibody, anti-Dlx 2 antibody and DAPI (nuclear dye) 24 hours after transfection with NXL-P20 (CE-pGfa 681-CI-hND1-P2A-hDlx2-WPRE-SV40 pA).
FIG. 51 depicts AAV9-P20 (CE-pGfa 681-CI-hDND1-P2A-hDlx2-WPRE-SV40 pA) at 2X10 10 RCA immunostained with anti-ND 1 antibody, anti-Dlx antibody and DAPI (nuclear dye) 6 days after vg/ml transduction.
FIG. 52 depicts AAV9-P20 (CE-pGfa 681-CI-hND1-P2A-hDlx2-bSV pA) at 2X10 10 RCA immunostained with anti-NeuN antibodies, anti-MAP 2 antibodies and DAPI (nuclear dye) 3 weeks after vg/ml transduction.
FIG. 53 depicts RCA immunostained with anti-ND 1 antibody, anti-Dlx 2 antibody and DAPI (nuclear dye) 24 hours after transfection with NXL-P31 (EE-pGfa 681-CI-hND1-P2A-hDlx2-WPRE-SV40 pA).
FIG. 54 depicts RCA immunostained with anti-ND 1 antibody, anti-Dlx antibody and DAPI (nuclear dye) 24 hours after transfection with NXL-P123 (EE-pGfa 681-CI-hDlx2-P2A-hND 1-bGHPA).
FIG. 55 depicts RCA immunostained with anti-ND 1 antibody, anti-Dlx antibody and DAPI (nuclear dye) 24 hours after transfection with NXL-P113 (EE-pGfa 681-CI-hDlx2-IRES-hND 1-bGHPA).
FIG. 56 depicts RCA immunostained with anti-ND 1 antibody, anti-Dlx antibody and DAPI (nuclear dye) 24 hours after transfection with NXL-P111 (CE-pGfa 681-CI-hDlx2-P2A-hND1-SV40 pA).
FIGS. 57A and 57B depict brain striatal tissue at 10dpi and 30dpi in AAV9-P12 (viral) infected mice. FIG. 57A shows extensive infection of the mouse striatum by AAV-P12 at 10dpi as demonstrated by GFP fluorescence. FIG. 57B shows infection of mice with AAV9-P12 at 30 dpi. Cells were immunostained with antibodies to GFAP and NeuN.
FIG. 58 depicts brain striatal tissue of mice co-infected with AAV9-P12 and AAV9-P112 (group P112) at 10 dpi. GFP fluorescence identified AAV 9-P112-infected cells. Cells were immunostained with antibodies to GFAP and NeuN.
FIG. 59 depicts brain striatal tissue of mice co-infected with AAV9-P12 and AAV9-P112 (group P112) at 30 dpi. GFP fluorescence identified AAV 9-P112-infected cells. Cells were immunostained with antibodies to GFAP and NeuN. White arrows indicate that AAV9-P12 and AAV9-P112 co-infected cells became NeuN positive neurons.
FIG. 60 depicts brain striatal tissue of mice co-infected with AAV9-P12 and AAV9-P122 (group P122) at 10 dpi. GFP fluorescence identified AAV 9-P112-infected cells. Cells were immunostained with antibodies to GFAP and NeuN.
FIG. 61 depicts brain striatal tissue of mice co-infected with AAV9-P12 and AAV9-P122 (group P122) at 30 dpi. GFP fluorescence identified AAV 9-P112-infected cells. Cells were immunostained with antibodies to GFAP and NeuN.
FIG. 62 depicts Rat Cortical Astrocytes (RCA) immunostained with anti-Dlx antibody and DAPI (nuclear dye) 24 hours after transfection with NXL-P104 (CE-pGfa 681-CGRI-Dlx 2-bGHPA) or NXL-P105 (CE-pGfa 681-CI-Dlx 2-oPRE-bGHPA).
FIG. 63 depicts Rat Cortical Astrocytes (RCA) immunostained with anti-Dlx antibody and DAPI (nuclear dye) 24 hours after transfection with NXL-P133 (EE-pGfa 681-CGRI-Dlx 2-oPRE-bGHPA), NXL-P137 (EE-pGfa 681-CGRI-Dlx 2-oPRE-bGHPA) or NXL-P131 (EE-pGfa 681-CI-Dlx 2-oPRE-bGHPA).
FIG. 64 depicts Rat Cortical Astrocytes (RCA) immunostained with anti-Dlx 2 antibody and DAPI (nuclear dye) after transduction with AAV9-P133 (CE-pGfa 681-CGRI-Dlx 2-oPRE-bGHPA).
Brief description of the sequence
A list of nucleic acid sequences and amino acid sequences is provided in table 1.
TABLE 1 nucleic acid sequences
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Detailed Description
Unless defined otherwise, all technical and scientific terms used have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Where a term is provided in the singular, the inventors of the present application also contemplate the various aspects of the present disclosure described in the plural of the term. Where there is a difference in terms and definitions used in the references incorporated by reference, the terms used in the present application shall have the definitions given herein. Other technical terms used have their ordinary meaning in the field in which they are used, as exemplified by various field-specific dictionaries, e.g. "AmericanScientific dictionary) ("edit of U.S. traditional dictionary (American Heritage Dictionaries)," 2011, boston and N.Y. Hoton Mivelin publishing company (Houghton Mifflin Harcourt, boston and New York)), "" McGraw-Hill Dictionary of Scientific and Technical Terms), "(6 th edition, 2002, N.Y. Magla-Hill, new York) or" [ oxford biology dictionary (Oxford Dictionary of Biology) ] (6 th edition, 2008, oxford and N.Y. oxford university publishing company (Oxford University Press, oxford and New York) ].
Any references cited herein, including, for example, all patents, published patent applications, and non-patent publications, are incorporated by reference in their entirety.
When a set of alternatives is presented, any and all combinations of the members making up the set of alternatives are specifically contemplated. For example, if the item is selected from the group consisting of A, B, C and D, the inventors will specifically contemplate each alternative individually (e.g., a alone, B alone, etc.) and such as A, B and D; a and C; b and C; etc. The term "and/or" when used in a list of two or more items means any one of the listed items by itself or in combination with any one or more other listed items. For example, the expression "a and/or B" is intended to mean either or both of a and B, i.e., a alone, B alone, or a combination of a and B. The expression "A, B and/or C" is intended to mean a alone, B alone, a combination of C, A and B alone, a combination of a and C, a combination of B and C, or a combination of A, B and C.
When numerical ranges are provided herein, the ranges are understood to include the edges of the ranges as well as any numbers between the defined edges of the ranges. For example, "between 1 and 10" includes any number between 1 and 10 as well as the numbers 1 and 10.
When the term "about" is used in reference to a number, it is understood to mean plus or minus 10%. For example, "about 100" will include from 90 to 110.
As used herein, "hND1" refers to a human neuro-derived differentiation factor (NeuroD 1) gene or protein.
As used herein, "CE" refers to a Cytomegalovirus (CMV) promoter enhancer sequence.
As used herein, "EE" refers to the Ef1 a promoter enhancer sequence.
As used herein, "pGfa681" refers to a human Glial Fibrillary Acidic Protein (GFAP) promoter truncated sequence of 681bp in size. As used herein, "pGfa681", "Gfa681", "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 branching and 3' -acceptor site from the heavy chain variable region intron of the immunoglobulin gene.
As used herein, "CRGI" refers to chimeric introns of rabbit β -globulin and chicken β -actin that are similar in the CAG promoter.
As used herein, "GI" refers to the human Glial Fibrillary Acidic Protein (GFAP) first intron.
As used herein, "WPRE" refers to Woodchuck Hepatitis Virus (WHV) post-transcriptional regulatory elements.
As used herein, "oPRE" refers to an optimized version of WPRE.
As used herein, "SV40pA" refers to the polyadenylation signal of SV40 virus.
As used herein, "bGHpA" refers to the polyadenylation signal of bovine growth hormone.
As used herein, "vg" refers to the viral genome.
As used herein, "hDlx2" refers to a human distantly related homeobox 2 gene or protein.
Any of the compositions or carriers provided herein are specifically contemplated for use in any of the methods provided herein.
In one aspect, the methods and compositions provided herein comprise a carrier. As used herein, the term "vector" refers to a circular double stranded DNA molecule that is physically separated from chromosomal DNA. It should be noted that the term "vector" may be used interchangeably with the term "plasmid".
In one aspect, the vectors provided herein are recombinant vectors. As used herein, the term "recombinant vector" refers to a vector comprising a recombinant nucleic acid. As used herein, "recombinant nucleic acid" refers to a nucleic acid molecule formed by laboratory methods of gene recombination (such as, but not limited to, molecular cloning). The gene vector may be formed by laboratory methods of genetic recombination, such as, but not limited to, molecular cloning. Furthermore, the person skilled in the art can create recombinant vectors de novo by synthesizing plasmids from a single nucleotide or by splicing together nucleic acid molecules from different pre-existing vectors, but is not limited thereto.
Adeno-associated viruses (AAV) are replication-defective, non-enveloped, dependent parvovirus (dependoparvorrus) viruses that infect humans and other primate species. AAV is known not to cause disease in any species, but they cause a mild immune response. AAV can infect dividing cells and resting cells. AAV stably integrates into the human genome at a specific site on chromosome 19 known as the AAVs1 locus (nucleotides 7774-11429 of GenBank accession No. AC 010327.8), although random integration of other loci in the human genome is possible.
AAV comprises a linear genome having single stranded DNA of about 4700 nucleotides in length. The genome of AAV also includes 145 nucleotide long Inverted Terminal Repeats (ITRs) at each end of the genome. Two viral genes flank the ITR: rep (for replication, encoding a non-structural protein) and cap (for capsid, encoding a structural protein). The ITR contains all cis-acting elements required for AAV genome rescue, replication and packaging.
When used in gene therapy methods, the rep and cap genes of the AAV genomic sequences are removed and replaced with the DNA of interest located between the two AAV ITRs. As used herein, an "AAV vector construct" refers to a DNA molecule comprising a desired sequence inserted between two AAV ITR sequences. As used herein, "AAV vector" refers to AAV packaged with a DNA vector construct.
As used herein, the term "AAV vector serotype" refers primarily to variation within the capsid protein of an AAV vector.
In one aspect, the AAV vector is selected from the group consisting of: AAV vector serotype 1, AAV vector serotype 2, AAV vector serotype 3, AAV vector serotype 4, AAV vector serotype 5, AAV vector serotype 6, AAV vector serotype 7, AAV vector serotype 8, AAV vector serotype 9, AAV vector serotype 10, AAV vector serotype 11, and AAV vector serotype 12. In one aspect, the AAV vector is selected from the group consisting of AAV serotype 2, AAV serotype 5, and AAV serotype 9. In one aspect, the AAV vector is AAV serotype 1. In one aspect, the AAV vector is AAV serotype 2. In one aspect, the AAV vector is AAV serotype 3. In one aspect, the AAV vector is AAV serotype 4. In one aspect, the AAV vector is AAV serotype 5. In one aspect, the AAV vector is AAV serotype 6. In one aspect, the AAV vector is AAV serotype 7. In one aspect, the AAV vector is AAV serotype 8. In one aspect, the AAV vector is AAV serotype 9. In one aspect, the AAV vector is AAV serotype 10. In one aspect, the AAV vector is AAV serotype 11. In one aspect, the AAV vector is AAV serotype 12.
In one aspect, the AAV vector ITR is selected from the group consisting of: AAV serotype 1 ITRs, AAV serotype 2 ITRs, AAV serotype 3 ITRs, AAV serotype 4 ITRs, AAV serotype 5 ITRs, AAV serotype 6 ITRs, AAV serotype 7 ITRs, AAV serotype 8 ITRs, AAV serotype 9 ITRs, AAV serotype 10 ITRs, AAV serotype 11 ITRs, and AAV serotype 12 ITRs. In one aspect, the AAV vector ITRs are AAV serotype 1 ITRs. In one aspect, the AAV vector ITRs are AAV serotype 2 ITRs. In one aspect, the AAV vector ITRs are AAV serotype 3 ITRs. In one aspect, the AAV vector ITRs are AAV serotype 4 ITRs. In one aspect, the AAV vector ITRs are AAV serotype 5 ITRs. In one aspect, the AAV vector ITRs are AAV serotype 6 ITRs. In one aspect, the AAV vector ITRs are AAV serotype 7 ITRs. In one aspect, the AAV vector ITRs are AAV serotype 8 ITRs. In one aspect, the AAV vector ITRs are AAV serotype 9 ITRs. In one aspect, the AAV vector ITRs are AAV serotype 10 ITRs. In one aspect, the AAV vector ITRs are AAV serotype 11 ITRs. In one aspect, the AAV vector ITRs are AAV serotype 12 ITRs.
In one aspect, the at least one AAV vector ITR nucleic acid sequence is selected from the group consisting of: SEQ ID NOS 1 and 9. In one aspect, the at least one AAV vector ITR nucleic acid sequence is SEQ ID NO 1. In one aspect, the at least one AAV vector ITR nucleic acid sequence is SEQ ID NO 9.
In one aspect, the AAV ITR nucleic acid sequence comprises a sequence at least 70% identical to: SEQ ID NO. 1, or a complement thereof. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence at least 75% identical to: SEQ ID NO. 1, or a complement thereof. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence at least 80% identical to: SEQ ID NO. 1, or a complement thereof. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence at least 85% identical to: SEQ ID NO. 1, or a complement thereof. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence at least 90% identical to: SEQ ID NO. 1, or a complement thereof. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence at least 91% identical to: SEQ ID NO. 1, or a complement thereof. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence at least 92% identical to: SEQ ID NO. 1, or a complement thereof. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence at least 93% identical to: SEQ ID NO. 1, or a complement thereof. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence at least 94% identical to: SEQ ID NO. 1, or a complement thereof. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence at least 95% identical to: SEQ ID NO. 1, or a complement thereof. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence at least 96% identical to: SEQ ID NO. 1, or a complement thereof. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence at least 97% identical to: SEQ ID NO. 1, or a complement thereof. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence at least 98% identical to: SEQ ID NO. 1, or a complement thereof. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence at least 99% identical to: SEQ ID NO. 1, or a complement thereof. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence at least 99.5% identical to: SEQ ID NO. 1, or a complement thereof. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence at least 99.8% identical to: SEQ ID NO. 1, or a complement thereof. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence at least 99.9% identical to: SEQ ID NO. 1, or a complement thereof. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence 100% identical to: SEQ ID NO. 1, or a complement thereof.
In one aspect, the AAV ITR nucleic acid sequence comprises a sequence at least 70% identical to: SEQ ID NO. 9, or a complement thereof. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence at least 75% identical to: SEQ ID NO. 9, or a complement thereof. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence at least 80% identical to: SEQ ID NO. 9, or a complement thereof. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence at least 85% identical to: SEQ ID NO. 9, or a complement thereof. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence at least 90% identical to: SEQ ID NO. 9, or a complement thereof. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence at least 91% identical to: SEQ ID NO. 9, or a complement thereof. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence at least 92% identical to: SEQ ID NO. 9, or a complement thereof. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence at least 93% identical to: SEQ ID NO. 9, or a complement thereof. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence at least 94% identical to: SEQ ID NO. 9, or a complement thereof. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence at least 95% identical to: SEQ ID NO. 9, or a complement thereof. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence at least 96% identical to: SEQ ID NO. 9, or a complement thereof. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence at least 97% identical to: SEQ ID NO. 9, or a complement thereof. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence at least 98% identical to: SEQ ID NO. 9, or a complement thereof. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence at least 99% identical to: SEQ ID NO. 9, or a complement thereof. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence at least 99.5% identical to: SEQ ID NO. 9, or a complement thereof. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence at least 99.8% identical to: SEQ ID NO. 9, or a complement thereof. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence at least 99.9% identical to: SEQ ID NO. 9, or a complement thereof. In one aspect, the AAV ITR nucleic acid sequence comprises a sequence 100% identical to: SEQ ID NO. 9, or a complement thereof.
As used herein, the term "percent identity" or "percent identity" with respect to two or more nucleotide or amino acid sequences is calculated by: (i) comparing the two optimally aligned sequences (nucleotides or amino acids) over a comparison window (one or more "alignable" regions), (ii) determining the number of positions at which the same nucleobase (for nucleotide sequences) or amino acid residue (for proteins and polypeptides) occurs in the two sequences to produce a number of matched positions, (iii) dividing the number of matched positions by the total number of positions in the comparison window, and then (iv) multiplying the quotient by 100% to produce a percentage of identity. If "percent identity" is calculated relative to the reference sequence without specifying a particular comparison window, the percent identity is determined by dividing the number of matching positions on the alignment area by the total length of the reference sequence. Thus, for the purposes of the present application, when two sequences (query and subject) are optimally aligned (gaps are allowed in their alignment), the "percent identity" of a query sequence is equal to the number of identical positions between the two sequences divided by the total number of positions of the query sequence over its length (or comparison window), and then multiplied by 100%.
When referring to amino acid use of sequence identity percentage, it is considered that the different residue positions usually by conservative amino acid substitution, which amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) other amino acid residues, thus not changing the molecular functional properties. When conservative substitutions of sequences are different, the percent sequence identity may be adjusted upward to correct for the conservation of the substitution. Sequences that differ due to such conservative substitutions are said to have "sequence similarity" or "similarity".
To optimally align sequences to calculate their percent identity, various pairwise or multiplex sequence alignment algorithms and procedures are known in the art, such as ClustalW or Basic Local Alignment Search, which can be used to compare sequence identity or similarity between two or more nucleotide or amino acid sequences(BLAST TM ) Etc. Although other alignment and comparison methods are known in the art, the alignment and percent identity between two sequences (including the percent identity ranges described above) can be determined by the ClustalW algorithm, see, e.g., chenna et al, "Multiple sequence alignment with the Clustal series of programs," Nucleic Acids Research31:3497-3500 (2003); thompson et al, "Clustal W Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice," Nucleic Acids Research 22:4673-4680 (1994); larkin MA et al, "Clustal W and Clustal X version 2.0," Bioinformation 23:2947-48 (2007); and Altschul et al, "Basic local alignment search tool." J.mol. Biol.215:403-410 (1990), the entire contents and disclosures of which are incorporated herein by reference.
As used herein, the term "percent complementarity" or "percent complementarity" with respect to two nucleotide sequences is similar to the concept of percent identity, but refers to the percent of nucleotides of a query sequence that optimally base pair or hybridize to nucleotides of a subject sequence when the query sequence and the subject sequence are aligned and optimally base pair without a secondary folding structure (such as a loop, stem, or hairpin). Such percent complementarity may be between two DNA strands, two RNA strands, or one DNA strand and one RNA strand. "percent complementarity" may be calculated by: (i) optimally base pairing or hybridizing two nucleotide sequences in a linear and fully extended arrangement (i.e., without folding or secondary structure) over a comparison window, (ii) determining the number of base pair positions between the two sequences over the comparison window to produce a number of complementary positions, (iii) dividing the number of complementary positions by the total number of positions in the comparison window, and (iv) multiplying the quotient by 100% to yield the percent complementarity of the two sequences. The optimal base pairing of two sequences can be determined by hydrogen bonding based on known pairing of nucleotide bases, such as G-C, A-T and A-U. If the "percent complementarity" is calculated relative to the reference sequence without specifying a particular comparison window, the percent identity is determined by dividing the number of positions of complementarity between the two linear sequences by the total length of the reference sequence. Thus, for the purposes of the present application, when two sequences (query and subject) are optimally base paired (nucleotides that allow for mismatch or non-base pairing), the "percent complementarity" of the query sequence is equal to the number of base pairing positions between the two sequences divided by the total number of positions of the query sequence over its length, and then multiplied by 100%.
The use of the terms "polynucleotide", "nucleic acid sequence" or "nucleic acid molecule" is not intended to limit the present disclosure to polynucleotides comprising deoxyribonucleic acid (DNA). For example, ribonucleic acid (RNA) molecules are also contemplated. One of ordinary skill in the art will recognize that polynucleotides and nucleic acid molecules may comprise a combination of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogs. Polynucleotides of the present disclosure also encompass all forms of sequences, including but not limited to single stranded forms, double stranded forms, hairpins, stem loop structures, and the like. In one aspect, the nucleic acid molecules provided herein are DNA molecules. In one aspect, the nucleic acid molecules provided herein are RNA molecules. In one aspect, the nucleic acid molecules provided herein are single stranded. In one aspect, the nucleic acid molecules provided herein are double-stranded. The nucleic acid molecule may encode a polypeptide or a small RNA.
As used herein, the term "polypeptide" refers to a chain of at least two covalently linked amino acids. The polypeptide may be encoded by a polynucleotide provided herein. The proteins provided herein may be encoded by the nucleic acid molecules provided herein. The protein may comprise a polypeptide provided herein. As used herein, "protein" refers to a chain of amino acid residues capable of providing a cell with structural or enzymatic activity. As used herein, "coding sequence" refers to a nucleic acid sequence that encodes a protein.
As used herein, the term "CpG site" or "CG site" refers to a region of DNA sequence in which cytosine and guanine are separated by only one phosphate group.
As used herein, the term "CpG island" of a "CG island" refers to CpG sites that occur at a high frequency.
As used herein, the term "codon" refers to a sequence of three nucleotides.
As used herein, the term "codon optimized" refers to codons modified to enhance expression in a host cell of interest by replacing at least one codon of the sequence with a more or most frequently used codon in the gene of the host cell while maintaining the original amino acid sequence.
As used herein, the term "enhancer" refers to a region of DNA sequence that operates to initiate, assist, influence, cause and/or promote transcription and expression of an associated transcribable DNA sequence or coding sequence in at least some tissues, developmental stages and/or conditions. In one aspect, the enhancer is a cis-enhancer. In one aspect, the enhancer is a trans-enhancer.
Enhancer sequences can be identified by utilizing genomic techniques well known in the art. Non-limiting examples include the use of reporter genes and next generation sequencing methods such as chromatin co-immunoprecipitation sequencing (ChIP-seq), DNase I hypersensitivity sequencing (DNase-seq), micrococcus nuclease sequencing (MNase-seq), formaldehyde assisted separation regulatory element sequencing (faie-seq), and chromatin transposase accessibility sequencing assays (ATAC-seq).
As used herein, the term "operably linked" refers to a functional linkage between a promoter or other regulatory element and an associated transcribable DNA sequence or coding sequence of a gene (or transgene) such that the promoter or the like operates to initiate, assist, affect, cause and/or promote transcription and expression of the associated transcribable DNA sequence or coding sequence at least in certain tissues, developmental stages and/or conditions. As used herein, "regulatory element" refers to any sequence element that positively or negatively regulates expression of an operably linked sequence. "regulatory elements" include, but are not limited to, promoters, enhancers, leader sequences, transcription initiation sites (TSS), linkers, 5 'and 3' untranslated regions (UTRs), introns, polyadenylation signals and termination regions or sequences, and the like, which are suitable, necessary or preferred for regulating or allowing expression of a gene or transcribable DNA sequence in a cell. Such additional regulatory elements may be optional and used to enhance or optimize expression of the gene or transcribable DNA sequence.
As used herein, the term "promoter" refers to a DNA sequence that contains an RNA polymerase binding site, a transcription initiation site, and/or a TATA box and that assists or facilitates transcription and expression of an associated transcribable polynucleotide sequence and/or gene (or transgene). Promoters may be synthetically produced, altered, or derived from known or naturally occurring promoter sequences or other promoter sequences. Promoters may also include chimeric promoters comprising a combination of two or more heterologous sequences. Thus, promoters of the application may include variants of promoter sequences that are similar in composition to, but not identical to, other promoter sequences known or provided herein.
As used herein, "intron" refers to a nucleotide sequence that is removed by RNA splicing when messenger RNA (mRNA) is mature from a pre-mRNA.
As used herein, "mRNA" or "messenger RNA" refers to single stranded RNA corresponding to the genetic sequence of a gene.
mRNA expression can be measured using any suitable method known in the art. Non-limiting examples of measuring mRNA expression include quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), northern blotting (e.g., northern blotting), and RNA sequencing. The expression difference may be described as absolute quantization or relative quantization. See, e.g., livak and Schmittgen, methods,25:402-408 (2001).
As used herein, "genome editing" or "gene editing" refers to targeted mutagenesis, insertion, deletion, inversion, substitution, or translocation of a nucleotide sequence of interest in a genome using targeted editing techniques. The nucleotide sequence of interest may be 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 techniques are small interfering RNA (siRNA) techniques, small hairpin RNA (shRNA) techniques, microrna (miRNA) techniques, antisense oligonucleotide (ASO) techniques or CRISPR/CAS techniques.
As used herein, an "ASO" or "antisense oligonucleotide" is a small single stranded nucleic acid that binds to a target RNA sequence within a cell and silences a gene.
As used herein, "coding region," "gene region," or "gene" refers to a polynucleotide that can produce a functional unit. Non-limiting examples include protein or non-coding RNA molecules. A "coding region," "gene," or "gene region" may comprise a promoter, enhancer sequence, leader sequence, transcription start site, transcription termination site, polyadenylation site, one or more exons, one or more introns, 5'-UTR, 3' -UTR, or any combination thereof.
In one aspect, the gene edits huntingtin (Htt) aggregates targeted to the mutation. In one aspect, gene editing is performed by non-coding RNA molecules. Non-limiting examples of non-coding RNA molecules include micrornas (mirnas), miRNA precursors (pre-mirnas), small interfering RNAs (sirnas), small RNAs (18-26 nucleotides in length) and precursors encoding them, heterochromatic sirnas (hc-sirnas), piwi interacting RNAs (pirnas), hairpin double-stranded RNAs (hairpin dsRNA), trans-acting sirnas (ta-sirnas), naturally-occurring antisense sirnas (nat-sirnas), CRISPR RNA (crrnas), tracer RNAs (tracrrnas), guide RNAs (grnas), and unidirectional guide RNAs (sgrnas). In one aspect, the shRNA targets the Htt gene. In one aspect, the siRNA targets the Htt gene. In one aspect, the ASO targets the Htt gene. In one aspect, the miRNA targets the Htt gene. In one aspect, the gRNA targets the Htt gene. In one aspect, the pre-miRNA targets the 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 NOS.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, a "donor molecule" or "donor sequence" is defined as a nucleic acid sequence that has been selected for site-directed, targeted insertion into the genome. In one aspect, the 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 molecule or donor sequence provided herein may be of any length. For example, a donor molecule or donor sequence 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 500 nucleotides, between 15 and 1000 nucleotides, between 15 and 5000 nucleotides, between 18 and 30 nucleotides, between 18 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 in length.
As used herein, "HTT" refers to Htt-specific guide RNA (gRNA) and/or donor sequences.
In one aspect, the present disclosure provides and includes a composition comprising an adeno-associated virus (AAV) vector, wherein the AAV vector comprises a Cas9 nuclease gene, htt-specific gRNA, and a donor sequence. In one aspect, the present disclosure provides and includes a composition comprising an adeno-associated virus (AAV) vector, wherein the AAV vector comprises a Cas9 nuclease gene, htt-specific gRNA, a donor sequence, and a Dlx gene sequence. In one aspect, the present disclosure provides and includes a composition comprising an adeno-associated virus (AAV) vector, wherein the AAV vector comprises a Cas9 nuclease gene, htt-specific shRNA, and a donor sequence. In one aspect, the present disclosure provides and includes a composition comprising an adeno-associated virus (AAV) vector, wherein the AAV vector comprises a Cas9 nuclease gene, htt-specific shRNA, donor sequence, and Dlx gene sequence.
The site-specific nucleases provided herein can be used as part of a targeted editing technique. Non-limiting examples of site-specific nucleases include meganucleases, zinc Finger Nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), RNA-guided nucleases (e.g., cas9 and Cpf 1), recombinases (e.g., without limitation, serine recombinases attached to DNA recognition motifs, tyrosine recombinases attached to DNA recognition motifs), transposases (e.g., without limitation, DNA transposases attached to DNA binding domains), or any combination thereof.
Site-specific nucleases such as meganucleases, ZFN, TALEN, argonaute proteins (non-limiting examples of Argonaute proteins include thermophilic thermus Argonaute (Thermus thermophilus Argonaute, ttAgo), thermophilic archaebacteria Argonaute (Pyrococcus furiosus Argonaute, pfAgo), halophila griseus Argonaute (Natronobacterium gregoryi Argonaute, ngAgo), homologues thereof or modified versions thereof), cas9 nuclease (non-limiting examples of RNA-guided nucleases include Cas1, cas1B, cas2, cas3, cas4, cas5, cas6, cas7, cas8, cas9 (also known as Csn1 and Csx 12), cas10, csy1, csy2, csy3, cse1, cse2, csc1, csc2, csa5, csn2, 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, sy, homologues thereof) at the genomic DNA sequence), and then repair the double stranded site by natural processes. Sequence modifications then occur at the cleavage site, which may include inversions, deletions or insertions resulting in gene disruption or nucleic acid sequence integration. In one aspect, the RNA-guided nucleases provided herein are selected from the group consisting of: cas1, cas1B, cas2, cas3, cas4, cas5, cas6, cas7, cas8, cas9 (also known as Csn1 and Csx 12), cas10, csy1, csy2, csy3, cse1, cse2, csc1, csc2, csa5, csn2, 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 include the use of RNA-guided nucleases.
