WO2021076983A1 - Regenerating functional neurons for treatment of spinal cord injury and als - Google Patents

Regenerating functional neurons for treatment of spinal cord injury and als Download PDF

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Publication number
WO2021076983A1
WO2021076983A1 PCT/US2020/056108 US2020056108W WO2021076983A1 WO 2021076983 A1 WO2021076983 A1 WO 2021076983A1 US 2020056108 W US2020056108 W US 2020056108W WO 2021076983 A1 WO2021076983 A1 WO 2021076983A1
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Prior art keywords
nucleic acid
neurod1
polypeptide
acid encoding
biologically active
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English (en)
French (fr)
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Gong Chen
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Penn State Research Foundation
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Penn State Research Foundation
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Priority to AU2020365130A priority Critical patent/AU2020365130A1/en
Priority to CA3157523A priority patent/CA3157523A1/en
Priority to EP20875773.2A priority patent/EP4045148A4/en
Priority to CN202511095971.3A priority patent/CN120899949A/zh
Priority to CN202080060115.3A priority patent/CN114364436A/zh
Priority to JP2022522966A priority patent/JP2022552003A/ja
Publication of WO2021076983A1 publication Critical patent/WO2021076983A1/en
Anticipated expiration legal-status Critical
Priority to JP2025124068A priority patent/JP2025184866A/ja
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0619—Neurons
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/072—Animals genetically altered by homologous recombination maintaining or altering function, i.e. knock in
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0306—Animal model for genetic diseases
    • A01K2267/0318—Animal model for neurodegenerative disease, e.g. non- Alzheimer's
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • this document provides methods and materials for administering a composition containing exogenous nucleic acid encoding a NeuroD1polypeptide (or a biologically active fragment thereof) alone or in combination with exogenous nucleic acid encoding a Dlx2 polypeptide (or a biologically active fragment thereof) to a mammal having SCI.
  • This document also relates to methods and materials involved in treating mammals having amyotrophic lateral sclerosis (ALS).
  • this document provides methods and materials for administering a composition containing exogenous nucleic acid encoding a NeuroD1 polypeptide (or a biologically active fragment thereof) to a mammal having ALS.
  • this document provides methods and materials for administering a composition containing exogenous nucleic acid encoding a NeuroD1 polypeptide (or a biologically active fragment thereof) alone or in combination with exogenous nucleic acid encoding an Isl1 polypeptide (or a biologically active fragment thereof) to a mammal having ALS.
  • SCI spinal cord injury
  • CNS central nervous system
  • reactive astrocytes also become proliferative and hypertrophic in cell morphology.
  • reactive astrocytes play important roles in repairing the blood-spinal cord barrier and restricting the size of the primary injury (Herrmann et al., J. Neurosci., 28:7231-7243 (2008); and Okada et al., Nature Med., 12:829- 834 (2006)).
  • reactive astrocytes constitute the major component of the glial scar, a dense tissue structure that is inhibitory to axonal regeneration (Silver and Miller, Nat. Rev. Neurosci., 5:146-156 (2004)).
  • ALS Amyotrophic lateral sclerosis
  • FALS familial ALS
  • SOD1 superoxide dismutase 1
  • This document also relates to methods and materials involved in treating mammals having amyotrophic lateral sclerosis (ALS).
  • this document provides methods and materials for administering a composition containing exogenous nucleic acid encoding a NeuroD1polypeptide (or a biologically active fragment thereof) to a mammal having ALS.
  • This document also provides methods and materials for administering a composition containing exogenous nucleic acid encoding a NeuroD1 polypeptide (or a biologically active fragment thereof) alone or in combination with exogenous nucleic acid encoding an Isl1 polypeptide (or a biologically active fragment thereof) to a mammal having ALS.
  • one aspect of this document features a method for treating a mammal having a spinal cord injury (SCI).
  • the method comprises (or consists essentially of or consists of) administering a composition comprising (or consisting essentially of or consisting of) exogenous nucleic acid encoding a Neuronal Differentiation 1 (NeuroD1) polypeptide or a biologically active fragment thereof to the mammal.
  • the mammal can be a human.
  • the spinal cord injury can be due to a condition selected from the group consisting of: ischemic stroke; hemorrhagic stroke; physical injury; concussion; contusion; blast; penetration; tumor; inflammation; infection; traumatic spinal injury; ischemic or hemorrhagic myelopathy (spinal cord infarction); global ischemia as caused by cardiac arrest or severe hypotension (shock); hypoxic-ischemic encephalopathy as caused by hypoxia, hypoglycemia, or anemia; CNS embolism as caused by infective endocarditis or atrial myxoma; fibrocartilaginous embolic myelopathy; CNS thrombosis as caused by pediatric leukemia; cerebral venous sinus thrombosis as caused by nephrotic syndrome (kidney disease), chronic inflammatory disease, pregnancy, use of estrogen-based contraceptives, meningitis, dehydration; or a combination of any two or more thereof.
  • ischemic stroke hemorrhagic stroke
  • the administering step can comprise delivering an expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof to the spinal cord.
  • the administering step can comprise delivering a recombinant viral expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof to the spinal cord.
  • the administering step can comprise delivering a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof to the spinal cord.
  • the adeno-associated virus can be an AAV.PHP.eB.
  • the administering step can comprise administering a recombinant expression vector comprising a nucleic acid sequence encoding NeuroD1 polypeptide, wherein the nucleic acid sequence encoding NeuroD1 polypeptide comprises a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence encoding SEQ ID NO:2 or a functional fragment thereof; a nucleic acid sequence encoding SEQ ID NO:4 or a functional fragment thereof; SEQ ID NO:1 or a functional fragment thereof; SEQ ID NO:3 or a functional fragment thereof; and a nucleic acid sequence encoding a protein which has 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater, identity to SEQ ID NO: 2 or
  • the administering step can comprise a stereotactic injection to the spinal cord.
  • the administering step can comprise an intravenous injection or intravenous infusion.
  • this document features a method of treating a mammal having a spinal cord injury.
  • the method comprises (or consists essentially of or consists of) administering a pharmaceutical composition comprising (or consisting essentially of or consisting of) a pharmaceutically acceptable carrier containing adeno-associated virus particles comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof to the spinal cord of the mammal.
  • the pharmaceutical composition can comprise about 1 ⁇ L to about 500 ⁇ L of a pharmaceutically acceptable carrier containing adeno-associated virus at a concentration of 10 10 -10 14 adeno-associated virus particles/mL of carrier comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof.
  • the pharmaceutical composition can be injected in the spinal cord of the mammal at a controlled flow rate of about 0.1 ⁇ L/minute to about 5 ⁇ L/minute.
  • this document features a method for treating a mammal having spinal cord injury.
  • the method comprises (or consists essentially of or consists of) administering a composition comprising (or consisting essentially of or consisting of) exogenous nucleic acid encoding mir124, exogenous nucleic acid encoding a ISL LIM Homeobox 1 (Isl1) polypeptide or a biologically active fragment thereof, and exogenous nucleic acid encoding a LIM Homeobox 3 (Lhx3) polypeptide or biologically active fragment thereof to the spinal cord of the mammal.
  • the mammal can be a human.
  • the administering step can comprise delivering (i) an expression vector comprising a nucleic acid encoding mir124, (ii) an expression vector comprising a nucleic acid encoding a Isl1 polypeptide or a biologically active fragment thereof, and (iii) an expression vector comprising a nucleic acid encoding a polypeptide or biologically active fragment thereof Lhx3 to the spinal cord of the mammal.
  • the administering step can comprise delivering (i) a recombinant viral expression vector comprising a nucleic acid encoding mir124, (ii) a recombinant viral expression vector comprising a nucleic acid encoding a Isl1 polypeptide or biologically active fragment thereof, and (iii) a recombinant viral expression vector comprising a nucleic acid encoding a Lhx3 polypeptide or a biologically active fragment thereof to the spinal cord of the mammal.
  • the administering step can comprise delivering (i) a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding mir124, (ii) a recombinant adeno- associated virus expression vector comprising a nucleic acid encoding a Isl1 polypeptide or a biologically active fragment thereof, and (iii) a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding a Lhx3 polypeptide or biologically active fragment thereof to the spinal cord of the mammal.
  • the administering step can comprise delivering an expression vector comprising a nucleic acid encoding mir124, a Isl1 polypeptide or a biologically active fragment thereof, and a Lhx3 polypeptide or a biologically active fragment thereof to the spinal cord of the mammal.
  • the administering step can comprise delivering a recombinant viral expression vector comprising a nucleic acid encoding mir124, a Isl1 polypeptide or a biologically active fragment thereof, and a Lhx3 polypeptide or a biologically active fragment thereof to the spinal cord of the mammal.
  • the administering step can comprise delivering a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding mir124, a Isl1 polypeptide or biologically active fragment thereof, and a Lhx3 polypeptide or a biologically active fragment thereof to the spinal cord of the mammal.
  • the administering step can further comprise administering therapeutically effective doses of one or more of exogenous nucleic acid encoding a Neurogenin 2 (Ngn2) polypeptide or a biologically active fragment thereof, mir218, and a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof in combination with any combination of mir124, a Isl1 polypeptide or a biologically active fragment thereof, or a Lhx3 polypeptide or a biologically active fragment thereof to the spinal cord of the mammal.
  • the adeno-associated virus can be an AAV.PHP.eB.
  • this document features a method for treating a mammal having Amyotrophic lateral sclerosis (ALS).
  • the method comprises (or consists essentially of or consists of) administering a composition comprising (or consisting essentially of or consisting of) exogenous nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof to the central nervous system of the mammal.
  • the mammal can be a human.
  • the administering step can comprise delivering an expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof to the brain.
  • the administering step can comprise delivering a recombinant viral expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof to the brain.
  • the administering step can comprise delivering a recombinant adeno- associated virus expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof to the brain.
  • the adeno-associated virus can be an AAV.PHP.eB.
  • the administering step can comprise administering a recombinant expression vector comprising a nucleic acid sequence encoding NeuroD1 protein, wherein the nucleic acid sequence encoding NeuroD1 protein comprises a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence encoding SEQ ID NO:2 or a functional fragment thereof; a nucleic acid sequence encoding SEQ ID NO:4 or a functional fragment thereof; SEQ ID NO:1 or a functional fragment thereof; SEQ ID NO:3 or a functional fragment thereof; and a nucleic acid sequence encoding a protein which has 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater, identity to SEQ ID NO: 2 or SEQ ID NO
  • the administering step can comprise a stereotactic intracranial injection.
  • the administering step can comprise two or more stereotactic intracranial injections.
  • the administering step can comprise a retro-orbital injection.
  • this document features a method of treating a mammal having ALS.
  • the method comprises (or consists essentially of or consists of) administering a pharmaceutical composition comprising (or consisting essentially of or consisting of) a pharmaceutically acceptable carrier containing adeno-associated virus particles comprising a nucleic acid encoding NeuroD1 to the central nervous system of the mammal.
  • the pharmaceutical composition can comprise about 1 ⁇ L to about 500 ⁇ L of a pharmaceutically acceptable carrier containing adeno-associated virus at a concentration of 10 10 -10 14 adeno- associated virus particles/ml of carrier comprising a nucleic acid encoding a NeuroD1 polypeptide.
  • the pharmaceutical composition can be injected in the central nervous system of the mammal at a controlled flow rate of about 0.1 ⁇ L/minute to about 5 ⁇ L/minute.
  • this document features a method for treating a mammal having Amyotrophic lateral sclerosis (ALS).
  • ALS Amyotrophic lateral sclerosis
  • the method comprises (or consists essentially of or consists of) administering a composition comprising (or consisting essentially of or consisting of) exogenous nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof, exogenous nucleic acid encoding an Isl1 polypeptide or a biologically active fragment thereof, and exogenous nucleic acid encoding a Lhx3 polypeptide or a biologically active fragment thereof to the central nervous system of the mammal.
  • the mammal can be a human.
  • the administering step can comprise delivering (i) an expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof, (ii) an expression vector comprising a nucleic acid encoding a Isl1 polypeptide or a biologically active fragment thereof, and (iii) an expression vector comprising a nucleic acid encoding a Lhx3 polypeptide or a biologically active fragment thereof to the central nervous system of the mammal.
  • the administering step can comprise delivering (i) a recombinant viral expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof, (ii) a recombinant viral expression vector comprising a nucleic acid encoding a Isl1 polypeptide or a biologically active fragment thereof, and (iii) a recombinant viral expression vector comprising a nucleic acid encoding a Lhx3 polypeptide or a biologically active fragment thereof to the central nervous system of the mammal.
  • the administering step can comprise delivering (i) a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof, (ii) a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding a Isl1 polypeptide or a biologically active fragment thereof, and (iii) a recombinant adeno- associated virus expression vector comprising a nucleic acid encoding a Lhx3 polypeptide or a biologically active fragment thereof to the central nervous system of the mammal.
  • the administering step can comprise delivering an expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof, a Isl1 polypeptide or a biologically active fragment thereof and a Lhx3 polypeptide or a biologically active fragment thereof to the central nervous system of the mammal.
  • the administering step can comprise delivering a recombinant viral expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof, a Isl1 polypeptide or a biologically active fragment thereof, and a Lhx3 polypeptide or a biologically active fragment thereof to the central nervous system of the mammal.
  • the administering step can comprise delivering a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof, a Isl1 polypeptide or a biologically active fragment thereof, and a Lhx3 polypeptide or a biologically active fragment thereof to the central nervous system of the mammal.
  • the administering step can further comprise administering therapeutically effective doses of one or more of exogenous nucleic acid encoding Ngn2, mir218, and mir124 in combination with any combination of a NeuroD1 polypeptide or a biologically active fragment thereof, a Isl1 polypeptide or a biologically active fragment thereof, and a Lhx3 polypeptide or a biologically active fragment thereof to the central nervous system of the mammal.
  • the adeno-associated virus can be an AAV.PHP.eB.
  • this document features a method for (1) regenerating dorsal spinal cord neurons, (2) generating new glutamatergic neurons, or (3) increasing circulation in the spinal cord within a mammal having a SCI and in need of the (1), (2), or (3).
  • the method comprises (or consists essentially of or consists of) administering a composition comprising (or consisting essentially of or consisting of) exogenous nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof to the mammal, wherein (a) the spinal cord neurons are regenerated, (b) new glutamatergic neurons are generated, or (c) spinal cord circulation is increased.
  • the mammal can be a human.
  • the administering step can comprise delivering an expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof to the spinal cord.
  • the administering step can comprise delivering a recombinant viral expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof to the spinal cord.
  • the administering step can comprise delivering a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof to the spinal cord.
  • the adeno-associated virus can be an AAV.PHP.eB.
  • the administering step can comprise administering a recombinant expression vector comprising a nucleic acid sequence encoding NeuroD1 polypeptide, wherein the nucleic acid sequence encoding NeuroD1 polypeptide comprises a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence encoding SEQ ID NO:2 or a functional fragment thereof; a nucleic acid sequence encoding SEQ ID NO:4 or a functional fragment thereof; SEQ ID NO:1 or a functional fragment thereof; SEQ ID NO:3 or a functional fragment thereof; and a nucleic acid sequence encoding a protein which has 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater, identity to SEQ ID NO: 2 or
  • the administering step can comprise a stereotactic injection to the spinal cord.
  • the administering step can comprise an intravenous injection or intravenous infusion.
  • this document features a method for (1) generating motor neurons, (2) reducing the number of microglia, or (3) reducing the number of reactive astrocytes within a mammal having ALS disease and in need of the (1), (2), or (3).
  • the method comprises (or consists essentially of or consists of) administering a composition comprising (or consisting essentially of or consisting of) exogenous nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof to the mammal, wherein (a) the motor neurons are generated, (b) the number of microglia is reduced, or (c) the number of reactive astrocytes is reduced.
  • the mammal can be a human.
  • the administering step can comprise delivering an expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof to the spinal cord.
  • the administering step can comprise delivering a recombinant viral expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof to the spinal cord.
  • the administering step can comprise delivering a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof to the spinal cord.
  • the adeno-associated virus can be an AAV.PHP.eB.
  • the administering step can comprise administering a recombinant expression vector comprising a nucleic acid sequence encoding NeuroD1 polypeptide, wherein the nucleic acid sequence encoding NeuroD1 polypeptide comprises a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence encoding SEQ ID NO:2 or a functional fragment thereof; a nucleic acid sequence encoding SEQ ID NO:4 or a functional fragment thereof; SEQ ID NO:1 or a functional fragment thereof; SEQ ID NO:3 or a functional fragment thereof; and a nucleic acid sequence encoding a protein which has 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater, identity to SEQ ID NO: 2 or
  • the administering step can comprise a stereotactic injection to the spinal cord.
