WO2025113676A1

WO2025113676A1 - Compositions and methods for treating stroke in primates

Info

Publication number: WO2025113676A1
Application number: PCT/CN2024/135809
Authority: WO
Inventors: Russell ADDIS; Anna Kabanova; Jie Xu; Gong Chen; Yuchen Chen; Ming Chen
Original assignee: Neuexcell Therapeutics Suzhou Co Ltd; NeuExcell Therapeutics Inc
Current assignee: Neuexcell Therapeutics Suzhou Co Ltd; NeuExcell Therapeutics Inc
Priority date: 2023-11-29
Filing date: 2024-11-29
Publication date: 2025-06-05
Anticipated expiration: 2026-05-29

Abstract

The present disclosure relates to compositions and methods of treating stroke, reducing neuroinflammation, and generating new neurons in a primate using an adeno-associated viral (AAV) vector encoding NeuroD1.

Description

COMPOSITIONS AND METHODS FOR TREATING STROKE IN PRIMATES

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to International Patent Application No.: PCT/CN2023/134880 filed on November 29, 2023, the content of which is incorporated by reference in its entirety.
SEQUENCE LISTING

This application contains an electronic Sequence Listing which has been submitted in XML file format with this application, the entire content of which is incorporated by reference herein in its entirety. The Sequence Listing XML file submitted with this application is entitled “14770-039-228_SEQLISTING. xml” , was created on November 25, 2024, and is 139,671 bytes in size.

1. FIELD OF THE INVENTION

The present disclosure provides and includes compositions for and methods of treating stroke in a primate using an adeno-associated viral (AAV) vector comprising a nucleic acid molecule encoding Neuronal Differentiation 1 (NeuroD1) .

2. BACKGROUND OF THE INVENTION

Neurons are often killed or damaged and unable to regenerate in subjects with a neurological condition or following an injury to the central nervous system (CNS) or peripheral nervous system (PNS) .

Glial cells can become reactive following an injury to the CNS or PNS, or in the case of a neurological condition. For instance, after stroke, reactive glial cells can proliferate and maintain a high number in the injury site, eventually forming a dense scar tissue that prevents the growth of neurons.

Currently there are no treatments available to regenerate functional new neurons in primate subjects, including human subjects, using AAVs.

3. SUMMARY OF THE INVENTION

In one aspect, this disclosure provides and includes a method of treating stroke in a primate, the method comprising administering to the primate a pharmaceutical composition comprising an adeno-associated viral (AAV) vector comprising a nucleic acid molecule encoding Neurogenic Differentiation 1 (NeuroD1) under the control of a Glial Fibrillary Acidic Protein (GFAP) promoter, where the pharmaceutical composition is administered by injecting the brain of the primate.

In one aspect, this disclosure provides and includes a method of treating stroke in a primate, the method comprising administering to the primate a pharmaceutical composition comprising an adeno-associated viral (AAV) vector comprising a nucleic acid molecule encoding Neurogenic Differentiation 1 (NeuroD1) under the control of a Glial Fibrillary Acidic Protein (GFAP) promoter, where the primate has a score of at least 21 on the National Institutes of Health Stroke Scale (NIHSS) or at least 25 on the Non-Human Primate Stroke Scale (NHPSS) , and where the score is improved by at least 1 unit after the primate is administered the pharmaceutical composition.

In one aspect, this disclosure provides and includes a method of treating stroke in a primate, the method comprising administering to the primate a pharmaceutical composition comprising an adeno-associated viral (AAV) vector comprising a nucleic acid molecule encoding Neurogenic Differentiation 1 (NeuroD1) under the control of a Glial Fibrillary Acidic Protein (GFAP) promoter, where the primate has a score of at least 4 on the Modified Rankin Scale, and where the score is improved by at least 1 unit after the primate is administered the pharmaceutical composition.

In one aspect, this disclosure provides and includes a method of partially or fully restoring neuronal pathways in the brain of a primate who has suffered a stroke, the method comprising administering to the primate a pharmaceutical composition comprising an adeno-associated viral (AAV) vector comprising a nucleic acid molecule encoding Neurogenic Differentiation 1 (NeuroD1) under the control of a Glial Fibrillary Acidic Protein (GFAP) promoter, where the restoring occurs within three to six months after the primate is administered the pharmaceutical composition

In one aspect, this disclosure provides and includes a method of reducing neuroinflammation in the brain of a primate who has suffered a stroke, the method comprising administering to the primate a pharmaceutical composition comprising an adeno-associated viral (AAV) vector comprising a nucleic acid molecule encoding Neurogenic Differentiation 1 (NeuroD1) under the control of a Glial Fibrillary Acidic Protein (GFAP) promoter, where the neuroinflammation is reduced within 14 to 21 days after the primate is administered the pharmaceutical composition

In one aspect, this disclosure provides and includes a method of generating new neurons in the brain of a primate who has suffered a stroke, the method comprising administering to the primate a pharmaceutical composition comprising an adeno-associated viral (AAV) vector comprising a nucleic acid molecule encoding Neurogenic Differentiation 1 (NeuroD1) under the control of a Glial Fibrillary Acidic Protein (GFAP) promoter, where the new neurons are generated within 14 to 28 days after the primate is administered the pharmaceutical composition.

In one aspect, this disclosure provides and includes a single-stranded nucleic acid molecule encoding a NeuroD1 polypeptide, wherein the nucleic acid molecule comprises an expression cassette comprising a coding sequence and one or more regulatory elements operably linked to the coding sequence, wherein the NeuroD1 polypeptide comprises an amino acid sequence having at least 90%sequence identity to the sequence set forth in SEQ ID NO: 15.

In some embodiments, the NeuroD1 polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the coding sequence comprises the nucleotide sequence set forth in SEQ ID NO: 4 or a codon-optimized variant thereof. In some embodiments, the one or more transcription regulatory elements comprise a chimeric intron. In some embodiments, the chimeric intron comprises the sequence set forth in SEQ ID NO: 19. In some embodiments, the one or more transcription regulatory elements further comprise a GFAP promoter comprising the sequence set forth in SEQ ID NO: 10. In some embodiments, the one or more transcription regulatory elements further comprises a CMV enhancer comprising the sequence set forth in SEQ ID NO: 8. In some embodiments, the one or more transcription regulatory elements further comprises an optimized WPRE comprising the sequence set forth in SEQ ID NO: 12. In some embodiments, the one or more transcription regulatory elements further comprise a polyadenylation (poly-A) signal comprising the sequence set forth in SEQ ID NO: 9.

In some embodiments, the nucleic acid molecule further comprises a first inverted terminal repeat (ITR) of a first AAV genome. In some embodiments, the first ITR is the 5’ ITR of the first AAV genome. In some embodiments, the first ITR comprises the sequence set forth in SEQ ID NO: 16. In some embodiments, the first ITR is the 5’ ITR of the first AAV genome. In some embodiments, the first ITR comprises the sequence set forth in SEQ ID NO: 58. In some embodiments, the nucleic acid molecule further comprises a second ITR of a second AAV genome. In some embodiments, the second ITR comprises the sequence set forth in SEQ ID NO: 23. In some embodiments, the second ITR comprises the sequence set forth in SEQ ID NO: 59. In some embodiments, the nucleic acid molecule further comprises the sequence set forth in SEQ ID NO: 24. In some embodiments, the nucleic acid molecule is DNA.

In one aspect, this disclosure provides and includes a single-stranded DNA molecule consists of the sequence set forth in SEQ ID NO: 24.

In one aspect, this disclosure provides and includes a recombinant adeno-associated virus (rAAV) comprising a single-stranded nucleic acid molecule as described herein. In some embodiments, the recombinant AAV comprises a AAV serotype 9 (AAV9) capsid. In some embodiments, the AAV9 capsid comprises capsid proteins selected from the group of AAV9 VP1 polypeptides, AAV9 VP2 polypeptides and AAV9 VP3 polypeptides. In some embodiments, the AAV9 capsid comprises AAV9 VP1 comprising the amino acid sequence set forth in SEQ ID NO: 40. In some embodiments, the AAV9 capsid further comprises AAV9 VP2 comprising the amino acid sequence set forth in SEQ ID NO: 41. In some embodiments, the AAV9 capsid further comprises AAV9 VP3 comprising the amino acid sequence set forth in SEQ ID NO: 42.

In one aspect, this disclosure provides and includes a pharmaceutical composition comprising the recombinant AAV of the present disclosure, wherein the pharmaceutical composition further comprises:
(a) potassium chloride,
(b) potassium phosphate monobasic,
(c) sodium chloride,
(d) sodium phosphate dibasic anhydrous, and
(e) poloxamer 188, polysorbate 20, or polysorbate 80.

In one aspect, this disclosure provides and includes a pharmaceutical composition consists of:
(a) a recombinant adeno-associated virus (AAV) ,
(b) sodium chloride at a concentration of about 180 mM;
(c) sodium phosphate at a concentration of about 10 mM; and
(d) poloxamer 188 at a concentration of about 0.001%weight/volume (0.01 g/L) ; and wherein
the pH of the pharmaceutical composition is about 7.3.

In one aspect, this disclosure provides and includes a pharmaceutical composition consists of:
(a) a recombinant adeno-associated virus (AAV) ,
(b) sodium chloride at a concentration of about 200 mM;
(c) magnesium chloride at a concentration of about 1 mM;
(d) Tris hydrochloride at a concentration of about 20 mM, and
(e) poloxamer 188 at a concentration of about 0.005%weight/volume (0.05 g/L) ; and wherein
the pH of the pharmaceutical composition is about 8.0.

In one aspect, this disclosure provides and includes a pharmaceutical composition consists of:
(a) a recombinant adeno-associated virus (AAV) ,
(b) sodium chloride at a concentration of about 150 mM;
(c) calcium chloride at a concentration of about 1.4 mM;
(d) magnesium chloride at a concentration of about 0.8 mM,
(e) sodium phosphate at a concentration of about 1 mM, and
(f) poloxamer 188 at a concentration of about 0.001%weight/volume (0.01 g/L) ; and wherein
the pH of the pharmaceutical composition is about 7.4.

In some embodiments, of the pharmaceutical compositions provided herein, the recombinant AAV is any of the recombinant AAV provided herein.

In some embodiments, a vector genome concentration of the recombinant AAV in the pharmaceutical composition is in the range of about 5×10¹¹ to about 2×10¹² viral genomes per mL (vg/mL) ; optionally, wherein the vector genome concentration is about 5 ×10¹¹ vg/mL, about 1 × 10¹² vg/mL, or about 2 × 10¹² vg/mL.

In one aspect, this disclosure provides and includes a method for treating stroke, comprising administering to a subject in need thereof a therapeutically effective amount of the recombinant AAV provided herein. In one aspect, this disclosure provides and includes a method for treating stroke, comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition provided herein.

In some embodiments of the method for treating stroke described herein, the pharmaceutical composition is administered to the subject intracerebrally. In some embodiments of the method for treating stroke described herein, the subject has a stroke and wherein the pharmaceutical composition is administered to the peri-infarct motor cortex region. In some embodiments of the method for treating stroke described herein, the subject has a stroke and wherein the pharmaceutical composition is administered to the area located around the infarct lesion.

In some embodiments of the method for treating stroke described herein, the pharmaceutical composition comprises from about 1×10¹¹ to about 1×10¹³ viral genomes (vg) of the recombinant AAV. In some embodiments of the method for treating stroke described herein, the pharmaceutical composition comprises about 3×10¹¹ vg of the recombinant AAV. In some embodiments of the method for treating stroke described herein, the pharmaceutical composition comprises about 6.0×10¹¹ vg of the recombinant AAV. In some embodiments of the method for treating stroke described herein, the pharmaceutical composition comprises about 1.2×10¹² vg of the recombinant AAV.

In some embodiments of the method for treating stroke described herein, the subject is administered intracerebrally the pharmaceutical composition comprising about 3×10¹¹ vg of the recombinant AAV once. In some embodiments, for each administration the subject is administered about 0.6 mL of the pharmaceutical composition comprising about 5×10¹¹ vg/mL of the recombinant AAV intracerebrally. In some embodiments, the pharmaceutical composition is administered stereo-tactically.

In some embodiments of the method for treating stroke described herein, the subject is administered intracerebrally the pharmaceutical composition comprising about 6×10¹¹ vg of the recombinant AAV once. In some embodiments, for each administration the subject is administered about 0.6 mL of the pharmaceutical composition comprising about 1×10¹² vg/mL of the recombinant AAV intracerebrally. In some embodiments, the pharmaceutical composition is administered stereo-tactically.

In some embodiments of the method for treating stroke described herein, the subject is administered intracerebrally the pharmaceutical composition comprising about 1.2×10¹² vg of the recombinant AAV once. In some embodiments, for each administration the subject is administered about 0.6 mL of the pharmaceutical composition comprising about 2×10¹² vg/mL of the recombinant AAV intracerebrally. In some embodiments, the pharmaceutical composition is administered stereo-tactically.

In some embodiments of the method for treating stroke described herein, the subject is a human.

In some embodiments, the stroke is ischemic stroke. In some embodiments, the stroke is hemorrhagic stroke.

In some embodiments of the method for treating stroke described herein, upon administering the pharmaceutical composition, the NeuroD1 polypeptide is expressed by a population of glial cells.

In some embodiments, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%of the glial cells in the population converts to neurons after the administering of the pharmaceutical composition. In some embodiments, the neurons are selected from glutamatergic neurons, GABAergic neurons, dopaminergic neurons; motor neurons, glycinergic neurons, serotonergic neurons, In some embodiments, the population of glial cells exhibit one or more neuronal phenotypes; optionally wherein the neuronal phenotype comprises expressing one or more neuronal markers selected from DCX, TUJ1, NeuN, and MAP2; optionally the population of glial cells exhibit the one or more neuronal phenotype after the administering of the pharmaceutical composition. In some embodiments, the population of glial cells stop expressing one or more glial marker; optionally the one or more glial marker is selected from GFAP, Aldh1l1, S100β and Sox9; optionally the population of glial cells stop expressing the one or more glial after the administering of the pharmaceutical composition. In some embodiments, neuroinflammation in the brain of subject is reduced; optionally wherein neuroinflammation in the brain of subject is reduced for at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%. In some embodiments, neuronal pathways are partially or fully restored in the brain of subject, optionally wherein neuronal pathways are partially or fully restored for at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%. In some embodiments, new neurons are generated in the brain of the subject after the administering of the pharmaceutical composition.

In one aspect, this disclosure provides and includes a gene-of-interest (GOI) plasmid comprising an expression cassette comprising a transgene of interest and a pair of AAV ITR sequences flanking the expression cassette, wherein the transgene encodes a NeuroD1 polypeptide, wherein the NeuroD1 polypeptide comprises an amino acid sequence having at least 90%sequence identity to the sequence set forth in SEQ ID NO: 15.

In one aspect, this disclosure provides and includes a gene-of-interest (GOI) plasmid comprising an expression cassette comprising a transgene of interest and a pair of AAV ITR sequences flanking the expression cassette, wherein the transgene encodes a NeuroD1 polypeptide, wherein the NeuroD1 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 13.

In one aspect, this disclosure provides and includes a gene-of-interest (GOI) plasmid comprising an expression cassette comprising a transgene of interest and a pair of AAV ITR sequences flanking the expression cassette, wherein the transgene encodes a NeuroD1 polypeptide, wherein the transgene comprises the nucleic acid sequence set forth in SEQ ID NO: 4, or a codon-optimized version thereof.

In one aspect, this disclosure provides and includes a host cell comprising the GOI plasmid provided herein.

In one aspect, this disclosure provides and includes a method of producing a recombinant AAV comprising:
(a) culturing a host cell containing:
(i) an artificial genome comprising a cis expression cassette, wherein the cis expression
cassette comprises a coding sequence encoding a NeuroD1 polypeptide, wherein the NeuroD1 polypeptide comprises an amino acid sequence having at least 90%sequence identity to the sequence set forth in SEQ ID NO: 15;
(ii) a trans expression cassette lacking AAV ITRs, wherein the trans expression cassette
encodes an AAV Rep and Capsid proteins operably linked to expression control element that drive expression of the AAV Rep and capsid proteins in the host cell in culture, and supply the Rep and Capsid proteins in trans;
(iii) sufficient adenovirus helper functions to permit replication and packaging of the
artificial genome by the AAV capsid proteins; and
(b) recovering the recombinant AAV encapsidating the artificial genome from the cell culture.

In one aspect, this disclosure provides and includes a method of producing a recombinant AAV comprising:
(a) culturing a host cell containing:
(i) an artificial genome comprising a cis expression cassette, wherein the cis expression
cassette comprises a coding sequence encoding a NeuroD1 polypeptide, wherein the NeuroD1 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 13;
(ii) a trans expression cassette lacking AAV ITRs, wherein the trans expression cassette
encodes an AAV Rep and Capsid proteins operably linked to expression control element that drive expression of the AAV Rep and capsid proteins in the host cell in culture, and supply the Rep and Capsid proteins in trans;
(iii) sufficient adenovirus helper functions to permit replication and packaging of the
artificial genome by the AAV capsid proteins; and
(b) recovering the recombinant AAV encapsidating the artificial genome from the cell culture.

In some embodiments of the method of producing recombinant AAV described herein, the artificial genome is synthesized by the host cell using a GOI plasmid sequence as a replication template, wherein the GOI plasmid comprises the cis expression cassette flanked by a pair of AAV ITR sequences.

In one aspect, this disclosure provides and includes a host cell comprising an artificial genome comprising the single-stranded nucleic acid molecule described herein.

4. BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing (s) will be provided by the Office upon request and payment of the necessary fee.

Figure 1 depicts magnetic resonance (MRI) images indicating the site of three injections (1, 2, and 3) in the brain of nonhuman primates.

Figure 2A illustrates the behavioral assessments performed daily on the Non-Human Primate Stroke Scale (NHPSS) for animal Vel-009 (treated with NeuroD1 14 days after the induction of stroke) and animal Kev-009 (an untreated control) .

Figure 2B illustrates the behavioral assessments performed daily on the Modified Rankin Scale (mRS) for animal Vel-009 (treated with NeuroD1 14 days after the induction of stroke) and animal Kev-009 (an untreated control) .

Figure 3 illustrates the behavioral assessments performed daily on the NHPSS and the mRS for animal Vel-009 (treated with NeuroD1 14 days after the induction of stroke) , animal Seb-006 (treated with NeuroD1 56 days after the induction of stroke) , and animal Kev-009 (an untreated control) . MCAO = Middle Cerebral Artery Occlusion.

Figures 4A, 4B, and 4C correspond to diffusion tensor imaging (DTI) assessments of the corticospinal white matter tracts (CST) for animal Velma (treated with NeuroD1 14 days post-MCAO) , animal Seb (treated with NeuroD1 56 days post-MCAO) , and animal Kev (an untreated control) at baseline (Fig. 4A) , 7 days post-MCAO (Figure 4B) , and 241 days post-MCAO (Figure 4C) .

Figure 5 corresponds to DTI assessments of the CST made for Velma (treated with NeuroD1 14 days post-MCAO) at baseline and different times post-MCAO.

Figure 6 depicts an immunohistochemical analysis of animal Vel-009 (treated with NeuroD1 14 days post-MCAO) showing a significantly increased neuronal density in the peri-infarct area of its brain. GFP (green) expression labels transduced cells. NeuN (NN) (red) expression labels neurons. The overlap panel (NN/GFP) depicts the presence of transduced glial cells (e.g., astrocytes) that have been converted into neurons.

Figure 7 depicts an immunohistochemical analysis of three different injection sites (1, 2, and 3) in the brain of animal Vel-009 (treated with NeuroD1 14 days post-MCAO) . The 3 injection sites (1, 2, and 3) correspond to the same 3 injection sites (1, 2, and 3) of the MRI panels of Figure 1. NN (red) and GFP (green) overlap can be observed in all three injection sites.

Figure 8 depicts an immunohistochemical analysis of the three different injection sites (1, 2, and 3) in the brain of animal Vel-009 (treated with NeuroD1 14 days post-MCAO) and further demonstrates the glial cell-to-neuron conversion. In the first row of panels (corresponding to injection site 1) , antibodies against NeuN/NN are used. The NN/GFP/DAPI and NN/GFP overlap figures demonstrate the presence of transduced glial cells (e.g., astrocytes) that have been converted into neurons. In the second row of panels (corresponding to injection site 2) , an SMI32 antibody (red) and an antibody against NeuN (NN) (green) are used. In the third row of panels (corresponding to injection site 2) , an SMI312 antibody (green) and an antibody against NeuN (NN) (red) are used. In the fourth row of panels (corresponding to injection site 3) , an SMI32 antibody (red) and an antibody against NeuN (NN) (green) are used. DAPI (blue) is a nuclear stain.

Figure 9 depicts an immunohistochemical analysis of the three different injection sites (1, 2, and 3) in the brain of animal Vel-009 (treated with NeuroD1 14 days post-MCAO) . The first and second rows of panels correspond to injection site 1, and the third and fourth rows of panels correspond to injection site 2. Antibodies against neuronal markers NeuN (NN) (green) and Parvalbumin (PV) (red) are used. DAPI (blue) is a nuclear stain.

Figure 10 is a schematic of the immunohistochemical analysis of animal Vel-009 (treated with NeuroD1 14 days post-MCAO) showing that newly formed neurons send axons to appropriate targets along the corticospinal tract. Figure 10 shows NeuroD1-converted neurons in the primary motor cortex and their distal axonal bundles in the striatum (internal capsule) and the brainstem (pons) .

Figure 11 depicts an immunohistochemical analysis of animal Vel-009 (treated with NeuroD1 14 days post-MCAO) in the internal capsule region, where distal axonal bundles are observed. GFP (green) expression identifies the transduced cells, and GFAP (red) expression traces the lineage of the newly converted neurons to GFAP-expressing cells (e.g., astrocytes) . DAPI (blue) is a nuclear stain.

Figures 12A and 12B depict an immunohistochemical analysis of animal Vel-009 (treated with NeuroD1 14 days post-MCAO) in the different cortical layers in the cortex of Vel-009. Figure 12A depicts the upper layers of the cortex of Vel-009. Figure 12B depicts the deep layers of the cortex of Vel-009 as well as regions of the white matter. Antibodies against neuronal marker NeuN (NN) (red) or glial cell marker GFAP (red) are used. GFP (green) expression identifies the transduced cells. DAPI (blue) is a nuclear stain. In Figure 12A, yellow arrows indicate co-staining of NeuN and GFP (top panels) or GFAP and GFP (bottom panels) .

Figure 13 is an immunohistochemical analysis of Vel-009’s and Kev-008’s non-stroked and peri-infarct areas. Vel-009 is an animal treated with NeuroD1 14 days post-MCAO. Kev-008 is a control untreated animal. Immunostaining of phosphorylated neurofilaments (Smi312) reveals significant differences in the relative axonal densities in the intact and stroked areas of Vel-009’s and Kev-008’s cortexes. Additionally, an antibody against microglia marker Iba1 (green) is used to detect neuroinflammation.

Figure 14 depicts representative images of the MRI data collection following stroke in the Vel-009 (treated with NeuroD1 at 14 days post-MCAO) and Kev-008 (untreated control) animals. Affected areas following MCAO are visible, allowing for selection of injection sites.

Figure 15 depicts photographs of the brain of Vel-009 (treated with NeuroD1 at 14 days post-MCAO) and sections thereof, indicating the location of injection sites 1, 2, and 3 (same injection sites as Figure 1) .

Figure 16 provides an anatomy comparison of the brains of the Vel-009 (treated with NeuroD1 at 14 days post-MCAO) and Kev-008 (untreated control) animals.

Figure 17 provides sequences of elements constituting the genomic sequence of a recombinant AAV vector encoding NeuroD1 (from left ITR to right ITR) .

Figures 18A, 18B, and 18C provide NeuroD1 expression comparison (AAV-NeuroD1 vs AAV9-Cre-FLEX-NeuroD1) . Figure 18A shows representative images showing NeuroD1 expression (in purple) in the left M1 region (AP +0.24, ML ±2, DV -2.2) of rats injected with 3μL of AAV-NeuroD1 (2E12vg/mL) and the right M1 region injected with 3μL of AAV9-Cre-FLEX-NeuroD1 (2E12vg/mL) . NeuroD1 expression was evaluated at 5 days post-injection (5 dpi) and 10 days post-injection (10 dpi) . Scale bar: 40 μm. Figure 18B shows quantification of NeuroD1 expression intensity in the AAV-NeuroD1 and AAV9-Cre-FLEX-NeuroD1 groups. There was no significant difference in NeuroD1 expression intensity between the two vectors at either 5 or 10 days post-injection. Figure 18C shows qPCR analysis of the NeuroD1 mRNA/DNA ratio in the injected M1 regions. The ratio of NeuroD1 mRNA to DNA showed no significant difference between AAV-NeuroD1 and AAV9-Cre-FLEX-NeuroD1 at both 5 and 10 days post-injection. *p < 0.05, **p < 0.01, two-way ANOVA.

Figures 19A, 19B, and 19C provide AAV-NeuroD1 Pharmacokinetic (PK) Study (Transduction Efficiency and NeuroD1 Expression) . Figure 19A shows representative images showing NeuroD1 expression (in purple) in the M1 regions of rats injected with 3μL of AAV-NeuroD1 (5E11vg/mL) at different time points: 3 dpi (days post injection) , 7 dpi, 15 dpi, 30 dpi, 3 mpi (months post injection) , 9 mpi, and 12 mpi. Scale bar: 100 μm. Figure 19B shows quantification of AAV-NeuroD1 transduction efficiency (NeuroD1+/DAPI+ × 100%) at 3, 7, 15, and 30 dpi. The results show a significant increase in transduction efficiency over time. *p < 0.05, **p < 0.01, ***p < 0.001, two-way ANOVA. Figure 19C shows quantification of NeuroD1 expression intensity over time. The expression of NeuroD1 increased from 3 to 30 days and then showed a declining trend at later time points (3, 9, and 12 months) . *p < 0.05, **p < 0.01, ****p < 0.0001, two-way ANOVA.

Figures 20A and 20B provide AAV-NeuroD1 Pharmacokinetic (PK) study (Conversion Efficiency) . Figure 20A shows representative images showing AAV-NeuroD1-mediated astrocyte-to-neuron conversion in the M1 regions of rats injected with 3μL of AAV-NeuroD1 (5E11vg/mL) mixed with AAV9-hGFAP: : GFP (1E11 vg/mL) at different time points: 3 dpi, 7 dpi, 15 dpi, 30 dpi, 3 mpi. Neuronal marker NeuN (red) , NeuroD1 (purple) , and GFP (green) . Scale bar: 50 μm. Figure 20B shows quantification of astrocyte-to-neuron conversion efficiency ( (GFP+NeuroD1+NeuN+) / (GFP+NeuroD1+) ) at 3, 7, 15, 30 days, and 3 months post-injection. Conversion efficiency showed a significant increase over time. (**p < 0.01, ****p < 0.0001, two-way ANOVA) .

Figure 21 shows no elevated cell division observed 12 -month post AAV-NeuroD1 injection. AAV-NeuroD1 was injected in M1 cortex and tissue was collected 12-month post injection. Immunostaining showed the expression of NeuroD1 (purple) and GFP (green) marked cells of neuron morphology. A normal level of Ki67 (red) expression indicated no elevated cell division. DAPI (blue) shows all cells.

Figures 22A, 22B, and 22C provide AAV-NeuroD1 Potency Assay. Figure 22A shows representative images showing NeuroD1 expression (in purple) in the M1 regions of rats injected with 3 μL of AAV-NeuroD1 at different titers (6E10, 3E11, 3E12 vg/mL) . NeuroD1 expression was evaluated at 7 days (7 dpi) and 14 days (14 dpi) post-injection. Scale bar: 200 μm. Figure 22B shows quantification of AAV-NeuroD1 transduction efficiency ( (NeuroD1+/DAPI+) *100%) at 7 and 14 days post-injection. The transduction efficiency was positively correlated with viral titer. (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, two-way ANOVA) . Figure 22C shows quantification of NeuroD1 expression intensity at 7 and 14 days post-injection. The intensity of NeuroD1 expression increased with higher viral titers. (*p < 0.05, **p < 0.01, two-way ANOVA)

Figures 23A and 23B show that AAV-NeuroD1 recovered neuronal loss in focal ischemic model. Figure 23A shows focal ischemic stroke models were established in the M1 region of rats injected with 0.2 μg endothelin-1 (ET-1) . Ten days post stroke, 5 μL of either control virus (AAV9-hGFAP: : GFP, 1E11 vg/mL) or AAV-NeuroD1 (5E11 vg/mL) mixed with control virus (1E11 vg/mL) was injected into the same coordinates. Brain samples were collected at 5, 10, 20, and 40 days post-injection (dpi) and stained with NeuN (red) to access neuronal loss. Scale bar: 500 μm. Figure 23B shows quantification of neuronal loss area shows significant neuronal loss in the control group (n≥3) at 5, 10, 20, and 40 dpi. In the AAV-NeuroD1-treated group (n≥3) , neuronal recovery was observed starting at 20 dpi, with significant recovery at 40 dpi compared to the control group (*p < 0.05, two-way ANOVA) .

Figures 24A and 24B show that AAV-NeuroD1 efficiently converted reactive astrocytes into neurons. Figure 24A shows focal ischemic stroke models were established in the M1 region of rats injected with 0.2 μg endothelin-1 (ET-1) . Ten days post stroke, 5 μL of either control virus (AAV9-hGFAP: : GFP, 1E11 vg/mL) or AAV-NeuroD1 (5E11 vg/mL) mixed with control virus (1E11 vg/mL) was injected into the same coordinates. Immunofluorescence staining of GFP (green) , NeuN (red) , and NeuroD1 (purple) shows that GFP+ cells were primarily glial in morphology at 5 and 10 dpi in both the control and AAV-NeuroD1-treated groups. However, at 20 and 40 dpi in the AAV-NeuroD1-treated group, significant conversion into neurons (GFP+/NeuN+/NeuroD1+) was observed. Scale bar: 20 μm. Figure 24B shows quantification of astrocyte-to-neuron conversion efficiency in a region 100 μm from the ischemic core shows a significant increase in conversion efficiency at 40 dpi (58.53 ± 5.85%) in the AAV-NeuroD1-treated group compared to both the control group (0.07%± 0.03) and at 20 dpi (13.63 ± 1.33%) (***p < 0.001, ****p < 0.0001, two-way ANOVA) .

Figures 25A, 25B, and 25C show AAV-NeuroD1 significantly reduced inflammation and reactive gliosis. Figure 25A shows focal ischemic stroke models were established in the M1 region of rats injected with 0.2 μg endothelin-1 (ET-1) . Ten days post stroke, 5 μL of either control virus (AAV9-hGFAP: : GFP, 1E11 vg/mL) or AAV-NeuroD1 (5E11 vg/mL) mixed with control virus (1E11 vg/mL) was injected into the same coordinates. Representative images show immunofluorescence staining for GFP (green) , astrocyte marker GFAP (red) , and microglia marker Iba-1 (purple) at different time points post-injection. Scale bar: 20 μm. Figure 25B and Figure 25C show quantification of GFAP and Iba-1 expression in a region 100 μm from the ischemic core. GFAP and Iba-1 levels were significantly elevated in the control group post-stroke compared to the uninjured region. At 40 dpi, GFAP and Iba-1 expression were significantly reduced in the AAV-NeuroD1-treated group compared to the control group (*p < 0.05, one-way ANOVA) .

Figures 26A and 26B provide results of axonal projection and neuronal distribution following AAV-NeuroD1 Treatment. Figure 26A shows focal ischemic stroke models were established in the M1 region of rats injected with 0.2 μg endothelin-1 (ET-1) . Ten days post stroke, 5 μL of either control virus (AAV9-hGFAP: : GFP, 1E11 vg/mL) or AAV-NeuroD1 (5E11 vg/mL) mixed with control virus (1E11 vg/mL) was injected into the same coordinates. GFP immunofluorescence staining (green) on sagittal brain sections at 40 dpi shows that in the control group, GFP expression is confined to the cortical injection site, with no GFP signal in deeper brain regions. In the AAV-NeuroD1 treated group, GFP-positive neurons and axonal projections are observed extending to multiple brain regions. Scale bar: 2 mm.Figure 26B shows Higher magnification images show detailed axonal projections from the newly generated neurons. Panel ① illustrates GFP-positive neuronal cell bodies and axons in the cortex. Panels ②, ③, and ④ show that the axonal projections from these neurons traverse through the striatum, thalamus, and hypothalamus, respectively. Scale bar: 100 μm.

Figure 27 provides magnetic resonance imaging (MRI) of the ischemic infarct over time in the NHP MCAO model. MRI horizontal scans of the monkey brains (Control animal C2, and C3, Treated animal T2 and T3) demonstrate pathophysiological changes including increased hemispheric swelling, midline shift, and hyperintense lesion at 7, 14, and 30 days after middle cerebral artery occlusion. Fluid-attenuated inversion recovery (FLAIR) axial images show hyperintense signal involving frontal and parietal lobes, as well as white matter of corona radiata. The reduction in the brain edema and infarct size in the first month post MCAO is associated with natural recovery of neurological deficits.

Figure 28 provides MRI-guided injection sites in the NHP cortex at two weeks post-stroke. T1-weighted coronal, sagittal and horizontal images show three injection sites (red dots) targeting peri-infarct cortical areas. R: right, A: anterior, S: sagittal

Figures 29A, 29B, and 29C show neurological function improvement after AAV-NeuroD1 treatment in MCAO NHP model. Figure 29A shows progressive NHPSS performance after MCAO in control and AAV-NeuroD1 treated animals. Both groups of animals showed spontaneous neurobehavioral recovery with the rapid improvement in the first two weeks and slow function improvement in the following two months post-stroke. While control group animals’ NHPSS score stopped improving around two months, AAV-NeuroD1 treated animals (T1, T2, T3 in red) exhibited improved NHPSS scores beyond that time point and showed nearly complete neurological function recovery. Figure 29B shows disability degree assessed with mRS scale showed moderately severe disability for animals in both groups after MCAO-induced ischemia. Interestingly, while control animals only improved by one point during the first month post stroke, treated animals achieved recovery with no significant disability after 6 months post treatment. Figure 29C shows motor subscores assessment measures the deficits in affected lower and upper extremity movement, grasp reflex and gait. After the first weeks of natural recovery, motor skill deficits in control group of animals plateau around 30 days post stroke, while AAV-NeuroD1 treated animals continued to show prolonged improvement.

Figures 30A and 30B show AAV-NeuroD1 drove consistent functional recovery in NHPs after MCAO-induced ischemic stroke. Figure 30A shows progressive NHPSS performance after MCAO in control and AAV-NeuroD1 treated animals. Both groups of animals showed spontaneous neurobehavioral recovery with the rapid improvement in the first two weeks and slow function improvement in the following two months post-stroke. Unlike control NHPs, AAV-NeuroD1 treated animals (red) exhibited improved NHPSS scores beyond that time point and showed nearly complete neurological function recovery. Figure 30B shows disability degree assessed with mRS scale showed moderately severe disability for animals in both groups after MCAO-induced ischemia. Interestingly, while control animals only improved by one point during the first month post stroke, treated animals achieved recovery with no significant disability after 6 months post treatment.

Figures 31A and 31B provide reconstitution of the NHP cortico-spinal tract after AAV-NeuroD1-mediated astrocyte to neuron conversion. Figure 31A shows schematic of corticospinal tract (CST) with the pyramidal neurons in the motor cortex and their descending axonal fibers in the brain stem and spinal cord. Immunohistochemical analysis shows GFP expressing reprogrammed neurons in the peri-infarct area of the motor cortex and their axonal projections along the CST. Figure 31B shows diffusion tensor imaging (DTI) and tractography reveals absence of fractional anisotropy (FA) due to severe brain edema at 7 days post MCAO. Interestingly, after 4-5 months post stroke, DTI results show axonal regrowth in AAV-NeuroD1 animals but not in control monkey. At 8 months post-MCAO, DTI of AAV-NeuroD1 animal shows nearly full restoration of the CST.

Figures 32A and 32B provide that AAV-NeuroD1 significantly reduced the neuroinflammation in the peri-infarct area following ischemic injury in NHP. Figure 32A shows representative images demonstrate a reduction of microglia (Iba1 positive) in the AAV-NeuroD1-infected peri-infarct areas following ischemic stroke, comparing to the control animal. Figure 32B shows quantification of the Iba1 intensity in AAV-NeuroD1 and control peri-infarct areas in animals injected at 14 days following ischemic injury. **p<0.01

Figure 33 provides AAV-NeuroD1 lead to astrocyte-to-neuron conversion in the peri-infarct area following ischemic injury in NHP. Representative images showing AAV-NeuroD1-mediated astrocyte-to-neuron conversion in the peri-infarct area of premotor cortex of NHPs injected with AAV-NeuroD1 mixed with AAV9-hGFAP: : GFP at 225 days post MCAO. Neuronal marker NeuN (red, A-A” and B-B” ) , axonal marker GFAP (red, C-C” ) , GFP (A-C” ) , and nuclear marker DAPI (A-C” ) . Yellow arrows (A-B” ) show the newly converted neurons that still express GFP indicating their astrocytic origin. Only a few astrocytes still express GFP (white arrows) , consistent with efficient astrocyte-to-neuron conversion.

Figure 34 shows the sequence alignment of NeuroD1 proteins from various species, including mouse (SEQ ID NO: 46) , zebrafish (SEQ ID NO: 47) , human (SEQ ID NO: 15) , rat (SEQ ID NO: 48) , chicken (SEQ ID NO: 49) , cattle (SEQ ID NO: 50) , hamster (SEQ ID NO: 51) , pig (SEQ ID NO: 52) , frog (SEQ ID NO: 53) , dog (SEQ ID NO: 54) , chimpanzee (SEQ ID NO: 55) and sheep (SEQ ID NO: 56) .

5. 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 also contemplate aspects of the disclosure described by the plural of that term. Where there are discrepancies in terms and definitions used in references that are incorporated by reference, the terms used in this application shall have the definitions given herein. Other technical terms used have their ordinary meaning in the art in which they are used, as exemplified by various art-specific dictionaries, for example, “The American Science Dictionary” (Editors of the American Heritage Dictionaries, 2011, Houghton Mifflin Harcourt, Boston and New York) , the “McGraw-Hill Dictionary of Scientific and Technical Terms” (6th edition, 2002, McGraw-Hill, New York) , or the “Oxford Dictionary of Biology” (6th edition, 2008, Oxford University Press, Oxford and New York) .

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

When a grouping of alternatives is presented, any and all combinations of the members that make up that grouping of alternatives is specifically envisioned. For example, if an item is selected from a group consisting of A, B, C, and D, the inventors specifically envision each alternative individually (e.g., A alone, B alone, etc. ) , as well as combinations such as A, B, and D; A and C; B and C; etc. The term “and/or” when used in a list of two or more items means any one of the listed items by itself or in combination with any one or more of the 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 and B in combination. The expression “A, B and/or C” is intended to mean A alone, B alone, C alone, A and B in combination, A and C in combination, B and C in combination, or A, B, and C in combination.

When a range of numbers is provided herein, the range is understood to be inclusive of the edges of the range as well as any number between the defined edges of the range. For example, “between 1 and 10” includes any number between 1 and 10, as well as the number 1 and the number 10.

When the term “about” is used in reference to a number, it is understood to mean plus or minus 10%. For example, “about 100” would include from 90 to 110. When “about” is used in reference to a numerical range, it is intended to be used to modify both the beginning and end values of the numerical range. For example, “about 1-10” means “about 1 to about 10. ”

As used herein, the term “NeuroD1 polypeptide” refers to NeuroD1 or a functional derivative of NeuroD1.

The term “neurogenic differentiation 1 protein” or “NeuroD1” as used herein, refers to any native NeuroD1 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats) , unless otherwise indicated. The term encompasses unprocessed NeuroD1 as well as any form of NeuroD1 that results from processing in the cell. The term also encompasses naturally occurring variants of NeuroD1, e.g., splice variants or allelic variants. The amino acid sequence of an exemplary human NeuroD1 is MTKSYSESGLMGEPQPQGPPSWTDECLSSQDEEHEADKKEDDLETMNAEEDSLRNGGEEEDEDEDLEEEEEEEEEDDDQKPKRRGPKKKKMTKARLERFKLRRMKANARERNRMHGLNAALDNLRKVVPCYSKTQKLSKIETLRLAKNYIWALSEILRSGKSPDLVSFVQTLCKGLSQPTTNLVAGCLQLNPRTFLPEQNQDMPPHLPTASASFPVHPYSYQSPGLPSPPYGTMDSSHVFHVKPPPHAYSAALEPFFESPLTDCTSPSFDGPLSPPLSINGNFSFKHEPSAEFEKNYAFTMHYPAATLAGAQSHGSIFSGTAAPRCEIPIDNIMSFDSHSHHERVMSAQLNAIFHD (SEQ ID NO: 15; GenBank Accession NP_002491.3) . A “full-length” NeuroD1 as used herein refers to the mature, natural length NeuroD1 molecule. For example, full-length human NeuroD1 refers to a molecule that has 356 amino acids (see e.g., SEQ ID NO: 1) . For example, a functional derivative of NeuroD1 is SEQ ID NO: 4, which has 357 amino acids.

An ortholog is a gene or genes that are related by vertical descent and are responsible for substantially the same or identical functions in different organisms. For example, mouse NeuroD1 and human NeuroD1 can be considered orthologs for the biological function of regulating neuronal differentiation and neurogenesis. See e.g., Cho, J. H. et al., Mol, Neurobiol., 30: 35-47, 2004; Kuwabara, T. et al., Nature Neurosci., 12: 1097-1105, 2009; and Gao, Z. et al., Nature Neurosci., 12: 1090-1092, 2009. Genes are related by vertical descent when, for example, they share sequence similarity of sufficient amount to indicate they are homologous, or related by evolution from a common ancestor. Genes can also be considered orthologs if they share three-dimensional structure but not necessarily sequence similarity, of a sufficient amount to indicate that they have evolved from a common ancestor to the extent that the primary sequence similarity is not identifiable. Genes that are orthologous can encode proteins with sequence similarity of about 25%to 100%amino acid sequence identity. Genes encoding proteins sharing an amino acid similarity less than 25%can also be considered to have arisen by vertical descent if their three-dimensional structure also shows similarities. Orthologs include genes or their encoded gene products that through, for example, evolution, have diverged in structure or overall activity. For example, where one species encodes a gene product exhibiting two functions and where such functions have been separated into distinct genes in a second species, the three genes and their corresponding products are considered to be orthologs. Those skilled in the art will understand how to identify orthologous genes harboring a biological function of interest. For example, a list of orthologous NeuroD1 genes and encoded NeuroD1 protein sequences can be found on GenBank website: www. ncbi. nlm. nih. gov/gene/4760/ortholog/? scope=89593&term=NEUROD1. Non-exhaustive examples of NeuroD1 proteins from various non-human organisms as identified by their respective GenBank accession numbers include Mus musculus (hose mouse) NP_035024.1, Danio rerio (zebrafish) NP_571053.1, Gallus gallus (chicken) NP_990251.2, Bos taurus (cattle) NP_001096758.1, Mesocricetus auratus (golden hamster) XP_005065174.1, Sus scrofa (pig) XP_020931169.1, Xenopus tropicalis (frog) NP_001090868.1, Canis lupus familiaris (dog) XP_005640434.2, Pan troglodytes (chimpanzee) XP_001158946.1, Ovis aries (sheep) XP_011987527.1. In some embodiments, A group orthologs genes encode protein products that can be considered functional derivatives of one another.

NeuroD1 is highly conserved in the vertebrate family. Figure 34 shows the sequence alignment of NeuroD1 proteins from various species, including mouse, zebrafish, human, rat, chicken, cattle, hamster, pig, frog, dog, chimpanzee and sheep. As shown, at least 95%amino acid residues in the NeuroD1 sequences are conserved across NeuroD1 orthologs from various species.

A “modification” of an amino acid residue/position refers to a change of a primary amino acid sequence as compared to a starting amino acid sequence, wherein the change results from a sequence alteration involving said amino acid residue/position. For example, typical modifications include substitution of the residue with another amino acid (e.g., a conservative or substantial substitution) , insertion of one or more (e.g., generally fewer than 5, 4, or 3) amino acids adjacent to said residue/position, and/or deletion of said residue/position.

Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been generally defined in the art, including basic side chains (e.g., lysine, arginine, histidine) , acidic side chains (e.g., aspartic acid, glutamic acid) , uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine) , nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) , beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine) . For example, substitution of a phenylalanine for a tyrosine is a conservative substitution. Alternatively, naturally occurring residues may be divided into groups based on common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe. Generally, conservative substitutions in the sequences of the peptides or polypeptides the disclosure do not abrogate the biological activity of interest of the peptide or polypeptide. Amino acid substitutions may be introduced into a polypeptide of interest and the products screened for a desired activity of interest, e.g., retained/improved ability of a NeuroD1 variant in producing one or more neuronal phenotypes in a glia cell, and methods for measuring such desired activity are well-known in the art.

In contrast, substantial modifications in the biological properties of a polypeptide are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.

In the context of a peptide or polypeptide, the term “derivative” as used herein refers to a peptide or polypeptide that comprises an amino acid sequence of the peptide or polypeptide, or a fragment of a peptide or polypeptide, which has been altered by the introduction of amino acid residue substitutions, deletions, or additions. The term “derivative” as used herein also refers to a peptide or polypeptide, or a fragment of a peptide or polypeptide, which has been chemically modified, e.g., by the covalent attachment of any type of molecule to the polypeptide. For example, but not by way of limitation, a peptide or polypeptide or a fragment of the peptide or polypeptide may be chemically modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, chemical cleavage, formulation, metabolic synthesis of tunicamycin, linkage to a cellular ligand or other protein, etc. The derivatives are modified in a manner that is different from naturally occurring or starting peptide or polypeptides, either in the type or location of the molecules attached. Derivatives further include deletion of one or more chemical groups which are naturally present on the peptide or polypeptide. Further, a derivative of a peptide or polypeptide or a fragment of a peptide or polypeptide may contain one or more non-classical amino acids. In specific embodiments, a derivative is a functional derivative of the native or unmodified peptide or polypeptide (e.g., a wild-type protein) from which it was derived. For example, a functional derivative of human NeuroD1 contains one or more modifications in its amino acid sequence with respect to the sequence shown in SEQ ID NO: 15. For example, in some embodiments, a functional derivative of human NeuroD1 comprises the amino acid sequence set forth in SEQ ID NO: 7 and 13.