While not being bound by any particular scientific theory, CRISPR/CAS nucleases are part of the bacterial and archaeal adaptive immune system that are protected from the invasion of nucleic acids such as viruses by cleaving the target DNA in a sequence-dependent manner. Immunity is obtained by integrating short stretches of invaded DNA, called spacers, between CRISPR repeats approximately 20 nucleotides long proximal to the CRISPR locus (CRISPR array). A well-described Cas protein is Cas9 nuclease (also known as Csn 1), which is part of a class 2 type II CRISPR/Cas system in streptococcus pyogenes (Streptococcus pyogenes). See Makarova et al Nature Reviews Microbiology (2015) doi 10.1038/nrmicro3569.Cas9 comprises a RuvC-like nuclease domain at its amino terminus and an HNH-like nuclease domain in the middle of the protein. Cas9 proteins also contain PAM Interaction (PI) domains, recognition leaf (REC) and BH domains. Cpf1 nuclease is another type II system that functions 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. Pat. nos. 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 by reference herein in its entirety.
As used herein, the term "glia" or "glial cell" refers to a non-neuronal cell in the CNS or PNS. In one aspect, the at least one glial cell is selected from the group consisting of: at least one oligodendrocyte, at least one astrocyte, at least one NG2 cell, at least one ependymal cell, and at least one microglial cell. In one aspect, the at least one glial cell is at least one oligodendrocyte. In one aspect, the at least one glial cell is at least one NG2 cell. In one aspect, the at least one glial cell is at least one ependymal cell. In one aspect, the at least one glial cell is at least one microglial cell. In one aspect, the at least one glial cell is at least one reactive astrocyte. In one aspect, the at least one astrocyte is at least one reactive astrocyte.
As used herein, the term "astrocyte" refers to a glial cell that is an important component of the brain. Astrocytes are involved in supporting neuronal functions such as synapse formation and plasticity, potassium buffering, nutrient supply, secretion and absorption of neural or glial transmitters, and maintenance of the blood brain barrier. As used herein, the term "reactive astrocytes" refers to an abnormal state of astrocytes after injury or disease.
As used herein, the term "NG2 cells" or "oligodendrocytes" refers to glial cells that express chondroitin sulfate proteoglycan (CSPG 4) and platelet derived growth factor alpha receptor (PDGFRA).
As used herein, the term "neuron" or "neuronal cell" refers to an electrically excitable cell that communicates with other neurons through synapses. In one aspect, the neuron is selected from the group consisting of a unipolar neuron, a bipolar neuron, a pseudounipolar neuron, and a multipolar neuron. In one aspect, the neuron is a unipolar neuron. In one aspect, the neuron is a bipolar neuron. In one aspect, the neuron is a pseudounipolar neuron. In one aspect, the neuron is a bipolar neuron. In one aspect, the neuron is selected from the group consisting of a sensory neuron, a motor neuron, and an interneuron. In one aspect, the neuron is a sensory neuron. In one aspect, the neuron is a motor neuron. In one aspect, the neuron is an interneuron.
As used herein, the term "functional neuron" refers to a neuron that can perform a biological process. Examples of biological processes include, but are not limited to, the processing and transmission of information and communication via chemical and electrical synapses.
As used herein, the term "glutamatergic neuron" refers to a subset of neurons that produce glutamate and establish excitatory synapses. As used herein, the term "excitatory synapse" refers to a synapse where an action potential in a presynaptic neuron increases the probability of an action potential occurring in a postsynaptic cell. As used herein, the term "action potential" or "nerve impulse" refers to an electrical pulse that passes through the axon membrane. As used herein, the term "axon" or "nerve fiber" refers to a neuron that conducts action potentials. As used herein, the term "gabaergic neuron" refers to a subset of neurons that produce GABA and establish inhibitory synapses. As used herein, the term "GABA" or "gamma-aminobutyric acid" refers to a compound that opens an ion channel to allow negatively charged chloride ions to flow into cells or positively charged potassium ions to flow out of cells. As used herein, the term "inhibitory synapse" refers to a synapse that brings the membrane potential of a post-synaptic neuron away from a threshold at which action potentials are generated. As used herein, the term "dopaminergic neuron" refers to a subset of neurons that produce dopamine. As used herein, the term "dopamine" refers to neurotransmitters. As used herein, the term "neurotransmitter" refers to endogenous chemicals that activate neurotransmission. As used herein, the term "neurotransmission" refers to the process of releasing neurotransmitters from the axon terminals of neurons. As used herein, the term "acetylcholine-producing neuron" or "cholinergic neuron" refers to a subset of neurons that secrete acetylcholine. As used herein, the term "acetylcholine" refers to neurotransmitters. As used herein, the term "serotonergic neuron" refers to a subset of neurons that synthesize serotonin. As used herein, the term "serotonin" refers to neurotransmitters. As used herein, "adrenergic neuron" refers to a neuron that releases epinephrine as a neurotransmitter. As used herein, the term "motor neuron" refers to a subset of neurons in which the cell body is located in the motor cortex, brain stem, or spinal cord and axons project outside the spinal cord or spinal cord and directly or indirectly control muscles and glands. As used herein, the term peptide energy neuron refers to a subset of neurons that utilize small peptide molecules as their neurotransmitters.
In one aspect, the neuron is a functional neuron. In one aspect, the functional neuron is selected from the group consisting of a glutamatergic neuron, a gabaergic neuron, a dopaminergic neuron, a cholinergic neuron, a serotonergic neuron, an adrenergic neuron, a motor neuron, and a peptidoergic neuron. In one aspect, the functional neuron is a glutamatergic neuron. In one aspect, the functional neuron is a gabaergic neuron. In one aspect, the functional neuron is a dopaminergic neuron. In one aspect, the functional neuron is a cholinergic neuron. In one aspect, the functional neuron is a serotonergic neuron. In one aspect, the functional neuron is an adrenergic neuron. In one aspect, the functional neuron is a motor neuron. In one aspect, the functional neuron is a peptide energy neuron.
As used herein, the term "transformed" or "transformed" refers to a cell type that changes its physical form and/or biological function to a different physical form and/or different biological function. In one aspect, the present disclosure provides for the conversion of at least one glial cell to at least one neuron. In one aspect, the conversion of at least one glial cell to at least one neuron occurs in the CNS or PNS. In one aspect, the conversion of at least one glial cell to at least one neuron occurs in the CNS. In one aspect, the conversion of at least one glial cell to at least one neuron occurs in PNS.
In one aspect, the present disclosure provides and includes an adeno-associated virus (AAV) vector comprising: a human neurogenic differentiation factor 1 (hNeuroD 1) sequence, the hNeuroD1 sequence comprising the nucleic acid sequence of SEQ ID NO. 6, and a human distantly related homeobox 2 (hDlx 2) sequence, the hDlx2 sequence comprising the 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 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.
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 (hNeuroD 1) protein, the hNeuroD1 protein comprising the 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 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 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 28; (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.
In one aspect, the present disclosure provides and includes an adeno-associated virus (AAV) vector comprising a NeuroD1 nucleic acid encoding a NeuroD1 (NeuroD 1) protein and a Dlx nucleic acid encoding a distantly related homeobox 2 (Dlx 2) protein, wherein the NeuroD1 encoding sequence and Dlx2 encoding sequence are separated by a linker sequence, wherein the NeuroD1 encoding sequence and Dlx encoding sequence are operably linked to a regulatory element comprising: (a) a Glial Fibrillary Acidic Protein (GFAP) promoter; (b) an enhancer; (c) a chimeric intron; (d) Woodchuck hepatitis virus posttranscriptional regulatory elements (WPREs); and (e) a polyadenylation signal sequence.
In one aspect, the present disclosure provides and includes a composition comprising an adeno-associated virus (AAV) vector for converting human glial cells to functional neurons, wherein the AAV vector comprises: a human neurogenic differentiation factor 1 (hNeuroD 1) sequence, the hNeuroD1 sequence having the nucleic acid sequence of SEQ ID NO. 6, and a human distantly related homeobox 2 (hDlx 2) sequence, the hDlx2 sequence having the 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.
In one aspect, the present disclosure provides and includes a composition comprising an adeno-associated virus (AAV) vector for converting human glial cells to functional neurons, wherein the AAV vector comprises: a nucleic acid coding sequence encoding a human neurogenic differentiation factor 1 (hNeuroD 1) protein, the hNeuroD1 protein comprising the amino acid sequence of SEQ ID No. 10, and a nucleic acid coding sequence encoding a human distantly related homeobox 2 (hDlx 2) 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 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.
In one aspect, the present disclosure provides and includes a composition comprising an adeno-associated virus (AAV) vector for treating a subject in need thereof, wherein the AAV vector comprises a neurogenic differentiation factor 1 (NeuroD 1) sequence and a 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 Dlx sequence are operably linked to an expression control element comprising: (a) a Glial Fibrillary Acidic Protein (GFAP) promoter; (b) an enhancer; (c) a chimeric intron; (d) Woodchuck hepatitis virus posttranscriptional regulatory elements (WPREs); and (e) a polyadenylation signal.
In one aspect, an AAV vector comprises a nucleic acid sequence encoding an AAV protein. In one aspect, the AAV vector comprises a nucleic acid sequence encoding a viral protein. Non-limiting examples of AAV proteins and viral proteins include rep and cap proteins.
NeuroD1 (also known as β2) is a basic helix-loop-helix (bHLH) transcription factor that forms heterodimers with other bHLH proteins to activate gene transcription containing a DNA sequence known as an E-cassette.
In one aspect, the NeuroD1 sequence is a human NeuroD1 (hNeuroD 1) sequence. In one aspect, the NeuroD1 sequence is selected from the group consisting of: a chimpanzee NeuroD1 sequence, a bonobo NeuroD1 sequence, a chimpanzee NeuroD1 sequence, a gorilla NeuroD1 sequence, a macaque NeuroD1 sequence, a marmoset NeuroD1 sequence, a pigtail monkey NeuroD1 sequence, a baboon NeuroD1 sequence, a gibbon NeuroD1 sequence, and a marmoset NeuroD1 sequence. In one aspect, the NeuroD1 sequence is a chimpanzee NeuroD1 sequence. In one aspect, the NeuroD1 sequence is a bonobo chimpanzee NeuroD1 sequence. In one aspect, the NeuroD1 sequence is a chimpanzee NeuroD1 sequence. In one aspect, the NeuroD1 sequence is a gorilla NeuroD1 sequence. In one aspect, the NeuroD1 sequence is a cynomolgus monkey NeuroD1 sequence. In one aspect, the NeuroD1 sequence is a marmoset NeuroD1 sequence. In one aspect, the NeuroD1 sequence is a cynomolgus monkey NeuroD1 sequence. In one aspect, the NeuroD1 sequence is a baboon NeuroD1 sequence. In one aspect, the NeuroD1 sequence is a gibbon NeuroD1 sequence. In one aspect, the NeuroD1 sequence is a cynomolgus monkey NeuroD1 sequence.
In one aspect, the NeuroD1 nucleic acid sequence comprises a sequence at least 70% identical to: SEQ ID NO. 6, or a complement thereof. In one aspect, the NeuroD1 nucleic acid sequence comprises a sequence at least 75% identical to: SEQ ID NO. 6, or a complement thereof. In one aspect, the NeuroD1 nucleic acid sequence comprises a sequence at least 80% identical to: SEQ ID NO. 6, or a complement thereof. In one aspect, the NeuroD1 nucleic acid sequence comprises a sequence at least 85% identical to: SEQ ID NO. 6, or a complement thereof. In one aspect, the NeuroD1 nucleic acid sequence comprises a sequence at least 90% identical to: SEQ ID NO. 6, or a complement thereof. In one aspect, the NeuroD1 nucleic acid sequence comprises a sequence at least 91% identical to: SEQ ID NO. 6, or a complement thereof. In one aspect, the NeuroD1 nucleic acid sequence comprises a sequence at least 92% identical to: SEQ ID NO. 6, or a complement thereof. In one aspect, the NeuroD1 nucleic acid sequence comprises a sequence at least 93% identical to: SEQ ID NO. 6, or a complement thereof. In one aspect, the NeuroD1 nucleic acid sequence comprises a sequence at least 94% identical to: SEQ ID NO. 6, or a complement thereof. In one aspect, the NeuroD1 nucleic acid sequence comprises a sequence at least 95% identical to: SEQ ID NO. 6, or a complement thereof. In one aspect, the NeuroD1 nucleic acid sequence comprises a sequence at least 96% identical to: SEQ ID NO. 6, or a complement thereof. In one aspect, the NeuroD1 nucleic acid sequence comprises a sequence at least 97% identical to: SEQ ID NO. 6, or a complement thereof. In one aspect, the NeuroD1 nucleic acid sequence comprises a sequence at least 98% identical to: SEQ ID NO. 6, or a complement thereof. In one aspect, the NeuroD1 nucleic acid sequence comprises a sequence at least 99% identical to: SEQ ID NO. 6, or a complement thereof. In one aspect, the NeuroD1 nucleic acid sequence comprises a sequence at least 99.5% identical to: SEQ ID NO. 6, or a complement thereof. In one aspect, the NeuroD1 nucleic acid sequence comprises a sequence at least 99.8% identical to: SEQ ID NO. 6, or a complement thereof. In one aspect, the NeuroD1 nucleic acid sequence comprises a sequence at least 99.9% identical to: SEQ ID NO. 6, or a complement thereof. In one aspect, the NeuroD1 nucleic acid sequence comprises a sequence 100% identical to: SEQ ID NO. 6, or a complement thereof.
In one aspect, the nucleic acid coding sequence encodes a NeuroD1 protein comprising an amino acid sequence at least 70% identical or similar to: SEQ ID NO. 10. In one aspect, the nucleic acid encoding sequence encodes a NeuroD1 protein comprising an amino acid sequence at least 75% identical or similar to: SEQ ID NO. 10. In one aspect, the nucleic acid encoding sequence encodes a NeuroD1 protein comprising an amino acid sequence at least 80% identical or similar to: SEQ ID NO. 10. In one aspect, the nucleic acid encoding sequence encodes a NeuroD1 protein comprising an amino acid sequence at least 85% identical or similar to: SEQ ID NO. 10. In one aspect, the nucleic acid encoding sequence encodes a NeuroD1 protein comprising an amino acid sequence at least 90% identical or similar to: SEQ ID NO. 10. In one aspect, the nucleic acid encoding sequence encodes a NeuroD1 protein comprising an amino acid sequence at least 91% identical or similar to: SEQ ID NO. 10. In one aspect, the nucleic acid encoding sequence encodes a NeuroD1 protein comprising an amino acid sequence at least 92% identical or similar to: SEQ ID NO. 10. In one aspect, the nucleic acid encoding sequence encodes a NeuroD1 protein comprising an amino acid sequence at least 93% identical or similar to: SEQ ID NO. 10. In one aspect, the nucleic acid encoding sequence encodes a NeuroD1 protein comprising an amino acid sequence at least 94% identical or similar to: SEQ ID NO. 10. In one aspect, the nucleic acid encoding sequence encodes a NeuroD1 protein comprising an amino acid sequence at least 95% identical or similar to: SEQ ID NO. 10. In one aspect, the nucleic acid coding sequence encodes a NeuroD1 protein comprising an amino acid sequence at least 96% identical or similar to: SEQ ID NO. 10. In one aspect, the nucleic acid encoding sequence encodes a NeuroD1 protein comprising an amino acid sequence at least 97% identical or similar to: SEQ ID NO. 10. In one aspect, the nucleic acid encoding sequence encodes a NeuroD1 protein comprising an amino acid sequence at least 98% identical or similar to: SEQ ID NO. 10. In one aspect, the nucleic acid coding sequence encodes a NeuroD1 protein comprising an amino acid sequence at least 99% identical or similar to: SEQ ID NO. 10. In one aspect, the nucleic acid encoding 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 encoding 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 encoding 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.
Non-distal homeobox 2 (Dlx; also known as TES 1) is a member of the Dlx gene family and is a homeobox containing genes that play a role in forebrain and craniofacial development.
In one aspect, the Dlx2 sequence is a human Dlx2 (hDlx 2) sequence. In one aspect, the Dlx sequence is selected from the group consisting of a chimpanzee Dlx2 sequence, a bonobo Dlx2 sequence, a chimpanzee Dlx2 sequence, a gorilla Dlx2 sequence, a macaque Dlx sequence, a marmoset Dlx2 sequence, a pigtail Dlx sequence, a baboon Dlx2 sequence, a gibbon Dlx2 sequence, and a marmoset Dlx2 sequence. In one aspect, the Dlx2 sequence is a chimpanzee Dlx2 sequence. In one aspect, the Dlx sequence is a bonobo Dlx sequence. In one aspect, the Dlx sequence is a chimpanzee Dlx2 sequence. In one aspect, the Dlx sequence is a gorilla Dlx2 sequence. In one aspect, the Dlx sequence is the macaque Dlx sequence. In one aspect, the Dlx sequence is a marmoset Dlx2 sequence. In one aspect, the Dlx sequence is a monkey Dlx2 sequence. In one aspect, the Dlx sequence is a baboon Dlx2 sequence. In one aspect, the Dlx sequence is an gibbon Dlx2 sequence. In one aspect, the Dlx sequence is the marmoset Dlx sequence.
In one aspect, the Dlx2 nucleic acid sequence comprises a sequence that is at least 70% identical to: SEQ ID NO. 13, or a complement thereof. In one aspect, the Dlx2 nucleic acid sequence comprises a sequence that is at least 75% identical to: SEQ ID NO. 13, or a complement thereof. In one aspect, the Dlx2 nucleic acid sequence comprises a sequence at least 80% identical to: SEQ ID NO. 13, or a complement thereof. In one aspect, the Dlx2 nucleic acid sequence comprises a sequence at least 85% identical to: SEQ ID NO. 13, or a complement thereof. In one aspect, the Dlx2 nucleic acid sequence comprises a sequence at least 90% identical to: SEQ ID NO. 13, or a complement thereof. In one aspect, the Dlx2 nucleic acid sequence comprises a sequence that is at least 91% identical to: SEQ ID NO. 13, or a complement thereof. In one aspect, the Dlx2 nucleic acid sequence comprises a sequence at least 92% identical to: SEQ ID NO. 13, or a complement thereof. In one aspect, the Dlx2 nucleic acid sequence comprises a sequence at least 93% identical to: SEQ ID NO. 13, or a complement thereof. In one aspect, the Dlx2 nucleic acid sequence comprises a sequence at least 94% identical to: SEQ ID NO. 13, or a complement thereof. In one aspect, the Dlx2 nucleic acid sequence comprises a sequence at least 95% identical to: SEQ ID NO. 13, or a complement thereof. In one aspect, the Dlx2 nucleic acid sequence comprises a sequence that is at least 913% identical to: SEQ ID NO. 13, or a complement thereof. In one aspect, the Dlx2 nucleic acid sequence comprises a sequence at least 97% identical to: SEQ ID NO. 13, or a complement thereof. In one aspect, the Dlx2 nucleic acid sequence comprises a sequence at least 98% identical to: SEQ ID NO. 13, or a complement thereof. In one aspect, the Dlx2 nucleic acid sequence comprises a sequence that is at least 99% identical to: SEQ ID NO. 13, or a complement thereof. In one aspect, the Dlx2 nucleic acid sequence comprises a sequence at least 99.5% identical to: SEQ ID NO. 13, or a complement thereof. In one aspect, the Dlx2 nucleic acid sequence comprises a sequence at least 99.8% identical to: SEQ ID NO. 13, or a complement thereof. In one aspect, the Dlx2 nucleic acid sequence comprises a sequence at least 99.9% identical to: SEQ ID NO. 13, or a complement thereof. In one aspect, the Dlx2 nucleic acid sequence comprises a sequence 100% identical to: SEQ ID NO. 13, or a complement thereof.
In one aspect, the nucleic acid encoding 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 encoding sequence encodes a Dlx 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 encoding sequence encodes a Dlx 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 encoding sequence encodes a Dlx 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 encoding sequence encodes a Dlx 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 encoding sequence encodes a Dlx 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 encoding sequence encodes a Dlx 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 encoding sequence encodes a Dlx 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 encoding sequence encodes a Dlx 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 encoding sequence encodes a Dlx 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 encoding sequence encodes a Dlx 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 encoding sequence encodes a Dlx 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 encoding sequence encodes a Dlx 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 encoding sequence encodes a Dlx 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 encoding sequence encodes a Dlx protein comprising an amino acid sequence that is at least 99.5% identical or similar to: SEQ ID NO. 14. In one aspect, the nucleic acid encoding sequence encodes a Dlx protein comprising an amino acid sequence that is at least 99.8% identical or similar to: SEQ ID NO. 14. In one aspect, the nucleic acid encoding sequence encodes a Dlx protein comprising an amino acid sequence that is at least 99.9% identical or similar to: SEQ ID NO. 14. In one aspect, the nucleic acid encoding sequence encodes a Dlx 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 Dlx sequence. In one aspect, the AAV vector comprises a NeuroD1 sequence. In one aspect, the AAV comprises a Dlx sequence.
As used herein, a "linker" or "spacer" is a short sequence that separates multiple proteins and coding domains. The linker may be cleavable or non-cleavable and facilitates the co-expression of multiple genes in a single vector.
As used herein, a "2A self-cleaving peptide" or "2A peptide" is a class of linkers that can induce cleavage of a recombinant protein in a cell.
As used herein, "P2A linker", "P2A" or "P2A" refers to a porcine teschovirus-1 (P2A) linker, which is a member of the 2A self-cleaving peptide.
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 the following nucleic acid sequence: SEQ ID NO. 15. In one aspect, the P2A linker has the following nucleic acid sequence: SEQ ID NO. 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 the following nucleic acid sequence: SEQ ID NO. 18. In one aspect, the P2A linker protein has the following nucleic acid coding sequence: SEQ ID NO. 20.
As used herein, a "T2A linker" is a linker that designates the vein flatworm virus 2A (thosea asigna virus a, T2A), which is a member of the 2A self-cleaving peptide.
In one aspect, the T2A linker has a nucleic acid sequence selected from the group consisting of: SEQ ID NOS 16 and 19. In one aspect, the T2A linker has the following nucleic acid sequence: SEQ ID NO. 16. In one aspect, the T2A linker has the following nucleic acid sequence: SEQ ID NO. 19. In one aspect, the T2A linker protein has the following nucleic acid coding sequence: SEQ ID NO. 21
As used herein, "E2A linker" refers to a equine rhinitis a virus (E2A) linker, which is a member of the 2A self-cleaving peptide.
As used herein, "F2A linker" refers to a hand-foot-and-mouth virus (F2A) linker, which is a member of the 2A self-cleaving peptide.
In one aspect, the linker is selected from the group consisting of a P2A linker, a T2A linker, an E2A linker, and an F2A linker. In one aspect, the linker is a P2A linker. In one aspect, the linker is a T2A linker. In one aspect, the linker is an E2A linker. In one aspect, the linker is an F2A linker.
In one aspect, the linker sequence 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 a complement thereof. In one aspect, the P2A linker nucleic acid sequence comprises a sequence that is at least 75% identical to: SEQ ID NO. 15, or a complement thereof. In one aspect, the P2A linker nucleic acid sequence comprises a sequence at least 80% identical to: SEQ ID NO. 15, or a complement thereof. In one aspect, the P2A linker nucleic acid sequence comprises a sequence that is at least 85% identical to: SEQ ID NO. 15, or a complement thereof. In one aspect, the P2A linker nucleic acid sequence comprises a sequence at least 90% identical to: SEQ ID NO. 15, or a complement thereof. In one aspect, the P2A linker nucleic acid sequence comprises a sequence that is at least 91% identical to: SEQ ID NO. 15, or a complement thereof. In one aspect, the P2A linker nucleic acid sequence comprises a sequence at least 92% identical to: SEQ ID NO. 15, or a complement thereof. In one aspect, the P2A linker nucleic acid sequence comprises a sequence at least 93% identical to: SEQ ID NO. 15, or a complement thereof. In one aspect, the P2A linker nucleic acid sequence comprises a sequence at least 94% identical to: SEQ ID NO. 15, or a complement thereof. In one aspect, the P2A linker nucleic acid sequence comprises a sequence at least 95% identical to: SEQ ID NO. 15, or a complement thereof. In one aspect, the P2A linker nucleic acid sequence comprises a sequence at least 96% identical to: SEQ ID NO. 15, or a complement thereof. In one aspect, the P2A linker nucleic acid sequence comprises a sequence at least 97% identical to: SEQ ID NO. 15, or a complement thereof. In one aspect, the P2A linker nucleic acid sequence comprises a sequence that is at least 98% identical to: SEQ ID NO. 15, or a complement thereof. In one aspect, the P2A linker nucleic acid sequence comprises a sequence that is at least 99% identical to: SEQ ID NO. 15, or a complement thereof. In one aspect, the P2A linker nucleic acid sequence comprises a sequence that is at least 99.5% identical to: SEQ ID NO. 15, or a complement thereof. In one aspect, the P2A linker nucleic acid sequence comprises a sequence that is at least 99.8% identical to: SEQ ID NO. 15, or a complement thereof. In one aspect, the P2A linker nucleic acid sequence comprises a sequence that is at least 99.9% identical to: SEQ ID NO. 15, or a complement thereof. In one aspect, the P2A linker nucleic acid sequence comprises a sequence 100% identical to: SEQ ID NO. 15, or a complement thereof.
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: 18, or the complement thereof. In one aspect, the P2A linker nucleic acid sequence comprises a sequence that is at least 75% identical to: 18, or the complement thereof. In one aspect, the P2A linker nucleic acid sequence comprises a sequence at least 80% identical to: 18, or the complement thereof. In one aspect, the P2A linker nucleic acid sequence comprises a sequence that is at least 85% identical to: 18, or the complement thereof. In one aspect, the P2A linker nucleic acid sequence comprises a sequence at least 90% identical to: 18, or the complement thereof. In one aspect, the P2A linker nucleic acid sequence comprises a sequence that is at least 91% identical to: 18, or the complement thereof. In one aspect, the P2A linker nucleic acid sequence comprises a sequence at least 92% identical to: 18, or the complement thereof. In one aspect, the P2A linker nucleic acid sequence comprises a sequence at least 93% identical to: 18, or the complement thereof. In one aspect, the P2A linker nucleic acid sequence comprises a sequence at least 94% identical to: 18, or the complement thereof. In one aspect, the P2A linker nucleic acid sequence comprises a sequence at least 95% identical to: 18, or the complement thereof. In one aspect, the P2A linker nucleic acid sequence comprises a sequence at least 96% identical to: 18, or the complement thereof. In one aspect, the P2A linker nucleic acid sequence comprises a sequence at least 97% identical to: 18, or the complement thereof. In one aspect, the P2A linker nucleic acid sequence comprises a sequence that is at least 98% identical to: 18, or the complement thereof. In one aspect, the P2A linker nucleic acid sequence comprises a sequence that is at least 99% identical to: 18, or the complement thereof. In one aspect, the P2A linker nucleic acid sequence comprises a sequence that is at least 99.5% identical to: 18, or the complement thereof. In one aspect, the P2A linker nucleic acid sequence comprises a sequence that is at least 99.8% identical to: 18, or the complement thereof. In one aspect, the P2A linker nucleic acid sequence comprises a sequence that is at least 99.9% identical to: 18, or the complement thereof. In one aspect, the P2A linker nucleic acid sequence comprises a sequence 100% identical to: 18, or the complement thereof.