  • the administering step can comprise an intravenous injection or intravenous infusion.
  • this document features a method for (1) regenerating dorsal spinal cord neurons, (2) generating new glutamatergic neurons, or (3) increasing circulation in the spinal cord within a mammal having a SCI and in need of the (1), (2), or (3).
  • the method comprises (or consists essentially of or consists of) administering a composition comprising (or consisting essentially of or consisting of) exogenous nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof, exogenous nucleic acid encoding a Isl1 polypeptide or a biologically active fragment thereof, or exogenous nucleic acid encoding a Lhx3 polypeptide or a biologically active fragment thereof to the mammal, wherein (a) the spinal cord neurons are regenerated, (b) new glutamatergic neurons are generated, or (c) spinal cord circulation is increased.
  • the mammal can be a human.
  • the administering step can comprise delivering (i) an expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof, (ii) an expression vector comprising a nucleic acid encoding a Isl1 polypeptide or a biologically active fragment thereof, or (iii) an expression vector comprising a nucleic acid encoding a Lhx3 polypeptide or a biologically active fragment thereof to the central nervous system of the mammal.
  • the administering step can comprise delivering (i) a recombinant viral expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof, (ii) a recombinant viral expression vector comprising a nucleic acid encoding a Isl1 polypeptide or a biologically active fragment thereof, or (iii) a recombinant viral expression vector comprising a nucleic acid encoding a Lhx3 polypeptide or a biologically active fragment thereof to the central nervous system of the mammal.
  • the administering step can comprise delivering (i) a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof, (ii) a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding a Isl1 polypeptide or a biologically active fragment thereof, or (iii) a recombinant adeno- associated virus expression vector comprising a nucleic acid encoding a Lhx3 polypeptide or a biologically active fragment thereof to the central nervous system of the mammal.
  • the adeno-associated virus can be an AAV.PHP.eB.
  • the administering step can comprise a stereotactic injection to the spinal cord.
  • the administering step can comprise an intravenous injection or intravenous infusion.
  • this document features a method for treating a mammal having spinal cord injury.
  • the method comprises (or consists essentially of or consists of) administering a composition comprising (or consisting essentially of or consisting of) (a) exogenous nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof and (b) exogenous nucleic acid encoding a Distal-Less Homeobox 2 (Dlx2) polypeptide or biologically active fragment thereof to the spinal cord of the mammal.
  • the mammal can be a human.
  • the administering step can comprise delivering (i) an expression vector comprising a nucleic acid a NeuroD1 polypeptide and (ii) an expression vector comprising a nucleic acid encoding a Dlx2 polypeptide or a biologically active fragment thereof to the spinal cord of the mammal.
  • the administering step can comprise delivering (i) a recombinant viral expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide and (ii) a recombinant viral expression vector comprising a nucleic acid encoding a Dlx2 polypeptide or biologically active fragment thereof to the spinal cord of the mammal.
  • the administering step can comprise delivering (i) a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide and (ii) a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding a Dlx2 polypeptide or a biologically active fragment thereof to the spinal cord of the mammal.
  • the administering step can comprise delivering an expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof and a Dlx2 polypeptide or a biologically active fragment thereof to the spinal cord of the mammal.
  • the administering step can comprise delivering a recombinant viral expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof and a Dlx2 polypeptide or a biologically active fragment thereof to the spinal cord of the mammal.
  • the administering step can comprise delivering a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or biologically active fragment thereof and a Dlx2 polypeptide or a biologically active fragment thereof to the spinal cord of the mammal.
  • the adeno-associated virus can be an AAV.PHP.eB.
  • the administering step can comprise administering a recombinant expression vector comprising a nucleic acid sequence encoding NeuroD1 polypeptide, wherein the nucleic acid sequence encoding NeuroD1 polypeptide comprises a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence encoding SEQ ID NO:2 or a functional fragment thereof; a nucleic acid sequence encoding SEQ ID NO:4 or a functional fragment thereof; SEQ ID NO:1 or a functional fragment thereof; SEQ ID NO:3 or a functional fragment thereof; and a nucleic acid sequence encoding a protein which has 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater, identity to SEQ ID NO: 2 or
  • the administering step can comprise administering a recombinant expression vector comprising a nucleic acid sequence encoding Dlx2 polypeptide, wherein the nucleic acid sequence encoding Dlx2 polypeptide comprises a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence encoding SEQ ID NO: 11 or a functional fragment thereof; a nucleic acid sequence encoding SEQ ID NO:13 or a functional fragment thereof; SEQ ID NO:10 or a functional fragment thereof; SEQ ID NO:12 or a functional fragment thereof; and a nucleic acid sequence encoding a protein which has 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater, identity to SEQ ID NO:
  • the administering step can comprise a stereotactic injection to the spinal cord.
  • the administering step can comprise an intravenous injection or intravenous infusion.
  • the adeno-associated virus can be an AAV serotype 5.
  • this document features a method for (1) regenerating dorsal spinal cord neurons, (2) generating new neurons, or (3) increasing circulation in the spinal cord within a mammal having a SCI and in need of the (1), (2), or (3).
  • the method comprises (or consists essentially of or consists of) administering a composition comprising (or consisting essentially of or consisting of) (i) exogenous nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof and (ii) exogenous nucleic acid encoding a Dlx2 polypeptide or a biologically active fragment thereof, wherein (a) the spinal cord neurons are regenerated, (b) new neurons are generated, or (c) spinal cord circulation is increased.
  • the mammal can be a human.
  • the administering step can comprise delivering (i) an expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof and (ii) an expression vector comprising a nucleic acid encoding a Dlx2 polypeptide or a biologically active fragment thereof to the central nervous system of the mammal.
  • the administering step can comprise delivering (i) a recombinant viral expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof and (ii) a recombinant viral expression vector comprising a nucleic acid encoding a Dlx2 polypeptide or a biologically active fragment thereof to the central nervous system of the mammal.
  • the administering step can comprise delivering (i) a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof and (ii) a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding a Dlx2 polypeptide or a biologically active fragment thereof to the central nervous system of the mammal.
  • the administering step can comprise delivering an expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof and a Dlx2 polypeptide or a biologically active fragment thereof to the spinal cord of the mammal.
  • the administering step can comprise delivering a recombinant viral expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof and a Dlx2 polypeptide or a biologically active fragment thereof to the spinal cord of the mammal.
  • the administering step can comprise delivering a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or biologically active fragment thereof and a Dlx2 polypeptide or a biologically active fragment thereof to the spinal cord of the mammal.
  • the adeno-associated virus can be an AAV.PHP.eB.
  • the administering step can comprise a stereotactic injection to the spinal cord.
  • the administering step can comprise an intravenous injection or intravenous infusion.
  • the new neurons can be selected from the group consisting of glutamatergic neurons and GABAergic neurons.
  • the new neurons can be glutamatergic neurons.
  • the new neurons can be GABAergic neurons.
  • the adeno-associated virus can be an AAV serotype 5.
  • this document features a method for treating a mammal having ALS.
  • the method comprises (or consists essentially of or consists of) administering a composition comprising (a) exogenous nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof and (b) exogenous nucleic acid encoding an Isl1 polypeptide or biologically active fragment thereof to the mammal.
  • the mammal can be a human.
  • the administering step can comprise delivering (i) an expression vector comprising a nucleic acid a NeuroD1 polypeptide and (ii) an expression vector comprising a nucleic acid encoding an Isl1 polypeptide or a biologically active fragment thereof to the mammal.
  • the administering step can comprise delivering (i) a recombinant viral expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide and (ii) a recombinant viral expression vector comprising a nucleic acid encoding an Isl1 polypeptide or biologically active fragment thereof to the mammal.
  • the administering step can comprise delivering (i) a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide and (ii) a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding an Isl1 polypeptide or a biologically active fragment thereof to the spinal cord of the mammal.
  • the administering step can comprise delivering an expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof and an Isl1 polypeptide or a biologically active fragment thereof to the mammal.
  • the administering step can comprise delivering a recombinant viral expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof and an Isl1 polypeptide or a biologically active fragment thereof to the mammal.
  • the administering step can comprise delivering a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or biologically active fragment thereof and an Isl1 polypeptide or a biologically active fragment thereof to the mammal.
  • the adeno-associated virus can be an AAV.PHP.eB.
  • the administering step can comprise administering a recombinant expression vector comprising a nucleic acid sequence encoding NeuroD1 polypeptide, wherein the nucleic acid sequence encoding NeuroD1 polypeptide comprises a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence encoding SEQ ID NO:2 or a functional fragment thereof; a nucleic acid sequence encoding SEQ ID NO:4 or a functional fragment thereof; SEQ ID NO:1 or a functional fragment thereof; SEQ ID NO:3 or a functional fragment thereof; and a nucleic acid sequence encoding a protein which has 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater, identity to SEQ ID NO: 2 or
  • the administering step can comprise administering a recombinant expression vector comprising a nucleic acid sequence encoding an Isl1 polypeptide, wherein the nucleic acid sequence encoding an Isl1 polypeptide comprises a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence encoding SEQ ID NO: 15 or a functional fragment thereof; a nucleic acid sequence encoding SEQ ID NO:17 or a functional fragment thereof; SEQ ID NO:14 or a functional fragment thereof; SEQ ID NO:16 or a functional fragment thereof; and a nucleic acid sequence encoding a protein which has 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater, identity to SEQ ID
  • this document features a method for treating a mammal having spinal cord injury.
  • the method comprises (or consists essentially of or consists of) administering a composition comprising (a) exogenous nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof and (b) exogenous nucleic acid encoding an Isl1 polypeptide or biologically active fragment thereof to the spinal cord of the mammal.
  • the mammal can be a human.
  • the administering step can comprise delivering (i) an expression vector comprising a nucleic acid a NeuroD1 polypeptide and (ii) an expression vector comprising a nucleic acid encoding an Isl1 polypeptide or a biologically active fragment thereof to the spinal cord of the mammal.
  • the administering step can comprise delivering (i) a recombinant viral expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide and (ii) a recombinant viral expression vector comprising a nucleic acid encoding an Isl1 polypeptide or biologically active fragment thereof to the spinal cord of the mammal.
  • the administering step can comprise delivering (i) a recombinant adeno- associated virus expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide and (ii) a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding an Isl1 polypeptide or a biologically active fragment thereof to the spinal cord of the mammal.
  • the administering step can comprise delivering an expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof and an Isl1 polypeptide or a biologically active fragment thereof to the spinal cord of the mammal.
  • the administering step can comprise delivering a recombinant viral expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof and an Isl1 polypeptide or a biologically active fragment thereof to the spinal cord of the mammal.
  • the administering step can comprise delivering a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or biologically active fragment thereof and an Isl1 polypeptide or a biologically active fragment thereof to the spinal cord of the mammal.
  • the adeno-associated virus can be an AAV.PHP.eB.
  • the administering step can comprise administering a recombinant expression vector comprising a nucleic acid sequence encoding NeuroD1 polypeptide, wherein the nucleic acid sequence encoding NeuroD1 polypeptide comprises a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence encoding SEQ ID NO:2 or a functional fragment thereof; a nucleic acid sequence encoding SEQ ID NO:4 or a functional fragment thereof; SEQ ID NO:1 or a functional fragment thereof; SEQ ID NO:3 or a functional fragment thereof; and a nucleic acid sequence encoding a protein which has 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater, identity to SEQ ID NO: 2 or
  • the administering step can comprise administering a recombinant expression vector comprising a nucleic acid sequence encoding an Isl1 polypeptide, wherein the nucleic acid sequence encoding an Isl1 polypeptide comprises a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence encoding SEQ ID NO: 15 or a functional fragment thereof; a nucleic acid sequence encoding SEQ ID NO:17 or a functional fragment thereof; SEQ ID NO:14 or a functional fragment thereof; SEQ ID NO:16 or a functional fragment thereof; and a nucleic acid sequence encoding a protein which has 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater, identity to SEQ ID
  • FIG.1A Experiment paradigm.
  • FIG.1B Schematic of dorsal horn injury and injection. Coordinates are 0.4 mm lateral of central artery and 0.4 mm below the tissue surface. Stab injury was performed with a 32-guage needle followed by stereotaxic injection at the injury site. Scale bar 500 ⁇ m.
  • FIG.1C Three main types of proliferating cells at 1 week post viral injection (wpi) after injection of Retro GFP: astrocytes, OPCs, and microglia. GFAP, Olig2, and Iba1 staining show markers for these cell types. Arrows show examples of each.
  • FIG.1D Quantification based on staining for Retro GFP, 1 wpi samples. Bars show the mean and standard deviation of three replicates.
  • FIG.1E Converting cells in the dorsal horn 1 wpi, 3 wpi, and 6 wpi after Retro ND1-GFP injection. Maturing cells gradually up-regulate NeuN, adapt neuronal morphology, and reorganize. Arrows show example NeuN+ cells. Scale bar 500 ⁇ m.
  • FIG.1F Quantification based on staining for Retro ND1-GFP, 1W samples. Bars show the mean and standard deviation of three replicates. Figures 2A-2F.
  • FIG.2A Experiment paradigm.
  • FIG.2B AAV9 FLEX-NeuroD1-mCherry/AAV9 GFAP::Cre system (abbreviated elsewhere as AAV9 ND1-mCh).
  • GFAP promoter restricts infected cells to astrocytes. Control virus replaces the ND1 transgene with an additional mCh.
  • FIG.2C Infected astrocytes in the dorsal horn 4 wpi after AAV9 mCh injection.
  • Arrows and inset (2 ⁇ mag) show an example GFAP+ cell. Scale bar 50 ⁇ m.
  • Fig.2D Converted neurons in the dorsal horn 4 wpi after AAV9-ND1-mChy injection. Arrows and inset (2 ⁇ mag) show an example NeuN+ cell. Scale bar 50 ⁇ m.
  • Fig.2E Converting cells in the dorsal horn 2 wpi after AAV9-ND1-mChy injection. Arrows and inset (4 ⁇ mag) show an example NeuN+/GFAP+ cell. Scale bar 50 ⁇ m.
  • FIG.2F Quantification based on staining for AAV9 ND1-mCh, 2 wpi and 4 wpi, and AAV9 ND1-GFP or AAV9 GFP (Control), 8 wpi samples. Bars show the mean and standard deviation of three replicates. Infected cells at 2 wpi are mostly transitional, staining positive for NeuN and GFAP, and by 4 wpi are mostly converted, staining positive for only NeuN. Figures 3A-3D. Subtypes of NeuroD1-converted neurons in the spinal cord dorsal horn.
  • FIG.3A Tlx3 (glutamatergic) and Pax2 (GABAergic) subtype staining for converted neurons in the dorsal horn 8 wpi after AAV9 ND1-GFP injection.
  • Z-projection targets an example Tlx3+ neuron.
  • FIG.3B Tlx3 and Pax2 subtype staining for converted neurons in the dorsal horn 6 wpi after Retro ND1-GFP injection.
  • Z-projection targets an example Pax2+ neuron. Scale bar 50 ⁇ m.
  • FIG.3C 24 Quantification based on subtype staining for AAV9 ND1-GFP, 8 wpi and Retro ND1-GFP, 6 wpi samples. Control data is based on NeuN+ cells in uninjured, untreated tissue. Bars show the mean and standard deviation of three replicates.
  • FIG.3D AAV9 ND1-mCy and CaMK2-GFP coinjection at 4 wpi with strong (89.5 ⁇ 5.2%) co-labeling of CaMK2 for converted, Tlx3+ cells. Z-projection targets an example Tlx3+, CaMK2+ neuron. Scale bar 50 ⁇ m. Figures 4A-4D.
  • FIG.4A Subtype staining for converted neurons in the cortex 4 wpi after AAV9 ND1-mCh injection. Arrows show examples of cells positive for each subtype. Scale bar 50 ⁇ m.
  • FIG.4B 25 Quantification based on subtype staining in the cortex for AAV9 ND1-mCh, 4 wpi samples. Bars show the mean and standard deviation of three replicates.