The term “functional derivative” refers to a derivative that retains one or more functions or activities of the naturally occurring or starting peptide or polypeptide (e.g. a wild-type protein) from which it is derived. For example, in some embodiments, a functional derivative of a reprograming protein factor as described herein (e.g., NeuroD1) may retain the activity of producing a neuronal phenotype in a glial cell after being expressed in a sufficient amount by the glial cell. In some embodiments, a functional derivative of a reprogramming protein factor may retain the activity of the reprogramming protein factor in reprogramming the glial cell to trans-differentiate into a neuron after being expressed in a sufficient amount by the glial cell. In some embodiments, a functional derivative of a peptide or polypeptide described herein shares at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%sequence identity with respect to the starting (e.g., wild-type) peptide or polypeptide.

A derivative of polypeptide can be prepared using methods well-known in the art, e.g., by modifying the corresponding nucleic acid molecules encoding the derivative. For example, derivatives may be a substitution, deletion, or insertion of one or more codons encoding the polypeptide that results in a change in the amino acid sequence as compared with the wild-type sequence of the polypeptide. The derivatives can be made using methods well-known in the art such as DNA synthesis, oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis (see, e.g., Carter, 1986, Biochem J. 237: 1-7; and Zoller et al., 1982, Nucl. Acids Res. 10: 6487-500) , cassette mutagenesis (see, e.g., Wells et al., 1985, Gene 34: 315-23) , or other known techniques can be performed on the cloned DNA to produce the derivatives DNA.

Those skilled in the art can determine the site (s) in an amino acid sequence of a given protein, where a modification (s) can be made in order to produce functional derivatives. In some embodiments, a functional derivative of a polypeptide comprises one or more modifications to one or more predicted non-essential amino acid residues in its sequence. In some embodiments, modifications made to non-essential amino acid residues can be a conservative substation as described herein. In some embodiments, modifications made to non-essential amino acid residues can be a substantial substation described herein. In some embodiments, modifications made to non-essential amino acid residues can be a deletion of the non-essential amino acid residue. In alternative embodiments, one or more modifications can be made to one or more predicted essential amino acid residues in its sequence. In particularly embodiments, the modifications made to essential amino acid residues in a protein sequence can be a conservative substitution as described herein. Methods well-known in the art can be used to analyze a protein (e.g., NeuroD1) sequence to identify essential and non-essential amino acid residues of the protein. For example, in some embodiments, an amino acid residue of a protein that is not conserved among orthologous gene products is predicted to be a non-essential amino acid residue, while another amino acid residue that is conserved among orthologous gene products is predicted to be an essential amino acid residue. For example, an alignment of twelve NeuroD1 orthologs is shown in Figure 34, and the conserved residues and non-conserved residues are marked with different shades, respectively.

In some embodiments, after making one or more modifications to the sequence of a polypeptide (e.g., by making insertions, deletions, or substitutions of amino acids in the original amino acid sequence either systematically, randomly, or at selected sites) , functional derivatives of the polypeptide can be identified by testing the resulting derivatives for activity exhibited by the original sequence. For example, to identify functional derivative of a reprograming protein factor (e.g., NeuroD1) as described herein, nucleic acid molecules encoding the derivative polypeptides can be delivered into a population of starting glial cells under a suitable condition to be expressed at a sufficient level, and assays can be conducted to detect and/or measure one or more neuronal phenotypes in the population of cells and compared the level at which the neuronal phenotype of interest is demonstrated by the population of cells to a control group of glial cells that express the original, unmodified (e.g., wild-type) reprogramming protein factor, and those derivatives that induce the neuronal phenotype in the testing cell population at a comparable level to that of the control population can be selected as functional derivatives. Alternatively, the comparison can be made to a control group of glial cells that do not express the reprogramming protein factor (e.g. transduced with a blank vector) , and those derivatives that induce the neuronal phenotype in the testing cell population at a greater level than that of the control population can be selected as functional derivatives.

As used herein “hND1” refers to a human neuronal differentiation (NeuroD1) gene or protein.

As used herein “CE” refers to a cytomegalovirus (CMV) promoter enhancer sequence.

As used herein “GfaABC1D promoter” or “pGfa681 promoter” refers to a human glial fibrillary acid protein (GFAP) promoter truncated sequence of 681 bp size.

As used herein “CRGI” refers to a chimeric intron of rabbit beta-globing and chicken beta actin similar in CAG promoter.

As used herein “oWPRE” refers to an optimized version of a Woodchuck Hepatitis Virus (WHV) Posttranscriptional Regulatory Element (WPRE) .

As used herein “bGHpA” refers to a poly A signal of bovine growth hormone.

As used herein “vg” refers to a viral genome.

Any composition or vector provided herein is specifically envisioned for use with any method provided herein.

The term “vector” refers to a substance that is used to carry or include a nucleic acid sequence, including for example, a nucleic acid sequence encoding a peptide or protein as described herein, in order to introduce a nucleic acid sequence into a host cell, or serve as a transcription template to carry out in vitro transcription reaction in a cell-free system to produce mRNA. Vectors applicable for use include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which can include selection sequences or markers operable for stable integration into a host cell’s chromosome. Additionally, the vectors can include one or more selectable marker genes and appropriate transcription or translation control sequences. Selectable marker genes that can be included, for example, provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not in the culture media. Transcription or translation control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like, which are well known in the art. When two or more nucleic acid molecules are to be co-transcribed or co-translated (e.g., nucleic acid molecules encoding two or more different peptides or proteins) , both nucleic acid molecules can be inserted, for example, into a single expression vector or in separate expression vectors. For single vector transcription and/or translation, the encoding nucleic acids can be operationally linked to one common transcription or translation control sequence or linked to different transcription or translation control sequences, such as one inducible promoter and one constitutive promoter. The introduction of nucleic acid molecules into a host cell can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA, immunoblotting for expression of gene products, or other suitable analytical methods to test the expression of an introduced nucleic acid sequence or its corresponding gene product. It is understood by those skilled in the art that the nucleic acid molecules are expressed in a sufficient amount to produce a desired product (e.g., a mRNA transcript of the nucleic acid as described herein) , and it is further understood that expression levels can be optimized to obtain sufficient expression using methods well known in the art.

In an aspect, methods and compositions provided herein comprise a vector. In some embodiments, a vector can be a circular, double-stranded DNA molecule that is physically separate from chromosomal DNA. It should be noted that in some embodiments, the term “vector” can be used interchangeably with the term “plasmid. ”

In an aspect, a vector provided herein is a recombinant vector. As used herein, the term “recombinant vector” refers to a vector that comprises a recombinant nucleic acid. As used herein, a “recombinant nucleic acid” refers to a nucleic acid molecule formed by laboratory methods of genetic recombination, such as, without being limiting, molecular cloning. A recombinant vector can be formed by laboratory methods of genetic recombination, such as, without being limiting, molecular cloning. Also, without being limiting, one skilled in the art can create a recombinant vector de novo via synthesizing a plasmid by individual nucleotides, or by splicing together nucleic acid molecules from different pre-existing vectors.

Adeno-associated viruses (AAVs) are replication-defective, non-enveloped Dependoparvovirus viruses that infect humans and additional primate species. AAVs are not known to cause disease in any species, although they can cause mild immune responses. AAVs can infect dividing and quiescent cells. AAVs are stably integrated into the human genome at a specific site in chromosome 19 termed the AAVS1 locus (nucleotides 7774-11429 of GenBank Accession No. AC010327.8) , although random integrations at other loci in the human genome are possible.

AAVs comprise a linear genome with a single-stranded DNA of about 4700 nucleotides in length. The genome of AAVs also includes a 145 nucleotide-long inverted terminal repeat (ITR) at each end of the genome. The ITRs flank two viral genes rep (for replication, encoding non-structural proteins) and cap (for capsid, encoding structural proteins) . The ITRs contain all of the cis-acting elements needed for genome rescue, replication, and packaging of the AAV.

As used herein, an AAV can be derived from a naturally occurring “wild-type” virus, or a recombinant AAV (rAAV) that is derived from a naturally occurring AAV, but having all or part of the AAV genome replaced with heterologous nucleotide sequences (e.g., expression cassettes disclosed in Section 5.1 (NeuroD1 Expression Cassette) of the present disclosure comprising a coding sequence and regulatory elements) . In certain embodiments, the rAAV comprises an AAV genome (e.g., an artificial genome) in which part or all of the Rep (Replication) and/or Cap (Capsid) genes have been replaced with heterologous nucleotide sequences, such as a transgene. In the absence of Rep proteins, the heterologous nucleotide sequences encoded within the rAAV can persist as episomes in the nucleus of transduced cells and does not integrate into host genomes. In certain embodiments, the rAAV further comprises a capsid comprising capsid proteins encoded by a naturally occurring or non-naturally occurring Cap gene. In certain embodiments, the non-naturally occurring Cap gene encodes a capsid protein comprising an insertion, deletion, or modification of the amino acid sequence of the naturally occurring capsid protein. For example, a rAAV can have an artificial genome packaged in a capsid having a viral protein 1 (VP1) , viral protein 2 (VP2) , or viral protein 3 (VP3) , where the VP1 sequences is different from the wild-type sequence, while VP2 and VP3 both have wild-type sequences. As used herein, a rAAV that carries a heterologous transgene of interest in the genome is sometimes referred to as a “AAV vector. ” In some embodiments, an “AAV vector” refers to an AAV packaged with a DNA vector construct.

The term “rep-cap packaging plasmid” refers to a plasmid that provides the viral rep and cap gene function and aids the production of AAVs from artificial genomes lacking functional rep and/or cap gene sequences.

The term “cap gene” refers to the nucleic acid sequences that encode capsid proteins that form or help form the capsid of the virus. In some embodiments, in a recombinant AAV virion, the capsid protein contains VP1, VP2, and/or VP3.

The term “rep gene” refers to the nucleic acid sequences that encode the non-structural proteins needed for replication and production of virus.

As used herein, the term “AAV vector serotype” mainly refers to a variation within the capsid proteins of an AAV vector.

In an aspect, an 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, an AAV vector is selected from the group consisting of AAV serotype 2, AAV serotype 5, and AAV serotype 9. In one aspect, an AAV vector is AAV serotype 1. In one aspect, an AAV vector is AAV serotype 2. In one aspect, an AAV vector is AAV serotype 3. In one aspect, an AAV vector is AAV serotype 4. In one aspect, an AAV vector is AAV serotype 5. In one aspect, an AAV vector is AAV serotype 6. In one aspect, an AAV vector is AAV serotype 7. In one aspect, an AAV vector is AAV serotype 8. In one aspect, an AAV vector is AAV serotype 9. In one aspect, an AAV vector is AAV serotype 10. In one aspect, an AAV vector is AAV serotype 11. In one aspect, an AAV vector is AAV serotype 12. In some embodiments, a AAV vector is AAV serotypes AAV1, AAV2, AAV2tYF, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVrh10, AAV. rh20, AAV. rh39, AAV. Rh74, AAV. RHM4-1, AAV. hu37, AAV. Anc80, AAV. Anc80L65, rAAV. 7m8, AAV. PHP. B, AAV. PHP. eB, AAV2.5, AAV2tYF, AAV3B, AAV. LK03, AAV. HSC1, AAV. HSC2, AAV. HSC3, AAV. HSC4, AAV. HSC5, AAV. HSC6, AAV. HSC7, AAV. HSC8, AAV. HSC9, AAV. HSC10 , AAV. HSC11, AAV. HSC12, AAV. HSC13, AAV. HSC14, AAV. HSC15, and AAV. HSC16

In an aspect, an AAV vector ITR is selected from the group consisting of an AAV serotype 1 ITR, an AAV serotype 2 ITR, an AAV serotype 3 ITR, an AAV serotype 4 ITR, an AAV serotype 5 ITR, an AAV serotype 6 ITR, an AAV serotype 7 ITR, an AAV serotype 8 ITR, an AAV serotype 9 ITR, an AAV serotype 10 ITR, an AAV serotype 11 ITR, and an AAV serotype 12 ITR. In one aspect, an AAV vector ITR is an AAV serotype 1 ITR. In one aspect, an AAV vector ITR is an AAV serotype 2 ITR. In one aspect, an AAV vector ITR is an AAV serotype 3 ITR. In one aspect, an AAV vector ITR is an AAV serotype 4 ITR. In one aspect, an AAV vector ITR is an AAV serotype 5 ITR. In one aspect, an AAV vector ITR is an AAV serotype 6 ITR. In one aspect, an AAV vector ITR is an AAV serotype 7 ITR. In one aspect, an AAV vector ITR is an AAV serotype 8 ITR. In one aspect, an AAV vector ITR is an AAV serotype 9 ITR. In one aspect, an AAV vector ITR is an AAV serotype 10 ITR. In one aspect, an AAV vector ITR is an AAV serotype 11 ITR. In one aspect, an AAV vector ITR is an AAV serotype 12 ITR. In some embodiments, a AAV vector ITR is selected from the ITRs of AAV serotypes AAV1, AAV2, AAV2tYF, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVrh10, AAV. rh20, AAV. rh39, AAV. Rh74, AAV. RHM4-1, AAV. hu37, AAV. Anc80, AAV. Anc80L65, rAAV. 7m8, AAV. PHP. B, AAV. PHP. eB, AAV2.5, AAV2tYF, AAV3B, AAV. LK03, AAV. HSC1, AAV. HSC2, AAV. HSC3, AAV. HSC4, AAV. HSC5, AAV. HSC6, AAV. HSC7, AAV. HSC8, AAV. HSC9, AAV. HSC10 , AAV. HSC11, AAV. HSC12, AAV. HSC13, AAV. HSC14, AAV. HSC15, and AAV. HSC16

In an aspect, at least one AAV vector ITR nucleic acid sequence is selected from the group consisting of SEQ ID NOs: 1, 2, 5, 6, 16, and 23. In one aspect, at least one AAV vector ITR nucleic acid sequence is SEQ ID NO: 1. In one aspect, at least one AAV vector ITR nucleic acid sequence is SEQ ID NO: 2. In one aspect, at least one AAV vector ITR nucleic acid sequence is SEQ ID NO: 5. In one aspect, at least one AAV vector ITR nucleic acid sequence is SEQ ID NO: 1. In one aspect, at least one AAV vector ITR nucleic acid sequence is SEQ ID NO: 6. In one aspect, at least one AAV vector ITR nucleic acid sequence is SEQ ID NO: 16. In one aspect, at least one AAV vector ITR nucleic acid sequence is SEQ ID NO: 58. In one aspect, at least one AAV vector ITR nucleic acid sequence is SEQ ID NO: 23. In one aspect, at least one AAV vector ITR nucleic acid sequence is SEQ ID NO: 59.

In an aspect, an AAV ITR nucleic acid sequence comprises a sequence at least 70%identical to any one of SEQ ID NOs: 1, 2, 5, 6, 16, or 23 or the complement thereof. In one aspect, an AAV ITR nucleic acid sequence comprises a sequence at least 75%identical to any one of SEQ ID NOs: 1, 2, 5, 6, 16, or 23, or the complement thereof. In one aspect, an AAV ITR nucleic acid sequence comprises a sequence at least 80%identical to any one of SEQ ID NOs: 1, 2, 5, 6, 16, or 23, or the complement thereof. In one aspect, an AAV ITR nucleic acid sequence comprises a sequence at least 85%identical to any one of SEQ ID NOs: 1, 2, 5, 6, 16, or 23, or the complement thereof. In one aspect, an AAV ITR nucleic acid sequence comprises a sequence at least 90%identical to any one of SEQ ID NOs: 1, 2, 5, 6, 16, or 23, or the complement thereof. In one aspect, an AAV ITR nucleic acid sequence comprises a sequence at least 91%identical to any one of SEQ ID NOs: 1, 2, 5, 6, 16, or 23, or the complement thereof. In one aspect, an AAV ITR nucleic acid sequence comprises a sequence at least 92%identical to any one of SEQ ID NOs: 1, 2, 5, 6, 16, or 23, or the complement thereof. In one aspect, an AAV ITR nucleic acid sequence comprises a sequence at least 93%identical to any one of SEQ ID NOs: 1, 2, 5, 6, 16, or 23, or the complement thereof. In one aspect, an AAV ITR nucleic acid sequence comprises a sequence at least 94%identical to any one of SEQ ID NOs: 1, 2, 5, 6, 16, or 23, or the complement thereof. In one aspect, an AAV ITR nucleic acid sequence comprises a sequence at least 95%identical to any one of SEQ ID NOs: 1, 2, 5, 6, 16, or 23, or the complement thereof. In one aspect, an AAV ITR nucleic acid sequence comprises a sequence at least 96%identical to any one of SEQ ID NOs: 1, 2, 5, 6, 16, or 23, or the complement thereof. In one aspect, an AAV ITR nucleic acid sequence comprises a sequence at least 97%identical to any one of SEQ ID NOs: 1, 2, 5, 6, 16, or 23, or the complement thereof. In one aspect, an AAV ITR nucleic acid sequence comprises a sequence at least 98%identical to any one of SEQ ID NOs: 1, 2, 5, or 6, or the complement thereof. In one aspect, an AAV ITR nucleic acid sequence comprises a sequence at least 99%identical to any one of SEQ ID NOs: 1, 2, 5, 6, 16, or 23, or the complement thereof. In one aspect, an AAV ITR nucleic acid sequence comprises a sequence at least 99.5%identical to any one of SEQ ID NOs: 1, 2, 5, 6, 16, or 23, or the complement thereof. In one aspect, an AAV ITR nucleic acid sequence comprises a sequence at least 99.8%identical to any one of SEQ ID NOs: 1, 2, 5, 6, 16, or 23, or the complement thereof. In one aspect, an AAV ITR nucleic acid sequence comprises a sequence at least 99.9%identical to any one of SEQ ID NOs: 1, 2, 5, 6, 16, or 23, or the complement thereof. In one aspect, an AAV ITR nucleic acid sequence comprises a sequence 100%identical to any one of SEQ ID NOs: 1, 2, 5, 6, 16, or 23, or the complement thereof.

The terms “percent identity” or “percent identical” as used herein in reference to two or more nucleotide or amino acid sequences is calculated by (i) comparing two optimally aligned sequences (nucleotide or amino acid) over a window of comparison (the “alignable” region or regions) , (ii) determining the number of positions at which the identical nucleic acid base (for nucleotide sequences) or amino acid residue (for proteins and polypeptides) occurs in both sequences to yield the number of matched positions, (iii) dividing the number of matched positions by the total number of positions in the window of comparison, and then (iv) multiplying this quotient by 100%to yield the percent identity. If the “percent identity” is being calculated in relation to a reference sequence without a particular comparison window being specified, then the percent identity is determined by dividing the number of matched positions over the region of alignment by the total length of the reference sequence. Accordingly, for purposes of the present application, when two sequences (query and subject) are optimally aligned (with allowance for gaps in their alignment) , the “percent identity” for the query sequence is equal to the number of identical positions between the two sequences divided by the total number of positions in the query sequence over its length (or a comparison window) , which is then multiplied by 100%.

When percentage of sequence identity is used in reference to amino acids it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity can be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have “sequence similarity” or “similarity. ”

For optimal alignment of sequences to calculate their percent identity, various pair-wise or multiple sequence alignment algorithms and programs are known in the art, such as ClustalW or Basic Local Alignment Search (BLAST^TM) , etc., that can be used to compare the sequence identity or similarity between two or more nucleotide or amino acid sequences. 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 as determined by the ClustalW algorithm, see, e.g., Chenna et al., “Multiple sequence alignment with the Clustal series of programs, ” Nucleic Acids Research 31: 3497-3500 (2003) ; Thompson et al., “Clustal W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice, ” Nucleic Acids Research 22: 4673-4680 (1994) ; Larkin MA et al., “Clustal W and Clustal X version 2.0, ” Bioinformatics 23: 2947-48 (2007) ; and Altschul et al. “Basic local alignment search tool. ” J. Mol. Biol. 215: 403-410 (1990) , the entire contents and disclosures of which are incorporated herein by reference.

The terms “percent complementarity” or “percent complementary” as used herein in reference to two nucleotide sequences is similar to the concept of percent identity but refers to the percentage of nucleotides of a query sequence that optimally base-pair or hybridize to nucleotides a subject sequence when the query and subject sequences are linearly arranged and optimally base paired without secondary folding structures, such as loops, stems or hairpins. Such a percent complementarity can be between two DNA strands, two RNA strands, or a DNA strand and a RNA strand. The “percent complementarity” can be calculated by (i) optimally base-pairing or hybridizing the two nucleotide sequences in a linear and fully extended arrangement (i.e., without folding or secondary structures) over a window of comparison, (ii) determining the number of positions that base-pair between the two sequences over the window of comparison to yield the number of complementary positions, (iii) dividing the number of complementary positions by the total number of positions in the window of comparison, and (iv) multiplying this quotient by 100%to yield the percent complementarity of the two sequences. Optimal base pairing of two sequences can be determined based on the known pairings of nucleotide bases, such as G-C, A-T, and A-U, through hydrogen binding. If the “percent complementarity” is being calculated in relation to a reference sequence without specifying a particular comparison window, then the percent identity is determined by dividing the number of complementary positions between the two linear sequences by the total length of the reference sequence. Thus, for purposes of the present application, when two sequences (query and subject) are optimally base-paired (with allowance for mismatches or non-base-paired nucleotides) , the “percent complementarity” for the query sequence is equal to the number of base-paired positions between the two sequences divided by the total number of positions in the query sequence over its length, which is then multiplied by 100%.

The use of the term “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 envisioned. Those of ordinary skill in the art will recognize that polynucleotides and nucleic acid molecules can comprise ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogues. The polynucleotides of the present disclosure also encompass all forms of sequences including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-and-loop structures, and the like. In an aspect, a nucleic acid molecule provided herein is a DNA molecule. In one aspect, a nucleic acid molecule provided herein is an RNA molecule. In one aspect, a nucleic acid molecule provided herein is single-stranded. In one aspect, a nucleic acid molecule provided herein is double-stranded. A nucleic acid molecule can 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. Polypeptides can be encoded by polynucleotides provided herein. Proteins provided herein can be encoded by nucleic acid molecules provided herein. Proteins can comprise polypeptides provided herein. As used herein, a “protein” refers to a chain of amino acid residues that is capable of providing structure or enzymatic activity to a cell. As used herein, a “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 where a cytosine and guanine is separated by only one phosphate group.

As used herein, the term “CpG island” of “CG island” refers to CpG sites that occur with a high frequency.

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

Codon substitution or codon replacement in the context of codon optimization refer to replacing a codon present in a candidate nucleotide sequence (e.g., an mRNA encoding a therapeutic agent) with another codon. Thus, a codon can be substituted in a candidate nucleic acid sequence, for example, via chemical peptide synthesis or through recombinant methods known in the art. Accordingly, references to a “substitution” or “replacement” at a certain location in a nucleic acid sequence (e.g., an mRNA) or within a certain region or subsequence of a nucleic acid sequence (e.g., an mRNA) refer to the substitution of a codon at such location or region with an alternative codon. As used herein, the term “codon-optimized variant” refers to a synonymous nucleotide sequence that encodes the same polypeptide sequence encoded by a candidate nucleotide sequence (e.g., a nucleotide sequence encoding a NeuroD1 polypeptide) . Thus, there are no amino acid substitutions in the polypeptide encoded by the codon optimized nucleotide sequence with respect to the polypeptide encoded by the candidate nucleotide sequence. A candidate nucleic acid sequence can be codon-optimized by replacing all or part of its codons according to a substitution table map. According to the present disclosure, a candidate nucleotide sequence can be codon-optimized, for example, to improve its translation efficacy of the encoded polypeptide. In some embodiments, the candidate nucleotide sequence is codon-optimized for improved translation efficacy after in vivo administration, e.g., administration as part of a recombinant AAV virion.

As used herein, the term “enhancer” refers to a region of DNA sequence that operates to initiate, assist, affect, cause, and/or promote the transcription and expression of the associated transcribable DNA sequence or coding sequence, at least in certain tissue (s) , developmental stage (s) and/or condition (s) . In an aspect, an enhancer is a cis enhancer. In one aspect, an enhancer is a trans enhancer.

Enhancer sequences can be identified by utilizing genomic techniques well known in the art. Non-limiting examples include use of a reporter gene and next-generation sequencing methods such as chromatin immunoprecipitation sequencing (ChIP-seq) , DNase I hypersensitivity sequencing (DNase-seq) , micrococcal nuclease sequencing (MNase-seq) , formaldehyde-assisted isolation of regulatory elements sequencing (FAIRE-seq) , and assay for transposase accessible chromatin sequencing (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, etc., operates to initiate, assist, affect, cause, and/or promote the transcription and expression of the associated transcribable DNA sequence or coding sequence, at least in certain tissue (s) , developmental stage (s) and/or condition (s) . As used herein, “regulatory elements” refer to any sequence elements that regulate, positively or negatively, the expression of an operably linked sequence. “Regulatory elements” include, without being limiting, a promoter, an enhancer, a leader, a transcription start site (TSS) , a linker, 5’a nd 3’ untranslated regions (UTRs) , an intron, a polyadenylation signal, and a termination region or sequence, etc., that are suitable, necessary or preferred for regulating or allowing expression of the gene or transcribable DNA sequence in a cell. Such additional regulatory element (s) can 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 start site, and/or a TATA box and assists or promotes the transcription and expression of an associated transcribable polynucleotide sequence and/or gene (or transgene) . A promoter can be synthetically produced, varied, or derived from a known or naturally occurring promoter sequence or other promoter sequence. A promoter can also include a chimeric promoter comprising a combination of two or more heterologous sequences. A promoter of the present application can thus include variants of promoter sequences that are similar in composition, but not identical to, other promoter sequence (s) known or provided herein.

As used herein, an “intron” refers to a nucleotide sequence that is removed by RNA splicing as a messenger RNA (mRNA) matures from a mRNA precursor.

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

Expression of mRNA can be measured using any suitable method known in the art. Non-limiting examples of measuring mRNA expression include quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) , RNA blot (e.g., a Northern blot) , and RNA sequencing. Differences in expression can be described as an absolute quantification or a relative quantification. See, for example, Livak and Schmittgen, Methods, 25: 402-408 (2001) .

As used herein, the term “glial” or “glial cell” refers to a non-neuronal cell in the CNS or the PNS. In an aspect, a glial cell is selected from the group consisting of an oligodendrocyte, an astrocyte, an NG2 cell, an ependymal cell, and a microglia. In one aspect, a glial cell is an oligodendrocyte. In one aspect, a glial cell is an NG2 cell. In one aspect, a glial cell is an ependymal cell. In one aspect, a glial cell is a microglia. In one aspect, a glial cell is an astrocyte. In one aspect, a glial cell is a reactive astrocyte. In one aspect, an astrocyte comprises a reactive astrocyte.

As used herein, the term “astrocyte” refers to a glial cell that is an important component of the brain. An astrocyte is involved in supporting neuronal functions such as synapse formation and plasticity, potassium buffering, nutrient supply, the 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 status of astrocytes after injury or disease.

As used herein, the term “NG2 cell” or “polydendrocyte” refers to a glial cell that expresses chondroitin sulfate proteoglycan (CSPG4) and the alpha receptor for platelet-derived growth factor (PDGFRA) .

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

As used herein, the term “functional neuron” refers to a neuron that can perform biological process. Without being limiting, examples of biological processes include processing and transmission of information and communication via chemical and electrical synapses.

As used herein, the term “glutamatergic neurons” refers to a subclass of neurons that produce glutamate and establish excitatory synapses. As used herein, the term “excitatory synapse” refers to a synapse in which an action potential in a presynaptic neuron increases the probability of an action potential occurring in a postsynaptic cell. As used herein, the term “action potential” or “nerve impulse” refers to an electrical impulse across the membrane of an axon. As used herein, the term “axon” or “nerve fiber” refers to a neuron that conducts action potentials. As used herein, the term “GABAergic neurons” 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 ion channels to allow the flow of negatively charged chloride ions into the cell or positively charged potassium ions out of the cell. As used herein, the term “inhibitory synapse” refers to a synapse that moves the membrane potential of a postsynaptic neuron away from the threshold for generating action potentials. As used herein, the term “dopaminergic neuron” refers to a subset of neurons that produce dopamine. As used herein, the term “dopamine” refers to a type of neurotransmitter. As used herein, the term “neurotransmitter” refers to a class of endogenous chemicals that activate neurotransmissions. As used herein, the term “neurotransmission” refers to a process where neurotransmitters are released by the axon terminal of a neuron. As used herein, the term “acetyl cholinergic neuron” or “cholinergic neuron” refers to a subset of neurons that secrete acetylcholine. As used herein, the term “acetylcholine” refers to a type of neurotransmitter. As used herein, the term “seratonergic neuron” refers to a subset of neurons that synthesizes serotonin. As used herein, the term “serotonin” refers to a type of neurotransmitter. As used herein, an “epinephrinergic neuron” refers to a neuron that releases epinephrine as the neurotransmitter. As used herein, the term “motor neuron” refers to a subset of neurons where the cell body is located in the motor cortex, brainstem, or the spinal cord and the axon projects to the spinal cord or outside the spinal cord and directly or indirectly controls muscles and glands. As used herein, the term “peptidergic neuron” refers to a subset of neurons that utilize small peptide molecules as their neurotransmitter.

In an aspect, a neuron is a functional neuron. In one aspect, a functional neuron is selected from the group consisting of glutamatergic neurons, GABAergic neurons, dopaminergic neurons, cholinergic neurons, seratonergic neurons, epinephrinergic neurons, motor neurons, and peptidergic neurons. In one aspect, a functional neuron is a glutamatergic neuron. In one aspect, a functional neuron is a GABAergic neuron. In one aspect, a functional neuron is a dopaminergic neuron. In one aspect, a functional neuron is a cholinergic neuron. In one aspect, a functional neuron is a seratonergic neuron. In one aspect, a functional neuron is an epinephrinergic neuron. In one aspect, a functional neuron is a motor neuron. In one aspect, a functional neuron is a peptidergic neuron.

As used herein, the term “converting” or “converted” refers to a cell type changing its physical morphology and/or biological function into a different physical morphology and/or different biological function. In an aspect, this disclosure provides the conversion of at least one glial cell into at least one neuron. In one aspect, conversion of at least one glial cell to at least one neuron occurs in the CNS or PNS. In one aspect, conversion of at least one glial cell to at least one neuron occurs in the CNS. In one aspect, conversion of at least one glial cell to at least one neuron occurs in the PNS. In one aspect, a glial cell is converted into a neuron after the glial cell is exposed to NeuroD1. In one aspect, a glial cell is converted into a neuron after it has been transduced with a vector encoding NeuroD1. In one aspect, a glial cell is converted into a neuron after it has been induced to express NeuroD1. In one aspect, the glial cell that is converted into a neuron is an astrocyte or a reactive astrocyte.

In an aspect, the present disclosure provides, and includes, methods of treating stroke in a subject. In an aspect, the present disclosure provides, and includes, methods of treating stroke in a subject by converting glial cells into neurons. In an aspect, the present disclosure provides, and includes, methods of treating stroke in a subject by converting glial cells into neurons via the expression of NeuroD1 in the glial cells.

In an aspect, the present disclosure provides, and includes, methods of generating new neurons in the brain of a subject who has suffered a stroke. In an aspect, the present disclosure provides, and includes, methods of generating new neurons in the brain of a subject who has suffered a stroke by converting glial cells into neurons. In an aspect, the present disclosure provides, and includes, methods of generating new neurons in the brain of a subject who has suffered a stroke by converting glial cells into neurons via the expression of NeuroD1 in the glial cells. In an aspect, new neurons are generated in the brain of the subject who has suffered a stroke within 14 to 28 days after the subject is administered a composition of the present disclosure. In an aspect, new neurons are generated in the brain of the subject who has suffered a stroke within 14 to 28 days after the subject is administered a composition comprising an AAV encoding NeuroD1. In an aspect, new neurons are generated in the brain of the subject who has suffered a stroke within 21 to 28 days after the subject is administered a composition comprising an AAV encoding NeuroD1. In an aspect, new neurons are generated in the brain of the subject who has suffered a stroke within 14 to 21 days after the subject is administered a composition comprising an AAV encoding NeuroD1.

In an aspect, the present disclosure provides, and includes, methods of partially or fully restoring neuronal pathways in the brain of a subject who has suffered a stroke. In an aspect, the present disclosure provides, and includes, methods of partially or fully restoring neuronal pathways in the brain of a subject who has suffered a stroke by converting glial cells into neurons. In an aspect, the present disclosure provides, and includes, methods of partially or fully restoring neuronal pathways in the brain of a subject who has suffered a stroke by converting glial cells into neurons via the expression of NeuroD1 in the glial cells. In an aspect, the partial or full restoration of the neuronal pathways in the brain of the subject can be assessed by MRI. In an aspect, the partial or full restoration of the neuronal pathways in the brain of the subject can be assessed by Diffusion Tensor Imaging (DTI) . In an aspect, the neuronal pathways are partially or fully restored within three to six months after the subject who has suffered a stroke is administered a composition of the present disclosure. In an aspect, the neuronal pathways are partially or fully restored within three to six months after the subject who has suffered a stroke is administered a composition comprising an AAV encoding NeuroD1. In an aspect, the neuronal pathways are partially or fully restored within four to six months after the subject who has suffered a stroke is administered a composition comprising an AAV encoding NeuroD1. In an aspect, the neuronal pathways are partially or fully restored within five to six months after the subject who has suffered a stroke is administered a composition comprising an AAV encoding NeuroD1. In an aspect, the neuronal pathways are partially or fully restored within three to five months after the subject who has suffered a stroke is administered a composition comprising an AAV encoding NeuroD1. In an aspect, the neuronal pathways are partially or fully restored within three to four months after the subject who has suffered a stroke is administered a composition comprising an AAV encoding NeuroD1.

In an aspect, the present disclosure provides, and includes, methods of reducing neuroinflammation in the brain of a subject who has suffered a stroke. In an aspect, the present disclosure provides, and includes, methods of reducing neuroinflammation in the brain of a subject who has suffered a stroke by converting glial cells into neurons. In an aspect, the present disclosure provides, and includes, methods of reducing neuroinflammation in the brain of a subject who has suffered a stroke by converting glial cells into neurons via the expression of NeuroD1 in the glial cells. In an aspect, reduction in neuroinflammation is determined by measuring the expression of Iba1 in a region of the brain of the subject. In an aspect, reduction in neuroinflammation is determined by measuring the abundance of microglia in a region of the brain of the subject. In an aspect, neuroinflammation is reduced in the brain of the subject who has suffered a stroke within 14 to 21 days after the subject is administered a composition of the present disclosure. In an aspect, neuroinflammation is reduced in the brain of the subject who has suffered a stroke within 14 to 21 days after the subject is administered a composition comprising an AAV encoding NeuroD1.

In an aspect, this disclosure provides, and includes, an adeno-associated virus (AAV) vector comprising a human neurogenic differentiation 1 (hNeuroD1) sequence comprising the nucleic acid sequence of SEQ ID NO: 3, where the hNeuroD1 sequence is operably linked to regulatory elements comprising: (a) a glial fibrillary acid protein (GFAP) promoter comprising the nucleic acid sequence of SEQ ID NO: 10; (b) a cytomegalovirus (CMV) enhancer comprising the nucleic acid sequence of SEQ ID NO: 8; (c) a chimeric intron comprising the nucleic acid sequence of SEQ ID NO: 11; (d) an optimized woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) comprising the nucleic acid sequence of SEQ ID NO: 12; and (e) a bGH polyadenylation sequence comprising the nucleic acid sequence of SEQ ID NO: 9.

In an aspect, this disclosure provides, and includes, an adeno-associated virus (AAV) vector comprising a human neurogenic differentiation 1 (hNeuroD1) sequence comprising the nucleic acid sequence of SEQ ID NO: 4, where the hNeuroD1 sequence is operably linked to regulatory elements comprising: (a) a glial fibrillary acid protein (GFAP) promoter comprising the nucleic acid sequence of SEQ ID NO: 10; (b) a cytomegalovirus (CMV) enhancer comprising the nucleic acid sequence of SEQ ID NO: 8; (c) a chimeric intron comprising the nucleic acid sequence of SEQ ID NO: 11; (d) an optimized woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) comprising the nucleic acid sequence of SEQ ID NO: 12; and (e) a bGH polyadenylation sequence comprising the nucleic acid sequence of SEQ ID NO: 9.

In an aspect, this disclosure provides, and includes, an adeno-associated virus (AAV) vector comprising a neurogenic differentiation 1 (NeuroD1) nucleic acid coding sequence encoding a NeuroD1 protein, where the coding sequence is operably linked to regulatory elements comprising: (a) a glial fibrillary acid protein (GFAP) promoter; (b) an enhancer; (c) a chimeric intron; (d) an optimized woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) ; and (e) a polyadenylation signal sequence.

In an aspect, this disclosure provides, and includes, an adeno-associated virus (AAV) vector comprising a human neurogenic differentiation 1 (hNeuroD1) sequence operably linked to regulatory elements comprising: (a) a glial fibrillary acid protein (GFAP) promoter; (b) a cytomegalovirus (CMV) enhancer; (c) a chimeric intron; (d) an optimized woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) ; and (e) a bGH polyadenylation sequence.

In an aspect, this disclosure provides, and includes, an adeno-associated viral (AAV) vector comprising a nucleic acid coding sequence encoding a human neurogenic differentiation 1 (hNeuroD1) protein comprising the amino acid sequence of SEQ ID NO: 7, wherein the coding sequence is operably linked to regulatory elements comprising: (a) a glial fibrillary acid protein (GFAP) promoter comprising the nucleic acid sequence of SEQ ID NO: 10; (b) a cytomegalovirus (CMV) enhancer comprising the nucleic acid sequence of SEQ ID NO: 8; (c) a chimeric intron comprising the nucleic acid sequence of SEQ ID NO: 11; (d) an optimized woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) comprising the nucleic acid sequence of SEQ ID NO: 12; and (e) a bGH polyadenylation sequence comprising the nucleic acid sequence of SEQ ID NO: 9.

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

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

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

It is appreciated in the art that it is possible that a fragment of a protein can retain the function, or part of the function, of the full length protein. For example, without being limiting, if a fragment of a NeuroD1 protein or polypeptide retained at least part of the function of a full length NeuroD1 protein or polypeptide, then such fragment can be referred to as a “functional fragment. ”

In an aspect, a NeuroD1 nucleic acid sequence comprises a sequence at least 70%identical to SEQ ID NO: 3, or the complement thereof. In one aspect, a NeuroD1 nucleic acid sequence comprises a sequence at least 75%identical to SEQ ID NO: 3, or the complement thereof. In one aspect, a NeuroD1 nucleic acid sequence comprises a sequence at least 80%identical to SEQ ID NO: 3, or the complement thereof. In one aspect, a NeuroD1 nucleic acid sequence comprises a sequence at least 85%identical to SEQ ID NO: 3, or the complement thereof. In one aspect, a NeuroD1 nucleic acid sequence comprises a sequence at least 90%identical to SEQ ID NO: 3, or the complement thereof. In one aspect, a NeuroD1 nucleic acid sequence comprises a sequence at least 91%identical to SEQ ID NO: 3, or the complement thereof. In one aspect, a NeuroD1 nucleic acid sequence comprises a sequence at least 92%identical to SEQ ID NO: 3, or the complement thereof. In one aspect, a NeuroD1 nucleic acid sequence comprises a sequence at least 93%identical to SEQ ID NO: 3, or the complement thereof. In one aspect, a NeuroD1 nucleic acid sequence comprises a sequence at least 94%identical to SEQ ID NO: 3, or the complement thereof. In one aspect, a NeuroD1 nucleic acid sequence comprises a sequence at least 95%identical to SEQ ID NO: 3, or the complement thereof. In one aspect, a NeuroD1 nucleic acid sequence comprises a sequence at least 96%identical to SEQ ID NO: 3, or the complement thereof. In one aspect, a NeuroD1 nucleic acid sequence comprises a sequence at least 97%identical to SEQ ID NO: 3, or the complement thereof. In one aspect, a NeuroD1 nucleic acid sequence comprises a sequence at least 98%identical to SEQ ID NO: 3, or the complement thereof. In one aspect, a NeuroD1 nucleic acid sequence comprises a sequence at least 99%identical to SEQ ID NO: 3, or the complement thereof. In one aspect, a NeuroD1 nucleic acid sequence comprises a sequence at least 99.5%identical to SEQ ID NO: 3, or the complement thereof. In one aspect, a NeuroD1 nucleic acid sequence comprises a sequence at least 99.8%identical to SEQ ID NO: 3, or the complement thereof. In one aspect, a NeuroD1 nucleic acid sequence comprises a sequence at least 99.9%identical to SEQ ID NO: 3, or the complement thereof. In one aspect, a NeuroD1 nucleic acid sequence comprises a sequence 100%identical to SEQ ID NO: 3, or the complement thereof.

In an aspect, a NeuroD1 nucleic acid sequence comprises a sequence at least 70%identical to SEQ ID NO: 4, or the complement thereof. In one aspect, a NeuroD1 nucleic acid sequence comprises a sequence at least 75%identical to SEQ ID NO: 4, or the complement thereof. In one aspect, a NeuroD1 nucleic acid sequence comprises a sequence at least 80%identical to SEQ ID NO: 4, or the complement thereof. In one aspect, a NeuroD1 nucleic acid sequence comprises a sequence at least 85%identical to SEQ ID NO: 4, or the complement thereof. In one aspect, a NeuroD1 nucleic acid sequence comprises a sequence at least 90%identical to SEQ ID NO: 4, or the complement thereof. In one aspect, a NeuroD1 nucleic acid sequence comprises a sequence at least 91%identical to SEQ ID NO: 4, or the complement thereof. In one aspect, a NeuroD1 nucleic acid sequence comprises a sequence at least 92%identical to SEQ ID NO: 4, or the complement thereof. In one aspect, a NeuroD1 nucleic acid sequence comprises a sequence at least 93%identical to SEQ ID NO: 4, or the complement thereof. In one aspect, a NeuroD1 nucleic acid sequence comprises a sequence at least 94%identical to SEQ ID NO: 4, or the complement thereof. In one aspect, a NeuroD1 nucleic acid sequence comprises a sequence at least 95%identical to SEQ ID NO: 4, or the complement thereof. In one aspect, a NeuroD1 nucleic acid sequence comprises a sequence at least 96%identical to SEQ ID NO: 4, or the complement thereof. In one aspect, a NeuroD1 nucleic acid sequence comprises a sequence at least 97%identical to SEQ ID NO: 4, or the complement thereof. In one aspect, a NeuroD1 nucleic acid sequence comprises a sequence at least 98%identical to SEQ ID NO: 4, or the complement thereof. In one aspect, a NeuroD1 nucleic acid sequence comprises a sequence at least 99%identical to SEQ ID NO: 4, or the complement thereof. In one aspect, a NeuroD1 nucleic acid sequence comprises a sequence at least 99.5%identical to SEQ ID NO: 4, or the complement thereof. In one aspect, a NeuroD1 nucleic acid sequence comprises a sequence at least 99.8%identical to SEQ ID NO: 4, or the complement thereof. In one aspect, a NeuroD1 nucleic acid sequence comprises a sequence at least 99.9%identical to SEQ ID NO: 4, or the complement thereof. In one aspect, a NeuroD1 nucleic acid sequence comprises a sequence 100%identical to SEQ ID NO: 4, or the complement thereof.

In an aspect, a nucleic acid coding sequence encodes a NeuroD1 protein comprising an amino acid sequence at least 70%identical or similar to SEQ ID NO: 7. In one aspect, a nucleic acid coding sequence encodes a NeuroD1 protein comprising an amino acid sequence at least 75%identical or similar to SEQ ID NO: 7. In one aspect, a nucleic acid coding sequence encodes a NeuroD1 protein comprising an amino acid sequence at least 80%identical or similar to SEQ ID NO: 7. In one aspect, a nucleic acid coding sequence encodes a NeuroD1 protein comprising an amino acid sequence at least 85%identical or similar to SEQ ID NO: 7. In one aspect, a nucleic acid coding sequence encodes a NeuroD1 protein comprising an amino acid sequence at least 90%identical or similar to SEQ ID NO: 7. In one aspect, a nucleic acid coding sequence encodes a NeuroD1 protein comprising an amino acid sequence at least 91%identical or similar to SEQ ID NO: 7. In one aspect, a nucleic acid coding sequence encodes a NeuroD1 protein comprising an amino acid sequence at least 92%identical or similar to SEQ ID NO: 7. In one aspect, a nucleic acid coding sequence encodes a NeuroD1 protein comprising an amino acid sequence at least 93%identical or similar to SEQ ID NO: 7. In one aspect, a nucleic acid coding sequence encodes a NeuroD1 protein comprising an amino acid sequence at least 94%identical or similar to SEQ ID NO: 7. In one aspect, a nucleic acid coding sequence encodes a NeuroD1 protein comprising an amino acid sequence at least 95%identical or similar to SEQ ID NO: 7. In one aspect, a nucleic acid coding sequence encodes a NeuroD1 protein comprising an amino acid sequence at least 96%identical or similar to SEQ ID NO: 7. In one aspect, a nucleic acid coding sequence encodes a NeuroD1 protein comprising an amino acid sequence at least 97%identical or similar to SEQ ID NO: 7. In one aspect, a nucleic acid coding sequence encodes a NeuroD1 protein comprising an amino acid sequence at least 98%identical or similar to SEQ ID NO: 7. In one aspect, a nucleic acid coding sequence encodes a NeuroD1 protein comprising an amino acid sequence at least 99%identical or similar to SEQ ID NO: 7. In one aspect, a nucleic acid coding sequence encodes a NeuroD1 protein comprising an amino acid sequence at least 99.5%identical or similar to SEQ ID NO: 7. In one aspect, a nucleic acid coding sequence encodes a NeuroD1 protein comprising an amino acid sequence at least 99.8%identical or similar to SEQ ID NO: 7. In one aspect, a nucleic acid coding sequence encodes a NeuroD1 protein comprising an amino acid sequence at least 99.9%identical or similar to SEQ ID NO: 7. In one aspect, a nucleic acid coding sequence encodes a NeuroD1 protein comprising an amino acid sequence 100%identical or similar to SEQ ID NO: 7.