In one aspect, the nucleic acid encoding sequence encodes a P2A protein comprising an amino acid sequence that is at least 70% identical or similar to: SEQ ID NO. 20. In one aspect, the nucleic acid encoding sequence encodes a P2A protein comprising an amino acid sequence that is at least 75% identical or similar to: SEQ ID NO. 20. In one aspect, the nucleic acid encoding sequence encodes a P2A protein comprising an amino acid sequence that is at least 80% identical or similar to: SEQ ID NO. 20. In one aspect, the nucleic acid encoding sequence encodes a P2A protein comprising an amino acid sequence that is at least 85% identical or similar to: SEQ ID NO. 20. In one aspect, the nucleic acid encoding sequence encodes a P2A protein comprising an amino acid sequence that is at least 90% identical or similar to: SEQ ID NO. 20. In one aspect, the nucleic acid encoding sequence encodes a P2A protein comprising an amino acid sequence that is at least 91% identical or similar to: SEQ ID NO. 20. In one aspect, the nucleic acid encoding sequence encodes a P2A protein comprising an amino acid sequence at least 92% identical or similar to: SEQ ID NO. 20. In one aspect, the nucleic acid encoding sequence encodes a P2A protein comprising an amino acid sequence that is at least 93% identical or similar to: SEQ ID NO. 20. In one aspect, the nucleic acid encoding sequence encodes a P2A protein comprising an amino acid sequence that is at least 94% identical or similar to: SEQ ID NO. 20. In one aspect, the nucleic acid encoding sequence encodes a P2A protein comprising an amino acid sequence at least 95% identical or similar to: SEQ ID NO. 20. In one aspect, the nucleic acid encoding sequence encodes a P2A protein comprising an amino acid sequence at least 96% identical or similar to: SEQ ID NO. 20. In one aspect, the nucleic acid encoding sequence encodes a P2A protein comprising an amino acid sequence that is at least 97% identical or similar to: SEQ ID NO. 20. In one aspect, the nucleic acid encoding sequence encodes a P2A protein comprising an amino acid sequence that is at least 98% identical or similar to: SEQ ID NO. 20. In one aspect, the nucleic acid encoding sequence encodes a P2A protein comprising an amino acid sequence that is at least 99% identical or similar to: SEQ ID NO. 20. In one aspect, the nucleic acid encoding sequence encodes a P2A protein comprising an amino acid sequence that is at least 99.5% identical or similar to: SEQ ID NO. 20. In one aspect, the nucleic acid encoding sequence encodes a P2A protein comprising an amino acid sequence that is at least 99.8% identical or similar to: SEQ ID NO. 20. In one aspect, the nucleic acid encoding sequence encodes a P2A protein comprising an amino acid sequence that is at least 99.9% identical or similar to: SEQ ID NO. 20. In one aspect, the nucleic acid encoding sequence encodes a P2A protein comprising an amino acid sequence 100% identical or similar to: SEQ ID NO. 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 a complement thereof. In one aspect, the T2A linker nucleic acid sequence comprises a sequence that is at least 75% identical to: SEQ ID NO. 16, or a complement thereof. In one aspect, the T2A linker nucleic acid sequence comprises a sequence at least 80% identical to: SEQ ID NO. 16, or a complement thereof. In one aspect, the T2A linker nucleic acid sequence comprises a sequence at least 85% identical to: SEQ ID NO. 16, or a complement thereof. In one aspect, the T2A linker nucleic acid sequence comprises a sequence at least 90% identical to: SEQ ID NO. 16, or a complement thereof. In one aspect, the T2A linker nucleic acid sequence comprises a sequence that is at least 91% identical to: SEQ ID NO. 16, or a complement thereof. In one aspect, the T2A linker nucleic acid sequence comprises a sequence at least 92% identical to: SEQ ID NO. 16, or a complement thereof. In one aspect, the T2A linker nucleic acid sequence comprises a sequence at least 93% identical to: SEQ ID NO. 16, or a complement thereof. In one aspect, the T2A linker nucleic acid sequence comprises a sequence at least 94% identical to: SEQ ID NO. 16, or a complement thereof. In one aspect, the T2A linker nucleic acid sequence comprises a sequence at least 95% identical to: SEQ ID NO. 16, or a complement thereof. In one aspect, the T2A linker nucleic acid sequence comprises a sequence at least 96% identical to: SEQ ID NO. 16, or a complement thereof. In one aspect, the T2A linker nucleic acid sequence comprises a sequence at least 97% identical to: SEQ ID NO. 16, or a complement thereof. In one aspect, the T2A linker nucleic acid sequence comprises a sequence that is at least 98% identical to: SEQ ID NO. 16, or a complement thereof. In one aspect, the T2A linker nucleic acid sequence comprises a sequence that is at least 99% identical to: SEQ ID NO. 16, or a complement thereof. In one aspect, the T2A linker nucleic acid sequence comprises a sequence that is at least 99.5% identical to: SEQ ID NO. 16, or a complement thereof. In one aspect, the T2A linker nucleic acid sequence comprises a sequence that is at least 99.8% identical to: SEQ ID NO. 16, or a complement thereof. In one aspect, the T2A linker nucleic acid sequence comprises a sequence that is at least 99.9% identical to: SEQ ID NO. 16, or a complement thereof. In one aspect, the T2A linker nucleic acid sequence comprises a sequence 100% identical to: SEQ ID NO. 16, or a complement thereof.
In one aspect, the linker sequence comprises a T2A linker. In one aspect, the T2A linker nucleic acid sequence comprises a sequence that is at least 70% identical to: SEQ ID NO. 19, or a complement thereof. In one aspect, the T2A linker nucleic acid sequence comprises a sequence that is at least 75% identical to: SEQ ID NO. 19, or a complement thereof. In one aspect, the T2A linker nucleic acid sequence comprises a sequence at least 80% identical to: SEQ ID NO. 19, or a complement thereof. In one aspect, the T2A linker nucleic acid sequence comprises a sequence at least 85% identical to: SEQ ID NO. 19, or a complement thereof. In one aspect, the T2A linker nucleic acid sequence comprises a sequence at least 90% identical to: SEQ ID NO. 19, or a complement thereof. In one aspect, the T2A linker nucleic acid sequence comprises a sequence that is at least 91% identical to: SEQ ID NO. 19, or a complement thereof. In one aspect, the T2A linker nucleic acid sequence comprises a sequence at least 92% identical to: SEQ ID NO. 19, or a complement thereof. In one aspect, the T2A linker nucleic acid sequence comprises a sequence at least 93% identical to: SEQ ID NO. 19, or a complement thereof. In one aspect, the T2A linker nucleic acid sequence comprises a sequence at least 94% identical to: SEQ ID NO. 19, or a complement thereof. In one aspect, the T2A linker nucleic acid sequence comprises a sequence at least 95% identical to: SEQ ID NO. 19, or a complement thereof. In one aspect, the T2A linker nucleic acid sequence comprises a sequence at least 96% identical to: SEQ ID NO. 19, or a complement thereof. In one aspect, the T2A linker nucleic acid sequence comprises a sequence at least 97% identical to: SEQ ID NO. 19, or a complement thereof. In one aspect, the T2A linker nucleic acid sequence comprises a sequence that is at least 98% identical to: SEQ ID NO. 19, or a complement thereof. In one aspect, the T2A linker nucleic acid sequence comprises a sequence that is at least 99% identical to: SEQ ID NO. 19, or a complement thereof. In one aspect, the T2A linker nucleic acid sequence comprises a sequence that is at least 99.5% identical to: SEQ ID NO. 19, or a complement thereof. In one aspect, the T2A linker nucleic acid sequence comprises a sequence that is at least 99.8% identical to: SEQ ID NO. 19, or a complement thereof. In one aspect, the T2A linker nucleic acid sequence comprises a sequence that is at least 99.9% identical to: SEQ ID NO. 19, or a complement thereof. In one aspect, the T2A linker nucleic acid sequence comprises a sequence 100% identical to: SEQ ID NO. 19, or a complement thereof.
In one aspect, the nucleic acid encoding sequence encodes a T2A protein comprising an amino acid sequence that is at least 70% identical or similar to: SEQ ID NO. 21. In one aspect, the nucleic acid encoding sequence encodes a T2A protein comprising an amino acid sequence that is at least 75% identical or similar to: SEQ ID NO. 21. In one aspect, the nucleic acid encoding sequence encodes a T2A protein comprising an amino acid sequence at least 80% identical or similar to: SEQ ID NO. 21. In one aspect, the nucleic acid encoding sequence encodes a T2A protein comprising an amino acid sequence that is at least 85% identical or similar to: SEQ ID NO. 21. In one aspect, the nucleic acid encoding sequence encodes a T2A protein comprising an amino acid sequence that is at least 90% identical or similar to: SEQ ID NO. 21. In one aspect, the nucleic acid encoding sequence encodes a T2A protein comprising an amino acid sequence that is at least 91% identical or similar to: SEQ ID NO. 21. In one aspect, the nucleic acid encoding sequence encodes a T2A protein comprising an amino acid sequence at least 92% identical or similar to: SEQ ID NO. 21. In one aspect, the nucleic acid encoding sequence encodes a T2A protein comprising an amino acid sequence that is at least 93% identical or similar to: SEQ ID NO. 21. In one aspect, the nucleic acid encoding sequence encodes a T2A protein comprising an amino acid sequence at least 94% identical or similar to: SEQ ID NO. 21. In one aspect, the nucleic acid encoding sequence encodes a T2A protein comprising an amino acid sequence at least 95% identical or similar to: SEQ ID NO. 21. In one aspect, the nucleic acid encoding sequence encodes a T2A protein comprising an amino acid sequence at least 96% identical or similar to: SEQ ID NO. 21. In one aspect, the nucleic acid encoding sequence encodes a T2A protein comprising an amino acid sequence at least 97% identical or similar to: SEQ ID NO. 21. In one aspect, the nucleic acid encoding sequence encodes a T2A protein comprising an amino acid sequence that is at least 98% identical or similar to: SEQ ID NO. 21. In one aspect, the nucleic acid encoding sequence encodes a T2A protein comprising an amino acid sequence that is at least 99% identical or similar to: SEQ ID NO. 21. In one aspect, the nucleic acid encoding sequence encodes a T2A protein comprising an amino acid sequence that is at least 99.5% identical or similar to: SEQ ID NO. 21. In one aspect, the nucleic acid encoding sequence encodes a T2A protein comprising an amino acid sequence that is at least 99.8% identical or similar to: SEQ ID NO. 21. In one aspect, the nucleic acid encoding sequence encodes a T2A protein comprising an amino acid sequence that is at least 99.9% identical or similar to: SEQ ID NO. 21. In one aspect, the nucleic acid encoding sequence encodes a T2A protein comprising an amino acid sequence 100% identical or similar to: SEQ ID NO. 21.
As used herein, "IRES" refers to an internal ribosome entry site of an encephalomyocarditis virus (EMCV).
In one aspect, an AAV or vector of the disclosure comprises an Internal Ribosome Entry Site (IRES) sequence of an encephalomyocarditis virus. In one aspect, the IRES sequence comprises SEQ ID NO. 3. In one aspect, the IRES sequence comprises a sequence that is at least 70% identical to: SEQ ID NO. 3, or a complement thereof. In one aspect, the IRES sequence comprises a sequence at least 75% identical to: SEQ ID NO. 3, or a complement thereof. In one aspect, the IRES sequence comprises a sequence at least 80% identical to: SEQ ID NO. 3, or a complement thereof. In one aspect, the IRES sequence comprises a sequence at least 85% identical to: SEQ ID NO. 3, or a complement thereof. In one aspect, the IRES sequence comprises a sequence at least 90% identical to: SEQ ID NO. 3, or a complement thereof. In one aspect, the IRES sequence comprises a sequence at least 91% identical to: SEQ ID NO. 3, or a complement thereof. In one aspect, the IRES sequence comprises a sequence at least 92% identical to: SEQ ID NO. 3, or a complement thereof. In one aspect, the IRES sequence comprises a sequence at least 93% identical to: SEQ ID NO. 3, or a complement thereof. In one aspect, the IRES sequence comprises a sequence at least 94% identical to: SEQ ID NO. 3, or a complement thereof. In one aspect, the IRES sequence comprises a sequence at least 95% identical to: SEQ ID NO. 3, or a complement thereof. In one aspect, the IRES sequence comprises a sequence at least 96% identical to: SEQ ID NO. 3, or a complement thereof. In one aspect, the IRES sequence comprises a sequence at least 97% identical to: SEQ ID NO. 3, or a complement thereof. In one aspect, the IRES sequence comprises a sequence at least 98% identical to: SEQ ID NO. 3, or a complement thereof. In one aspect, the IRES sequence comprises a sequence at least 99% identical to: SEQ ID NO. 3, or a complement thereof. In one aspect, the IRES sequence comprises a sequence at least 99.5% identical to: SEQ ID NO. 3, or a complement thereof. In one aspect, the IRES sequence comprises a sequence at least 99.8% identical to: SEQ ID NO. 3, or a complement thereof. In one aspect, the IRES sequence comprises a sequence at least 99.9% identical to: SEQ ID NO. 3, or a complement thereof. In one aspect, the IRES sequence comprises a sequence 100% identical to: SEQ ID NO. 3, or a complement thereof.
Glial fibrillary acidic protein (glial fibrillary acid protein, GFAP); also known as glial fibrillary acidic protein (glial fibrillary acidic protein) is a member of the class III intermediate filamin family, which is expressed in the central nervous system and plays a role in cellular communication and blood brain barrier function.
In one aspect, the promoter is selected from the group consisting of: GFAP promoter, sox9 promoter, S100b promoter, aldh1l1 promoter, lipocalin 2 (Lcn 2) promoter, glutamine synthetase promoter, aquaporin-4 (AQP 4) promoter, oligodendrocyte transcription factor (Olig 2) promoter and synaptoprotein promoter, NG2 promoter, ionized calcium binding adapter molecule 1 (Iba 1) promoter, cluster of differentiation 86 (CD 86) promoter, platelet Derived Growth Factor Receptor Alpha (PDGFRA) promoter, platelet Derived Growth Factor Receptor Beta (PDGFRB) promoter, elongation factor 1-alpha (EF 1 a) promoter, CAG promoter, cytomegalovirus (CMV) promoter, ubiquitin promoter. In one aspect, the promoter is a GFAP promoter. In one aspect, the promoter is a truncated GFAP promoter. In one aspect, the promoter is the Sox9 promoter. In one aspect, the promoter is an S100b promoter. In one aspect, the promoter is an Aldh1l1 promoter. In one aspect, the promoter is an Lcn2 promoter. In one aspect, the promoter is a glutamine synthetase promoter. In one aspect, the promoter is the AQP4 promoter. In one aspect, the promoter is an Olig2 promoter. In one aspect, the promoter is a synapsin promoter. In one aspect, the promoter is the Iba1 promoter. In one aspect, the promoter is a CD86 promoter. In one aspect, the promoter is a PDGFRA promoter. In one aspect, the promoter is a PDGFRB promoter. In one aspect, the promoter is an EF1a promoter. In one aspect, the promoter is a CAG promoter. In one aspect, the promoter is a CMV promoter. In one aspect, the promoter is a ubiquitin promoter.
In one aspect, the 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 Acidic Protein (GFAP) in a cell. In one aspect, the GFAP promoter sequence is a human GFAP (hGFAP) promoter sequence. In one aspect, the GFAP promoter is selected from the group consisting of GfaABC1D (also referred to as "pGfa 681"), gfa1.6 and hgfa2.2. In one aspect, the GFAP promoter is GfaABC1D (also referred to as "pGfa 681"). In one aspect, the GFAP promoter is gfa1.6. In one aspect, the GFAP promoter is hgfa2.2. In one aspect pGfa681 is SEQ ID NO 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 NOS 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, a chimpanzee GFAP promoter sequence, a gorilla GFAP promoter sequence, a macaque GFAP promoter sequence, a marmoset GFAP promoter sequence, a pigtail GFAP promoter sequence, a baboon GFAP promoter sequence, a gibbon GFAP promoter sequence, and a marmoset GFAP promoter sequence. In one aspect, the GFAP promoter sequence is a chimpanzee GFAP promoter sequence. In one aspect, the GFAP promoter sequence is a bonobo GFAP promoter sequence. In one aspect, the GFAP promoter sequence is a chimpanzee GFAP promoter sequence. In one aspect, the GFAP promoter sequence is a gorilla GFAP promoter sequence. In one aspect, the GFAP promoter sequence is a macaque GFAP promoter sequence. In one aspect, the GFAP promoter sequence is a marmoset GFAP promoter sequence. In one aspect, the GFAP promoter sequence is a monkey GFAP promoter sequence. In one aspect, the GFAP promoter sequence is a baboon GFAP promoter sequence. In one aspect, the GFAP promoter sequence is a gibbon GFAP promoter sequence. In one aspect, the GFAP promoter sequence is a cynomolgus GFAP promoter sequence.
In one aspect, the GFAP promoter sequence comprises at least 100 nucleotides. In one aspect, the GFAP promoter comprises at least 500 nucleotides. In another aspect, the GFAP promoter comprises at least 1000 nucleotides. In yet another aspect, the GFAP promoter comprises at least 1400 nucleotides.
It is understood in the art that fragments of a promoter sequence may function to drive transcription of an operably linked nucleic acid molecule. For example, but not limited to, if a 1000 nucleotide promoter is truncated to 500 nucleotides and the 500 nucleotide fragment is capable of driving transcription, the 500 nucleotide fragment is referred to as a "functional fragment".
In one aspect, the promoter comprises at least 10 nucleotides. In one aspect, the promoter comprises at least 50 nucleotides. In one aspect, the promoter comprises at least 100 nucleotides. In one aspect, the intron comprises at least 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 at least 70% identical to a sequence selected from the group consisting of seq id no: SEQ ID NOS 4, 12, 26 and functional fragments thereof. In one aspect, the GFAP promoter nucleic acid sequence comprises a sequence at least 75% identical to a sequence selected from the group consisting of seq id nos: SEQ ID NOS 4, 12, 26 and functional fragments thereof. In one aspect, the GFAP promoter nucleic acid sequence comprises a sequence at least 80% identical to a sequence selected from the group consisting of seq id nos: SEQ ID NOS 4, 12, 26 and functional fragments thereof. In one aspect, the GFAP promoter nucleic acid sequence comprises a sequence at least 85% identical to a sequence selected from the group consisting of seq id nos: SEQ ID NOS 4, 12, 26 and functional fragments thereof. In one aspect, the GFAP promoter nucleic acid sequence comprises a sequence at least 90% identical to a sequence selected from the group consisting of seq id nos: SEQ ID NOS 4, 12, 26 and functional fragments thereof. In one aspect, the GFAP promoter nucleic acid sequence comprises a sequence at least 91% identical to a sequence selected from the group consisting of seq id nos: SEQ ID NOS 4, 12, 26 and functional fragments thereof. In one aspect, the GFAP promoter nucleic acid sequence comprises a sequence at least 92% identical to a sequence selected from the group consisting of seq id nos: SEQ ID NOS 4, 12, 26 and functional fragments thereof. In one aspect, the GFAP promoter nucleic acid sequence comprises a sequence at least 93% identical to a sequence selected from the group consisting of seq id nos: SEQ ID NOS 4, 12, 26 and functional fragments thereof. In one aspect, the GFAP promoter nucleic acid sequence comprises a sequence at least 94% identical to a sequence selected from the group consisting of seq id nos: SEQ ID NOS 4, 12, 26 and functional fragments thereof. In one aspect, the GFAP promoter nucleic acid sequence comprises a sequence at least 95% identical to a sequence selected from the group consisting of seq id nos: SEQ ID NOS 4, 12, 26 and functional fragments thereof. In one aspect, the GFAP promoter nucleic acid sequence comprises a sequence at least 96% identical to a sequence selected from the group consisting of seq id nos: SEQ ID NOS 4, 12, 26 and functional fragments thereof. In one aspect, the GFAP promoter nucleic acid sequence comprises a sequence at least 97% identical to a sequence selected from the group consisting of seq id nos: SEQ ID NOS 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 nos: SEQ ID NOS 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 nos: SEQ ID NOS 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 nos: SEQ ID NOS 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 nos: SEQ ID NOS 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 nos: SEQ ID NOS 4, 12, 26 and functional fragments thereof. In one aspect, the GFAP promoter nucleic acid sequence comprises a sequence 100% identical to a sequence selected from the group consisting of seq id nos: SEQ ID NOS 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 CpG site-depleted.
As used herein, the term "brain" refers to an organ that serves as a central nervous system. In one aspect, the brain comprises the brain, cortex, cerebellum, and/or brain stem.
As used herein, the term "cortex" refers to the outer layer of brain nerve tissue.
As used herein, the term "striatum" or "striatum" (or "striatum") refers to clusters of neurons in the basal ganglia under the anterior cerebral cortex, and includes the ventral striatum and the dorsal striatum.
As used herein, the term "substantia nigra" refers to the clusters of neurons in the basal ganglia under the mesocortex and comprises dense and reticular portions.
As used herein, the term "forebrain" refers to the forefront of the brain.
As used herein, the term "putamen" refers to the circular structure of the forebrain base and is an integral part of the dorsal striatum.
As used herein, the term "caudate nucleus" refers to the structure of the anterior brain base and is an integral part of the dorsal striatum.
As used herein, the term "subcortical basal ganglia" refers to clusters of neurons in the deep brain hemisphere.
As used herein, the term "spinal cord" refers to structures that function in transmitting nerve signals from the motor cortex to the body.
As used herein, the term "motor cortex" refers to the area of the frontal lobe of the cerebral cortex involved in the planning, control and execution of voluntary movements.
In one aspect, the methods provided herein convert reactive astrocytes into functional neurons in the brain. In one aspect, the methods provided herein convert reactive astrocytes into functional neurons in the cerebral cortex of the brain. In one aspect, the methods provided herein convert reactive astrocytes into functional neurons in the striatum of the brain. In one aspect, the methods provided herein convert reactive astrocytes into functional neurons in the dorsal striatum of the brain. In one aspect, the methods provided herein convert reactive astrocytes into functional neurons in the spinal cord of the brain. In one aspect, the methods provided herein convert reactive astrocytes into functional neurons in the putamen of the brain. In one aspect, the methods provided herein convert reactive astrocytes into functional neurons in the caudate nucleus of the brain. In one aspect, the methods provided herein convert reactive astrocytes into functional neurons in the substantia nigra of the brain.
Elongation factor 1 alpha (EF-1 alpha; also known as eEF1a 1) is an isoform of the alpha subunit of the elongation factor 1 complex. The complex is involved in the enzymatic delivery of aminoacyltRNA to the ribosome. EF-1 alpha isoforms are expressed in brain, placenta, lung, liver, kidney and pancreas.
In one aspect, the enhancer sequence from the EF-1 alpha promoter is a human enhancer sequence from the EF-1 alpha promoter. In one aspect, the enhancer sequence from the EF-1 alpha promoter is selected from the group consisting of: a chimpanzee enhancer sequence from an EF-1 a promoter, a bonobo enhancer sequence from an EF-1 a promoter, a chimpanzee enhancer sequence from an EF-1 a promoter, a gorilla enhancer sequence from an EF-1 a promoter, a macaque enhancer sequence from an EF-1 a promoter, a marmoset enhancer sequence from an EF-1 a promoter, a pigtail enhancer sequence from an EF-1 a promoter, a baboon enhancer sequence from an EF-1 a promoter, a gibbon enhancer sequence from an EF-1 a promoter, and a marmoset enhancer sequence from an EF-1 a promoter. In one aspect, the enhancer sequence from the EF-1 alpha promoter is a chimpanzee enhancer sequence from the EF-1 alpha promoter. In one aspect, the enhancer sequence from the EF-1. Alpha. Promoter is a bonobo enhancer sequence from the EF-1. Alpha. Promoter. In one aspect, the enhancer sequence from the EF-1 alpha promoter is a chimpanzee enhancer sequence from the EF-1 alpha promoter. In one aspect, the enhancer sequence from the EF-1 alpha promoter is a gorilla enhancer sequence from the EF-1 alpha promoter. In one aspect, the enhancer sequence from the EF-1. Alpha. Promoter is a cynomolgus monkey enhancer sequence from the EF-1. Alpha. Promoter. In one aspect, the enhancer sequence from the EF-1 alpha promoter is a marmoset enhancer sequence from the EF-1 alpha promoter. In one aspect, the enhancer sequence from the EF-1. Alpha. Promoter is a monkey enhancer sequence from the EF-1. Alpha. Promoter. In one aspect, the enhancer sequence from the EF-1. Alpha. Promoter is a baboon enhancer sequence from the EF-1. Alpha. Promoter. In one aspect, the enhancer sequence from the EF-1 alpha promoter is a gibbon enhancer sequence from the EF-1 alpha promoter. In one aspect, the enhancer sequence from the EF-1 alpha promoter is a cynomolgus monkey enhancer sequence from the EF-1 alpha promoter.
In one aspect, the enhancer from the EF-1 alpha promoter nucleic acid sequence comprises a sequence at least 70% identical to: SEQ ID NO. 2, or a complement thereof. In one aspect, the enhancer from the EF-1 alpha promoter nucleic acid sequence comprises a sequence at least 75% identical to: SEQ ID NO. 2, or a complement thereof. In one aspect, the enhancer from the EF-1 alpha promoter nucleic acid sequence comprises a sequence at least 80% identical to: SEQ ID NO. 2, or a complement thereof. In one aspect, the enhancer from the EF-1 alpha promoter nucleic acid sequence comprises a sequence at least 85% identical to: SEQ ID NO. 2, or a complement thereof. In one aspect, the enhancer from the EF-1 alpha promoter nucleic acid sequence comprises a sequence at least 90% identical to: SEQ ID NO. 2, or a complement thereof. In one aspect, the enhancer from the EF-1 alpha promoter nucleic acid sequence comprises a sequence at least 91% identical to: SEQ ID NO. 2, or a complement thereof. In one aspect, the enhancer from the EF-1 alpha promoter nucleic acid sequence comprises a sequence at least 92% identical to: SEQ ID NO. 2, or a complement thereof. In one aspect, the enhancer from the EF-1 alpha promoter nucleic acid sequence comprises a sequence at least 93% identical to: SEQ ID NO. 2, or a complement thereof. In one aspect, the enhancer from the EF-1 alpha promoter nucleic acid sequence comprises a sequence at least 94% identical to: SEQ ID NO. 2, or a complement thereof. In one aspect, the enhancer from the EF-1 alpha promoter nucleic acid sequence comprises a sequence at least 95% identical to: SEQ ID NO. 2, or a complement thereof. In one aspect, the enhancer from the EF-1 alpha promoter nucleic acid sequence comprises a sequence at least 96% identical to: SEQ ID NO. 2, or a complement thereof. In one aspect, the enhancer from the EF-1 alpha promoter nucleic acid sequence comprises a sequence at least 97% identical to: SEQ ID NO. 2, or a complement thereof. In one aspect, the enhancer from the EF-1 alpha promoter nucleic acid sequence comprises a sequence at least 98% identical to: SEQ ID NO. 2, or a complement thereof. In one aspect, the enhancer from the EF-1 alpha promoter nucleic acid sequence comprises a sequence at least 99% identical to: SEQ ID NO. 2, or a complement thereof. In one aspect, the enhancer from the EF-1 alpha promoter nucleic acid sequence comprises a sequence at least 99.5% identical to: SEQ ID NO. 2, or a complement thereof. In one aspect, the enhancer from the EF-1 alpha promoter nucleic acid sequence comprises a sequence at least 99.8% identical to: SEQ ID NO. 2, or a complement thereof. In one aspect, the enhancer from the EF-1 alpha promoter nucleic acid sequence comprises a sequence at least 99.9% identical to: SEQ ID NO. 2, or a complement thereof. In one aspect, the enhancer from the EF-1 alpha promoter nucleic acid sequence comprises a sequence 100% identical to: SEQ ID NO. 2, or a complement thereof.
Cytomegalovirus (CMV) is a genus of viruses in the order herpesviridae (Herpesvirale).
In one aspect, the enhancer sequence from CMV is a human enhancer sequence from CMV. In one aspect, the enhancer sequence from CMV is selected from the group consisting of: a chimpanzee enhancer sequence from CMV, a bonobo enhancer sequence from CMV, a chimpanzee enhancer sequence from CMV, a gorilla enhancer sequence from CMV, a macaque enhancer sequence from CMV, a marmoset enhancer sequence from CMV, a pigtail enhancer sequence from CMV, a baboon enhancer sequence from CMV, a gibbon enhancer sequence from CMV, and a marmoset enhancer sequence from CMV. In one aspect, the enhancer sequence from CMV is a chimpanzee enhancer sequence from CMV. In one aspect, the enhancer sequence from CMV is a bonobo enhancer sequence from CMV. In one aspect, the enhancer sequence from CMV is a chimpanzee enhancer sequence from CMV. In one aspect, the enhancer sequence from CMV is a gorilla enhancer sequence from CMV. In one aspect, the enhancer sequence from CMV is a cynomolgus monkey enhancer sequence from CMV. In one aspect, the enhancer sequence from CMV is a marmoset enhancer sequence from CMV. In one aspect, the enhancer sequence from CMV is a monkey enhancer sequence from CMV. In one aspect, the enhancer sequence from CMV is a baboon enhancer sequence from CMV. In one aspect, the enhancer sequence from CMV is a gibbon enhancer sequence from CMV. In one aspect, the enhancer sequence from CMV is a armpit enhancer sequence from CMV.
In one aspect, the enhancer from the CMV nucleic acid sequence comprises a sequence at least 70% identical to: SEQ ID NO. 11, or a complement thereof. In one aspect, the enhancer from the CMV nucleic acid sequence comprises a sequence at least 75% identical to: SEQ ID NO. 11, or a complement thereof. In one aspect, the enhancer from the CMV nucleic acid sequence comprises a sequence at least 80% identical to: SEQ ID NO. 11, or a complement thereof. In one aspect, the enhancer from the CMV nucleic acid sequence comprises a sequence at least 85% identical to: SEQ ID NO. 11, or a complement thereof. In one aspect, the enhancer from the CMV nucleic acid sequence comprises a sequence at least 90% identical to: SEQ ID NO. 11, or a complement thereof. In one aspect, the enhancer from the CMV nucleic acid sequence comprises a sequence at least 91% identical to: SEQ ID NO. 11, or a complement thereof. In one aspect, the enhancer from the CMV nucleic acid sequence comprises a sequence at least 911% identical to: SEQ ID NO. 11, or a complement thereof. In one aspect, the enhancer from the CMV nucleic acid sequence comprises a sequence at least 93% identical to: SEQ ID NO. 11, or a complement thereof. In one aspect, the enhancer from the CMV nucleic acid sequence comprises a sequence at least 94% identical to: SEQ ID NO. 11, or a complement thereof. In one aspect, the enhancer from the CMV nucleic acid sequence comprises a sequence at least 95% identical to: SEQ ID NO. 11, or a complement thereof. In one aspect, the enhancer from the CMV nucleic acid sequence comprises a sequence at least 96% identical to: SEQ ID NO. 11, or a complement thereof. In one aspect, the enhancer from the CMV nucleic acid sequence comprises a sequence at least 97% identical to: SEQ ID NO. 11, or a complement thereof. In one aspect, the enhancer from the CMV nucleic acid sequence comprises a sequence at least 98% identical to: SEQ ID NO. 11, or a complement thereof. In one aspect, the enhancer from the CMV nucleic acid sequence comprises a sequence at least 99% identical to: SEQ ID NO. 11, or a complement thereof. In one aspect, the enhancer from the CMV nucleic acid sequence comprises a sequence at least 99.5% identical to: SEQ ID NO. 11, or a complement thereof. In one aspect, the enhancer from the CMV nucleic acid sequence comprises a sequence at least 99.8% identical to: SEQ ID NO. 11, or a complement thereof. In one aspect, the enhancer from the CMV nucleic acid sequence comprises a sequence at least 99.9% identical to: SEQ ID NO. 11, or a complement thereof. In one aspect, the enhancer from the CMV nucleic acid sequence comprises a sequence 100% identical to: SEQ ID NO. 11, or a complement thereof.