  • FIG.4C Subtype staining for converted neurons in the spinal cord dorsal horn 4 wpi after AAV9 ND1-mChy injection. Arrows show examples of cells positive for each subtype. Scale bar 50 ⁇ m.
  • FIG.4D Quantification based on subtype staining in the spinal cord for AAV9 ND1-mCh, 4 wpi samples. Bars show the mean and standard deviation of three replicates.
  • Figures 5A-5I Maturation and functionality of NeuroD1-converted neurons in the spinal cord dorsal horn.
  • FIG.5A Fluorescent/transmitted light image of a patch-clamped converted neuron.
  • FIG.5B Sample action potentials of a converted neuron.
  • FIG.5C Sample Na+ and K+ currents of a converted neuron.
  • Fig.5D Sample EPSCs of a converted neuron.
  • FIG.5E Na+ current amplitudes for converted and native neurons.
  • FIG.5F EPSC amplitudes and frequencies for converted and native neurons.
  • FIG.5G Synaptic SV2 and VGluT1 puncta for converted neurons in the dorsal horn 8 wpi after AAV9 ND1-GFP injection. Arrows and inset (4 ⁇ mag) show an example cell with puncta visible on its soma and processes. Scale bar 50 ⁇ m.
  • FIG.5H Synaptic SV2 and VGluT2 puncta for converted neurons in the dorsal horn 8 wpi after AAV9 ND1-GFP injection. Arrows and inset (4 ⁇ mag) show an example cell with puncta visible on its soma and processes. Scale bar 50 ⁇ m.
  • FIG.5G Synaptic SV2 and VGluT1 puncta for converted neurons in the dorsal horn 8 wpi after AAV9 ND1-GFP injection. Arrows and inset (4 ⁇ mag) show an example cell with puncta visible on its soma and processes. Scale bar 50
  • FIG. 5I Integration of converted neurons into local network in the dorsal horn 8 wpi after AAV9 ND1-GFP injection. Activated neurons indicated by cFos staining are a subset of all neurons.
  • Figures 6A-6F NeuroD1 converts reactive astrocytes into neurons around the injury core with a short delay of viral injection after contusive SCI.
  • FIG.6A AAV9 FLEX expressing either GFP reporter alone or NeuroD1-GFP were injected along with AAV9 GFAP::Cre to target reactive astrocytes at 10 days after a contusive SCI (30 Kdyn force). Spinal cords were analyzed at 6 wpi.
  • FIG.6B 28 Experiment paradigm.
  • FIG.6C Many infected cells survived around the injury core (indicated by *) and showing distinct cellular morphology between the two groups. Immunostaining of the neuronal markers GFAP and NeuN indicates successful neuronal conversion from reactive astrocytes by ND1-GFP. Scale bars 50 ⁇ m at low-mag, 20 ⁇ m at high-mag.
  • Figures 7A-7I NeuroD1-mediated neuronal conversion with a long delay of viral injection after contusive SCI.
  • FIG.7A AAV9 FLEX expressing either GFP reporter alone or NeuroD1-GFP were injected along with AAV9 GFAP::Cre to target reactive astrocytes at 16 weeks after a contusive SCI (30 Kdyn force). The spinal cords were analyzed at 10 wpi.
  • FIG.7C Co-expression of the astrocyte marker S100b in control GFP+ cells. Scale bar 50 ⁇ m.
  • FIG.7D Immunostaining of the neuronal marker NeuN indicates successful neuronal conversion from reactive astrocytes by ND1-GFP with high efficiency. Scale bar 50 ⁇ m.
  • FIG.7F Co-expression of ND1 protein in the ND1-GFP+ cells with a typical neuronal morphology. Scale bar 20 ⁇ m.
  • FIG.7G Co-expression of the mature neuronal marker SV2 in the ND1-GFP+ cells.
  • Fig.7H Co-expression of the neuronal activity marker cFos in the ND1-GFP+ cells. Scale bar 20 ⁇ m.
  • Fig.7I Co- expression of the glutamatergic subtype marker Tlx3 in ND1-GFP+ neurons in the spinal cord dorsal horn. Arrows show an example Tlx3+ cell. Scale bar 20 ⁇ m.
  • Figures 8A-8B Infected cells by AAV9-ND1-mChy overexpress ND1 protein in the injured spinal cord.
  • FIG.8A Immunostaining analysis confirmed that the infected cells by AAV9 ND1-mCh expressed the neuronal marker NeuN indicating neuronal conversion in the dorsal horn of injured spinal cord at 4 wpi. Scale bar 200 ⁇ m.
  • FIG.8B Infected cells overexpressed ND1 protein at 4 wpi. Scale bar 50 ⁇ m.
  • Figure 9. NeuroD1-mediated neuronal conversion does not involve apoptosis. TUNEL assays were performed to detect apoptotic cells at different stages of neuronal conversion by AAV9 ND1-mCh in the injured spinal cord. Arrows show infected cells that are NeuN+ but TUNEL-. Figures 10A-10B.
  • CaMK2-GFP virus and GAD-GFP mice can be used to confirm neuronal subtype.
  • Fig.10A CaMK2-GFP (from co-injected AAV9 virus) co-stains with Tlx3 while
  • Fig.10B GAD-GFP (from GAD-GFP transgenic mouse) co-stains with Pax2, indicating that these markers can be used to confirm glutamatergic and GABAergic subtypes in the dorsal horn. Scale bar 200 ⁇ m. Dotted boxes at 4 ⁇ mag below.
  • Figures 11A-11D (Fig.11A) Schematic of stab injury and viral infection sites.
  • FIG.11C Staining following infection shows co- stains for RFP, NeuN and GFAP.
  • FIG.11D Panel of markers following different combinations of infections.
  • Figures 12A-12E Panel of markers following infection with control (mCherry) or MIL (mir124, Isl1, and Lhx3) at 1 week post infection (wpi) and 3 weeks post infection (wpi).
  • Fig.12B Histogram of NeuN + RFP + /RFP + for mCherry and MIL at 1 wpi.
  • FIG.12C Histogram of NeuN + RFP + /RFP + for mCherry and MIL at 3 wpi.
  • FIG.12D Histogram of GFAP + RFP + /RFP + for mCherry and MIL at 1 wpi.
  • FIG.12E Histogram of GFAP + RFP + /RFP + for mCherry and MIL at 3 wpi.
  • Figures 13A-13G NeuN immunostaining for mir124, Isl1, and Lhx3. Both Isl1 and Lhx3 can efficiently convert astrocytes into neurons. * indicates p ⁇ 0.05.
  • Figures 14A-14C Immunostaining with motor neuron marker, ChAT, found that Isl1 can convert astrocytes into motoneurons.
  • Figures 15A-15C Immunostaining with motor neuron marker, ChAT, found that Isl1 can convert astrocytes into motoneurons.
  • ND1 expression increases mobility and leg movement in SOD1-G93A mice at 24 weeks.
  • Figures 24A-24F Expression of GFP, NeuroD1, and SOD1 in spinal cord
  • Fig.23B dorsal horn
  • Fig.23C ventral horn
  • FIG.24A-Fig.24C a motor neuron marker
  • Fig.24D-Fig.24E a motor neuron marker
  • cerebra a cerebellum
  • Fig.24F a motor neuron marker
  • Figures 25A-25L Expression analysis of SOD1 mice injected with ND1/Isl1/Lhx expressing viruses (DIL group) or control viruses (Con group) via retro-orbital injection.
  • the DIL group received the following viruses: AAV.PHP.eB-GFAP-Cre (1.6x10 10 genome copies) plus AAV.PHP.eB-Flex-ND1-GFP (1.9x10 10 genome copies) plus AAV.PHP.eB- Flex-Isl1-mCherry (1.3x10 10 genome copies) plus AAV.PHP.eB-Flex-Lhx31-mCherry (1.5x10 10 genome copies).
  • the Con group received the following viruses: AAV.PHP.eB- GFAP-Cre (1.8x10 10 genome copies) plus AAV.PHP.eB-Flex-GFP (0.8x10 10 genome copies) plus AAV.PHP.eB-Flex-mCherry (3.4x10 10 genome copies).
  • AAV.PHP.eB-GFAP-Cre 1.8x10 10 genome copies
  • AAV.PHP.eB-Flex-GFP 0.8x10 10 genome copies
  • AAV.PHP.eB-Flex-mCherry 3.4x10 10 genome copies.
  • Control group exhibited about 30-40% leakage in cortex and brainstem, and greater than 90% leakage in thalamus. mCherry exhibited a little more leakage than GFP.
  • DIL group demonstrated that all neurons express GFP and RFP signals. Infected cells were more located in the ventral horn of the spinal cord (Fig.25B). Cre signals were located in astrocytes with the Con group exhibiting much more Cre signal (Fig.25C). In the DIL group, GFP + cells expressed ND1 signal, and RFP + cells expressed Isl1 signal (Fig.25D). The intensity of Isl1 expression varied in different regions in the brain (Fig.25E). The distribution of Tbr1 signals exhibited little difference between the Control (CON) and DIL groups (Fig.25F).
  • Iba1 signals had higher density and brightness in the Control (CON) group in brain (Fig.25G) and in spinal cord (Fig.25H). There were more motor neurons in the DIL group in cervical spinal cord, ventral horn (Fig.25I). Neuron loss in the motor column was severe in both the CON and DIL groups in the lumbar (Fig.25J). Motor neurons were hard to detect in the lumbar spinal cord, and neurodegeneration was too severe in the lumbar spinal cord at the end stage (22.1 weeks). The blood vessels did not show huge differences in high magnification images (Fig. 25K). The blood vessels had higher density and thickness in the DIL group (Fig.25L). Figures 26A-26D. (Fig.25K).
  • FIG. 1 A) Staining for NeuroD1 (ND1) and Dlx2 at 4 wpi following infection with AAV5-ND1-mCherry (AAV5-ND1-mCh) and AAV5-Dlx2-mCherry (AAV5- Dlx2-mCh).
  • FIG. B Panel of markers (Tlx3 and Pax2) at 4 wpi following co-infection with AAV5-ND1-mCh and AAV5-Dlx2-mCh.
  • FIG. C Histogram of Tlx3 + cells and Pax2 + cells for AAV5-ND1 alone and AAV5-ND1-mCh+AAV5-Dlx2-mCh at 4 wpi.
  • FIG. 27A-27B NeuroD1-Mediated Astrocyte-to-Neuron Conversion in ALS mice.
  • Fig.27A Injecting control AAV expressing mCherry (RFP staining) into spinal cord revealed infection of GFAP-positive reactive astrocytes in the ventral horn of ALS mice.
  • Fig.27B AAV NeuroD1-mCherry infected cells were immunopositive for NeuroD1 (ND1), NeuN (neuronal marker), and ChAT (motor neuron marker).
  • NeuroD1-converted neurons were ChAT-positive motor neurons in the spinal cord ventral horn. Scale bar, 50 ⁇ m.
  • Figures 28A-28B Intrathecal injection of viruses designed to express ND1 + Isl1 and viruses designed to express ND1 + Lhx3 converts astrocytes into neurons in the ventral horn of ALS mice. (Fig.28A) Following intrathecal injection, AAV PHP.eB-GFAP::Cre plus AAV PHP.eB-Flex-GFP specifically targeted astrocytes in the ventral horn, as shown by left column GFP control group.
  • FIG.29A Immunostaining with a motor neuron-specific marker, ChAT, revealed the motor neuron number in wild type (WT) mice and mice from the ND1 + Isl1, ND1 + Lhx3, and GFP control groups. A substantial reduction of motor neurons was observed in the GFP control group. Scale bar: 100 ⁇ m.
  • FIG. 29B Quantitative analyses of motor neurons in different segments of the spinal cord of WT, ND1 + Isl1, ND1 + Lhx3, and GFP control groups.
  • Figures 30A-30B Viral delivery of NeuroD1 + Isl1 reduces microglia-mediated inflammation in ALS mice.
  • FIG.30A GFAP immunostaining revealed repressed astroglial activation in the cervical cord treated by viruses designed to express ND1 + Isl1. Scale bar: 40 ⁇ m.
  • FIG.30B Repression of microglial activation was observed by decreased Iba1 and CD11b immunostaining in the ND1 + Isl1 group in ALS mice. Scale bar: 100 ⁇ m.
  • Figures 31A-31G Partial rescue of body weight and mobility after delivery of viruses designed to express NeuroD1 + Isl1.
  • Fig.31A The ND1 + Isl1 treatment partially rescued the body weight in ALS mice.
  • FIG.31B The ND1+Lhx3-treated ALS mice exhibited higher mortality rate after intrathecal AAV injection, although motor neuron number was increased.
  • FIG.31C Leg stretching measurement at the week 22.
  • FIG.31D-31F Quantified results on leg stretching experiments: the average distance of leg stretching (Fig.31D), the number of leg stretching (Fig.31E), and the total mobility time (Fig.31F).
  • the ALS mice treated with viruses designed to express ND1 + Isl1 exhibited better motor function.
  • FIG.31G The ALS mice treated with viruses designed to express ND1 + Isl1 exhibited longer duration in the hanging wire test.
  • Figures 32A-32C Open field test showing moderate improvement after gene therapy treatment.
  • Fig.33B-33C Quantified data showing that ALS mice treated with viruses designed to express ND1 + Isl1 displayed increased paw print area in either the contralateral (Fig.33B) or the ipsilateral (Fig.33C) comparison.
  • DETAILED DESCRIPTION This document provides methods and materials for treating mammals having a SCI. For example, this document provides methods and materials for administering a composition containing exogenous nucleic acid encoding a NeuroD1polypeptide to a mammal having a SCI. In another example, this document provides methods and materials for administering a composition containing exogenous nucleic acid encoding a NeuroD1 polypeptide and a Dlx2 polypeptide to a mammal having a SCI.
  • this document provides methods and materials for administering a composition containing exogenous nucleic acid encoding a mir124 microRNA, exogenous nucleic acid encoding an Isl1 polypeptide, and/or exogenous nucleic acid encoding an Lhx3 polypeptide to a mammal having a SCI.
  • this document provides methods and materials for administering a composition containing exogenous nucleic acid encoding NeuroD1, exogenous nucleic acid encoding a mir124 microRNA, exogenous nucleic acid encoding an Isl1 polypeptide, and/or exogenous nucleic acid encoding an Lhx3 polypeptide to a mammal having a SCI.
  • This document also provides methods and materials involved in treating mammals having ALS. For example, this document provides methods and materials for administering a composition containing exogenous nucleic acid encoding a NeuroD1 polypeptide to a mammal having ALS. In another example, this document provides methods and materials for administering a composition containing exogenous nucleic acid encoding a NeuroD1 polypeptide, exogenous nucleic acid encoding an Isl1 polypeptide, and exogenous nucleic acid encoding a Lhx3 polypeptide to a mammal having ALS.
  • this document provides methods and materials for administering a composition containing exogenous nucleic acid encoding a NeuroD1 polypeptide and exogenous nucleic acid encoding an Isl1 polypeptide to a mammal having ALS.
  • a neurological disorder e.g., ALS
  • humans and other primates such as monkeys can be identified as having ALS.
  • humans, non-human primates, cats, dogs, sheep, goats, horses, cows, pigs and rodents (e.g., mice and rats) having a neurological disorder (e.g., ALS) in the brain and/or central nervous system can be treated as described herein.
  • any appropriate mammal can be identified as having a spinal cord injury.
  • humans and other primates such as monkeys can be identified as having a spinal cord injury.
  • humans, non-human primates, cats, dogs, sheep, goats, horses, cows, pigs and rodents (e.g., mice and rats) having a spinal cord injury can be treated as described herein.
  • administration of a therapeutically effective amount of (i) exogenous nucleic acid encoding any of the polypeptides, or any combination of polypeptides, described herein (e.g., NeuroD1, Isl1, Lhx3, and Ngn2) and (ii) exogenous nucleic acid encoding mir124 and mir218 to a subject affected by a neurological disorder (e.g., ALS) in the brain mediates: the generation of new glutamatergic neurons by conversion of reactive astrocytes to glutamatergic neurons; reduction of the number of reactive astrocytes; survival of injured neurons including GABAergic and glutamate
  • administering a therapeutically effective amount of (i) exogenous nucleic acid encoding any of the polypeptides, or any combination of polypeptides, described herein (e.g., NeuroD1, Isl1, Lhx3, Dlx2, and Ngn2) and/or (ii) exogenous nucleic acid encoding mir124 and mir218 to a subject affected by a spinal cord injury mediates: the generation of new glutamatergic neurons by conversion of reactive astrocytes to glutamatergic neurons; reduction of the number of reactive astrocytes; survival of injured neurons including GABAergic and glutamatergic neurons; the generation of new non-reactive astrocytes; the reduction of reactivity of non-converted reactive astrocytes; reintegration of blood vessels into the injured region, and regenerating dorsal spinal cord neurons.