Glial fibrillary acid protein (GFAP) , also referred to as glial fibrillary acidic protein, is a member of the type III intermediate filament family of proteins that is expressed in the central nervous system and plays a role in cell communication and the functioning of the blood–brain barrier.

In an aspect, the promoter is selected from the group consisting of GFAP promoter, Sox9 promoter, S100b promoter, Aldh1l1 promoter, Lipocalin 2 (Lcn2) promoter, glutamine synthetase promoter, Aquaporin-4 (AQP4) promoter, oligodendrocyte transcription factor (Olig2) promoter, synapsin promoter, NG2 promoter, ionized calcium binding adaptor molecule 1 (Iba1) promoter, cluster of differentiation 86 (CD86) promoter, platelet-derived growth factor receptor alpha (PDGFRA) promoter, platelet-derived growth factor receptor beta (PDGFRB) promoter, elongation factor 1-alpha (EF1a) promoter, CAG promoter, cytomegalovirus (CMV) promoter, ubiquitin promoter. In one aspect, the promoter is a GFAP promoter. In one aspect, the promoter is a truncated GFAP promoter. In one aspect, the promoter is a Sox9 promoter. In one aspect, the promoter is an S100b promoter. In one aspect, the promoter is an Aldhl1l 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 an 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 an 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 an aspect, a GFAP promoter is a promoter directing astrocyte-specific expression of a protein called glial fibrillary acidic protein (GFAP) in cells. In one aspect, a GFAP promoter sequence is a human GFAP (hGFAP) promoter sequence. In one aspect, a GFAP promoter comprises a GfaABC1D promoter (also called pGfa681 promoter) . In one aspect, GfaABC1D promoter comprises SEQ ID NO: 10.

In one aspect, a GFAP promoter sequence is a non-human primate GFAP promoter sequence. In one aspect, a GFAP promoter sequence is a cynomolgus monkey GFAP promoter sequence. In one aspect, a GFAP promoter sequence is selected from the group consisting of a cynomolgus monkey GFAP promoter sequence, a chimpanzee GFAP promoter sequence, a bonobo GFAP promoter sequence, an orangutan GFAP promoter sequence, a gorilla GFAP promoter sequence, a macaque GFAP promoter sequence, a marmoset GFAP promoter sequence, a capuchin GFAP promoter sequence, a baboon GFAP promoter sequence, a gibbon GFAP promoter sequence, and a lemur GFAP promoter sequence. In one aspect, a GFAP promoter sequence is a chimpanzee GFAP promoter sequence. In one aspect, a GFAP promoter sequence is a bonobo GFAP promoter sequence. In one aspect, a GFAP promoter sequence is an orangutan GFAP promoter sequence. In one aspect, a GFAP promoter sequence is a gorilla GFAP promoter sequence. In one aspect, a GFAP promoter sequence is a macaque GFAP promoter sequence. In one aspect, a GFAP promoter sequence is a marmoset GFAP promoter sequence. In one aspect, a GFAP promoter sequence is a capuchin GFAP promoter sequence. In one aspect, a GFAP promoter sequence is a baboon GFAP promoter sequence. In one aspect, a GFAP promoter sequence is a gibbon GFAP promoter sequence. In one aspect, a GFAP promoter sequence is a lemur GFAP promoter sequence.

In an aspect, a GFAP promoter sequence comprises at least 100 nucleotides. In one aspect, a GFAP promoter comprises at least 500 nucleotides. In a further aspect, a GFAP promoter comprises at least 1000 nucleotides. In still another aspect, a GFAP promoter comprises at least 1500 nucleotides. In one aspect, a GFAP promoter comprises about 681 nucleotides. In one aspect, a GFAP promoter comprises 681 nucleotides.

It is appreciated in the art that a fragment of a promoter sequence can function to drive transcription of an operably linked nucleic acid molecule. For example, without being limiting, if a 1000 nucleotides promoter is truncated to 500 nucleotides, and the 500 nucleotides fragment is capable of driving transcription, the 500 nucleotides fragment is referred to as a “functional fragment. ”

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

In an aspect, a GFAP promoter nucleic acid sequence comprises a sequence at least 70%identical to the sequence of SEQ ID NO: 10, or a functional fragment thereof. In one aspect, a GFAP promoter nucleic acid sequence comprises a sequence at least 75%identical to the sequence of SEQ ID NO: 10 or a functional fragment thereof. In one aspect, a GFAP promoter nucleic acid sequence comprises a sequence at least 80%identical to the sequence of SEQ ID NO: 10 or a functional fragment thereof. In one aspect, a GFAP promoter nucleic acid sequence comprises a sequence at least 85%identical to the sequence of SEQ ID NO: 10 or a functional fragment thereof. In one aspect, a GFAP promoter nucleic acid sequence comprises a sequence at least 90%identical to the sequence of SEQ ID NO: 10 or a functional fragment thereof. In one aspect, a GFAP promoter nucleic acid sequence comprises a sequence at least 91%identical to the sequence of SEQ ID NO: 10 or a functional fragment thereof. In one aspect, a GFAP promoter nucleic acid sequence comprises a sequence at least 92%identical the sequence of SEQ ID NO: 10 or a functional fragment thereof. In one aspect, a GFAP promoter nucleic acid sequence comprises a sequence at least 93%identical to the sequence of SEQ ID NO: 10 or a functional fragment thereof. In one aspect, a GFAP promoter nucleic acid sequence comprises a sequence at least 94%identical to the sequence of SEQ ID NO: 10 or a functional fragment thereof. In one aspect, a GFAP promoter nucleic acid sequence comprises a sequence at least 95%identical to the sequence of SEQ ID NO: 10 or a functional fragment thereof. In one aspect, a GFAP promoter nucleic acid sequence comprises a sequence at least 96%identical to the sequence of SEQ ID NO: 10 or a functional fragment thereof. In one aspect, a GFAP promoter nucleic acid sequence comprises a sequence at least 97%identical to the sequence of SEQ ID NO: 10 or a functional fragment thereof. In one aspect, a GFAP promoter nucleic acid sequence comprises a sequence at least 98%identical to the sequence of SEQ ID NO: 10 or a functional fragment thereof. In one aspect, a GFAP promoter nucleic acid sequence comprises a sequence at least 99%identical to the sequence of SEQ ID NO: 10 or a functional fragment thereof. In one aspect, a GFAP promoter nucleic acid sequence comprises a sequence at least 99.5%identical to the sequence of SEQ ID NO: 10 or a functional fragment thereof. In one aspect, a GFAP promoter nucleic acid sequence comprises a sequence at least 99.8%identical to the sequence of SEQ ID NO: 10 or a functional fragment thereof. In one aspect, a GFAP promoter nucleic acid sequence comprises a sequence at least 99.9%identical to the sequence of SEQ ID NO: 10 or a functional fragment thereof. In one aspect, a GFAP promoter nucleic acid sequence comprises a sequence 100%identical to the sequence of SEQ ID NO: 10 or a functional fragment thereof.

In an aspect, a nucleic acid sequence as provided herein is codon optimized.

In an aspect, a nucleic acid sequence as provided herein is CpG site depleted.

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

As used herein, the term “cerebral cortex” refers to the outer layer of neural tissue of the cerebrum.

As used herein, the term “striatum” or “corpus striatum” refers to a cluster of neurons in the subcortical basal ganglia of the forebrain and comprises the ventral striatum and dorsal striatum.

As used herein, the term “substantia nigra” refers to a cluster of neurons in the subcortical basal ganglia of the midbrain and comprises the pars compacta and the pars reticulata.

As used herein, the term “forebrain” refers to the forward-most portion of the brain.

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

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

As used herein, the term “subcortical basal ganglia” refers to a cluster of neurons in the deep cerebral hemispheres of the brain.

As used herein, the term “spinal cord” refers to a structure that functions in the transmission of nerve signals from the motor cortex to the body.

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

In one aspect, a method provided herein converts glial cells to functional neurons in the brain. In an aspect, a method provided herein converts glial cells to functional neurons in a cerebral cortex of the brain. In one aspect, a method provided herein converts glial cells to functional neurons in a striatum of the brain. In one aspect, a method provided herein converts glial cells to functional neurons in a dorsal striatum of the brain. In one aspect, a method provided herein converts glial cells to functional neurons in a spinal cord of the brain. In one aspect, a method provided herein converts glial cells to functional neurons in a putamen of the brain. In one aspect, a method provided herein converts glial cells to functional neurons in a caudate nucleus of the brain. In one aspect, a method provided herein converts glial cells to functional neurons in a substantia nigra of the brain. In one aspect, a method provided herein converts glial cells to functional neurons in the primary motor cortex. In one aspect, newly formed neurons in the primary motor cortex send axons to appropriate targets along the corticospinal tract (e.g., the striatum and the brainstem) .

In one aspect, a method provided herein converts astrocytes to functional neurons in the brain. In an aspect, a method provided herein converts astrocytes to functional neurons in a cerebral cortex of the brain. In one aspect, a method provided herein converts astrocytes to functional neurons in a striatum of the brain. In one aspect, a method provided herein converts astrocytes to functional neurons in a dorsal striatum of the brain. In one aspect, a method provided herein converts astrocytes to functional neurons in a spinal cord of the brain. In one aspect, a method provided herein converts astrocytes to functional neurons in a putamen of the brain. In one aspect, a method provided herein converts astrocytes to functional neurons in a caudate nucleus of the brain. In one aspect, a method provided herein converts astrocytes to functional neurons in a substantia nigra of the brain. In one aspect, a method provided herein converts astrocytes to functional neurons in the primary motor cortex. In one aspect, newly formed neurons in the primary motor cortex send axons to appropriate targets along the corticospinal tract (e.g., the striatum and the brainstem) .

In one aspect, a method provided herein converts reactive astrocytes to functional neurons in the brain. In an aspect, a method provided herein converts reactive astrocytes to functional neurons in a cerebral cortex of the brain. In one aspect, a method provided herein converts reactive astrocytes to functional neurons in a striatum of the brain. In one aspect, a method provided herein converts reactive astrocytes to functional neurons in a dorsal striatum of the brain. In one aspect, a method provided herein converts reactive astrocytes to functional neurons in a spinal cord of the brain. In one aspect, a method provided herein converts reactive astrocytes to functional neurons in a putamen of the brain. In one aspect, a method provided herein converts reactive astrocytes to functional neurons in a caudate nucleus of the brain. In one aspect, a method provided herein converts reactive astrocytes to functional neurons in a substantia nigra of the brain. In one aspect, a method provided herein converts reactive astrocytes to functional neurons in the primary motor cortex. In one aspect, newly formed neurons in the primary motor cortex send axons to appropriate targets along the corticospinal tract (e.g., the striatum and the brainstem) .

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

In an aspect, an enhancer sequence from the CMV, or a CMV enhancer (CE) , is a human enhancer sequence from the CMV. In an aspect, an enhancer sequence from the CMV is a non-human primate enhancer sequence from the CMV. In an aspect, an enhancer sequence from the CMV is a cynomolgus monkey enhancer sequence from the CMV. In one aspect, an enhancer sequence from the CMV is selected form the group consisting of a chimpanzee enhancer sequence from the CMV, a bonobo enhancer sequence from the CMV, an orangutan enhancer sequence from the CMV, a gorilla enhancer sequence from the CMV, a macaque enhancer sequence from the CMV, a marmoset enhancer sequence from the CMV, a capuchin enhancer sequence from the CMV, a baboon enhancer sequence from the CMV, a gibbon enhancer sequence from the CMV, and a lemur enhancer sequence from the CMV. In one aspect, an enhancer sequence from the CMV is a chimpanzee enhancer sequence from the CMV. In one aspect, an enhancer sequence from the CMV is a bonobo enhancer sequence from the CMV. In one aspect, an enhancer sequence from the CMV is an orangutan enhancer sequence from the CMV. In one aspect, an enhancer sequence from the CMV is a gorilla enhancer sequence from the CMV. In one aspect, an enhancer sequence from the CMV is a macaque enhancer sequence from the CMV. In one aspect, enhancer sequence from the CMV is a marmoset enhancer sequence from the CMV. In one aspect, enhancer sequence from the CMV is a capuchin enhancer sequence from the CMV. In one aspect, enhancer sequence from the CMV is a baboon enhancer sequence from the CMV. In one aspect, enhancer sequence from the CMV is a gibbon enhancer sequence from the CMV. In one aspect, enhancer sequence from the CMV is a lemur enhancer sequence from the CMV.

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

In an aspect, a vector of the present disclosures comprises a chimeric intron. In an aspect the chimeric intron is composed of the 5′-donor site from the first intron of the human β-globin gene and the branch and 3′-acceptor site from the intron of an immunoglobulin gene heavy chain variable region. In an aspect, the chimeric intron is a chimeric intron of a rabbit beta-globing and a chicken beta actin similar in CAG promoter. In an aspect, the chimeric intron is a CRGI chimeric intron.

Introns can be grouped into at least five classes, including: spliceosomal introns; transfer RNA introns; group I introns; group II introns; and group III introns. An intron can be synthetically produced, varied, or derived from a known or naturally occurring intron sequence or other intron sequence. An intron can also include a chimeric intron comprising a combination of two or more heterologous sequences. An intron of the present application can thus include variants of intron sequences that are similar in composition, but not identical to, other intron sequence (s) known or provided herein. In an aspect, an intron comprises at least 10 nucleotides. In one aspect, an intron comprises at least 50 nucleotides. In one aspect, an intron comprises at least 100 nucleotides. In one aspect, an intron comprises at least 150 nucleotides. In one aspect, an intron comprises at least 200 nucleotides. In one aspect, an intron comprises at least 250 nucleotides. In one aspect, an intron comprises at least 300 nucleotides. In one aspect, an intron comprises at least 350 nucleotides. In one aspect, an intron comprises at least 400 nucleotides. In one aspect, an intron comprises at least 450 nucleotides. In one aspect, an intron comprises at least 500 nucleotides. In one aspect, an intron comprises between 50 nucleotides and 7500 nucleotides. In one aspect, an intron comprises between 50 nucleotides and 5000 nucleotides. In one aspect, an intron comprises between 50 nucleotides and 2500 nucleotides. In one aspect, an intron comprises between 50 nucleotides and 1000 nucleotides. In one aspect, an intron comprises between 50 nucleotides and 500 nucleotides. In one aspect, an intron comprises between 10 nucleotides and 7500 nucleotides. In one aspect, an intron comprises between 10 nucleotides and 5000 nucleotides. In one aspect, an intron comprises between 10 nucleotides and 2500 nucleotides. In one aspect, an intron comprises between 10 nucleotides and 1000 nucleotides. In one aspect, an intron comprises between 10 nucleotides and 500 nucleotides.

In an aspect, a chimeric intron nucleic acid sequence comprises a sequence at least 70%identical to SEQ ID NO: 11, or the complement thereof. In one aspect, a chimeric intron nucleic acid sequence comprises a sequence at least 75%identical to SEQ ID NO: 11, or the complement thereof. In one aspect, a chimeric intron nucleic acid sequence comprises a sequence at least 80%identical to SEQ ID NO: 11, or the complement thereof. In one aspect, a chimeric intron nucleic acid sequence comprises a sequence at least 85%identical to SEQ ID NO: 11, or the complement thereof. In one aspect, a chimeric intron nucleic acid sequence comprises a sequence at least 90%identical to SEQ ID NO: 11, or the complement thereof. In one aspect, a chimeric intron nucleic acid sequence comprises a sequence at least 91%identical to SEQ ID NO: 11, or the complement thereof. In one aspect, a chimeric intron nucleic acid sequence comprises a sequence at least 92%identical to SEQ ID NO: 11, or the complement thereof. In one aspect, a chimeric intron nucleic acid sequence comprises a sequence at least 93%identical to SEQ ID NO: 11, or the complement thereof. In one aspect, a chimeric intron nucleic acid sequence comprises a sequence at least 94%identical to SEQ ID NO: 11, or the complement thereof. In one aspect, a chimeric intron nucleic acid sequence comprises a sequence at least 95%identical to SEQ ID NO: 11, or the complement thereof. In one aspect, a chimeric intron nucleic acid sequence comprises a sequence at least 96%identical to SEQ ID NO: 11, or the complement thereof. In one aspect, a chimeric intron nucleic acid sequence comprises a sequence at least 97%identical to SEQ ID NO: 11, or the complement thereof. In one aspect, a chimeric intron nucleic acid sequence comprises a sequence at least 98%identical to SEQ ID NO: 11, or the complement thereof. In one aspect, a chimeric intron nucleic acid sequence comprises a sequence at least 99%identical to SEQ ID NO: 11, or the complement thereof. In one aspect, a chimeric intron nucleic acid sequence comprises a sequence at least 99.5%identical to SEQ ID NO: 11, or the complement thereof. In one aspect, a chimeric intron nucleic acid sequence comprises a sequence at least 99.8%identical to SEQ ID NO: 11, or the complement thereof. In one aspect, a chimeric intron nucleic acid sequence comprises a sequence at least 99.9%identical to SEQ ID NO: 11, or the complement thereof. In one aspect, a chimeric intron nucleic acid sequence comprises a sequence 100%identical to SEQ ID NO: 11, or the complement thereof.

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

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

In an aspect, an optimized WPRE nucleic acid sequence comprises a sequence at least 70%identical to SEQ ID NO: 12, or the complement thereof. In one aspect, an optimized WPRE nucleic acid sequence comprises a sequence at least 75%identical to SEQ ID NO: 12, or the complement thereof. In one aspect, an optimized WPRE nucleic acid sequence comprises a sequence at least 80%identical to SEQ ID NO: 12, or the complement thereof. In one aspect, an optimized WPRE nucleic acid sequence comprises a sequence at least 85%identical to SEQ ID NO: 12, or the complement thereof. In one aspect, an optimized WPRE nucleic acid sequence comprises a sequence at least 90%identical to SEQ ID NO: 12, or the complement thereof. In one aspect, an optimized WPRE nucleic acid sequence comprises a sequence at least 91%identical to SEQ ID NO: 12, or the complement thereof. In one aspect, an optimized WPRE nucleic acid sequence comprises a sequence at least 92%identical to SEQ ID NO: 12, or the complement thereof. In one aspect, an optimized WPRE nucleic acid sequence comprises a sequence at least 93%identical to SEQ ID NO: 12, or the complement thereof. In one aspect, an optimized WPRE nucleic acid sequence comprises a sequence at least 94%identical to SEQ ID NO: 12, or the complement thereof. In one aspect, an optimized WPRE nucleic acid sequence comprises a sequence at least 95%identical to SEQ ID NO: 12, or the complement thereof. In one aspect, an optimized WPRE nucleic acid sequence comprises a sequence at least 96%identical to SEQ ID NO: 12, or the complement thereof. In one aspect, an optimized WPRE nucleic acid sequence comprises a sequence at least 97%identical to SEQ ID NO: 12, or the complement thereof. In one aspect, an optimized WPRE nucleic acid sequence comprises a sequence at least 98%identical to SEQ ID NO: 12, or the complement thereof. In one aspect, an optimized WPRE nucleic acid sequence comprises a sequence at least 99%identical to SEQ ID NO: 12, or the complement thereof. In one aspect, an optimized WPRE nucleic acid sequence comprises a sequence at least 99.5%identical to SEQ ID NO: 12, or the complement thereof. In one aspect, an optimized WPRE nucleic acid sequence comprises a sequence at least 99.8%identical to SEQ ID NO: 12, or the complement thereof. In one aspect, an optimized WPRE nucleic acid sequence comprises a sequence at least 99.9%identical to SEQ ID NO: 12, or the complement thereof. In one aspect, an optimized WPRE nucleic acid sequence comprises a sequence 100%identical to SEQ ID NO: 12, or the complement thereof.

A bGH polyadenylation signal sequence (also referred as bGH PolyA or bGHpA) refers to a Poly A signal or PolyA tail of a bovine growth hormone. The bGH polyadenylation signal sequence is a DNA sequence the can terminate transcription and add a PolyA tail to the 3′ end of a messenger RNA (mRNA) .

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

In an aspect, a bGH polyadenylation signal nucleic acid sequence comprises a sequence at least 70%identical to SEQ ID NO: 9, or the complement thereof. In one aspect, a bGH polyadenylation signal nucleic acid sequence comprises a sequence at least 75%identical to SEQ ID NO: 9, or the complement thereof. In one aspect, a bGH polyadenylation signal nucleic acid sequence comprises a sequence at least 80%identical to SEQ ID NO: 9, or the complement thereof. In one aspect, a bGH polyadenylation signal nucleic acid sequence comprises a sequence at least 85%identical to SEQ ID NO: 9, or the complement thereof. In one aspect, a bGH polyadenylation signal nucleic acid sequence comprises a sequence at least 90%identical to SEQ ID NO: 9, or the complement thereof. In one aspect, a bGH polyadenylation signal nucleic acid sequence comprises a sequence at least 91%identical to SEQ ID NO: 9, or the complement thereof. In one aspect, a bGH polyadenylation signal nucleic acid sequence comprises a sequence at least 92%identical to SEQ ID NO: 9, or the complement thereof. In one aspect, a bGH polyadenylation signal nucleic acid sequence comprises a sequence at least 93%identical to SEQ ID NO: 9, or the complement thereof. In one aspect, a bGH polyadenylation signal nucleic acid sequence comprises a sequence at least 94%identical to SEQ ID NO: 9, or the complement thereof. In one aspect, a bGH polyadenylation signal nucleic acid sequence comprises a sequence at least 95%identical to SEQ ID NO: 9, or the complement thereof. In one aspect, a bGH polyadenylation signal nucleic acid sequence comprises a sequence at least 96%identical to SEQ ID NO: 9, or the complement thereof. In one aspect, a bGH polyadenylation signal nucleic acid sequence comprises a sequence at least 97%identical to SEQ ID NO: 9, or the complement thereof. In one aspect, a bGH polyadenylation signal nucleic acid sequence comprises a sequence at least 98%identical to SEQ ID NO: 9, or the complement thereof. In one aspect, a bGH polyadenylation signal nucleic acid sequence comprises a sequence at least 99%identical to SEQ ID NO: 9, or the complement thereof. In one aspect, a bGH polyadenylation signal nucleic acid sequence comprises a sequence at least 99.5%identical to SEQ ID NO: 9, or the complement thereof. In one aspect, a bGH polyadenylation signal nucleic acid sequence comprises a sequence at least 99.13%identical to SEQ ID NO: 9, or the complement thereof. In one aspect, a bGH polyadenylation signal nucleic acid sequence comprises a sequence at least 99.9%identical to SEQ ID NO: 9, or the complement thereof. In one aspect, a bGH polyadenylation signal nucleic acid sequence comprises a sequence 100%identical to SEQ ID NO: 9, or the complement thereof.

As used herein, the term “central nervous system” or “CNS” refers to the brain and spinal cord of a bilaterally symmetric animal. The CNS also includes the retina, the optic nerve, olfactory nerves, and olfactory epithelium.

As used herein, the term “peripheral nervous system” or “PNS” refers to nerves and ganglia outside of the brain and spinal cord, excluding the retina, the optic nerve, olfactory nerves, and olfactory epithelium. In an 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 parts of the PNS that are associated with voluntary control of body movements.

As used herein, the term “autonomic nervous system” refers to the parts of the PNS that regulate the function of internal organs

As used herein, the term “GFAP positive” refers to a cell having detectable protein accumulation of human glial fibrillary acid protein (GFAP) or detectable accumulation of GFAP mRNA expression using techniques standard in the art. In one aspect, a glial cell is GFAP positive cell. In one aspect, an astrocyte is GFAP positive. In one aspect, a reactive astrocyte is a GFAP positive cell.

As used herein, the term “detectable” refers to protein or mRNA accumulation that is identifiable.

Protein accumulation can be identified using antibodies. Non limiting examples of measuring protein accumulation include Western blots, enzyme linked immunosorbent assays (ELISAs) , immunoprecipitations and immunofluorescence. An antibody provided herein can be a polyclonal antibody or a monoclonal antibody. An antibody having specific binding affinity for a protein provided herein can be generated using methods well known in the art. An antibody provided herein can be attached to a solid support such as a microtiter plate using methods known in the art.

As used herein, the term “multiplicity of infection” and “MOI” refers to the number of virions that are added per cell during infection.

As used herein, the term “virion” refers to the infective form of a virus outside a host cell.

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

As used herein, the term “injury” refers to damage to the central nervous system or peripheral nervous system.

In one aspect, a neurological condition is a stroke. In one aspect, a neurological condition is ischemic stroke. In one aspect, a neurological condition is hemorrhagic stroke.

In an aspect, a neurological condition comprises an injury to the CNS or to the PNS. In one aspect, a neurological condition comprises an injury to the CNS. In one aspect, a neurological condition comprises an injury to the PNS.

In an aspect, this disclosure provides, and includes, a method of treating stroke in a subject, the method comprising administering to the subject a pharmaceutical composition comprising an adeno-associated viral (AAV) vector comprising a nucleic acid molecule encoding neurogenic differentiation 1 (NeuroD1) , wherein the nucleic acid molecule is operably linked to expression control elements comprising: (a) a glial fibrillary acid protein (GFAP) promoter; (b) an enhancer; (c) a chimeric intron; (d) an optimized woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) ; and (e) a polyadenylation signal sequence.

In an aspect, this disclosure provides, and includes a method of treating stroke in a primate, the method comprising administering to the primate a pharmaceutical composition comprising an adeno-associated virus (AAV) vector comprising a human neurogenic differentiation 1 (hNeuroD1) sequence comprising the nucleic acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4, where the hNeuroD1 sequence is operably linked to regulatory elements comprising: (a) a glial fibrillary acid protein (GFAP) promoter comprising the nucleic acid sequence of SEQ ID NO: 10; (b) a cytomegalovirus (CMV) enhancer comprising the nucleic acid sequence of SEQ ID NO: 8; (c) a chimeric intron comprising the nucleic acid sequence of SEQ ID NO: 11; (d) an optimized woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) comprising the nucleic acid sequence of SEQ ID NO: 12; and (e) a bGH polyadenylation sequence comprising the nucleic acid sequence of SEQ ID NO: 9.

In an aspect, this disclosure provides, and includes, a method of partially or fully restoring neuronal pathways in the brain of a subject who has suffered a stroke, the method comprising administering to the subject a pharmaceutical composition comprising an adeno-associated viral (AAV) vector comprising a nucleic acid molecule encoding neurogenic differentiation 1 (NeuroD1) , wherein the nucleic acid molecule is operably linked to expression control elements comprising: (a) a glial fibrillary acid protein (GFAP) promoter; (b) an enhancer; (c) a chimeric intron; (d) an optimized woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) ; and (e) a polyadenylation signal sequence.

In an aspect, this disclosure provides, and includes, a method of partially or fully restoring neuronal pathways in the brain of a primate who has suffered a stroke, the method comprising administering to the primate a pharmaceutical composition comprising an adeno-associated virus (AAV) vector comprising a human neurogenic differentiation 1 (hNeuroD1) sequence comprising the nucleic acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4, where the hNeuroD1 sequence is operably linked to regulatory elements comprising: (a) a glial fibrillary acid protein (GFAP) promoter comprising the nucleic acid sequence of SEQ ID NO: 10; (b) a cytomegalovirus (CMV) enhancer comprising the nucleic acid sequence of SEQ ID NO: 8; (c) a chimeric intron comprising the nucleic acid sequence of SEQ ID NO: 11; (d) an optimized woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) comprising the nucleic acid sequence of SEQ ID NO: 12; and (e) a bGH polyadenylation sequence comprising the nucleic acid sequence of SEQ ID NO: 9.

In an aspect, this disclosure provides, and includes, a method of reducing neuroinflammation in the brain of a subject who has suffered a stroke, the method comprising administering to the subject a pharmaceutical composition comprising an adeno-associated viral (AAV) vector comprising a nucleic acid molecule encoding neurogenic differentiation 1 (NeuroD1) , wherein the nucleic acid molecule is operably linked to expression control elements comprising: (a) a glial fibrillary acid protein (GFAP) promoter; (b) an enhancer; (c) a chimeric intron; (d) an optimized woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) ; and (e) a polyadenylation signal sequence.

In an aspect, this disclosure provides, and includes, a method of reducing neuroinflammation in the brain of a primate who has suffered a stroke, the method comprising administering to the primate a pharmaceutical composition comprising an adeno-associated virus (AAV) vector comprising a human neurogenic differentiation 1 (hNeuroD1) sequence comprising the nucleic acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4, where the hNeuroD1 sequence is operably linked to regulatory elements comprising: (a) a glial fibrillary acid protein (GFAP) promoter comprising the nucleic acid sequence of SEQ ID NO: 10; (b) a cytomegalovirus (CMV) enhancer comprising the nucleic acid sequence of SEQ ID NO: 8; (c) a chimeric intron comprising the nucleic acid sequence of SEQ ID NO: 11; (d) an optimized woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) comprising the nucleic acid sequence of SEQ ID NO: 12; and (e) a bGH polyadenylation sequence comprising the nucleic acid sequence of SEQ ID NO: 9.

In an aspect, this disclosure provides, and includes, a method of generating new neurons in the brain of a subject who has suffered a stroke, the method comprising administering to the subject a pharmaceutical composition comprising an adeno-associated viral (AAV) vector comprising a nucleic acid molecule encoding neurogenic differentiation 1 (NeuroD1) , wherein the nucleic acid molecule is operably linked to expression control elements comprising: (a) a glial fibrillary acid protein (GFAP) promoter; (b) an enhancer; (c) a chimeric intron; (d) an optimized woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) ; and (e) a polyadenylation signal sequence.

In an aspect, this disclosure provides, and includes, a method of generating new neurons in the brain of a primate who has suffered a stroke, the method comprising administering to the primate a pharmaceutical composition comprising an adeno-associated virus (AAV) vector comprising a human neurogenic differentiation 1 (hNeuroD1) sequence comprising the nucleic acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4, where the hNeuroD1 sequence is operably linked to regulatory elements comprising: (a) a glial fibrillary acid protein (GFAP) promoter comprising the nucleic acid sequence of SEQ ID NO: 10; (b) a cytomegalovirus (CMV) enhancer comprising the nucleic acid sequence of SEQ ID NO: 8; (c) a chimeric intron comprising the nucleic acid sequence of SEQ ID NO: 11; (d) an optimized woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) comprising the nucleic acid sequence of SEQ ID NO: 12; and (e) a bGH polyadenylation sequence comprising the nucleic acid sequence of SEQ ID NO: 9.

In an aspect, this disclosure provides, and includes, a method of treating a neurological condition in a subject, the method comprising administering to the subject a pharmaceutical composition comprising an adeno-associated viral (AAV) vector comprising a nucleic acid molecule encoding neurogenic differentiation 1 (NeuroD1) , wherein the nucleic acid molecule is operably linked to expression control elements comprising: (a) a glial fibrillary acid protein (GFAP) promoter; (b) an enhancer; (c) a chimeric intron; (d) an optimized woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) ; and (e) a polyadenylation signal sequence.

In an aspect, this disclosure provides, and includes, a method of treating a neurological condition in a primate, the method comprising administering to the primate a pharmaceutical composition comprising an adeno-associated virus (AAV) vector comprising a human neurogenic differentiation 1 (hNeuroD1) sequence comprising the nucleic acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4, where the hNeuroD1 sequence is operably linked to regulatory elements comprising: (a) a glial fibrillary acid protein (GFAP) promoter comprising the nucleic acid sequence of SEQ ID NO: 10; (b) a cytomegalovirus (CMV) enhancer comprising the nucleic acid sequence of SEQ ID NO: 8; (c) a chimeric intron comprising the nucleic acid sequence of SEQ ID NO: 11; (d) an optimized woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) comprising the nucleic acid sequence of SEQ ID NO: 12; and (e) a bGH polyadenylation sequence comprising the nucleic acid sequence of SEQ ID NO: 9.

In an aspect, a method as provided herein, is capable of converting at least one glial cell into a neuron. In one aspect, a method as provided herein converts at least one glial cell into a neuron. In an aspect, a method as provided herein, is capable of converting at least one astrocyte into a neuron. In one aspect, a method as provided herein converts at least one astrocyte into a neuron. In an aspect, a method as provided herein, is capable of converting at least one reactive astrocyte into a neuron. In one aspect, a method as provided herein converts at least one reactive astrocyte into a neuron.

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

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

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

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

In an aspect, a method as provided herein uses an AAV vector comprising a NeuroD1 coding sequence in accordance with the present disclosure. In one aspect, a method as provided herein uses an AAV vector comprising a NeuroD1 coding sequence in combination with a second AAV vector comprising a second transcription factor coding sequence. In one aspect, a method as provided herein uses an AAV vector comprising a NeuroD1 coding sequence and a second transcription factor coding sequence. In one aspect, a second transcription factor is selected from the group consisting of Dlx2, Ascl1, ISL1, and LHX3. In one aspect, a second transcription factor is Dlx2. In one aspect, a second transcription factor is Ascl1. In one aspect, a second transcription factor is ISL1. In one aspect, a second transcription factor is LHX3. In one aspect, a method as provided herein uses an AAV vector comprising a NeuroD1 coding sequence and second NeuroD1 coding sequence. In one aspect, a method as provided herein uses an AAV vector comprising a NeuroD1 coding sequence in combination with a second AAV vector comprising a NeuroD1 coding sequence.

In an aspect, an AAV vector as provided herein, is measured for functionality by assessing transcription levels and/or protein levels of NeuN, Parvalbumin, and ionized calcium binding adaptor molecule (Iba1) , or by the detection of neurofilaments, dendrites, or cell bodies indicating the presence of neurons.

As used herein, the term “NeuN” or “Fox-3” or “Rbfox2” or “Hexaribonucleotide Binding Protein-3” refers to a protein which is a homologue to the protein product of a sex-determining gene in Caenorhabditis elegans and is a neuronal nuclear antigen.

As used herein, the term “Parvalbumin” refers to a calcium-binding protein found in some neurons, e.g., interneurons.

As used herein, the term “Iba1” refers to a microglia macrophage-specific calcium binding protein. Iba1 can be used as a neuroinflammation marker, indicating the presence of high levels of microglia in a region of the nervous system.

As used herein, the term “SMI312” refers to a mixture of monoclonal antibodies that react against complex networks of axons. It is directed against extensively phosphorylated axonal epitopes on neurofilaments M and H. Neurofilaments (NF) are approximately 10 nanometer intermediate filaments found in neurons. They are a major component of the neuronal cytoskeleton and their function is primarily to provide structural support for the axon and to regulate axon diameter. There are three major NF subunits, and the names given to these subunits are based upon the apparent molecular mass of the mammalian subunits on SDS-PAGE. The light or lowest (NF-L) runs at 68-70 kD, the medium or middle (NF-M) runs at about 145-160 kD, and the heavy or highest (NF-H) runs at 200-220 kD.

As used herein, the term “SMI32” refers to an antibody that reacts against a non-phosphorylated epitope in neurofilament H of most mammalian species. The reaction is masked when the epitope is phosphorylated. Immunocytochemically, SMI32 visualizes neuronal cell bodies, dendrites, and some thick axons in the central and peripheral nervous systems. However, thin axons are not revealed.

In an aspect, a composition as provided herein, is capable of converting at least one glial cell into a neuron. In one aspect, a composition as provided herein converts at least one glial cell into a neuron. In an aspect, conversion of a glial cell into a neuron is measured via the detection of the expression of NeuN or Parvalbumin in the converted cells. Particularly, when glial cells are converted into neurons they may express neuronal markers such as NeuN or Parvalbumin. Additionally, SMI312 and SMI32 antibodies can be used to confirm the neuronal or neuron-like characteristics of the converted cells.

As used herein, the term “mammal” refers to any species classified in the class Mammalia.

As used herein, the term “human” refers to a Homo sapiens. In an aspect, a human has a neurological disorder.

As used herein, the term “living human” refers to a human that has heart, respiration and brain activity.

As used herein, the term “non-human primate” refers to any species or subspecies classified in the order Primates that are not Homo sapiens. Non-limiting examples of non-human primates include chimpanzee, bonobo, orangutan, gorilla, macaque (e.g., cynomolgus monkey) , marmoset, capuchin, baboon, gibbon, and lemur.

In an aspect, because of the similarities and evolutionary relationship among primates, clinically relevant effects that the compositions of the present disclosure have on non-human primates could also be reasonably expected in humans. For instance, compared with mice, rats, or dogs, cynomolgus macaque monkeys have a closer evolutionary relationship to humans (Shen et al., Drug Metabolism and Disposition, March 1, 2022, 50 (3) 299-319) . Macaque monkeys (genus Macaca) have a gyrencephalic brain with similar cortical and subcortical anatomy to humans and they have been used in permanent and transient Middle Cerebral Artery Occlusion (MCAO) models (Cook et. al, Neurotherapeutics. 2012 Apr; 9 (2) : 371-9) . Additionally, the vascular anatomy of the macaque closely resembles that of a human. Id.

As used herein, the term “delivering” or “delivery” refers to treating a mammal with an AAV vector or composition as provided herein. In an aspect, an AAV vector or composition as provided herein is delivered to a subject in need thereof. In one aspect, an AAV vector or composition as provided herein is formulated to be delivered to a subject in need thereof. In one aspect, delivering comprises local delivery. In one aspect, an AAV vector or composition as provided herein is formulated for local delivery. In one aspect, delivering comprises systemic delivery. In one aspect, an AAV vector or composition as provided herein is formulated for systemic delivery. In one aspect, delivery comprises injecting an AAV vector or composition as provided herein into a subject in need thereof. In one aspect, delivering is selected from the group consisting of intraperitoneal, intramuscular, intravenous, intrathecal, intracerebral, intracranial, intra lateral ventricle of the brain, intra cisterna magna, intra vitreous, intra-subretina, intraparenchymal, intranasal, or oral administration. In one aspect, delivery comprises intraperitoneal delivery. In one aspect, delivery comprises intramuscular delivery. In one aspect, delivery comprises intravenous delivery. In one aspect, delivery comprises intrathecal delivery. In one aspect, delivery comprises intracerebral delivery. In one aspect, delivery comprises intracranial delivery. In one aspect, delivery comprises intra lateral ventricle of the brain delivery. In one aspect, delivery comprises intra cisterna magna delivery. In one aspect, delivery comprises intra vitreous delivery. In one aspect, delivery comprises intra-subretinal delivery. In one aspect, delivery comprises intraparenchymal delivery. In one aspect, delivery comprises intranasal delivery. In one aspect, delivery comprises oral administration.

In an aspect, an AAV vector or composition as provided herein is delivered to a brain of a subject who has suffered a stroke. In an aspect, an AAV vector or composition as provided herein is delivered to an area of the brain adjacent to the core region of the stroke. In an aspect, an AAV vector or composition as provided herein is delivered to a peri-infarct region of the stroke.

As used herein, the term “injecting” refers to delivering an AAV vector or composition as provided herein under pressure and with force. As a non-limiting example, injecting can comprise the use of a syringe and needle. In an aspect, an AAV vector or composition is injected into a subject, e.g., into the brain of a subject. In an aspect, an AAV vector or composition is injected using a 33-gauge needle that is 1.5 inches in length and has a 30° bevel. In an aspect, an AAV vector or composition is injected using a 100 μL syringe equipped with a 33-gauge needle, 1.5 in length, with a 30° bevel. In an aspect, an AAV vector or composition is injected using a syringe pump. In an aspect, an AAV vector or composition is injected using a syringe pump mounted on a stereotaxic arm. In an aspect, an injection site is determined prior to the injecting via a magnetic resonance imaging (MRI) scan. In an aspect, coordinates of the determined injection site are used for the injecting, such for injecting the brain of a subject. In an aspect, an AAV vector or composition is injected using a surgical navigation system to target an injection site.

As used herein, the term “flow rate” or “controlled infusion rate” refer to the rate of delivery of an AAV vector or composition.

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

In an aspect, an AAV vector or composition as provided herein is injected into a brain of a subject who has suffered a stroke. In an aspect, an AAV vector or composition as provided herein is injected into an area of the brain adjacent to the core region of the stroke. In an aspect, an AAV vector or composition as provided herein is injected into a peri-infarct region of the stroke. In an aspect, an AAV vector or composition as provided herein is injected into a brain of a subject at one injection site. In an aspect, an AAV vector or composition as provided herein is injected into a brain of a subject at multiple injection sites. In an aspect, an AAV vector or composition as provided herein is injected into a brain of a subject at 1, 2, 3, 4, or 5 injection sites. In an aspect, an AAV vector or composition as provided herein is injected into a brain of a subject at 3 injection sites.

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

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

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

In an aspect, an AAV vector or composition as provided herein has spread in the striatum between about 1%and about 100%. In one aspect, an AAV vector or composition as provided herein has spread 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 50%and about 80%, between about 60%and about 70%, between about 60%and about 80%, between about 60%and about 90%, between about 70%and about 80%, between about 70%and about 90%, between about 70%and about 100%, between about 80%and about 90%, between about 80%and about 100%, or between about 90%and about 100%.

In an aspect, an AAV vector or composition as provided herein has spread in the dorsal striatum between about 1%and about 100%. In one aspect, an AAV vector or composition as provided herein has spread in the dorsal 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 50%and about 80%, between about 60%and about 70%, between about 60%and about 80%, between about 60%and about 90%, between about 70%and about 80%, between about 70%and about 90%, between about 70%and about 100%, between about 80%and about 90%, between about 80%and about 100%, or between about 90%and about 100%.

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

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

In an aspect, an AAV vector or composition as provided herein has a spread at from injection site between about 1%and about 100%. In one aspect, an AAV vector or composition as provided herein has a spread from 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 50%and about 80%, between about 60%and about 70%, between about 60%and about 80%, between about 60%and about 90%, between about 70%and about 80%, between about 70%and about 90%, between about 70%and about 100%, between about 80%and about 90%, between about 80%and about 100%, or between about 90%and about 100%.

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

As used herein, the term “AAV particle” refers to packaged capsid forms of the AAV virus that transmits its nucleic acid genome to cells.

As used herein, the unit “vg/mL” refers to viral genomes per mL. AAV titers are often given as vg/mL. Alternatively viral titers can also be expressed as the number of viral particles per mL (VP/mL) .

In an aspect, an AAV particle or composition as provided herein can be provided together with a pharmaceutically acceptable carrier. As used herein, a “pharmaceutically acceptable carrier” refers to a non-toxic solvent, dispersant, excipient, adjuvant, or other material which is mixed with an AAV particles or composition as provided herein.

The term “administer” or “administration” refers to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g., a recombinant AAV as described herein) into a patient, such as by intracranial, mucosal, intradermal, intravenous, intramuscular delivery, and/or any other method of physical delivery described herein or known in the art. When a disease, disorder, condition, or a symptom thereof, is being treated, administration of the substance typically occurs after the onset of the disease, disorder, condition, or symptoms thereof. When a disease, disorder, condition, or symptoms thereof, are being prevented, administration of the substance typically occurs before the onset of the disease, disorder, condition, or symptoms thereof.

An “effective amount” is generally an amount sufficient to produce a desirable outcome, such as, producing one or more neuronal phenotypes in a population of cells, or in the context of disease management, to reduce the severity and/or frequency of symptoms, eliminate the symptoms and/or underlying cause, prevent the occurrence of symptoms and/or their underlying cause, and/or improve or remediate the damage that results from or is associated with a disease, disorder, or condition, including, for example, stroke.

The term “therapeutically effective amount” as used herein refers to the amount of an agent (e.g., a recombinant AAV described herein or any other agent described herein) that is sufficient to reduce and/or ameliorate the severity and/or duration of a given disease, disorder or condition, and/or a symptom related thereto. A therapeutically effective amount of an agent, including a therapeutic agent, can be an amount necessary for (i) reduction, delay or amelioration of the advancement or progression of a given disease, disorder, or condition, (ii) reduction, delay or amelioration of the recurrence, development or onset of a given disease, disorder or conditions, and/or (iii) to improve or enhance the prophylactic or therapeutic effect of another therapy (e.g., a therapy other than the administration of an agent described herein) . A “therapeutically effective amount” of a substance/molecule/agent of the present disclosure (e.g., a recombinant AAV) may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the substance/molecule/agent, to elicit a desired response in the individual. A therapeutically effective amount encompasses an amount in which any toxic or detrimental effects of the substance/molecule/agent are outweighed by the therapeutically beneficial effects. In certain embodiments, the term “therapeutically effective amount” refers to an amount of a recombinant AAV effective to “treat” a disease, disorder, or condition, in a subject or mammal.

The term “treating” or any grammatical variation thereof refers to reducing and/or ameliorating the severity and/or duration of a given disease, disorder or condition, and/or a symptom related thereto, such as (i) reduction, delay or amelioration of the advancement or progression of a given disease, disorder, or condition, (ii) reduction, delay or amelioration of the recurrence, development or onset of a given disease, disorder or conditions, and/or (iii) to improve or enhance the prophylactic or therapeutic effect of another therapy (e.g., a therapy other than the administration of a recombinant AAV described herein) .