In one aspect, the enhancer is selected from the group consisting of an enhancer from the EF 1-alpha promoter and a CMV enhancer. In one aspect, the enhancer is from the EF 1-alpha promoter. In one aspect, the enhancer is a CMV enhancer.
In one aspect, the vectors of the present disclosure comprise a chimeric intron. In one aspect, the chimeric intron consists of a 5 '-donor site from the first intron of the human β -globulin gene and a branching and 3' -acceptor site from the heavy chain variable region intron of the immunoglobulin gene. In one aspect, the chimeric intron is a chimeric intron of rabbit β -globulin and chicken β -actin that are similar in the CAG promoter. In one aspect, the vectors of the present disclosure comprise a Glial Fibrillary Acidic Protein (GFAP) intron. In one aspect, the vectors of the present disclosure comprise a Glial Fibrillary Acidic Protein (GFAP) first intron.
Introns can be divided into at least five classes, including: splicing the introns; transferring the RNA intron; group I introns; group II introns; and group III introns. Introns may be synthetically generated, altered, or derived from known or naturally occurring intron sequences or other intron sequences. Introns may also include chimeric introns comprising a combination of two or more heterologous sequences. Thus, introns of the application may include variants of intron sequences that are similar in composition to, but not identical to, other intron sequences known or provided herein. In one aspect, the intron comprises at least 10 nucleotides. In one aspect, the intron comprises at least 50 nucleotides. In one aspect, the intron comprises at least 100 nucleotides. In one aspect, the intron comprises at least 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, the intron comprises between 10 nucleotides and 500 nucleotides.
In one aspect, the chimeric intron nucleic acid sequence is selected from the group consisting of SEQ ID NOs 5 and 27. In one aspect, the chimeric intron nucleic acid sequence is SEQ ID NO. 5. In one aspect, the chimeric intron nucleic acid sequence is SEQ ID NO. 27.
In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 70% identical to: SEQ ID NO. 5, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 75% identical to: SEQ ID NO. 5, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 80% identical to: SEQ ID NO. 5, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 85% identical to: SEQ ID NO. 5, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 90% identical to: SEQ ID NO. 5, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 91% identical to: SEQ ID NO. 5, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 92% identical to: SEQ ID NO. 5, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 93% identical to: SEQ ID NO. 5, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 94% identical to: SEQ ID NO. 5, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 95% identical to: SEQ ID NO. 5, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 96% identical to: SEQ ID NO. 5, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 97% identical to: SEQ ID NO. 5, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 98% identical to: SEQ ID NO. 5, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 99% identical to: SEQ ID NO. 5, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 99.5% identical to: SEQ ID NO. 5, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 99.8% identical to: SEQ ID NO. 5, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 99.9% identical to: SEQ ID NO. 5, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence 100% identical to: SEQ ID NO. 5, or a complement thereof.
In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 70% identical to: SEQ ID NO. 27, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 75% identical to: SEQ ID NO. 27, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 80% identical to: SEQ ID NO. 27, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 85% identical to: SEQ ID NO. 27, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 90% identical to: SEQ ID NO. 27, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 91% identical to: SEQ ID NO. 27, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 92% identical to: SEQ ID NO. 27, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 93% identical to: SEQ ID NO. 27, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 94% identical to: SEQ ID NO. 27, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 95% identical to: SEQ ID NO. 27, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 96% identical to: SEQ ID NO. 27, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 97% identical to: SEQ ID NO. 27, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 98% identical to: SEQ ID NO. 27, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 99% identical to: SEQ ID NO. 27, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 99.5% identical to: SEQ ID NO. 27, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 99.8% identical to: SEQ ID NO. 27, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence at least 99.9% identical to: SEQ ID NO. 27, or a complement thereof. In one aspect, the chimeric intron nucleic acid sequence comprises a sequence 100% identical to: SEQ ID NO. 27, or a complement thereof.
The woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) is a DNA sequence that creates a tertiary structure that enhances expression of a delivery gene in a viral vector.
In one aspect, the WPRE nucleic acid sequence is an optimized version of WPRE.
In one aspect, the WPRE nucleic acid sequence is selected from the group consisting of: SEQ ID NOS.7 and 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 at least 70% identical to: SEQ ID NO. 7, or a complement thereof. In one aspect, the WPRE nucleic acid sequence comprises a sequence at least 75% identical to: SEQ ID NO. 7, or a complement thereof. In one aspect, the WPRE nucleic acid sequence comprises a sequence at least 80% identical to: SEQ ID NO. 7, or a complement thereof. In one aspect, the WPRE nucleic acid sequence comprises a sequence at least 85% identical to: SEQ ID NO. 7, or a complement thereof. In one aspect, the WPRE nucleic acid sequence comprises a sequence at least 90% identical to: SEQ ID NO. 7, or a complement thereof. In one aspect, the WPRE nucleic acid sequence comprises a sequence at least 91% identical to: SEQ ID NO. 7, or a complement thereof. In one aspect, the WPRE nucleic acid sequence comprises a sequence at least 92% identical to: SEQ ID NO. 7, or a complement thereof. In one aspect, the WPRE nucleic acid sequence comprises a sequence at least 93% identical to: SEQ ID NO. 7, or a complement thereof. In one aspect, the WPRE nucleic acid sequence comprises a sequence at least 94% identical to: SEQ ID NO. 7, or a complement thereof. In one aspect, the WPRE nucleic acid sequence comprises a sequence at least 95% identical to: SEQ ID NO. 7, or a complement thereof. In one aspect, the WPRE nucleic acid sequence comprises a sequence at least 96% identical to: SEQ ID NO. 7, or a complement thereof. In one aspect, the WPRE nucleic acid sequence comprises a sequence at least 97% identical to: SEQ ID NO. 7, or a complement thereof. In one aspect, the WPRE nucleic acid sequence comprises a sequence at least 98% identical to: SEQ ID NO. 7, or a complement thereof. In one aspect, the WPRE nucleic acid sequence comprises a sequence at least 99% identical to: SEQ ID NO. 7, or a complement thereof. In one aspect, the WPRE nucleic acid sequence comprises a sequence at least 99.5% identical to: SEQ ID NO. 7, or a complement thereof. In one aspect, the WPRE nucleic acid sequence comprises a sequence at least 99.8% identical to: SEQ ID NO. 7, or a complement thereof. In one aspect, the WPRE nucleic acid sequence comprises a sequence at least 99.9% identical to: SEQ ID NO. 7, or a complement thereof. In one aspect, the WPRE nucleic acid sequence comprises a sequence 100% identical to: SEQ ID NO. 7, or a complement thereof.
In one aspect, the WPRE nucleic acid sequence comprises a sequence at least 70% identical to: SEQ ID NO. 29, or a complement thereof. In one aspect, the WPRE nucleic acid sequence comprises a sequence at least 75% identical to: SEQ ID NO. 29, or a complement thereof. In one aspect, the WPRE nucleic acid sequence comprises a sequence at least 80% identical to: SEQ ID NO. 29, or a complement thereof. In one aspect, the WPRE nucleic acid sequence comprises a sequence at least 85% identical to: SEQ ID NO. 29, or a complement thereof. In one aspect, the WPRE nucleic acid sequence comprises a sequence at least 90% identical to: SEQ ID NO. 29, or a complement thereof. In one aspect, the WPRE nucleic acid sequence comprises a sequence at least 91% identical to: SEQ ID NO. 29, or a complement thereof. In one aspect, the WPRE nucleic acid sequence comprises a sequence at least 92% identical to: SEQ ID NO. 29, or a complement thereof. In one aspect, the WPRE nucleic acid sequence comprises a sequence at least 93% identical to: SEQ ID NO. 29, or a complement thereof. In one aspect, the WPRE nucleic acid sequence comprises a sequence at least 94% identical to: SEQ ID NO. 29, or a complement thereof. In one aspect, the WPRE nucleic acid sequence comprises a sequence at least 95% identical to: SEQ ID NO. 29, or a complement thereof. In one aspect, the WPRE nucleic acid sequence comprises a sequence at least 96% identical to: SEQ ID NO. 29, or a complement thereof. In one aspect, the WPRE nucleic acid sequence comprises a sequence at least 97% identical to: SEQ ID NO. 29, or a complement thereof. In one aspect, the WPRE nucleic acid sequence comprises a sequence at least 98% identical to: SEQ ID NO. 29, or a complement thereof. In one aspect, the WPRE nucleic acid sequence comprises a sequence at least 99% identical to: SEQ ID NO. 29, or a complement thereof. In one aspect, the WPRE nucleic acid sequence comprises a sequence at least 99.5% identical to: SEQ ID NO. 29, or a complement thereof. In one aspect, the WPRE nucleic acid sequence comprises a sequence at least 99.8% identical to: SEQ ID NO. 29, or a complement thereof. In one aspect, the WPRE nucleic acid sequence comprises a sequence at least 99.9% identical to: SEQ ID NO. 29, or a complement thereof. In one aspect, the WPRE nucleic acid sequence comprises a sequence 100% identical to: SEQ ID NO. 29, or a complement thereof.
SV40 polyadenylation signal sequences (also known as SV40 PolyA; simian Virus 40PolyA; and PolyA) are DNA sequences that terminate transcription and add the 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 can terminate transcription and add the PolyA tail to the 3' end of the messenger RNA (mRNA).
The bGH polyadenylation signal sequence (also known as bGH PolyA or bGHpA) refers to the PolyA signal or PolyA tail of bovine growth hormone.
As used herein, "PolyA tail" refers to a stretch of RNA that contains only nucleobase adenine. In one aspect, an RNA molecule transcribed from an AAV vector construct provided herein comprises a poly a tail. In one aspect, the PolyA tail comprises at least two adenine. In one aspect, the PolyA tail comprises at least ten adenine. In one aspect, the PolyA tail comprises at least 50 adenine. In one aspect, the polyA tail comprises at least 100 adenine. In one aspect, the PolyA tail comprises at least 140 adenine. In one aspect, the PolyA tail comprises at least 200 adenine. In one aspect, the polyA tail comprises at least 250 adenine. In one aspect, the PolyA tail comprises between 50 adenine and 300 adenine.
In one aspect, the SV40 polyadenylation signal nucleic acid sequence comprises a sequence at least 70% identical to: SEQ ID NO. 8, or a complement thereof. In one aspect, the SV40 polyadenylation signal nucleic acid sequence comprises a sequence at least 75% identical to: SEQ ID NO. 8, or a complement thereof. In one aspect, the SV40 polyadenylation signal nucleic acid sequence comprises a sequence at least 80% identical to: SEQ ID NO. 8, or a complement thereof. In one aspect, the SV40 polyadenylation signal nucleic acid sequence comprises a sequence at least 85% identical to: SEQ ID NO. 8, or a complement thereof. In one aspect, the SV40 polyadenylation signal nucleic acid sequence comprises a sequence at least 90% identical to: SEQ ID NO. 8, or a complement thereof. In one aspect, the SV40 polyadenylation signal nucleic acid sequence comprises a sequence at least 91% identical to: SEQ ID NO. 8, or a complement thereof. In one aspect, the SV40 polyadenylation signal nucleic acid sequence comprises a sequence at least 92% identical to: SEQ ID NO. 8, or a complement thereof. In one aspect, the SV40 polyadenylation signal nucleic acid sequence comprises a sequence at least 93% identical to: SEQ ID NO. 8, or a complement thereof. In one aspect, the SV40 polyadenylation signal nucleic acid sequence comprises a sequence at least 94% identical to: SEQ ID NO. 8, or a complement thereof. In one aspect, the SV40 polyadenylation signal nucleic acid sequence comprises a sequence at least 95% identical to: SEQ ID NO. 8, or a complement thereof. In one aspect, the SV40 polyadenylation signal nucleic acid sequence comprises a sequence at least 96% identical to: SEQ ID NO. 8, or a complement thereof. In one aspect, the SV40 polyadenylation signal nucleic acid sequence comprises a sequence at least 97% identical to: SEQ ID NO. 8, or a complement thereof. In one aspect, the SV40 polyadenylation signal nucleic acid sequence comprises a sequence at least 98% identical to: SEQ ID NO. 8, or a complement thereof. In one aspect, the SV40 polyadenylation signal nucleic acid sequence comprises a sequence at least 99% identical to: SEQ ID NO. 8, or a complement thereof. In one aspect, the SV40 polyadenylation signal nucleic acid sequence comprises a sequence at least 99.5% identical to: SEQ ID NO. 8, or a complement thereof. In one aspect, the SV40 polyadenylation signal nucleic acid sequence comprises a sequence at least 99.8% identical to: SEQ ID NO. 8, or a complement thereof. In one aspect, the SV40 polyadenylation signal nucleic acid sequence comprises a sequence at least 99.9% identical to: SEQ ID NO. 8, or a complement thereof. In one aspect, the SV40 polyadenylation signal nucleic acid sequence comprises a sequence 100% identical to: SEQ ID NO. 8, or a complement thereof.
In one aspect, the hGH polyadenylation signal nucleic acid sequence comprises a sequence at least 70% identical to: SEQ ID NO. 17, or a complement thereof. In one aspect, the hGH polyadenylation signal nucleic acid sequence comprises a sequence at least 75% identical to: SEQ ID NO. 17, or a complement thereof. In one aspect, the hGH polyadenylation signal nucleic acid sequence comprises a sequence at least 80% identical to: SEQ ID NO. 17, or a complement thereof. In one aspect, the hGH polyadenylation signal nucleic acid sequence comprises a sequence at least 85% identical to: SEQ ID NO. 17, or a complement thereof. In one aspect, the hGH polyadenylation signal nucleic acid sequence comprises a sequence at least 90% identical to: SEQ ID NO. 17, or a complement thereof. In one aspect, the hGH polyadenylation signal nucleic acid sequence comprises a sequence at least 91% identical to: SEQ ID NO. 17, or a complement thereof. In one aspect, the hGH polyadenylation signal nucleic acid sequence comprises a sequence at least 92% identical to: SEQ ID NO. 17, or a complement thereof. In one aspect, the hGH polyadenylation signal nucleic acid sequence comprises a sequence at least 93% identical to: SEQ ID NO. 17, or a complement thereof. In one aspect, the hGH polyadenylation signal nucleic acid sequence comprises a sequence at least 94% identical to: SEQ ID NO. 17, or a complement thereof. In one aspect, the hGH polyadenylation signal nucleic acid sequence comprises a sequence at least 95% identical to: SEQ ID NO. 17, or a complement thereof. In one aspect, the hGH polyadenylation signal nucleic acid sequence comprises a sequence at least 96% identical to: SEQ ID NO. 17, or a complement thereof. In one aspect, the hGH polyadenylation signal nucleic acid sequence comprises a sequence at least 97% identical to: SEQ ID NO. 17, or a complement thereof. In one aspect, the hGH polyadenylation signal nucleic acid sequence comprises a sequence at least 917% identical to: SEQ ID NO. 17, or a complement thereof. In one aspect, the hGH polyadenylation signal nucleic acid sequence comprises a sequence at least 99% identical to: SEQ ID NO. 17, or a complement thereof. In one aspect, the hGH polyadenylation signal nucleic acid sequence comprises a sequence at least 99.5% identical to: SEQ ID NO. 17, or a complement thereof. In one aspect, the hGH polyadenylation signal nucleic acid sequence comprises a sequence at least 99.17% identical to: SEQ ID NO. 17, or a complement thereof. In one aspect, the hGH polyadenylation signal nucleic acid sequence comprises a sequence at least 99.9% identical to: SEQ ID NO. 17, or a complement thereof. In one aspect, the hGH polyadenylation signal nucleic acid sequence comprises a sequence 100% identical to: SEQ ID NO. 17, or a complement thereof
In one aspect, the bGH polyadenylation signal nucleic acid sequence comprises a sequence at least 70% identical to: SEQ ID NO. 30, or a complement thereof. In one aspect, the bGH polyadenylation signal nucleic acid sequence comprises a sequence at least 75% identical to: SEQ ID NO. 30, or a complement thereof. In one aspect, the bGH polyadenylation signal nucleic acid sequence comprises a sequence at least 80% identical to: SEQ ID NO. 30, or a complement thereof. In one aspect, the bGH polyadenylation signal nucleic acid sequence comprises a sequence at least 85% identical to: SEQ ID NO. 30, or a complement thereof. In one aspect, the bGH polyadenylation signal nucleic acid sequence comprises a sequence at least 90% identical to: SEQ ID NO. 30, or a complement thereof. In one aspect, the bGH polyadenylation signal nucleic acid sequence comprises a sequence at least 91% identical to: SEQ ID NO. 30, or a complement thereof. In one aspect, the bGH polyadenylation signal nucleic acid sequence comprises a sequence at least 92% identical to: SEQ ID NO. 30, or a complement thereof. In one aspect, the bGH polyadenylation signal nucleic acid sequence comprises a sequence at least 93% identical to: SEQ ID NO. 30, or a complement thereof. In one aspect, the bGH polyadenylation signal nucleic acid sequence comprises a sequence at least 94% identical to: SEQ ID NO. 30, or a complement thereof. In one aspect, the bGH polyadenylation signal nucleic acid sequence comprises a sequence at least 95% identical to: SEQ ID NO. 30, or a complement thereof. In one aspect, the bGH polyadenylation signal nucleic acid sequence comprises a sequence at least 96% identical to: SEQ ID NO. 30, or a complement thereof. In one aspect, the bGH polyadenylation signal nucleic acid sequence comprises a sequence at least 97% identical to: SEQ ID NO. 30, or a complement thereof. In one aspect, the bGH polyadenylation signal nucleic acid sequence comprises a sequence at least 917% identical to: SEQ ID NO. 30, or a complement thereof. In one aspect, the bGH polyadenylation signal nucleic acid sequence comprises a sequence at least 99% identical to: SEQ ID NO. 30, or a complement thereof. In one aspect, the bGH polyadenylation signal nucleic acid sequence comprises a sequence at least 99.5% identical to: SEQ ID NO. 30, or a complement thereof. In one aspect, the bGH polyadenylation signal nucleic acid sequence comprises a sequence at least 99.17% identical to: SEQ ID NO. 30, or a complement thereof. In one aspect, the bGH polyadenylation signal nucleic acid sequence comprises a sequence at least 99.9% identical to: SEQ ID NO. 30, or a complement thereof. In one aspect, the bGH polyadenylation signal nucleic acid sequence comprises a sequence 100% identical to: SEQ ID NO. 30, or a complement thereof
As used herein, the term "central nervous system" or "CNS" refers to the brain and spinal cord of bilaterally symmetric animals. The CNS also includes retina, optic nerve, olfactory nerve and olfactory epithelial cells.
As used herein, the term "peripheral nervous system" or "PNS" refers to nerves and ganglia outside the brain and spinal cord, excluding retina, optic nerve, olfactory nerve and olfactory epithelial cells. In one aspect, the peripheral nervous system is divided into the somatic nervous system and the autonomic nervous system.
As used herein, the term "somatic nervous system" refers to the portion of PNS that is associated with the autonomous control of body movement.
As used herein, the term "autonomic nervous system" refers to the PNS portion that regulates internal organ function
As used herein, the term "GFAP positive" refers to a cell having an accumulation of protein of human Glial Fibrillary Acidic Protein (GFAP) detectable using techniques standard in the art or an accumulation of mRNA expression of GFAP detectable using techniques standard in the art. In one aspect, the glial cells are GFAP positive.
As used herein, the term "detectable" refers to the accumulation of a recognizable protein or mRNA.
Antibodies can be used to identify protein accumulation. Non-limiting examples of measuring protein accumulation include Western blotting, enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, and immunofluorescence. The antibodies provided herein may be polyclonal or monoclonal. Antibodies provided herein having specific binding affinity for a protein can be generated using methods well known in the art. The antibodies provided herein can be attached to a solid support (such as a microtiter plate) using methods known in the art.
As used herein, the terms "multiplicity of infection" and "MOI" refer to the number of virions added per cell during infection.
As used herein, the term "virion" refers to a form of viral infection outside of a host cell.
As used herein, the term "neurological disorder" refers to a disorder, disease, condition, injury, or disease in the central or peripheral nervous system. Non-limiting examples of neurological disorders can be found in Neurological Disorders: course and treatment, version 2 (2002) (Academic Press Inc.) and Christopher Goetz, textbook of Clinical Neurology, version 3 (2007) (Saunders).
As used herein, the term "injury" refers to an injury to the central or peripheral nervous system.
In one aspect, the neurological disorder is selected from the group consisting of: alzheimer's disease, parkinson's disease, amyotrophic Lateral Sclerosis (ALS), huntington's disease, epilepsy, physical injury, stroke, cerebral aneurysms, traumatic brain injury, concussion, tumors, inflammation, infection, ataxia, brain atrophy, spinal cord atrophy, multiple sclerosis, traumatic spinal cord injury, ischemic or hemorrhagic myelopathy (myelopathy), global cerebral ischemia, hypoxic ischemic encephalopathy, embolism, fibrocartilage embolic myelopathy, thrombosis, kidney disease, chronic inflammatory diseases, meningitis, and cerebral venous sinus thrombosis. In one aspect, the neurological disorder is alzheimer's disease. In one aspect, the neurological disorder is parkinson's disease. In one aspect, the neurological disorder is ALS. In one aspect, the neurological disorder is huntington's disease. In one aspect, the neurological disorder is epilepsy. In one aspect, the neurological condition is a physical injury. In one aspect, the neurological condition is stroke. In one aspect, the neurological disorder is ischemic stroke. In one aspect, the neurological disorder is hemorrhagic stroke. In one aspect, the neurological disorder is a cerebral aneurysm. In one aspect, the neurological disorder is traumatic brain injury. In one aspect, the neurological disorder is concussion. In one aspect, the neurological disorder is a tumor. In one aspect, the neurological disorder is inflammation. In one aspect, the neurological disorder is an infection. In one aspect, the neurological disorder is ataxia. In one aspect, the neurological disorder is brain atrophy. In one aspect, the neurological disorder is spinal atrophy. In one aspect, the neurological disorder is multiple sclerosis. In one aspect, the neurological disorder is traumatic spinal cord injury. In one aspect, the neurological disorder is ischemic or hemorrhagic myelopathy (myelopathy). In one aspect, the neurological disorder is global cerebral ischemia. In one aspect, the neurological disorder is hypoxic ischemic encephalopathy. In one aspect, the neurological disorder is embolism. In one aspect, the neurological disorder is fibrocartilage embolic spinal cord disease. In one aspect, the neurological disorder is thrombosis. In one aspect, the neurological disorder is kidney disease. In one aspect, the neurological disorder is a chronic inflammatory disease. In one aspect, the neurological disorder is meningitis. In one aspect, the neurological disorder is cerebral sinus thrombosis.
In one aspect, the neurological disorder includes damage to the CNS or PNS. In one aspect, the neurological condition includes damage to the CNS. In one aspect, the neurological condition includes damage to PNS.
In one aspect, the present disclosure provides and includes a method of converting reactive astrocytes into functional neurons in a living human brain, comprising: injecting an adeno-associated virus (AAV) into a subject in need thereof, wherein the AAV comprises a DNA vector construct comprising: a human neurogenic differentiation factor 1 (hNeuroD 1) sequence, the hNeuroD1 sequence comprising a nucleic acid sequence of SEQ ID NO. 6, and a 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 said hNeuroD1 sequence and said 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.
In one aspect, the present disclosure provides and includes a method of converting reactive astrocytes into functional neurons in a living human brain, comprising: injecting an adeno-associated virus (AAV) into a subject in need thereof, wherein the AAV comprises a DNA vector construct comprising: a nucleic acid coding sequence encoding a human neurogenic differentiation factor 1 (hNeuroD 1) protein, the hNeuroD1 protein comprising the amino acid sequence of SEQ ID No. 10, and a nucleic acid coding sequence encoding a human distantly related homeobox 2 (hDlx 2) 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 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.
In one aspect, the present disclosure provides and includes a method of converting glial cells into neurons in a subject in need thereof, comprising: delivering an adeno-associated virus (AAV) to 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) a Glial Fibrillary Acidic Protein (GFAP) promoter; (b) an enhancer; (c) a chimeric intron; (d) Woodchuck hepatitis virus posttranscriptional regulatory elements (WPREs); and (e) a polyadenylation signal sequence, wherein the AAV vector is capable of converting at least one glial cell to a neuron in the subject in need thereof.
In one aspect, the present disclosure provides and includes a method of treating a neurological disorder in a subject in need thereof, comprising: delivering an adeno-associated virus (AAV) to the subject, wherein the AAV administered to the subject in need thereof comprises a DNA vector 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 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) Woodchuck hepatitis virus posttranscriptional regulatory elements (WPREs); 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 convert at least one glial cell to a neuron.
Achaete-scale family BHLH transcription factor 1 (Ascl 1; also known as ASH1, HASH1, MASH-1, and bHLHa 46) encodes members of the basic helix-loop-helix family of transcription factors and is a gene that plays a role in neuronal commitment and differentiation.
Insulin gene enhancer proteins (ISL 1; also known as ISL LIM homeobox 1 and ISLET 1) are genes encoding transcription factors that contain two N-terminal LIM domains and one C-terminal homeodomain. The encoded protein plays a role in embryogenesis of langerhans islets.
LIM-homeobox 3 (LHX 3; also referred to as LIM3 and CPHD 3) genes encode proteins from a family of proteins with unique cysteine-rich zinc binding domains (LIM domains).
The huntingtin (Htt; also known as huntington's disease gene) gene encodes huntingtin. Wild type contains 6-35 glutamine residues and 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 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 Dlx coding sequence. In one aspect, the methods provided herein use a combination of an AAV vector comprising a NeuroD1 or Dlx 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 Dlx coding sequence, a second NeuroD1 coding sequence, and a second Dlx coding sequence. In one aspect, the methods provided herein use a combination of an AAV vector comprising NeuroD1 and Dlx coding sequences and a second AAV vector comprising NeuroD1 and Dlx coding sequences.
In one aspect, the methods provided herein use a combination of an AAV vector comprising NeuroD1 and Dlx coding sequences and an AAV vector comprising Htt-targeting shRNA sequences. In one aspect, the methods provided herein use a combination of an AAV vector comprising NeuroD1 and Dlx coding sequences and an AAV vector comprising an Htt-targeting shRNA sequence and an Htt-targeting second shRNA sequence. In one aspect, the methods provided herein use an AAV vector comprising NeuroD1 and Dlx coding sequences in combination with an AAV vector comprising an Htt-targeting shRNA sequence, an Htt-targeting second shRNA sequence, and an Htt-targeting third shRNA. In one aspect, the methods provided herein use an AAV vector comprising NeuroD1 and Dlx coding sequences and Htt-targeting shRNA sequences. In one aspect, the methods provided herein use an AAV vector comprising NeuroD1 and Dlx coding sequences and an Htt-targeting shRNA sequence and an Htt-targeting second shRNA sequence. In one aspect, the methods provided herein use an AAV vector comprising NeuroD1 and Dlx coding sequences and an Htt-targeting shRNA sequence, an Htt-targeting second shRNA sequence, and an Htt-targeting third shRNA.
In one aspect, the methods provided herein use an AAV vector comprising a combination of NeuroD1 and Dlx coding sequences and an Htt-targeting ASO sequence. In one aspect, the methods provided herein use an AAV vector comprising a combination of NeuroD1 and Dlx coding sequences with an Htt-targeting ASO sequence and an Htt-targeting second ASO sequence. In one aspect, the methods provided herein use an AAV vector comprising a combination of NeuroD1 and Dlx coding sequences with an Htt-targeting ASO sequence, an Htt-targeting second ASO sequence, and an Htt-targeting third ASO.
In one aspect, the methods provided herein use an AAV vector comprising a combination of NeuroD1 and Dlx coding sequences and Htt-targeting siRNA sequences. In one aspect, the methods provided herein use an AAV vector comprising a combination of NeuroD1 and Dlx coding sequences with an Htt-targeting siRNA sequence and an Htt-targeting second siRNA sequence. In one aspect, the methods provided herein use an AAV vector comprising a combination of NeuroD1 and Dlx coding sequences with an Htt-targeting siRNA sequence, an Htt-targeting second siRNA sequence, and an Htt-targeting third siRNA.
In one aspect, the methods provided herein use a combination of an AAV vector comprising NeuroD1 and Dlx coding sequences and an AAV vector comprising Htt-targeting miRNA sequences. In one aspect, the methods provided herein use a combination of an AAV vector comprising NeuroD1 and Dlx coding sequences and an AAV vector comprising an Htt-targeting miRNA sequence and an Htt-targeting second miRNA sequence. In one aspect, the methods provided herein use a combination of an AAV vector comprising NeuroD1 and Dlx coding sequences with an AAV vector comprising an Htt-targeting miRNA sequence, an Htt-targeting second miRNA sequence, and an Htt-targeting third miRNA. In one aspect, the methods provided herein use an AAV vector comprising NeuroD1 and Dlx coding sequences and Htt-targeting miRNA sequences. In one aspect, the methods provided herein use an AAV vector comprising NeuroD1 and Dlx coding sequences and an Htt-targeting miRNA sequence and an Htt-targeting second miRNA sequence. In one aspect, the methods provided herein use an AAV vector comprising NeuroD1 and Dlx coding sequences and an Htt-targeting miRNA sequence, an Htt-targeting second miRNA sequence, and an Htt-targeting third miRNA.