  • exogenous nucleic acid encoding any of the polypeptides, or any combination of polypeptides, described herein e.g., NeuroD1, Isl
  • a method or composition provided herein regenerates dorsal spinal cord neurons, increasing the number of dorsal spinal cord neurons from a baseline level by between about 1% and 100% after administration of a composition provided herein.
  • a method or composition provided herein generates regenerates dorsal spinal cord neurons, increasing the number of dorsal spinal cord neurons from a baseline level by 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 80%, between about 60% and about 90%, between about 70% and about 80%,
  • a method or composition provided herein generates new glutamatergic neurons, increasing the number of glutamatergic neurons from a baseline level by between about 1% and 500% after administration of a composition provided herein. In some cases, a method or composition provided herein generates new glutamatergic neurons, increasing the number of glutamatergic neurons from a baseline level by between about 1% and 50%, between about 1% and 100%, between about 1% and 150%, between about 50% and 100%, between about 50% and 150%, between about 50% and 200%, between about 100% and 150%, between about 100% and 200%, between 100% and 200%, between 100% and 250%, between about 150% and 200%, between about 150% and 250%, between about 150% and 300%, between 200% and 250%, between 200% and 300%, between 200% and 350%, between 250% and 300%, between 250% and 350%, between about 250% and 400%, between about 300% and 350%, between about 300% and 400%, between about 300% and 450%, between about 350% and 400%, between about
  • a method or composition provided herein increases circulation in the spinal cord between about 1% and 100% after administration of a composition provided herein. In some cases, a method or composition provided herein increases circulation 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% after administration of a composition provided herein.
  • a method or composition provided herein generates motor neurons, increasing the number of motor neurons from a baseline level by between about 1% and 500% after administration of a composition provided herein. In some cases, a method or composition provided herein generates motor neurons, increasing the number of motor neurons from a baseline level by between about 1% and 50%, between about 1% and 100%, between about 1% and 150%, between about 50% and 100%, between about 50% and 150%, between about 50% and 200%, between about 100% and 150%, between about 100% and 200%, between 100% and 250%, between about 150% and 200%, between about 150% and 250%, between about 150% and 300%, between 200% and 350%, between 250% and 300%, between 250% and 350%, between about 250% and 400%, between about 300% and 350%, between about 300% and 400%, between about 300% and 350%, between about 300% and 400%, between about 300% and 450%, between about 350% and 400%, between about 350% and 450%, between about 350% and 400%, between about 350%
  • a method or composition provided herein reduces microglia, reducing the number of microglia from a baseline level by between about 1% and 100% after administration of a composition provided herein. In some cases, a method or composition provided herein reduces the microglia, reducing the number of microglia from a baseline level by between by 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
  • a method or composition provided herein reduces the number of reactive astrocytes by between about 1% and 100% after administration of a composition provided herein. In some cases, a method or composition provided herein reduces the number of reactive astrocytes by 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%
  • administering a therapeutically effective amount of (i) exogenous nucleic acid encoding any of the polypeptides, or any combination of polypeptides, described herein (e.g., NeuroD1, Isl1, Lhx3, Dlx2, and Ngn2) and/or (ii) exogenous nucleic acid encoding mir124 and mir218 to a subject affected by a neurological disorder (e.g., ALS) in the brain or having a spinal cord injury mediates: reduced inflammation at the injury site; reduced neuroinhibition at the injury site; re-establishment of normal microglial morphology at the injury site; re-establishment of neural circuits at the injury site, increased blood vessels at the injury site; re-establishment of blood-brain-barrier at the injury site; re-establishment of normal tissue structure at the injury site; and improvement of motor deficits due to the disruption of normal blood flow.
  • a neurological disorder e.g., ALS
  • administration of a therapeutically effective amount of (i) exogenous nucleic acid encoding any of the polypeptides, or any combination of polypeptides, described herein (e.g., NeuroD1, Isl1, Lhx3, Dlx2, and Ngn2) and/or (ii) exogenous nucleic acid encoding mir124 and mir218 or any combination thereof to ameliorate the effects of a neurological disorder (e.g., ALS) in the brain in an individual subject in need thereof has greater beneficial effects when administered to reactive astrocytes than to quiescent astrocytes.
  • a neurological disorder e.g., ALS
  • administering a therapeutically effective amount of (i) exogenous nucleic acid encoding any of the polypeptides, or any combination of polypeptides, described herein (e.g., NeuroD1, Isl1, Lhx3, Dlx2, and Ngn2) and/or (ii) exogenous nucleic acid encoding mir124 and mir218 to ameliorate the effects of a spinal cord injury in an individual subject in need thereof has greater beneficial effects when administered to reactive astrocytes than to quiescent astrocytes.
  • exogenous nucleic acid encoding any of the polypeptides, or any combination of polypeptides, described herein e.g., NeuroD1, Isl1, Lhx3, Dlx2, and Ngn2
  • exogenous nucleic acid encoding mir124 and mir218 to ameliorate the effects of a spinal cord injury in an individual subject in need thereof has greater beneficial effects when administered to reactive astrocytes than to quiescent astrocytes.
  • a method for treating a mammal having spinal cord injury can include administrating a therapeutically effective amount of a composition, expression vector, or adeno-associated expression vector including a nucleic acid sequence encoding a NeuroD1 polypeptide (or a biologically active fragment thereof) and a nucleic acid sequence encoding a Dlx2 polypeptide (or biologically active fragment thereof).
  • nucleic acid sequence encoding a NeuroD1 polypeptide (or a biologically active fragment thereof) and a nucleic acid sequence encoding a Dlx2 polypeptide (or biologically active fragment thereof) are subcloned into one expression vector.
  • nucleic acid sequence encoding a NeuroD1 polypeptide (or a biologically active fragment thereof) and a nucleic acid sequence encoding a Dlx2 polypeptide (or biologically active fragment thereof) are subcloned into separate expression vectors.
  • a method for (1) regenerating dorsal spinal cord neurons, (2) generating new neurons, and/or (3) increasing circulation in the spinal cord within a mammal having a SCI and in need of said (1), (2), and/or (3) can include administering a composition including exogenous nucleic acid encoding a NeuroD1 polypeptide (or a biologically active fragment thereof) and exogenous nucleic acid encoding a Dlx2 polypeptide (or a biologically active fragment thereof) under conditions wherein (a) the spinal cord neurons are regenerated, (b) new neurons are generated, and/or (c) spinal cord circulation is increased.
  • new neurons are selected from the group consisting of glutamatergic neurons and GABAergic neurons.
  • a method for treating a mammal having spinal cord injury can include administrating a therapeutically effective amount of a composition, expression vector, or adeno-associated expression vector including a nucleic acid sequence encoding a NeuroD1 polypeptide (or a biologically active fragment thereof) and a nucleic acid sequence encoding an Isl1 polypeptide (or biologically active fragment thereof).
  • nucleic acid sequence encoding a NeuroD1 polypeptide (or a biologically active fragment thereof) and a nucleic acid sequence encoding an Isl1 polypeptide (or biologically active fragment thereof) are subcloned into one expression vector. In some cases, nucleic acid sequence encoding a NeuroD1 polypeptide (or a biologically active fragment thereof) and a nucleic acid sequence encoding an Isl1 polypeptide (or biologically active fragment thereof) are subcloned into separate expression vectors.
  • a spinal cord injury can be due to a condition selected from the group consisting of ischemic stroke, hemorrhagic stroke, physical injury, concussion, contusion, blast, penetration, tumor, inflammation, infection, traumatic spinal injury, ischemic or hemorrhagic myelopathy (spinal cord infarction), global ischemia, hypoxic-ischemic encephalopathy, CNS embolism as caused by, fibrocartilaginous embolic myelopathy, CNS thrombosis, and cerebral venous sinus thrombosis.
  • global ischemia is caused by cardiac arrest or severe hypotension (shock).
  • hypoxic-ischemic encephalopathy is caused by hypoxia, hypoglycemia, or anemia.
  • CNS embolism is caused by infective endocarditis or atrial myxoma.
  • CNS thrombosis is caused by pediatric leukemia.
  • cerebral venous sinus thrombosis is caused by nephrotic syndrome (kidney disease), chronic inflammatory disease, pregnancy, use of estrogen-based contraceptives, meningitis, or dehydration.
  • a spinal cord injury is due to ischemic stroke.
  • a spinal cord injury is due to hemorrhagic stroke.
  • a spinal cord injury is due to a physical injury.
  • a spinal cord injury is due to concussion. In some cases, a spinal cord injury is due to contusion. In some cases, a spinal cord injury is due to a blast. In some cases, a spinal cord injury is due to penetration. In some cases, a spinal cord injury is due to a tumor. In some cases, a spinal cord injury is due to inflammation. In some cases, a spinal cord injury is due to infection. In some cases, a spinal cord injury is due traumatic spinal injury. In some cases, a spinal cord injury is due to ischemic or hemorrhagic myelopathy (spinal cord infaction). In some cases, a spinal cord injury is due to global ischemia.
  • a spinal cord injury is due to hypoxic-ischemic encephalopathy. In some cases, a spinal cord injury is due to CNS embolism. In some cases, a spinal cord injury is due to fibrocartilaginous embolic myelopathy. In some cases, a spinal cord injury is due to CNS thrombosis. In some cases, a spinal cord injury is due to cerebral venous sinus. In some cases, a spinal cord injury is due to thrombosis.
  • a method for treating a mammal having ALS can include administrating a therapeutically effective amount of a composition, expression vector, or adeno-associated expression vector including a nucleic acid sequence encoding a NeuroD1 polypeptide (or a biologically active fragment thereof) and a nucleic acid sequence encoding an Isl1 polypeptide (or biologically active fragment thereof).
  • nucleic acid sequence encoding a NeuroD1 polypeptide (or a biologically active fragment thereof) and a nucleic acid sequence encoding an Isl1 polypeptide (or biologically active fragment thereof) are subcloned into one expression vector.
  • nucleic acid sequence encoding a NeuroD1 polypeptide (or a biologically active fragment thereof) and a nucleic acid sequence encoding an Isl1 polypeptide (or biologically active fragment thereof) are subcloned into separate expression vectors.
  • Treatment with (i) exogenous nucleic acid encoding any of the polypeptides, or any combination of polypeptides, described herein (e.g., NeuroD1, Isl1, Lhx3, Dlx2, and Ngn2) and/or (ii) exogenous nucleic acid encoding mir124 and mir218 can be administered to the region of injury as diagnosed by magnetic resonance imaging (MRI). Electrophysiology can assess functional changes in neural firing as caused by neural cell death or injury.
  • MRI magnetic resonance imaging
  • Non- invasive methods to assay neural damage include EEG.
  • Disruption of blood flow to a point of injury may be non-invasively assayed via Near Infrared Spectroscopy and functional magnetic resonance (fMRI).
  • Blood flow within the region may either be increased, as seen in aneurysms, or decreased, as seen in ischemia.
  • Injury to the CNS caused by disruption of blood flow additionally causes short-term and long-term changes to tissue structure that can be used to diagnose point of injury. In the short term, injury will cause localized swelling. In the long term, cell death will cause points of tissue loss.
  • Non-invasive methods to assay structural changes caused by tissue death include MRI, position emission tomography (PET) scan, computerized axial tomography (CAT) scan, or ultrasound.
  • PET position emission tomography
  • CAT computerized axial tomography
  • non-invasive methods to assay structural changes caused by tissue death include MRI, CAT scan, or ultrasound.
  • Functional assay may include EEG recording.
  • exogenous nucleic acid encoding any of the polypeptides, or any combination of polypeptides, described herein e.g., NeuroD1, Isl1, Lhx3, Dlx2, and Ngn2
  • exogenous nucleic acid encoding mir124 and mir218 is administered as an expression vector containing a nucleic acid sequence encoding any of the polypeptides described herein or mir218 and/or mir214.
  • a viral vector e.g., an AAV
  • exogenous nucleic acid encoding any of the polypeptides, or any combination of polypeptides, described herein e.g., NeuroD1, Isl1, Lhx3, Dlx2, and Ngn2
  • exogenous nucleic acid encoding mir124 and/or mir218 is delivered by injection into the brain of a subject, such as stereotaxic intracranial injection or retro-orbital injection.
  • composition containing the adeno-associated virus including (i) exogenous nucleic acid encoding any of the polypeptides, or any combination of polypeptides, described herein (e.g., NeuroD1, Isl1, Lhx3, Dlx2, and Ngn2) and/or (ii) exogenous nucleic acid encoding mir124 and/or mir218 is administered to the brain using two more intracranial injections at the same location in the brain.
  • composition containing the adeno-associated virus including (i) exogenous nucleic acid encoding any of the polypeptides, or any combination of polypeptides, described herein (e.g., NeuroD1, Isl1, Lhx3, Dlx2, and Ngn2) and/or (ii) exogenous nucleic acid encoding mir124 and/or mir218 is administered to the brain using two more intracranial injections at two or more different locations in the brain.
  • a viral vector e.g., an AAV
  • exogenous nucleic acid encoding any of the polypeptides, or any combination of polypeptides, described herein e.g., NeuroD1, Isl1, Lhx3, Dlx2, and Ngn2
  • exogenous nucleic acid encoding mir124 and/or mir218 is delivered by injection into spinal cord of a subject, such as stereotaxic injection, or by intravenous infusion or intravenous injection.
  • the gene delivery vector can be an AAV vector.
  • an AAV vector can be selected from the group of: an AAV2 vector, an AAV5 vector, and an AAV8 vector, an AAV1 vector, an AAV7 vector, an AAV9 vector, an AAV3 vector, an AAV6 vector, an AAV10 vector, and an AAV11 vector.
  • expression vector refers to a recombinant vehicle for introducing (i) exogenous nucleic acid encoding any of the polypeptides, or any combination of polypeptides, described herein (e.g., NeuroD1, Isl1, Lhx3, Dlx2, and Ngn2) and/or (ii) exogenous nucleic acid encoding mir124 and mir218 into a host cell in vitro or in vivo where the nucleic acid is expressed to produce the polypeptide as described herein.
  • an expression vector including SEQ ID NO: 1 or 3 or a substantially identical nucleic acid sequence is expressed to produce NeuroD1 in cells containing the expression vector.
  • an expression vector including SEQ ID NO: 10 or 12 or a substantially identical nucleic acid sequence is expressed to produce Dlx2 in cells containing the expression vector.
  • the term “recombinant” is used to indicate a nucleic acid construct in which two or more nucleic acids are linked and which are not found linked in nature.
  • Expression vectors include, but are not limited to plasmids, viruses, BACs and YACs.
  • Particular viral expression vectors illustratively include those derived from adenovirus, adeno-associated virus, retrovirus, and lentivirus.
  • This document describes material and methods for treating a neurological disorder (e.g., ALS) or a spinal cord injury in a subject in need thereof according to the methods described which include providing a viral vector including (i) exogenous nucleic acid encoding any of the polypeptides, or any combination of polypeptides, described herein (e.g., NeuroD1, Isl1, Lhx3, Dlx2, and Ngn2) and/or (ii) exogenous nucleic acid encoding mir124 and mir218; and delivering the viral vector to the brain or spinal cord of the subject, whereby the viral vector infects glial cells of the central nervous system, respectively, producing infected glial cells and whereby the (i) exogenous nucleic acid encoding any of the polypeptides, or any combination of polypeptides, described herein (e.g., NeuroD1, Isl1, Lhx3, Dlx2, and Ngn2) and/or (ii) exogenous nucleic acid encoding
  • adeno-associated vectors can be used in a method described herein and will infect both dividing and non-dividing cells, at an injection site.
  • Adeno-associated viruses are ubiquitous, noncytopathic, replication-incompetent members of ssDNA animal virus of parvoviridae family.
  • any of various recombinant adeno-associated viruses can be used as described herein.
  • an AAV- PHP.eb is used to administer the exogenous NeuroD1.
  • an AAV-PHP.eb is used to administer the exogenous Dlx2.
  • an AAV-PHP.eb is used to administer the exogenous Isl1.
  • an AAV serotype 5 is used to administer the exogenous NeuroD1.
  • an AAV serotype 5 is used to administer the exogenous Dlx2.