A “prophylactically effective amount” is an amount of a pharmaceutical composition that, when administered to a subject, will have the intended prophylactic effect, e.g., preventing or delaying the onset (or reoccurrence) of a disease, disorder or condition, or reducing the likelihood of the onset (or reoccurrence) of a disease, disorder, or condition or associated symptom (s) .

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

As used herein, “asubject in need thereof” refers to a subject with a neurological condition.
In an aspect, a subject in need thereof is a subject who has suffered a stroke.

In an aspect, a subject in need thereof is a mammal. In one aspect, a 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 chimpanzee, bonobo, orangutan, gorilla, macaque (e.g., cynomolgus monkey) , marmoset, capuchin, baboon, gibbon, and lemur. In one aspect, a subject in need thereof is a chimpanzee. In one aspect, a subject in need thereof is a bonobo. In one aspect, a subject in need thereof is orangutan. In one aspect, a subject in need thereof is gorilla. In one aspect, a subject in need thereof is a macaque. In one aspect, a subject in need thereof is marmoset. In one aspect, a subject in need thereof is a capuchin. In one aspect, a subject in need thereof is a baboon. In one aspect, a subject in need thereof is a gibbon. In one aspect, a subject in need thereof is lemur.

In one aspect, a subject in need thereof is a male. In one aspect, a subject in need thereof is a female. In one aspect, a subject in need thereof is gender neutral. In one aspect, a subject in need thereof is between 1 year and 5 years, between 2 years and 10 years, between 3 years and 18 years, between 21 years and 50 years, between 21 years and 40 years, between 21 years and 30 years, between 50 years and 90 years, between 60 years and 90 years, between 70 years and 90 years, between 60 years and 80 years, or between 65 years and 75 years old. In one aspect, a subject in need thereof is a young old subject (65 to 74 years old) . In one aspect, a subject in need thereof is a middle old subject (75 to 84 years old) . In one aspect, a subject in need thereof is an old subject (>85 years old) .

In an aspect, a subject or a subject in need thereof comprises a subject who has suffered a stroke. In an aspect, a subject or a subject in need thereof comprises a subject who has suffered a severe stroke. Methods of determining the severity of a stroke are known in the field. For instance, the National Institutes of Health Stroke Scale or NIH Stroke Scale (NIHSS) is a tool used to objectively quantify the impairment caused by a stroke. The NIHSS is composed of 11 items, each of which scores a specific ability between a 0 and 4. For each item, a score of 0 typically indicates normal function in that specific ability, while a higher score is indicative of some level of impairment. The individual scores from each item are summed in order to calculate a patient's total NIHSS score. The maximum possible score is 42, with the minimum score being a 0. In the NIHSS, a total score of 0 equals no stroke symptoms, a total score of 1–4 equals a minor stroke, a total score of 5–15 equals a moderate stroke, a total score of 6–20 equals a moderate to severe stroke, and a total score of 21–42 equals a severe stroke. A comparable scale used in stroke studies involving non-human primates is the Non-Human Primate Stroke Scale (NHPSS) . The NHPSS is comprised of 11 categories, each scored independently to produce a composite score out of 41 points (0 being normal, 41 being severely impaired) . The categories include state of consciousness, defense reaction, grasp reflex, extremity movement, gait, circling behavior, bradykinesia, balance, neglect, visual field defect, and facial weakness. In the NHPSS, a total score of 25 or above indicates a severe stroke. Moreover, the Modified Rankin Scale (mRS) is another tool used to measure the degree of disability in patients who have had a stroke. The scale runs from 0 to 6, running from perfect health without symptoms (0) to death (6) .

In an aspect, a subject or a subject in need thereof is a subject who has suffered a stroke and has a score of at least 21 on the NIHSS. In an aspect, the NIHSS score of the subject is improved by at least 1 unit after the subject is administered a composition of the present disclosure. In an aspect, the NIHSS score of the subject is improved by at least 1 unit, 2 units, 3 units, 4 units, 5 units, 6 units, 7 units, 8 units, 9 units, 10 units, 11 units, 12 units, 13 units, 14 units, 15 units, 16 units, 17 units, 18 units, 19 units, 20 units, or 21 units after the subject is administered a composition of the present disclosure. In an aspect, the NIHSS score of the subject is improved by at least 1 unit, 2 units, 3 units, 4 units, 5 units, 6 units, 7 units, 8 units, 9 units, 10 units, 11 units, 12 units, 13 units, 14 units, 15 units, 16 units, 17 units, 18 units, 19 units, 20 units, or 21 units after the subject is administered a composition comprising an AAV vector encoding NeuroD1. In an aspect, the NIHSS score is improved within 30 days to 100 days after the subject has been administered a composition of the present disclosure. In an aspect, the NIHSS score is improved within 40 days to 100 days, within 40 days to 90 days, within 40 days to 80 days, within 40 days to 70 days, within 40 days to 60 days, within 40 days to 50 days, within 50 days to 100 days, within 60 days to 100 days, within 70 days to 100 days, within 80 days to 100 days, within 90 days to 100 days, within 50 days to 90 days, or within 60 days to 80 days after the subject has been administered a composition of the present disclosure.

In an aspect, a subject or a subject in need thereof is a subject who has suffered a stroke and has a score of at least 25 on the NHPSS. In an aspect, the NHPSS score of the subject is improved by at least 1 unit after the subject is administered a composition of the present disclosure. In an aspect, the NHPSS score of the subject is improved by at least 1 unit, 2 units, 3 units, 4 units, 5 units, 6 units, 7 units, 8 units, 9 units, 10 units, 11 units, 12 units, 13 units, 14 units, 15 units, 16 units, 17 units, 18 units, 19 units, 20 units, 21 units, 22 units, 23 units, 24 units, or 25 units after the subject is administered a composition of the present disclosure. In an aspect, the NHPSS score of the subject is improved by at least 1 unit, 2 units, 3 units, 4 units, 5 units, 6 units, 7 units, 8 units, 9 units, 10 units, 11 units, 12 units, 13 units, 14 units, 15 units, 16 units, 17 units, 18 units, 19 units, 20 units, 21 units, 22 units, 23 units, 24 units, or 25 units after the subject is administered a composition comprising an AAV vector encoding NeuroD1. In an aspect, the NHPSS score is improved within 30 days to 100 days after the subject has been administered a composition of the present disclosure. In an aspect, the NHPSS score is improved within 40 days to 100 days, within 40 days to 90 days, within 40 days to 80 days, within 40 days to 70 days, within 40 days to 60 days, within 40 days to 50 days, within 50 days to 100 days, within 60 days to 100 days, within 70 days to 100 days, within 80 days to 100 days, within 90 days to 100 days, within 50 days to 90 days, or within 60 days to 80 days after the subject has been administered a composition of the present disclosure.

In an aspect, a subject or a subject in need thereof is a subject who has suffered a stroke and has a score of at least 4 on the mRS. In an aspect, a subject or a subject in need thereof is a subject who has suffered a stroke and has a score of at least 5 on the mRS. In an aspect, the mRS score of the subject is improved by at least 1 unit after the subject is administered a composition of the present disclosure. In an aspect, the mRS score of the subject is improved by at least 1 unit, 2 units, 3 units, 4 units, or 5 units after the subject is administered a composition of the present disclosure. In an aspect, the mRS score of the subject is improved by at least 1 unit, 2 units, 3 units, 4 units, or 5 units after the subject is administered a composition comprising an AAV vector encoding NeuroD1. In an aspect, the mRS score is improved within 30 days to 100 days after the subject has been administered a composition of the present disclosure. In an aspect, the mRS score is improved within 40 days to 100 days, within 40 days to 90 days, within 40 days to 80 days, within 40 days to 70 days, within 40 days to 60 days, within 40 days to 50 days, within 50 days to 100 days, within 60 days to 100 days, within 70 days to 100 days, within 80 days to 100 days, within 90 days to 100 days, within 50 days to 90 days, or within 60 days to 80 days after the subject has been administered a composition of the present disclosure.

In an aspect, a therapeutically effective dose of a composition described herein is delivered to a subject in need thereof once. In an aspect, a therapeutically effective dose of a composition described herein is delivered to a subject in need thereof more than once. In an aspect, a therapeutically effective dose of a composition described herein is delivered to a subject in need thereof at one injection site in the brain of the subject. In an aspect, a therapeutically effective dose of a composition described herein is delivered to a subject in need thereof at multiple injection sites in the brain of the subject. In an aspect, a therapeutically effective dose of a composition described herein is delivered to a subject in need thereof at 1 to 5 injection sites in the brain of the subject. In an aspect, a therapeutically effective dose of a composition described herein is delivered to a subject in need thereof at 3 injection sites in the brain of the subject.

In an aspect, a therapeutically effective dose of a composition described herein is delivered to subject who has suffered a stroke. In an aspect, a therapeutically effective dose of a composition described herein is delivered to subject who has suffered a stroke within 7 days after the stroke occurs. In an aspect, a therapeutically effective dose of a composition described herein is delivered to subject who has suffered a stroke within 14 days after the stroke occurs. In an aspect, a therapeutically effective dose of a composition described herein is delivered to subject who has suffered a stroke within 21 days after the stroke occurs. In an aspect, a therapeutically effective dose of a composition described herein is delivered to subject who has suffered a stroke within 28 days after the stroke occurs. In an aspect, a therapeutically effective dose of a composition described herein is delivered to subject who has suffered a stroke within 7 to 28 days after the stroke occurs.

In an aspect, the present disclosure provides, and includes, methods of generating new neurons in the brain of a subject who has suffered a stroke. In an aspect, the present disclosure provides, and includes, methods of generating new neurons in the brain of a subject who has suffered a stroke by converting glial cells into neurons. In an aspect, the present disclosure provides, and includes, methods of generating new neurons in the brain of a subject who has suffered a stroke by converting glial cells into neurons via the expression of NeuroD1 in the glial cells. In an aspect, new neurons are generated in the brain of the subject who has suffered a stroke within 14 to 28 days after the subject is administered a composition of the present disclosure. In an aspect, new neurons are generated in the brain of the subject who has suffered a stroke within 14 to 28 days after the subject is administered a composition comprising an AAV encoding NeuroD1. In an aspect, new neurons are generated in the brain of the subject who has suffered a stroke within 21 to 28 days after the subject is administered a composition comprising an AAV encoding NeuroD1. In an aspect, new neurons are generated in the brain of the subject who has suffered a stroke within 14 to 21 days after the subject is administered a composition comprising an AAV encoding NeuroD1. In an aspect, new neurons are generated in the brain of the subject who has suffered a stroke within 14 to 28 days, 15 to 28 days, 16 to 28 days, 17 to 28 days, 18 to 28 days, 19 to 28 days, 20 to 28 days, 21 to 28 days, 22 to 28 days, 23 to 28 days, 24 to 28 days, 25 to 28, 26 to 28 days, or 27 to 28 days after the subject is administered a composition of the present disclosure. In an aspect, new neurons are generated in the brain of the subject who has suffered a stroke within 14 to 28 days, 15 to 28 days, 16 to 28 days, 17 to 28 days, 18 to 28 days, 19 to 28 days, 20 to 28 days, 21 to 28 days, 22 to 28 days, 23 to 28 days, 24 to 28 days, 25 to 28, 26 to 28 days, or 27 to 28 days after the subject is administered a composition comprising an AAV encoding NeuroD1. In an aspect, new neurons are generated in the brain of the subject who has suffered a stroke within 14 to 28 days, 14 to 27 days, 14 to 26 days, 14 to 25 days, 14 to 24 days, 14 to 23 days, 14 to 22 days, 14 to 21 days, 14 to 20 days, 14 to 19 days, 14 to 18 days, 14 to 17 days, 14 to 16 days, or 14 to 15 days after the subject is administered a composition of the present disclosure. In an aspect, new neurons are generated in the brain of the subject who has suffered a stroke within 14 to 28 days, 14 to 27 days, 14 to 26 days, 14 to 25 days, 14 to 24 days, 14 to 23 days, 14 to 22 days, 14 to 21 days, 14 to 20 days, 14 to 19 days, 14 to 18 days, 14 to 17 days, 14 to 16 days, or 14 to 15 days after the subject is administered a composition comprising an AAV encoding NeuroD1. In an aspect, new neurons are generated in the brain of the subject who has suffered a stroke within 14 to 28 days, 15 to 27 days, 16 to 26 days, 15 to 25 days, 16 to 24 days, 17 to 23 days, 18 to 22 days, or 19 to 21 days after the subject is administered a composition of the present disclosure. In an aspect, new neurons are generated in the brain of the subject who has suffered a stroke within 14 to 28 days, 15 to 27 days, 16 to 26 days, 15 to 25 days, 16 to 24 days, 17 to 23 days, 18 to 22 days, or 19 to 21 days after the subject is administered a composition comprising an AAV encoding NeuroD1.

In an aspect, the present disclosure provides, and includes, methods of partially or fully restoring neuronal pathways in the brain of a subject who has suffered a stroke. In an aspect, the present disclosure provides, and includes, methods of partially or fully restoring neuronal pathways in the brain of a subject who has suffered a stroke by converting glial cells into neurons. In an aspect, the present disclosure provides, and includes, methods of partially or fully restoring neuronal pathways in the brain of a subject who has suffered a stroke by converting glial cells into neurons via the expression of NeuroD1 in the glial cells. In an aspect, the partial or full restoration of the neuronal pathways in the brain of the subject can be assessed by MRI. In an aspect, the partial or full restoration of the neuronal pathways in the brain of the subject can be assessed by Diffusion Tensor Imaging (DTI) . In an aspect, the neuronal pathways are partially or fully restored within 3 to 6 months after the subject who has suffered a stroke is administered a composition of the present disclosure. In an aspect, the neuronal pathways are partially or fully restored within 3 to 6 months after the subject who has suffered a stroke is administered a composition comprising an AAV encoding NeuroD1. In an aspect, the neuronal pathways are partially or fully restored within 4 to 6 months after the subject who has suffered a stroke is administered a composition comprising an AAV encoding NeuroD1. In an aspect, the neuronal pathways are partially or fully restored within 5 to 6 months after the subject who has suffered a stroke is administered a composition comprising an AAV encoding NeuroD1. In an aspect, the neuronal pathways are partially or fully restored within 3 to 5 months after the subject who has suffered a stroke is administered a composition comprising an AAV encoding NeuroD1. In an aspect, the neuronal pathways are partially or fully restored within 3 to 4 months after the subject who has suffered a stroke is administered a composition comprising an AAV encoding NeuroD1.

In an aspect, the neuronal pathways are partially or fully restored within 12 to 24 weeks, 12 to 23 weeks, 12 to 22 weeks, 12 to 21 weeks, 12 to 20 weeks, 12 to 19 weeks, 12 to 18 weeks, 12 to 17 weeks, 12 to 16 weeks, 12 to 15 weeks, 12 to 14 weeks, 12 to 13 weeks, 13 to 24 weeks, 14 to 24 weeks, 15 to 24 weeks, 16 to 24 weeks, 17 to 24 weeks, 18 to 24 weeks, 19 to 24 weeks, 20 to 24 weeks, 21 to 24 weeks, 22 to 24 weeks, or 23 to 24 weeks after the subject who has suffered a stroke is administered a composition of the present disclosure. In an aspect, the neuronal pathways are partially or fully restored within 12 to 24 weeks, 12 to 23 weeks, 12 to 22 weeks, 12 to 21 weeks, 12 to 20 weeks, 12 to 19 weeks, 12 to 18 weeks, 12 to 17 weeks, 12 to 16 weeks, 12 to 15 weeks, 12 to 14 weeks, 12 to 13 weeks, 13 to 24 weeks, 14 to 24 weeks, 15 to 24 weeks, 16 to 24 weeks, 17 to 24 weeks, 18 to 24 weeks, 19 to 24 weeks, 20 to 24 weeks, 21 to 24 weeks, 22 to 24 weeks, or 23 to 24 weeks after the subject who has suffered a stroke is administered a composition comprising an AAV encoding NeuroD1. In an aspect, the neuronal pathways are partially or fully restored within 12 to 24 weeks, 13 to 23 weeks, 14 to 22 weeks, 15 to 21 weeks, 16 to 20 weeks, or 17 to 19 weeks after the subject who has suffered a stroke is administered a composition of the present disclosure. In an aspect, the neuronal pathways are partially or fully restored within 12 to 24 weeks, 13 to 23 weeks, 14 to 22 weeks, 15 to 21 weeks, 16 to 20 weeks, or 17 to 19 weeks after the subject who has suffered a stroke is administered a composition comprising an AVV encoding NeuroD1.

In an aspect, the present disclosure provides, and includes, methods of reducing neuroinflammation in the brain of a subject who has suffered a stroke. In an aspect, the present disclosure provides, and includes, methods of reducing neuroinflammation in the brain of a subject who has suffered a stroke by converting glial cells into neurons. In an aspect, the present disclosure provides, and includes, methods of reducing neuroinflammation in the brain of a subject who has suffered a stroke by converting glial cells into neurons via the expression of NeuroD1 in the glial cells. In an aspect, reduction in neuroinflammation is determined by measuring the expression of Iba1 in a region of the brain of the subject. In an aspect, reduction in neuroinflammation is determined by measuring the abundance of microglia in a region of the brain of the subject. In an aspect, neuroinflammation is reduced in the brain of the subject who has suffered a stroke within 14 to 21 days after the subject is administered a composition of the present disclosure. In an aspect, neuroinflammation is reduced in the brain of the subject who has suffered a stroke within 14 to 21 days after the subject is administered a composition comprising an AAV encoding NeuroD1. In an aspect, neuroinflammation is reduced in the brain of the subject who has suffered a stroke within 14 to 21 days, 15 to 21 days, 16 to 21 days, 17 to 21 days, 18 to 21 days, 19 to 21 days, or 20 to 21 days after the subject is administered a composition of the present disclosure. In an aspect, neuroinflammation is reduced in the brain of the subject who has suffered a stroke within 14 to 21 days, 14 to 20 days, 14 to 19 days, 14 to 18 days, 14 to 17 days, 14 to 16 days, or 14 to 15 days after the subject is administered a composition of the present disclosure. In an aspect, neuroinflammation is reduced in the brain of the subject who has suffered a stroke within 14 to 21 days, 15 to 20 days, 16 to 19 days, or 17 to 18 days after the subject is administered a composition of the present disclosure. In an aspect, neuroinflammation is reduced in the brain of the subject who has suffered a stroke within 14 to 21 days, 15 to 21 days, 16 to 21 days, 17 to 21 days, 18 to 21 days, 19 to 21 days, or 20 to 21 days after the subject is administered a composition comprising an AAV encoding NeuroD1. In an aspect, neuroinflammation is reduced in the brain of the subject who has suffered a stroke within 14 to 21 days, 14 to 20 days, 14 to 19 days, 14 to 18 days, 14 to 17 days, 14 to 16 days, or 14 to 15 days after the subject is administered a composition comprising an AAV encoding NeuroD1. In an aspect, neuroinflammation is reduced in the brain of the subject who has suffered a stroke within 14 to 21 days, 15 to 20 days, 16 to 19 days, or 17 to 18 days after the subject is administered a composition comprising an AAV encoding NeuroD1.

As used herein, the term “remission” , “cure, ” or “resolution rate” refers to the percentage of subjects in need thereof that are cured or obtain remission or complete resolution of a neurological condition in response to a therapeutically effective dose.

As used herein, the term “response rate” refers to the percentage of subjects in need thereof that respond positively (e.g., reduced severity or frequency of one or more symptoms) to a therapeutically effective dose.

Non-limiting examples of tests to evaluate the brain of a subject in need thereof before and after treatment include Nissel staining, MRI, Diffusion tensor imaging (DTI) , functional magnetic resonance fMRI, and PET scanning.
5.1. NeuroD1 Expression Cassette

In one aspect, provided herein are functional nucleic acid molecules for treating stroke. In some embodiments, the functional nucleic acid comprises an expression cassette encoding a NeuroD1 polypeptide, which upon contacting with the glial cell, is expressed by the glial cell to produce the encoded NeuroD1 polypeptide. In some embodiment, the expression cassette comprises at least one coding region encoding a NeuroD1 polypeptide (e.g., an open reading frame (ORF) ) . In some embodiment, the expression cassette further comprises at least one untranslated region (UTR) . In some embodiments, the UTR comprises one or more regulatory elements as described herein. In some embodiments, the expression cassette can comprise any coding sequences as described in this Section 5.1.1 (Coding Region) . In some embodiments, the expression cassette can comprise any regulatory elements described in Section 5.1.2 (Untranslated Regions (UTRs) ) .

In some embodiments, the NeuroD1 expression cassette is part of a single-stranded nucleic acid molecule, including a single-stranded DNA molecule. In some embodiments, the single-stranded DNA molecule is an artificial AAV genome that can be packaged into a recombinant AAV capsid.

In other embodiments, the NeuroD1 expression cassette is part of a nucleic acid molecule that is configured to produce a linear nucleic acid molecule, including a single-stranded DNA molecule. In some embodiments, the NeuroD1 expression cassette is part of a vector (e.g., a plasmid) that can be processed into a linear recombinant AAV genome in a host cell in the presence of sufficient adenovirus helper functions to permit replication and packaging of the linear recombinant AAV genome by the AAV capsid proteins.
5.1.1. Coding Region

In some embodiments, the NeuroD1 expression cassette of the present disclosure comprises at least one coding region. In some embodiments, the coding region is an open reading frame (ORF) that encodes for a NeuroD1 polypeptide. In some embodiments, the coding region comprises at least two ORFs, each encoding a NeuroD1 polypeptide. In those embodiments where the coding region comprises more than one ORFs, the encoded NeuroD1 polypeptides can be the same as or different from each other. In some embodiments, the multiple ORFs in a coding region are separated by non-coding sequences.

In specific embodiments, the coding sequences or amino acid sequences of NeuroD1 polypeptides can be any NeuroD1 polypeptide as described herein. Table 5.1.1 shows exemplary NeuroD1 polypeptides and encoding nucleic acid sequences thereof.
Table 5.1.1 Exemplary NeuroD1 polypeptide and encoding nucleic acid sequences.

In particular embodiments, the NeuroD1 expression cassette encodes a NeuroD1 polypeptide. In some embodiments, the encoded NeuroD1 polypeptide is a wild-type NeuroD1. In some embodiments, the encoded NeuroD1 is human NeuroD1 having the amino acid sequence of SEQ ID NO: 15. In some embodiments, the encoded NeuroD1 is a NeuroD1 polypeptide having the amino acid sequence of SEQ ID NO: 13, where an extra V is located at the second residue. In some embodiments, the encoded NeuroD1 is a NeuroD1 polypeptide having the amino acid sequence of SEQ ID NO: 7 having a T to A mutation at position 45.

In alternative embodiments, the encoded NeuroD1 polypeptide is a functional derivative of NeuroD1. In some embodiments, a functional derivative of NeuroD1 shares at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%sequence identity with respect to the native (e.g., wild-type) NeuroD1 protein from which it derives.

In some embodiments, a functional derivative of NeuroD1 comprises one or more modifications to one or more predicted non-essential amino acid residues in the NeuroD1 sequence. Methods well-known in the art can be used to analyze a protein (e.g., NeuroD1) sequence to identify essential and non-essential amino acid residues of the protein. For example, in some embodiments, an amino acid residue of a protein that is not conserved among orthologous gene products is predicted to be a non-essential amino acid residue, while another amino acid residue that is conserved among orthologous gene products is predicted to be an essential amino acid residue. An exemplary alignment of NeuroD1 orthologs is shown in Figure 34, and the conserved residues and non-conserved residues are marked with different shades, respectively.

In specific embodiments, a functional derivative of NeuroD1 comprises one or more conservative amino acid substitutions at one or more predicted non-essential amino acid residues of NeuroD1. In specific embodiments, a functional derivative of NeuroD1 comprises one or more conservative amino acid substitutions at one or more predicted essential amino acid residues of NeuroD1.

In some embodiments, a functional derivative of NeuroD1 retains the NeuroD1 function in producing one or more neuronal phenotypes in a glial cell, which neuronal phenotypes include but are not limited to neuronal morphology, expression of one or more neuronal marker, electrophysiologic characteristics of neurons, synapse formation and release of neurotransmitters. Methods disclosed herein (see e.g., Example section) and/or well-known in the art can be used to measure the one or more neuronal phenotypes. In some embodiments, a functional derivative of NeuroD1 retains the NeuroD1 function in reprogramming a glial cell to trans-differentiate into a neuron.

In specific embodiments, a functional derivative of NeuroD1 comprises one or more conservative amino acid substitutions at one or more predicted non-essential amino acid residues, and shares at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%sequence identity with respect to a wild-type NeuroD1 protein. In some embodiments, the wild-type NeuroD1 protein from which the functional derivative is derived is a wild-type human NeuroD1 having SEQ ID NO: 15. In some embodiments, the NeuroD1 protein from which the functional derivative is derived is a NeuroD1 polypeptide having the amino acid sequence of SEQ ID NO: 13. In some embodiments, the NeuroD1 protein from which the functional derivative is derived is a NeuroD1 polypeptide having the amino acid sequence of SEQ ID NO: 7.

In specific embodiments, a functional derivative of NeuroD1 comprises one or more conservative amino acid substitutions at one or more predicted non-essential amino acid residues, and shares at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%sequence identity with respect to the native (e.g., wild-type) NeuroD1 protein from which it derives, and further retains the function in producing one or more neuronal phenotypes in a glial cell when expressed in a sufficient amount by the glial cell. In some embodiments, the wild-type NeuroD1 protein from which the functional derivative is derived is a wild-type human NeuroD1 having the amino acid sequence of SEQ ID NO: 15. In some embodiments, the NeuroD1 protein from which the functional derivative is derived is a NeuroD1 polypeptide having the amino acid sequence of SEQ ID NO: 13. In some embodiments, the NeuroD1 protein from which the functional derivative is derived is a NeuroD1 polypeptide having the amino acid sequence of SEQ ID NO: 7.

In specific embodiments, a functional derivative of NeuroD1 comprises one or more conservative amino acid substitutions at one or more predicted non-essential amino acid residues, and shares at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%sequence identity with respect to the native (e.g., wild-type) NeuroD1 protein from which it derives, and further retains the function in reprogramming a glial cell to trans-differentiate into a neuron when expressed in a sufficient amount by the glial cell. In some embodiments, the wild-type NeuroD1 protein from which the functional derivative is derived is a wild-type human NeuroD1 having the amino acid sequence of SEQ ID NO: 15. In some embodiments, the NeuroD1 protein from which the functional derivative is derived is a NeuroD1 polypeptide having the amino acid sequence of SEQ ID NO: 13. In some embodiments, the NeuroD1 protein from which the functional derivative is derived is a NeuroD1 polypeptide having the amino acid sequence of SEQ ID NO: 7.

In specific embodiments, a functional derivative of NeuroD1 comprises one or more conservative amino acid substitutions at one or more predicted essential amino acid residues, and shares at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%sequence identity with respect to the native (e.g., wild-type) NeuroD1 protein from which it derives, and further retains the function in producing one or more neuronal phenotypes in a glial cell when expressed in a sufficient amount by the glial cell. In some embodiments, the wild-type NeuroD1 protein from which the functional derivative is derived is a wild-type human NeuroD1 having the amino acid sequence of SEQ ID NO: 15. In some embodiments, the NeuroD1 protein from which the functional derivative is derived is a NeuroD1 polypeptide having the amino acid sequence of SEQ ID NO: 13. In some embodiments, the NeuroD1 protein from which the functional derivative is derived is a NeuroD1 polypeptide having the amino acid sequence of SEQ ID NO: 7.

In specific embodiments, a functional derivative of NeuroD1 comprises one or more conservative amino acid substitutions at one or more predicted essential amino acid residues, and shares at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%sequence identity with respect to the native (e.g., wild-type) NeuroD1 protein from which it derives, and further retains the function in reprogramming a glial cell to trans-differentiate into a neuron when expressed in a sufficient amount by the glial cell. In some embodiments, the wild-type NeuroD1 protein from which the functional derivative is derived is a wild-type human NeuroD1 having the amino acid sequence of SEQ ID NO: 15. In some embodiments, the NeuroD1 protein from which the functional derivative is derived is a NeuroD1 polypeptide having the amino acid sequence of SEQ ID NO: 13. In some embodiments, the NeuroD1 protein from which the functional derivative is derived is a NeuroD1 polypeptide having the amino acid sequence of SEQ ID NO: 7.

In some embodiments, the encoded NeuroD1 polypeptide is encoded by (a) a DNA sequence of SEQ ID NO: 4, SEQ ID NO: 14, or SEQ ID NO: 3, (b) a codon-optimized variant of (a) , or (c) a transcribed RNA sequence of (a) or (b) . In some embodiments, the codon-optimized variant shares at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%sequence identity to SEQ ID NO: 4. In some embodiments, the codon-optimized variant shares at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%sequence identity to SEQ ID NO: 14. In some embodiments, the codon-optimized variant shares at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%sequence identity to SEQ ID NO: 3. In some embodiments, the transcribed RNA sequence has the same sequence as the DNA coding sequences except that thymine bases in the DNA sequence are replaced by uracil bases in the RNA sequence. In particular embodiments, the NeuroD1 expression cassette is mono-cistronic and encodes only one NeuroD1 polypeptide as described herein.

In alternative embodiments, the NeuroD1 expression cassette is multi-cistronic and encodes multiple NeuroD1 polypeptides as described herein. In some embodiments, a multi-cistronic expression sequence encoding at least two NeuroD1 polypeptides further encodes an internal ribosome entry site (IRES) that separate two ORFs. Without being bound by the theory, it is contemplated that an internal ribosome entry sites (IRES) can act as the sole ribosome binding site, or serve as one of multiple ribosome binding sites of an mRNA. An mRNA molecule containing more than one functional ribosome binding site can encode several peptides or proteins that are translated independently by the ribosomes (e.g., multicistronic mRNA) . Accordingly, in some embodiments, the nucleic acid molecule of the present disclosure (e.g., mRNA) comprises one or more internal ribosome entry sites (IRES) . Examples of IRES sequences that can be used in connection with the present disclosure include, without limitation, those from picomaviruses (e.g., FMDV) , pest viruses (CFFV) , polio viruses (PV) , encephalomyocarditis viruses (ECMV) , foot-and-mouth disease viruses (FMDV) , hepatitis C viruses (HCV) , classical swine fever viruses (CSFV) , murine leukemia virus (MLV) , simian immune deficiency viruses (SIV) or cricket paralysis viruses (CrPV) .

In particular embodiments, the IRES has a sequence of an IRES from Taura syndrome virus, Triatoma virus, Theiler's encephalomyelitis virus, Simian Virus 40, Solenopsis invicta virus 1, Rhopalosiphum padi virus, Reticuloendotheliosis virus, Human poliovirus 1, Plautia stall intestine virus, Kashmir bee virus, Human rhinovirus 2, Homalodisca coagulata virus-1, Human Immunodeficiency Virus type 1, Homalodisca coagulata virus-1, Himetobi P virus, Hepatitis C virus, Hepatitis A virus, Hepatitis GB virus, Foot and mouth disease virus, Human enterovirus 71, Equine rhinitis virus, Ectropis obliqua picorna-like virus, Encephalomyocarditis virus, Drosophila C Virus, Human coxsackievirus B3, Crucifer tobamovirus, Cricket paralysis virus, Bovine viral diarrhea virus 1, Black Queen Cell Virus, Aphid lethal paralysis virus, Avian encephalomyelitis virus, Acute bee paralysis virus, Hibiscus chlorotic ringspot virus, Classical swine fever virus, Human FGF2, Human SFTPA1, Human AML1/RUNX1, Drosophila antennapedia, Human AQP4, Human AT1R, Human BAG-1, Human BCL2, Human BiP, Human c-IAP1, Human c-myc, Human eIF4G, Mouse NDST4L, Human LEF1, Mouse HIF1 alpha, Human n. myc, Mouse Gtx, Human p27kip1, Human PDGF2/c-sis, Human p53, Human Pim-1, Mouse Rbm3, Drosophila reaper, Canine Scamper, Drosophila Ubx, Human UNR, Mouse UtrA, Human VEGF-A, Human XIAP, Drosophila hairless, S. cerevisiae TFIID, S. cerevisiae YAP1, tobacco etch virus, turnip crinkle virus, EMCV-A, EMCV-B, EMCV-Bf, EMCV-Cf, EMCV pEC9, Picobirnavirus, HCV QC64, Human Cosavirus E/D, Human Cosavirus F, Human Cosavirus JMY, Rhinovirus NAT001, HRV14, HRV89, HRVC-02, HRV-A21, Salivirus A SH1, Salivirus FHB, Salivirus NG-J1, Human Parechovirus 1, Crohivirus B, Yc-3, Rosavirus M-7, Shanbavirus A, Pasivirus A, Pasivirus A 2, Echovirus E14, Human Parechovirus 5, Aichi Virus, Hepatitis A Virus HA16, Phopivirus, CVA10, Enterovirus C, Enterovirus D, Enterovirus J, Human Pegivirus 2, GBV-C GT110, GBV-C K1737, GBV-C Iowa, Pegivirus A 1220, Pasivirus A 3, Sapelovirus, Rosavirus B, Bakunsa Virus, Tremovirus A, Swine Pasivirus 1, PLV-CHN, Pasivirus A, Sicinivirus, Hepacivirus K, Hepacivirus A, BVDV1, Border Disease Virus, BVDV2, CSFV-PK15C, SF573 Dicistrovirus, Hubei Picorna-like Virus, CRPV, Apodemus Agrarius Picornavirus, Caprine Kobuvirus, Parabovirus, Salivirus A BN5, Salivirus A BN2, Salivirus A 02394, Salivirus A GUT, Salivirus A CH, Salivirus A SZ1, Salivirus FHB, CVB3, CVB1, Echovirus 7, CVB5, EVA71, CVA3, CVA12, EV24, or an aptamer to eIF4G.
5.1.2. Untranslated Regions (UTRs)

In some embodiments, the NeuroD1 expression cassette comprises one or more untranslated regions (UTRs) .

In the particular embodiments, the untranslated region (UTR) located upstream (to the 5’ -end) of the coding region is referred to herein as the 5’ -UTR, and the UTR located upstream (to the 3’ -end) of the coding region is referred to herein as the 3’ -UTR. In particular embodiments, the NeuroD1 expression cassette comprises both a 5’ -UTR and a 3’ -UTR. In some embodiments, the NeuroD1 expression cassette comprises a Kozak sequence (e.g., in the 5’ -UTR) . In some embodiments, the NeuroD1 expression cassette comprises a polyadenylation signal (e.g., in the 3’ -UTR) . In particular embodiments, the NeuroD1 expression cassette comprises a polyadenylation signal having the sequence set forth in SEQ ID NO: 9 located in the 3’ -UTR. In particular embodiments, the NeuroD1 expression cassette comprises a SV40 polyadenylation signal having the sequence set forth in SEQ ID NO: 28 located in the 3’ -UTR. In some embodiments, the NeuroD1 expression cassette comprises stabilizing region (e.g., in the 3’ -UTR) . In some embodiments, the NeuroD1 expression cassette comprises a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) having the sequence set forth in SEQ ID NO: 12 located in the 3’ -UTR. In some embodiments, the NeuroD1 expression cassette comprises a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) having the sequence set forth in SEQ ID NO: 27 located in the 3’ -UTR. In some embodiments, the NeuroD1 expression cassette comprises one or more intronic regions capable of being excised during splicing (e.g., in the 5’ -UTR) . In one specific embodiment, the NeuroD1 expression cassette comprises a chimeric intron comprising the sequence set forth in SEQ ID NO: 11 located in the 5’ -UTR. In one specific embodiment, the NeuroD1 expression cassette comprises a chimeric intron comprising the sequence set forth in SEQ ID NO: 19 located in the 5’ -UTR. In one specific embodiment, the NeuroD1 expression cassette comprises a chimeric intron comprising the sequence set forth in SEQ ID NO: 26 located in the 5’ -UTR. In some embodiments, the NeuroD1 expression cassette comprises a promoter (e.g., in the 5’ -UTR) . In a specific embodiment, the NeuroD1 expression cassette comprises a glial fibrillary acid protein (GFAP) promoter comprising the sequence set forth in SEQ ID NO: 10 located in the 5’ -UTR. In some embodiments, the NeuroD1 expression cassette comprises a transcription enhancer element (e.g., in the 5’ -UTR or 3’ -UTR) . In a specific embodiment, the NeuroD1 expression cassette comprises a CMV enhancer comprising the sequence set forth in SEQ ID NO: 8 located in the 5’ -UTR. In a specific embodiment, the NeuroD1 expression cassette comprises a EF1α enhancer comprising the sequence set forth in SEQ ID NO: 25 located in the 5’ -UTR. In a specific embodiment, the nucleic acid molecule comprises one or more region selected from a 5’ -UTR, and a coding region. In a specific embodiment, the nucleic acid molecule comprises a coding region and one or more region selected from a 3’ -UTR. In a specific embodiment, the nucleic acid molecule comprises one or more region selected from a 5’ -UTR, a coding region, and one or more region selected from a 3’ -UTR.

In some embodiments, the sequence of an UTR can be homologous or heterologous to the sequence of the coding region found in a nucleic acid molecule. Multiple UTRs can be included in a nucleic acid molecule and can be of the same or different sequences, and/or genetic origin. According to the present disclosure, any portion of UTRs in a nucleic acid molecule (including none) can be codon optimized and any may independently contain one or more different structural or chemical modification, before and/or after codon optimization.

In some embodiments, a NeuroD1 expression cassette of the present disclosure comprises UTRs and coding regions that are homologous with respect to each other. In other embodiments, a NeuroD1 expression cassette of the present disclosure comprises UTRs and coding regions that are heterologous with respect to each other. In some embodiments, to monitor the activity of a UTR sequence, a nucleic acid molecule comprising the UTR and a coding sequence of a detectable probe can be administered in vitro (e.g., cell or tissue culture) or in vivo (e.g., to a subject) , and an effect of the UTR sequence (e.g., modulation on the expression level, cellular localization of the encoded product, or half-life of the encoded product) can be measured using methods known in the art.

Combinations of regulatory elements may be used to drive expression of an operably linked coding sequence. According to the present disclosure, homologues and functional variants of ubiquitous or cell type-specific promoters may be used in expressing the operably linked coding sequence as described herein. The terms “promoter homologue” and “promoter variant” refer to a promoter which has substantially similar functional properties to confer the desired type of expression, such as cell type-specific expression of the NeuroD1 polypeptide or ubiquitous expression of the NeuroD1 polypeptide, of an operably linked coding sequence of the NeuroD1 polypeptide compared to a given promoter disclosed herein. For example, a promoter homologue or promoter variant has substantially similar functional properties to confer cell type-specific expression of an operably linked coding sequence encoding the NeuroD1 polypeptide compared to any of a GFAP, AldhlL1, NG2, lcn2, S100b, Sox9, CAG, CMV, ubiquitin, or EF-1a promoter.

One of skill in the art will recognize that one or more nucleic acid mutations can be introduced without altering the functional properties of a given promoter. Mutations can be introduced using standard molecular biology techniques, such as DNA synthesis, site-directed mutagenesis and PCR-mediated mutagenesis, to produce promoter variants. As used herein, the term “promoter variant” refers to either an isolated naturally occurring or a recombinantly prepared variation of a reference promoter, such as, but not limited to GFAP, AldhlL1, NG2, lcn2, S100b, Sox9, CAG, CMV, ubiquitin, or EF-1a promoter.

It is known in the art that promoters from other species are functional, e.g. the mouse AldhlLl promoter is known to be functional in human cells. Homologues and homologous promoters from other species can be identified using bioinformatics tools known in the art, see for example, Xuan et al., 2005, Genome Biol 6: R72; Zhao et al., 2005, Nucl Acid Res 33: D103-107; and Halees et al. 2003, Nucl. Acids. Res. 2003 31: 3554-3559.

Structurally, homologues and variants of a cell type-specific promoter or an ubiquitous promoter can have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater, nucleic acid sequence identity to the reference promoter and include a site for binding of RNA polymerase and, optionally, one or more binding sites for transcription factors.

In some embodiments, the UTR of a nucleic acid molecule of the present disclosure (e.g., NeuroD1 expression cassette) comprises at least one translation enhancer element (TEE) that functions to increase the amount of polypeptide or protein produced from the nucleic acid molecule. In some embodiments, the TEE is located in the 5’ -UTR of the nucleic acid molecule. In other embodiments, the TEE is located at the 3’ -UTR of the nucleic acid molecule. In yet other embodiments, at least two TEE are located at the 5’ -UTR and 3’ -UTR of the nucleic acid molecule respectively. In some embodiments, a nucleic acid molecule of the present disclosure can comprise one or more copies of a TEE sequence or comprise more than one different TEE sequences. In some embodiments, different TEE sequences that are present in a nucleic acid molecule of the present disclosure can be homologues or heterologous with respect to one another.

In some embodiments, the TEE sequence is derived from a promoter sequence of a gene. In some embodiments, a promoter can be derived entirely from a single gene. In other embodiments, a promoter can be chimeric, having portions derived from more than one gene.

In some embodiments, the TEE sequence used in connection with the present disclosure can drive expression of an operably linked expression sequence preferentially in glial cells. In some embodiments, the TEE sequence drives expression of an operably linked expression sequence preferentially in astrocytes. In some embodiments, the TEE sequence drives expression of an operably linked expression sequence preferentially in reactive astrocytes. In some embodiments, the TEE sequence drives expression of an operably linked expression sequence preferentially in NG2 cells. In some embodiments, the TEE sequence drives expression of an operably linked expression sequence preferentially in reactive NG2 cells. In some embodiments, the TEE sequence drives expression of an operably linked expression sequence preferentially in Müller glia cells.

Additionally, various TEE sequences that are known in the art and can be used in connection with the present disclosure. For example, in some embodiments, the TEE can be an internal ribosome entry site (IRES) , HCV-IRES or an IRES element. Chappell et al. Proc. Natl. Acad. Sci. USA 101: 9590-9594, 2004; Zhou et al. Proc. Natl. Acad. Sci. 102: 6273-6278, 2005. Additional internal ribosome entry site (IRES) that can be used in connection with the present disclosure include but are not limited to those described in U.S. Patent No. 7,468,275, U.S. Patent Publication No. 2007/0048776 and U.S. Patent Publication No. 2011/0124100 and International Patent Publication No. WO2007/025008 and International Patent Publication No. WO2001/055369, the content of each of which is enclosed herein by reference in its entirety. In some embodiments, the TEE can be those described in Supplemental Table 1 and in Supplemental Table 2 of Wellensiek et al Genome-wide profiling of human cap-independent translation-enhancing elements, Nature Methods, 2013 Aug; 10 (8) : 747–750; the content of which is incorporated by reference in its entirety.

Additional exemplary TEEs that can be used in connection with the present disclosure include but are not limited to the TEE sequences disclosed in U.S. Patent No. 6,310,197, U.S. Patent No. 6,849,405, U.S. Patent No. 7,456,273, U.S. Patent No. 7,183,395, U.S. Patent Publication No. 2009/0226470, U.S. Patent Publication No. 2013/0177581, U.S. Patent Publication No. 2007/0048776, U.S. Patent Publication No. 2011/0124100, U.S. Patent Publication No. 2009/0093049, International Patent Publication No. WO2009/075886, International Patent Publication No. WO2012/009644, and International Patent Publication No. WO1999/024595, International Patent Publication No.WO2007/025008, International Patent Publication No. WO2001/055371, European Patent No. 2610341, European Patent No. 2610340, the content of each of which is enclosed herein by reference in its entirety.

In various embodiments, a nucleic acid molecule of the present disclosure (e.g., an NeuroD1 expression cassette) comprises at least one UTR that comprises 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 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18 at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55 or more than 60 TEE sequences. In some embodiments, the TEE sequences in the UTR of a nucleic acid molecule are copies of the same TEE sequence. In other embodiments, at least two TEE sequences in the UTR of a nucleic acid molecule are of different TEE sequences. In some embodiments, multiple different TEE sequences are arranged in one or more repeating patterns in the UTR region of a nucleic acid molecule. For illustrating purpose only, a repeating pattern can be, for example, ABABAB, AABBAABBAABB, ABCABCABC, or the like, where in these exemplary patterns, each capitalized letter (A, B, or C) represents a different TEE sequence. In some embodiments, at least two TEE sequences are consecutive with one another (i.e., no spacer sequence in between) in a UTR of a nucleic acid molecule. In other embodiments, at least two TEE sequences are separated by a spacer sequence. In some embodiments, a UTR can comprise a TEE sequence-spacer sequence module that is repeated at least once, at least twice, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, or more than 9 times in the UTR. In any of the embodiments described in this paragraph, the UTR can be a 5’ -UTR, a 3’ -UTR or both 5’ -UTR and 3’ -UTR of a nucleic acid molecule.

In some embodiments, the UTR of a nucleic acid molecule of the present disclosure comprises at least one translation suppressing element that functions to decrease the amount of polypeptide or protein produced from the nucleic acid molecule. In some embodiments, the UTR of the nucleic acid molecule comprises one or more miR sequences or fragment thereof (e.g., miR seed sequences) that are recognized by one or more microRNA. Other mechanisms for suppressing translational activities associated with nucleic acid molecules are known in the art. In some of the embodiments described in this paragraph, the nucleic acid molecule is linear, and the UTR can be a 5’ -UTR, a 3’ -UTR or both 5’ -UTR and 3’ -UTR of a nucleic acid molecule.

Table 5.1.2 (A) shows exemplary 5’ -UTR and 3’ -UTR sequences that can be operably linked to a NeuroD1 coding sequence as described herein.
Table 5.1.2 (A) Examples of UTRs.