In one aspect, the methods provided herein use a combination of an AAV vector comprising NeuroD1 and Dlx coding sequences and an AAV vector comprising gRNA sequences that target Htt and CAS nucleases. In one aspect, the methods provided herein use AAV vectors comprising NeuroD1 and Dlx coding sequences and gRNA sequences targeting Htt and CAS nucleases.
In one aspect, the functionality of the AAV vectors provided herein is measured by assessing transcript levels and protein levels of NeuN, bisdermatan (DCX), β3-tubulin, (neurofilament 200) NF-200, (microtubule-associated protein 2) MAP2, ionized calcium binding adapter molecule (Iba 1).
As used herein, the term "NeuN" or "Fox-3" or "Rbfox2" or "hexaribonucleotide binding protein-3" refers to a protein that is homologous to the protein product of a sex determining gene in caenorhabditis elegans (Caenorhabditis elegans) and is a neuronal nuclear antigen.
As used herein, the term "DCX" or "doubering" or "lissencephalin-X" refers to microtubule-associated proteins expressed by neuronal precursor cells and immature neurons in embryonic and adult cortical structures.
As used herein, the term "β3-tubulin" or "class III β -tubulin" or "β -tubulin III" refers to the tubulin family of tubulin elements found in neurons.
As used herein, the term "NF-200" refers to a class of proteins that are type IV intermediate filars found in the cytoplasm of neurons.
As used herein, the term "MAP2" refers to a protein that belongs to the microtubule-associated protein family and that plays a role in determining and stabilizing neuronal morphology during neuronal development.
As used herein, the term "Iba1" refers to microglial macrophage-specific calcium binding protein.
In one aspect, the methods provided herein incorporate gene editing techniques to convert glial cells into neurons. In one aspect, the gene editing technique targets mutant Htt. In one aspect, the gene editing technique is selected from the group consisting of siRNA, miRNA, ASO and CRISPR/CAS. In one aspect, the gene editing technique is siRNA. In one aspect, the gene editing technique is miRNA. In one aspect, the gene editing technique is ASO. In one aspect, the gene editing technique 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 convert at least one glial cell to a neuron
As used herein, the term "mammal" refers to any species classified as a class of mammals.
As used herein, the term "human" refers to Homo sapiens. In one aspect, the human suffers from a neurological disorder.
As used herein, the term "living human" refers to a person having cardiac, respiratory and brain activity.
As used herein, the term "non-human primate" refers to any species or subspecies classified as primates but not homo sapiens. Non-limiting examples of non-human primates include chimpanzees, bonobo, red chimpanzees, gorillas, macaques, marmosets, pigtails, baboons, gibbons, and lemkeys.
As used herein, the term "delivery" or "delivery" refers to the treatment of a mammal with an AAV vector or composition provided herein. In one aspect, an AAV vector or composition provided herein is delivered to a subject in need thereof. In one aspect, an AAV vector or composition provided herein is formulated for delivery to a subject in need thereof. In one aspect, delivering includes local delivery. In one aspect, an AAV vector or composition provided herein is formulated for topical delivery. In one aspect, the delivery comprises systemic delivery. In one aspect, an AAV vector or composition provided herein is formulated for systemic delivery. In one aspect, delivery comprises injecting an AAV vector or composition provided herein into a subject in need thereof. In one aspect, the delivery is selected from the group consisting of: intraperitoneal, intramuscular, intravenous, intrathecal, intracerebral, intracranial, lateral cerebral compartments of the brain, intracavitary, intravitreal, subretinal, intraparenchymal, intranasal or oral administration. In one aspect, the delivery comprises intraperitoneal delivery. In one aspect, the delivery comprises intramuscular delivery. In one aspect, the delivery comprises intravenous delivery. In one aspect, the delivery comprises intrathecal delivery. In one aspect, the delivery comprises intra-brain delivery. In one aspect, the delivery comprises intracranial delivery. In one aspect, the delivery comprises lateral intraventricular delivery of the brain. In one aspect, the delivery comprises intracavitary delivery. In one aspect, the delivery comprises intravitreal delivery. In one aspect, the delivery comprises subretinal intradelivery. In one aspect, the delivery comprises intraparenchymal delivery. In one aspect, the delivery comprises intranasal delivery. In one aspect, the delivery comprises oral administration.
As used herein, the term "injection" refers to delivery of an AAV vector or composition provided herein under pressure and effort. By way of non-limiting example, injection may include the use of a syringe and needle.
In one aspect, an AAV vector or composition provided herein is injected into the brain of a subject. In one aspect, the AAV vector or composition is injected into the cerebral cortex of a subject. In one aspect, an AAV vector or composition provided herein is injected into the striatum of a subject. In one aspect, an AAV vector or composition provided herein is injected into the spinal cord or subject. In one aspect, the AAV vector or composition is injected into the 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, an AAV vector or composition provided herein has spread between about 1% and about 100% in the brain. In one aspect, an AAV vector or composition provided herein has spread in the brain between about 1% and about 10%, between 1% and about 20%, between 1% and about 30%, between 10% and about 20%, between 10% and about 30%, between about 10% and about 40%, between about 20% and about 30%, between about 20% and about 40%, between about 20% and about 50%, between about 30% and about 40%, between about 30% and about 50%, between about 30% and about 60%, between about 40% and about 50%, between about 40% and about 60%, between about 40% and about 70%, between about 50% and about 60%, between about 50% and about 70%, between about 60% and about 80%, between about 60% and about 90%, between about 70% and about 80%, between about 70% and about 90%, between about 100% and about 100%, or between about 100%.
In one aspect, an AAV vector or composition provided herein has spread between about 1% and about 100% in the cerebral cortex. In one aspect of the present invention, AAV vectors or compositions provided herein have spread between about 1% and about 10%, between 1% and about 20%, between 1% and about 30%, between 10% and about 20%, between 10% and about 30%, between about 10% and about 40%, between about 20% and about 30%, between about 20% and about 40%, between about 20% and about 50%, between about 30% and about 40%, between about 30% and about 50%, between about 30% and about 60%, between about 40% and about 50%, between about 40% and about 60%, between about 40% and about 70%, between about 50% and about 60%, between about 50% and about 70%, between about 50% and about 80%, between about 60% and about 90%, between about 70% and about 80%, between about 70% and about 70%, between about 90% and about 100%, between about 100% and about 100% or between about 100%.
In one aspect, an AAV vector or composition provided herein has spread between about 1% and about 100% in the spinal cord. In one aspect, an AAV vector or composition provided herein has spread between about 1% and about 10%, between 1% and about 20%, between 1% and about 30%, between 10% and about 20%, between 10% and about 30%, between about 10% and about 40%, between about 20% and about 30%, between about 20% and about 40%, between about 20% and about 50%, between about 30% and about 40%, between about 30% and about 50%, between about 30% and about 60%, between about 40% and about 50%, between about 40% and about 60%, between about 40% and about 70%, between about 50% and about 60%, between about 50% and about 70%, between about 60% and about 80%, between about 60% and about 90%, between about 70% and about 80%, between about 70% and about 90%, between about 100% and about 100%, or between about 100%.
In one aspect, an AAV vector or composition provided herein has spread between about 1% and about 100% in the striatum. In one aspect of the present invention, AAV vectors or compositions provided herein have diffused in the striatum between about 1% and about 10%, between 1% and about 20%, between 1% and about 30%, between 10% and about 20%, between 10% and about 30%, between about 10% and about 40%, between about 20% and about 30%, between about 20% and about 40%, between about 20% and about 50%, between about 30% and about 40%, between about 30% and about 50%, between about 30% and about 60%, between about 40% and about 50%, between about 40% and about 60%, between about 40% and about 70%, between about 50% and about 60%, between about 50% and about 70%, between about 60% and about 80%, between about 60% and about 90%, between about 70% and about 80%, between about 70%, about 40% and about 90% and about 100%, or between about 100% and about 100%.
In one aspect, an AAV vector or composition provided herein has spread between about 1% and about 100% in the dorsal striatum. In one aspect of the present invention, AAV vectors or compositions provided herein have diffused between about 1% and about 10%, between 1% and about 20%, between 1% and about 30%, between 10% and about 20%, between 10% and about 30%, between about 10% and about 40%, between about 20% and about 30%, between about 20% and about 40%, between about 20% and about 50%, between about 30% and about 40%, between about 30% and about 50%, between about 30% and about 60%, between about 40% and about 50%, between about 40% and about 60%, between about 40% and about 70%, between about 50% and about 60%, between about 50% and about 70%, between about 60% and about 80%, between about 60% and about 90%, between about 70% and about 80%, between about 70% and about 90%, between about 100% and about 100%, or between about 100% and about 100%.
In one aspect, an AAV vector or composition provided herein has been spread between about 1% and about 100% in the putamen. In one aspect, an AAV vector or composition provided herein has diffused between about 1% and about 10%, between 1% and about 20%, between 1% and about 30%, between 10% and about 20%, between 10% and about 30%, between about 10% and about 40%, between about 20% and about 30%, between about 20% and about 40%, between about 20% and about 50%, between about 30% and about 40%, between about 30% and about 50%, between about 30% and about 60%, between about 40% and about 50%, between about 40% and about 60%, between about 40% and about 70%, between about 50% and about 60%, between about 50% and about 70%, between about 60% and about 80%, between about 60% and about 90%, between about 70% and about 70%, between about 90% and about 100%, between about 100% and about 100%.
In one aspect, an AAV vector or composition provided herein has been spread between about 1% and about 100% in the caudate nucleus. In one aspect of the present invention, AAV vectors or compositions provided herein have diffused between about 1% and about 10%, between 1% and about 20%, between 1% and about 30%, between 10% and about 20%, between 10% and about 30%, between about 10% and about 40%, between about 20% and about 30%, between about 20% and about 40%, between about 20% and about 50%, between about 30% and about 40%, between about 30% and about 50%, between about 30% and about 60%, between about 40% and about 50%, between about 40% and about 60%, between about 40% and about 70%, between about 50% and about 60%, between about 50% and about 70%, between about 50% and about 80%, between about 60% and about 90%, between about 70% and about 80%, between about 70% and about 70%, between about 90% and about 100%, between about 100% and about 100% or between about 100%.
In one aspect, an AAV vector or composition provided herein diffuses between about 1% and about 100% from the injection site. In one aspect, an AAV vector or composition provided herein has diffused from an injection site between about 1% and about 10%, between 1% and about 20%, between 1% and about 30%, between 10% and about 20%, between 10% and about 30%, between about 10% and about 40%, between about 20% and about 30%, between about 20% and about 40%, between about 20% and about 50%, between about 30% and about 40%, between about 30% and about 50%, between about 30% and about 60%, between about 40% and about 50%, between about 40% and about 60%, between about 40% and about 70%, between about 50% and about 60%, between about 50% and about 70%, between about 60% and about 80%, between about 60% and about 90%, between about 70% and about 80%, between about 70% and about 90%, between about 100% and about 100%, or between about 100%.
In one aspect, an AAV vector or composition provided herein has between about 1% and about 100% spread in the substantia nigra. In one aspect, an AAV vector or composition provided herein has diffused between about 1% and about 10%, between 1% and about 20%, between 1% and about 30%, between 10% and about 20%, between 10% and about 30%, between about 10% and about 40%, between about 20% and about 30%, between about 20% and about 40%, between about 20% and about 50%, between about 30% and about 40%, between about 30% and about 50%, between about 30% and about 60%, between about 40% and about 50%, between about 40% and about 60%, between about 40% and about 70%, between about 50% and about 60%, between about 50% and about 70%, between about 60% and about 80%, between about 60% and about 90%, between about 70% and about 70%, between about 90% and about 100%, between about 100% and about 100%.
As used herein, the term "AAV particle" refers to the packaged capsid form of an AAV virus that delivers its nucleic acid genome to a cell.
In one aspect, an AAV particle comprising an AAV particle encoded by an AAV vector provided hereinThe composition is between 10 10 AAV particles/mL and 10 14 Concentration between AAV particles/mL. In one aspect, a composition comprising AAV particles encoded by an AAV vector provided herein is provided to be between 10 10 AAV particles/mL and 10 11 Between 10 AAV particles/mL 10 AAV particles/mL and 10 12 Between 10 AAV particles/mL 10 AAV particles/mL and 10 13 Between 10 AAV particles/mL 11 AAV particles/mL and 10 12 Between 10 AAV particles/mL 11 AAV particles/mL and 10 13 Between 10 AAV particles/mL 11 AAV particles/mL and 10 14 Between 10 AAV particles/mL 12 AAV particles/mL and 10 13 Between 10 AAV particles/mL 12 AAV particles/mL and 10 14 Between or between 10 AAV particles/mL 13 AAV particles/mL and 10 14 Concentration between AAV particles/mL.
In one aspect, a composition comprising AAV particles encoded by an AAV vector provided herein is injected in a volume of between 10 μl and 1000 μl. In one aspect of the present invention, a composition comprising AAV particles encoded by an AAV vector provided herein is injected at a volume between 10 μl and 100 μl, between 10 μl and 200 μl, between 10 μl and 300 μl, between 100 μl and 200 μl, between 100 μl and 300 μl, between 100 μl and 400 μl, between 200 μl and 300 μl, between 200 μl and 400 μl, between 200 μl and 500 μl, between 300 μl and 400 μl, between 300 μl and 500 μl, between 300 μl and 600 μl, between 400 μl and 500 μl, between 400 μl and 600 μl, between 400 μl and 700 μl, between 500 μl and 600 μl, between 500 μl and 700 μl, between 500 μl and 900 μl, between 500 μl and 800 μl, between 600 μl and 900 μl, between 700 μl and 700 μl, between 700 μl and 900 μl, between 1000 μl and 900 μl, between 700 μl and 900 μl, between 1000 μl, between 700 μl and 900 μl, between 400 μl and 900 μl).
As used herein, the term "subject" refers to any animal subject. Non-limiting examples of animal subjects include humans, laboratory animals (e.g., primates, rats, mice), domestic animals (e.g., cows, sheep, goats, pigs, turkeys, chickens), and domestic pets (e.g., dogs, cats, rodents, etc.).
As used herein, "a subject in need thereof" refers to a subject suffering from a neurological disorder. In one aspect, a subject in need thereof has a neurological disorder selected from the group consisting of: alzheimer's disease, parkinson's disease, amyotrophic Lateral Sclerosis (ALS), huntington's disease, epilepsy, physical injury, stroke, cerebral aneurysms, traumatic brain injury, concussion, tumors, inflammation, infection, ataxia, brain atrophy, spinal cord atrophy, multiple sclerosis, traumatic spinal cord injury, ischemic or hemorrhagic myelopathy (myelopathy), global cerebral ischemia, hypoxic ischemic encephalopathy, embolism, fibrocartilage embolic myelopathy, thrombosis, kidney disease, chronic inflammatory diseases, meningitis, and cerebral venous sinus thrombosis. In one aspect, a subject in need thereof suffers from alzheimer's disease. In one aspect, a subject in need thereof suffers from parkinson's disease. In one aspect, a subject in need thereof has ALS. In one aspect, a subject in need thereof suffers from huntington's disease. In one aspect, a subject in need thereof suffers from epilepsy. In one aspect, a subject in need thereof suffers from physical injury. In one aspect, a subject in need thereof suffers from stroke. In one aspect, a subject in need thereof has ischemic stroke. In one aspect, a subject in need thereof suffers from hemorrhagic stroke. In one aspect, a subject in need thereof has a cerebral aneurysm. In one aspect, a subject in need thereof suffers from traumatic brain injury. In one aspect, a subject in need thereof suffers from concussion. In one aspect, a subject in need thereof has a tumor. In one aspect, a subject in need thereof suffers from inflammation. In one aspect, a subject in need thereof suffers from an infection. In one aspect, a subject in need thereof suffers from ataxia. In one aspect, a subject in need thereof suffers from brain atrophy. In one aspect, a subject in need thereof suffers from spinal atrophy. In one aspect, a subject in need thereof suffers from multiple sclerosis. In one aspect, a subject in need thereof suffers from a traumatic spinal cord injury. In one aspect, a subject in need thereof suffers from ischemic or hemorrhagic myelopathy (myelopathy). In one aspect, a subject in need thereof suffers from global cerebral ischemia. In one aspect, a subject in need thereof suffers from hypoxic ischemic encephalopathy. In one aspect, a subject in need thereof suffers from embolism. In one aspect, a subject in need thereof suffers from fibrocartilage embolic myelopathy. In one aspect, a subject in need thereof suffers from thrombosis. In one aspect, a subject in need thereof suffers from kidney disease. In one aspect, a subject in need thereof suffers from a chronic inflammatory disease. In one aspect, a subject in need thereof has meningitis. In one aspect, a subject in need thereof is suffering from cerebral venous sinus thrombosis.
In one aspect, the subject in need thereof is a mammal. In one aspect, the subject in need thereof is a human. In one aspect, a subject in need thereof is a non-human primate. In one aspect, a subject in need thereof is selected from the group consisting of chimpanzees, bonobes, chimpanzees, gorillas, macaques, marmosets, pigtails, baboons, gibbons, and lemkeys. In one aspect, the subject in need thereof is a chimpanzee. In one aspect, the subject in need thereof is a bonobo. In one aspect, the subject in need thereof is an gorilla. In one aspect, the subject in need thereof is a gorilla. In one aspect, the subject in need thereof is cynomolgus monkey. In one aspect, the subject in need thereof is a marmoset. In one aspect, the subject in need thereof is a cynomolgus monkey. In one aspect, the subject in need thereof is a baboon. In one aspect, the subject in need thereof is a gibbon. In one aspect, the subject in need thereof is a cynomolgus monkey.
In one aspect, the subject in need thereof is a male. In one aspect, the subject in need thereof is a female. In one aspect, the subject in need thereof is neutral. In one aspect, the subject in need thereof is a premature infant. In one aspect, the premature infant is born before 36 weeks of gestation. In one aspect, the subject in need thereof is a term infant. In one aspect, the term infant is less than about 2 months of age. In one aspect, the subject in need thereof is a neonate. In one aspect, the neonate is less than about 1 month old. In one aspect, the subject in need thereof is an infant. In one aspect, the infant is between 2 months of age and 24 months of age. In one aspect of the present invention, infants between 2 and 3 months of age, between 2 and 4 months of age, between 2 and 5 months of age, between 3 and 4 months of age, between 3 and 5 months of age, between 3 and 6 months of age, between 4 and 5 months of age, between 4 and 6 months of age, between 4 and 7 months of age, between 5 and 6 months of age, between 5 and 7 months of age between 5 and 8 months of age, between 6 and 7 months of age, between 6 and 8 months of age, between 6 and 9 months of age, between 7 and 8 months of age, between 7 and 9 months of age, between 7 and 10 months of age, between 8 and 9 months of age, between 8 and 10 months of age, between 8 and 11 months of age, between 9 and 10 months of age between 5 and 8 months of age, between 6 and 7 months of age, between 6 and 8 months of age, between 6 and 9 months of age, between 7 and 8 months of age, between 7 and 9 months of age between 7 and 10 months of age, between 8 and 9 months of age, between 8 and 10 months of age, between 8 and 11 months of age, between 9 and 10 months of age, between 16 and 18 months of age, between 16 and 19 months of age, between 17 and 18 months of age, between 17 and 19 months of age, between 17 and 20 months of age, between 18 and 19 months of age, between 18 and 20 months of age, between 18 and 21 years of age, between 19 and 20 months of age, between 19 and 21 months of age, between 19 and 22 months of age, between 20 and 21 months of age, between 20 and 22 months of age, between 20 and 23 months of age, between 21 and 22 months of age, between 21 and 23 months of age, between 21 and 24 months of age, between 22 and 23 months of age, and between 23 and 24 years of age. In one aspect, the subject in need thereof is a pediatric subject. In one aspect, the young child is between 1 year and 4 years old. In one aspect, the young child is between 1 and 2 years old, between 1 and 3 years old, between 1 and 4 years old, between 2 and 3 years old, between 2 and 4 years old, and between 3 and 4 years old. In one aspect, the subject in need thereof is a pediatric subject. In one aspect, the child is between 2 and 5 years of age. In one aspect, the child is between 2 and 3 years old, between 2 and 4 years old, between 2 and 5 years old, between 3 and 4 years old, between 3 and 5 years old, and between 4 and 5 years old. In one aspect, the subject in need thereof is a child. In one aspect, the child is between 6 and 12 years old. In one aspect, the child is between 6 and 7 years old, between 6 and 8 years old, between 6 and 9 years old, between 7 and 8 years old, between 7 and 9 years old, between 7 and 10 years old, between 8 and 9 years old, between 8 and 10 years old, between 8 and 11 years old, between 9 and 10 years old, between 9 and 11 years old, between 9 and 12 years old, between 10 and 11 years old, between 10 and 12 years old, and between 11 and 12 years old. In one aspect, the subject in need thereof is an adolescent. In one aspect, the teenager is between 13 and 19 years of age. In one aspect, teenagers are between 13 and 14 years old, between 13 and 16 years old, between 14 and 14 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 of age and 18 years of age. In one aspect of the present invention, pediatric subjects are between 1 day old and 1 year old, between 1 day old and 2 years old, between 1 day old and 3 years old, between 1 year old and 2 years old, between 1 year old and 3 years old, between 1 year old and 4 years old, between 2 years old and 3 years old, between 2 years old and 4 years old, between 2 years old and 5 years old, between 3 years old and 4 years old, between 3 years old and 5 years old, between 3 years old and 6 years old between 4 and 5 years old, between 4 and 6 years old, between 4 and 7 years old, between 5 and 6 years old, between 5 and 7 years old, between 5 and 8 years old, between 6 and 7 years old, between 6 and 8 years old, between 6 and 9 years old, between 7 and 8 years old, between 7 and 9 years old, between 7 and 10 years old, between 8 and 9 years old between 4 and 5 years, between 4 and 6 years, between 4 and 7 years, between 5 and 6 years, between 5 and 7 years, between 5 and 8 years, between 6 and 7 years between 6 and 8 years, between 6 and 9 years, between 7 and 8 years, between 7 and 9 years, between 7 and 10 years, between 8 and 9 years, between 16 and 18 years and between 17 and 18 years. In one aspect, the subject in need thereof is an elderly subject. In one aspect, the elderly subject is between 65 and 95 years old or higher. In one aspect, an elderly subject is between 65 and 70 years old, between 65 and 75 years old, between 65 and 80 years old, between 70 and 75 years old, between 70 and 80 years old, between 70 and 85 years old, between 75 and 80 years old, between 75 and 85 years old, between 75 and 90 years old, between 80 and 85 years old, between 80 and 90 years old, between 80 and 95 years old, between 85 and 90 years old, and between 85 and 95 years old. In one aspect, the subject in need thereof is an adult subject. In one aspect, the adult subject is between 20 years and 95 years or more. In one aspect of the present invention, an adult subject is between 20 and 25 years old, between 20 and 30 years old, between 20 and 35 years old, between 25 and 30 years old, between 25 and 35 years old, between 25 and 40 years old, between 30 and 35 years old, between 30 and 40 years old, between 30 and 45 years old, between 35 and 40 years old, between 35 and 45 years old, between 35 and 50 years old, between 40 and 45 years old, between 40 and 50 years old, between 40 and 55 years old, between 45 and 50 years old, between 45 and 55 years old, between 45 and 60 years old, between 50 and 55 years old, between 50 and 60 years old between 50 and 65, between 55 and 60, between 55 and 65, between 55 and 70, between 60 and 65, between 60 and 70, between 60 and 75, between 65 and 70, between 65 and 75, between 65 and 80, between 70 and 75, between 70 and 80, between 70 and 85, between 75 and 80, between 75 and 85, between 75 and 90, between 80 and 95, between 85 and 90. In one aspect, a subject in need thereof is between 1 and 5 years old, between 2 and 10 years old, between 3 and 18 years old, between 21 and 50 years old, between 21 and 40 years old, between 21 and 30 years old, between 50 and 90 years old, between 60 and 90 years old, between 70 and 90 years old, between 60 and 80 years old, or between 65 and 75 years old. In one aspect, the subject in need thereof is a young aged subject (65 to 74 years old). In one aspect, the subject in need thereof is an elderly subject (75 to 84 years old). In one aspect, the subject in need thereof is an elderly subject (> 85 years).
As used herein, the term "flow rate" refers to the rate of delivery of an AAV vector or composition. In one aspect, the flow rate is between 0.1 μl/min and 5.0 μl/min. In one aspect, the flow rate is between 0.1 and 0.2 μL/min, between 0.1 and 0.3 μL/min, between 0.1 and 0.4 μL/min, between 0.2 and 0.3 μL/min, between 0.2 and 0.4 μL/min, between 0.2 and 0.5 μL/min, between 0.3 and 0.4 μL/min, between 0.3 and 0.5 μL/min, between 0.3 and 0.6 μL/min, between 0.3 and 0.5 μL/min, between 0.4 and 0.6 μL/min, between 0.4 and 0.7 μL/min, between 0.5 and 6 μL/min, between 0.5 and 0.6 μL/min, between 0.7 μL/min, between 0.3 and 0.6 μL/min, between 0.7 and 0.8 and 7 μL/min, between 0.7 and 7 μL/min, between 0.3 and 0.6 μL/min, between 0.7 and 0.8.7 μL/min, between 0.3 and 0.6 μL/min, between 0.7 and 0.5 μL/min, between 0.8 and 1.1. Mu.L/min, between 0.9 and 1.0. Mu.L/min, between 0.9 and 1.1. Mu.L/min, between 0.9 and 1.2. Mu.L/min, between 1.0 and 1.1. Mu.L/min, between 1.0 and 1.2. Mu.L/min, between 1.0 and 1.3. Mu.L/min, between 1.1 and 1.2. Mu.L/min, between 1.3 and 1.3. Mu.L/min, between 1.1 and 1.1. Mu.L/min, between 1.2 and 1.2. Mu.L/min, between 1.2 and 1.4. Mu.L/min, between 1.2 and 1.5. Mu.5 and 1.5 and 6.1.1 and 6.1.1.1 and 6.1.1.1.4. Mu.L/min, between 1.3 and 1.1.1 and 1.1.4. Mu.L/min, between 1.1 and 1.1.1 and 1.1.1.1 and 6. Mu.L/min, between 1.1.1 and 6.1.1.1 and 6. Mu.L/1.L/min Between 1.6 and 1.9. Mu.L/min, between 1.7 and 1.8. Mu.L/min, between 1.7 and 1.9. Mu.L/min, between 1.7 and 2.0. Mu.L/min, between 1.8 and 1.9. Mu.L/min, between 1.8 and 2.0. Mu.L/min, between 1.8 and 2.1. Mu.L/min, between 1.9 and 2.0. Mu.L/min, between 1.9 and 2.9. Mu.L/min, between 1.9 and 2.2. Mu.L/min, between 2.0 and 2.0. Mu.L/min, between 2.0 and 2.2. Mu.L/min, between 2.0 and 2.3. Mu.3 and 2.3, between 2.4 and 2.4. Mu.4. Mu.L/min, between 1 and 2.4. Mu.3, between 1 and 2.4. Mu.L/min, between 1.9 and 2.2.2.2 and 2.2. Mu.L/min, between 1 and 2.4. Mu.L/min Between 2.4 and 2.7 μL/min, between 2.5 and 2.6 μL/min, between 2.5 and 2.7 μL/min, between 2.5 and 2.8 μL/min, between 2.6 and 2.7 μL/min, between 2.6 and 2.8 μL/min, between 2.6 and 2.9 μL/min, between 2.7 and 2.8 μL/min, between 2.7 and 2.9 μL/min, between 2.7 and 3.0 μL/min, between 2.8 and 2.9 μL/min, between 2.8 and 3.0 μL/min between 2.8 and 3.1 μL/min, between 2.9 and 3.0 μL/min, between 2.9 and 3.1 μL/min, between 2.9 and 3.2 μL/min, between 3.0 and 3.1 μL/min, between 3.0 and 3.2 μL/min, between 3.0 and 3.3 μL/min, between 3.1 and 3.2 μL/min, between 3.1 and 3.3 μL/min, between 3.1 and 3.4 μL/min, between 3.2 and 3.3 μL/min, between 3.2 and 3.2 μL/min, between 3.4 μL/min, between 3.2 and 3.4 μL/min, between 3.2 and 3.5 μL/min, between 3.3 and 3.4 μL/min, between 3.3 and 3.5 μL/min, between 3.3 and 3.6 μL/min, between 3.4 and 3.5 μL/min, between 3.4 and 3.6 μL/min, between 3.4 and 3.7 μL/min, between 3.5 and 3.6 μL/min, between 3.5 and 3.7 μL/min, between 3.5 and 3.8 μL/min, between 3.6 and 3.7 μL/min, between 3.6 and 3.8 μL/min between 3.6 and 3.9 μL/min, between 3.7 and 3.8 μL/min, between 3.7 and 3.9 μL/min, between 3.7 and 4.0 μL/min, between 3.8 and 3.9 μL/min, between 3.8 and 4.0 μL/min, between 3.8 and 4.1 μL/min, between 3.9 and 4.0 μL/min, between 3.9 and 4.1 μL/min, between 3.9 and 4.2 μL/min, between 4.0 and 4.1 μL/min, between 4.0 and 4.0 μL/min, between 4.2 μL/min, between 3.9 and 4.2 μL/min, between 4.0 and 4.3 μL/min, between 4.1 and 4.2 μL/min, between 4.1 and 4.3 μL/min, between 4.1 and 4.4 μL/min, between 4.2 and 4.3 μL/min, between 4.2 and 4.4 μL/min, between 4.2 and 4.2 μL/min, between 4.2 and 4.5 μL/min, between 4.3 and 4.4 μL/min, between 4.3 and 4.5 μL/min, between 4.3 and 4.6 μL/min, between 4.4 and 4.5 μL/min, between 4.4 and 4.6 μL/min, between 4.4 and 4.7 μL/min, between 4.7 and 4.7 μL/min, between 4.5 and 4.6 μL/min, between 4.7 and 4.6 μL/min, between 4.3 and 4.4 and 4.6 μL/min, between 4.3 and 4.5 μL/min, between 4.3 and 4.6 μL/min, between 4.6 μL/min and 4.6 μL/min, between 4.7 and 4.7 μL/min, between 4.7 and 4.6 μL/min Between 4.8 and 5.0 or between 4.9 and 5.0. Mu.L/min.