  • an AAV serotype 5 is used to administer the exogenous Isl1.
  • a “FLEX” switch approach is used to express, for example, NeuroD1 in infected cells according to some aspects described herein.
  • the terms “FLEX” and “flip-excision” are used interchangeably to indicate a method in which two pairs of heterotypic, antiparallel loxP-type recombination sites are disposed on either side of an inverted NeuroD1 coding sequence which first undergo an inversion of the coding sequence followed by excision of two sites, leading to one of each orthogonal recombination site oppositely oriented and incapable of further recombination, achieving stable inversion, see for example Schnutgen et al., Nature Biotechnology, 21:562-565 (2003); and Atasoy et al, J.
  • exogenous nucleic acid encoding any of the polypeptides, or any combination of polypeptides, described herein e.g., NeuroD1, Isl1, Lhx3, Dlx2, and Ngn2
  • exogenous nucleic acid encoding mir124 and mir218 is administered to a subject in need thereof by administration of 1) an adeno-associated virus expression vector including a DNA sequence encoding a site-specific recombinase under transcriptional control of an astrocyte-specific promoter such as GFAP or S100b or Aldh1L1; and 2) an adeno-associated virus expression vector including (i) exogenous nucleic acid encoding any of the polypeptides, or any combination of polypeptides, described herein (e.g., NeuroD1, Isl1, Lhx3, Dlx2, and Ngn2) and/or (ii) exogenous nucleic acid encoding mir124 and mir218
  • Site-specific recombinases and their recognition sites include, for example, Cre recombinase along with recognition sites loxP and lox2272 sites, or FLP-FRT recombination, or their combinations.
  • a therapeutically effective amount of the composition including an exogenous nucleic acid encoding a NeuroD1 polypeptide can be formulated with one or more pharmaceutically acceptable carriers (additives) and/or diluents.
  • a therapeutically effective amount of the composition including an exogenous nucleic acid encoding a Dlx2 polypeptide can be formulated with one or more pharmaceutically acceptable carriers (additives) and/or diluents.
  • a pharmaceutical composition including an exogenous nucleic acid encoding a NeuroD1 polypeptide can be formulated for various routes of administration, for example, for oral administration as a capsule, a liquid or the like.
  • a pharmaceutical composition including an exogenous nucleic acid encoding a Dlx2 polypeptide can be formulated for various routes of administration, for example, for oral administration as a capsule, a liquid or the like.
  • a pharmaceutical composition including an exogenous nucleic acid encoding a Isl1 polypeptide can be formulated for various routes of administration, for example, for oral administration as a capsule, a liquid or the like.
  • a viral vector including (i) exogenous nucleic acid encoding any of the polypeptides, or any combination of polypeptides, described herein (e.g., NeuroD1, Isl1, Lhx3, Dlx2, and Ngn2) and/or (ii) exogenous nucleic acid encoding mir124 and mir218 is administered parenterally, preferably by intravenous injection or intravenous infusion.
  • the administration can be, for example, by intravenous infusion, for example for 60 minutes, for 30 minutes or for 15 minutes. In some cases, the administration can be between 1 minute and 60 minutes.
  • the administration can be between 1 minute and 5 minutes, between 1 minute and 10 minutes, between 1 minute and 15 minutes, between 5 minutes and 10 minutes, between 5 minutes and 15 minutes, between 5 minutes and 20 minutes, between 10 minutes and 15 minutes, between 10 minutes and 20 minutes, between 10 minutes and 25 minutes, between 15 minutes and 20 minutes, between 15 minutes and 25 minutes, between 15 minutes and 30 minutes, between 20 minutes and 25 minutes, between 20 minutes and 30 minutes, between 20 minutes and 35 minutes, between 25 minutes and 30 minutes, between 25 minutes and 35 minutes, between 25 minutes and 40 minutes, between 30 minutes and 35 minutes, between 30 minutes and 40 minutes, between 30 minutes and 45 minutes, between 35 minutes and 40 minutes, between 35 minutes and 45 minutes, between 35 minutes and 50 minutes, between 40 minutes and 45 minutes, between 40 minutes and 50 minutes, between 40 minutes and 55 minutes, between 45 minutes and 50 minutes, between 45 minutes and 55 minutes, between 45 minutes and 60 minutes, between 50 minutes and 60 minutes, or between 55 minutes and 60 minutes.
  • the viral vector ⁇ e.g., AAV including (i) exogenous nucleic acid encoding any of the polypeptides, or any combination of polypeptides, described herein (e.g., NeuroD1, Isl1, Lhx3, Dlx2, and Ngn2) and/or (ii) exogenous nucleic acid encoding mir124 and mir218 ⁇ is administered locally by injection to the brain during a surgery.
  • Compositions which are suitable for administration by injection and/or infusion include solutions and dispersions, and powders from which corresponding solutions and dispersions can be prepared. Such compositions will comprise the viral vector and at least one suitable pharmaceutically acceptable carrier.
  • Suitable pharmaceutically acceptable carriers for intravenous administration include, but not limited to, bacterostatic water, Ringer’s solution, physiological saline, phosphate buffered saline (PBS) and Cremophor ELTM.
  • Sterile compositions for the injection and/or infusion can be prepared by introducing the viral vector ⁇ e.g., AAV including (i) exogenous nucleic acid encoding any of the polypeptides, or any combination of polypeptides, described herein (e.g., NeuroD1, Isl1, Lhx3, Dlx2, and Ngn2) and/or (ii) exogenous nucleic acid encoding mir124 and mir218 ⁇ in the required amount into an appropriate carrier, and then sterilizing by filtration.
  • AAV including (i) exogenous nucleic acid encoding any of the polypeptides, or any combination of polypeptides, described herein (e.g., NeuroD1, Isl1, Lhx3, Dlx2, and Ng
  • compositions for administration by injection or infusion should remain stable under storage conditions after their preparation over an extended period of time.
  • the compositions can contain a preservative for this purpose. Suitable preservatives include, but not limited to, chlorobutanol, phenol, ascorbic acid and thimerosal.
  • a pharmaceutical composition can be formulated for administration in solid or liquid form including, without limitation, sterile solutions, suspensions, sustained-release formulations, tablets, capsules, pills, powders, and granules.
  • the formulations can be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials, and may be stored in a freeze dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water for injections, immediately prior to use.
  • sterile liquid carrier for example, water for injections, immediately prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.
  • Additional pharmaceutically acceptable carriers, fillers, and vehicles that may be used in a pharmaceutical composition described herein include, without limitation, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.
  • ion exchangers alumina, aluminum stearate, lecithin
  • serum proteins such as human serum albumin
  • buffer substances such as phosphates,
  • the term “adeno-associated virus particle” refers to packaged capsid forms of the AAV virus that transmits its nucleic acid genome to cells.
  • An effective amount of composition containing an (i) exogenous nucleic acid encoding any of the polypeptides, or any combination of polypeptides, described herein (e.g., NeuroD1, Isl1, Lhx3, Dlx2, and Ngn2) and/or (ii) exogenous nucleic acid encoding mir124 and mir218 can be any amount that ameliorates the symptoms of the neurological disorder within a mammal (e.g., a human) without producing severe toxicity to the mammal.
  • an effective amount of adeno-associated virus encoding a NeuroD1 polypeptide can be a concentration from about 10 10 to 10 14 adeno-associated virus particles/mL.
  • an effective amount of adeno-associated virus encoding a NeuroD1 polypeptide can be between 10 10 adeno-associated virus particles/mL and 10 11 adeno-associated virus particles/mL, between 10 10 adeno-associated virus particles/mL and 10 12 adeno-associated virus particles/mL, between 10 10 adeno-associated virus particles/mL and 10 13 adeno- associated virus particles/mL, between 10 11 adeno-associated virus particles/mL and 10 12 adeno-associated virus particles/mL, between 10 11 adeno-associated virus particles/mL and 10 13 adeno-associated virus particles/mL, between 10 11 adeno-associated virus particles/mL and 10 14 adeno-associated virus particles/mL, between 10 12
  • an effective amount of adeno-associated virus encoding a Dlx2 polypeptide can be a concentration from about 10 10 to 10 14 adeno-associated virus particles/mL. In some cases, an effective amount of adeno-associated virus encoding a Dlx2 polypeptide can be between 10 10 adeno-associated virus particles/mL and 10 11 adeno-associated virus particles/mL, between 10 10 adeno-associated virus particles/mL and 10 12 adeno-associated virus particles/mL, between 10 10 adeno-associated virus particles/mL and 10 13 adeno-associated virus particles/mL, between 10 11 adeno-associated virus particles/mL and 10 12 adeno-associated virus particles/mL, between 10 11 adeno-associated virus particles/mL and 10 13 adeno-associated virus particles/mL, between 10 11 adeno-associated virus particles/mL and 10 14 adeno-associated virus particles/mL, between 10 11
  • an effective amount of adeno-associated virus encoding an Isl1 polypeptide can be a concentration from about 10 10 to 10 14 adeno-associated virus particles/mL. In some cases, an effective amount of adeno-associated virus encoding an Isl1 polypeptide can be between 10 10 adeno-associated virus particles/mL and 10 11 adeno-associated virus particles/mL, between 10 10 adeno- associated virus particles/mL and 10 12 adeno-associated virus particles/mL, between 10 10 adeno-associated virus particles/mL and 10 13 adeno-associated virus particles/mL, between 10 11 adeno-associated virus particles/mL and 10 12 adeno-associated virus particles/mL, between 10 11 adeno-associated virus particles/mL and 10 13 adeno-associated virus particles/mL, between 10 11 adeno-associated virus particles/mL and 10 14 adeno-associated virus particles/mL, between 10 12
  • the amount of the AAV encoding a NeuroD1 polypeptide can be increased. In some case, if a particular mammal fails to respond to a particular amount, then the amount of the AAV encoding a polypeptide can be increased.
  • Factors that are relevant to the amount of viral vector (e.g., an AAV encoding an exogenous nucleic acid encoding a NeuroD1 polypeptide) to be administered are, for example, the route of administration of the viral vector, the nature and severity of the disease, the disease history of the patient being treated, and the age, weight, height, and health of the patient to be treated.
  • the expression level of the polypeptides or miRNA as described herein, which is required to achieve a therapeutic effect, the immune response of the patient, as well as the stability of the gene product are relevant for the amount to be administered.
  • the administration of the viral vector e.g., an AAV encoding a NeuroD1 polypeptide
  • an effective amount of composition containing (i) exogenous nucleic acid encoding any of the polypeptides, or any combination of polypeptides, described herein (e.g., NeuroD1, Isl1, Lhx3, Dlx2, and Ngn2) and/or (ii) exogenous nucleic acid encoding mir124 and mir218 can be any administered at a controlled flow rate of about 0.1 ⁇ L/minute to about 5 ⁇ L/minute.
  • the controlled flow rate is between 0.1 ⁇ L/minute and 0.2 ⁇ L/minute, between 0.1 ⁇ L/minute and 0.3 ⁇ L/minute, between 0.1 ⁇ L/minute and 0.4 ⁇ L/minute, between 0.2 ⁇ L/minute and 0.3 ⁇ L/minute, between 0.2 ⁇ L/minute and 0.4 ⁇ L/minute, between 0.2 ⁇ L/minute and 0.5 ⁇ L/minute, between 0.3 ⁇ L/minute and 0.4 ⁇ L/minute, between 0.3 ⁇ L/minute and 0.5 ⁇ L/minute, between 0.3 ⁇ L/minute and 0.6 ⁇ L/minute, between 0.4 ⁇ L/minute and 0.5 ⁇ L/minute, between 0.4 ⁇ L/minute and 0.6 ⁇ L/minute, between 0.4 ⁇ L/minute and 0.7 ⁇ L/minute, between 0.5 ⁇ L/minute and 0.6 ⁇ L/minute, between 0.5 ⁇ L/minute and 0.7 ⁇ L/minute, between 0.5 ⁇ L/minute and 0.6
  • the viral vector ⁇ e.g., an AAV including (i) exogenous nucleic acid encoding any of the polypeptides, or any combination of polypeptides, described herein (e.g., NeuroD1, Isl1, Lhx3, Dlx2, and Ngn2) and/or (ii) exogenous nucleic acid encoding mir124 and mir218 can be administered in an amount corresponding to a dose of virus in the range of about 1.0 ⁇ 10 10 vg/kg to about 1.0 ⁇ 10 14 vg/kg (virus genomes per kg body weight).
  • the viral vector e.g., an AAV including (i) exogenous nucleic acid encoding any of the polypeptides, or any combination of polypeptides, described herein (e.g., NeuroD1, Isl1, Lhx3, Dlx2, and Ngn2) and/or (ii) exogenous nucleic acid encoding mir124 and mir218 ⁇
  • the viral vector e.g., an AAV including (i) exogenous nucleic acid encoding any of the polypeptides, or any combination of polypeptides, described herein (e.g., NeuroD1, Isl1, Lhx3, Dlx2, and Ngn2) and/or (ii) exogenous nucleic acid encoding mir124 and mir218 ⁇
  • the viral vector is administered in an amount corresponding to a dose of about 2.5 ⁇ 10 12 vg/kg.
  • the effective amount of the viral vector ⁇ e.g., an AAV including (i) exogenous nucleic acid encoding any of the polypeptides, or any combination of polypeptides, described herein (e.g., NeuroD1, Isl1, Lhx3, Dlx2, and Ngn2) and/or (ii) exogenous nucleic acid encoding mir124 and mir218 ⁇ can be a volume of about 1 ⁇ L to about 500 ⁇ L, corresponding to the volume for the vg/kg (virus genomes per kg body weight) doses described herein.
  • the amount of the viral vector to be administered ⁇ e.g., an AAV including (i) exogenous nucleic acid encoding any of the polypeptides, or any combination of polypeptides, described herein (e.g., NeuroD1, Isl1, Lhx3, Dlx2, and Ngn2) and/or (ii) exogenous nucleic acid encoding mir124 and mir218 ⁇ is adjusted according to the strength of the expression of one or more transgenes (e.g., NeuroD1).
  • an AAV including (i) exogenous nucleic acid encoding any of the polypeptides, or any combination of polypeptides, described herein (e.g., NeuroD1, Isl1, Lhx3, Dlx2, and Ngn2) and/or (ii) exogenous nucleic acid encoding mir124 and mir218 ⁇ is adjusted according to the strength of the expression of one or more transgenes (e.g., NeuroD1).
  • the effective volume administered of the viral vector is between 1 ⁇ L and 25 ⁇ L, between 1 ⁇ L and 50 ⁇ L, between 1 ⁇ L and 75 ⁇ L, between 25 ⁇ L and 50 ⁇ L, between 25 ⁇ L and 75 ⁇ L, between 25 ⁇ L and 100 ⁇ L, between 50 ⁇ L and 75 ⁇ L, between 50 ⁇ L and 100 ⁇ L, between 50 ⁇ L and 125 ⁇ L, between 75 ⁇ L and 100 ⁇ L, between 75 ⁇ L and 125 ⁇ L, between 75 ⁇ L and 150 ⁇ L, between 100 ⁇ L and 125 ⁇ L, between 100 ⁇ L and 150 ⁇ L, between 100 ⁇ L and 150 ⁇ L, between 100 ⁇ L and 175 ⁇ L, between 125 ⁇ L and 150 ⁇ L, between 125 ⁇ L and 175 ⁇ L, between 125 ⁇ L and 200 ⁇ L, between 150 ⁇ L and 175 ⁇ L, between 150 ⁇ L and 200 ⁇ L, between 150 ⁇ L
  • an adeno-associated virus vector including (i) exogenous nucleic acid encoding any of the polypeptides, or any combination of polypeptides, described herein ⁇ e.g., NeuroD1, Isl1, Lhx3, Dlx2, and Ngn2) and/or (ii) exogenous nucleic acid encoding mir124 and mir218 under transcriptional control of a ubiquitous (constitutive) promoter or a neuron- specific promoter wherein the nucleic acid sequence encoding the polypeptides and miRNA (e.g., NeuroD1, Isl1, Lhx3, Dlx2, mir124, Ngn2, and mir218 ⁇ or any combination thereof is inverted and in the wrong orientation for expression of the transgene and further includes sites for recombinase activity by a site specific recombinase, until the site-specific recombinase inverts the inverted nucleic acid sequence encoding the transgene, thereby allowing
  • an adeno-associated virus vector including (i) exogenous nucleic acid encoding any of the polypeptides, or any combination of polypeptides, described herein (e.g., NeuroD1, Isl1, Lhx3, Dlx2, and Ngn2) and/or (ii) exogenous nucleic acid encoding mir124 and mir218 under transcriptional control of a ubiquitous (constitutive) promoter or a neuron- specific promoter wherein the nucleic acid sequence encoding the transgene is inverted and in the wrong orientation for expression of the transgene and further includes sites for recombinase activity by a site specific recombinase, until the site-specific recombinase inverts the inverted nucleic acid sequence encoding the transgene, thereby allowing expression of the transgene, is delivered by stereotactic injection into the brain or by local delivery to the spinal cord of a subject along with an adeno-associated virus en
  • the site-specific recombinase is Cre recombinase and the sites for recombinase activity are recognition sites loxP and lox2272 sites.