In some embodiments, the expression cassette is mono-cistronic and encodes one copy of a NeuroD1 polypeptide. In some embodiments, the encoded NeuroD1 polypeptide can be any NeuroD1 polypeptide as described in Section 5.1.1 (Coding Region) . In some embodiments, the encoded NeuroD1 polypeptide comprises an amino acid sequence having at least 95%sequence identity to the sequence set forth in SEQ ID NO: 15. In some embodiments, the encoded NeuroD1 polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the encoded NeuroD1 polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, coding sequence that encodes the NeuroD1 polypeptide comprises the nucleic acid sequence as set forth in SEQ ID NO: 14 or a codon-optimized variant thereof. In some embodiments, coding sequence that encodes the NeuroD1 polypeptide comprises the nucleic acid sequence as set forth in SEQ ID NO: 4 or a codon-optimized variant thereof. In some embodiments, coding sequence that encodes the NeuroD1 polypeptide comprises the nucleic acid sequence as set forth in SEQ ID NO: 3 or a codon-optimized variant thereof.

In some embodiments, the expression cassette further comprises one or more untranslated regions (UTRs) . In some embodiments, the UTR comprises one or more regulatory elements operably linked to the coding sequence that encodes the NeuroD1 polypeptide. In some embodiments, the UTRs can be any UTR as described in Section 5.1.2 (Untranslated Regions (UTRs) ) . In some embodiments, the expression cassette comprises a 5’ UTR located upstream (to the 5’ end) of the coding sequence that encodes the NeuroD1 polypeptide. In some embodiments, the 5’ -UTR comprises, from the 5’ to 3’ direction, a CMV enhancer, a glial fibrillary acid protein (GFAP) promoter, and a chimeric intron. In some embodiments, the 5’ -UTR comprises, from the 5’ to 3’ direction, an EF1α enhancer, a GFAP promoter, and a chimeric intron.

In some embodiments, the CMV enhancer comprises the sequence set forth in SEQ ID NO: 8, or a functional variant having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity thereof. In some embodiments, the CMV enhancer consists of the sequence set forth in SEQ ID NO: 8.

In some embodiments, the EF1α enhancer comprises the sequence set forth in SEQ ID NO: 25, or a functional variant having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity thereof. In some embodiments, the EF1α enhancer consists of the sequence set forth in SEQ ID NO: 25.

In some embodiments, the GFAP promoter comprises the sequence set forth in SEQ ID NO: 10, or a functional variant having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity thereof. In some embodiments, the GFAP promoter consists of the sequence set forth in SEQ ID NO: 10.

In some embodiments, the chimeric intron comprises the sequence set forth in SEQ ID NO: 11, or a functional variant having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity thereof. In some embodiments, the chimeric intron consists of the sequence set forth in SEQ ID NO: 11.

In some embodiments, the chimeric intron comprises the sequence set forth in SEQ ID NO: 19, or a functional variant having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity thereof. In some embodiments, the chimeric intron consists of the sequence set forth in SEQ ID NO: 19.

In some embodiments, the chimeric intron comprises the sequence set forth in SEQ ID NO: 26, or a functional variant having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity thereof. In some embodiments, the chimeric intron consists of the sequence set forth in SEQ ID NO: 26.

In some embodiments, the 5’ UTR of the expression cassette comprises the sequence set forth in SEQ ID NO: 31. In some embodiments, the expression cassette comprises a 5’ UTR comprising the sequence set forth in SEQ ID NO: 31, wherein the 5’ UTR is linked to the 5’ end of a coding sequence that encodes a NeuroD1 polypeptide comprising an amino acid sequence having at least 95%sequence identity to the sequence set forth in SEQ ID NO: 15. In some embodiments, the expression cassette comprises a 5’ UTR comprising the sequence set forth in SEQ ID NO: 31, wherein the 5’ UTR is linked to the 5’ end of a coding sequence that encodes a NeuroD1 polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the expression cassette comprises a 5’ UTR comprising the sequence set forth in SEQ ID NO: 31, wherein the 5’ UTR is linked to the 5’ end of a coding sequence that encodes a NeuroD1 polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the expression cassette comprises a 5’ UTR comprising the sequence set forth in SEQ ID NO: 31, wherein the 5’ UTR is linked to the 5’ end of a coding sequence that encodes a NeuroD1 polypeptide comprising the nucleic acid sequence set forth in SEQ ID NO: 14, or a codon optimized variant thereof. In some embodiments, the expression cassette comprises a 5’ UTR comprising the sequence set forth in SEQ ID NO: 31, wherein the 5’ UTR is linked to the 5’ end of a coding sequence that encodes a NeuroD1 polypeptide comprising the nucleic acid sequence set forth in SEQ ID NO: 4, or a codon optimized variant thereof. In some embodiments, the expression cassette comprises a 5’ UTR comprising the sequence set forth in SEQ ID NO: 31, wherein the 5’ UTR is linked to the 5’ end of a coding sequence that encodes a NeuroD1 polypeptide comprising the nucleic acid sequence set forth in SEQ ID NO: 3, or a codon optimized variant thereof.

In some embodiments, the expression cassette comprises a 3’ UTR located downstream (to the 3’ end) of the coding sequence that encodes the NeuroD1 polypeptide.

In some embodiments, the 3’ -UTR comprises a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) . In some embodiments, a WPRE nucleic acid sequence is an optimized version of WPRE. In some embodiments, the optimized WPRE comprises the sequence set forth in SEQ ID NO: 12, or a functional variant having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity thereof. In some embodiments, the optimized WPRE consists of the sequence set forth in SEQ ID NO: 12. In some embodiments, the optimized WPRE comprises the sequence set forth in SEQ ID NO: 27, or a functional variant having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity thereof. In some embodiments, the chimeric intron consists of the sequence set forth in SEQ ID NO: 27.

In some embodiments, the 3’ -UTR comprises a polyadenylation signal comprising the sequence set forth in SEQ ID NO: 9, or a functional variant having at least 90%sequence identity thereof. In some embodiments, the polyadenylation signal consists of the sequence set forth in SEQ ID NO: 9. In some embodiments, the polyadenylation signal comprises a SV40 polyadenylation signal. In some embodiments, the SV40 polyadenylation signal comprising the sequence set forth in SEQ ID NO: 28, or a functional variant having at least 90%sequence identity thereof. In some embodiments, the SV40 polyadenylation signal consists of the sequence set forth in SEQ ID NO: 28. In some embodiments, the polyadenylation signal comprises a human beta globin polyadenylation signal. In some embodiments, the polyadenylation signal comprises a polyadenylation signal originated from a human gene. In some embodiments, the polyadenylation signal comprises a polyadenylation signal originated from a non-human gene.

In some embodiments, the 3’ UTR of the expression cassette comprises the sequence set forth in SEQ ID NO: 32. In some embodiments, the expression cassette comprises a 3’ UTR comprising the sequence set forth in SEQ ID NO: 32, wherein the 3’ UTR is linked to the 3’ end of a coding sequence that encodes a NeuroD1 polypeptide comprising an amino acid sequence having at least 95%sequence identity to the sequence set forth in SEQ ID NO: 15. In some embodiments, the expression cassette comprises a 3’ UTR comprising the sequence set forth in SEQ ID NO: 32, wherein the 3’ UTR is linked to the 3’ end of a coding sequence that encodes a NeuroD1 polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 13. In some embodiments, the expression cassette comprises a 3’ UTR comprising the sequence set forth in SEQ ID NO: 32, wherein the 3’ UTR is linked to the 3’ end of a coding sequence that encodes a NeuroD1 polypeptide comprising an amino acid sequence having at least 95%sequence identity to the sequence set forth in SEQ ID NO: 7. In some embodiments, the expression cassette comprises a 3’ UTR comprising the sequence set forth in SEQ ID NO: 32, wherein the 3’ UTR is linked to the 3’ end of a coding sequence that encodes a NeuroD1 polypeptide comprising the nucleic acid sequence set forth in SEQ ID NO: 14, or a codon optimized variant thereof. In some embodiments, the expression cassette comprises a 3’ UTR comprising the sequence set forth in SEQ ID NO: 32, wherein the 3’UTR is linked to the 3’ end of a coding sequence that encodes a NeuroD1 polypeptide comprising the nucleic acid sequence set forth in SEQ ID NO: 4 or codon-optimized variant thereof. In some embodiments, the expression cassette comprises a 3’ UTR comprising the sequence set forth in SEQ ID NO:32, wherein the 3’ UTR is linked to the 3’ end of a coding sequence that encodes a NeuroD1 polypeptide comprising the nucleic acid sequence set forth in SEQ ID NO: 3 or codon-optimized variant thereof.

Table 5.1.2 (B) below lists the sequences of functional fragments in a NeuroD1 expression cassette according to some embodiments described herein. In some embodiments, the expression cassette comprises, from the 5’ to 3’ direction, a CMV enhancer (SEQ ID NO: 8) , a GFAP promoter (SEQ ID NO: 10) , a chimeric intron (SEQ ID NO: 19) , a coding sequence (SEQ ID NO: 4) that encodes a NeuroD1 polypeptide (SEQ ID NO: 13) , an optimized WPRE (SEQ ID NO: 12) and a polyadenylation signal (SEQ ID NO: 9) . In specific embodiments, the CMV enhancer (SEQ ID NO: 8) , GFAP promoter (SEQ ID NO: 10) , chimeric intron (SEQ ID NO: 19) , coding sequence that encodes a NeuroD1 polypeptide (SEQ ID NO: 4) , optimized WPRE (SEQ ID NO: 12) and polyadenylation signal (SEQ ID NO: 9) are connected directly to each other in the 5’ to 3’ order. In specific embodiments, the CMV enhancer (SEQ ID NO: 8) , GFAP promoter (SEQ ID NO: 10) , chimeric intron (SEQ ID NO: 19) , coding sequence that encodes a NeuroD1 polypeptide (SEQ ID NO: 4) , optimized WPRE (SEQ ID NO: 12) and polyadenylation signal (SEQ ID NO: 9) are connected to each other in the 5’ to 3’ order via linkers.
Table 5.1.2 (B) Example of a NeuroD1 Expression Cassette

5.2. AAV Genome

In some embodiments, the expression cassette described herein is part of a single-stranded nucleic acid molecule. In some embodiments, the single-stranded nucleic acid molecule is a DNA molecule. In some embodiments, the single-stranded nucleic acid molecule is an artificial AAV genome that can be packaged into a recombinant AAV capsid.

In some embodiments, the single-stranded nucleic acid molecule comprises a first inverted terminal repeat (ITR) of a first AAV genome located at 5’ UTR. In some embodiments, the single-stranded nucleic acid molecule comprises a second inverted terminal repeat (ITR) of a second AAV genome located at 3’ UTR. In some embodiments, the first and the second ITRs are selected from the genomic ITR sequences of AAV serotypes AAV1, AAV2, AAV2tYF, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVrh10, AAV. rh20, AAV. rh39, AAV. Rh74, AAV. RHM4-1, AAV. hu37, AAV. Anc80, AAV. Anc80L65, rAAV. 7m8, AAV. PHP. B, AAV. PHP. eB, AAV2.5, AAV2tYF, AAV3B, AAV. LK03, AAV. HSC1, AAV. HSC2, AAV. HSC3, AAV. HSC4, AAV. HSC5, AAV. HSC6, AAV. HSC7, AAV. HSC8, AAV. HSC9, AAV. HSC10 , AAV. HSC11, AAV. HSC12, AAV. HSC13, AAV. HSC14, AAV. HSC15, and AAV. HSC16. In some embodiments, the first and the second ITRs are selected from the genomic ITR sequences of the same AAV serotype.

In some embodiments, the first ITR is selected from the genomic ITR sequences of AAV serotypes 1 to 8. In some embodiments, the first ITR comprises the 5’ ITR sequence from the AAV1 genome. In some embodiments, the first ITR comprises the 5’ ITR sequence from the AAV2 genome. In some embodiments, the first ITR comprises the 5’ ITR sequence from the AAV3 genome. In some embodiments, the first ITR comprises the 5’ ITR sequence from the AAV4 genome. In some embodiments, the first ITR comprises the 5’ ITR sequence from the AAV5 genome. In some embodiments, the first ITR comprises the 5’ ITR sequence from the AAV6 genome. In some embodiments, the first ITR comprises the 5’ ITR sequence from the AAV7 genome. In some embodiments, the first ITR comprises the 5’ ITR sequence from the AAV8 genome. In some embodiments, the first ITR comprises the full-length ITR sequence from the AAV genome. In some embodiments, the first ITR comprises a truncated version of the ITR sequence from the AAV genome. In some embodiments, the first ITR comprises the wild-type ITR sequence from the AAV genome. In some embodiments, the first ITR comprises a mutated ITR sequence from the AAV genome. In some embodiments, the first ITR comprises the sequence set forth in SEQ ID NO: 1, or a functional variant having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity thereof. In some embodiments, the first ITR consists of the sequence set forth in SEQ ID NO: 1. In some embodiments, the first ITR comprises the sequence set forth in SEQ ID NO: 2, or a functional variant having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity thereof. In some embodiments, the first ITR consists of the sequence set forth in SEQ ID NO: 2. In some embodiments, the first ITR comprises the sequence set forth in SEQ ID NO: 16, or a functional variant having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity thereof. In some embodiments, the first ITR consists of the sequence set forth in SEQ ID NO: 16. In some embodiments, the first ITR comprises the sequence set forth in SEQ ID NO: 58, or a functional variant having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity thereof. In some embodiments, the first ITR consists of the sequence set forth in SEQ ID NO: 58

In some embodiments, the single-stranded nucleic acid molecule comprises a second inverted terminal repeat (ITR) of a second AAV genome located at 3’ UTR. In some embodiments, the second ITR is selected from the genomic ITR sequences of AAV serotypes 1 to 8. In some embodiments, the second ITR comprises the 3’ ITR sequence from the AAV1 genome. In some embodiments, the second ITR comprises the 3’ ITR sequence from the AAV2 genome. In some embodiments, the second ITR comprises the 3’ ITR sequence from the AAV3 genome. In some embodiments, the second ITR comprises the 3’ ITR sequence from the AAV4 genome. In some embodiments, the second ITR comprises the 3’ ITR sequence from the AAV5 genome. In some embodiments, the second ITR comprises the 3’ ITR sequence from the AAV6 genome. In some embodiments, the second ITR comprises the 3’ ITR sequence from the AAV7 genome. In some embodiments, the second ITR comprises the 3’ ITR sequence from the AAV8 genome. In some embodiments, the second ITR comprises the full-length ITR sequence from the AAV genome. In some embodiments, the second ITR comprises a truncated version of the ITR sequence from the AAV genome. In some embodiments, the second ITR comprises the wild-type ITR sequence from the AAV genome. In some embodiments, the second ITR comprises a mutated ITR sequence from the AAV genome.

In some embodiments, the second ITR comprises the sequence set forth in SEQ ID NO: 5, or a functional variant having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity thereof. In some embodiments, the second ITR consists of the sequence set forth in SEQ ID NO: 5. In some embodiments, the second ITR comprises the sequence set forth in SEQ ID NO: 6, or a functional variant having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity thereof. In some embodiments, the second ITR consists of the sequence set forth in SEQ ID NO: 6. In some embodiments, the second ITR comprises the sequence set forth in SEQ ID NO: 23, or a functional variant having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity thereof. In some embodiments, the second ITR consists of the sequence set forth in SEQ ID NO: 23. In some embodiments, the second ITR comprises the sequence set forth in SEQ ID NO: 59, or a functional variant having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity thereof. In some embodiments, the second ITR consists of the sequence set forth in SEQ ID NO: 59.

ITR sequences for various AAV serotypes are known in the art. See, for example, GenBank: ITR1: NC_002077.1, nts 1-143, nts 4574-4718, ITR2: NC_001401.2, nts 1-145, nts 4535-4679, ITR3: NC_001729, nts 1-143, 4582-4726, ITR4: NC_001829.1, nts 1-146, nts 4623-4767, ITR5: NC_006152, nts 1-145, nts 4498-4642, ITR6: AF028704.1, nts 1-145, nts 4539-4683, ITR7: NC_006260.1, nts 1-145, nts 4577-4721 for the 5’ (left) ITR sequences.
Table 5.2 Example of a recombinant AAV genome encoding NeuroD1 (AAV-NeuroD1)

5.3. Recombinant AAV Vectors

In some embodiments, the single-stranded nucleic acid molecule is an artificial AAV genome that can be packaged into a AAV capsid to produce a recombinant AAV virion. In some embodiments, such recombinant AAV carries a transgene encoding NeuroD1 in its genome and is sometimes referred to as a AAV vector encoding NeuroD1 in the present disclosure.

In certain embodiments, the AAV vector encoding NeuroD1 further comprises an AAV capsid protein. In certain embodiments, the AAV capsid protein has a serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16, AAV. rh8, AAV. rh10, AAV. rh20, AAV. rh39, AAV. Rh74, AAV. RHM4-1, AAV. hu37, AAV. Anc80, AAV. Anc80L65, AAV. 7m8, AAV. PHP. B, AAV2.5, AAV2tYF, AAV3B, AAV. LK03, AAV. HSC1, AAV. HSC2, AAV. HSC3, AAV. HSC4, AAV. HSC5, AAV. HSC6, AAV. HSC7, AAV. HSC8, AAV. HSC9, AAV. HSC10, AAV. HSC11, AAV. HSC12, AAV. HSC13, AAV. HSC14, AAV. HSC15, and AAV. HSC16, derivatives thereof, modifications thereof, pseudotypes thereof, and combinations thereof. In certain embodiments, the AAV capsid protein comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%or at least about 99.5%, or 100%homologous or identical to the amino acid sequence of viral protein 1 (VP1) , viral protein 2 (VP2) , or viral protein 3 (VP3) of an AAV capsid serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16, AAV. rh8, AAV. rh10, AAV. rh20, AAV. rh39, AAV. Rh74, AAV. RHM4-1, AAV. hu37, AAV. Anc80, AAV. Anc80L65, AAV. 7m8, AAV. PHP. B, AAV2.5, AAV2tYF, AAV3B, AAV. LK03, AAV. HSC1, AAV. HSC2, AAV. HSC3, AAV. HSC4, AAV. HSC5, AAV. HSC6, AAV. HSC7, AAV. HSC8, AAV. HSC9, AAV. HSC10, AAV. HSC11, AAV. HSC12, AAV. HSC13, AAV. HSC14, AAV. HSC15, and AAV. HSC16.

In specific embodiments, the AAV vector encoding NeuroD1 further comprises a AAV serotype 9 (AAV9) capsid. In some embodiments, the AAV9 capsid comprises at least one capsid protein comprises or consists of an amino acid sequence that is about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%or at least about 99.5%, or 100%homologous or identical to the amino acid sequence of viral protein 1 (VP1) , viral protein 2 (VP2) , or viral protein 3 (VP3) of AAV9.

In some embodiments, the AAV9 capsid comprises a capsid protein that is a functional derivative of AAV9 VP1 having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%or at least about 99.5%, or 100%sequence identity to SEQ ID NO: 40. These functional derivative of AAV9 VP1 is collected referred to as “AAV9 VP1 polypeptides. ” In specific embodiments, the AAV9 capsid comprises a capsid protein comprising the amino acid sequence set forth in SEQ ID NO: 40.

In some embodiments, the AAV9 capsid comprises a capsid protein that is a functional derivative of AAV9 VP2 having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%or at least about 99.5%, or 100%sequence identity to SEQ ID NO: 41. These functional derivative of AAV9 VP2 is collected referred to as “AAV9 VP2 polypeptides. ” In specific embodiments, the AAV9 capsid comprises a capsid protein comprising the amino acid sequence set forth in SEQ ID NO: 41.

In some embodiments, the AAV9 capsid comprises a capsid protein that is a functional derivative of AAV9 VP3 having at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%or at least about 99.5%, or 100%sequence identity to SEQ ID NO: 42. These functional derivative of AAV9 VP3 is collected referred to as “AAV9 VP3 polypeptides. ” In specific embodiments, the AAV9 capsid comprises a capsid protein comprising the amino acid sequence set forth in SEQ ID NO: 42.

In some embodiments, the AAV9 capsid comprises an AAV VP1 polypeptide and an AAV VP2 polypeptide. In some embodiments, the AAV9 capsid comprises an AAV VP1 polypeptide and an AAV VP3 polypeptide. In some embodiments, the AAV9 capsid comprises an AAV VP2 polypeptide and an AAV VP3 polypeptide. In some embodiments, the AAV9 capsid comprises an AAV VP1 polypeptide, an AAV VP2 polypeptide, and an AAV VP3 polypeptide.

In yet a particular embodiment, the AAV vector encoding NeuroD1 further comprises a AAV9 capsid, wherein the AAV9 capsid comprises a VP1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 40, a VP2 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 41, and a VP3 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 42.

In certain embodiments, the AAV vector comprises capsid of Anc80 or Anc80L65, as described in Zinn et al., 2015, Cell Rep. 12 (6) : 1056-1068, the content of which is incorporated by reference in its entirety. In certain embodiments, the AAV vector comprises the capsid with one of the following amino acid insertions: LGETTRP or LALGETTRP, as described in U.S. Patent Nos. 9,193,956; 9,458,517; and 9,587,282 and U.S. patent application publication no. 2016/0376323, the content of each of which is incorporated herein by reference in its entirety. In certain embodiments, the AAV vector comprises the capsid of AAV. 7m8, as described in U.S. Patent Nos. 9,193,956; 9,458,517; and 9,587,282 and U.S. patent application publication no. 2016/0376323, the content of each of which is incorporated herein by reference in its entirety. In certain embodiments, the AAV vector comprises any AAV capsid disclosed in U.S. Patent No. 9,585,971 (e.g., AAV-PHP. B) , the content of which is incorporated by reference in its entirety. In certain embodiments, the AAV vector comprises any AAV capsid disclosed in U.S. Patent No. 9,840,719 and WO 2015/013313, such as AAV. Rh74 and RHM4-1, the content of each of which is incorporated herein by reference in its entirety. In certain embodiments, the AAV vector comprises any AAV capsid disclosed in International Publication No. WO 2014/172669, such as AAV rh.74, the content of which is incorporated herein by reference in its entirety. In certain embodiments, the AAV vector comprises the capsid of AAV2/5, as described in Georgiadis et al., 2016, Gene Therapy 23: 857-862 and Georgiadis et al., 2018, Gene Therapy 25: 450, the content of each of which is incorporated by reference in its entirety. In certain embodiments, the AAV vector comprises any AAV capsid disclosed in International Publication No. WO 2017/070491, such as AAV2tYF, the content of which is incorporated herein by reference in its entirety. In certain embodiments, the AAV vector comprises the capsids of AAVLK03 or AAV3B, as described in Puzzo et al., 2017, Sci. Transl. Med. 29 (9) : 418, the content of which is incorporated by reference in its entirety. In certain embodiments, the AAV vector comprises any AAV capsid disclosed in U.S. Patent Nos. 8,628,966; 8,927,514; and 9,923, 120 and International Publication No. WO 2016/049230, such as HSC1, HSC2, HSC3, HSC4, HSC5, HSC6, HSC7, HSC8, HSC9, HSC10, HSC11, HSC12, HSC13, HSC14, HSC15, or HSC16, the content of each of which is incorporated by reference in its entirety.

In certain embodiments, the AAV vector comprises an AAV capsid disclosed in any of the following patents and patent applications, the content of each of which is incorporated herein by reference in its entirety: U.S. Patent Nos. 7,282,199; 7,906,111; 8,524,446; 8,999,678; 8,628,966; 8,927,514; 8,734,809; 9,284,357; 9,409,953; 9,169,299; 9,193,956; 9458517; and 9,587,282; U.S. Patent Publication Nos. 2015/0374803; 2015/0126588; 2017/0067908; 2013/0224836; 2016/0215024; 2017/0051257; and International Patent Publication Nos. WO2015191508; WO2015121501. In certain embodiments, the AAV vector comprises a capsid protein that is at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%or at least about 99.5%, or 100%homologous or identical to the amino acid sequence of the VP1, VP2, or VP3 of an AAV capsid disclosed in any of the following patents and patent applications, the content each of which is incorporated herein by reference in its entirety: U.S. Patent Nos. 7,282,199; 7,906,111; 8,524,446; 8,999,678; 8,628,966; 8,927,514; 8,734,809; 9,284,357; 9,409,953; 9,169,299; 9,193,956; 9458517; and 9,587,282; U.S. Patent Publication Nos. 2015/0374803; 2015/0126588; 2017/0067908; 2013/0224836; 2016/0215024; 2017/0051257; and International Patent Publication Nos. WO2015191508; WO2015121501.

In certain embodiments, the AAV vector comprises a capsid protein disclosed in International Patent Publication Nos. WO 2003/052051 (see, e.g., SEQ ID NO: 2 of WO 2003/052051) , WO 2005/033321 (see, e.g., SEQ ID NOs: 123 and 88 of WO 2005/033321) , WO 03/042397 (see, e.g., SEQ ID NOs: 2, 81, 85, and 97 of WO 03/042397) , WO 2006/068888 (see, e.g., SEQ ID NOs: 1 and 3-6 of WO 2006/068888) , WO 2006/110689 (see, e.g., SEQ ID NOs: 5-38 of WO 2006/110689) , WO2009/104964 (see, e.g., SEQ ID NOs: 1-5, 7, 9, 20, 22, 24 and 31 of WO2009/104964) , WO 2010/127097 (see, e.g., SEQ ID NOs: 5-38 of WO 2010/127097) , and WO 2015/191508 (see, e.g., SEQ ID NOs: 80-294 of WO 2015/191508) , and U.S. Patent Publication No. 20150023924 (see, e.g., SEQ ID NOs: 1, 5-10 of 20150023924) , the contents of each of which is herein incorporated by reference in its entirety. In certain embodiments, the AAV vector comprises a capsid protein at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%or at least about 99.5%, or 100%homologous or identical to the amino acid sequence of the VP1, VP2, or VP3 protein of an AAV capsid disclosed in International Patent Publication Nos. WO 2003/052051 (see, e.g., SEQ ID NO: 2 of WO 2003/052051) , WO 2005/033321 (see, e.g., SEQ ID NOs: 123 and 88 of WO 2005/033321) , WO 03/042397 (see, e.g., SEQ ID NOs: 2, 81, 85, and 97 of WO 03/042397) , WO 2006/068888 (see, e.g., SEQ ID NOs: 1 and 3-6 of WO 2006/068888) , WO 2006/110689 (see, e.g., SEQ ID NOs: 5-38 of WO 2006/110689) , WO2009/104964 (see, e.g., SEQ ID NOs: 1-5, 7, 9, 20, 22, 24 and 31 of WO2009/104964) , WO 2010/127097 (see, e.g., SEQ ID NOs: 5-38 of WO 2010/127097) , and WO 2015/191508 (see, e.g., SEQ ID NOs: 80-294 of WO 2015/191508) , and U.S. Patent Publication No. 20150023924 (see, e.g., SEQ ID NOs: 1, 5-10 of 20150023924) .

In certain embodiments, the AAV vector comprises a pseudotyped AAV capsid. In certain embodiments, the pseudotyped AAV capsids are rAAV2/8 or rAAV2/9 pseudotyped AAV capsids. Methods for producing and using pseudotyped AAV vectors are known in the art (see, e.g., Duan et al., J. Virol., 75: 7662-7671 (2001) ; Halbert et al., J. Virol., 74: 1524-1532 (2000) ; Zolotukhin et al., Methods 28: 158-167 (2002) ; and Auricchio et al., Hum. Molec. Genet. 10: 3075-3081, (2001) , the content of each of which is incorporated by reference in its entirety) .

In certain embodiments, the AAV vector comprises a capsid comprising a capsid protein chimeric of two or more AAV capsid serotypes. In certain embodiments, the capsid protein is a chimeric of two or more AAV capsid proteins of AAV serotypes selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16, AAV. rh8, AAV. rh10, AAV. rh20, AAV. rh39, AAV. Rh74, AAV. RHM4-1, AAV. hu37, AAV. Anc80, AAV. Anc80L65, AAV. 7m8, AAV. PHP. B, AAV2.5, AAV2tYF, AAV3B, AAV. LK03, AAV. HSC1, AAV. HSC2, AAV. HSC3, AAV. HSC4, AAV. HSC5, AAV. HSC6, AAV. HSC7, AAV. HSC8, AAV. HSC9, AAV. HSC10, AAV. HSC11, AAV. HSC12, AAV. HSC13, AAV. HSC14, AAV. HSC15, and AAV. HSC16.

In certain embodiments, the AAV vector comprises an AAV capsid protein chimeric of AAV8 capsid protein and one or more AAV capsid proteins from an AAV serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16, AAV. rh8, AAV. rh10, AAV. rh20, AAV. rh39, AAV. Rh74, AAV. RHM4-1, AAV. hu37, AAV. Anc80, AAV. Anc80L65, AAV. 7m8, AAV. PHP. B, AAV2.5, AAV2tYF, AAV3B, AAV. LK03, AAV. HSC1, AAV. HSC2, AAV. HSC3, AAV. HSC4, AAV. HSC5, AAV. HSC6, AAV. HSC7, AAV. HSC8, AAV. HSC9, AAV. HSC10, AAV. HSC11, AAV. HSC12, AAV. HSC13, AAV. HSC14, AAV. HSC15, and AAV. HSC16. In certain embodiments, the AAV vector comprises an AAV capsid protein chimeric of AAV8 capsid protein and one or more AAV capsid proteins from an AAV serotype selected from the group consisting of AAV1, AAV2, AAV5, AAV6, AAV7, AAV9, AAV10, AAVrh. 8, and AAVrh. 10.

In certain embodiments, the AAV vector comprises an AAV capsid protein chimeric of AAV9 capsid protein the capsid protein of one or more AAV capsid serotypes selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16, AAV. rh8, AAV. rh10, AAV. rh20, AAV. rh39, AAV. Rh74, AAV. RHM4-1, AAV. hu37, AAV. Anc80, AAV. Anc80L65, AAV. 7m8, AAV. PHP. B, AAV2.5, AAV2tYF, AAV3B, AAV. LK03, AAV. HSC1, AAV. HSC2, AAV. HSC3, AAV. HSC4, AAV. HSC5, AAV. HSC6, AAV. HSC7, AAV. HSC8, AAV. HSC9, AAV. HSC10, AAV. HSC11, AAV. HSC12, AAV. HSC13, AAV. HSC14, AAV. HSC15, and AAV. HSC16

In certain embodiments, the AAV vectors comprises a mosaic capsid. In certain embodiments, the mosaic capsid comprises a mixture of viral capsid proteins from different AAV serotypes. In certain embodiments, the mosaic capsid comprises capsid proteins of serotypes selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16, AAV. rh8, AAV. rh10, AAV. rh20, AAV. rh39, AAV. Rh74, AAV. RHM4-1, AAV. hu37, AAV. Anc80, AAV. Anc80L65, AAV. 7m8, AAV. PHP. B, AAV2.5, AAV2tYF, AAV3B, AAV. LK03, AAV. HSC1, AAV. HSC2, AAV. HSC3, AAV. HSC4, AAV. HSC5, AAV. HSC6, AAV. HSC7, AAV. HSC8, AAV. HSC9, AAV. HSC10, AAV. HSC11, AAV. HSC12, AAV. HSC13, AAV. HSC14, AAV. HSC15, and AAV. HSC16. In certain embodiments, the mosaic capsid comprises capsid proteins of serotypes selected from the group consisting of AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh. 8, and AAVrh. 10.

In certain embodiments, the AAV vector comprises a pseudotyped AAV vector. In certain embodiments, the pseudotyped AAV vector comprises a capsid protein of an AAV serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16, AAV. rh8, AAV. rh10, AAV. rh20, AAV. rh39, AAV. Rh74, AAV. RHM4-1, AAV. hu37, AAV. Anc80, AAV. Anc80L65, AAV. 7m8, AAV. PHP. B, AAV2.5, AAV2tYF, AAV3B, AAV. LK03, AAV. HSC1, AAV. HSC2, AAV. HSC3, AAV. HSC4, AAV. HSC5, AAV. HSC6, AAV. HSC7, AAV. HSC8, AAV. HSC9, AAV. HSC10, AAV. HSC11, AAV. HSC12, AAV. HSC13, AAV. HSC14, AAV. HSC15, and AAV. HSC16. In certain embodiments, the pseudotyped AAV vector are AAV2/8 or AAV2/9 pseudotyped vectors. Methods for producing and using pseudotyped AAV vectors are known in the art (see, e.g., Duan et al., J. Virol., 75: 7662-7671 (2001) ; Halbert et al., J. Virol., 74: 1524-1532 (2000) ; Zolotukhin et al., Methods 28: 158-167 (2002) ; and Auricchio et al., Hum. Molec. Genet. 10: 3075-3081, (2001, the content of each of which is incorporated herein by reference in its entirety) .

Nucleotide sequences of AAV vectors and methods of making thereof are taught, for example, in U.S. Patent Nos. 7,282,199; 7,906,111; 8,524,446; 8,999,678; 8,628,966; 8,927,514; 8,734,809; US 9,284,357; 9,409,953; 9,169,299; 9,193,956; 9458517; and 9,587,282; U.S. Patent Publication Nos. 2015/0374803; 2015/0126588; 2017/0067908; 2013/0224836; and 2016/0215024; 2017/0051257; 2015/0023924; International Patent Publication Nos. WO2015191508; WO2015121501; WO 2003/052051, WO 2005/033321, WO 03/042397, WO 2006/068888, WO 2006/110689, WO2009/104964, WO 2010/127097, and WO 2015/191508, the content of each of which is incorporated herein by reference in its entirety.
5.4. Methods and Compositions for Making Recombinant AAV

In one aspect, the present disclosure provides plasmids comprising a presently disclosed expression cassettes (e.g., expression cassettes disclosed in Section 5.1 (NeuroD1 Expression Cassette) of the present disclosure) . The presently disclosed plasmids can be used for producing AAV vectors (e.g., AAV vectors disclosed in Section 5.3 (Recombinant AAV Vectors) of the present disclosure) by being delivered into host cell for AAV packaging.

In some embodiments, provided herein is a gene-of-interest (GOI) plasmid that carries a transgene encoding a NeuroD1 polypeptide. In some embodiments, the GOI plasmid comprises a NeuroD1 expression cassette as described in Section 5.1 (NeuroD1 Expression Cassette) and a pair of AAV ITR sequences flanking the NeuroD1 expression cassette.

In some embodiments, the pair of AAV ITRs are independently selected from the genomic ITR sequences of AAV serotypes AAV1, AAV2, AAV2tYF, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVrh10, AAV. rh20, AAV. rh39, AAV. Rh74, AAV. RHM4-1, AAV. hu37, AAV. Anc80, AAV. Anc80L65, rAAV. 7m8, AAV. PHP. B, AAV. PHP. eB, AAV2.5, AAV2tYF, AAV3B, AAV. LK03, AAV. HSC1, AAV. HSC2, AAV. HSC3, AAV. HSC4, AAV. HSC5, AAV. HSC6, AAV. HSC7, AAV. HSC8, AAV. HSC9, AAV. HSC10 , AAV. HSC11, AAV. HSC12, AAV. HSC13, AAV. HSC14, AAV. HSC15, and AAV. HSC16.

In some embodiments, the ITR sequence located 5’ to the NeuroD1 expression cassette comprises the full-length 5’ ITR sequence of the AAV genome. In some embodiments, the ITR sequence located 5’ to the NeuroD1 expression cassette comprises the wild-type 5’ ITR sequence of the AAV genome. In some embodiments, the ITR sequence located 5’ to the NeuroD1 expression cassette is a truncated version of the 5’ ITR sequence of the AAV genome. In some embodiments, the ITR sequence located 5’ to the NeuroD1 expression cassette is a mutated version of the 5’ ITR sequence of the AAV genome.

In some embodiments, the ITR sequence located 3’ to the NeuroD1 expression cassette comprises the wild-type 3’ ITR sequence of the AAV genome. In some embodiments, the ITR sequence located 3’ to the NeuroD1 expression cassette is a truncated version of the 3’ ITR sequence of the AAV genome. In some embodiments, the ITR sequence located 3’ to the NeuroD1 expression cassette is a mutated version of the 3’ ITR sequence of the AAV genome.

In some embodiments, the ITR sequence located 5’ to the NeuroD1 expression cassette (i.e., the 5’ ITR sequence in the plasmid) is selected from the genomic ITR sequences of AAV serotypes 1 to 8. The 5’ ITR sequence in the plasmid comprises the 5’ ITR sequence from the AAV1 genome. The 5’ ITR sequence in the plasmid comprises the 5’ ITR sequence from the AAV2 genome. The 5’ ITR sequence in the plasmid comprises the 5’ ITR sequence from the AAV3 genome. The 5’ ITR sequence in the plasmid comprises the 5’ ITR sequence from the AAV4 genome. The 5’ ITR sequence in the plasmid comprises the 5’ ITR sequence from the AAV5 genome. The 5’ ITR sequence in the plasmid comprises the 5’ ITR sequence from the AAV6 genome. The 5’ ITR sequence in the plasmid comprises the 5’ ITR sequence from the AAV7 genome. The 5’ ITR sequence in the plasmid comprises the 5’ ITR sequence from the AAV8 genome. In some embodiments, the ITR sequence located 5’ to the NeuroD1 expression cassette comprises the full-length 5’ ITR sequence of the AAV genome. In some embodiments, the ITR sequence located 5’ to the NeuroD1 expression cassette comprises the wild-type 5’ ITR sequence of the AAV genome. In some embodiments, the ITR sequence located 5’ to the NeuroD1 expression cassette is a truncated version of the 5’ ITR sequence of the AAV genome. In some embodiments, the ITR sequence located 5’ to the NeuroD1 expression cassette is a mutated version of the 5’ ITR sequence of the AAV genome.

In some embodiments, the ITR sequence located 3’ to the NeuroD1 expression cassette (i.e., the 3’ ITR sequence in the plasmid) is selected from the genomic ITR sequences of AAV serotypes 1 to 8. In some embodiments, the 3’ ITR sequence in the plasmid comprises the 3’ ITR sequence from the AAV1 genome. In some embodiments, the 3’ ITR sequence in the plasmid comprises the 3’ ITR sequence from the AAV2 genome. In some embodiments, the 3’ ITR sequence in the plasmid comprises the 3’ ITR sequence from the AAV3 genome. In some embodiments, the 3’ ITR sequence in the plasmid comprises the 3’ ITR sequence from the AAV4 genome. In some embodiments, the 3’ ITR sequence in the plasmid comprises the 3’ ITR sequence from the AAV5 genome. In some embodiments, the 3’ ITR sequence in the plasmid comprises the 3’ ITR sequence from the AAV6 genome. In some embodiments, the 3’ ITR sequence in the plasmid comprises the 3’ ITR sequence from the AAV7 genome. In some embodiments, the 3’ ITR sequence in the plasmid comprises the 3’ ITR sequence from the AAV8 genome. In some embodiments, the ITR sequence located 3’ to the NeuroD1 expression cassette comprises the wild-type 3’ ITR sequence of the AAV genome. In some embodiments, the ITR sequence located 3’ to the NeuroD1 expression cassette is a truncated version of the 3’ ITR sequence of the AAV genome. In some embodiments, the ITR sequence located 3’ to the NeuroD1 expression cassette is a mutated version of the 3’ITR sequence of the AAV genome.

ITR sequences for various AAV serotypes are known in the art. See, for example, GenBank: ITR1: NC_002077.1, nts 1-143, ITR2: NC_001401.2, nts 1-145, ITR3: JB292182.1, nts 1-143, ITR4: NC_001829.1, nts 1-146, ITR6: AF028704.1, nts 1-145, ITR7: NC_006260.1, nts 1-145, for the 5’ (left) ITR sequences.

In specific embodiments, the plasmid comprises a pair of ITR sequences located on each end of an NeuroD1 expression cassette, and wherein the ITR located 5’ to the NeuroD1 expression cassette comprises the sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 16, SEQ ID NO: 58, and the ITR located 3’ to the NeuroD1 expression cassette comprises the sequence selected from SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 23, and SEQ ID NO: 59, and wherein the NeuroD1 expression cassette comprises a coding sequence that encodes a NeuroD1 polypeptide comprising an amino acid sequence having at least 90%sequence identity to the sequence set forth in SEQ ID NO: 15, SEQ ID NO: 13 or SEQ ID NO: 7.

In specific embodiments, the plasmid comprises a pair of ITR sequences located on each end of an NeuroD1 expression cassette, and wherein the ITR located 5’ to the NeuroD1 expression cassette comprises the sequence of SEQ ID NO: 16, and the ITR located 3’ to the NeuroD1 expression cassette comprises the sequence of SEQ ID NO: 23, and wherein the NeuroD1 expression cassette comprises a coding sequence that encodes a NeuroD1 polypeptide comprising an amino acid sequence having at least 90%sequence identity to the sequence set forth in SEQ ID NO: 15.

In specific embodiments, the plasmid comprises a pair of ITR sequences located on each end of an NeuroD1 expression cassette, and wherein the ITR located 5’ to the NeuroD1 expression cassette comprises the sequence of SEQ ID NO: 16, and the ITR located 3’ to the NeuroD1 expression cassette comprises the sequence of SEQ ID NO: 23, and wherein the NeuroD1 expression cassette comprises a coding sequence that encodes a NeuroD1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 13.

In specific embodiments, the plasmid comprises a pair of ITR sequences located on each end of an NeuroD1 expression cassette, and wherein the ITR located 5’ to the NeuroD1 expression cassette comprises the sequence of SEQ ID NO: 16, and the ITR located 3’ to the NeuroD1 expression cassette comprises the sequence of SEQ ID NO: 23, and wherein the NeuroD1 expression cassette comprises a coding sequence that encodes a NeuroD1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 7.

In yet a specific embodiment, the plasmid comprises a pair of ITR sequences located on each end of an NeuroD1 expression cassette, and wherein the ITR located 5’ to the NeuroD1 expression cassette comprises the sequence of SEQ ID NO: 16, and the ITR located 3’ to the NeuroD1 expression cassette comprises the sequence of SEQ ID NO: 23, and wherein the NeuroD1 expression cassette comprises a coding sequence comprising the sequence set forth in SEQ ID NO: 14 or codon-optimized variant thereof. In yet a specific embodiment, the NeuroD1 expression cassette further comprises one or more regulatory elements operably linked to the coding sequence. In specific embodiments, the regulatory elements are one or more selected from a CMV enhancer sequence, a GFAP promoter sequence, a chimeric intron, an optimized WPRE, and a polyadenylation signal. In specific embodiments, the regulatory elements are one or more selected from a CMV enhancer sequence comprising the sequence set forth in SEQ ID NO: 8, a GFAP promoter sequence comprising the sequence set forth in SEQ ID NO: 10, a chimeric intron comprising the sequence set forth in SEQ ID NO: 19, an optimized WPRE comprising the sequence set forth in SEQ ID NO: 12, and a polyadenylation signal comprising the sequence set forth in SEQ ID NO: 9.

In yet a specific embodiment, the plasmid comprises a pair of ITR sequences located on each end of an NeuroD1 expression cassette, and wherein the ITR located 5’ to the NeuroD1 expression cassette comprises the sequence of SEQ ID NO: 16, and the ITR located 3’ to the NeuroD1 expression cassette comprises the sequence of SEQ ID NO: 23, and wherein the NeuroD1 expression cassette comprises a coding sequence comprising the sequence set forth in SEQ ID NO: 4 or codon-optimized variant thereof. In yet a specific embodiment, the NeuroD1 expression cassette further comprises one or more regulatory elements operably linked to the coding sequence. In specific embodiments, the regulatory elements are one or more selected from a CMV enhancer sequence, a GFAP promoter sequence, a chimeric intron, an optimized WPRE, and a polyadenylation signal. In specific embodiments, the regulatory elements are one or more selected from a CMV enhancer sequence comprising the sequence set forth in SEQ ID NO: 8, a GFAP promoter sequence comprising the sequence set forth in SEQ ID NO: 10, a chimeric intron comprising the sequence set forth in SEQ ID NO: 19, an optimized WPRE comprising the sequence set forth in SEQ ID NO: 12, and a polyadenylation signal comprising the sequence set forth in SEQ ID NO: 9.

In yet a specific embodiment, the plasmid comprises a pair of ITR sequences located on each end of an NeuroD1 expression cassette, and wherein the ITR located 5’ to the NeuroD1 expression cassette comprises the sequence of SEQ ID NO: 16, and the ITR located 3’ to the NeuroD1 expression cassette comprises the sequence of SEQ ID NO: 23, and wherein the NeuroD1 expression cassette comprises a coding sequence comprising the sequence set forth in SEQ ID NO: 3 or codon-optimized variant thereof. In yet a specific embodiment, the NeuroD1 expression cassette further comprises one or more regulatory elements operably linked to the coding sequence. In specific embodiments, the regulatory elements are one or more selected from a CMV enhancer sequence, a GFAP promoter sequence, a chimeric intron, an optimized WPRE, and a polyadenylation signal. In specific embodiments, the regulatory elements are one or more selected from a CMV enhancer sequence comprising the sequence set forth in SEQ ID NO: 8, a GFAP promoter sequence comprising the sequence set forth in SEQ ID NO: 10, a chimeric intron comprising the sequence set forth in SEQ ID NO: 19, an optimized WPRE comprising the sequence set forth in SEQ ID NO: 12, and a polyadenylation signal comprising the sequence set forth in SEQ ID NO: 9.

In yet a specific embodiment, the plasmid comprises a pair of ITR sequences located on each end of an NeuroD1 expression cassette, and wherein the ITR located 5’ to the NeuroD1 expression cassette comprises the sequence of SEQ ID NO: 16, and the ITR located 3’ to the NeuroD1 expression cassette comprises the sequence of SEQ ID NO: 23, and wherein the NeuroD1 expression cassette comprises the sequence set forth in SEQ ID NO: 33. In yet a specific embodiment, the plasmid comprises a pair of ITR sequences located on each end of an NeuroD1 expression cassette, and wherein the ITR located 5’ to the NeuroD1 expression cassette comprises the sequence of SEQ ID NO: 16, and the ITR located 3’ to the NeuroD1 expression cassette comprises the sequence of SEQ ID NO: 23, and wherein the NeuroD1 expression cassette consists of the sequence set forth in SEQ ID NO: 33.