As used herein, the term "therapeutically effective dose" or "pharmaceutically active dose" refers to an amount of an AAV particle or composition provided herein that is effective to treat a neurological disorder. In one aspect, the AAV particles or compositions provided herein may be provided with a pharmaceutically acceptable carrier. As used herein, a "pharmaceutically acceptable carrier" refers to a non-toxic solvent, dispersant, excipient, adjuvant, or other material that is mixed with an AAV particle or composition provided herein.
Non-limiting examples of pharmaceutically acceptable carriers include diluents, adjuvants, excipients or acid resistant encapsulating ingredients in liquid (e.g., saline), gel, nanoparticle, exosome, lipid vesicles or solid form. Non-limiting examples of suitable diluents and excipients include pharmaceutical grade physiological saline, dextrose, glycerol, mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like, and combinations thereof. In one aspect, the therapeutically effective dose contains auxiliary substances such as wetting or emulsifying agents, stabilizing agents, or pH buffering agents. In one aspect, a therapeutically effective dose of an AAV particle or composition provided herein is injected into a subject. In one aspect, a therapeutically effective dose of an AAV particle or composition provided herein is delivered to a subject. In one aspect, a therapeutically effective dose is administered with at least one pharmaceutically acceptable carrier. In one aspect, a therapeutically effective dose contains between about 1% and about 5%, between about 5% and about 10%, between about 10% and about 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 95%, between about 25% and about 95%, between about 30% and about 95%, between about 35% and about 95%, between about 40% and about 95%, between about 45% and about 95%, between about 50% and about 95%, between about 55% and about 95%, between about 60% and about 95%, between about 45% and about 95%, between about 80% and about 95%, or between about 95% and about 80% of the AAV.
In one aspect, a therapeutically effective dose is delivered to a subject in need thereof at least once daily or at least once weekly for at least two consecutive days or weeks. In one aspect, a therapeutically effective dose is delivered to a subject in need thereof at least once daily or at least once weekly for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 14 consecutive days or weeks. In one aspect, a therapeutically effective dose is delivered to a subject in need thereof at least once daily or at least once weekly for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 consecutive weeks. In one aspect, a therapeutically effective dose is delivered to a subject in need thereof at least once daily or at least once weekly for up to 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, 19, or 20 consecutive days or weeks. In one aspect, a therapeutically effective dose is delivered to a subject in need thereof at least once daily or at least once weekly for up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 consecutive weeks or months. In one aspect, a therapeutically effective dose is delivered to a subject in need thereof for at least one administration for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 consecutive months or years, for a prolonged period of time throughout the subject's life cycle, or for an indefinite period of time. In one aspect, a therapeutically effective dose is delivered once a year to a subject in need thereof for 2 years, 3 years, or 5 years. In one aspect, a therapeutically effective dose is delivered once a year to a subject in need thereof for 2 consecutive years. In one aspect, a therapeutically effective dose is delivered once a year to a subject in need thereof for 3 consecutive years. In one aspect, a therapeutically effective dose is delivered once a year to a subject in need thereof for 5 consecutive years.
As used herein, the terms "alleviating", "cure" or "resolution" refer to the percentage of a subject in need thereof that is cured of or achieves relief or complete resolution of a neurological disorder in response to a therapeutically effective dose.
As used herein, the term "response rate" refers to the percentage of a therapeutically effective dose that a subject in need thereof responds positively (e.g., the severity or frequency of one or more symptoms decreases).
In one aspect, a therapeutically effective dose achieves at least about 50% relief, cure, response rate, or resolution of the neurological disorder. In one aspect, a therapeutically effective dose eliminates, reduces, slows or delays symptoms of one or more neurological disorders. Non-limiting examples of neurological disorder symptoms include tremors, bradykinesia (bradykinesia), muscle stiffness, impaired posture and balance, automatic loss of motion, uncoordinated motion, uncontrolled motion, spontaneous percussive motion, speech changes, numbness, writing changes, involuntary movements (such as chorea-like movements), uncontrolled postures, mood changes, and sleep disorders. In one aspect, the symptom of the neurological disorder is a motor symptom. Non-limiting examples of movement symptoms include involuntary movement disorders or autonomic movement disorders. In one aspect, the symptom of the neurological disorder is a cognitive symptom. Non-limiting examples of cognitive symptoms include fine motor skills, tremors, epilepsy, chorea, dystonia, dyskinesia, slow or abnormal eye movements, impaired gait, impaired posture, impaired balance, speech difficulties, dysphagia, tissue difficulties, difficulty in prioritizing (difficulty prioritizing), difficulty concentrating on tasks, lack of flexibility, lack of impulse control, outbreak, lack of consciousness on their own behavior and/or ability, slow processing ideas, difficulty learning new information, difficulty memory things, difficulty communicating, difficulty following commands, difficulty performing tasks.
In one aspect, the symptom of the neurological disorder is a psychotic symptom. Non-limiting examples of psychotic symptoms include depression, irritability, sadness or apathy, social withdrawal, insomnia, fatigue, lack of energy, obsessive compulsive disorder, mania, bipolar disorders and weight loss. In one aspect, the symptom of the neurological disorder is an impairment of at least one blood vessel. In one aspect, the symptom of the neurological disorder is an impaired blood brain barrier. In one aspect, the symptom of the neurological disorder is impaired blood flow. Non-limiting examples of tests for evaluating the elimination, reduction, slowing or delay of neurological condition symptoms include the Unified Huntington's Disease Rating Scale (UHDRS) score, the UHDRS Total Functional Capacity (TFC), the UHDRS functional assessment, the UHDRS gait score, the UHDRS Total Motor Score (TMS), the hamilton depression scale (HAM-D), the columbian suicide severity rating scale (C-SSRS), the montreal cognitive assessment (MoCA), the modified rank scale (mRS), the National Institutes of Health Stroke Scale (NIHSS) and the pap index (BI), the rising-walking timing Test (TUG), the Chedoke upper limb and hand functional activity scale (CAHAI), the symbolic digital modality test, the controlled oral word association task (Controlled Oral Word Association tasks), magnetic Resonance Imaging (MRI), functional magnetic resonance imaging (fMRI) and Positron Emission Tomography (PET) scans.
In one aspect, a therapeutically effective dose achieves a relief, cure, response rate, or resolution of between about 10% and about 99% or more of the neurological disorder. In one aspect, a therapeutically effective dose achieves between 10% and 100% relief, cure, response rate, or regression rate of the neurological condition, such as between 10% and 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 25% and 35%, between 25% and 40%, between 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 50% and 60% 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 between 40% and 55%, between 45% and 50%, between 45% and 55%, between 45% and 60%, between 50% and 55%, between 50% and 60%, between 90% and 100% or between 95% and 100%.
In one aspect, the therapeutically effective dose eliminates, reduces, slows or delays between 10% and 100% of the symptoms of one or more neurological disorders, such as between 10% and about 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 25% and 35%, between 25% and 40%, between 30% and 35%, between 30% and 40%, between 35% and 45%, between 35% and 50%, between 40% and 45%, between 40% and 50%, between 40% and 55%, between 45% and 50%, between 45% and 55%, between 45% and 60%, between 50% and 55%, between between 50% and 60%, between 50% and 65%, between 55% and 60%, between 55% and 65%, between 55% and 70%, between 60% and 65%, between 60% and 70%, between 60% and 75%, between 65% and 70%, between 65% and 75%, between 65% and 80%, between 70% and 75%, between between 70% and 80%, between 70% and 85%, between 75% and 80%, between 75% and 85%, between 75% and 90%, between 80% and 85%, between 80% and 90%, between 80% and 95%, between 85% and 90%, between 85% and 95%, between 85% and 100%, between 90% and 95%, between, between 90% and 100% or between 95% and 100%.
In one aspect, the symptoms of the neurological disorder are assessed annually on the day of treatment, 1 day after treatment, 3 months after treatment, 6 months after treatment, 1 year after treatment, and after treatment.
In one aspect, the symptoms of the neurological disorder are assessed between 1 day after treatment and 7 days after treatment. In one aspect, symptoms can be assessed between 1 day and 2 days post-treatment, between 1 day and 3 days post-treatment, between 1 day and 4 days post-treatment, between 2 days post-treatment and 3 days post-treatment, between 2 days post-treatment and 4 days post-treatment, between 2 days post-treatment and 5 days post-treatment, between 3 days post-treatment and 4 days post-treatment, between 3 days post-treatment and 5 days post-treatment, between 3 days post-treatment and 6 days post-treatment, between 4 days post-treatment and 5 days post-treatment, between 4 days post-treatment and 6 days post-treatment, between 4 days post-treatment and 7 days post-treatment, between 5 days post-treatment and 6 days post-treatment, between 5 days post-treatment and 7 days post-treatment, or between 6 days post-treatment and 7 days post-treatment. In one aspect, symptoms can be assessed between 1 week after treatment and 4 weeks after treatment. In one aspect, symptoms can be assessed between 1 week and 2 weeks after treatment, between 1 week and 3 weeks after treatment, between 1 week and 4 weeks after treatment, between 2 weeks and 3 weeks after treatment, between 2 weeks and 4 weeks after treatment, or between 3 weeks and 4 weeks after treatment. In one aspect, symptoms can be assessed between 1 month after treatment and 12 months after treatment. In one aspect of the present invention, symptoms can be between 1 month and 2 months after treatment, between 1 month and 3 months after treatment, between 1 month and 4 months after treatment, between 2 months and 3 months after treatment, between 2 months and 4 months after treatment, between 2 months and 5 months after treatment, between 3 months and 4 months after treatment, between 3 months and 5 months after treatment, between 3 months and 6 months after treatment, between 4 months and 5 months after treatment, between 4 months and 6 months after treatment, between 4 months and 7 months after treatment, between 5 months and 6 months after treatment, between 5 months and 7 months after treatment, between 5 months and between 5 months and 8 months, between 6 months and 7 months, between 6 months and 8 months, between 6 months and 9 months, between 7 months and 8 months, between 7 months and 9 months, between 7 months and 10 months, between 8 months and 9 months, between 8 months and 10 months, between 8 months and 11 months, between 9 months and 10 months, between 9 months and 11 months, between 9 months and 12 months, between 10 months and 11 months Assessment between 10 months and 12 months after treatment or between 11 months and 12 months after treatment. In one aspect, symptoms can be assessed between 1 year after treatment and about 20 years after treatment. In one aspect, symptoms may be assessed between 1 year and 5 years post-treatment, between 1 year and 10 years post-treatment, between 1 year and 14 years post-treatment, between 5 years post-treatment and 10 years post-treatment, between 5 years post-treatment and 14 years post-treatment, between 5 years post-treatment and 20 years post-treatment, between 10 years post-treatment and 14 years post-treatment, between 10 years post-treatment and 20 years post-treatment, or between 14 years post-treatment and 20 years post-treatment.
As used herein, the term "survival rate" refers to the survival of a subject cohort in a treatment group after a given period of time following diagnosis of a neurological disorder.
In one aspect, a therapeutically effective dose achieves an increase in survival rate of between about 10% and 99% or more. In one aspect, a therapeutically effective dose achieves an increase in survival rate of between 10% and 100%, such as between 10% and 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 25% and 35%, between 25% and 40%, between 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 50% and 60% 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 between 40% and 55%, between 45% and 50%, between 45% and 55%, between 45% and 60%, between 50% and 55%, between 50% and 60%, between 90% and 100% or between 95% and 100%.
As used herein, the term "life expectancy" refers to the period of time in which the subject is expected to survive.
In one aspect, a therapeutically effective dose increases the life expectancy by between about 10% and 99% or more. In one aspect, a therapeutically effective dose increases the life expectancy by between 10% and 100%, such as between 10% and 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 25% and 35%, between 25% and 40%, between 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 50% and 60% 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 between 40% and 55%, between 45% and 50%, between 45% and 55%, between 45% and 60%, between 50% and 55%, between 50% and 60%, 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 25% and 35%, between 25% and 40%, between 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 50% and 60% 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 between 40% and 55%, between 45% and 50%, between 45% and 55%, between 45% and 60%, between 50% and 55%, between 50% and 60%, between 90% and 100% or between 95% and 100%.
In one aspect, the amount of atrophy in the brain of a subject in need thereof is assessed annually on the day of treatment, 1 day after treatment, 3 months after treatment, 6 months after treatment, 1 year after treatment, and after treatment.
In one aspect, the amount of atrophy in the brain of a subject in need thereof is assessed between 1 day post-treatment and 7 days post-treatment. In one aspect, symptoms can be assessed between 1 day and 2 days post-treatment, between 1 day and 3 days post-treatment, between 1 day and 4 days post-treatment, between 2 days post-treatment and 3 days post-treatment, between 2 days post-treatment and 4 days post-treatment, between 2 days post-treatment and 5 days post-treatment, between 3 days post-treatment and 4 days post-treatment, between 3 days post-treatment and 5 days post-treatment, between 3 days post-treatment and 6 days post-treatment, between 4 days post-treatment and 5 days post-treatment, between 4 days post-treatment and 6 days post-treatment, between 4 days post-treatment and 7 days post-treatment, between 5 days post-treatment and 6 days post-treatment, between 5 days post-treatment and 7 days post-treatment, or between 6 days post-treatment and 7 days post-treatment. In one aspect, symptoms can be assessed between 1 week after treatment and 4 weeks after treatment. In one aspect, symptoms can be assessed between 1 week and 2 weeks after treatment, between 1 week and 3 weeks after treatment, between 1 week and 4 weeks after treatment, between 2 weeks and 3 weeks after treatment, between 2 weeks and 4 weeks after treatment, or between 3 weeks and 4 weeks after treatment. In one aspect, symptoms can be assessed between 1 month after treatment and 12 months after treatment. In one aspect of the present invention, symptoms can be between 1 month and 2 months after treatment, between 1 month and 3 months after treatment, between 1 month and 4 months after treatment, between 2 months and 3 months after treatment, between 2 months and 4 months after treatment, between 2 months and 5 months after treatment, between 3 months and 4 months after treatment, between 3 months and 5 months after treatment, between 3 months and 6 months after treatment, between 4 months and 5 months after treatment, between 4 months and 6 months after treatment, between 4 months and 7 months after treatment, between 5 months and 6 months after treatment, between 5 months and 7 months after treatment, between 5 months and between 5 months and 8 months, between 6 months and 7 months, between 6 months and 8 months, between 6 months and 9 months, between 7 months and 8 months, between 7 months and 9 months, between 7 months and 10 months, between 8 months and 9 months, between 8 months and 10 months, between 8 months and 11 months, between 9 months and 10 months, between 9 months and 11 months, between 9 months and 12 months, between 10 months and 11 months Assessment between 10 months and 12 months after treatment or between 11 months and 12 months after treatment. In one aspect, symptoms can be assessed between 1 year after treatment and about 20 years after treatment. In one aspect, symptoms may be assessed between 1 year and 5 years post-treatment, between 1 year and 10 years post-treatment, between 1 year and 14 years post-treatment, between 5 years post-treatment and 10 years post-treatment, between 5 years post-treatment and 14 years post-treatment, between 5 years post-treatment and 20 years post-treatment, between 10 years post-treatment and 14 years post-treatment, between 10 years post-treatment and 20 years post-treatment, or between 14 years post-treatment and 20 years post-treatment.
Non-limiting examples of tests to assess the amount of atrophy in the brain of a subject in need thereof include Nissle staining, MRI, functional magnetic resonance fMRI, and PET scanning.
Although the present disclosure has been described with reference to preferred embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof to accommodate specific cases without departing from the scope of the present disclosure. Therefore, it is intended that the disclosure not be limited to the particular embodiment disclosed as the best mode contemplated for carrying out this disclosure, but that the disclosure will include all embodiments falling within the scope and spirit of the appended claims.
The examples set forth herein illustrate several embodiments of the disclosure, but should not be construed as limiting the scope of the disclosure in any way.
Examples
Example 1 AAV vector constructs
Forty-eight AAV vector constructs were constructed:
EF-1α: gfaABC1D: neuroD1: P2A: dlx2: WPRE: SV40 (FIG. 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) (FIG. 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 (FIG. 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 (FIG. 3A);
CE:Gfa1.6:NeuroD1:T2A:Dlx2:WPRE:SV40；
CE:GFA2.2:NeuroD1:T2A:Dlx2:WPRE:SV40；
CE: gfaABC1D: neuroD1: T2A: dlx2: WPRE: hGH (FIG. 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 (FIG. 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 (FIG. 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 (FIG. 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 (FIG. 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 (FIG. 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 (FIG. 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 (FIG. 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 used PHSG-299 (Takara, mountain View, calif.), a pUC-based vector construct containing replication origin, kanamycin resistance gene and multiple cloning site (MSC), with lacZ gene as backbone.
The 5 'end of the expression cassette is an enhancer from the human elongation factor 1 alpha promoter (EF-1 alpha enhancer; SEQ ID NO: 2) or the cytomegalovirus enhancer (CMV enhancer; SEQ ID NO: 11) which places the 5' on the following promoters: 758 nucleotide GFAP promoter (GfaABC 1D; SEQ ID NO: 26), 1667 nucleotide GFAP promoter (Gfa1.6; SEQ ID NO: 4), or 2214 nucleotide GFAP promoter (GFA 2.2 SEQ ID NO: 12).
After the enhancer/GFAP promoter (e.g., 3' of the enhancer/GFAP promoter), several additional sequences are introduced into the expression cassette in a 5' to 3' orientation, including: chimeric intron (SEQ ID NO: 5); human NeuroD1 coding sequence (hNeuroD 1; SEQ ID NO: 6); human Dlx coding sequence (hDlx 2; SEQ ID NO: 13); linker sequences (P2A; SEQ ID NO: 15), (GSG-P2A; SEQ ID NO: 18), (T2A; SEQ ID NO: 16) or (GSG-T2A; SEQ ID NO: 19); and woodchuck hepatitis virus posttranscriptional regulatory elements (WPRE; SEQ ID NO: 7). These sequences are each 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). Enhancers, GFAP promoter, chimeric introns, hNeuroD1 coding sequence, hDlx2 coding sequence, linker, WPRE and SV40 poly (A) signals are flanked by two AAVITR sequences.
Example 2 AAV Virus production
Each of the 24 plasmids was co-transfected into 293AAV cells using polyethylenimine together with a Rep-Cap plasmid (a plasmid comprising a promoter driving expression of AAV Rep and Cap genes) and a helper plasmid (a plasmid comprising a promoter driving expression of adenovirus E2A, E and VA RNA) to produce recombinant AAV viral particles.
The transfected cells were scraped and centrifuged 72 hours after transfection. The cell pellet was frozen and thawed, i.e., placed in a dry ice/ethanol mixture, then placed in a 37 ℃ water bath. The freeze/thaw cycle was repeated three more times. AAV lysates were purified (e.g., cell debris removed) by ultracentrifugation at 350,000g for 1 hour in a discontinuous iodixanol gradient. The virus-containing layer was collected and then concentrated using a Millipore Amicon ultrafiltration centrifuge. Viral titers were then determined by qPCR using primers or gene/expression cassette specific sequences that amplified the ITR region.
EXAMPLE 3 astrocyte cultures
Human cortical astrocytes (HA 1800; scienCell Research Laboratories, inc., carlsbad, california) were subcultured at confluence exceeding 90%. For subculture, trypLE was used TM Select (Invitrogen, carlsbad, california) trypsinized cells, centrifuged at 200×g for 5 min, then resuspended and inoculated in a culture medium containing DMEM/F12 (Gibco); 10% fetal bovine serum (Gibco); penicillin/streptomycin (Gibco); 3.5mM glucose (Sigma-Aldrich); b27 (Gibco); 10ng/mL epidermal growth factor (Invitrogen); and 10ng/mL fibroblast growth factor 2 (Invitrogen). Astrocytes were cultured on poly-D-lysine (Sigma-Aldrich) coated coverslips (12 mm) of 24 well plates (BD Biosciences) at a density of about 20,000 cells per coverslip.
Rat primary astrocytes (isolated from Sprague Dawley rat cortex or striatum) in a culture medium containing DMEM/F12 (Gibco); 10% fetal bovine serum (Gibco), penicillin/streptomycin (Gibco); cultured in a medium of 3.5mM glucose (Gibco).
All cells were kept in humid air containing 5% carbon dioxide at 37 ℃.
Example 4 testing of AAV vectors in astrocyte cultures (in vitro)
10 using the recombinant AAV obtained from the method of example 2 10 Individual particles/mL and 10 14 The concentration range of individual particles/ml infects human cortical astrocytes and rat primary astrocytes of example 3. Twenty-four hours after infection of the cells, the medium was replaced with one containing DMEM/F12 (Gibco); n2 additive (Gibco); and 20ng/mL of a differentiation medium of brain-derived neurotrophic factor (Invitrogen). Differentiation medium was added to the cell culture every four days. See Song et al, nature,417:39-44 (2002).
The voids in the cell culture are filled with additional human astrocytes to support functional development of the transformed neurons when astrocytes or rat primary astrocytes are transformed into neurons.
Example 5 test of AAV vector efficacy
10 using the recombinant AAV obtained from the method of example 2 10 Individual particles/mL and 10 14 The concentration range of individual particles/mL infects the 4 th to 7 th passages of the human cortical astrocytes and rat primary astrocytes (or astrocytes of other brain regions or spinal cord) of example 3. qPCR, enzyme-linked immunosorbent assay (ELISA) and western blot were performed to determine expression of NeuroD1 transcripts and protein levels.
Expression of NeuN, bisdermatan (DCX), β3-tubulin, NF-200, and MAP2 was assessed by qPCR, ELISA, western blotting and immunostaining to determine the functional output of recombinant AAV.
Example 6 test of AAV vector titres and infection Rate
The purified AAV vector was treated with dnase I to eliminate residual plasmid contamination. AAV vectors were serially diluted 100-fold, 500-fold, 2500-fold and 12500-fold. AAV plasmids were diluted to generate standard curves by serial dilution. Plasmid dilution to 10 4 、10 5 、10 6 、10 7 And 10 8 Molecules/ul. qPCR was performed on diluted AAV vector and diluted AAV plasmid. The primers used were directed to the ITR region (forward ITR primer 5'-GGAACCCCTAGTGATGGAGTT, reverse ITR primer 5' -CGGCCTCAGTGAGCGA). The qPCR mixture contained 10uL Universal SYBR Master Mix X, 2uL 5uM forward ITR primer, 2uL 5uM reverse ITR primer, 5uL test sample or dilution standard and 1uL H 2 O. The qPCR procedure was 95℃for 10 minutes, followed by 40 cycles of 95℃for 15 seconds, 60℃for 30 seconds, and then the melting curve. The data were analyzed using qPCR cycler software. The physical titer (viral genome (vg)/ml) of AAV samples was calculated from the standard curve.
AAV vector infection rates were tested by a 50% tissue culture infection dose (TCID 50) assay using standard protocols of the american type culture collection (ATCC; manassas, VA).
Example 7 testing of AAV dose ranges (in vivo)
Recombinant AAV obtained from the method of example 2 was injected into C57/BL6 mice by bilateral intracranial injection into the motor cortex. Each AAV is 1x10 11 、3x10 11 、1x10 12 、3x10 12 、1x10 12 、3x10 12 、1x10 13 The dose of each viral genome/mL was injected in a volume of 1 uL. Each dose was evaluated 4, 20 and 60 days post injection to determine the Optimal Effective Dose (OED), maximum Tolerated Dose (MTD) and Minimum Effective Dose (MED) at the cellular and tissue level. Three mice per time point. OED, MTD and MED conversion efficiency and potential toxicity of astrocytes into neurons by immunostaining via NeuroD1, GFAP, neuN and Iba1Sex is determined. If the first dose range is insufficient to determine OED, MTD and MED, then 1x10 10 Individual viral genomes/mL to 1x10 14 GC/mL was performed in a second dose range at a volume of 1 uL.
Example 8 comparison of neuronal conversion of recombinant AAV obtained from various AAV 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 AAV was obtained as described in example 2: (1) an AAV vector construct expressing NeuroD1 alone; (2) AAV vector constructs expressing Dlx2 alone; (3) a combination of AAV vector constructs (1) and (2); and (4) AAV vector constructs expressing NeuroD1 and linker Dlx2. The resulting recombinant AAV was used to infect human cortical astrocytes and rat primary astrocytes of example 3. Twenty-four hours after infection of the cells, the medium was replaced with one containing DMEM/F12 (Gibco); n2 additive (Gibco); and 20ng/mL of a differentiation medium of brain-derived neurotrophic factor (Invitrogen). Differentiation medium was added to the cell culture every four days. See Song et al, nature,417:39-44 (2002). The voids in the cell culture are filled with additional human astrocytes to support functional development of the transformed neurons when astrocytes or microglia are transformed into neurons. Neuronal conversion levels for each treatment were measured and compared.
Example 9 testing of AAV vectors in human subjects (in vivo)
Human brain or spinal astrocytes were infected in vivo using the recombinant AAV obtained from the method of example 2. Recombinant AAV at 10 10 Individual particles/mL and 10 14 The concentration range of individual particles/mL is injected into the brain or spinal cord of a human subject suffering from a neurological disorder in a volume range of 10 μl to 1 mL. The symptoms of neurological disorders, brain imaging (including MRI, PET scan, or a combination of MRI and PET) and behavioral indicators of the human subject are observed before, during, and after injection. Post-injection observations were made weekly until the first month post-injection. After the first month after injection, observations were made once a month for the next 11 months and can be extended to 2 years after virus injection.
Example 10 dose-scale determination of non-human primate
The volume of NeuroD1 expressing brain tissue from example 7 divided by the number of vector genomes (mm 3 Vector genome) is used to determine the viral infection rate of brain tissue. Calculation of specific brain region volume to be treated (mm) in non-human primate 3 ) And the dose range of the vector genome was scaled according to the infection rate obtained in example 7. Dose-range studies were performed as in example 7 and OED, MTD and MED were determined by assessing astrocyte to neuron conversion efficiency and potential toxicity via immunostaining of NeuroD1, GFAP, neuN and Iba 1.
Example 11 treatment (in vivo) of a subject in need thereof suffering from Huntington's disease
Subjects with huntington's disease are treated with recombinant AAV obtained from the method of example 2. Neurological symptoms of a subject include involuntary movements (such as chorea-like movements), uncontrolled postures, mood changes, sleep disorders, language changes, dysphagia, and impaired cognitive functions (such as learning and memory deficits). Recombinant AAV at 10 10 Individual particles/mL and 10 14 The concentration range of individual particles/mL is injected into the striatum (nucleocapsid and caudate nucleus) of a human subject suffering from a neurological disorder in a volume range of 10 μl to 1000 μl. The symptoms of neurological disorders, brain imaging (including MRI, PET scan, or a combination of MRI and PET) and behavioral indicators of the human subject are observed before, during, and after injection. Post-injection observations were made weekly until the first month post-injection. After the first month after injection, observations were made once a month for the next 11 months and can be extended to 2 years after virus injection.
Example 12 combinatorial methods for direct conversion of glial cells to neurons in combination with shRNA for knockdown of Htt Gene expression
Identifying a target sequence complementary to the Htt gene. shRNA was designed to target Htt gene. NeuroD1, dlx2 and target shRNA were packaged into AAV vectors (hU 6:: htt shRNA-hGFAP:: hNeuroD1-P2A-hDlx 2) (FIGS. 5A-8D) and recombinant AAV was produced as described in example 2. Recombinant AAV was injected into the striatum of mice with mutant Htt genes. Mice receiving treatment are tested for behavioral indicators such as cat walking, open field testing, gripping, mouse weight and grip strength, and brain imaging (including MRI, PET scanning, or a combination of MRI and PET). The behavioral test results of the following groups were compared to brain imaging: (i) received no treatment, (ii) received recombinant AAV from example 2, and (iii) received recombinant AAV (hU 6:: htt shRNA-hGFAP::: hNeuroD1-P2A-hDlx 2) (FIGS. 5A-8D).