  • treatment of a subject with (i) exogenous nucleic acid encoding any of the polypeptides, or any combination of polypeptides, described herein (e.g., NeuroD1, Isl1, Lhx3, Dlx2, and Ngn2) and/or (ii) exogenous nucleic acid encoding mir124 and mir218 is monitored during or after treatment to monitor progress and/or final outcome of the treatment.
  • Post-Treatment Assay for successful neuronal cell integration and restoration of tissue microenvironment is diagnosed by restoration or near-restoration of normal electrophysiology, blood flow, tissue structure, and function.
  • Non-invasive methods to assay neural function include EEG. Blood flow may be non-invasively assayed via Near Infrared Spectroscopy and fMRI.
  • Non-invasive methods to assay tissue structure include MRI, CAT scan, PET scan, or ultrasound.
  • Behavioral assays may be used to non-invasively assay for restoration of brain function. The behavioral assay should be matched to the loss of function caused by original brain injury. For example, if injury caused paralysis, the patient's mobility and limb dexterity should be tested.
  • Assays to evaluate treatment may be performed at any point, such as 1 day, 2 days, 3 days, one week, 2 weeks, 3 weeks, one month, two months, three months, six months, one year, or later, after NeuroD1 treatment alone or in combination with Dlx2. Such assays may be performed prior to NeuroD1 treatment alone or in combination with Dlx2 in order to establish a baseline comparison if desired.
  • RNA Interference Nuts and Bolts of RNAi Technology, DNA Press LLC, Eagleville, PA, 2003; Herdewijn, p. (Ed.), Oligonucleotide Synthesis: Methods and Applications, Methods in Molecular Biology, Humana Press, 2004; A. Nagy, M. Gertsenstein, K. Vintersten, R.
  • NeuroD1 protein refers to a bHLH proneural transcription factor involved in embryonic brain development and in adult neurogenesis, see Cho et al., Mol. Neurobiol., 30:35-47 (2004); Kuwabara et al., Nature Neurosci., 12:1097-1105 (2009); and Gao et al., Nature Neurosci., 12:1090-1092 (2009).
  • NeuroD1 is expressed late in development, mainly in the nervous system and is involved in neuronal differentiation, maturation and survival.
  • Dlx2 protein refers to a distal-less Homeobox 2 transcription factor that has a role in forebrain and craniofacial development.
  • a human Dlx2 polypeptide includes, without limitation, NCBI reference sequence: NP_004396.1 or a biologically active fragment thereof.
  • the Dlx2 polypeptide includes an amino acid substitution, insertion, or deletion that results in increased activity of the mutated Dlx2 as compared to the wildtype Dlx2 polypeptide (e.g., NCBI reference sequence: NP_004396.1).
  • the term “Isl1” refers to ISL LIM Homeobox 1 transcription factor involved in regulating insulin gene expression and required for motor neuron generation.
  • An example of a human Isl1 polypeptide includes, without limitation, NCBI reference sequence: NP_002193.2 or a biologically active fragment thereof.
  • the Isl1 polypeptide includes an amino acid substitution, insertion, or deletion that results in increased activity of the mutated Isl1 as compared to the wildtype Isl1 polypeptide (e.g., NCBI reference sequence: NP_002193.2).
  • the term “NeuroD1 protein” or “exogenous NeuroD1” encompasses human NeuroD1 protein, identified here as SEQ ID NO: 2 and mouse NeuroD1 protein, identified here as SEQ ID NO: 4.
  • NeuroD1 protein encompasses variants of NeuroD1 protein, such as variants of SEQ ID NO: 2 and SEQ ID NO: 4, which may be included in a method described herein.
  • Dlx2 protein or “exogenous Dlx2” encompasses human Dlx2 protein, identified here as SEQ ID NO: 11 and mouse Dlx2 protein, identified here as SEQ ID NO: 13.
  • Dlx2 protein encompasses variants of Dlx2 protein, such as variants of SEQ ID NO: 11 and SEQ ID NO: 13, which may be included in a method described herein.
  • Isl1 protein or “exogenous Isl1” encompasses human Isl1 protein, identified here as SEQ ID NO: 15 and mouse Isl1 protein, identified here as SEQ ID NO: 17.
  • Isl1 protein encompasses variants of Isl1 protein, such as variants of SEQ ID NO: 15 and SEQ ID NO: 17, which may be included in a method described herein.
  • an Isl1 protein can include the sequence set forth in GenBank Accession No. EAW54861, NP_002193.2, or AAH31213.1.
  • variant refers to naturally occurring genetic variations and recombinantly prepared variations, each of which contain one or more changes in its amino acid sequence compared to a reference NeuroD1 protein, such as SEQ ID NO: 2 or SEQ ID NO: 4.
  • the term “variant” refers to naturally occurring genetic variations and recombinantly prepared variations, each of which contain one or more changes in its amino acid sequence compared to a reference Dlx2 protein, such as SEQ ID NO: 11 or SEQ ID NO: 13.
  • the term “variant” refers to naturally occurring genetic variations and recombinantly prepared variations, each of which contain one or more changes in its amino acid sequence compared to a reference Isl1 protein, such as SEQ ID NO: 15 or SEQ ID NO: 17.
  • Such changes include those in which one or more amino acid residues have been modified by amino acid substitution, addition or deletion.
  • variants encompasses orthologs of human NeuroD1, including for example mammalian and bird NeuroD1, such as, but not limited to NeuroD1 orthologs from a non-human primate, cat, dog, sheep, goat, horse, cow, pig, bird, poultry animal and rodent such as but not limited to mouse and rat.
  • mouse NeuroD1, exemplified herein as amino acid sequence SEQ ID NO: 4 is an ortholog of human NeuroD1.
  • preferred variants have at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 2 or SEQ ID NO: 4.
  • the term “variant” encompasses orthologs of human Dlx2, including for example mammalian and bird Dlx2, such as, but not limited to Dlx2 orthologs from a non-human primate, cat, dog, sheep, goat, horse, cow, pig, bird, poultry animal and rodent such as but not limited to mouse and rat.
  • mouse Dlx2, exemplified herein as amino acid sequence SEQ ID NO: 13 is an ortholog of human Dlx2.
  • preferred variants have at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 11 or SEQ ID NO: 13.
  • Mutations can be introduced using standard molecular biology techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • one or more amino acid mutations can be introduced without altering the functional properties of the NeuroD1 protein, Dlx2 protein, or Isl1 protein.
  • one or more amino acid substitutions, additions, or deletions can be made without altering the functional properties of the NeuroD1 protein of SEQ ID NO: 2 or 4.
  • one or more amino acid substitutions, additions, or deletions can be made without altering the functional properties of the Dlx2 protein of SEQ ID NO: 11 or 13. In some cases, one or more amino acid substitutions, additions, or deletions can be made without altering the functional properties of the Isl1 protein of SEQ ID NO: 15 or 17. Conservative amino acid substitutions can be made in a NeuroD1 protein to produce a NeuroD1 protein variant. In some cases, conservative amino acid substitutions can be made in a Dlx2 protein to produce a Dlx2 protein variant. In some cases, conservative amino acid substitutions can be made in a Isl1 protein to produce a Isl1 protein variant.
  • Conservative amino acid substitutions are art recognized substitutions of one amino acid for another amino acid having similar characteristics.
  • each amino acid may be described as having one or more of the following characteristics: electropositive, electronegative, aliphatic, aromatic, polar, hydrophobic and hydrophilic.
  • a conservative substitution is a substitution of one amino acid having a specified structural or functional characteristic for another amino acid having the same characteristic.
  • Acidic amino acids include aspartate, glutamate; basic amino acids include histidine, lysine, arginine; aliphatic amino acids include isoleucine, leucine and valine; aromatic amino acids include phenylalanine, glycine, tyrosine and tryptophan; polar amino acids include aspartate, glutamate, histidine, lysine, asparagine, glutamine, arginine, serine, threonine and tyrosine; and hydrophobic amino acids include alanine, cysteine, phenylalanine, glycine, isoleucine, leucine, methionine, proline, valine and tryptophan; and conservative substitutions include substitution among amino acids within each group.
  • NeuroD1 variants can include synthetic amino acid analogs, amino acid derivatives and/or non-standard amino acids, illustratively including, without limitation, alpha- aminobutyric acid, citrulline, canavanine, cyanoalanine, diaminobutyric acid, diaminopimelic acid, dihydroxy-phenylalanine, djenkolic acid, homoarginine, hydroxyproline, norleucine, norvaline, 3-phosphoserine, homoserine, 5-hydroxytryptophan, 1-methylhistidine, 3- methylhistidine, and ornithine.
  • Dlx2 variants can include synthetic amino acid analogs, amino acid derivatives and/or non-standard amino acids, illustratively including, without limitation, alpha-aminobutyric acid, citrulline, canavanine, cyanoalanine, diaminobutyric acid, diaminopimelic acid, dihydroxy-phenylalanine, djenkolic acid, homoarginine, hydroxyproline, norleucine, norvaline, 3-phosphoserine, homoserine, 5- hydroxytryptophan, 1-methylhistidine, 3-methylhistidine, and ornithine.
  • synthetic amino acid analogs amino acid derivatives and/or non-standard amino acids
  • amino acid derivatives illustratively including, without limitation, alpha-aminobutyric acid, citrulline, canavanine, cyanoalanine, diaminobutyric acid, diaminopimelic acid, dihydroxy-phenylalanine, djenkolic acid, homoarginine,
  • Isl1 variants can include synthetic amino acid analogs, amino acid derivatives and/or non-standard amino acids, illustratively including, without limitation, alpha-aminobutyric acid, citrulline, canavanine, cyanoalanine, diaminobutyric acid, diaminopimelic acid, dihydroxy- phenylalanine, djenkolic acid, homoarginine, hydroxyproline, norleucine, norvaline, 3- phosphoserine, homoserine, 5-hydroxytryptophan, 1-methylhistidine, 3-methylhistidine, and ornithine.
  • synthetic amino acid analogs amino acid derivatives and/or non- standard amino acids
  • amino acid derivatives illustratively including, without limitation, alpha-aminobutyric acid, citrulline, canavanine, cyanoalanine, diaminobutyric acid, diaminopimelic acid, dihydroxy- phenylalanine, djenkolic acid, homoargin
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence).
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the determination of percent identity between two sequences can also be accomplished using a mathematical algorithm.
  • a preferred, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, PNAS, 87:2264-2268 (1990), modified as in Karlin and Altschul, PNAS, 90:5873-5877 (1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., J. Mol. Biol., 215:403 (1990).
  • Gapped BLAST are utilized as described in Altschul et al. (Nucleic Acids Res., 25:3389-3402 (1997)).
  • PSI BLAST is used to perform an iterated search which detects distant relationships between molecules.
  • BLAST Gapped BLAST
  • PSI Blast programs the default parameters of the respective programs (e.g., of XBLAST and NBLAST) are used (see, e.g., the NCBI website).
  • Another preferred, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller (CABIOS, 4:11-17 (1988)). Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package.
  • ALIGN program version 2.0
  • NeuroD1 protein encompasses fragments of the NeuroD1 protein, such as fragments of SEQ ID NOs.2 and 4 and variants thereof, operable in a method or composition described herein.
  • Dlx2 protein encompasses fragments of the Dlx2 protein, such as fragments of SEQ ID NOs: 11 and 13 and variants thereof, operable in a method or composition described herein.
  • Isl1 protein encompasses fragments of the Isl1 protein, such as fragments of SEQ ID NOs: 15 and 17 and variants thereof, operable in a method or composition described herein.
  • NeuroD1 proteins and nucleic acids may be isolated from natural sources, such as the brain of an organism or cells of a cell line which expresses NeuroD1. Alternatively, NeuroD1 protein or nucleic acid may be generated recombinantly, such as by expression using an expression construct, in vitro or in vivo. NeuroD1 proteins and nucleic acids may also be synthesized by well-known methods.
  • Dlx2 proteins and nucleic acids may be isolated from natural sources, such as the brain of an organism or cells of a cell line which expresses Dlx2.
  • Dlx2 protein or nucleic acid may be generated recombinantly, such as by expression using an expression construct, in vitro or in vivo.
  • Dlx2 proteins and nucleic acids may also be synthesized by well-known methods.
  • Isl1 proteins and nucleic acids may be isolated from natural sources, such as the brain of an organism or cells of a cell line which expresses Isl1.
  • Isl1 protein or nucleic acid may be generated recombinantly, such as by expression using an expression construct, in vitro or in vivo.
  • Isl1 proteins and nucleic acids may also be synthesized by well-known methods.
  • NeuroD1 included in a method or composition described herein can be produced using recombinant nucleic acid technology.
  • Recombinant NeuroD1 production includes introducing a recombinant expression vector encompassing a DNA sequence encoding NeuroD1 into a host cell.
  • Dlx2 included in a method or composition described herein can be produced using recombinant nucleic acid technology.
  • Recombinant Dlx2 production includes introducing a recombinant expression vector encompassing a DNA sequence encoding Dlx2 into a host cell.
  • Isl1 included in a method or composition described herein can be produced using recombinant nucleic acid technology.
  • Recombinant Isl1 production includes introducing a recombinant expression vector encompassing a DNA sequence encoding Isl1 into a host cell.
  • a nucleic acid sequence encoding NeuroD1 introduced into a host cell to produce NeuroD1 can encode SEQ ID NO: 2, SEQ ID NO: 4, or a variant thereof.
  • a nucleic acid sequence encoding Dlx2 introduced into a host cell to produce Dlx2 can encode SEQ ID NO: 11, SEQ ID NO: 13, or a variant thereof.
  • a nucleic acid sequence encoding Isl1 introduced into a host cell to produce Isl1 can encode SEQ ID NO: 15, SEQ ID NO: 17, or a variant thereof.
  • the nucleic acid sequence identified herein as SEQ ID NO: 1 encodes SEQ ID NO: 2 and is included in an expression vector and expressed to produce NeuroD1.
  • the nucleic acid sequence identified herein as SEQ ID NO: 10 encodes SEQ ID NO: 11 and is included in an expression vector and expressed to produce Dlx2.
  • the nucleic acid sequence identified herein as SEQ ID NO: 14 encodes SEQ ID NO: 15 and is included in an expression vector and expressed to produce Isl1.
  • the nucleic acid sequence identified herein as SEQ ID NO: 3 encodes SEQ ID NO: 4 and is included in an expression vector and expressed to produce NeuroD1.
  • the nucleic acid sequence identified herein as SEQ ID NO: 12 encodes SEQ ID NO: 13 and is included in an expression vector and expressed to produce Dlx2.
  • the nucleic acid sequence identified herein as SEQ ID NO: 16 encodes SEQ ID NO: 17 and is included in an expression vector and expressed to produce Isl1.
  • nucleic acid sequences substantially identical to SEQ ID NOs.1 and 3 encode NeuroD1 and variants of NeuroD1, and that such alternate nucleic acids may be included in an expression vector and expressed to produce NeuroD1 and variants of NeuroD1.
  • nucleic acid sequences substantially identical to SEQ ID NOs: 10 and 12 encode Dlx2 and variants of Dlx2, and that such alternate nucleic acids may be included in an expression vector and expressed to produce Dlx2 and variants of Dlx2.
  • nucleic acid sequences substantially identical to SEQ ID NOs: 14 and 16 encode Isl1 and variants of Isl1, and that such alternate nucleic acids may be included in an expression vector and expressed to produce Isl1 and variants of Isl1.
  • a fragment of a nucleic acid encoding NeuroD1 protein can be used to produce a fragment of a NeuroD1 protein.
  • a fragment of a nucleic acid encoding Dlx2 protein can be used to produce a fragment of a Dlx2 protein.
  • Lhx3 refers to LIM Homeobox 3 transcription factor involved in pituitary development and motor neuron specification.