In some embodiments, the plasmid further comprises a backbone sequence. Any suitable plasmid backbone known in the art for the production of AAV vectors can be used with the presently disclosed subject matter, and an exemplary plasmid backbone sequence is provided in Table 5.4 (B) (see e.g., SEQ ID NO: 43) .

The present disclosure provides host cells comprising a presently disclosed plasmid. Any suitable host cells for AAV vector production can be used with the presently disclosed subject matter. In certain embodiments, the host cell is a mammalian cell. In certain embodiments, the host cell from humans, monkeys, mice, rats, rabbits, or hamsters. In certain embodiments, the host cell is an insect cell.

Non-limiting examples of suitable host cells that can be used with the presently disclosed subject matter include A549 cells, WEHI cells, 10T1/2 cells, MDCK cells, COS1 cells, COS7 cells, BSC 1 cells, BSC 40 cells, BMT 10 cells, VERO cells, W138 cells, Saos cells, C2C12 cells, L cells, HT1080 cells, HepG2 cells, HeLa cells, HEK293 cells, HEK293 derived cells (e.g., HEK293T cells, HEK293F cells) , CHO cells, CHO-K1 cells, CHO derived cells, EB66 cells, BSC cells, HepG2 cells, LLC-MK cells, CV-1 cells, COS cells, MDBK cells, MDCK cells, CRFK cells, RAF cells, RK cells, TCMK-1 cells, LLCPK cells, PK15 cells, LLC-RK cells, BHK cells, BHK-21 cells, NS-1 cells, MRC-5 cells, WI-38 cells, 3T3 cells, 293 cells, RK cells, Per. C6 cells, chicken embryo cells, SF-9 cells, primary fibroblasts, primary hepatocytes, primary myoblast cells, primary hippocampal neurons, primary cerebral cortical neurons, primary cerebellar neurons, and primary striatal neurons.

Any suitable methods known in the art for producing recombinant AAV can be used with the presently disclosed subject matter for producing the recombinant AAV as described herein (e.g., recombinant AAV disclosed in Section 5.3 (Recombinant AAV Vectors) of the present disclosure) . In certain embodiments, the methods comprise: (a) transfecting a host cell described herein with a presently disclosed GOI plasmid, (b) culturing the host cell in a culturing medium; and (c) isolating the recombinant AAV virions from the culturing medium. In certain embodiments, the methods further comprise transfecting the host cell with a plasmid comprising an expression cassette encoding AAV rep proteins and capsid proteins. In certain embodiments, the methods further comprise transfecting the host cell with a plasmid encoding adenovirus regions (e.g., VA, E2A and E4) that mediate AAV vector replication. In certain embodiments, the methods comprise: (a) culturing a host cell comprising a cis expression cassette (e.g., expression cassettes disclosed in Section 5.1 (NeuroD1 Expression Cassette) of the present disclosure) in a culture medium, and (b) isolating the recombinant AAV virions from the cell culture. In certain embodiments, the host cell further comprises (ii) a trans expression cassette encoding one or more AAV rep proteins and capsid proteins. In certain embodiments, the host cell further comprises (iii) nucleic acid sequence encoding adenovirus regions (e.g., VA, E2A and E4) that mediate AAV vector replication. Exemplary plasmid sequences for the GOI plasmid, rep-cap packaging plasmid and helper plasmid that can be used in connection with the present disclosure are provided in Table 6.2 (B) and Table 6.2 (C) .

Exemplary methods of producing recombinant AAV vectors are disclosed in International Patent Publication Nos. WO20220033842 and WO2019079496, the content of each of which is incorporated by reference in its entirety.

Genome copy titers of the recombinant AAV vectors may be determined, for example, by analysis. Virions may be recovered, for example, by CsCl2 sedimentation. Alternatively, baculovirus expression systems in insect cells may be used to produce AAV vectors. For a review, see Aponte-Ubillus et al., 2018, Appl. Microbiol. Biotechnol. 102: 1045-1054, which is incorporated by reference herein in its entirety for manufacturing techniques.

In vitro assays, e.g., cell culture assays, can be used to measure coding nucleotide sequence expression from the recombinant AAV vector, thus indicating, e.g., potency of the recombinant vector. For example, the HeLa cell, a cell line derived from human cervical cancer cells (available from) , can be used to assess coding nucleotide sequence expression. Alternatively, cell lines derived from liver or muscle or other cell types may be used, for example, but not limited, to HuH-7, HEK293, fibrosarcoma HT-1080, HKB-11, C2C12 myoblasts, and CAP cells. Once expressed, characteristics of the expressed product (e.g., protein) can also be determined, including serum half-life, functional activity of the protein (e.g., enzymatic activity or binding to a target) , determination of the glycosylation and tyrosine sulfation patterns, and other assays known in the art for determining protein characteristics.
5.5. Pharmaceutical Composition and Kit

In one aspect, the present disclosure provides a pharmaceutical composition comprising a recombinant AAV vector (e.g., recombinant AAV vectors disclosed in Section 5.3 (Recombinant AAV Vectors) ) and a pharmaceutically acceptable carrier.

In certain embodiments, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. As used herein, the term “carrier” refers to a diluent, an adjuvant (e.g., Freund’s complete and incomplete adjuvant) , an excipient, or vehicle with which the AAV vector is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, including, e.g., peanut oil, soybean oil, mineral oil, sesame oil or the like. Water is a common carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include but not limited to starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. Additional examples of pharmaceutically acceptable carriers, excipients, and stabilizers include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight polypeptides; proteins, such as serum albumin and gelatin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEENTM, polyethylene glycol (PEG) , and PLURONICSTM as known in the art. In certain embodiments, the pharmaceutical composition further comprises a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifier, a suspending agent, or a preservative, in addition to the above ingredients. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations or the like.

In certain embodiments, the pharmaceutical composition is provided for use in accordance with the presently disclosed methods of treatment (e.g., methods of treatment disclosed in Section 5.6 (Method of Treatment) of the present disclosure) , said pharmaceutical compositions comprise a therapeutically and/or prophylactically effective amount of the presently disclosed recombinant AAV vector and a pharmaceutically acceptable carrier.

In certain embodiments, the AAV vector is substantially purified (i.e., substantially free from substances that limit its effect or produce undesired side-effects) . In certain embodiments, the subject receiving the pharmaceutical composition is a mammal such as non-primate (e.g., cows, pigs, horses, cats, dogs, rats etc. ) and a primate (e.g., monkey such as, a cynomolgus monkey and a human) . In certain embodiments, the subject is a human.

In some embodiments, the pharmaceutical composition is in a fluidic formulation. In some embodiments, the pharmaceutical composition is a solution. In some embodiments, the pharmaceutical composition comprises a recombinant AAV described in Section 5.4 (Recombinant AAV Vectors) , and further comprises:
(a) potassium chloride,
(b) potassium phosphate monobasic,
(c) sodium chloride,
(d) sodium phosphate dibasic anhydrous, and
(e) poloxamer 188, polysorbate 20, or polysorbate 80.

In some embodiments, the pharmaceutical composition comprises a recombinant AAV described in Section 5.4 (Recombinant AAV Vectors) , and further comprises:
(a) sodium chloride at a concentration of about 180 mM;
(b) sodium phosphate at a concentration of about 10 mM; and
(c) poloxamer 188 at a concentration of about 0.001%weight/volume (0.01 g/L) ; and wherein
the pH of the pharmaceutical composition is about 7.3.

In some embodiments, the pharmaceutical composition comprises a recombinant AAV described in Section 5.4 (Recombinant AAV Vectors) , and further comprises:
(a) sodium chloride at a concentration of about 200 mM;
(b) magnesium chloride at a concentration of about 1 mM;
(c) Tris hydrochloride at a concentration of about 20 mM, and
(d) poloxamer 188 at a concentration of about 0.005%weight/volume (0.05 g/L) ; and wherein
the pH of the pharmaceutical composition is about 8.0.

In some embodiments, the pharmaceutical composition comprises a recombinant AAV described in Section 5.4 (Recombinant AAV Vectors) , and further comprises:
(a) sodium chloride at a concentration of about 150 mM;
(b) calcium chloride at a concentration of about 1.4 mM;
(c) magnesium chloride at a concentration of about 0.8 mM,
(d) sodium phosphate at a concentration of about 1 mM, and
(e) poloxamer 188 at a concentration of about 0.001%weight/volume (0.01 g/L) ; and wherein
the pH of the pharmaceutical composition is about 7.4.

The present disclosure also provides kits for treating stroke in a subject in need thereof.

In certain embodiment, the kit comprises the presently disclosed recombinant AAV vector, e.g., in a container. Such containers can be boxes, ampules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments. Optionally associated with such container (s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use, or sale for human administration.

In certain embodiments, the kit further comprises instructions for administering to a subject having stroke. The instructions generally include information about the use of the composition for the treatment and/or prevention of stroke. In certain embodiments, the instructions include at least one of the following: description of the therapeutic agent; dosage schedule and administration for treatment or prevention of stroke or symptoms thereof; precautions; warnings; indications; counter-indications; over-dosage information; adverse reactions; animal pharmacology; clinical studies; and/or references. The instructions may be printed directly on the container (when present) , or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.
5.6. Method of Treatment

The present disclosure provides methods for selectively expressing an encoding nucleic acid in glial cells. The present disclosure also provides methods for treating neurological condition in a subject in need thereof. In certain embodiments, the methods comprise delivering to the subject a presently disclosed recombinant AAV (e.g., AAV vectors disclosed in Section 5.3 (Recombinant AAV Vectors) of the present disclosure) . In certain embodiments, the methods comprise delivering to the subject a presently disclosed pharmaceutical composition comprising recombinant AAV (e.g., pharmaceutical composition disclosed in Section 5.5 (Pharmaceutical Composition and Kit) of the present disclosure) .

In some embodiments, the methods of treating a stroke comprise delaying, preventing, treating, and/or managing the disease or disorder. In certain embodiments, the methods prevent occurrence or recurrence of the disease or disorder. In certain embodiments, the methods alleviate one or more symptoms of the disease or disorder. In certain embodiments, the methods diminish any direct or indirect pathological consequences of the disease or disorder. In certain embodiments, the methods decrease the rate of disease progression. In certain embodiments, the methods delay remission or improves prognosis of the disease or disorder.

In some embodiments, the method for treating stroke comprises administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a recombinant AAV, wherein the recombinant AAV comprises a genome comprising a transgene encoding a NeuroD1 polypeptide. In some embodiments, the genome is a single-stranded DNA molecule. In some embodiments, the genome is one described in Section 5.4 (Recombinant AAV Vectors) of the present disclosure. In some embodiments, the genome comprises the sequence set forth in SEQ ID NO: 24 or a sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%sequence identity to SEQ ID NO: 24. In some embodiments, the genome comprises the sequence set forth in SEQ ID NO: 24. In some embodiments, the genome consists of the sequence set forth in SEQ ID NO: 24.

In some embodiments, the method for treating stroke comprises administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a recombinant AAV, wherein the recombinant AAV comprises a genome comprising a transgene encoding a NeuroD1 polypeptide and further comprises a AAV capsid protein. In some embodiments, the AAV capsid protein is one described in Section 5.4 (Recombinant AAV Vectors) of the present disclosure. In some embodiments, the AAV capsid is AAV9.

In some embodiments, the method for treating stroke comprises administering to a subject in need thereof a pharmaceutical composition comprising from about 1×10¹¹ viral genomes (vg) to about 1×10¹³ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 1×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 1.1×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 1.2×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 1.3×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 1.4×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 1.5×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 1.6×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 1.7×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 1.8×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 1.9×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 2×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 2.1×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 2.2×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 2.3×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 2.4×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 2.5×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 2.6×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 2.7×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 2.8×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 2.9×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 3×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 3.1×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 3.2×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 3.3×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 3.4×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 3.5×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 3.6×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 3.7×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 3.8×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 3.9×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 4×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 4.1×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 4.2×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 4.3×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 4.4×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 4.5×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 4.6×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 4.7×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 4.8×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 4.9×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 5×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 5.1×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 5.2×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 5.3×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 5.4×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 5.5×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 5.6×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 5.7×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 5.8×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 5.9×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 6×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 6.1×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 6.2×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 6.3×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 6.4×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 6.5×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 6.6×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 6.7×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 6.8×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 6.9×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 7×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 7.1×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 7.2×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 7.3×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 7.4×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 7.5×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 7.6×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 7.7×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 7.8×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 7.9×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 8×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 8.1×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 8.2×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 8.3×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 8.4×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 8.5×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 8.6×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 8.7×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 8.8×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 8.9×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 9×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 9.1×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 9.2×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 9.3×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 9.4×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 9.5×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 9.6×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 9.7×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 9.8×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 9.9×10¹¹ vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 1×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 1.1×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 1.2×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 1.3×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 1.4×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 1.5×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 1.6×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 1.7×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 1.8×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 1.9×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 2×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 2.1×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 2.2×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 2.3×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 2.4×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 2.5×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 2.6×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 2.7×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 2.8×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 2.9×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 3×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 3.1×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 3.2×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 3.3×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 3.4×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 3.5×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 3.6×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 3.7×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 3.8×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 3.9×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 4.0×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 4.1×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 4.2×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 4.3×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 4.4×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 4.5×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 4.6×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 4.7×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 4.8×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 4.9×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 5×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 5.1×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 5.2×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 5.3×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 5.4×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 5.5×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 5.6×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 5.7×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 5.8×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 5.9×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 6×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 6.1×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 6.2×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 6.3×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 6.4×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 6.5×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 6.6×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 6.7×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 6.8×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 6.9×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 7×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 7.1×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 7.2×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 7.3×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 7.4×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 7.5×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 7.6×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 7.7×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 7.8×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 7.9×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 8×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 8.1×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 8.2×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 8.3×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 8.4×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 8.5×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 8.6×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 8.7×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 8.8×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 8.9×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 9×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 9.1×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 9.2×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 9.3×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 9.4×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 9.5×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 9.6×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 9.7×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 9.8×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 9.9×10¹² vg of the recombinant AAV. In some embodiments, the method for treating stroke comprises administering to the subject a pharmaceutical composition comprising about 1×10¹³ vg of the recombinant AAV.

In some embodiments, the subject is administered the pharmaceutical composition comprising about 1×10¹¹ viral genomes (vg) to about 1×10¹³ vg once. In some embodiments, each time the pharmaceutical composition that is administered to the subject comprises about 1×10¹¹ vg, about 1.1×10¹¹ vg, about 1.2×10¹¹ vg, about 1.3×10¹¹ vg, about 1.4×10¹¹ vg, about 1.5×10¹¹ vg, about 1.6×10¹¹ vg, about 1.7×10¹¹ vg, about 1.8×10¹¹ vg, about 1.9×10¹¹ vg, about 2×10¹¹ vg, about 2.1×10¹¹ vg, about 2.2×10¹¹ vg, about 2.3×10¹¹ vg, about 2.4×10¹¹ vg, about 2.5×10¹¹ vg, about 2.6×10¹¹ vg, about 2.7×10¹¹ vg, about 2.8×10¹¹ vg, about 2.9×10¹¹ vg, 3×10¹¹ vg, about 3.1×10¹¹ vg, about 3.2×10¹¹ vg, about 3.3×10¹¹ vg, about 3.4×10¹¹ vg, about 3.5×10¹¹ vg, about 3.6×10¹¹ vg, about 3.7×10¹¹ vg, about 3.8×10¹¹ vg, about 3.9×10¹¹ vg, 4×10¹¹ vg, about 4.1×10¹¹ vg, about 4.2×10¹¹ vg, about 4.3×10¹¹ vg, about 4.4×10¹¹ vg, about 4.5×10¹¹ vg, about 4.6×10¹¹ vg, about 4.7×10¹¹ vg, about 4.8×10¹¹ vg, about 4.9×10¹¹ vg, 5×10¹¹ vg, about 5.1×10¹¹ vg, about 5.2×10¹¹ vg, about 5.3×10¹¹ vg, about 5.4×10¹¹ vg, about 5.5×10¹¹ vg, about 5.6×10¹¹ vg, about 5.7×10¹¹ vg, about 5.8×10¹¹ vg, about 5.9×10¹¹ vg, about 6×10¹¹ vg, about 6.1×10¹¹ vg, about 6.2×10¹¹ vg, about 6.3×10¹¹ vg, about 6.4×10¹¹ vg, about 6.5×10¹¹ vg, about 6.6×10¹¹ vg, about 6.7×10¹¹ vg, about 6.8×10¹¹ vg, about 6.9×10¹¹ vg, about 7.0×10¹¹ vg, about 7.1×10¹¹ vg, about 7.2×10¹¹ vg, about 7.3×10¹¹ vg, about 7.4×10¹¹ vg, about 7.5×10¹¹ vg, about 7.6×10¹¹ vg, about 7.7×10¹¹ vg, about 7.8×10¹¹ vg, about 7.9×10¹¹ vg, about 8×10¹¹ vg, about 8.1×10¹¹ vg, about 8.2×10¹¹ vg, about 8.3×10¹¹ vg, about 8.4×10¹¹ vg, about 8.5×10¹¹ vg, about 8.6×10¹¹ vg, about 8.7×10¹¹ vg, about 8.8×10¹¹ vg, about 8.9×10¹¹ vg, about 9×10¹¹ vg, about 9.1×10¹¹ vg, about 9.2×10¹¹ vg, about 9.3×10¹¹ vg, about 9.4×10¹¹ vg, about 9.5×10¹¹ vg, about 9.6×10¹¹ vg, about 9.7×10¹¹ vg, about 9.8×10¹¹ vg, about 9.9×10¹¹ vg, about 1×10¹² vg, about 1.1×10¹² vg, about 1.2×10¹² vg, about 1.3×10¹² vg, about 1.4×10¹² vg, about 1.5×10¹² vg, about 1.6×10¹² vg, about 1.7×10¹² vg, about 1.8×10¹² vg, about 1.9×10¹² vg, about 2×10¹² vg, about 2.1×10¹² vg, about 2.2×10¹² vg, about 2.3×10¹² vg, about 2.4×10¹² vg, about 2.5×10¹² vg, about 2.6×10¹² vg, about 2.7×10¹² vg, about 2.8×10¹² vg, about 2.9×10¹² vg, about 3×10¹² vg, about 3.1×10¹² vg, about 3.2×10¹² vg, about 3.3×10¹² vg, about 3.4×10¹² vg, about 3.5×10¹² vg, about 3.6×10¹² vg, about 3.7×10¹² vg, about 3.8×10¹² vg, about 3.9×10¹² vg, about 4×10¹² vg, about 4.1×10¹² vg, about 4.2×10¹² vg, about 4.3×10¹² vg, about 4.4×10¹² vg, about 4.5×10¹² vg, about 4.6×10¹² vg, about 4.7×10¹² vg, about 4.8×10¹² vg, about 4.9×10¹² vg, about 5×10¹² vg, about 5.1×10¹² vg, about 5.2×10¹² vg, about 5.3×10¹² vg, about 5.4×10¹² vg, about 5.5×10¹² vg, about 5.6×10¹² vg, about 5.7×10¹² vg, about 5.8×10¹² vg, about 5.9×10¹² vg, about 6×10¹² vg, about 6.1×10¹² vg, about 6.2×10¹² vg, about 6.3×10¹² vg, about 6.4×10¹² vg, about 6.5×10¹² vg, about 6.6×10¹² vg, about 6.7×10¹² vg, about 6.8×10¹² vg, about 6.9×10¹² vg, about 7×10¹² vg, about 7.1×10¹² vg, about 7.2×10¹² vg, about 7.3×10¹² vg, about 7.4×10¹² vg, about 7.5×10¹² vg, about 7.6×10¹² vg, about 7.7×10¹² vg, about 7.8×10¹² vg, about 7.9×10¹² vg, about 8×10¹² vg, about 8.1×10¹² vg, about 8.2×10¹² vg, about 8.3×10¹² vg, about 8.4×10¹² vg, about 8.5×10¹² vg, about 8.6×10¹² vg, about 8.7×10¹² vg, about 8.8×10¹² vg, about 8.9×10¹² vg, about 9×10¹² vg, about 9.1×10¹² vg, about 9.2×10¹² vg, about 9.3×10¹² vg, about 9.4×10¹² vg, about 9.5×10¹² vg, about 9.6×10¹² vg, about 9.7×10¹² vg, about 9.8×10¹² vg, about 9.9×10¹² vg, or about 1×10¹³ vg of the recombinant AAV described herein.

In some embodiments, the fluid formulation of the pharmaceutical composition is a solution. In some embodiments, the fluid formulation of the pharmaceutical composition comprises the recombinant AAV at a concentration in the range of from about 1×10¹¹ vg/mL to about 1×10¹³ vg/mL, and further comprises potassium chloride, potassium phosphate monobasic, sodium chloride, sodium phosphate dibasic anhydrous, and a surfactant selected from poloxamer 188, polysorbate 20, or polysorbate 80. In some embodiments, the fluid formulation of the pharmaceutical composition comprises the recombinant AAV at a concentration in the range of from about 1×10¹¹ vg/mL to about 1×10¹³ vg/mL, and further comprises potassium chloride, potassium phosphate monobasic, sodium chloride, sodium phosphate dibasic anhydrous, and poloxamer 188.

In some embodiments, the fluid formulation of the pharmaceutical composition comprises the recombinant AAV at a concentration in the range of from about 1×10¹¹ vg/mL to about 1×10¹³ vg/mL, and further comprises sodium chloride, sodium phosphate, and poloxamer 188. In specific embodiments, the fluid formulation of the pharmaceutical composition comprises sodium chloride at a concentration of about 180 mM. In some embodiments, the fluid formulation comprises sodium phosphate at a concentration of about 10 mM. In some embodiments, the fluid formulation comprises poloxamer 188 at a concentration of about 0.001%weight/volume (0.01 g/L) . In some embodiments, the pH of the pharmaceutical composition is about 7.3.

In some embodiments, the fluid formulation of the pharmaceutical composition comprises the recombinant AAV at a concentration in the range of from about 1×10¹¹vg/mL to about 1×10¹³ vg/mL, sodium chloride at a concentration of about 180 mM, sodium phosphate at a concentration of about 10 mM, poloxamer 188 at a concentration of about 0.001%weight/volume (0.01 g/L) , and the pH of the pharmaceutical composition is about 7.3.

In some embodiments, the fluid formulation of the pharmaceutical composition comprises the recombinant AAV at a concentration in the range of from about 1×10¹¹ vg/mL to about 1×10¹³ vg/mL, and further comprises sodium chloride, magnesium chloride, Tris hydrochloride, and poloxamer 188. In some embodiments, the fluid formulation of the pharmaceutical composition comprises sodium chloride at a concentration of about 200 mM. In some embodiments, the fluid formulation of the pharmaceutical composition comprises magnesium chloride at a concentration of about 1 mM. In some embodiments, the fluid formulation of the pharmaceutical composition comprises Tris hydrochloride at a concentration of about 20 mM. In some embodiments, the fluid formulation of the pharmaceutical composition comprises poloxamer 188 at a concentration of about 0.005%weight/volume (0.05 g/L) . In some embodiments, and wherein the pH of the pharmaceutical composition is about 8.0.

In some embodiments, the fluid formulation of the pharmaceutical composition comprises the recombinant AAV at a concentration in the range of from about 1×10¹¹ vg/mL to about 1×10¹³ vg/mL, sodium chloride at a concentration of about 200 mM, magnesium chloride at a concentration of about 1 mM, Tris hydrochloride at a concentration of about 20 mM, poloxamer 188 at a concentration of about 0.005%weight/volume (0.05 g/L) , and wherein the pH of the pharmaceutical composition is about 8.0.

In some embodiments, the fluid formulation of the pharmaceutical composition comprises the recombinant AAV at a concentration in the range of from about 1×10¹¹ vg/mL to about 1×10¹³ vg/mL, and further comprises sodium chloride, calcium chloride, magnesium chloride, sodium phosphate, and poloxamer 188. In some embodiments, the fluid formulation of the pharmaceutical composition comprises sodium chloride at a concentration of about 150 mM. In some embodiments, the fluid formulation of the pharmaceutical composition comprises calcium chloride at a concentration of about 1.4 mM. In some embodiments, the fluid formulation of the pharmaceutical composition comprises magnesium chloride at a concentration of about 0.8 mM. In some embodiments, the fluid formulation of the pharmaceutical composition comprises sodium phosphate at a concentration of about 1 mM. In some embodiments, the fluid formulation of the pharmaceutical composition comprises poloxamer 188 at a concentration of about 0.001%weight/volume (0.01 g/L) . In some embodiments, the pH of the pharmaceutical composition is about 7.4.

In some embodiments, the fluid formulation of the pharmaceutical composition comprises the recombinant AAV at a concentration in the range of from about 1×10¹¹ vg/mL to about 1×10¹³ vg/mL, sodium chloride at a concentration of about 150 mM; calcium chloride at a concentration of about 1.4 mM; magnesium chloride at a concentration of about 0.8 mM, sodium phosphate at a concentration of about 1 mM, and poloxamer 188 at a concentration of about 0.001%weight/volume (0.01 g/L) ; and wherein the pH of the pharmaceutical composition is about 7.4.

In some embodiments, the pharmaceutical composition is administered to the subject intracerebrally. In some embodiments, the pharmaceutical composition is administered to the subject by intracerebral injection of a fluid formulation of the pharmaceutical composition. In some embodiments, the pharmaceutical composition is delivered to a brain of a subject who has suffered a stroke. In some embodiments, the pharmaceutical composition is delivered to an area of the brain adjacent to the core region of the stroke. In some embodiments, the pharmaceutical composition is delivered to an area of the brain around the infarct lesion. In an aspect, an AAV vector or composition as provided herein is delivered to a peri-infarct region of the stroke. In some embodiments, an injection site is determined prior to the injecting via a magnetic resonance imaging (MRI) scan.

In some embodiments, the pharmaceutical composition comprising the recombinant AAV as described herein is administered intracerebrally to the subject, wherein the intracerebral administration is performed by injecting a fluid formulation of the pharmaceutical composition comprising the recombinant AAV at a concentration in the range of from about 1×10¹¹ vg/mL to about 1×10¹³ vg/mL. In some embodiments, the fluid formulation of the pharmaceutical composition administered intracerebrally comprises the recombinant AAV at a concentration of about 1×10¹¹ vg/mL, about 1.1 ×10¹¹ vg/mL, about 1.2 ×10¹¹ vg/mL, about 1.3 ×10¹¹ vg/mL, about 1.4 ×10¹¹ vg/mL, about 1.5 ×10¹¹ vg/mL, about 1.6 ×10¹¹ vg/mL, about 1.7 ×10¹¹ vg/mL, about 1.8 ×10¹¹ vg/mL, about 1.9 ×10¹¹ vg/mL, about 2 ×10¹¹ vg/mL, about 2.1 ×10¹¹ vg/mL, about 2.2 ×10¹¹ vg/mL, about 2.3 ×10¹¹ vg/mL, about 2.4 ×10¹¹ vg/mL, about 2.5 ×10¹¹ vg/mL, about 2.6 ×10¹¹ vg/mL, about 2.7 ×10¹¹ vg/mL, about 2.8 ×10¹¹ vg/mL, about 2.9 ×10¹¹ vg/mL, about 3×10¹¹ vg/mL, about 3.1×10¹¹ vg/mL, about 3.2×10¹¹ vg/mL, about 3.3×10¹¹ vg/mL, about 3.4×10¹¹ vg/mL, about 3.5×10¹¹ vg/mL, about 3.6×10¹¹ vg/mL, about 3.7×10¹¹ vg/mL, about 3.8×10¹¹ vg/mL, about 3.9×10¹¹ vg/mL, 4×10¹¹ vg/mL, about 4.1×10¹¹ vg/mL, about 4.2×10¹¹ vg/mL, about 4.3×10¹¹ vg/mL, about 4.4×10¹¹ vg/mL, about 4.5×10¹¹ vg/mL, about 4.6×10¹¹ vg/mL, about 4.7×10¹¹ vg/mL, about 4.8×10¹¹ vg/mL, about 4.9×10¹¹ vg/mL, 5×10¹¹ vg/mL, about 5.1×10¹¹ vg/mL, about 5.2×10¹¹ vg/mL, about 5.3×10¹¹ vg/mL, about 5.4×10¹¹ vg/mL, about 5.5×10¹¹ vg/mL, about 5.6×10¹¹ vg/mL, about 5.7×10¹¹ vg/mL, about 5.8×10¹¹ vg/mL, about 5.9×10¹¹ vg/mL, about 6×10¹¹ vg/mL, about 6.1×10¹¹ vg/mL, about 6.2×10¹¹ vg/mL, about 6.3×10¹¹ vg/mL, about 6.4×10¹¹ vg/mL, about 6.5×10¹¹ vg/mL, about 6.6×10¹¹ vg/mL, about 6.7×10¹¹ vg/mL, about 6.8×10¹¹ vg/mL, about 6.9×10¹¹ vg/mL, about 7×10¹¹ vg/mL, about 7.1×10¹¹ vg/mL, about 7.2×10¹¹ vg/mL, about 7.3×10¹¹ vg/mL, about 7.4×10¹¹ vg/mL, about 7.5×10¹¹ vg/mL, about 7.6×10¹¹ vg/mL, about 7.7×10¹¹ vg/mL, about 7.8×10¹¹ vg/mL, about 7.9×10¹¹ vg/mL, about 8×10¹¹ vg/mL, about 8.1×10¹¹ vg/mL, about 8.2×10¹¹ vg/mL, about 8.3×10¹¹ vg/mL, about 8.4×10¹¹ vg/mL, about 8.5×10¹¹ vg/mL, about 8.6×10¹¹ vg/mL, about 8.7×10¹¹ vg/mL, about 8.8×10¹¹ vg/mL, about 8.9×10¹¹ vg/mL, about 9×10¹¹ vg/mL, about 9.1×10¹¹ vg/mL, about 9.2×10¹¹ vg/mL, about 9.3×10¹¹ vg/mL, about 9.4×10¹¹ vg/mL, about 9.5×10¹¹ vg/mL, about 9.6×10¹¹ vg/mL, about 9.7×10¹¹ vg/mL, about 9.8×10¹¹ vg/mL, about 9.9×10¹¹ vg/mL, about 1×10¹² vg/mL, about 1.1×10¹² vg/mL, about 1.2×10¹² vg/mL, about 1.3×10¹² vg/mL, about 1.4×10¹² vg/mL, about 1.5×10¹² vg/mL, about 1.6×10¹² vg/mL, about 1.7×10¹² vg/mL, about 1.8×10¹² vg/mL, about 1.9×10¹² vg/mL, about 2×10¹² vg/mL, about 2.1×10¹² vg/mL, about 2.2×10¹² vg/mL, about 2.3×10¹² vg/mL, about 2.4×10¹² vg/mL, about 2.5×10¹² vg/mL, about 2.6×10¹² vg/mL, about 2.7×10¹² vg/mL, about 2.8×10¹² vg/mL, about 2.9×10¹² vg/mL, about 3×10¹² vg/mL, about 3.1×10¹² vg/mL, about 3.2×10¹² vg/mL, about 3.3×10¹² vg/mL, about 3.4×10¹² vg/mL, about 3.5×10¹² vg/mL, about 3.6×10¹² vg/mL, about 3.7×10¹² vg/mL, about 3.8×10¹² vg/mL, about 3.9×10¹² vg/mL, about 4×10¹² vg/mL, about 4.1×10¹² vg/mL, about 4.2×10¹² vg/mL, about 4.3×10¹² vg/mL, about 4.4×10¹² vg/mL, about 4.5×10¹² vg/mL, about 4.6×10¹² vg/mL, about 4.7×10¹² vg/mL, about 4.8×10¹² vg/mL, about 4.9×10¹² vg/mL, about 5×10¹² vg/mL, about 5.1×10¹² vg/mL, about 5.2×10¹² vg/mL, about 5.3×10¹² vg/mL, about 5.4×10¹² vg/mL, about 5.5×10¹² vg/mL, about 5.6×10¹² vg/mL, about 5.7×10¹² vg/mL, about 5.8×10¹² vg/mL, about 5.9×10¹² vg/mL, about 6×10¹² vg/mL, about 6.1×10¹² vg/mL, about 6.2×10¹² vg/mL, about 6.3×10¹² vg/mL, about 6.4×10¹² vg/mL, about 6.5×10¹² vg/mL, about 6.6×10¹² vg/mL, about 6.7×10¹² vg/mL, about 6.8×10¹² vg/mL, about 6.9×10¹² vg/mL, about 7×10¹² vg/mL, about 7.1×10¹² vg/mL, about 7.2×10¹² vg/mL, about 7.3×10¹² vg/mL, about 7.4×10¹² vg/mL, about 7.5×10¹² vg/mL, about 7.6×10¹² vg/mL, about 7.7×10¹² vg/mL, about 7.8×10¹² vg/mL, about 7.9×10¹² vg/mL, about 8×10¹² vg/mL, about 8.1×10¹² vg/mL, about 8.2×10¹² vg/mL, about 8.3×10¹² vg/mL, about 8.4×10¹² vg/mL, about 8.5×10¹² vg/mL, about 8.6×10¹² vg/mL, about 8.7×10¹² vg/mL, about 8.8×10¹² vg/mL, about 8.9×10¹² vg/mL, about 9×10¹² vg/mL, about 9.1×10¹² vg/mL, about 9.2×10¹² vg/mL, about 9.3×10¹² vg/mL, about 9.4×10¹² vg/mL, about 9.5×10¹² vg/mL, about 9.6×10¹² vg/mL, about 9.7×10¹² vg/mL, about 9.8×10¹² vg/mL, about 9.9×10¹² vg/mL, or about 1×10¹³ vg/mL.

In some embodiments, the fluid formulation of the pharmaceutical composition comprising the recombinant AAV at a concentration in the range of from about 1×10¹¹ vg/mL to about 1×10¹³ vg/mL is administered intracerebrally once. In some embodiments, the intracerebral injection volume of the fluid formulation of the pharmaceutical composition comprising the recombinant AAV at a concentration in the range of from about 1×10¹¹ vg/mL to about 1×10¹³ vg/mL is about 0.3 mL to about 1 mL. In some embodiments, each time the intracerebral injection volume of the fluid formulation of the pharmaceutical composition comprising the recombinant AAV at a concentration in the range of from about 1×10¹¹ vg/mL to about 1×10¹³ vg/mL is about 0.3 mL, about 0.4 mL, about 0.5 mL, about 0.6 mL, about 0.7 mL, about 0.8 mL, about 0.9 mL, or about 1 mL. In specific embodiments, for at least one of the sequential administrations, 0.6 mL of a fluid formulation of the pharmaceutical composition comprising the recombinant AAV at a concentration of about 5×10¹¹ vg/mL is injected intracerebrally. In specific embodiments, for at least one of the sequential administrations, 0.6 mL of a fluid formulation of the pharmaceutical composition comprising the recombinant AAV at a concentration of about 1×10¹² vg/mL is injected intracerebrally. In specific embodiments, for at least one of the sequential intracerebral injections, 0.6 mL of a fluid formulation of the pharmaceutical composition comprising the recombinant AAV at a concentration of about 2×10¹² vg/mL is injected intracerebrally.

In some embodiments, the subject has stroke, and wherein the pharmaceutical composition is administered into an area of the brain adjacent to the core region of the stroke. In some embodiments, the administration is by injecting a fluid formulation of the pharmaceutical composition comprising the recombinant AAV at a concentration in the range of from about 1×10¹¹ vg/mL to about 1×10¹³ vg/mL. In some embodiments, the fluid formulation of the pharmaceutical composition administered into the area of the brain adjacent to the core region of the stroke comprises the recombinant AAV at a concentration of about 1×10¹¹ vg/mL, about 1.1×10¹¹ vg/mL, about 1.2×10¹¹ vg/mL, about 1.3×10¹¹ vg/mL, about 1.4×10¹¹ vg/mL, about 1.5×10¹¹ vg/mL, about 1.6×10¹¹ vg/mL, about 1.7×10¹¹ vg/mL, about 1.8×10¹¹ vg/mL, about 1.9×10¹¹ vg/mL, about 2×10¹¹ vg/mL, about 2.1×10¹¹ vg/mL, about 2.2×10¹¹ vg/mL, about 2.3×10¹¹ vg/mL, about 2.4×10¹¹ vg/mL, about 2.5×10¹¹ vg/mL, about 2.6×10¹¹ vg/mL, about 2.7×10¹¹ vg/mL, about 2.8×10¹¹ vg/mL, about 2.9×10¹¹ vg/mL, about 3×10¹¹ vg/mL, about 3.1×10¹¹ vg/mL, about 3.2×10¹¹ vg/mL, about 3.3×10¹¹ vg/mL, about 3.4×10¹¹ vg/mL, about 3.5×10¹¹ vg/mL, about 3.6×10¹¹ vg/mL, about 3.7×10¹¹ vg/mL, about 3.8×10¹¹ vg/mL, about 3.9×10¹¹ vg/mL, 4×10¹¹ vg/mL, about 4.1×10¹¹ vg/mL, about 4.2×10¹¹ vg/mL, about 4.3×10¹¹ vg/mL, about 4.4×10¹¹ vg/mL, about 4.5×10¹¹ vg/mL, about 4.6×10¹¹ vg/mL, about 4.7×10¹¹ vg/mL, about 4.8×10¹¹ vg/mL, about 4.9×10¹¹ vg/mL, 5×10¹¹ vg/mL, about 5.1×10¹¹ vg/mL, about 5.2×10¹¹ vg/mL, about 5.3×10¹¹ vg/mL, about 5.4×10¹¹ vg/mL, about 5.5×10¹¹ vg/mL, about 5.6×10¹¹ vg/mL, about 5.7×10¹¹ vg/mL, about 5.8×10¹¹ vg/mL, about 5.9×10¹¹ vg/mL, about 6×10¹¹ vg/mL, about 6.1×10¹¹ vg/mL, about 6.2×10¹¹ vg/mL, about 6.3×10¹¹ vg/mL, about 6.4×10¹¹ vg/mL, about 6.5×10¹¹ vg/mL, about 6.6×10¹¹ vg/mL, about 6.7×10¹¹ vg/mL, about 6.8×10¹¹ vg/mL, about 6.9×10¹¹ vg/mL, about 7×10¹¹ vg/mL, about 7.1×10¹¹ vg/mL, about 7.2×10¹¹ vg/mL, about 7.3×10¹¹ vg/mL, about 7.4×10¹¹ vg/mL, about 7.5×10¹¹ vg/mL, about 7.6×10¹¹ vg/mL, about 7.7×10¹¹ vg/mL, about 7.8×10¹¹ vg/mL, about 7.9×10¹¹ vg/mL, about 8×10¹¹ vg/mL, about 8.1×10¹¹ vg/mL, about 8.2×10¹¹ vg/mL, about 8.3×10¹¹ vg/mL, about 8.4×10¹¹ vg/mL, about 8.5×10¹¹ vg/mL, about 8.6×10¹¹ vg/mL, about 8.7×10¹¹ vg/mL, about 8.8×10¹¹ vg/mL, about 8.9×10¹¹ vg/mL, about 9×10¹¹ vg/mL, about 9.1×10¹¹ vg/mL, about 9.2×10¹¹ vg/mL, about 9.3×10¹¹ vg/mL, about 9.4×10¹¹ vg/mL, about 9.5×10¹¹ vg/mL, about 9.6×10¹¹ vg/mL, about 9.7×10¹¹ vg/mL, about 9.8×10¹¹ vg/mL, about 9.9×10¹¹ vg/mL, about 1×10¹² vg/mL, about 1.1×10¹² vg/mL, about 1.2×10¹² vg/mL, about 1.3×10¹² vg/mL, about 1.4×10¹² vg/mL, about 1.5×10¹² vg/mL, about 1.6×10¹² vg/mL, about 1.7×10¹² vg/mL, about 1.8×10¹² vg/mL, about 1.9×10¹² vg/mL, about 2×10¹² vg/mL, about 2.1×10¹² vg/mL, about 2.2×10¹² vg/mL, about 2.3×10¹² vg/mL, about 2.4×10¹² vg/mL, about 2.5×10¹² vg/mL, about 2.6×10¹² vg/mL, about 2.7×10¹² vg/mL, about 2.8×10¹² vg/mL, about 2.9×10¹² vg/mL, about 3×10¹² vg/mL, about 3.1×10¹² vg/mL, about 3.2×10¹² vg/mL, about 3.3×10¹² vg/mL, about 3.4×10¹² vg/mL, about 3.5×10¹² vg/mL, about 3.6×10¹² vg/mL, about 3.7×10¹² vg/mL, about 3.8×10¹² vg/mL, about 3.9×10¹² vg/mL, about 4×10¹² vg/mL, about 4.1×10¹² vg/mL, about 4.2×10¹² vg/mL, about 4.3×10¹² vg/mL, about 4.4×10¹² vg/mL, about 4.5×10¹² vg/mL, about 4.6×10¹² vg/mL, about 4.7×10¹² vg/mL, about 4.8×10¹² vg/mL, about 4.9×10¹² vg/mL, about 5×10¹² vg/mL, about 5.1×10¹² vg/mL, about 5.2×10¹² vg/mL, about 5.3×10¹² vg/mL, about 5.4×10¹² vg/mL, about 5.5×10¹² vg/mL, about 5.6×10¹² vg/mL, about 5.7×10¹² vg/mL, about 5.8×10¹² vg/mL, about 5.9×10¹² vg/mL, about 6×10¹² vg/mL, about 6.1×10¹² vg/mL, about 6.2×10¹² vg/mL, about 6.3×10¹² vg/mL, about 6.4×10¹² vg/mL, about 6.5×10¹² vg/mL, about 6.6×10¹² vg/mL, about 6.7×10¹² vg/mL, about 6.8×10¹² vg/mL, about 6.9×10¹² vg/mL, about 7×10¹² vg/mL, about 7.1×10¹² vg/mL, about 7.2×10¹² vg/mL, about 7.3×10¹² vg/mL, about 7.4×10¹² vg/mL, about 7.5×10¹² vg/mL, about 7.6×10¹² vg/mL, about 7.7×10¹² vg/mL, about 7.8×10¹² vg/mL, about 7.9×10¹² vg/mL, about 8×10¹² vg/mL, about 8.1×10¹² vg/mL, about 8.2×10¹² vg/mL, about 8.3×10¹² vg/mL, about 8.4×10¹² vg/mL, about 8.5×10¹² vg/mL, about 8.6×10¹² vg/mL, about 8.7×10¹² vg/mL, about 8.8×10¹² vg/mL, about 8.9×10¹² vg/mL, about 9×10¹² vg/mL, about 9.1×10¹² vg/mL, about 9.2×10¹² vg/mL, about 9.3×10¹² vg/mL, about 9.4×10¹² vg/mL, about 9.5×10¹² vg/mL, about 9.6×10¹² vg/mL, about 9.7×10¹² vg/mL, about 9.8×10¹² vg/mL, about 9.9×10¹² vg/mL, or about 1×10¹³ vg/mL.

In some embodiments, the fluid formulation of the pharmaceutical composition comprising the recombinant AAV at a concentration in the range of from about 1×10¹¹ vg/mL to about 1×10¹³ vg/mL is administered into an area of the brain adjacent to the core region of the stroke once. In some embodiments, the fluid formulation of the pharmaceutical composition comprising the recombinant AAV at a concentration in the range of from about 1×10¹¹ vg/mL to about 1×10¹³ vg/mL is administered into a peri-infarct region of the stroke once.

In specific embodiments, the subject is administered intracerebrally the pharmaceutical composition comprising about 3×10¹¹ vg of the recombinant AAV once. In specific embodiments, the subject receives intracerebral injection of 0.6 mL of the pharmaceutical composition comprising about 5×10¹¹ vg/mL of the recombinant AAV. In some embodiments, the pharmaceutical composition is administered intracerebrally through stereotactic brain injection. In some embodiments, the injection sites are selected based on MRI scan. In some embodiments, injection sites should be located around the infarct lesion. In some embodiments, injection sites should be located in peri-infarct motor cortex.

In specific embodiments, the subject is administered intracerebrally the pharmaceutical composition comprising about 6×10¹¹ vg of the recombinant AAV once. In specific embodiments, the subject receives intracerebral injection of 0.6 mL of the pharmaceutical composition comprising about 1×10¹² vg/mL of the recombinant AAV. In some embodiments, the pharmaceutical composition is administered intracerebrally through stereotactic brain injection. In some embodiments, the injection sites are selected based on MRI scan. In some embodiments, injection sites should be located around the infarct lesion. In some embodiments, injection sites should be located in peri-infarct motor cortex.

In specific embodiments, the subject is administered intracerebrally the pharmaceutical composition comprising about 1.2×10¹² vg of the recombinant AAV once. In specific embodiments, the subject receives intracerebral injection of 0.6 mL of the pharmaceutical composition comprising about 2×10¹² vg/mL of the recombinant AAV. In some embodiments, the pharmaceutical composition is administered intracerebrally through stereotactic brain injection. In some embodiments, the injection sites are selected based on MRI scan. In some embodiments, injection sites should be located around the infarct lesion. In some embodiments, injection sites should be located in peri-infarct motor cortex.

As a non-limiting example, injecting can comprise the use of a syringe and needle. In an aspect, an AAV vector or composition is injected into a subject, e.g., into the brain of a subject. In an aspect, an AAV vector or composition is injected using a 33-gauge needle that is 1.5 inches in length and has a 30° bevel. In an aspect, an AAV vector or composition is injected using a 100 μL syringe equipped with a 33-gauge needle, 1.5 in length, with a 30° bevel. In an aspect, an AAV vector or composition is injected using a syringe pump. In an aspect, an AAV vector or composition is injected using a syringe pump mounted on a stereotaxic arm. In an aspect, an injection site is determined prior to the injecting via a magnetic resonance imaging (MRI) scan. In an aspect, coordinates of the determined injection site are used for the injecting, such for injecting the brain of a subject. In an aspect, an AAV vector or composition is injected using a surgical navigation system to target an injection site.