Alternatively, the target shRNA is packaged into an AAV vector (hU 6:: hHtt shRNA) and another recombinant AAV is produced as described in example 2. Two recombinant AAV were injected into the striatum of mice with mutant Htt. Mice receiving treatment are tested for behavioral indicators such as cat walking, open field testing, gripping, mouse weight and grip strength, and brain imaging (including MRI, PET scanning, or a combination of MRI and PET). The behavioral test results of the following groups were compared to brain imaging: (i) not treated, (ii) only recombinant AAV from example 2, and (iii) a combination of recombinant AAV (hU 6:: hHtt shRNA) and recombinant AAV from example 2.
Example 13 combination method for direct conversion of glial cells into neurons in combination with CRISPR/CAS Gene editing of the Htt Gene
Identifying a target sequence complementary to the Htt gene. The guide RNA (gRNA) sequence was designed to target the Htt gene. The donor sequence was designed to modify the CAG repeat sequence number of the Htt gene to less than 36. Cas9 nuclease, htt-specific gRNA and donor sequences are packaged into an AAV vector (AAV-Cas 9-Htt). Recombinant AAV is produced as described in example 2.
Recombinant AAV (AAV-Cas 9-HTT) was injected into the striatum of mice with mutant HTT simultaneously with the recombinant AAV from example 2. Mice receiving treatment are tested for behavioral indicators such as cat walking, open field testing, gripping, mouse weight and grip strength, and brain imaging (including MRI, PET scanning, or a combination of MRI and PET). The behavioral test results of the following groups were compared to brain imaging: (i) receiving no treatment, (ii) receiving recombinant AAV from example 2, and (iii) receiving recombinant AAV-Cas9-HTT and recombinant AAV from example 2, to identify a synergistic effect between mutant HTT gene editing and glial to neuronal conversion. The recombinant AAV-Cas9-HTT and the recombinant AAV from example 2 may be injected simultaneously or at different times.
Alternatively, the NeuroD1, linker (P2A), dlx2, second linker (P2A), cas9 nuclease, htt-specific gRNA, and donor sequences are packaged into an AAV vector (AAV-hNeuroD 1-P2A-hDlx2-P2A-Cas 9-Htt). Recombinant AAV is produced as described in example 2. Recombinant AAV (AAV-hNeuroD 1-P2A-hDlx2-P2A-Cas 9-HTT) was injected into the striatum of mice with mutant Htt simultaneously with the recombinant AAV from example 2. Mice receiving treatment are tested for behavioral indicators such as cat walking, open field testing, gripping, mouse weight and grip strength, and brain imaging (including MRI, PET scanning, or a combination of MRI and PET). The behavioral test results of the following groups were compared to brain imaging: (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 a synergistic effect between mutant HTT gene editing and glial to neuronal conversion.
Example 14 combination method for direct conversion of glial cells into neurons in combination with antisense oligonucleotides (ASOs) knockdown of Htt Gene expression
Identifying a target sequence complementary to the Htt gene. ASO knocked down expression of Htt gene was designed and synthesized. The recombinant AAV from example 2 was injected into the striatum of mice with mutant Htt along with Htt ASO. Mice receiving treatment are tested for behavioral indicators such as cat walking, open field testing, gripping, mouse weight and grip strength, and brain imaging (including MRI, PET scanning, or a combination of MRI and PET). The behavioral test results of the following groups were compared to brain imaging: (i) not receiving treatment, (ii) receiving recombinant AAV from example 2, and (iii) receiving recombinant AAV and Htt ASO from example 2.
Example 15 combination method for direct conversion of glial cells to neurons in combination with siRNA knockdown of Htt Gene expression
Identifying a target sequence complementary to the Htt gene. siRNA for knocking down Htt gene expression is designed and synthesized. The recombinant AAV from example 2 was injected into the striatum of mice with mutant Htt along with Htt siRNA. Mice receiving treatment are tested for behavioral indicators such as cat walking, open field testing, gripping, mouse weight and grip strength, and brain imaging (including MRI, PET scanning, or a combination of MRI and PET). The behavioral test results of the following groups were compared to brain imaging: (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 methods of direct conversion of glial cells to neurons in combination with miRNAs knockdown of Htt Gene expression
Mirnas were identified that regulated Htt gene expression. NeuroD1, dlx and miRNA were packaged into AAV vectors (CAG:: htt miRNA-hGFAP:: hNeuroD1-P2A-hDlx 2) and recombinant AAV was produced as described in example 2. Recombinant AAV was injected into the striatum of mice with mutant Htt. Mice receiving treatment are tested for behavioral indicators such as cat walking, open field testing, gripping, mouse weight and grip strength, and brain imaging (including MRI, PET scanning, or a combination of MRI and PET). The behavioral test results of the following groups were compared to brain imaging: (i) receiving no treatment, (ii) receiving recombinant AAV from example 2, and (iii) receiving recombinant AAV (CAG:: htt miRNA-hGFAP::: hNeuroD1-P2A-hDlx 2).
Alternatively, the target miRNA is packaged into an AAV vector (CAG:: hHtt miRNA) and recombinant AAV is produced as described in example 2. Recombinant AAV was injected into the striatum of mice with mutant Htt. Mice receiving treatment are tested for behavioral indicators such as cat walking, open field testing, gripping, mouse weight and grip strength, and brain imaging (including MRI, PET scanning, or a combination of MRI and PET). The behavioral test results of the following groups were compared to brain imaging: (i) not treated, (ii) only recombinant AAV from example 2, and (iii) a combination of recombinant AAV (CAG:: hHtt miRNA) and 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 the Rep-Cap plasmid expressing serotype 5 capsid protein and Helper plasmid P40Helper (P40H) or pALD-X80 (X80) to produce recombinant AAV viral particles (P31-P40H or P31-X80). Viral titers were determined by qPCR using primers that amplify the gene of interest (GOI), primers specific for the P31 plasmid and the ITR region. Reverse package primers were used to evaluate non-specific packages. An increase in viral yield was observed for the X80 helper plasmid compared to the P40H helper plasmid (FIG. 9).
EXAMPLE 18 successful establishment of Primary culture of rat astrocytes
Cortex and striatal tissue were isolated from the brain of SpragueDawley rats 3 days postnatal. Tissues were treated with papain to generate a single cell suspension and inoculated into poly-D-lysine coated flasks. Cells were immunostained with GFAP antibody and SOX9 antibody. Cells were counterstained with DAPI antibody. Over 95% of the cells were astrocytes identified by GFAP and SOX9 staining (fig. 10). The leftmost panel presents images of GFAP stained cells. The middle left panel presents an image of SOX9 stained cells. The middle right panel presents images of DAPI stained cells. The rightmost panel presents a pooled image of GFAP, SOX9 and DAPI stained cells.
EXAMPLE 19 comparison of plasmid transfection
Primary rat astrocytes were inoculated and transfected with expression vectors P14 (CE: gfaABC1D: hNeuroD1-P2A-Dlx2: WPRE: SV 40), P31 (EF-1α: gfaABC1D: neuroD 1-P2A-Dlx: WPRE: SV 40) and P63 (CE: gfaABC1D: neuroD1-GSG P2A-Dlx2: WPRE: SV 40) as described in example 18 to test the expression efficiency of NeuroD1 and Dlx2 in transfected cells. P14 resulted in expression of neuroD1 and Dlx2 as indicated by neuroD1 staining and Dlx staining of the cells (FIG. 11; upper panel shows neuroD1 staining of the cells, middle panel shows Dlx staining of the cells, and lower panel shows pooled neuroD1, dlx2 and DAPI staining of the cells).
Example 20 successful transduction of AAV viral particles into Primary rat astrocytes
AAV9-P12 (pGfaABC 1D: GFP) at 3X10 10 vg/well, 1x10 10 vg/well and 2.5x10 9 Different doses of vg/well transduced recombinant AAV obtained from the method of example 2 into primary rat astrocytes seeded in 96-well plates. 24 hours prior to transduction, RCA of generations 5-7 were inoculated onto poly-D-lysine (PDL) -coated glass coverslips in 96-well plates at a confluency of 50%. Cells were transduced with the indicated titers of virus in fresh astrocyte medium. The medium was refreshed the next day and every 3-4 days. At the position ofImages obtained six days after GFP positive cell transduction showed higher transduction rates when the viral titer was higher (fig. 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 seeded in 24-well or 96-well plates with the viral particles AAV9-P12 (pGfaABC 1D: GFP) and AAV5-P7 (pEF-1. Alpha.: GFP). Cells were harvested 7 days after infection by trypsinization. Cells were fixed, washed and suspended in PBS. Viral transduction was analyzed using flow cytometry to calculate GFP positive cells compared to all cells (fig. 13A and 13B). Fig. 13A shows the percent transduction at different MOIs. At 5x10 5 Cell, 2x10 5 Cell and 5x10 4 MOI infection of cells at 1X10 5 Cells/well were seeded in 24-well plates. Viral transduction decreases with decreasing MOI. FIG. 13B shows the density (2 x 10) 4 Individual cells/well, 1.5x10 4 Individual cells/well, 1x10 4 Individual cells/well and 5x10 3 Individual cells/well) were inoculated in 96-well plates and incubated with a series of amounts of virus (1 x10 in 100 μl of medium 13 Transduction of AAV viral particles in 2. Mu.l, 1. Mu.l, 0.5. Mu.l, 0.25. Mu.l, 0.125. Mu.l of/ml virus). This corresponds to 2x10 per well, respectively 10 vg、1x10 10 vg、5x10 9 vg、2.5x10 9 vg and 1.25x10 9 vg. The viral transduction rate was unchanged as the number of cells per well was reduced.
Example 22 neurod1 vector-induced transgene expression in vitro and astrocyte to neuron transformation.
Materials and methods
Primary rat astrocyte culture:rat Cortical Astrocytes (RCA) were isolated from Sprague Dawley rat cortical tissue 3 days after birth. Cells were maintained in Astrocyte Medium (AM) consisting of DMEM supplemented with 10% FBS, 2.5mM glutamine, 3.5mM glucose, penicillin/streptomycin. Subculturing fine at low cell density at a ratio of 1:3-1:4 The first two passages of cells to promote differentiation of residual progenitor cells. When 90% -100% confluence is reached, the ratio of subsequent subcultures is 1:2 or 1:3. The 5 th-7 th generation cells were used for transfection and transduction. Immunostaining with GFAP antibodies showed>90% of the cells were GFAP positive astrocytes. The 6 th generation astrocyte cultures were immunostained with astrocyte markers GFAP and Sox9 (fig. 14).
Carrier body: AAV was produced from the selected vector and tested in vitro using rat astrocytes:
·NXL-P9(CE-pGfa681-CI-hND1-p2A-GFP-WPRE-SV40pA)
·NXL-P22(CE-pGfa681-CI-hND1-WRPE-SV40pA)
·NXL-P35(EE-pGfa681-CI-hND1-WRPE-SV40pA)
·NXL-P37(EE-pGfa681-CI-hND1-p2A-GFP-WPRE-SV40pA
·NXL-P107(CE-pGfa681-CI-hND1-bGHpA)
·NXL-P108(CE-pGfa681-CI-hND1-oPRE-bGHpA)
·NXL-P109(CE-pGfa681-CRGI-hND1-bGHpA)
·NXL-P130(CE-pGfa681-GI-hND1-oPRE-bGHpA)
·NXL-P134(CE-pGfa681-CRGI-hND1-oPRE-bGHpA)
·NXL-P136(EE-Gfa681-CRGI-hND1-bGHpA)
·NXL-P138(EE-Gfa681-CRGI-hND1-oPRE-bGHpA)
virus production:viruses for in vitro studies were generated using Polyethylenimine (PEI) by triple transfection (GOI, helper and Rep/Cap plasmids) using adherent AAV293 cells. Virus recovery and purification is achieved by ultracentrifugation or using commercial purification kits.
Specifically, AAV293 cells (Cell Biolabs, cat# AAV-100) were seeded in 15-cm dishes 24 hours prior to transfection. Cells with 70% -85% confluence were transfected with 10ug GOI, 10ug Rep/Cap and 14ug pALD-X80 (Alveron) or pHelper (Cell Biolabs) in each dish using Polyethylenimine (PEI) at a DNA to PEI ratio of 1:4. Multiple dishes were transfected for production according to the desired scale. The medium was refreshed daily. Seventy-two hours after transfection, cells were collected and lysed to harvest virus using AAVpro purification kit (Takara, cat #6666,6675,6235) according to the manufacturer's protocol.
Viral titers were determined by real-time quantitative PCR using primer pairs in the ITR region, primers to amplify the gene of interest (GOI), or vector-specific primers. Plasmid DNA was used as a standard. Production yield of about 10 3 -10 4 vg/cell level. 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 helper plasmid pALD-X80 (X80) produced recombinant AAV viral particles as measured by qPCR.
Transfection and immunofluorescence:24-48 hours prior to transfection, 5-7 th generation Rat Cortical Astrocytes (RCA) were seeded onto poly-D-lysine (PDL) -coated glass coverslips in 24-well plates at a confluency of 30% -50%. Cells were transfected with 300ng vector DNA using Lipofectamine reagent (Thermo Fisher Cat # 15338) according to the manufacturer's protocol. 24-48 hours after transfection, cells were fixed with 4% paraformaldehyde in PBS, followed by washing and immunostaining with anti-NeuroD 1 (anti-ND 1) antibody (Abcam cat#ab 60704) followed by immunostaining with a secondary antibody conjugated with a fluorescent dye (Invitrogen, alexa Fluor). Images were taken under a fluorescence microscope (Zeiss Axiovert A1, zen Blue). The gene expression level was evaluated by comparing the fluorescence intensities.
Transduction and immunofluorescence:24-48 hours prior to transduction, RCA of generations 5-7 were inoculated onto poly-D-lysine (PDL) -coated glass coverslips in 24-well plates at a confluency of 30% -50%. 2-6X10 in fresh astrocyte medium 10 AAV transduced cells were isolated from each viral genome (vg)/ml. The medium was refreshed the next day and every 3-4 days. Three to six days post transduction, cells were fixed with 4% paraformaldehyde in PBS, followed by washing and immunostaining with anti-ND 1 antibody (Abcam cat#ab 60704), followed by immunostaining with a secondary antibody conjugated with a fluorescent dye (Invitrogen, alexafluor) for viewing and imaging under a fluorescent microscope (Zeiss Axiovert A1, zen Blue). Assessment of genes by comparing fluorescence intensitiesExpression level.
Astrocyte to neuron transformation assessment24-48 hours prior to transduction, RCA of generations 5-7 were inoculated onto poly-D-lysine (PDL) -coated glass coverslips in 24-well plates at a confluency of 30% -50%. 2-6X10 in 500ul fresh astrocyte medium (DMEM supplemented with 10% FBS, 2.5mM glutamine, 3.5mM glucose, penicillin/streptomycin) 10 vg/ml of virus transduced cells. At 48 hours post transduction, the medium was replaced with 5% FBS astrocyte medium. Subsequently, 100ul of transformation medium (DMEM/F12+1% FBS+B27+N2 and 1uM Rock inhibitor and 10ng/ml BDNF) was added daily for 4 days. After 4 days, the medium was completely replaced with transformation medium.
Cells were fixed with 4% paraformaldehyde in PBS at different desired time points (three days, one to five weeks post transduction), followed by washing and immunostaining with antibodies to ND1 (Abcam cat#ab 60704), to NeuN (Millipore, cat#abn 78), to Map2 (Invitrogen, cat#pa 5-17646), followed by immunostaining with a secondary antibody conjugated with a fluorescent dye (Invitrogen, alexafluor) for viewing and imaging under a fluorescent microscope (Zeiss Axiovert A1, zen Blue).
Results of in vitro studies:
all the NeuroD1 (ND 1) plasmids tested were effective in driving expression of NeuroD1 (fig. 16-31). The expression level of NeuroD1 is affected by the elements in the vector. Among the three versions of the GFA promoter, the 681bp promoter showed the highest expression level of neuroD1, and the 1.6kb promoter showed the weakest expression level of neuroD 1. Promoter enhancer elements significantly affect the expression level of NeuroD 1. The CMV enhancer increases the expression level of NeuroD1 over the ef1α enhancer. Chimeric introns and WPRE also increased expression levels of NeuroD 1.
As shown by positive staining of NeuN and/or MAP2 (fig. 17, 30, 22, 25 and 28), all AAVs tested containing ND1 were effective in driving expression of ND1 and inducing astrocyte to neuronal conversion in cultured rat astrocytes. When astrocytes were transduced with vectors driving higher ND1 expression, the conversion was higher. Vectors NXL-P134 and NXL-P138, and viruses produced using these vectors (i.e., AAV9-P134 and AAV9-P138, respectively) are most effective in driving ND1 expression and inducing astrocyte to neuronal conversion, with AAV-P134 being the most effective (FIGS. 15-20). Plasmid AAV9-P21 (CE-pGFA 681-CI-GFP-WPRE-SV40 pA) without ND1 sequence served as a control and it did not induce astrocyte to neuronal transformation as shown by the lack of positive staining for NeuN and/or Map2 (FIG. 14).
NeuN/RBFOX3 (neuronal nucleoprotein) is a marker of neuronal differentiation that stains the nuclear and perinuclear cytoplasm of neurons. MAP2 (microtubule-associated protein 2) is another neuronal marker for chromatin microtubules including dendrites in neurons.
One week after transduction with AAV containing ND1, a small number of NeuN and MAP2 positive cells (neurons) were observed. After two and three weeks, more NeuN/MAP2 positive cells were observed. Some NeuN/MAP2 positive cells showed typical neuronal morphology.
Example 23 neurod1 viral vector-induced transgene expression in vivo and astrocyte to neuronal transformation.
AAV9-P134 and AAV9-P138 viruses were used for in vivo studies. AAV9-P12, driving GFP expression alone (no ND 1) under GFAP promoter, was used for control and to identify GFAP expressing cells (astrocytes).
Single-stranded adeno-associated virus (ssAAV, abbreviated AAV) vectors NXL-P12, NXL-P134 and NXL-P138 were packaged into AAV serotype 9 (AAV 9) and then subjected to subsequent iodixanol gradient ultracentrifugation and concentration. Titration of purified AAV virus was performed using a quantitative PCR-based method. All AAV used in this study were prepared in 0.001% Pluronic F-68 (Poloxamer 188 solution, PFL01-100ML,Caisson Laboratories,Smithfield,UT,USA) in PBS (pH 7.4)
Normal C57BL/6 mice over 8 weeks of age were injected with AAV9-P134, AAV-P138 and AAV9-P12 viruses as follows:
p12 control group: AAV9-P12 5x10 11 GC/ml,1 μl, cortical injection 1 time (unilateral) (n=6)
Group P134: AAV9-P12 2.5x10 11 GC/ml+AAV9-P134 2.5x10 11 GC/ml,1 μl, cortical injection 1 time (unilateral) (n=6)
P138 group: AAV 9-P12.5x10 11 GC/ml+AAV9-P138 2.5x10 11 GC/ml,1 μl, cortical injection 1 time (unilateral) (n=6)
Mice were sacrificed 10 days (dpi) and 30dpi post infection and analyzed for cortical tissue. Animals were anesthetized with 1.25% avetin and then sequentially perfused intracardially with saline (0.9% NaCl) followed by 4% Paraformaldehyde (PFA). Brains were collected and, after overnight fixation in 4% PFA, placed in 20% and 30% sucrose at 4 ℃ in sequence until the tissues subsided. Embedding dehydrated brain into optimal cutting temperature (Tissue-O.C.T. compounds, +.>Finetek, torrance, CA, USA) and then serially sectioned at 30 μm thickness on the coronal plane on a cryostat (Thermo Scientific, shanghai, china). For immunofluorescence, free-floating brain sections were first washed with PBS and blocked for 1 hour in 5% normal donkey serum, 3% bovine serum albumin and 0.3% triton x-100 prepared in PBS at Room Temperature (RT), then incubated overnight at 4 ℃ with primary antibody diluted in blocking solution. After an additional wash 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 donkey anti-mouse/rabbit secondary antibodies conjugated to Alexa Fluor 555, goat anti-chicken secondary antibodies conjugated to Alexa Fluor 488 (1:1000,Life technologies,Carlsbad,CA,USA) and goat anti-rat (Life technologies)/guinea pig (Jackson immune research) secondary antibodies conjugated to Alexa Fluor 647 (1:500) for 2 hours at room temperature, followed by extensive washing with PBS. Sample end use- >The caplets (VECTOR Laboratories, burlingame, CA, USA) were capped and sealed with nail polish. Photographing with confocal microscope (LSM 880, zeiss, jena, germany)Representative images are photographed. The primary antibodies used were as follows: rat anti-GFAP (astrocyte marker, 1:1000, cat#13-0300, invitrogen), guinea pig anti-NeuN (neuronal marker, 1:1000, cat#ABN90, millipore), mouse anti-neuroD 1 (1:500, cat#ab60704, abcam) and chicken anti-GFP (1:1000, cat#ab13970, abcam). The fluorescence microscope (Axio image Z2, zeiss, & gt was used by Zeiss Axioplan>Germany) or confocal microscopy (LSM 880, zeiss, jena, germany). Quantitative analysis was performed from 3 brain sections per mouse (3 mice per group) based on 4 randomly selected fields (212 μm x μ, 212 μm, obtained from LSM880 confocal microscopy at 400 x magnification). Data are shown as mean ± SEM.
Control virus P12 expressing GFP reporter alone was first compared to NeuroD1 expressing viruses P134 and P138 (both P12 were added together to track transformed neurons). When control virus P12 was injected into the uninjured mouse cortex, the infected cells were predominantly astrocytes without NeuroD1 expression at 10dpi (days post injection) (fig. 36). In contrast, neuroD1 expression was clearly detected in both P134 and P138 groups. While most of the NeuroD 1-expressing cells in the P138 group were still astrocytes at 10dpi, some of the NeuroD 1-expressing cells in the P134 group were already neun+ neurons (fig. 36), indicating that P134 may have better conversion capacity than P138. Furthermore, analysis of cortical brain tissue at 10dpi in group P134 mice showed high levels of astrocyte conversion to neurons, as evidenced by morphological changes in GFP positive cells such as the presence of long projections (fig. 32). Group P138 showed lower levels of conversion.
30 days after virus injection, the infected cells in the control group (P12) were still astrocytes, but most GFP positive cells in the P134 group were neurons expressing NeuN (fig. 37). However, the conversion rate was lower for group P138 than for group P134. Most of the infected cells in this stage P138 group remained astrocytes and GFP signal in the transformed neurons was weak (fig. 37). Furthermore, analysis of cortical brain tissue at 30dpi in group P134 mice showed even higher levels of astrocyte conversion to neurons, as evidenced by the presence of long projections in GFP positive cells (FIG. 33)
AAV9-P134 virus was also effective in a bilateral lesion mouse model. In normal C57BL/6J mice (over 8 weeks old), ischemic stroke was induced by injecting 1. Mu.L of Endothelin 1,1-31aa (1. Mu.g/. Mu.L) on each side of the cortex. Mice were anesthetized with 20mg/kg of 1.25% Avertin (a mixture of 12.5mg/mL 2, 2-tribromoethanol and 25 μl/mL 2-methyl-2-butanol, sigma, st.Louis, MO, USA) by intraperitoneal injection, and then placed on a stereotactic frame in the prone position. Endothelin-1 (ET-1) and virus were injected by a glass pipette into the motor cortex at +0.2mm anterior and posterior (AP, from the bregma), -1.5mm medial-lateral (ML, from the bregma, left), -0.7mm dorsal-ventral (DV, from the dura). The injection rate was 80nL/min. The pipette is kept in place for about 10 minutes after injection and then slowly withdrawn. Seven days after injection of Endothelin 1, mice were injected with AAV9-P12 and AAV9-P134 viruses as follows:
Group P12: AAV9-P12 5x10 11 GC/ml, 1. Mu.L, 1 injection per side of cortex (bilateral)
Group P14: AAV 9-P12.5x10 11 GC/ml+AAV9-P134 2.5x10 11 GC/ml, 1. Mu.L, 1 injection per side of cortex (bilateral)
Mice were sacrificed 10 days (dpi) after virus injection and cortical tissue was analyzed. When control virus P12 was injected into the cortex of ET-1 injured mice, the infected cells were predominantly astrocytes without NeuroD1 expression at 10dpi (days post injection) (fig. 38). In contrast, neuroD1 expression was detected in the P134 group (fig. 38). Mice were sacrificed 10 days (dpi) after virus injection and cortical tissue was analyzed. Analysis of cortical brain tissue from group P134 mice at 10dpi showed high levels of astrocyte conversion to neurons, as evidenced by the morphological changes observed in GFP positive cells, such as the presence of long projections (fig. 34).
Example 24 expression of neurod1 and Dlx 2-induced transgene expression in vitro and conversion of astrocytes into neurons
Materials and methods
Carrier body: the vector was tested by transfection of Rat Cortical Astrocytes (RCA). Furthermore, AAV is produced from the selected vector 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)
FIG. 40 depicts two general diagrams of constructs.
Cell culture:24-48 hours prior to transduction, 5-7 th generation Rat Cortical Astrocytes (RCA) were seeded onto poly-D-lysine (PDL) -coated glass coverslips in 24-well plates at a confluency of 30% -50%.
Transduction and immunofluorescence:2-6X10 in 500ul fresh astrocyte medium (DMEM supplemented with 10% FBS, 2.5mM glutamine, 3.5mM glucose, penicillin/streptomycin) 10 vg/ml of virus transduced cells. At 48 hours post transduction, the medium was replaced with 5% FBS astrocyte medium. On the next 4 days, 100ul of transformation medium (DMEM/F12+1% FBS+B27+N2 and 1uM Rock inhibitor and 10ng/ml BDNF) was added daily. The medium is then completely replaced with the transformation medium.
Cells were fixed with 4% paraformaldehyde in PBS at various desired time points (e.g., three days, one to five weeks after transduction), followed by washing and immunostaining with antibodies to ND1 (Abcam cat#ab 60704), antibodies to Dlx2 (Millipore cat#ab 5726), antibodies to NeuN (Millipore, cat#abn78), antibodies to Map2 (Invitrogen, cat#pa 5-17646), and then with secondary antibodies conjugated with fluorescent dyes (Invitrogen, alexafluor). Images were taken under a fluorescence microscope (Zeiss Axiovert A1, zen Blue). The gene expression level was evaluated by comparing the fluorescence intensities.
Results of in vitro studies:
the ND1-Dlx2 construct tested effectively driven expression of ND1 and Dlx2 by transfected and/or transduced cultured rat astrocytes, as evidenced by positive staining of ND1 and Dlx2 in these cells (FIGS. 41-56). Furthermore, AAV (AAV 9-P122, AAV-P124, and AAV-P20) containing ND1/Dlx2 was effective in driving expression of ND1, dlx2 in cultured rat astrocytes and inducing astrocyte to neuronal transformation as indicated by positive staining of the neuronal markers NeuN and/or MAP2 (FIGS. 43, 46, and 49 and 52). Some NeuN/MAP2 positive cells showed typical neuronal morphology.
Example 25 expression of neurod1 and Dlx 2-induced transgene expression in vivo and conversion of astrocytes into neurons
AAV9-P112 and AAV9-P122 viruses were used for in vivo studies. AAV9-P12, driving GFP expression alone (without ND1 and Dlx 2) under GFAP promoter, was used for control and to identify GFAP expressing cells (astrocytes).
Normal C57BL/6 mice over 8 weeks of age were injected with AAV-P12, AAV9-P112 and AAV9-P122 virus as follows:
p12 control group: AAV 9-P12.5x10 11 GC/ml,2 μl, striatal injections 1 time (n=6)
P112 group: AAV 9-P12.5x10 11 GC/ml+AAV9-P112 2.5x10 11 GC/ml,2 μl, striatal injections 1 time (n=6)
Group P122: AAV 9-P12.5x10 11 GC/ml+AAV9-P122 2.5x 11 GC/ml,2 μl, striatal injections 1 time (n=6)
Mice were sacrificed 10 days (dpi) and 30dpi post infection and analyzed for cortical tissue. Infection of the mouse striatum by AAV9-P12 was confirmed by the presence of GFP fluorescence at 10 dpi. At 30dpi, AAV 9-P12-infected cells were still GFAP positive astrocytes, indicating that AAV9-P12 was specific in infecting astrocytes (FIGS. 57A-B). In group P112, at 10dpi, most of the cells co-infected with AAV9-P12 and AAV9-P112 were GFAP-positive stained astrocytes (FIG. 58). By 30 days after viral infection, many cells co-infected with AAV9-P12 and AAV9-P112 became NeuN positive neurons (FIG. 59, white arrow). In group P122, most of the cells co-infected with AAV9-P12 and AAV9-P122 were GFAP-positive stained astrocytes at 10dpi (FIG. 60). By 30 days after viral infection, many cells co-infected with AAV9-P12 and AAV9-P122 became NeuN positive neurons (FIG. 61, white arrow).
Example 19 expression of Dlx2 in vitro transgenes
Carrier body: vectors were tested by transfection of rat cortical astrocytes (RAC). Furthermore, AAV is produced with the selected vector 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, the NXL-P104 and NXL-P105 constructs effectively driven the expression of Dlx2, as evidenced by positive Dlx2 staining in these cells (FIG. 62). 24 hours after transfection of cultured RACs, NXL-P133, NXL-P137 and NXL-P131 constructs effectively driven expression of Dlx2, as evidenced by positive Dlx2 staining in these cells (FIG. 63). This was demonstrated by positive Dlx2 staining in these cells after transduction of cultured RAC with AAV9-P133 (AAV produced with NLX-P133), which was effectively driven by the virus for expression of Dlx2 (fig. 64).