  • An example of a human Lhx3 polypeptide includes, without limitation, NCBI reference sequence: NP_001350675.1 or a biologically active fragment thereof.
  • the Lhx3 polypeptide includes an amino acid substitution, insertion, or deletion that results in increased activity of the mutated Lhx3 as compared to the wildtype Lhx3 polypeptide (e.g., NCBI reference sequence: NP_001350675.1).
  • the term “mir124” refers to microRNA 124.
  • MicroRNAs (miRNAs) are short (20-24 nt) non-coding RNAs that are involved in post-transcriptional regulation of gene expression in multicellular organisms by affecting both the stability and translation of mRNAs.
  • the term “mir218” refers to microRNA 218.
  • MicroRNAs are short (20-24 nt) non-coding RNAs that are involved in post-transcriptional regulation of gene expression in multicellular organisms by affecting both the stability and translation of mRNAs.
  • Ngn2 refers to neural-specific basic helix-loop-helix (bHLH) transcription factor that can specify a neuronal fate on ectodermal cells and is expressed in neural progenitor cells within the developing central and peripheral nervous systems.
  • An example of a human Ngn2 polypeptide includes, without limitation, NCBI reference sequence: NP_076924.1 or a biologically active fragment thereof.
  • the Ngn2 polypeptide includes an amino acid substitution, insertion, or deletion that results in increased activity of the mutated Ngn2 as compared to the wildtype Ngn2 polypeptide (e.g., NCBI reference sequence: NP_076924.1).
  • An expression vector contains a nucleic acid that includes segment encoding a polypeptide of interest operably linked to one or more regulatory elements that provide for transcription of the segment encoding the polypeptide of interest.
  • operably linked refers to a nucleic acid in functional relationship with a second nucleic acid.
  • operably linked encompasses functional connection of two or more nucleic acid molecules, such as a nucleic acid to be transcribed and a regulatory element.
  • regulatory element refers to a nucleotide sequence which controls some aspect of the expression of an operably linked nucleic acid.
  • Exemplary regulatory elements include an enhancer, such as, but not limited to: woodchuck hepatitis virus posttranscriptional regulatory element (WPRE); an internal ribosome entry site (IRES) or a 2A domain; an intron; an origin of replication; a polyadenylation signal (pA); a promoter; a transcription termination sequence; and an upstream regulatory domain, which contribute to the replication, transcription, post-transcriptional processing of an operably linked nucleic acid sequence.
  • WPRE woodchuck hepatitis virus posttranscriptional regulatory element
  • IVS internal ribosome entry site
  • promoter refers to a DNA sequence operably linked to a nucleic acid sequence to be transcribed such as a nucleic acid sequence encoding NeuroD1.
  • a promoter is generally positioned upstream of a nucleic acid sequence to be transcribed and provides a site for specific binding by RNA polymerase and other transcription factors.
  • a promoter is generally positioned upstream of the nucleic acid sequence transcribed to produce the desired molecule, and provides a site for specific binding by RNA polymerase and other transcription factors.
  • the 5’ non-coding region of a gene can be isolated and used in its entirety as a promoter to drive expression of an operably linked nucleic acid.
  • a portion of the 5’ non-coding region can be isolated and used to drive expression of an operably linked nucleic acid.
  • about 500-6000 bp of the 5’ non-coding region of a gene is used to drive expression of the operably linked nucleic acid.
  • a portion of the 5’ non-coding region of a gene containing a minimal amount of the 5’ non-coding region needed to drive expression of the operably linked nucleic acid is used.
  • Assays to determine the ability of a designated portion of the 5’ non-coding region of a gene to drive expression of the operably linked nucleic acid are well-known in the art.
  • exogenous nucleic acid encoding any of the polypeptides, or any combination of polypeptides, described herein e.g., NeuroD1, Isl1, Lhx3, Dlx2, and Ngn2
  • exogenous nucleic acid encoding mir124 and mir218 according to methods described herein are “ubiquitous” or “constitutive” promoters, that drive expression in many, most, or all cell types of an organism into which the expression vector is transferred.
  • Non-limiting examples of ubiquitous promoters that can be used in expression of (i) exogenous nucleic acid encoding any of the polypeptides, or any combination of polypeptides, described herein (e.g., NeuroD1, Isl1, Lhx3, Dlx2, and Ngn2) and/or (ii) exogenous nucleic acid encoding mir124 and mir218 are cytomegalovirus promoter; simian virus 40 (SV40) early promoter; rous sarcoma virus promoter; adenovirus major late promoter; beta actin promoter; glyceraldehyde 3-phosphate dehydrogenase; glucose-regulated protein 78 promoter; glucose-regulated protein 94 promoter; heat shock protein 70 promoter; beta-kinesin promoter; ROSA promoter; ubiquitin B promoter; eukaryotic initiation factor 4A1 promoter and elongation Factor I promoter; all of which are well-known in the art and which can be isolated from
  • Promoters can be derived entirely from a single gene or can be chimeric, having portions derived from more than one gene. Combinations of regulatory sequences may be included in an expression vector and used to drive expression of NeuroD1.
  • a non-limiting example included in an expression vector to drive expression of NeuroD1 is the CAG promoter which combines the cytomegalovirus CMV early enhancer element and chicken beta-actin promoter.
  • Particular promoters used to drive expression of NeuroD1 are those that drive expression preferentially in glial cells, particularly astrocytes and/or NG2 cells.
  • Such promoters are termed “astrocyte-specific” and/or “NG2 cell-specific” promoters.
  • Non-limiting examples of astrocyte-specific promoters are glial fibrillary acidic protein (GFAP) promoter and aldehyde dehydrogenase 1 family, member L1 (Aldh1L1) promoter.
  • Human GFAP promoter is shown herein as SEQ ID NO:6.
  • Mouse Aldh1L1 promoter is shown herein as SEQ ID NO:7.
  • a non-limiting example of an NG2 cell-specific promoter is the promoter of the chondroitin sulfate proteoglycan 4 gene, also known as neuron-glial antigen 2 (NG2).
  • NG2 promoter is shown herein as SEQ ID NO:8.
  • Particular promoters used to drive expression of NeuroD1 according to methods described herein are those that drive expression preferentially in reactive glial cells, particularly reactive astrocytes and/or reactive NG2 cells. Such promoters are termed “reactive astrocyte-specific” and/or “reactive NG2 cell-specific” promoters. Particular promoters used to drive expression of Dlx2 according to methods described herein are those that drive expression preferentially in reactive glial cells, particularly reactive astrocytes and/or reactive NG2 cells. Such promoters are termed “reactive astrocyte- specific” and/or “reactive NG2 cell-specific” promoters.
  • a non-limiting example of a “reactive astrocyte-specific” promoter is the promoter of the lipocalin 2 (lcn2) gene.
  • Mouse lcn2 promoter is shown herein as SEQ ID NO:5.
  • Homologues and variants of ubiquitous and cell type-specific promoters may be used in expressing NeuroD1.
  • promoter homologues and promoter variants can be included in an expression vector for expressing NeuroD1.
  • promoter homologues and promoter variants can be included in an expression vector for expressing Dlx2.
  • promoter homologues and promoter variants can be included in an expression vector for expressing Isl1.
  • 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 NeuroD1 or ubiquitous expression of NeuroD1, on an operably linked nucleic acid encoding NeuroD1 compared to those disclosed herein.
  • a promoter homologue or variant has substantially similar functional properties to confer cell type-specific expression on an operably linked nucleic acid encoding NeuroD1 compared to GFAP, S100b, Aldh1L1, NG2, lcn2 and CAG promoters.
  • nucleic acid mutations can be introduced without altering the functional properties of a given promoter.
  • 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, S100b, Aldh1L1, NG2, lcn2 and pCAG promoters. It is known in the art that promoters from other species are functional, e.g. the mouse Aldh1L1promoter is 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., Genome Biol., 6:R72 (2005); Zhao et al., Nucl. Acid Res., 33:D103-107 (2005); and Halees et al., Nucl. Acids. Res., 31:3554-3559 (2003).
  • homologues and variants of cell type-specific promoters of NeuroD1 or and/or ubiquitous promoters have at least 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 developmentally regulated and/or ubiquitous promoter and include a site for binding of RNA polymerase and, optionally, one or more binding sites for transcription factors.
  • a nucleic acid sequence which is substantially identical to SEQ ID NO:1 or SEQ ID NO:3 is characterized as having a complementary nucleic acid sequence capable of hybridizing to SEQ ID NO:1 or SEQ ID NO:3 under high stringency hybridization conditions.
  • additional proteins include non-NeuroD1 proteins such as reporters, including, but not limited to, beta-galactosidase, green fluorescent protein and antibiotic resistance reporters.
  • the recombinant expression vector encodes at least NeuroD1 of SEQ ID NO:2, a protein having at least 95% identity to SEQ ID NO:2, or a protein encoded by a nucleic acid sequence substantially identical to SEQ ID NO:1.
  • the recombinant expression vector encodes at least NeuroD1 of SEQ ID NO:4, a protein having at least 95% identity to SEQ ID NO:4, or a protein encoded by a nucleic acid sequence substantially identical to SEQ ID NO:2.
  • SEQ ID NO:9 is an example of a nucleic acid including CAG promoter operably linked to a nucleic acid encoding NeuroD1, and further including a nucleic acid sequence encoding EGFP and an enhancer, WPRE.
  • An IRES separates the nucleic acid encoding NeuroD1 and the nucleic acid encoding EGFP.
  • SEQ ID NO:9 is inserted into an expression vector for expression of NeuroD1 and the reporter gene EGFP.
  • the IRES and nucleic acid encoding EGFP are removed and the remaining CAG promoter and operably linked nucleic acid encoding NeuroD1 is inserted into an expression vector for expression of NeuroD1.
  • the WPRE or another enhancer is optionally included.
  • a reporter gene is included in a recombinant expression vector encoding NeuroD1.
  • a reporter gene may be included to produce a peptide or protein that serves as a surrogate marker for expression of NeuroD1 from the recombinant expression vector.
  • a reporter gene is included in a recombinant expression vector encoding Dlx2.
  • a reporter gene is included in a recombinant expression vector encoding Isl1.
  • a reporter gene may be included to produce a peptide or protein that serves as a surrogate marker for expression of Dlx2 from the recombinant expression vector.
  • reporter gene refers to gene that is easily detectable when expressed, for example by chemiluminescence, fluorescence, colorimetric reactions, antibody binding, inducible markers and/or ligand binding assays.
  • exemplary reporter genes include, but are not limited to, green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (eYFP), cyan fluorescent protein (CFP), enhanced cyan fluorescent protein (eCFP), blue fluorescent protein (BFP), enhanced blue fluorescent protein (eBFP), MmGFP (Zernicka-Goetz et al., Development, 124:1133-1137 (1997)), dsRed, luciferase and beta-galactosidase (lacZ).
  • transfection The process of introducing genetic material into a recipient host cell, such as for transient or stable expression of a desired protein encoded by the genetic material in the host cell is referred to as “transfection.”
  • Transfection techniques are well-known in the art and include, but are not limited to, electroporation, particle accelerated transformation also known as “gene gun” technology, liposome-mediated transfection, calcium phosphate or calcium chloride co-precipitation-mediated transfection, DEAE-dextran-mediated transfection, microinjection, polyethylene glycol mediated transfection, heat shock mediated transfection and virus-mediated transfection.
  • virus-mediated transfection may be accomplished using a viral vector such as those derived from adenovirus, adeno-associated virus and lentivirus.
  • a host cell is transfected ex-vivo and then re-introduced into a host organism.
  • cells or tissues may be removed from a subject, transfected with an expression vector encoding NeuroD1 and then returned to the subject.
  • cells or tissues may be removed from a subject, transfected with an expression vector encoding NeuroD1 and Dlx2 and then returned to the subject.
  • cells or tissues may be removed from a subject, transfected with an expression vector encoding NeuroD1 and an expression vector encoding Dlx2 and then returned to the subject.
  • cells or tissues may be removed from a subject, transfected with an expression vector encoding NeuroD1 and Isl1 and then returned to the subject.
  • cells or tissues may be removed from a subject, transfected with an expression vector encoding NeuroD1 and an expression vector encoding Isl1 and then returned to the subject.
  • Introduction of a recombinant expression vector including a nucleic acid encoding NeuroD1, or a functional fragment thereof, into a host glial cell in vitro or in vivo for expression of exogenous NeuroD1 in the host glial cell to convert the glial cell to a neuron is accomplished by any of various transfection methodologies.
  • introduction of a recombinant expression vector including a nucleic acid encoding Dlx2, or a functional fragment thereof, into a host glial cell in vitro or in vivo for expression of exogenous Dlx2 in the host glial cell to convert the glial cell to a neuron is accomplished by any of various transfection methodologies.
  • introduction of a recombinant expression vector including a nucleic acid encoding Isl1, or a functional fragment thereof, into a host glial cell in vitro or in vivo for expression of exogenous Isl1 in the host glial cell to convert the glial cell to a neuron is accomplished by any of various transfection methodologies.
  • exogenous nucleic acid encoding any of the polypeptides, or any combination of polypeptides, described herein (e.g., NeuroD1, Isl1, Lhx3, Dlx2, and Ngn2) and/or (ii) exogenous nucleic acid encoding mir124 and mir218 in the host glial cell to convert the glial cell to a neuron is optionally achieved by introduction of mRNA encoding NeuroD1 (or any other polypeptide described herein), or a functional fragment thereof, to the host glial cell in vitro or in vivo.
  • exogenous nucleic acid encoding any of the polypeptides, or any combination of polypeptides, described herein (e.g., NeuroD1, Isl1, Lhx3, Dlx2, and Ngn2) and/or (ii) exogenous nucleic acid encoding mir124 and mir218 in the host glial cell to convert the glial cell to a neuron is optionally achieved by introduction of polypeptides to the host glial cell in vitro or in vivo. Details of these and other techniques are known in the art, for example, as described in J. Sambrook and D.W. Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 3rd Ed., 2001; F.M.
  • RNA Interference Nuts and Bolts of RNAi Technology, DNA Press LLC, Eagleville, PA, 2003.
  • An expression vector including (i) exogenous nucleic acid encoding any of the polypeptides, or any combination of polypeptides, described herein (e.g., NeuroD1, Isl1, Lhx3, Dlx2, and Ngn2) and/or (ii) exogenous nucleic acid encoding mir124 and mir218 or a functional fragment thereof, mRNA encoding the polypeptides or a functional fragment thereof, full-length or a functional fragment thereof, is optionally associated with a carrier for introduction into a host cell in vitro or in vivo.
  • exogenous nucleic acid encoding any of the polypeptides, or any combination of polypeptides, described herein (e.g., NeuroD1, Isl1, Lhx3, Dlx2, and Ngn2) and/or (ii) exogenous nucleic acid encoding mir124 and mir218 or a functional fragment thereof, mRNA encoding the polypeptides or a functional fragment thereof, full-length
  • the carrier is a particulate carrier such as lipid particles including liposomes, micelles, unilamellar or mulitlamellar vesicles; polymer particles such as hydrogel particles, polyglycolic acid particles or polylactic acid particles; inorganic particles such as calcium phosphate particles such as described in for example U.S. Patent No.5,648,097; and inorganic/organic particulate carriers such as described for example in U.S. Patent No. 6,630,486.
  • a particulate carrier can be selected from among a lipid particle; a polymer particle; an inorganic particle; and an inorganic/organic particle.
  • a mixture of particle types can also be included as a particulate pharmaceutically acceptable carrier.
  • a particulate carrier is typically formulated such that particles have an average particle size in the range of about 1 nm - 10 microns. In particular aspects, a particulate carrier is formulated such that particles have an average particle size in the range of about 1 nm - 100 nm.
  • liposomes and methods relating to their preparation and use may be found in Liposomes: A Practical Approach (The Practical Approach Series, 264), V. P. Torchilin and V. Weissig (Eds.), Oxford University Press; 2nd ed., 2003. Further aspects of nanoparticles are described in S.M. Moghimi et al., FASEB J.2005, 19, 311-30.
  • exogenous nucleic acid encoding any of the polypeptides, or any combination of polypeptides, described herein (e.g., NeuroD1, Isl1, Lhx3, Dlx2, and Ngn2) and/or (ii) exogenous nucleic acid encoding mir124 and mir218 using a recombinant expression vector is accomplished by introduction of the expression vector into a eukaryotic or prokaryotic host cell expression system such as an insect cell, mammalian cell, yeast cell, bacterial cell or any other single or multicellular organism recognized in the art.