In one aspect, all injection sites are located around the infarct lesion and cover the peri-infarct motor cortex and are determined by MRI scan. The injection volume of each site is approximately 25-50 μL. The injection rate is no more than 10 μL/min.

In some embodiments, upon administration of the recombinant AAV encoding a NeuroD1 polypeptide as described herein, or a pharmaceutical composition thereof, to a subject suffering from stroke, the NeuroD1 polypeptide encoded by the recombinant AAV genome is expressed in a population of glial cells in the subject. In an aspect, the present disclosure provides, and includes, methods of treating stroke in a subject by converting glial cells into neurons via the expression of NeuroD1 in the glial cells. In some embodiments, the neurons are selected from glutamatergic neurons, GABAergic neurons, dopaminergic neurons; motor neurons, glycinergic neurons, serotonergic neurons, norepinephrinergic neurons, and sensory neurons.

In some embodiments, upon administration of the recombinant AAV encoding a NeuroD1 polypeptide as described herein, or a pharmaceutical composition thereof, to the subject suffering from stroke, the NeuroD1 polypeptide encoded by the recombinant AAV genome is expressed in a population of glial cells in the subject, and the population of glial cells is converted into neurons. In specific embodiments, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%of the glial cells are converted into neurons. In some embodiments, the conversion of glial cells into neurons is measure by the expression level of neuronal marker. In some embodiments, conversion of a glial cell into a neuron is measured via the detection of the expression level of neuronal marker, such as DCX, TUJ1, NeuN, MAP2, or Parvalbumin, in the converted cells. Additionally, SMI312 and SMI32 antibodies can be used to confirm the neuronal or neuron-like characteristics of the converted cells. In specific embodiments, conversion of glial cells into neurons occurs in less than about 21 days, less than about 14 days, less than about 13 days, less than about 12 days, less than about 11 days, less than about 10 days, less than about 9 days, less than about 8 days, less than about 7 days, less than about 6 days, less than about 5 days, less than about 4 days, less than about 3 days, less than about 2 days, or less than about 1 day after the subject received administration of the recombinant AAV encoding a NeuroD1 polypeptide as described herein, or a pharmaceutical composition thereof.

In some embodiments, upon administration of the recombinant AAV encoding a NeuroD1 polypeptide as described herein, or a pharmaceutical composition thereof, to the subject suffering from stroke, the NeuroD1 polypeptide encoded by the recombinant AAV genome is expressed in a population of glial cells in the subject, and the population of glial cells start to exhibit one or more neuronal phenotypes. In some embodiments, the one or more neuronal phenotypes comprise expression of one or more neuronal markers selected from DCX, TUJ1, NeuN, MAP2, and Parvalbumin. In some embodiments, the one or more neuronal phenotypes comprise ability of firing action potentials. In some embodiments, the one or more neuronal phenotypes comprise formation of dendrites and/or exons on the cell surface. In some embodiments, the one or more neuronal phenotypes comprise formation of synapses with a neighboring cell. In some embodiments, the one or more neuronal phenotypes comprise the ability of releasing synaptic currents. In some embodiments, the synaptic currents are glutamatergic current, GABAergic current, Dopaminergic current, glycinergic current, serotonergic current or norepinephrinergic current. In specific embodiments, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%of the glial cells in the population start to exhibit one or more neuronal phenotypes. In specific embodiments, the population of glial cells start to exhibit one or more neuronal phenotypes in less than about 21 days, less than about 14 days, less than about 13 days, less than about 12 days, less than about 11 days, less than about 10 days, less than about 9 days, less than about 8 days, less than about 7 days, less than about 6 days, less than about 5 days, less than about 4 days, less than about 3 days, less than about 2 days, or less than about 1 day after the subject received administration of the recombinant AAV encoding a NeuroD1 polypeptide as described herein, or a pharmaceutical composition thereof.

In some embodiments, upon administration of the recombinant AAV encoding a NeuroD1 polypeptide as described herein, or a pharmaceutical composition thereof, to the subject suffering from stroke, the NeuroD1 polypeptide encoded by the recombinant AAV genome is expressed in a population of glial cells in the subject, and the population of glial cells stop expressing one or more glial markers. In some embodiments, the one or more glial markers is selected from GFAP, Aldh1l1, S100β and Sox9. In specific embodiments, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%of the glial cells in the population stop to express one or more glial markers. In specific embodiments, the population of glial cells stop to express one or more glial markers in less than about 21 days, less than about 14 days, less than about 13 days, less than about 12 days, less than about 11 days, less than about 10 days, less than about 9 days, less than about 8 days, less than about 7 days, less than about 6 days, less than about 5 days, less than about 4 days, less than about 3 days, less than about 2 days, or less than about 1 day after the subject received administration of the recombinant AAV encoding a NeuroD1 polypeptide as described herein, or a pharmaceutical composition thereof.

In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a primate (e.g., a monkey such as, a cynomolgus monkey and a human) or a non-primate (e.g., a cow, a pig, a horse, a cat, a dog, a rat, a mouse) . In certain embodiments, the subject is a mouse or a rat. In certain embodiments, the subject is a human.

In some embodiments, the population of glial cells are located in the brain of a subject who suffered a stroke. In some embodiments, the population of glial cells are located in the grey matter of the brain. In some embodiments, the population of glial cells are located in the white matter of the brain. In some embodiments, the population of glial cells are located in the brain striatum. In some embodiments, the population of glial cells are located in the cortex of the brain. In some embodiments, the population of glial cells are located in the hippocampus of the brain. In some embodiments, the population of glial cells are located in the cerebellum of the brain. In some embodiments, the population of glial cells are located around the infarct lesion of the brain. In some embodiments, the population of glial cells are located in the peri-infarct motor cortex of the brain. In some embodiments, the population of glial cells comprises one or more glial cell types selected from astrocytes, reactive astrocytes, NG-2 cells, reactive NG-2 cells, and microglial cells that are undergoing pathogenic neoplasm.

In one aspect, a method provided herein converts glial cells to functional neurons in the brain of a subject who suffered a stroke. In an aspect, a method provided herein converts glial cells to functional neurons in a cerebral cortex of the brain. In one aspect, a method provided herein converts glial cells to functional neurons in a striatum of the brain. In one aspect, a method provided herein converts glial cells to functional neurons in a dorsal striatum of the brain. In one aspect, a method provided herein converts glial cells to functional neurons in a spinal cord of the brain. In one aspect, a method provided herein converts glial cells to functional neurons in a putamen of the brain. In one aspect, a method provided herein converts glial cells to functional neurons in a caudate nucleus of the brain. In one aspect, a method provided herein converts glial cells to functional neurons in a substantia nigra of the brain. In one aspect, a method provided herein converts glial cells to functional neurons in the primary motor cortex. In one aspect, newly formed neurons in the primary motor cortex send axons to appropriate targets along the corticospinal tract (e.g., the striatum and the brainstem) .

In one aspect, a method provided herein converts astrocytes to functional neurons in the brain of a subject who suffered a stroke. In an aspect, a method provided herein converts astrocytes to functional neurons in a cerebral cortex of the brain. In one aspect, a method provided herein converts astrocytes to functional neurons in a striatum of the brain. In one aspect, a method provided herein converts astrocytes to functional neurons in a dorsal striatum of the brain. In one aspect, a method provided herein converts astrocytes to functional neurons in a spinal cord of the brain. In one aspect, a method provided herein converts astrocytes to functional neurons in a putamen of the brain. In one aspect, a method provided herein converts astrocytes to functional neurons in a caudate nucleus of the brain. In one aspect, a method provided herein converts astrocytes to functional neurons in a substantia nigra of the brain. In one aspect, a method provided herein converts astrocytes to functional neurons in the primary motor cortex. In one aspect, newly formed neurons in the primary motor cortex send axons to appropriate targets along the corticospinal tract (e.g., the striatum and the brainstem) .

In one aspect, a method provided herein converts reactive astrocytes to functional neurons in the brain of a subject who suffered a stroke. In an aspect, a method provided herein converts reactive astrocytes to functional neurons in a cerebral cortex of the brain. In one aspect, a method provided herein converts reactive astrocytes to functional neurons in a striatum of the brain. In one aspect, a method provided herein converts reactive astrocytes to functional neurons in a dorsal striatum of the brain. In one aspect, a method provided herein converts reactive astrocytes to functional neurons in a spinal cord of the brain. In one aspect, a method provided herein converts reactive astrocytes to functional neurons in a putamen of the brain. In one aspect, a method provided herein converts reactive astrocytes to functional neurons in a caudate nucleus of the brain. In one aspect, a method provided herein converts reactive astrocytes to functional neurons in a substantia nigra of the brain. In one aspect, a method provided herein converts reactive astrocytes to functional neurons in the primary motor cortex. In one aspect, newly formed neurons in the primary motor cortex send axons to appropriate targets along the corticospinal tract (e.g., the striatum and the brainstem) .

In an aspect, the present disclosure provides, and includes, methods of partially or fully restoring neuronal pathways in the brain of a subject who has suffered a stroke. In some embodiments, upon administration of the recombinant AAV encoding a NeuroD1 polypeptide as described herein, or a pharmaceutical composition thereof, to a subject suffering from stroke, partially or fully neuronal pathways are restored in the brain of the subject. In an aspect, the present disclosure provides, and includes, methods of partially or fully restoring neuronal pathways in the brain of a subject who has suffered a stroke by converting glial cells into neurons. In an aspect, the present disclosure provides, and includes, methods of partially or fully restoring neuronal pathways in the brain of a subject who has suffered a stroke by converting glial cells into neurons via the expression of NeuroD1 in the glial cells. In an aspect, the partial or full restoration of the neuronal pathways in the brain of the subject can be assessed by MRI. In an aspect, the partial or full restoration of the neuronal pathways in the brain of the subject can be assessed by Diffusion Tensor Imaging (DTI) .

In an aspect, the present disclosure provides, and includes, methods of reducing neuroinflammation in the brain of a subject who has suffered a stroke. In some embodiments, upon administration of the recombinant AAV encoding a NeuroD1 polypeptide as described herein, or a pharmaceutical composition thereof, to a subject suffering from stroke, neuroinflammation in the brain of the subject are reduced. In an aspect, the present disclosure provides, and includes, methods of reducing neuroinflammation in the brain of a subject who has suffered a stroke by converting glial cells into neurons. In an aspect, the present disclosure provides, and includes, methods of reducing neuroinflammation in the brain of a subject who has suffered a stroke by converting glial cells into neurons via the expression of NeuroD1 in the glial cells. In an aspect, reduction in neuroinflammation is determined by measuring the expression of Iba1 in a region of the brain of the subject. In an aspect, reduction in neuroinflammation is determined by measuring the abundance of microglia in a region of the brain of the subject. In specific embodiments, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%neuroinflammation is reduced in the brain of the subject who has suffered a stroke. In specific embodiments, neuroinflammation is reduced in the brain of the subject who has suffered a stroke in less than about 21 days, less than about 14 days, less than about 13 days, less than about 12 days, less than about 11 days, less than about 10 days, less than about 9 days, less than about 8 days, less than about 7 days, less than about 6 days, less than about 5 days, less than about 4 days, less than about 3 days, less than about 2 days, or less than about 1 day after the subject received administration of the recombinant AAV encoding a NeuroD1 polypeptide as described herein, or a pharmaceutical composition thereof.

In some embodiments, the life span of the subject suffering from stroke is increased. In some embodiments, the life span of the subject suffering from stroke is increased for at least about10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%.

In some embodiments, upon administration of the recombinant AAV encoding a NeuroD1 polypeptide as described herein, or a pharmaceutical composition thereof, to a subject suffering from stroke, one or more neurological condition symptoms are eliminated, reduced, slowed or delayed. In some embodiments, the one or more neurological condition symptoms are eliminated, reduced, slowed or delayed for at least about10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%.

Non-limiting examples of symptoms of stoke (e.g., ischemic or hemorrhagic stroke) include tremor, slowed movement (bradykinesia) , rigid muscles, impaired posture and balance, loss of automatic movements, uncoordinated movement, uncontrolled movement, spontaneous jerking movement, speech changes, numbness, and writing changes. In an aspect, a symptom of stoke is a movement symptom. Non-limiting examples of movement symptoms include impairment of an involuntary movement or an impairment of a voluntary movement. In one aspect, a neurological condition symptom is a cognitive symptom. Non-limiting examples of cognitive symptoms include fine motor skills, tremors, seizures, chorea, dystonia, dyskinesia, slow or abnormal eye movements, impaired gait, impaired posture, impaired balance, difficulty with speech, difficulty with swallowing, difficulty organizing, difficulty prioritizing, difficulty focusing on tasks, lack of flexibility, lack of impulse control, outbursts, lack of awareness of one's own behaviors and/or abilities, slowness in processing thoughts, difficulty in learning new information, difficulty in remember things, difficulty in communications, difficulty in following orders, difficulty in executing tasks. In an aspect, the symptom of stoke is a psychiatric symptom. Non-limiting examples of psychiatric symptoms include depression, irritability, sadness or apathy, social withdrawal, insomnia, fatigue, lack of energy, obsessive-compulsive disorder, mania, bipolar disorder, and weight loss. In one aspect, the symptom of stoke is at least one damaged blood vessel. In one aspect, the symptom of stoke is a damaged blood brain barrier. In one aspect, the symptom of stoke is damaged blood flow. Non-limiting examples of tests to evaluate the elimination, reduction, slow, or delay, of symptoms of stoke (e.g., ischemic or hemorrhagic stroke) include the unified Huntington's disease rating scale (UHDRS) score, UHDRS Total Functional Capacity (TFC) , UHDRS Functional Assessment, UHDRS Gait score, UHDRS Total Motor Score (TMS) , Hamilton depression scale (HAM-D) , Columbia-suicide severity rating scale (C-SSRS) , Montreal cognitive assessment (MoCA) , modified Rankin Scale (mRS) , National Institutes of Health Stroke Scale (NIHSS) , and Barthel Index (BI) , Timed Up and Go Test (TUG) , Chedoke Arm and Hand Activity Inventory (CAHAI) , Symbol Digit Modalities Test, Controlled Oral Word Association tasks, magnetic resonance imaging (MRI) , functional magnetic resonance imaging (fMRI) , and positron emission tomography (PET) scanning.

In some embodiments, the recombinant AAV encoding a NeuroD1 polypeptide as described herein, or a pharmaceutical composition thereof is administered to a subject suffering from stroke and has a score of at least 20 on the NHPSS. In some embodiments, the recombinant AAV encoding a NeuroD1 polypeptide as described herein, or a pharmaceutical composition thereof, is administered to a subject suffering from stroke and has a score of at least 25 on the NHPSS. In an aspect, the NHPSS score of the subject is improved by at least 1 unit after the subject is administered the recombinant AAV encoding a NeuroD1 polypeptide as described herein, or a pharmaceutical composition thereof. In an aspect, the NHPSS score of the subject is improved by at least 1 unit, 2 units, 3 units, 4 units, 5 units, 6 units, 7 units, 8 units, 9 units, 10 units, 11 units, 12 units, 13 units, 14 units, 15 units, 16 units, 17 units, 18 units, 19 units, 20 units, 21 units, 22 units, 23 units, 24 units, or 25 units after the subject is administered the recombinant AAV encoding a NeuroD1 polypeptide as described herein, or a pharmaceutical composition thereof. In an aspect, the NHPSS score is improved within 30 days to 100 days after the subject has been administered the recombinant AAV encoding a NeuroD1 polypeptide as described herein, or a pharmaceutical composition thereof. In an aspect, the NHPSS score is improved within 40 days to 100 days, within 40 days to 90 days, within 40 days to 80 days, within 40 days to 70 days, within 40 days to 60 days, within 40 days to 50 days, within 50 days to 100 days, within 60 days to 100 days, within 70 days to 100 days, within 80 days to 100 days, within 90 days to 100 days, within 50 days to 90 days, or within 60 days to 80 days after the subject has been administered the recombinant AAV encoding a NeuroD1 polypeptide as described herein, or a pharmaceutical composition thereof.

In some embodiments, the recombinant AAV encoding a NeuroD1 polypeptide as described herein, or a pharmaceutical composition thereof is administered to a subject suffering from stroke and has a score of at least 3 on the mRS. In some embodiments, the recombinant AAV encoding a NeuroD1 polypeptide as described herein, or a pharmaceutical composition thereof, is administered to a subject suffering from stroke and has a score of at least 4 on the mRS. In an aspect, the mRS score of the subject is improved by at least 1 unit after the subject is administered the recombinant AAV encoding a NeuroD1 polypeptide as described herein, or a pharmaceutical composition thereof. In an aspect, the mRS score of the subject is improved by at least 1 unit, 2 units, 3 units, 4 units, or 5 units after the subject is administered the recombinant AAV encoding a NeuroD1 polypeptide as described herein, or a pharmaceutical composition thereof. In an aspect, the mRS score is improved within 30 days to 100 days after the subject has been administered the recombinant AAV encoding a NeuroD1 polypeptide as described herein, or a pharmaceutical composition thereof. In an aspect, the mRS score is improved within 40 days to 100 days, within 40 days to 90 days, within 40 days to 80 days, within 40 days to 70 days, within 40 days to 60 days, within 40 days to 50 days, within 50 days to 100 days, within 60 days to 100 days, within 70 days to 100 days, within 80 days to 100 days, within 90 days to 100 days, within 50 days to 90 days, or within 60 days to 80 days after the subject has been administered the recombinant AAV encoding a NeuroD1 polypeptide as described herein, or a pharmaceutical composition thereof.

In some embodiments, the recombinant AAV encoding a NeuroD1 polypeptide as described herein, or a pharmaceutical composition thereof is administered to a subject suffering from stroke and has a score of at least 10 on the Motor subscore. In some embodiments, the recombinant AAV encoding a NeuroD1 polypeptide as described herein, or a pharmaceutical composition thereof, is administered to a subject suffering from stroke and stroke and has a score of at least 12 on the Motor subscore. In an aspect, the Motor subscore of the subject is improved by at least 1 unit after the subject is administered the recombinant AAV encoding a NeuroD1 polypeptide as described herein, or a pharmaceutical composition thereof. In an aspect, the mRS score of the subject is improved by at least 1 unit, 2 units, 3 units, 4 units, 5 units, 6 units, 7 units, 8 units, 9 units, or 10 units after the subject is administered the recombinant AAV encoding a NeuroD1 polypeptide as described herein, or a pharmaceutical composition thereof. In an aspect, the mRS score is improved within 30 days to 100 days after the subject has been administered the recombinant AAV encoding a NeuroD1 polypeptide as described herein, or a pharmaceutical composition thereof. In an aspect, the mRS score is improved within 40 days to 100 days, within 40 days to 90 days, within 40 days to 80 days, within 40 days to 70 days, within 40 days to 60 days, within 40 days to 50 days, within 50 days to 100 days, within 60 days to 100 days, within 70 days to 100 days, within 80 days to 100 days, within 90 days to 100 days, within 50 days to 90 days, or within 60 days to 80 days after the subject has been administered the recombinant AAV encoding a NeuroD1 polypeptide as described herein, or a pharmaceutical composition thereof.

In some embodiments, the subject is a human patient suffering from stroke (e.g., ischemic stroke or hemorrhagic stroke) . In some embodiments, the subject has clinical diagnosis of stroke is confirmed by neuro-imaging, such as a CT or MRI scan. In some embodiments, the subject suffered from stroke for at least about 1 month. In some embodiments, the subject suffered from stroke for at least about 2 months. In some embodiments, the subject suffered from stroke for at least about 3 months. In some embodiments, the subject suffered from stroke for at least about 4 months. In some embodiments, the subject suffered from stroke for about 2 to about 4 months.

In some embodiments, the subject has a stroke lesion with a size of about 20 to about 80ml. In some embodiments, the subject has a stroke lesion that is affecting motor cortex. In some embodiments, the subject has a stroke lesion that is causing damage to corticospinal tract. In some embodiments, the subject has a stroke lesion that is identifiable by neuro-imaging such as an MRI scan.

In some embodiments, the subject suffers from moderate to severe motor dysfunction after receiving standardized or guide-recommended recommended rehabilitation therapy after the episode of stroke (e.g., ischemic stroke) . In some embodiments, the motor dysfunction the subject is suffering is characterized by a NIHSS score of about 6-20 points. In some embodiments, the motor dysfunction the subject is suffering is characterized by an affected upper or lower limb motor score of about 3-4.

In some embodiments, the subject does not have motor deficit due to ischemic stroke of posterior circulation. In some embodiments, the subject does not have motor deficit due to any causes other than the stroke. In some embodiments, the subject does not have a history of epilepsy. In some embodiments, the subject does not have a history of encephalitis. In some embodiments, the subject does not have a history of meningitis. In some embodiments, the subject does not have a history of multiple sclerosis. In some embodiments, the subject does not have a history of central nervous system infection. In some embodiments, the subject does not have a history of intracranial hemorrhage. In some embodiments, the subject does not have a history of subarachnoid hemorrhage. In some embodiments, the subject does not have a severe history of head trauma. In some embodiments, the subject does not have a history of malignant tumors within 5 years. In some embodiments, the subject does not have a history of malignant tumors within 5 years, except for one or more selected from adequately treated cervical carcinoma in situ, papillary thyroid cancer, basal cell or squamous epithelial cell skin cancer, localized prostate cancer after radical surgery, and breast ductal carcinoma in situ.

In some embodiments, the subject has no serum anti-AAV9 antibody. In some embodiments, the subject has serum anti-AAV9 antibody at a title less than 1: 100.

In some embodiments, the subject has not active infections. In some embodiments, the subject does not have HIV infection. In some embodiments, the subject does not have infection by hepatitis A, B, or C.In some embodiments, the subject does not have syphilis.

In some embodiments, the subject is treatment for gene or cell therapy for stroke.

In some embodiments, the subject does not have a condition requiring anticoagulant treatment.

In some embodiments, the subject does not have a condition requiring intermittent use of oral anti-spasticity medications during the period starting from about 1-month prior to receiving treatment with the present AAV vectors and ending after about 3 months after the treatment with the present AAV vectors.

In some embodiments, the subject does not suffer from insufficient reserved functions of liver, kidney and bone marrow, characterized by one or more parameters selected from Neutrophil count <1,500/mm ³ ; platelets <100, 000/mm ³ ; hemoglobin <9.0 g/dL; serum creatinine >1.5 times the upper limit of normal range (ULN) ; renal function eGFR < 60mL/min/1.73m² ; Bilirubin, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) >2.5 times ULN; activated partial prothrombin time (APTT ) or international normalized ratio (INR ) >1.3 times ULN.

In certain embodiments, the recombinant AAV vector may be administered alone or in combination with other prophylactic and/or therapeutic agents. In certain embodiments, the presently disclosed AAV vectors are administered intravenously and may be administered together with other biologically active agents.

The dosage amounts and frequencies of administration provided herein are encompassed by the terms therapeutically effective and prophylactically effective. The dosage and frequency typically vary according to factors specific for each patient depending on the specific therapeutic or prophylactic AAV vectors administered, the severity and type of disease, the route of administration, as well as age, body weight, response, and the past medical history of the patient, and should be decided according to the judgment of the practitioner and each patient’s circumstances. Suitable regimens can be selected by one skilled in the art by considering such factors and by following, for example, dosages reported in the literature and recommended in the Physician’s Desk Reference. Prophylactic and/or therapeutic AAV vectors can be administered repeatedly. Several aspects of the procedure may vary such as the temporal regimen of administering the prophylactic or therapeutic AAV vectors, and whether such AAV vectors are administered separately or as an admixture.

Effective doses of the AAV vector can be determined by standard clinical techniques. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems. In certain embodiments, the therapeutically effective dose can be estimated initially from cell culture assays.

The presently disclosed AAV vectors, as well as combinations thereof, can be tested in suitable animal model systems prior to use in humans. Such animal model systems include, but are not limited to, rats, mice, chicken, cows, monkeys, pigs, dogs, rabbits, etc. Any animal system known in the art may be used. Such model systems are widely used and well known to the skilled artisan. In certain embodiments, animal model systems for a CNS condition are used that are based on rats, mice, or other small mammal other than a primate.

Once the presently disclosed AAV vectors have been tested in an animal model, they can be tested in clinical trials to establish their efficacy. Establishing clinical trials will be done in accordance with common methodologies known to one skilled in the art, and the optimal dosages and routes of administration as well as toxicity profiles of the presently disclosed AAV vectors can be established. For example, a clinical trial can be designed to test the presently disclosed AAV vectors for efficacy and toxicity in human patients.

Toxicity and efficacy of the presently disclosed AAV vectors can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50%of the population) and the ED50 (the dose therapeutically effective in 50%of the population) . The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. AAV vectors that exhibit large therapeutic indices are preferred. While AAV vectors that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such AAV vectors to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

The presently disclosed AAV vectors generally will be administered for a time and in an amount effective for obtain a desired therapeutic and/or prophylactic benefit. The data obtained from the cell culture assays and animal studies can be used in formulating a range and/or schedule for dosage of the presently disclosed AAV vectors for use in humans. The dosage of such AAV vectors lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
6. Examples
6.1. Example 1. Stroke model: Middle cerebral artery occlusion in nonhuman primates.

Middle Cerebral Artery Occlusion (MCAO) Ischemic Stroke Induction: Monkeys were subjected to transient cerebral ischemia by temporarily occluding the right side middle cerebral artery M2 (MCAO-M2) for 120 minutes. To prepare for the surgery, monkeys were fasted overnight and anesthetized with ketamine (8 mg/kg, IM) , followed by administration of atropine (0.04 mg/kg, IM) , iron dextran (30.0 mg/kg IM) , Dura Penn (60, 000 units/kg IM) , meloxicam (0.20 mg/kg SC) , or equivalent. Animals were shaved and prepped aseptically for an off-midline craniotomy to expose the temporal region of the calveria. Animals were intubated and maintained on isoflurane anesthesia combined with O₂, to effect. Supplemental heat/cooling were supplied via a circulating water blanket or warm air blanket. Body temperature was maintained at about 33 ℃ to mitigate the potential for an increase in intracranial pressure and accelerated neuroendangerment. Heart rate, respiratory rate, rectal temperature, electrocardiogram (ECG) , SpO₂ (oxygen saturation) , CO₂, capillary refill time (CRT) , and mucous membrane color were monitored during the procedure. A peripheral catheter was placed for administration of lactated Ringer’s solution at a 3.0 mL/hr rate by drip or infusion pump. The animals were then transferred to the surgical suite, prepped, and draped, then placed on assisted ventilation.

Using a surgical drill and an approximately 2 mm burr bit, a craniotomy was performed over the right temporal bone to form a roughly 7 mm × 10 mm craniectomy. The dura mater was opened to expose the brain surface. The Sylvian fissure (lateral sulcus) was located and, using a microscope and microsurgical technique, the arachnoid was teased away from the vessels in the Sylvian fissure to expose the Middle Cerebral Artery (MCA) . With careful retraction along the Sylvian fissure, the MCA was visualized down in the Sylvian cistern and the M2-M3 bifurcation visualized and isolated proximal to the bifurcation. An MRI-compatible aneurysm clip was placed occluding the M2 segment of the MCA proximal to the M3 bifurcation and occluding both branches of the M3.

Once clamped, the incision was temporally closed, and the animal transported to a Magnetic Resonance Imaging (MRI) machine for an interoperative scan. The interoperative scan provided an early timepoint on the status of the infarct, and in some cases can help guide the duration of the occlusion. If there was insufficient evidence of infarct, the occlusion duration may be extended up to the 180 min occlusion maximum. Following the scan, the animal was returned to the operating suite, the incision reopened, the clip removed, and reperfusion visually verified. The dura opening was then covered with moist gelfoam. The muscles and skin were closed in layers with 3-0 Vicryl suture to complete the craniectomy. Tissue adhesive was applied to prevent migration of cage debris into the incision site.

Post-surgery, the animals received prophylactic anti-seizure medication (Phenytoin 5.0 mg/kg, administered as a slow IV bolus over 2 minutes, or equivalent as directed by a facility veterinarian) . Bupivacaine (1 mg/kg ID) was infused into the incision sites to minimize local pain and discomfort. Buprenorphine (0.02 mg/kg IM) was administered, or an alternative, if recommended by a facility veterinarian.
6.2. Example 2. Adeno-Associated Virus (AAV) Production

AAV9-GFAP-NeuroD1 (aka AAV-NeuroD1) was produced from plasmid CE-pGfa681-CRGI-hND1-oWPRE-bGHpA and packaged by Packgene. The stock viral titer of AAV9-GFAP-NeuroD1 is 1 × 10¹³ vg/mL. A reporter vector, AAV9-GFAP-GFP was produced from plasmid pGfaABC1D: GFP and packaged by Packgene. The stock viral titer of AAV9-GFAP-GFP was 1 × 10¹³ vg/mL.

AAV particles were produced by the triple transfection method using a HEK293 production cell line. Production and quality control of viral preparations for clinical use strictly followed cGMP standards. Particularly, HEK293 cells were seeded and expanded, and co-transfected with three plasmids:
(1) a gene of interest (GOI) plasmid containing the transgene expression elements flanked by AAV ITRs;
(2) a helper plasmid encoding adenovirus regions (VA, E2A and E4) that mediate AAV vector
replication; and (3) a rep-cap packaging plasmid encoding the AAV capsid proteins (VP1, VP2, and VP3) through alternative splicing and initiation of translation, and AAV life cycle machinery Rep proteins (Rep78, Rep68, Rep52 and Rep40) through two promoters and alternative splicing.

Table 6.2 (A) shows the sequences of functional fragments of the GOI plasmid used to produce AAV encoding NeuroD1 (AAV-NeuroD1) , and the full-length sequence of the GOI plasmid used in the study.
Table 6.2 (A) GOI Plasmid

Rep genes from AAV2 serotype were used for packaging AAV9 serotype virus, while capsid protein genes were serotype specific. Rep gene and encoded Rep protein sequences can be found in Table 6.2 (B) , SEQ ID NOS: 34-38. Cap gene and encoded capsid protein sequences for serotype 9 AAV virus can be found in Table 6.2 (B) , SEQ ID NOS: 39-42. The full-length sequence of a Rep (serotype 2) /Cap (serotype 9) packaging plasmid used in the study can be found in Table 6.2 (C) , SEQ ID NO: 43. Helper plasmid sequences used in this study can be found in Table 6.2 (C) SEQ ID NO: 44 and SEQ ID NO: 45.
Table 6.2 (B) Rep/Cap sequences

Table 6.2 (C) Packaging and Helper Plasmids

After triple-transfection, the cells were harvested in lysis buffer at 48-72 hours post transfection. For clinical use, the viral particles were purified by affinity purification, followed by ultracentrifugation, and ion exchange filtration after treatment of Benzonase and clear out cell debris. For research use, viral particles were purified by PEG as a preliminary purification step, followed by ultracentrifugation, and Ultrafiltration after treatment of Benzonase and clear out cell debris.

Final virus suspensions were produced after buffer exchange, concentration, and sterile filtration steps and were ready for in vitro or in vivo applications. The viral preparations were aliquoted before use.
6.3. Example 3. Selection of AAV Injection Sites

One day prior to AAV injection, subjects underwent a targeting MRI session to visualize the stroke region and borders. T1 MRI scans were used to delineate the core and the peri-infarct region of the stroke. The peri-infarct area was the region of the brain adjacent to the stroke. Three injection sites were selected, targeting the peri-infarct cortex. These sites were at mid-cortical depth, ideally 3-4 mm below the pia to target layer V. Alternatively, an injection can be dispensed at two sites along the same needle track, with a superficial injection (in layer II-IV) followed by a deeper injection (in layer V/VI) . No injections were delivered to the core of the stroke, as the core does not contain viable reactive astrocytes. The peri-infarct region contains abundant reactive astrocytes that can be converted into neurons. At least one injection was made in the cortex immediately adjacent to the primary motor cortex. Injected AAV may spread into white matter although the primary objective was to target grey matter in the cortex. Coordinates of each injection site were saved and targeted by either stereotaxis (ROSA robot or similar) or (ideally) neuro-navigation using a Stryker iNav, StealthStation, or other comparable system (Figure 1) .
6.4. Example 4. AAV Delivery

AAV9-GFAP-NeuroD1 and AAV9-GFAP-GFP were co-injected to permit labeling of the AAV-transduced cells. This allowed for the quantification of astrocyte-to-neuron conversion in transduced cells. The AAV9-GFAP-GFP vector was administered at 1/5th the dose of the AAV9-GFAP-NeuroD1 vector. The number of burr holes or injection sites was 3 per animal.

Delivery device: A 100 μL Hamilton Gastight Syringe equipped with a 33-gauge needle, 1.5 in length, with a 30° bevel was used. The syringe was placed into a syringe pump (Pump 11 Elite Nanomite, Harvard Apparatus) that was mounted on a stereotaxic arm. The rate of controlled infusion was 1 μL per minute. MRI guidance of injections was performed using a Stryker Nav3i system. The dose titer and volume were 20 μL per injection site at 5 x 10¹¹ vg/mL of AAV9-GFAP-NeuroD1 and 1 x 10¹¹ vg/mL of AAV9-GFAP-GFP.
6.5. Example 5. Behavioral Data Collection

Non-Human Primate Stroke Scale (NHPSS) : This scale has been previously validated in cynomolgus macaques following middle cerebral artery occlusion. (Roitberg et al, Neurol Res. 2003 Jan; 25 (1) : 68-78; Swieten et al., Stroke vol. 19, 5 (1988) : 604-7, and Wilson et al., Stroke vol. 36, 4 (2005) : 777-81) . The scale is comprised of 11 categories, each scored independently to produce a composite score out of 41 points (0 being normal, 41 being severely impaired) . The categories include state of consciousness, defense reaction, grasp reflex, extremity movement, gait, circling behavior, bradykinesia, balance, neglect, visual field defect, and facial weakness.

Modified Rankin Scale (mRS) : This is a 6 point disability scale with possible scores ranging from 0 to 5. A separate category of 6 is usually added for patients who expire. The Modified Rankin Score (mRS) is the most widely used outcome measure in stroke clinical trials. The scoring system used in humans is as follows:
● 0: No symptoms at all
● 1: No significant disability despite symptoms; able to carry out all usual duties and activities
● 2: Slight disability; unable to carry out all previous activities, but able to look after own
affairs without assistance
● 3: Moderate disability; requiring some help, but able to walk without assistance
● 4: Moderately severe disability; unable to walk without assistance and unable to attend to
own bodily needs without assistance
● 5: Severe disability; bedridden, incontinent and requiring constant nursing care and attention
● 6: Dead

NHPSS and (mRS assessments are performed daily on the subjects for the duration of the study.

Subject Vel-009 (injected with AAV9-GFAP-NeuroD1 and reporter vector at 14 days post-MCAO) and subject Kev-008 (untreated control, injected only with reporter vector) were matched since they had similarly sized stroke regions and similar starting NHPSS and mRS scores (Figure 2A and 2B) . Kev-008 exhibited recovery in the first 30-60 days post-MCAO, as was typical for this model and in patients. Vel-009 exhibited essentially full recovery (final NHPSS = 1; final mRS = 1) . Figure 3 further included the NHPSS and mRS assessments performed for an animal that was treated with AAV9-GFAP-NeuroD1 at 56 days post-MCAO (Seb-006) . Animals treated at 14 days post-MCAO showed neurobehavioral improvement beyond 30-40 days post stroke, which was consistent with the timing of astrocyte-to-neuron conversion. This was not observed in the animals treated at 56 days post-MCAO. Improvement in the early phase, i.e., 7-30 days post-MCAO may be driven by NeuroD1 anti-inflammatory/neuroprotective effects. These results suggest that administering AAV9-GFAP-NeuroD1 earlier in the process increases the likelihood of a full recovery.
6.6. Example 6. MRI Data Collection

MRI was performed throughout the study, at 2–4 week intervals. See the able below:
MRI Scans

Representative images of the MRI data collection following stroke in the Vel-009 and Kev-008 animals is shown in Figure 14. Affected areas following MCAO were visible, allowing for selection of injection sites.

Diffusion tensor imaging (DTI) : DTI is a magnetic resonance imaging (MRI) technique that measures the diffusion of hydrogen. DTI of corticospinal white matter tracts (CST) was performed in the animals at various timepoints throughout the study. Figures 4A-4C depict DTI assessments made for Vel-009 (Velma) , Seb-006 (Seb) , and Kev-008 (Kev) at baseline (Figure 4A) , 7 days post-MCAO (Figure 4B) , and 241 days post-MCAO (Figure 4C) . Vel-009 was treated with NeuroD1 at 14 days post-MCAO, Seb-006 was treated with NeuroD1 at 56 days post-MCAO, and Kev-008 did not receive treatment. Following MCAO, activity in the CST was completely lost in all animals as seen in the assessments done 7 days post-MCAO (Figure 4B) . However, at 241 days post-MCAO, CST connectivity appears to be fully restored in the Vel-009 subject in comparison to Seb-006 and Kev-008 (Figure 4C) .

Moreover, significant CST connectivity can be observed in Vel-009 as early as 99 days post-MCAO (Figure 5) (the signal at 21 days post-MCCAO was likely an artifact) . By 241 days post-MCAO, CST connectivity in Vel-009 was indistinguishable from baseline, consistent with complete recovery of the CST (Figure 5) .
6.7. Example 7. Termination and Brain Collection

At terminus, animals were euthanized with a lethal dose of sodium pentobarbital and then perfused transcardially with 0.9%buffered saline. Brains were extracted, sectioned in thick slabs (2cm) and post-fixed in 4%paraformaldehyde in 0.1 M phosphate buffer (PBS, pH 7.4) for 5–7 days, then transferred to a 30%sucrose solution in PBS at 4℃ for at least 72 hours before sectioning at 30 μm thickness on a sliding microtome (Epredia HM 440E) .
6.8. Example 8. Immunohistochemical analysis

Free-floating sections were pretreated with 0.3%Triton X-100 prepared in PBS (0.3%PBST) for 30 min and subsequently incubated in blocking buffer containing a mixture of 5%normal goat serum and 5%normal horse serum in 0.1%PBST for 1 hour at room temperature, followed by incubation with primary antibodies at 4℃ overnight. The next day, the sections were washed three times (10 min each) in 0.1%PBST and incubated with secondary antibodies at room temperature for 2 hours. To counterstain the nuclei, slides were then mounted with mounting medium with DAPI and covered with coverslips. Immunofluorescence labeling was observed and acquired with a fluorescent microscope (AxioVision, Carl Zeiss) . Images were acquired using a 20x objective (Axiovision Zeiss) with structured illumination and analyzed using ImageJ software.

Immunohistochemical analysis of Vel-009 showed newly formed neurons in appropriate regions of cerebral cortex. Figure 6 is a representative image illustrating a significantly increased neuronal density in the peri-infarct area in brain of the animal. GFP (green) expression in the cortical neurons (NeuN (NN) , red) demonstrate an astrocyte to neuron conversion. Both NeuN signal and cortical tissue integrity were significantly compromised in the non-treated animals (data not shown) .

Figure 7 further depicts an immunohistochemical analysis of three different injection sites (1, 2, and 3) in the brain of Vel-009. The 3 injection sites (1, 2, and 3) corresponded to the same 3 injection sites (1, 2, and 3) of the MRI panels of Figure 1. NN (red) and GFP overlap can be observed in all three injection sites. Figure 8 further depicts the expression of axonal (SMI312) and dendrite (SMI32) markers in the newly converted neurons in injection sites 1, 2, and 3 (each row of panels corresponding to an injection site) . Figure 9 shows the expression of Parvalbumin (PV) in newly generated and existing cortical neurons. The first two rows correspond to injection site 1 and the second two rows correspond to injection site 2. In all instances, the expression of NeuN (NN) , SMI312, SMI32, and Parvalbumin (PV) was readily observed together with the expression of GFP, confirming the glial cell-to-neuron conversion induced in the cells transduced with NeuroD1.

Moreover, Figure 10 depicts an immunohistochemical analysis of Vel-009 showing that newly formed neurons send axons to appropriate targets along the corticospinal tract. Figure 10 illustrates NeuroD1-converted neurons in the primary motor cortex and their distal axonal bundles in the striatum (internal capsule) and the brainstem (pons) . Figure 11 depicts an image of the distal axonal bundles in the striatum (internal capsule) , where GFP expression identifies the transduced cells, and GFAP expression traces the lineage of the newly converted neurons to GFAP-expressing cells (e.g., astrocytes) .

Figures 12A and 12B further demonstrate that astrocyte-to-neuron converted cells (GFP expressing neurons) were found throughout the cortical layers in the cortex of Vel-009. Figure 12A depicts the upper layers of the cortex of Vel-009. Figure 12B depicts the deep layers of the cortex of Vel-009 as well as regions of the white matter. The overlap of NeuN (NN) expression (identifying neurons) and GFP expression (identifying transduced cells) demonstrate the astrocyte-to-neuron conversion. The majority of GFP positive cells in the white matter are neither GFAP nor NeuN positive, but morphologically resembled neurons. This suggests the presence of cells in a transitional stage between astrocytes and neurons. Yellow arrows indicate co-staining of NeuN and GFP (top panels) or GFAP and GFP (bottom panels) .

Figure 13 is an immunohistochemical analysis of Vel-009’s and Kev-008’s non-stroked and peri-infarct areas. Immunostaining of phosphorylated neurofilaments (Smi312) revealed significant differences in the relative axonal densities in the intact and stroked areas of Vel-009’s and Kev-008’s cortexes. Additionally, reduced inflammation was seen in Vel-009 after NeuroD1 treatment and cell conversion. Particularly, Iba1 was a marker for microglia, whose activation was a part of the brain’s immune response and thus, it served as a specific marker of neuroinflammation. Figure 13 shows that in the peri-infarct area of an untreated animal (Kev-008) , there was substantial neuroinflammation (as indicated by the presence of Iba1+ microglia) . However, in an animal that receives NeuroD1 treatment, there was significantly less inflammation.
6.9. Example 9. Post-mortem gross anatomy examination

Figure 15 depicts photographs of the brain of Vel-009 and sections thereof, indicating the location of injection sites 1, 2, and 3 (same injection sites as Figure 1) . Figure 16 provides an anatomy comparison of the brains of Vel-009 and Kev-008. Grey matter loss in the frontal lobe (frontal and precentral gyri) and severe damage of the insula and thalamic structures were observed in the control animal (Kev-008) . Tissue regeneration in frontal and precentral gyri and preserved thalamic structures were observed in the treated animal (Vel-009) .
6.10. Example 10. Additional Non-human Animal Model Study
6.10.1. Materials and Methods.

AAV-NeuroD1 and related sequences are shown in Figure 17.

Research Grade Virus Production. Research grade viruses were produced using broadly used triple transfection of HEK293T cells. Steps in virus production: Briefly, seeding and expansion of HEK293T cells followed by triple transfection with three plasmids (GOI, RepCap, and helper) . Cells harvested and lysed at 48-72 hours post transfection. Virus particles were purified and concentrated by Benzonase treatment, PEG precipitation followed by ultracentrifugation, buffer exchange and concentration and sterile filtration.

Clinical and Pre-clinical Virus Production. Viruses used for clinical and pre-clinical studies were produced by triple transfection of HEK293 cells. Briefly, the process commenced with seeding and expansion of HEK293 cells, followed by triple transfection with three plasmids (GOI, RepCap, and helper) . Cells were harvested and lysed at 48-72 hours post transfection. Virus particles were purified by affinity purification, ultracentrifugation, and ion exchange filtration after treatment of Benzonase and clear out cell debris. Buffer exchange and concentration were used to produce final virus suspension. Viruses were sterile filtrated and aliquoted for drug substance and product. All procedures adhered to GMP standards for clinical-use virus production.

Virus formulation: virus at a titer of 1x10¹³ vg/mL in a phosphate-based buffer (e.g., containing potassium, potassium phosphate monobasic, sodium chloride, and sodium phosphate dibasic anhydrous) with poloxamer 188 were used.

Method for study in normal and stroke model brain of rodents. All animal experiment procedures strictly follow IACUC protocol approved by the institute. Adult Sprague-Dawley rats (over 8-week year old) were used.

Viral vector injection. Rats were anesthetized with 187.5mg/kg 1.25 %Avertin (amixture of 12.5 mg/mL of 2, 2, 2-Tribromoethanol and 25μL/mL 2-Methyl-2-butanol, Sigma, St. Louis, MO, USA) through intraperitoneal injection and then placed in a prone position in the stereotaxic frame. Virus was injected through glass pipette into motor cortex at the coordinate +0.24mm anterior-posterior (AP from Bregma) , f 2.2 mm medial-lateral (ML from Bregma, left and/or right side) , -2.2 mm dorsal-lateral (DV from dura) . The injection speed was 500 nL/min. The pipette was kept in place after injection for about 5 minutes and then slowly withdrawn.

Rodent stroke model induced by Endothelin-1 (ET-1) and treatment. Adult wild-type rats were anesthetized same as above described. 0.2 μg endothelin-1 (ET-1) was injected through glass pipette into motor cortex at the coordinate +0.24 mm anterior-posterior (AP from Bregma) , ± 2.2 mm medial-lateral (ML from Bregma, left side) , -2.2-1.8 mm dorsal-lateral (DV from dura) . The injection speed was 500 nL/min. The pipette was kept in place after injection for about 5 minutes and then slowly withdrawn. Virus was injected 10 days after ET-1 injection.