Various further modifications and improvements to the compositions and methods of the present disclosure will be apparent to those skilled in the art. The following non-limiting examples are contemplated:
1. an adeno-associated virus (AAV) vector comprising: a human neurogenic differentiation factor 1 (hNeuroD 1) sequence, the hNeuroD1 sequence comprising a nucleic acid sequence of SEQ ID NO. 6, and a 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 modulator
A control element, the control 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 operable
Operatively connected 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 embodiments 1-3 or composition of any one of embodiments 4-6, wherein the AAV vector is selected from the group consisting of AAV serotype 2, AAV serotype 5, and AAV serotype 9.
8. The AAV vector or composition of embodiment 7, wherein the AAV vector is AAV serotype 2.
9. The AAV vector or composition of embodiment 7, wherein the AAV vector is AAV serotype 5.
10. The AAV vector or composition of embodiment 7, wherein the AAV vector is AAV serotype 9.
11. The composition of embodiment 4 or 5, wherein the glial cell is a reactive astrocyte.
12. The composition of embodiment 4 or 5, wherein the functional neuron is selected from the group consisting of a glutamatergic neuron, a gabaergic neuron, a dopaminergic neuron, a cholinergic neuron, a serotonergic neuron, an adrenergic neuron, a motor neuron, and a peptidoergic neuron.
13. The composition of embodiments 4 or 5, wherein the human suffers from a neurological disorder.
14. The AAV vector of example 3 or the composition of example 6, wherein the NeuroD1 is human NeuroD1 (hNeuroD 1).
15. The AAV vector of example 3 or the composition of example 6, wherein the Dlx2 is human Dlx2 (hDlx 2).
16. The AAV vector of example 3 or the composition of example 6, wherein the NeuroD1 is selected from the group consisting of chimpanzee NeuroD1, bonobo NeuroD1, red-chimpanzee NeuroD1, gorilla NeuroD1, macaque NeuroD1, marmoset NeuroD1, pigtail NeuroD1, baboon NeuroD1, gibbon NeuroD1, and marmoset NeuroD 1.
17. The AAV vector of example 3 or the composition of example 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 lemur Dlx 2.
18. The AAV vector or composition of embodiment 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 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 complement thereof.
21. The AAV vector or composition of example 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 example 3 or the composition of example 6, wherein the linker is selected from the group consisting of P2A and T2A.
23. The AAV vector or composition of 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 the complements 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 the complements thereof.
27. The AAV vector of example 3 or composition of example 6, wherein the GFAP promoter is a human GFAP (hGFAP) promoter.
28. The AAV vector of example 3 or the composition of example 6, wherein the GFAP promoter is selected from the group consisting of a chimpanzee GFAP promoter, a bonoban GFAP promoter, a chimpanzee GFAP promoter, a macaque GFAP promoter, a marmoset GFAP promoter, a pigtail GFAP promoter, a baboon GFAP promoter, a gibbon GFAP promoter, and a lemur GFAP promoter.
29. The AAV vector or composition of any one of the preceding embodiments, wherein the IRES sequence comprises a nucleic acid sequence at least 80% identical to: SEQ ID NO. 3, or a complement thereof.
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 complement 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 complement 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 complement thereof.
33. The AAV vector of example 3 or the composition of example 6, wherein the enhancer is selected from the group consisting of an enhancer from the human elongation factor-1 alpha (EF 1-alpha) promoter and a Cytomegalovirus (CMV) enhancer
34. The AAV vector or composition of embodiment 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 embodiment 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 embodiment 3 or composition of embodiment 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 embodiment 3 or composition of embodiment 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 example 3 or composition of example 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 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 complement 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 complement 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 complement thereof.
42. The AAV vector of example 3, or composition of example 6, wherein the AAV vector further comprises a nucleic acid sequence encoding an AAV protein sequence.
43. The AAV vector of any one of embodiments 1-3, or composition of any one of embodiments 4-6, wherein the AAV vector comprises an AAV serotype 2 Inverted Terminal Repeat (ITR).
44. The AAV vector of any one of embodiments 1-3, or composition of any one of embodiments 4-6, wherein the AAV vector comprises an AAV serotype 5 Inverted Terminal Repeat (ITR).
45. The AAV vector of any one of embodiments 1-3, or composition of any one of embodiments 4-6, wherein the AAV vector comprises an AAV serotype 9 Inverted Terminal Repeat (ITR).
46. The AAV vector of any one of embodiments 1-3, or composition of any one of embodiments 4-6, wherein the AAV vector comprises at least one ITR nucleic acid sequence at least 80% identical to: SEQ ID NO. 1.
47. The AAV vector of any one of embodiments 1-3, or composition of any one of embodiments 4-6, wherein the AAV vector comprises at least one ITR nucleic acid sequence at least 80% identical to: SEQ ID NO. 9.
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 has a neurological disorder.
52. The composition of embodiment 13 or 51, wherein the neurological disorder comprises an injury to the Central Nervous System (CNS) or peripheral nervous system.
53. The composition of embodiment 13 or 51, wherein the neurological condition comprises an injury to the CNS.
54. The composition of embodiment 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 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 hemorrhagic stroke.
62. The composition of embodiment 51, wherein the composition is capable of converting at least one glial cell into neurons.
63. The composition of embodiment 62, wherein the glial cell is selected from the group consisting of an astrocyte and a NG2 cell.
64. The composition of embodiment 62, wherein the glial cell is an astrocyte.
65. The composition of embodiment 62, wherein the astrocytes are reactive astrocytes.
66. The composition of embodiment 62, wherein the glial cells are GFAP positive.
67. The composition of embodiment 62, wherein the neuron is a functional neuron.
68. The composition of embodiment 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 embodiment 68, wherein the functional neuron is a glutamatergic neuron.
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 70-72, wherein the composition is formulated for delivery via intraperitoneal, intramuscular, intravenous, intrathecal, intracerebral, intracranial, intraventricular of the brain, intracavitary of the cerebellum, intravitreal, subretinal, intraparenchymal, intranasal, or oral administration.
74. A method comprising delivering the composition of example 6 to the 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 74-77, 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 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. The method of embodiment 79 or 80, wherein the functional neuron is a glutamatergic neuron, a gabaergic neuron, a dopaminergic neuron, a cholinergic neuron, a serotonergic neuron, an adrenergic neuron, a motor neuron, and a peptidoergic neuron.
88. The method of embodiment 81 or 82 wherein the NeuroD1 is human NeuroD1 (hNeuroD 1).
89. The method of embodiment 81 or 82 wherein the Dlx2 is human Dlx2 (hDlx 2).
90. The method of embodiment 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 embodiment 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 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 example 89, wherein the 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 complement thereof.
95. The method of embodiment 89, wherein the hDlx2 coding sequence comprises a nucleic acid sequence that is at least 80% identical to: SEQ ID NO. 13, or a complement thereof.
96. The method of embodiment 81 or 82, wherein the GFAP promoter is a human GFAP (hGFAP) promoter.
97. The method of embodiment 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 embodiments 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 embodiment 96, wherein the hGFAP promoter comprises a nucleic acid sequence that is at least 80% identical to: SEQ ID NO. 4, or a complement 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 complement 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 complement 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 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 embodiment 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 embodiment 81 or 82, wherein the enhancer is selected from the group consisting of an enhancer from the human elongation factor-1 alpha (EF 1-alpha) promoter and a Cytomegalovirus (CMV) enhancer.
106. The method of embodiment 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 embodiment 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 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 NOS 5 and 27, or the complements 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 NOS.7 and 29, or the complements 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 complement 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 complement thereof.
113. The method of 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 an AAV serotype 2 Inverted Terminal Repeat (ITR).
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 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 embodiments 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 embodiment 81, wherein the transformation occurs in the Central Nervous System (CNS) or peripheral nervous system.
120. The method of embodiment 81, wherein the transforming 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 the 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 delivering comprises administration selected from the group consisting of: intraperitoneal administration, intramuscular administration, intravenous administration, intrathecal administration, intracerebral administration, intracranial, lateral cerebral compartments of the brain, intracavitary, intravitreal, subretinal, intraparenchymal administration, intranasal administration, and oral administration.
127. The method of embodiment 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 embodiment 81 or 82, wherein the delivering comprises injection.
129. The method of any one of embodiments 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 embodiment 129, wherein the injecting further comprises a flow rate of 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. The method of embodiment 131, wherein the at least one glial cell is at least one astrocyte.
133. The method of embodiment 131 or 132, wherein the at least one astrocyte is a reactive astrocyte.
134. The method of embodiment 81, wherein the neuron is a functional neuron.
135. The method of any one of embodiments 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 embodiment 81, wherein the subject exhibits an improvement in at least one symptom of a neurological disorder as compared to the subject prior to the delivering.
137. The method of embodiment 136, wherein the improvement is measured within 1 year of the 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 the transformation is in the cortex of the 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 a injury 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 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 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 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 cell is selected from the group consisting of an astrocyte and a NG2 cell.
152. The method of embodiment 150, wherein the glial cell is an astrocyte.
153. The method of embodiment 152, wherein the astrocytes are reactive astrocytes.
154. The method of embodiment 150, wherein the glial cell is GFAP positive.
155. The method of embodiment 150, wherein the neuron is a functional neuron.
156. The method of embodiment 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 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 into 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 (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 embodiment 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 embodiment 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.
<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> patent in version 3.5
<210> 1
<211> 128
<212> DNA
<213> artificial sequence
<220>
<223> manual sequence description: synthesis of polynucleotides
<400> 1
tgcaggcagc tgcgcgctcg ctcgctcact gaggccgccc gggcgtcggg cgacctttgg 60
tcgcccggcc tcagtgagcg agcgagcgcg cagagaggga gtggccaact ccatcactag 120
gggttcct 128
<210> 2
<211> 153
<212> DNA
<213> artificial sequence
<220>
<223> manual sequence description: synthesis of polynucleotides
<400> 2
tgcaaagatg gataaagttt taaacagaga ggaatctttg cagctaatgg accttctagg 60
tcttgaaagg agtgggaatt ggctccggtg cccgtcagtg ggcagagcgc acatcgccca 120
cagtccccga gaagttgggg ggaggggtcg gca 153
<210> 3
<211> 553
<212> DNA
<213> artificial sequence
<220>
<223> manual sequence description: synthesis of polynucleotides
<400> 3
acgttactgg ccgaagccgc ttggaataag gccggtgtgc gtttgtctat atgttatttt 60
ccaccatatt gccgtctttt ggcaatgtga gggcccggaa acctggccct gtcttcttga 120
cgagcattcc taggggtctt tcccctctcg ccaaaggaat gcaaggtctg ttgaatgtcg 180
tgaaggaagc agttcctctg gaagcttctt gaagacaaac aacgtctgta gcgacccttt 240
gcaggcagcg gaacccccca cctggcgaca ggtgcctctg cggccaaaag ccacgtgtat 300
aagatacacc tgcaaaggcg gcacaacccc agtgccacgt tgtgagttgg atagttgtgg 360
aaagagtcaa atggctctcc tcaagcgtat tcaacaaggg gctgaaggat gcccagaagg 420
taccccattg tatgggatct gatctggggc ctcggtgcac atgctttaca tgtgtttagt 480
cgaggttaaa aaaacgtcta ggccccccga accacgggga cgtggttttc ctttgaaaaa 540
cacgatgata ata 553
<210> 4
<211> 1667
<212> DNA
<213> artificial sequence
<220>
<223> manual sequence description: synthesis of polynucleotides
<400> 4
ctgcaagcag acctggcagc attgggctgg ccgcccccca gggcctcctc ttcatgccca 60
gtgaatgact caccttggca cagacacaat gttcggggtg ggcacagtgc ctgcttcccg 120
ccgcacccca gcccccctca aatgccttcc gagaagccca ttgagtaggg ggcttgcatt 180
gcaccccagc ctgacagcct ggcatcttgg gataaaagca gcacagcccc ctaggggctg 240
cccttgctgt gtggcgccac cggcggtgga gaacaaggct ctattcagcc tgtgcccagg 300
aaaggggatc aggggatgcc caggcatgga cagtgggtgg caggggggga gaggagggct 360
gtctgcttcc cagaagtcca aggacacaaa tgggtgaggg gactgggcag ggttctgacc 420
ctgtgggacc agagtggagg gcgtagatgg acctgaagtc tccagggaca acagggccca 480
ggtctcaggc tcctagttgg gcccagtggc tccagcgttt ccaaacccat ccatccccag 540
aggttcttcc catctctcca ggctgatgtg tgggaactcg aggaaataaa tctccagtgg 600
gagacggagg ggtggccagg gaaacggggc gctgcaggaa taaagacgag ccagcacagc 660
cagctcatgc gtaacggctt tgtggagctg tcaaggcctg gtctctggga gagaggcaca 720
gggaggccag acaaggaagg ggtgacctgg agggacagat ccaggggcta aagtcctgat 780
aaggcaagag agtgccggcc ccctcttgcc ctatcaggac ctccactgcc acatagaggc 840
catgattgac ccttagacaa agggctggtg tccaatccca gcccccagcc ccagaactcc 900
agggaatgaa tgggcagaga gcaggaatgt gggacatctg tgttcaaggg aaggactcca 960
ggagtctgct gggaatgagg cctagtagga aatgaggtgg cccttgaggg tacagaacag 1020
gttcattctt cgccaaattc ccagcacctt gcaggcactt acagctgagt gagataatgc 1080
ctgggttatg aaatcaaaaa gttggaaagc aggtcagagg tcatctggta cagcccttcc 1140
ttcccttttt tttttttttt ttttgtgaga caaggtctct ctctgttgcc caggctggag 1200
tggcgcaaac acagctcact gcagcctcaa cctactgggc tcaagcaatc ctccagcctc 1260
agcctcccaa agtgctggga ttacaagcat gagccacccc actcagccct ttccttcctt 1320
tttaattgat gcataataat tgtaagtatt catcatggtc caaccaaccc tttcttgacc 1380
caccttccta gagagagggt cctcttgatt cagcggtcag ggccccagac ccatggtctg 1440
gctccaggta ccacctgcct catgcaggag ttggcgtgcc caggaagctc tgcctctggg 1500
cacagtgacc tcagtggggt gaggggagct ctccccatag ctgggctgcg gcccaacccc 1560
accccctcag gctatgccag ggggtgttgc caggggcacc cgggcatcgc cagtctagcc 1620
cactccttca taaagccctc gcatcccagg agcgagcaga gccagag 1667
<210> 5
<211> 133
<212> DNA
<213> artificial sequence
<220>
<223> manual sequence description: synthesis of polynucleotides
<400> 5
gtaagtatca aggttacaag acaggtttaa ggagaccaat agaaactggg cttgtcgaga 60
cagagaagac tcttgcgttt ctgataggca cctattggtc ttactgacat ccactttgcc 120
tttctctcca cag 133
<210> 6
<211> 1068
<212> DNA
<213> Chile person
<400> 6
atgaccaaat cgtacagcga gagtgggctg atgggcgagc ctcagcccca aggtcctcca 60
agctggacag acgagtgtct cagttctcag gacgaggagc acgaggcaga caagaaggag 120
gacgacctcg aagccatgaa cgcagaggag gactcactga ggaacggggg agaggaggag 180
gacgaagatg aggacctgga agaggaggaa gaagaggaag aggaggatga cgatcaaaag 240
cccaagagac gcggccccaa aaagaagaag atgactaagg ctcgcctgga gcgttttaaa 300
ttgagacgca tgaaggctaa cgcccgggag cggaaccgca tgcacggact gaacgcggcg 360
ctagacaacc tgcgcaaggt ggtgccttgc tattctaaga cgcagaagct gtccaaaatc 420
gagactctgc gcttggccaa gaactacatc tgggctctgt cggagatcct gcgctcaggc 480
aaaagcccag acctggtctc cttcgttcag acgctttgca agggcttatc ccaacccacc 540
accaacctgg ttgcgggctg cctgcaactc aatcctcgga cttttctgcc tgagcagaac 600
caggacatgc ccccccacct gccgacggcc agcgcttcct tccctgtaca cccctactcc 660
taccagtcgc ctgggctgcc cagtccgcct tacggtacca tggacagctc ccatgtcttc 720
cacgttaagc ctccgccgca cgcctacagc gcagcgctgg agcccttctt tgaaagccct 780
ctgactgatt gcaccagccc ttcctttgat ggacccctca gcccgccgct cagcatcaat 840
ggcaacttct ctttcaaaca cgaaccgtcc gccgagtttg agaaaaatta tgcctttacc 900
atgcactatc ctgcagcgac actggcaggg gcccaaagcc acggatcaat cttctcaggc 960
accgctgccc ctcgctgcga gatccccata gacaatatta tgtccttcga tagccattca 1020
catcatgagc gagtcatgag tgcccagctc aatgccatat ttcatgat 1068
<210> 7
<211> 589
<212> DNA
<213> artificial sequence
<220>
<223> manual sequence description: synthesis of polynucleotides
<400> 7
aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct 60
ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt 120
atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg 180
tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact 240
ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct 300
attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg 360
ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc 420
gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc 480
aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt 540
cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgc 589
<210> 8
<211> 251
<212> DNA
<213> artificial sequence
<220>
<223> manual sequence description: synthesis of polynucleotides
<400> 8
cgatccaccg gatctagata actgatcata atcagccata ccacatttgt agaggtttta 60
cttgctttaa aaaacctccc acacctcccc ctgaacctga aacataaaat gaatgcaatt 120
gttgttgtta acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca 180
aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc 240
aatgtatctt a 251
<210> 9
<211> 139
<212> DNA
<213> artificial sequence
<220>
<223> manual sequence description: synthesis of polynucleotides
<400> 9
aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 60
ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc 120
gagcgcgcag ctgcctgca 139
<210> 10
<211> 356
<212> PRT
<213> Chile person
<400> 10
Met Thr Lys Ser Tyr Ser Glu Ser Gly Leu Met Gly Glu Pro Gln Pro
1 5 10 15
Gln Gly Pro Pro Ser Trp Thr Asp Glu Cys Leu Ser Ser Gln Asp Glu
20 25 30
Glu His Glu Ala Asp Lys Lys Glu Asp Asp Leu Glu Ala Met Asn Ala
35 40 45
Glu Glu Asp Ser Leu Arg Asn Gly Gly Glu Glu Glu Asp Glu Asp Glu
50 55 60
Asp Leu Glu Glu Glu Glu Glu Glu Glu Glu Glu Asp Asp Asp Gln Lys
65 70 75 80
Pro Lys Arg Arg Gly Pro Lys Lys Lys Lys Met Thr Lys Ala Arg Leu
85 90 95
Glu Arg Phe Lys Leu Arg Arg Met Lys Ala Asn Ala Arg Glu Arg Asn
100 105 110
Arg Met His Gly Leu Asn Ala Ala Leu Asp Asn Leu Arg Lys Val Val
115 120 125
Pro Cys Tyr Ser Lys Thr Gln Lys Leu Ser Lys Ile Glu Thr Leu Arg
130 135 140
Leu Ala Lys Asn Tyr Ile Trp Ala Leu Ser Glu Ile Leu Arg Ser Gly
145 150 155 160
Lys Ser Pro Asp Leu Val Ser Phe Val Gln Thr Leu Cys Lys Gly Leu
165 170 175
Ser Gln Pro Thr Thr Asn Leu Val Ala Gly Cys Leu Gln Leu Asn Pro
180 185 190
Arg Thr Phe Leu Pro Glu Gln Asn Gln Asp Met Pro Pro His Leu Pro
195 200 205
Thr Ala Ser Ala Ser Phe Pro Val His Pro Tyr Ser Tyr Gln Ser Pro
210 215 220
Gly Leu Pro Ser Pro Pro Tyr Gly Thr Met Asp Ser Ser His Val Phe
225 230 235 240
His Val Lys Pro Pro Pro His Ala Tyr Ser Ala Ala Leu Glu Pro Phe
245 250 255
Phe Glu Ser Pro Leu Thr Asp Cys Thr Ser Pro Ser Phe Asp Gly Pro
260 265 270
Leu Ser Pro Pro Leu Ser Ile Asn Gly Asn Phe Ser Phe Lys His Glu
275 280 285
Pro Ser Ala Glu Phe Glu Lys Asn Tyr Ala Phe Thr Met His Tyr Pro
290 295 300
Ala Ala Thr Leu Ala Gly Ala Gln Ser His Gly Ser Ile Phe Ser Gly
305 310 315 320
Thr Ala Ala Pro Arg Cys Glu Ile Pro Ile Asp Asn Ile Met Ser Phe
325 330 335
Asp Ser His Ser His His Glu Arg Val Met Ser Ala Gln Leu Asn Ala
340 345 350
Ile Phe His Asp
355
<210> 11
<211> 380
<212> DNA
<213> artificial sequence
<220>
<223> manual sequence description: synthesis of polynucleotides
<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
tagtcatcgc tattaccatg 380
<210> 12
<211> 2214
<212> DNA
<213> Chile person
<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 tcttgggata 720
aaagcagcac agccccctag gggctgccct tgctgtgtgg cgccaccggc ggtggagaac 780
aaggctctat tcagcctgtg cccaggaaag gggatcaggg gatgcccagg catggacagt 840
gggtggcagg gggggagagg agggctgtct gcttcccaga agtccaagga cacaaatggg 900
tgaggggact gggcagggtt ctgaccctgt gggaccagag tggagggcgt agatggacct 960
gaagtctcca gggacaacag ggcccaggtc tcaggctcct agttgggccc agtggctcca 1020
gcgtttccaa acccatccat ccccagaggt tcttcccatc tctccaggct gatgtgtggg 1080
aactcgagga aataaatctc cagtgggaga cggaggggtg gccagggaaa cggggcgctg 1140
caggaataaa gacgagccag cacagccagc tcatgtgtaa cggctttgtg gagctgtcaa 1200
ggcctggtct ctgggagaga ggcacaggga ggccagacaa ggaaggggtg acctggaggg 1260
acagatccag gggctaaagt cctgataagg caagagagtg ccggccccct cttgccctat 1320
caggacctcc actgccacat agaggccatg attgaccctt agacaaaggg ctggtgtcca 1380
atcccagccc ccagccccag aactccaggg aatgaatggg cagagagcag gaatgtggga 1440
catctgtgtt caagggaagg actccaggag tctgctggga atgaggccta gtaggaaatg 1500
aggtggccct tgagggtaca gaacaggttc attcttcgcc aaattcccag caccttgcag 1560
gcacttacag ctgagtgaga taatgcctgg gttatgaaat caaaaagttg gaaagcaggt 1620
cagaggtcat ctggtacagc ccttccttcc cttttttttt tttttttttt gtgagacaag 1680
gtctctctct gttgcccagg ctggagtggc gcaaacacag ctcactgcag cctcaaccta 1740
ctgggctcaa gcaatcctcc agcctcagcc tcccaaagtg ctgggattac aagcatgagc 1800
caccccactc agccctttcc ttccttttta attgatgcat aataattgta agtattcatc 1860
atggtccaac caaccctttc ttgacccacc ttcctagaga gagggtcctc ttgcttcagc 1920
ggtcagggcc ccagacccat ggtctggctc caggtaccac ctgcctcatg caggagttgg 1980
cgtgcccagg aagctctgcc tctgggcaca gtgacctcag tggggtgagg ggagctctcc 2040
ccatagctgg gctgcggccc aaccccaccc cctcaggcta tgccaggggg tgttgccagg 2100
ggcacccggg catcgccagt ctagcccact ccttcataaa gccctcgcat cccaggagcg 2160
agcagagcca gagcaggttg gagaggagac gcatcacctc cgctgctcgc cggg 2214
<210> 13
<211> 987
<212> DNA
<213> Chile person
<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 cggggacgat tttctga 987
<210> 14
<211> 328
<212> PRT
<213> Chile person
<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 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 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 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> manual sequence description: synthetic oligonucleotides
<400> 15
gctactaact tcagcctgct gaagcaggct ggagacgtgg aggagaaccc tggacct 57
<210> 16
<211> 54
<212> DNA
<213> artificial sequence
<220>
<223> manual sequence description: synthetic oligonucleotides
<400> 16
gaggggagag gaagtcttct gacctgcgga gacgtcgaag agaatcctgg accc 54
<210> 17
<211> 477
<212> DNA
<213> Chile person
<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> manual sequence description: synthetic oligonucleotides
<400> 18
ggaagcggag ctactaactt cagcctgctg aagcaggctg gagacgtgga ggagaaccct 60
ggacct 66
<210> 19
<211> 66
<212> DNA
<213> artificial sequence
<220>
<223> manual sequence description: synthetic oligonucleotides
<400> 19
ggaagcggag ctactaactt cagcctgctg aagcaggctg gagacgtgga ggagaaccct 60
ggacct 66
<210> 20
<211> 22
<212> PRT
<213> artificial sequence
<220>
<223> manual sequence description: synthetic peptides
<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> manual sequence description: synthetic peptides
<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> manual sequence description: synthesis of polynucleotides
<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> manual sequence description: synthetic oligonucleotides
<400> 23
ccggtggttc agttacgggt taattctcga gaattaaccc gtaactgaac catttttg 58
<210> 24
<211> 60
<212> DNA
<213> artificial sequence
<220>
<223> manual sequence description: synthetic oligonucleotides
<400> 24
ccggcagtta cgggttaatt aatacctgac ccatattaat taacccgtaa ctgctttttg 60
<210> 25
<211> 57
<212> DNA
<213> artificial sequence
<220>
<223> manual sequence description: synthetic oligonucleotides
<400> 25
ccggtgttgc cgcagcatca ctaatctcga gattagtgat gctgcggcaa cattttg 57
<210> 26
<211> 681
<212> DNA
<213> artificial sequence
<220>
<223> manual sequence description: synthesis of polynucleotides
<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> manual sequence description: synthesis of polynucleotides
<400> 27
ggagtcgctg cgttgccttc gccccgtgcc ccgctccgcg ccgcctcgcg ccgcccgccc 60
cggctctgac tgaccgcgtt actcccacag gtgagcgggc gggacggccc ttctccctcc 120
gggctgtaat tagcgcttgg tttaatgacg gctcgtttct tttctgtggc tgcgtgaaag 180
ccttaaaggg ctccgggagg gcctttgtgc gggggggagc ggctcggggg gtgcgtgcgt 240
gtgtgtgtgc gtggggagcg ccgcgtgcgg cccgcgctgc ccggcggctg tgagcgctgc 300
gggcgcggcg cggggctttg tgcgctccgc gtgtgcgcga ggggagcgcg ggccgggggc 360
ggtgccccgc ggtgcggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg 420
tgggggggtg agcagggggt gtgggcgcgg cggtcgggct gtaacccccc cctggcaccc 480
ccctccccga gttgctgagc acggcccggc ttcgggtgcg gggctccgtg cggggcgtgg 540
cgcggggctc gccgtgccgg gcggggggtg gcggcaggtg ggggtgccgg gcggggcggg 600
gccgcctcgg gccggggagg gctcggggga ggggcgcggc ggccccggag cgccggcggc 660
tgtcgaggcg cggcgagccg cagccattgc cttttatggt aatcgtgcga gagggcgcag 720
ggacttcctt tgtcccaaat ctggcggagc cgaaatctgg gaggcgccgc cgcaccccct 780
ctagcgggcg cgggcgaagc ggtgcggcgc cggcaggaag gaaatgggcg gggagggcct 840
tcgtgcgtcg ccgcgccgcc gtccccttct ccatctccag cctcggggct gccgcagggg 900
gacggctgcc ttcggggggg acggggcagg gcggggttcg gcttctggcg tgtgaccggc 960
ggctttagag cctctgctaa ccatgttcat gccttcttct ttttcctaca gctcctgggc 1020
aacgtgctgg ttgttgtgct gtctcatcat tttggcaaag at 1062
<210> 28
<211> 978
<212> DNA
<213> artificial sequence
<220>
<223> manual sequence description: synthesis of polynucleotides
<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 catgagggag attggattct 420
ggggtgaaga aagtgaggga aagagcaggc aggtctgggc gcaaagcaca ggtgactgcc 480
tgccaccagc ttgtgacccc catcaagtta ctttgacttg cacagctgtg aagcggtggt 540
cataataaaa ttcatttcaa aaggtggtta cctgggatca gaggaatccc caggggcatg 600
gcgcttcact gagctgacag gacatgcatg tgtgccttca agtgcaggag gacatgtgcg 660
tgtgtgtgtg tgtgtgtgca acagtgagtg tatgcttgtg gatgcgcctg tgtgagcaga 720
agcaggtgca ccaaccctga taaggcacct tagtaatgag ttaaggcaaa agcccacatc 780
tgctcatcct ccagacaagt cctctgtcta aggcccccca acccttaatc ctcctgctgc 840
tctactggtc ctgggtgggg gtggtctctg tgacagctgc ctcaagggag actgaggcag 900
gtattcaagt gtcctcagaa gagcctggac ccaggaatgt gtccccccac tccaggctcc 960
aggatgaaac caacctga 978
<210> 29
<211> 581
<212> DNA
<213> artificial sequence
<220>
<223> manual sequence description: synthesis of polynucleotides
<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> manual sequence description: synthesis of polynucleotides
<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.
CN202180080014.7A 2020-09-29 2021-09-28 NEUROD1 and DLX2 vectors Pending CN116761812A (en)

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US202163247442P 2021-09-23 2021-09-23
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