Immunostaining. The animals were anesthetized with 1.25 %Avertin and then sequentially perfused intracardially first with saline solution (0.9 %NaCl) and then with 4 %paraformaldehyde (PFA) . The brains were collected and processed for immunostaining with desired antibodies to detect NeuroD1 expression and astrocyte to neuron conversion. The antibodies included anti-NeuroD1, anti-GFAP (amarker for astrocytes) , anti-NeuN (amarker for neurons) , anti-GFP, anti-Iba1. Images were captured by fluorescent microscope (OLYMPUS, VS200) .

Reverse transcription-quantitative PCR (RT-qPCR) . The brain tissues within 5 mm diameter of the injection site were harvested for RT-qPCR analysis. RNA and DNA were extracted using AllPrep DNA/RNA Mini Kit (Qiagen) , and reverse transcription was performed on RNA using the Transcriptor First Strand cDNA Synthesis kit (Roche) . qPCR was then performed using the QuantiNova SYBR Green PCR kit on the QuantStudio 6 Pro Real-Time PCR System.

Statistics. All statistical analyses were conducted using GraphPad Prism Software 9.3.1. The RT-qPCR data was analyzed using one-way or two-way ANOVA test to determine the significance of differences between experimental groups accordingly.

Method for study in non-human primates (NHP) stroke model. All animal experiment procedures strictly follow IACUC protocol approved by the institute. Adult NHPs (rhesus or cynomolgus macaque 3–5-year-old, both sexes) were used.

Animal stroke model and virus delivery. Animals underwent a surgical model of stroke, referred to as MCAO (middle cerebral artery occlusion) . Anesthesia was induced with a mixture of ketamine (5mg/kg) and dexmedetomidine (0.05mg/kg) . Animals were then intubated, and anesthetically maintained by Isoflurane anesthesia (1.0-2.5%, 2L/min O2 flow rate) . Blood pressure, end-tidal CO2, O2 saturation, and EEG were monitored throughout surgical procedure. Temperature was monitored throughout surgery and MRI scanning by rectal probe and maintained at ～36.6℃ by heating blanket and heating disks. The incision was carried through the skin and temporalis muscle to expose the skull. A right pterional craniotomy was used to expose the frontal and temporal lobes and visualize the right MCA in the Sylvian fissure. The MCA was exposed by careful dissection of the arachnoid covering the Sylvian fissure and subsequently following the MCA branch proximally to the MCA bifurcation. A 5-mm titanium aneurysm clip was placed proximal to the orbitofrontal branch and occlusion was confirmed by direct visualization. Animals were transferred to the MRI to confirm occlusion by perfusion weighted imaging. The ischemia duration was 120 min. By the end of the occlusion duration, the incision was reopened, and the aneurysm clip was removed to restore blood flow. Following artery occlusion, the craniotomy was irrigated with 0.9%NaCl solution and the dura, temporalis muscle, fascia and skin were closed.

Virus was delivered through intracranial injection. One day prior to AAV injection, animals underwent a targeting MRI session to visualize the stroke region and borders. T1 MRI scans were used to delineate the core and penumbra of the stroke region. Three injection sites were selected, targeting the penumbra in the cerebral cortex at mid-cortical depth (example Figure 11) . At least one injection site was within the primary motor cortex. Coordinates of each injection site were saved and targeted by a Stryker iNav neuro-navigation system. Anesthesia in NHPS was induced with a mixture of ketamine (5mg/kg) and dexmedetomidine (0.05mg/kg) and maintained by Isoflurane (1.0-2.5%, 2L/min O2 flow rate) . The animals were placed in a prone position in the stereotaxic frame and three burr holes were drilled. 20 μl of virus per injection site was injected using 100 μl Hamilton Gastight Syringe equipped with a 33-gauge needle. The syringe was placed into a syringe pump that was mounted on the stereotaxic arm. The injection speed was 1μl /min. The needle was kept in place after injection for about 5 minutes and then slowly withdrawn.

Animal recovery assessment. Neurological function was assessed prior to, and following, MCAO using the Non-Human Primate Stroke Scale (NHPSS) and modified Rankin Scale (mRS) daily for the duration of the study. The NHPSS is a composite score based on the NIH Stroke Scale in humans that measures unilaterally level of consciousness, defense reaction, gait, circling, bradykinesia, balance, and bilaterally grasp reflex, extremity movement (upper and lower limbs) , neglect, hemianopsia, and facial weakness. It is composed of 11 domains (19 sub-scores from 5 unilateral and 7 bilateral domains) and yields a total score of 41 points, where 0 corresponds to normal behavior, and 41 to severe bilateral neurological impairment. mRS measures the level of disability with 6-point scale ranging from no symptoms to dead.

Neuro Imaging. Magnetic resonance scanning was performed using a 3 T Siemens Trio scanner with a 32-channel head coil. For the acquisition of MRI images, animals were intubated, and anesthetized (Isoflurane 1.0–2.5%, O₂ flow rate of 2 L/min) throughout scanning. The induction of anesthesia was performed in the same way as in the surgical procedure with a mixture of ketamine (5 mg/kg) and dexmedetomidine (0.05 mg/kg) . Baseline MRI acquisitions were made 14 days prior to MCAO. Post-stroke MRI acquisitions occurred throughout the study, at 2–4-week intervals.

Immunostaining. The animals were euthanized with lethal dose of sodium pentobarbital and then sequentially perfused intracardially with saline solution (0.9 %NaCl) followed by 4 %PFA in 0.1 M phosphate buffer. The brains were extracted and processed for immunostaining with desired antibodies to detect NeuroD1 expression and astrocyte to neuron conversion. The antibodies included anti-NeuroD1, anti-GFAP, anti-NeuN, anti-GFP, and anti-Iba1. Images were acquired using a 20X objective (Axiovision Zeiss) with structured illumination and analyzed using ImageJ software.
6.10.2. Results

Rodent study. Wild-type Sprague-Dawley rats and animals with ET-1 induced stroke were used to investigate NeuroD1 expression induced astrocyte to neuron conversion and efficacy in rodent stroke model. In previous study, virus system AAV9-Cre-FLEX-NeuroD1 was used in in stroke treatment and had shown efficacy in both tissue repair and behavior improvement. To compare the expression level and conversion efficiency of AAV-NeuroD1 (AAV9-GFAP-ND1) with AAV9-Cre-FLEX-NeuroD1, viruses of same titer were delivered by intracranial injection to cortex. Brain tissues were collected and analyzed with immunostaining and qPCR. The result showed no significant difference in NeuroD1 level at mRNA or protein levels assessed by RT-qPCR and immunostaining. (Figures 18A-18C)

Time course of NeuroD1 expression delivered by AAV-NeuroD1 in wild type rats was investigated up to 12 months (Figure 19A) . The transduction (Figure 19B) and expression (Figure 19C) can be detected 3 days post injection and the level gradually increase until 30 days. The expression level decreased by 3 and 9 months and became much lower by 12 months. (Figure 19C) . The astrocyte to neuron conversion efficiency increases gradually with time, from around 10%at 7 dpi to about 40%at 30dpi and over 80%at three months post injection at dose of 1.5E9 vg (3 ml of 5E11vg/ml) (Figures 20 A and 20B) . A normal, low Ki67 expression level at 12-month post injection addressed the concerns of tumorigenesis possibility caused by long term NeuroD1 expression (Figure 21) .

Quantitative analysis at 7 and 14 dpi of three doses of AAV-NeuroD1 delivered (low, medium, and high) showed that both transduction efficiency and NeuroD1 expression level were positively correlated with AAV-NeuroD1 dosage delivered (Figures 22A-22C) .

In the ET-1 induced rat focal ischemic stroke model, expression of NeuroD1 in the inflicted region through intracranial injection of AAV-NeuroD1 significantly mitigate the neural tissue loss comparing to control group (Figures 23A and 23B) . The astrocyte to neuron conversion efficiency at 40 dpi is close to 60% (Figures 24A and 24B) . AAV-NeuroD1 treatment also reduced the inflammation level indicated by gradual decrease of GFAP and IBa1 levels comparing to animals treated with control vector (Figures 25A-25C) .

In the AAV-NeuroD1 treated stroke animal brain, the axonal projections of newly converted neurons were observed extending from cortical to multiple brain regions, including striatum, thalamus, and hypothalamus, demonstrating the maturation and integration of the new neurons in the network. (Figures 26A and 26B) .

Non-human primate (NHP) study.

The animals with MCAO-induced ischemia with NHPSS score 20 and above were randomly assigned to the treatment and the control groups. The monkeys in the treatment group received injections of AAV-NeuroD1, co-injected with reporter AAV vector (GFAP-GFP) to allow for the quantification of astrocyte-to-neuron (AtN) conversion in transduced cells, whereas control group only received injections of AAV-GFAP-GFP. Analysis of the lesions (Figure 27) showed that they were consistent across the animals and the affected regions comprised parts of the right hemisphere temporal and frontoparietal poles and underlying white matter. Pathophysiological changes included increased hemispheric swelling, midline shift, and hyperintense lesion. The brain tissue atrophy could be seen in the insular cortex, claustrum, lateral parts of the somatosensory and premotor cortices and the putamen. Fluid-attenuated inversion recovery (FLAIR) axial images showed hyperintense signal involving frontal and parietal lobes, as well as white matter of corona radiata. The reduction in brain edema and infarct size in the first month post MCAO was associated with natural recovery of neurological deficits. This MCAO in NHPs resulted in severe functional deficits that closely matched the deficits observed in patients with moderate to severe stroke with motor deficits in upper and lower extremities. The neurological deficits were assessed daily after the MCAO procedure using the NHPSS and mRS scales. The NHPSS score for the animals in the control and treatment group at the first post-operative evaluation was 25.3±3.2 and 26.3±3.1, respectively. Salient features exhibited by the monkeys receiving stroke initially included spastic left hemiparesis, left facial weakness, left visual field deficit, impaired gait and balance, decrease in defense reaction and responsiveness to their environment. The average score declined significantly over the first two months in both control and treatment animals (to 10±0.5 and 9.1±0.7, respectively) . Neurological improvement as measured by NHPSS, showed the spontaneous neurobehavioral recovery in the first two months following MCAO with improvements in defense reaction, visual field and facial paralysis in both groups. Gait, extremity movements and grasp also improved in both groups in the first 60 days as shown by motor subscore analysis.

While the control group's NHPSS scores plateaued after initial recovery, indicating stable long-term neurological deficits, AAV-NeuroD1 treated group exhibited ongoing improvement in NHPSS scores beyond two months and ultimately showed nearly complete neurological function and motor skill recovery (Figures 29A-29C and Figures 30A and 30B) .

Immunohistochemical analysis confirmed that AAV-NeuroD1-mediated astrocyte-to-neuron conversion led to regeneration of cortical neurons and reconstitution of the cortico-spinal tract (Figures 31A and 31B) . Immunohistochemical staining showed newly converted neurons that were positively stained for both the neuronal marker NeuN and tracking marker GFP in the peri-infarct areas after NeuroD1 injections (Figure 33) . Moreover, GFP expression was also observed in the axonal fibers of the newly generated pyramidal cells in the premotor cortex, the internal capsule, the pons and medulla, indicating regrowth and incorporation of these neurons into the local neuronal network (Figure 31A) . In addition, histological findings were confirmed with diffusion tensor imaging (DTI) and tractography of the cortico-spinal tract (CST) . The DTI results indicate axonal regrowth of CST fibers after 5 months post MCAO in the AAV-NeuroD1 treated animal but not control (T1 and C1, Figure 31B) . At 8 months post stroke, DTI of AAV-NeuroD1 animal showed nearly full restoration of the CST.

In addition, at 8 months following viral injection, a significant reduction of Iba1 positive microglia and macrophage in the peri-infarct area of AAV-NeuroD1 treated group was observed, in comparison to the control group (Figures 32A and 32B) , indicating that NeuroD1-mediated in vivo AtN conversion not only generated new neurons in the ischemic injured areas in the NHP cortex, but also reduced neuroinflammation after ischemic injury.
6.11. Example 11. An exploratory clinical study to evaluate safety and preliminary efficacy of
NXL-001 for ischemic stroke.

Number of Sites and Product. This is a single-site trial studying NXL-001, a gene therapy that uses an AAV9 vector to deliver and express the NEUROD1 gene in astrocytes, converting them into neurons

Objectives and Trial Design. The study aims to evaluate the safety and tolerability of NXL-001 in patients with chronic neuronal deficits from ischemic stroke, while preliminarily assessing its efficacy. The trial is designed as an open-label, single-center, dose-escalation clinical study.

Patient Population.

Inclusion criteria:
● Age of 18 or above, below 80 years, male or female.
● Clinical diagnosis of ischemic stroke confirmed by neuro-imaging (CT, MRI, et al) .
● Between 2-4 months post-stroke.
● Stroke lesion with a size of 20-80ml, affecting motor cortex and causing damage to corticospinal
tract, identified by MRI scan.
● Moderate to severe motor dysfunction remains after standardized and guide-recommended
rehabilitation therapy after ischemic stroke, characterized by baseline NIHSS score of 6-20 points, and affected upper or lower limb motor score of 3-4.
● Expected survival ≥ 12 months.
● Patient or legal authorized representative was able to understand and sign an informed consent
form.
● Willing and able to return for follow-up visits as required by the trial protocol.
● Able to undergo rehabilitation training and treatment;
● Male and female subjects participating in clinical trial must agree to use an adequate birth control
method for at least 6 months after administration.

Exclusion criteria:
● Motor deficit due to ischemic stroke of posterior circulation.
● Motor deficit due to any other causes.
● History of epilepsy.
● History of encephalitis, meningitis, multiple sclerosis or other central nervous system infections.
● History of intracranial hemorrhage and subarachnoid hemorrhage.
● History of severe head trauma within the past 5 years.
● Any contraindications to MRI scanning (such as implanted pacemaker, infusion pump etc. ) .
● Serum anti-AAV9 antibody titers ≥ 1: 100
● History of malignant tumors within 5 years before screening (except for adequately treated
cervical carcinoma in situ, papillary thyroid cancer, basal cell or squamous epithelial cell skin cancer, localized prostate cancer after radical surgery, and breast ductal carcinoma in situ) .
● Active infections, including but not limited to human immunodeficiency virus (HIV) , hepatitis A,
B or C, syphilis, etc.
● Received any investigational drugs within 3 months (or 5 half-lives of the investigational drug,
whichever is longer) of initial screening.
● Received any other cell and/or gene therapy for stroke.
● Requirement for anticoagulants.
● Requirement for intermittent use of oral anti-spasticity medications (stop/start date from 1-month
prior-to and 3 month post-NXL-001 administration) . Use of oral anti-spasticity medications are acceptable if they have been taken regularly for at least one month prior to NXL-001 administration) .
● Pregnant or lactating female subjects.
● Insufficient reserved functions of liver, kidney and bone marrow: Neutrophil count <1, 500/mm
3 ;platelets <100, 000/mm 3 ; hemoglobin <9.0 g/dL; serum creatinine >1.5 times the upper limit of normal range (ULN) ; renal function eGFR < 60mL/min/1.73m2 ; Bilirubin, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) >2.5 times ULN; activated partial prothrombin time (APTT ) or international normalized ratio (INR ) >1.3 times ULN.
● Poorly controlled illness judged by the investigator at screening, including cardiovascular system
(decompensated heart failure (NYHA classification III and IV) , unstable angina, acute myocardial infarction) , Respiratory system, digestive system, endocrine metabolic system, neuropsychiatric system, blood system and immune system diseases, etc.
● Based on the medical history and the investigator's judgment, the subject is at significant risk for
suicide.
● In the investigator's judgment, the subject has any other factors deemed inappropriate for
participation in this trial.

Sample Size. The total number of patients to be enrolled are based on the observed toxicity and efficacy of the treatment. At least 9 patients are expected to be enrolled.

Treatment groups. The study include 3 cohorts at escalating doses, with 3 subjects in each cohort, all receiving a single intracerebral injection of NXL-001.

Cohort 1: 3.0x10¹¹ vg (5.0x10¹¹ vg/mL x 0.6mL)

Cohort 2: 6.0x10¹¹ vg (1.0x10¹² vg/mL x 0.6mL)

Cohort 3: 1.2x10¹² vg (2.0x10¹² vg/mL x 0.6mL)

Treatment Administration. Based on MRI scan, injection sites are selected before surgery. All injection sites should be located around the infarct lesion and cover the peri-infarct motor cortex.

During the surgery, NXL-001 is administered stereo-tactically by an experienced neurosurgeon, through several burr-holes, targeting the pre-selected sites, the injection volume of each site is approximately 25-50 μL, and the injection rate is no more than 10 μL/min.

Study Procedures. Subjects are screened at baseline, and those who meet the inclusion/exclusion criteria are hospitalized and receive administration within 28 days of screening.

NXL-001 is administered by intracerebral injection at 3 escalating dose levels.

There is at least a 2-week intra-cohort dosing interval between dosing of the first patient and others within a cohort to allow review of the safety analysis. Based on the observed AE/DLT, additional subjects may be enrolled at a given dose level.

There is at least a 2-week inter-cohort dosing interval between dosing of patients between cohorts to allow time for review of the safety analysis from all the patients within a cohort.

Dose escalation is based on dose-limiting toxicity (DLT) .

Subjects are followed up for 1 year after dosing to evaluate safety and efficacy.

Endpoints.

The primary endpoint focuses on safety and tolerability of NXL-001 within 3 months after intracerebral administration. Incidence of adverse events (AE) and serious adverse events (SAE) are according to CTCAE 5.0.

Secondary endpoints evaluate the efficacy of NXL-001 in restoring function following an ischemic stroke:
● Changes in MRI images of the cerebral infarction area before and after treatment (structure,
volume, function, DTI)
● FDG-PET, SPECT
● mRS
● NIHSS
● Fugl-Meyer Assessment (FMA)
● Modified Ashworth Scale
● Functional gait assessment
● Action Research Arm Test (ARAT)
● EQ-5D-5L
● Electroencephalography (EEG)
● Motor Evoked Potential (MEP)

Statistical Methods. This study adopted a single-arm design, so the analysis of the results was primarily a summary of descriptive statistics and did not involve formal hypothesis testing.

Statistical analysis set.

Full analysis set (FAS) : According to the intention-to-treat (ITT) principle, all subjects who are enrolled and receive at least 1 dose of study drug. FAS is mainly used to report the distribution, demographic and baseline characteristics of subjects and efficacy analysis.

mITT analysis set: subjects who were enrolled and received at least 1 dose of study drug and had at least 1 post-baseline efficacy assessment. This analysis set is used for the analysis of efficacy indicators.

Safety analysis set (SS) : All subjects who were enrolled and received at least 1 dose of the study drug and for whom post-medication safety data were collected. This analysis set is used for analysis of safety data.

General principles of statistical analysis: In this study, unless otherwise stated, the data will be analyzed with descriptive statistics in accordance with the following general principles. Measurement data is summarized by calculating the number of non-missing cases, mean, standard deviation, median, maximum value, and minimum value; count data is calculated by frequency and percentage.

All adverse events (AEs) are coded using MedDRA and graded according to the NCI CTCAE v5.0 grading system. The analysis of adverse events are based on the safety analysis set, and the AE data of different dose groups/stratifications are summarized in terms of number of subjects, frequency and incidence; and classified by system organ classification and preferred terminology, as well as severity. The above adverse events are summarized separately.
7. Embodiments

A variety of further modifications and improvements in and to the compositions and methods of the present disclosure will be apparent to those skilled in the art based. The following non-limiting embodiments are envisioned:
1. A method of treating stroke in a primate, the method comprising administering to the primate a
pharmaceutical composition comprising an adeno-associated viral (AAV) vector comprising a nucleic acid molecule encoding Neurogenic Differentiation 1 (NeuroD1) under the control of a Glial Fibrillary Acidic Protein (GFAP) promoter, wherein the pharmaceutical composition is administered by injecting the brain of the primate with a dosage between about 10 μL and about 50 μL of between about 1 x 10¹¹ vg/mL and about 20 x 10¹¹ vg/mL of the AAV per injection site.
2. The method of embodiment 1, wherein the primate is a human.
3. The method of embodiment 1 or 2, wherein the pharmaceutical composition is injected into a
peri-infarct region of the stroke.
4. The method of any one of embodiments 1–3, wherein an injection site is determined prior to the
administering via a magnetic resonance imaging (MRI) scan.
5. The method of embodiment 4, wherein coordinates of the determined injection site are used for
injecting the brain of the primate.
6. The method of any one of embodiments 1–5, wherein a surgical navigation system is used to
target an injection site on the brain of the primate.
7. The method of any one of embodiments 1–6, wherein a 33-gauge needle that is 1.5 inches in
length and has a 30° bevel is used for the injecting.
8. The method of any one of embodiments 1–7, wherein the pharmaceutical composition is
administered at a flow rate between about 0.4 μL per minute and about 2 μL per minute.
9. The method of any one of embodiments 1–7, wherein the pharmaceutical composition is
administered via a syringe pump at a controlled infusion rate between about 0.4 μL per minute and about 2 μL per minute.
10. The method of any one of embodiments 1–9, wherein the dosage comprises between about 10 μL
and about 50 μL of 5 x 10¹¹ vg/mL of the AAV per injection site.
11. The method of any one of embodiments 1–10, wherein the pharmaceutical composition is
administered in three to five injection sites in the brain.
12. The method of any one of embodiments 1–11, wherein the treating comprises converting glial
cells to neurons in the brain of the primate.
13. The method of any one of embodiments 1–12, wherein the treating comprises reducing
neuroinflammation in the brain of the primate.
14. The method of embodiment 13, wherein reduction in neuroinflammation is determined by
measuring the expression of Iba1 in a region of the brain of the primate.
15. The method of embodiment 14, wherein a reduced expression of Iba1 in a region of the brain of
the primate indicates a reduction in neuroinflammation.
16. The method of embodiment 13, wherein reduction in neuroinflammation is determined by
measuring the abundance of microglia in a region of the brain of the primate.
17. The method of embodiment 16, wherein a decreased abundance of microglia in a region of the
brain of the primate indicates a reduction in neuroinflammation.
18. The method of any one of embodiments 1–17, wherein neuronal pathways are partially or fully
restored in the brain of the primate after the primate is administered the pharmaceutical composition.
19. The method of embodiment 18, wherein the neuronal pathways are partially or fully restored
within three to six months after the primate is administered the pharmaceutical composition.
20. The method of any one of embodiments 1–18, wherein the treating comprises reducing
neuroinflammation in the brain of the primate within 14 to 21 days after the primate is administered the pharmaceutical composition.
21. The method of any one of embodiments 1–18, wherein the treating comprises generating new
neurons in the brain of the primate within 14 to 28 days after the primate is administered the pharmaceutical composition.
22. The method of any one of embodiments 1–21, wherein recovery of the primate after the stroke is
assessed via Diffusion Tensor Imaging.
23. The method of any one of embodiments 1–22, wherein the pharmaceutical composition is
administered to the primate within 7 to 28 days after the stroke occurs.
24. A method of treating stroke in a primate, the method comprising administering to the primate a
pharmaceutical composition comprising an adeno-associated viral (AAV) vector comprising a nucleic acid molecule encoding Neurogenic Differentiation 1 (NeuroD1) under the control of a Glial Fibrillary Acidic Protein (GFAP) promoter, wherein the primate has a score of at least 21 on the National Institutes of Health Stroke Scale (NIHSS) or at least 25 on the Non-Human Primate Stroke Scale (NHPSS) , and wherein the score is improved by at least 1 unit after the primate is administered the pharmaceutical composition.
25. A method of treating stroke in a primate, the method comprising administering to the primate a
pharmaceutical composition comprising an adeno-associated viral (AAV) vector comprising a nucleic acid molecule encoding Neurogenic Differentiation 1 (NeuroD1) under the control of a Glial Fibrillary Acidic Protein (GFAP) promoter, wherein the primate has a score of at least 4 on the Modified Rankin Scale (mRS) , and wherein the score is improved by at least 1 unit after the primate is administered the pharmaceutical composition.
26. The method of embodiment 24 or 25, wherein the primate is a human.
27. The method of any one of embodiments 24–26, wherein the pharmaceutical composition is
injected into a peri-infarct region of the stroke.
28. The method of embodiment 27, wherein an injection site is determined prior to the administering
via a magnetic resonance imaging (MRI) scan.
29. The method of embodiment 28, wherein coordinates of the determined injection site are used for
injecting the brain of the primate.
30. The method of any one of embodiments 27–29, wherein a surgical navigation system is used to
target an injection site on the brain of the primate.
31. The method of any one of embodiments 27–30, wherein a 33-gauge needle that is 1.5 inches in
length and has a 30° bevel is used for the injecting.
32. The method of any one of embodiments 27–31, wherein the pharmaceutical composition is
administered in three to five injection sites.
33. The method of any one of embodiments 24–32, wherein the pharmaceutical composition is
administered at a flow rate between about 0.4 μL per minute and about 2 μL per minute.
34. The method of any one of embodiments 24–32, wherein the pharmaceutical composition is
administered via a syringe pump at a controlled infusion rate between about 0.4 μL per minute and about 2 μL per minute.
35. The method of any one of embodiments 24–33, wherein the pharmaceutical composition is
injected into the brain of the primate at a dosage between about 10 μL and about 50 μL of between about 1 x 10¹¹ vg/mL and about 20 x 10¹¹ vg/mL of the AAV per injection site.
36. The method of embodiment 35, wherein the dosage comprises about 20 μL of 5 x 10¹¹ vg/mL of
the AAV per injection site.
37. The method of any one of embodiments 24–36, wherein the treating comprises converting glial
cells to neurons in the brain of the primate.
38. The method of any one of embodiments 24–37, wherein neuronal pathways are partially or fully
restored in the brain of the primate after the primate is administered the pharmaceutical composition.
39. The method of any one of embodiments 24–38, wherein recovery of the primate after the stroke is
assessed via Diffusion Tensor Imaging.
40. The method of any one of embodiments 24–39, wherein the treating comprises reducing
neuroinflammation in the brain of the primate.
41. The method of embodiment 40, wherein reduction in neuroinflammation is determined by
measuring the expression of Iba1 in a region of the brain of the primate.
42. The method of embodiment 41, wherein a reduced expression of Iba1 in a region of the brain of
the primate indicates a reduction in neuroinflammation.
43. The method of embodiment 40, wherein reduction in neuroinflammation is determined by
measuring the abundance of microglia in a region of the brain of the primate.
44. The method of embodiment 43, wherein a decreased abundance of microglia in a region of the
brain of the primate indicates a reduction in neuroinflammation.
45. The method of any one of embodiments 24–44, wherein the score is improved within 30 to 100
days after the primate is administered the pharmaceutical composition.
46. The method of any one of embodiments 24–44, wherein the treating comprises reducing
neuroinflammation in the brain of the primate within 14 to 21 days after the primate is administered the pharmaceutical composition.
47. The method of any one of embodiments 24–44, wherein the treating comprises generating new
neurons in the brain of the primate within 14 to 28 days after the primate is administered the pharmaceutical composition.
48. The method of any one of embodiments 24–44, wherein the pharmaceutical composition is
administered to the primate within 7 to 28 days after the stroke occurs.
49. A method of partially or fully restoring neuronal pathways in the brain of a primate who has
suffered a stroke, the method comprising administering to the primate a pharmaceutical composition comprising an adeno-associated viral (AAV) vector comprising a nucleic acid molecule encoding Neurogenic Differentiation 1 (NeuroD1) under the control of a Glial Fibrillary Acidic Protein (GFAP) promoter, wherein the restoring occurs within three to six months after the primate is administered the pharmaceutical composition.
50. A method of reducing neuroinflammation in the brain of a primate who has suffered a stroke, the
method comprising administering to the primate a pharmaceutical composition comprising an adeno-associated viral (AAV) vector comprising a nucleic acid molecule encoding Neurogenic Differentiation 1 (NeuroD1) under the control of a Glial Fibrillary Acidic Protein (GFAP) promoter, wherein the neuroinflammation is reduced within 14 to 21 days after the primate is administered the pharmaceutical composition.
51. A method of generating new neurons in the brain of a primate who has suffered a stroke, the
method comprising administering to the primate a pharmaceutical composition comprising an adeno-associated viral (AAV) vector comprising a nucleic acid molecule encoding Neurogenic Differentiation 1 (NeuroD1) under the control of a Glial Fibrillary Acidic Protein (GFAP) promoter, wherein the new neurons are generated within 14 to 28 days after the primate is administered the pharmaceutical composition.
52. The method of any one of embodiments 49–51, wherein the primate is human.
53. The method of any one of embodiments 49–51, wherein the pharmaceutical composition is
administered by injecting the brain of the primate with a dosage between about 10 μL and about 50 μL of between about 1 x 10¹¹ vg/mL and about 20 x 10¹¹ vg/mL of the AAV per injection site.
54. The method of any one of embodiments 49–53, wherein the pharmaceutical composition is
injected into a peri-infarct region of the stroke.
55. The method of embodiment 53, wherein an injection site is determined prior to the administering
via a magnetic resonance imaging (MRI) scan.
56. The method of embodiment of any one of embodiments 53–55, wherein a surgical navigation
system is used to target an injection site on the brain of the primate.
57. The method of any one of embodiments 53–56, wherein a 33-gauge needle that is 1.5 inches in
length and has a 30° bevel is used for the injecting.
58. The method of any one of embodiments 53–57, wherein the pharmaceutical composition is
administered at a flow rate between about 0.4 μL per minute and about 2 μL per minute.
59. The method of any one of embodiments 53–58, wherein the pharmaceutical composition is
administered in three to five injection sites in the brain of the primate.
60. The method of any one of embodiments 53–58, wherein recovery of the primate after the stroke is
assessed via Diffusion Tensor Imaging.
8. Exemplary elements in or encoded by AAV vectors of the present disclosure.

Claims

A single-stranded nucleic acid molecule encoding a NeuroD1 polypeptide, wherein the nucleic acid molecule comprises an expression cassette comprising a coding sequence and one or more regulatory elements operably linked to the coding sequence, wherein the NeuroD1 polypeptide comprises an amino acid sequence having at least 90%sequence identity to the sequence set forth in SEQ ID NO: 15.
The nucleic acid molecule of claim 1, wherein the NeuroD1 polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 13.
The nucleic acid molecule of claim 2, wherein the coding sequence comprises the nucleotide sequence set forth in SEQ ID NO: 4 or a codon-optimized variant thereof.
The nucleic acid molecule of any one of claims 1 to 3, wherein the one or more transcription regulatory elements comprise a chimeric intron.
The nucleic acid molecule of claim 4, wherein the chimeric intron comprises the sequence set forth in SEQ ID NO: 19.
The nucleic acid molecule of any one of claims 1 to 5, wherein the one or more transcription regulatory elements further comprise a GFAP promoter comprising the sequence set forth in SEQ ID NO: 10.
The nucleic acid molecule of any one of claims 1 to 6, wherein the one or more transcription regulatory elements further comprises a CMV enhancer comprising the sequence set forth in SEQ ID NO: 8.
The nucleic acid molecule of any one of claims 1 to 7, wherein the one or more transcription regulatory elements further comprises an optimized WPRE comprising the sequence set forth in SEQ ID NO: 12.
The nucleic acid molecule of any one of claims 1 to 8, wherein the one or more transcription regulatory elements further comprise a polyadenylation (poly-A) signal comprising the sequence set forth in SEQ ID NO: 9.
The nucleic acid molecule of any one of claim 1 to 9, further comprises a first inverted terminal repeat (ITR) of a first AAV genome.
The nucleic acid molecule of claim 10, wherein the first ITR is the 5’ ITR of the first AAV genome.
The nucleic acid molecule of claims 10 or 11, wherein the first ITR comprises the sequence set forth in SEQ ID NO: 16.
The nucleic acid molecule of any one of claim 1 to 12 further comprises a second ITR of a second AAV genome.
The nucleic acid molecule of claim 13, wherein the second ITR comprises the sequence set forth in SEQ ID NO: 23.
The nucleic acid molecule of any one of claim 1 to 14, comprising the sequence set forth in SEQ ID NO: 24.
The nucleic acid molecule of any one of claims 1 to 15, wherein the nucleic acid molecule is DNA.
A single-stranded DNA molecule consists of the sequence set forth in SEQ ID NO: 24.
A recombinant adeno-associated virus (rAAV) comprising a single-stranded nucleic acid molecule of any one of claims 1 to 17.
The recombinant AAV of claim 18, wherein the recombinant AAV comprises a AAV serotype 9 (AAV9) capsid.
The recombinant AAV of claim 18 or 19, wherein the AAV9 capsid comprises capsid proteins selected from the group of AAV9 VP1 polypeptides, AAV9 VP2 polypeptides and AAV9 VP3 polypeptides.
The recombinant AAV of claim 20, wherein the AAV9 capsid comprises AAV9 VP1 comprising the amino acid sequence set forth in SEQ ID NO: 40.
The recombinant AAV of claim 20 or 21, wherein the AAV9 capsid further comprises AAV9 VP2 comprising the amino acid sequence set forth in SEQ ID NO: 41.
The recombinant AAV of any one of claims 20-22, wherein the AAV9 capsid further comprises AAV9 VP3 comprising the amino acid sequence set forth in SEQ ID NO: 42.
A pharmaceutical composition comprising the recombinant AAV of any one of claims 18 to 23, wherein the pharmaceutical composition further comprises:

(a) potassium chloride,

(b) potassium phosphate monobasic,

(c) sodium chloride,

(d) sodium phosphate dibasic anhydrous, and

(e) poloxamer 188, polysorbate 20, or polysorbate 80.
A pharmaceutical composition consists of:

(a) a recombinant adeno-associated virus (AAV) ,

(b) sodium chloride at a concentration of about 180 mM;

(c) sodium phosphate at a concentration of about 10 mM; and

(d) poloxamer 188 at a concentration of about 0.001%weight/volume (0.01 g/L) ; and wherein the pH of the pharmaceutical composition is about 7.3.
A pharmaceutical composition consists of:

(a) a recombinant adeno-associated virus (AAV) ,

(b) sodium chloride at a concentration of about 200 mM;

(c) magnesium chloride at a concentration of about 1 mM;

(d) Tris hydrochloride at a concentration of about 20 mM, and

(e) poloxamer 188 at a concentration of about 0.005%weight/volume (0.05 g/L) ; and wherein the pH of the pharmaceutical composition is about 8.0.
A pharmaceutical composition consists of:

(a) a recombinant adeno-associated virus (AAV) ,

(b) sodium chloride at a concentration of about 150 mM;

(c) calcium chloride at a concentration of about 1.4 mM;

(d) magnesium chloride at a concentration of about 0.8 mM,

(e) sodium phosphate at a concentration of about 1 mM, and

(f) poloxamer 188 at a concentration of about 0.001%weight/volume (0.01 g/L) ; and wherein the pH of the pharmaceutical composition is about 7.4.
The pharmaceutical composition of any one of claims 25 to 27, wherein recombinant AAV is the recombinant AAV of any one of claims 18 to 23.
The pharmaceutical composition of any one of claims 25 to 28, wherein a vector genome concentration of the recombinant AAV in the pharmaceutical composition is in the range of about 5×10¹¹ to about 2×10¹² viral genomes per mL (vg/mL) ; optionally, wherein the vector genome concentration is about 5 ×10¹¹ vg/mL, about 1 × 10¹² vg/mL, or about 2 × 10¹² vg/mL.
A method for treating stroke, comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a recombinant AAV, wherein the recombinant AAV comprises a genome comprising a transgene encoding a NeuroD1 polypeptide.
The method of claim 30, wherein the NeuroD1 polypeptide comprises an amino acid sequence having at least 90%sequence identity to the sequence set forth in SEQ ID NO: 15.
The method of claim 30, wherein the NeuroD1 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 13.
The method of any one of claims 30 to 32, wherein the coding sequence of the transgene comprises the nucleotide sequence set forth in SEQ ID NO: 4 or a codon-optimized version thereof.
The method of any one of claims 30 to 33, wherein the expression cassette further comprises one or more transcription regulatory elements operably linked to the coding sequence of the transgene.
The method of claim 34, wherein the one or more transcription regulatory elements comprise a chimeric intron.
The method of claim 35, wherein the chimeric intron comprises the sequence of SEQ ID NO: 19.
The method of any one of claims 34 to 36, wherein the one or more transcription regulatory elements further comprise a GFAP promoter comprising the sequence of SEQ ID NO: 10.
The method of any one of claims 34 to 37, wherein the one or more transcription regulatory elements further comprise a CMV enhancer comprising the sequence of SEQ ID NO: 8.
The method of any one of claims 34 to 38, wherein the one or more transcription regulatory elements further comprise an optimized WPRE comprising the sequence of SEQ ID NO: 12.
The method of any one of claims 34 to 39, wherein the one or more transcription regulatory elements further comprise a polyadenylation (poly-A) signal comprising the sequence of SEQ ID NO: 9.
The method of any one of claims 30 to 40, wherein the genome further comprises a first inverted terminal repeat (ITR) of a first AAV genome.
The method of claim 41, wherein the first ITR comprises the sequence set forth in SEQ ID NO: 16.
The method of claim 41 or 42, further comprises a second ITR of a second AAV genome.
The method of claim 43, wherein the second ITR comprises the sequence set forth in SEQ ID NO: 23.
The method of any one of claims 30 to 44, wherein the recombinant AAV comprises an AAV serotype 6 (AAV9) capsid.
The method of claim 45, wherein the AAV9 capsid comprises capsid proteins selected from the group of AAV9 VP1 polypeptides, AAV9 VP2 polypeptides and AAV9 VP3 polypeptides.
The method of claim 45, wherein the AAV9 capsid comprises AAV9 VP1 comprising the amino acid sequence set forth in SEQ ID NO: 40.
The method of claim 45 or 47, wherein the AAV9 capsid further comprises AAV9 VP2 comprising the amino acid sequence set forth in SEQ ID NO: 41.
The method of any one of claims 45, 47and 48, wherein the AAV9 capsid further comprises AAV9 VP3 comprising the amino acid sequence set forth in SEQ ID NO: 42.
The method of any one of claims 30 to 49, wherein the pharmaceutical composition is administered to the subject intracerebrally.
The method of any one of claims 30 to 50, wherein the subject has a stroke and wherein the pharmaceutical composition is administered to the peri-infarct motor cortex region.
The method of any one of claims 30 to 50, wherein the subject has a stroke and wherein the pharmaceutical composition is administered to the area located around the infarct lesion.
The method of any one of claims 30 to 52, wherein the pharmaceutical composition comprises from about 1×10¹¹ to about 1×10¹³ viral genomes (vg) of the recombinant AAV.
The method of any one of claims 30 to 53, wherein the pharmaceutical composition comprises about 3×10¹¹ vg of the recombinant AAV.
The method of any one of claims 30 to 53, wherein the pharmaceutical composition comprises about 6.0×10¹¹ vg of the recombinant AAV.
The method of any one of claims 30 to 53, wherein the pharmaceutical composition comprises about 1.2×10¹² vg of the recombinant AAV.
The method of any one of claims 30 to 53, wherein the subject is administered intracerebrally the pharmaceutical composition comprising about 3×10¹¹ vg of the recombinant AAV once; optionally wherein for each administration the subject is administered about 0.6 mL of the pharmaceutical composition comprising about 5×10¹¹ vg/mL of the recombinant AAV intracerebrally; optionally wherein the pharmaceutical composition is administered stereo-tactically.
The method of any one of claims 30 to 53, wherein the subject is administered intracerebrally the pharmaceutical composition comprising about 6×10¹¹ vg of the recombinant AAV once; optionally wherein for each administration the subject is administered about 0.6 mL of the pharmaceutical composition comprising about 1×10¹² vg/mL of the recombinant AAV intracerebrally; optionally wherein the pharmaceutical composition is administered stereo-tactically.
The method of any one of claims 30 to 53, wherein the subject is administered intracerebrally the pharmaceutical composition comprising about 1.2×10¹² vg of the recombinant AAV once; optionally wherein for each administration the subject is administered about 0.6 mL of the pharmaceutical composition comprising about 2×10¹² vg/mL of the recombinant AAV intracerebrally; optionally wherein the pharmaceutical composition is administered stereo-tactically.
The method of any one of claims 30 to 59, wherein the pharmaceutical composition further comprises:

(a) potassium chloride,

(b) potassium phosphate monobasic,

(c) sodium chloride,

(d) sodium phosphate dibasic anhydrous, and

(e) poloxamer 188, polysorbate 20, or polysorbate 80.
The method of any one of claims 30 to 59, wherein the pharmaceutical composition further comprises:

(a) sodium chloride at a concentration of about 180 mM;

(b) sodium phosphate at a concentration of about 10 mM; and

(c) poloxamer 188 at a concentration of about 0.001%weight/volume (0.01 g/L) ; and wherein the pH of the pharmaceutical composition is about 7.3.
The method of any one of claims 30 to 59, wherein the pharmaceutical composition further comprises:

(a) sodium chloride at a concentration of about 200 mM;

(b) magnesium chloride at a concentration of about 1 mM;

(c) Tris hydrochloride at a concentration of about 20 mM, and

(d) poloxamer 188 at a concentration of about 0.005%weight/volume (0.05 g/L) ; and wherein the pH of the pharmaceutical composition is about 8.0.
The method of any one of claims 30 to 59, wherein the pharmaceutical composition further comprises:

(a) sodium chloride at a concentration of about 150 mM;

(b) calcium chloride at a concentration of about 1.4 mM;

(c) magnesium chloride at a concentration of about 0.8 mM,

(d) sodium phosphate at a concentration of about 1 mM, and

(e) poloxamer 188 at a concentration of about 0.001%weight/volume (0.01 g/L) ; and wherein the pH of the pharmaceutical composition is about 7.4.
The method of any one of claims 30 to 63, wherein the subject is a human.
The method of any one of claims 30 to 64, wherein upon administering the pharmaceutical composition, the NeuroD1 polypeptide is expressed by a population of glial cells.
The method of claim 65, wherein at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%of the glial cells in the population converts to neurons after the administering of the pharmaceutical composition.
The method of claim 66, wherein the neurons are selected from glutamatergic neurons, GABAergic neurons, dopaminergic neurons; motor neurons, glycinergic neurons, serotonergic neurons,
The method of claim 65, wherein the population of glial cells exhibit one or more neuronal phenotypes; optionally wherein the neuronal phenotype comprises expressing one or more neuronal markers selected from DCX, TUJ1, NeuN, and MAP2; optionally the population of glial cells exhibit the one or more neuronal phenotype after the administering of the pharmaceutical composition.
The method of claim 65, wherein the population of glial cells stop expressing one or more glial marker; optionally the one or more glial marker is selected from GFAP, Aldh1l1, S100β and Sox9; optionally the population of glial cells stop expressing the one or more glial after the administering of the pharmaceutical composition.
The method of claim 65, wherein neuroinflammation in the brain of subject is reduced; optionally wherein neuroinflammation in the brain of subject is reduced for at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%.
The method of claim 65, wherein neuronal pathways are partially or fully restored in the brain of subject, optionally wherein neuronal pathways are partially or fully restored for at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%.
The method of claim 65, wherein new neurons are generated in the brain of the subject after the administering of the pharmaceutical composition.
The method of any one of claims 18 to 72, wherein the stroke is ischemic stroke or hemorrhagic stroke.
A gene-of-interest (GOI) plasmid comprising an expression cassette comprising a transgene of interest and a pair of AAV ITR sequences flanking the expression cassette, wherein the transgene encodes a NeuroD1 polypeptide, wherein the NeuroD1 polypeptide comprises an amino acid sequence having at least 90%sequence identity to the sequence set forth in SEQ ID NO: 15.
A gene-of-interest (GOI) plasmid comprising an expression cassette comprising a transgene of interest and a pair of AAV ITR sequences flanking the expression cassette, wherein the transgene encodes a NeuroD1 polypeptide, wherein the NeuroD1 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 13.
A gene-of-interest (GOI) plasmid comprising an expression cassette comprising a transgene of interest and a pair of AAV ITR sequences flanking the expression cassette, wherein the transgene encodes a NeuroD1 polypeptide, wherein the transgene comprises the nucleic acid sequence set forth in SEQ ID NO: 4, or a codon-optimized version thereof.
A host cell comprising the GOI plasmid of any one of claims 74 to 75.
A method of producing a recombinant AAV comprising:

(a) culturing a host cell containing:

(i) an artificial genome comprising a cis expression cassette, wherein the cis expression cassette comprises a coding sequence encoding a NeuroD1 polypeptide, wherein the NeuroD1 polypeptide comprises an amino acid sequence having at least 90%sequence identity to the sequence set forth in SEQ ID NO: 15;

(ii) a trans expression cassette lacking AAV ITRs, wherein the trans expression cassette encodes an AAV Rep and Capsid proteins operably linked to expression control element that drive expression of the AAV Rep and capsid proteins in the host cell in culture, and supply the Rep and Capsid proteins in trans;

(iii) sufficient adenovirus helper functions to permit replication and packaging of the artificial genome by the AAV capsid proteins; and

(b) recovering the recombinant AAV encapsidating the artificial genome from the cell culture.
A method of producing a recombinant AAV comprising:

(a) culturing a host cell containing:

(i) an artificial genome comprising a cis expression cassette, wherein the cis expression cassette comprises a coding sequence encoding a NeuroD1 polypeptide, wherein the NeuroD1 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 13;

(ii) a trans expression cassette lacking AAV ITRs, wherein the trans expression cassette encodes an AAV Rep and Capsid proteins operably linked to expression control element that drive expression of the AAV Rep and capsid proteins in the host cell in culture, and supply the Rep and Capsid proteins in trans;

(iii) sufficient adenovirus helper functions to permit replication and packaging of the artificial genome by the AAV capsid proteins; and

(b) recovering the recombinant AAV encapsidating the artificial genome from the cell culture.
The method of claim 76 or 77, wherein the artificial genome is synthesized by the host cell using a GOI plasmid sequence as a replication template, wherein the GOI plasmid comprises the cis expression cassette flanked by a pair of AAV ITR sequences.
A host cell comprising an artificial genome comprising the single-stranded nucleic acid molecule of any one of claims 1 to 17.