  • Host cells are optionally primary cells or immortalized derivative cells. Immortalized cells are those which can be maintained in-vitro for at least 5 replication passages.
  • Host cells containing the recombinant expression vector are maintained under conditions wherein NeuroD1 is produced. In some cases, host cells containing the recombinant expression vector are maintained under conditions wherein Dlx2 is produced. Host cells may be cultured and maintained using known cell culture techniques such as described in Celis, Julio, ed., 1994, Cell Biology Laboratory Handbook, Academic Press, N.Y. Various culturing conditions for these cells, including media formulations with regard to specific nutrients, oxygen, tension, carbon dioxide and reduced serum levels, can be selected and optimized by one of skill in the art.
  • a recombinant expression vector including (i) exogenous nucleic acid encoding any of the polypeptides, or any combination of polypeptides, described herein (e.g., NeuroD1, Isl1, Lhx3, Dlx2, and Ngn2) and/or (ii) exogenous nucleic acid encoding mir124 and mir218 is introduced into glial cells of a subject.
  • a recombinant expression vector including (i) exogenous nucleic acid encoding any of the polypeptides, or any combination of polypeptides, described herein (e.g., NeuroD1, Isl1, Lhx3, Dlx2, and Ngn2) and/or (ii) exogenous nucleic acid encoding mir124 and mir218 is introduced into astrocytes of a subject.
  • exogenous nucleic acid encoding any of the polypeptides, or any combination of polypeptides, described herein (e.g., NeuroD1, Isl1, Lhx3, Dlx2, and Ngn2) and/or (ii) exogenous nucleic acid encoding mir124 and mir218 in the glial cells “converts” the astrocytes into neurons.
  • a recombinant expression vector (i) exogenous nucleic acid encoding any of the polypeptides, or any combination of polypeptides, described herein (e.g., NeuroD1, Isl1, Lhx3, Dlx2, and Ngn2) and/or (ii) exogenous nucleic acid encoding mir124 and mir218 is introduced into reactive astrocytes of a subject.
  • exogenous nucleic acid encoding any of the polypeptides, or any combination of polypeptides, described herein (e.g., NeuroD1, Isl1, Lhx3, Dlx2, and Ngn2) and/or (ii) exogenous nucleic acid encoding mir124 and mir218 in the reactive astrocytes “converts” the reactive astrocytes into neurons.
  • a recombinant expression vector including (i) exogenous nucleic acid encoding any of the polypeptides, or any combination of polypeptides, described herein (e.g., NeuroD1, Isl1, Lhx3, Dlx2, and Ngn2) and/or (ii) exogenous nucleic acid encoding mir124 and mir218 is introduced into NG2 cells of a subject.
  • exogenous nucleic acid encoding any of the polypeptides, or any combination of polypeptides, described herein (e.g., NeuroD1, Isl1, Lhx3, Dlx2, and Ngn2) and/or (ii) exogenous nucleic acid encoding mir124 and mir218 in the NG2 cells “converts” the NG2 cells into neurons.
  • Detection of expression of (i) exogenous nucleic acid encoding any of the polypeptides, or any combination of polypeptides, described herein (e.g., NeuroD1, Isl1, Lhx3, Dlx2, and Ngn2) and/or (ii) exogenous nucleic acid encoding mir124 and mir218 following introduction of a recombinant expression vector including a nucleic acid encoding (i) exogenous nucleic acid encoding any of the polypeptides, or any combination of polypeptides, described herein (e.g., NeuroD1, Isl1, Lhx3, Dlx2, and Ngn2) and/or (ii) exogenous nucleic acid encoding mir124 and mir218 is accomplished using any of various standard methodologies including, but not limited to, immunoassays, nucleic acid detection assays and detection of a reporter gene co-expressed with the exogenous nucleic acids.
  • neuroD1 and Dlx2 are used herein to describe the effect of expression of NeuroD1 or a functional fragment thereof alone or in combination with Dlx2 or a functional fragment thereof resulting in a change of a glial cell, astrocyte or reactive astrocyte phenotype to a neuronal phenotype.
  • neuronal phenotype a glial cell, astrocyte or reactive astrocyte phenotype to a neuronal phenotype.
  • neuronal neuroD1 converted neurons “NeuroD1 and Dlx2 converted neurons” and “converted neurons” are used herein to designate a cell including exogenous NeuroD1 protein or a functional fragment thereof alone or in combination with exogenous Dlx2 protein or a functional fragment thereof which has consequent neuronal phenotype.
  • neuronal phenotype refers to well-known detectable characteristics of the cells referred to herein.
  • the neuronal phenotype can be, but is not limited to, one or more of: neuronal morphology, expression of one or more neuronal markers, electrophysiological characteristics of neurons, synapse formation and release of neurotransmitter.
  • neuronal phenotype encompasses but is not limited to: characteristic morphological aspects of a neuron such as presence of dendrites, an axon and dendritic spines; characteristic neuronal protein expression and distribution, such as presence of synaptic proteins in synaptic puncta, presence of MAP2 in dendrites; and characteristic electrophysiological signs such as spontaneous and evoked synaptic events.
  • glial phenotype such as astrocyte phenotype and reactive astrocyte phenotypes encompasses but is not limited to: characteristic morphological aspects of astrocytes and reactive astrocytes such as a generally “star-shaped” morphology; and characteristic astrocyte and reactive astrocyte protein expression, such as presence of glial fibrillary acidic protein (GFAP).
  • GFAP glial fibrillary acidic protein
  • nucleic acid refers to RNA or DNA molecules having more than one nucleotide in any form including single-stranded, double-stranded, oligonucleotide or polynucleotide.
  • nucleotide sequence refers to the ordering of nucleotides in an oligonucleotide or polynucleotide in a single-stranded form of nucleic acid.
  • NeuroD1 nucleic acid refers to an isolated NeuroD1 nucleic acid molecule and encompasses isolated NeuroD1 nucleic acids having a sequence that has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the DNA sequence set forth in SEQ ID NO:1 or SEQ ID NO:3, or the complement thereof, or a fragment thereof, or an isolated DNA molecule having a sequence that hybridizes under high stringency hybridization conditions to the nucleic acid set forth as SEQ ID NO:1 or SEQ ID NO:3, a complement thereof or a fragment thereof.
  • the nucleic acid of SEQ ID NO:3 is an example of an isolated DNA molecule having a sequence that hybridizes under high stringency hybridization conditions to the nucleic acid set forth in SEQ ID NO:1.
  • a fragment of a NeuroD1 nucleic acid is any fragment of a NeuroD1 nucleic acid that is operable in an aspect described herein including a NeuroD1 nucleic acid.
  • a nucleic acid probe or primer able to hybridize to a target NeuroD1 mRNA or cDNA can be used for detecting and/or quantifying mRNA or cDNA encoding NeuroD1 protein.
  • a nucleic acid probe can be an oligonucleotide of at least 10, 15, 30, 50 or 100 nucleotides in length and sufficient to specifically hybridize under stringent conditions to NeuroD1 mRNA or cDNA or complementary sequence thereof.
  • a nucleic acid primer can be an oligonucleotide of at least 10, 15 or 20 nucleotides in length and sufficient to specifically hybridize under stringent conditions to the mRNA or cDNA, or complementary sequence thereof.
  • nucleic acid refers to Watson-Crick base pairing between nucleotides and specifically refers to nucleotides hydrogen bonded to one another with thymine or uracil residues linked to adenine residues by two hydrogen bonds and cytosine and guanine residues linked by three hydrogen bonds.
  • a nucleic acid includes a nucleotide sequence described as having a “percent complementarity” to a specified second nucleotide sequence.
  • a nucleotide sequence may have 80%, 90%, or 100% complementarity to a specified second nucleotide sequence, indicating that 8 of 10, 9 of 10 or 10 of 10 nucleotides of a sequence are complementary to the specified second nucleotide sequence.
  • the nucleotide sequence 3’-TCGA-5’ is 100% complementary to the nucleotide sequence 5’-AGCT-3’.
  • the nucleotide sequence 3’- TCGA- is 100% complementary to a region of the nucleotide sequence 5’-TTAGCTGG-3’.
  • hybridization and “hybridizes” refer to pairing and binding of complementary nucleic acids.
  • Hybridization occurs to varying extents between two nucleic acids depending on factors such as the degree of complementarity of the nucleic acids, the melting temperature, Tm, of the nucleic acids and the stringency of hybridization conditions, as is well known in the art.
  • stringency of hybridization conditions refers to conditions of temperature, ionic strength, and composition of a hybridization medium with respect to particular common additives such as formamide and Denhardt’s solution. Determination of particular hybridization conditions relating to a specified nucleic acid is routine and is well known in the art, for instance, as described in J. Sambrook and D.W. Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 3rd Ed., 2001; and F.M.
  • High stringency hybridization conditions are those which only allow hybridization of substantially complementary nucleic acids. Typically, nucleic acids having about 85-100% complementarity are considered highly complementary and hybridize under high stringency conditions. Intermediate stringency conditions are exemplified by conditions under which nucleic acids having intermediate complementarity, about 50-84% complementarity, as well as those having a high degree of complementarity, hybridize. In contrast, low stringency hybridization conditions are those in which nucleic acids having a low degree of complementarity hybridize.
  • hybridization and “specifically hybridizes” refer to hybridization of a particular nucleic acid to a target nucleic acid without substantial hybridization to nucleic acids other than the target nucleic acid in a sample.
  • Stringency of hybridization and washing conditions depends on several factors, including the Tm of the probe and target and ionic strength of the hybridization and wash conditions, as is well-known to the skilled artisan.
  • Hybridization and conditions to achieve a desired hybridization stringency are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2001; and Ausubel, F. et al., (Eds.), Short Protocols in Molecular Biology, Wiley, 2002.
  • An example of high stringency hybridization conditions is hybridization of nucleic acids over about 100 nucleotides in length in a solution containing 6X SSC, 5X Denhardt’s solution, 30% formamide, and 100 micrograms/ml denatured salmon sperm at 37oC overnight followed by washing in a solution of 0.1X SSC and 0.1% SDS at 60oC for 15 minutes.
  • SSC is 0.15M NaCl/0.015M Na citrate.
  • Denhardt’s solution is 0.02% bovine serum albumin/0.02% FICOLL/0.02% polyvinylpyrrolidone.
  • SEQ ID NO:1 and SEQ ID NO:3 will hybridize to the complement of substantially identical targets and not to unrelated sequences.
  • Methods of treating a neurological condition in a subject in need thereof are provided according to aspects of the disclosure which include delivering a therapeutically effective (i) exogenous nucleic acid encoding any of the polypeptides, or any combination of polypeptides, described herein (e.g., NeuroD1, Isl1, Lhx3, Dlx2, and Ngn2) and/or (ii) exogenous nucleic acid encoding mir124 and mir218 to glial cells of the central nervous system or peripheral nervous system of the subject, the therapeutically effective amount of (i) exogenous nucleic acid encoding any of the polypeptides, or any combination of polypeptides, described herein (e.g., NeuroD1, Isl1, Lhx3, Dlx2, and Ngn2) and/or (ii) exogenous nucleic acid encoding mir124 and mir218 in the glial cells results in a greater number of neurons in the subject compared to an untreated subject having the same neurological condition, whereby the neurological
  • neurodegenerative disease The conversion of reactive glial cells into neurons also reduces neuroinflammation and neuroinhibitory factors associated with reactive glial cells, thereby making the glial scar tissue more permissive to neuronal growth so that neurological condition is alleviated.
  • the term “neurological condition” or “neurological disorder” as used herein refers to any condition of the central nervous system of a subject which is alleviated, ameliorated or prevented by additional neurons. Injuries or diseases which result in loss or inhibition of neurons and/or loss or inhibition of neuronal function are neurological conditions for treatment by methods described herein. Injuries or diseases which result in loss or inhibition of glutamatergic neurons and/or loss or inhibition of glutaminergic neuronal functions are neurological conditions that can be treated as described herein.
  • a therapeutically effective amount as used herein is intended to mean an amount of an inventive composition which is effective to alleviate, ameliorate or prevent a symptom or sign of a neurological condition to be treated.
  • a therapeutically effective amount is an amount which has a beneficial effect in a subject having signs and/or symptoms of a neurological condition.
  • treat refers to alleviating, inhibiting or ameliorating a neurological condition, symptoms or signs of a neurological condition, and preventing symptoms or signs of a neurological condition, and include, but are not limited to therapeutic and/or prophylactic treatments. Signs and symptoms of neurological conditions are well-known in the art along with methods of detection and assessment of such signs and symptoms. In some cases, combinations of therapies for a neurological condition of a subject can be administered.
  • an additional pharmaceutical agent or therapeutic treatment administered to a subject to treats the effects of disruption of normal blood flow in the CNS in an individual subject in need thereof include treatments such as, but not limited to, removing a blood clot, promoting blood flow, administration of one or more anti- inflammation agents, administration of one or more anti-oxidant agents, and administration of one or more agents effective to reduce excitotoxicity
  • subject refers to humans and also to non-human mammals such as, but not limited to, non-human primates, cats, dogs, sheep, goats, horses, cows, pigs and rodents, such as but not limited to, mice and rats; as well as to non-mammalian animals such as, but not limited to, birds, poultry, reptiles, amphibians.
  • inventive compositions and methods are illustrated in the following examples. These examples are provided for illustrative purposes and are not considered limitations on the scope of inventive compositions and methods.
  • IACUC Institutional Animal Care and Use Committee
  • Retrovirus and AAV production Retroviral vectors expressing GFP and NeuroD1-GFP under the CAG promoter (pCAG) were previously described (Guo et al., Cell Stem Cell, 14:188-202 (2014)). Retrovirus packaging, purification and titering were performed as previously described (Guo et al., Cell Stem Cell, 14:188-202 (2014)).
  • Cre-Flex system was applied to target transgene expression specifically to reactive astrocytes using the human GFAP (hGFAP) promoter.
  • the hGFAP promoter was first amplified from pDRIVE-hGFAP plasmid (InvivoGen) by PCR and inserted into pAAV-MCS (Cell Biolab) between the MluI and SacII sites to replace the CMV promoter.
  • the Cre gene coding fragment was then similarly subcloned from phGFAP-Cre (Addgene plasmid #40591) and inserted into pAAV MCS between the EcoRI and SalI sites.
  • pAAV-FLEX vectors expressing transgenes the coding sequences of NeuroD1, mCherry or GFP were amplified by PCR from the corresponding retroviral constructs.
  • the NeuroD1 fragment was fused with either P2A-mCherry or P2A-GFP and subcloned into the pAAV-FLEX-GFP vector (Addgene plasmid #28304) between the KpnI and XhoI sites. All plasmid constructs were confirmed by sequencing.
  • the AAV-CamKII-GFP plasmid was purchased from Addgene (#64545).
  • HEK 293T cells were transfected with the pAAV expression vectors, pAAV9-RC vector (Cell Biolab), and pHelper vector (Cell Biolab) to generate AAV particles carrying our transgenes.
  • the cells were scraped in their medium and centrifuged. The supernatant was then discarded and the cell pellet was frozen and thawed four times, resuspended in a discontinuous iodixanol gradient, and centrifuged at 54,000 rpm for two hours. Finally, the virus-containing layer was extracted, and the viruses were concentrated using Millipore Amicon Ultra Centrifugal Filters.
  • the viral titers were determined using the QuickTiterTM AAV Quantitation Kit (Cell Biolabs) and then diluted to a final concentration of 1 ⁇ 10 10 genome copy (GC)/mL for injection.
  • Laminectomy, injury, and stereotaxic viral injection Mice were anesthetized by intraperitoneal injection of ketamine/xylazine (80-120 mg/kg ketamine; 10-16 mg/kg xylazine). A laminectomy was then performed at the T11-T12 vertebrae to expose dorsal surface of the spinal cord, and a stab or contusion injury was performed.
  • the stab injury was conducted with a 31-gauge needle at the center of the exposed surface, 0.4 mm lateral to the central artery with a depth of 0.4 mm, while the contusion injury was generated with a force of 45 kdyn on an Infinite Horizon Impactor (IH- 0400, Precision Systems and Instrumentation) directly at the center of the exposed surface. Either immediately following the injury or at a specified delay, 1.0 ⁇ L of concentrated virus was injected using a 50 ⁇ L Hamilton syringe with a 34-gauge injection needle at a rate of 0.05 ⁇ L/minute at the same coordinates for the stab injury or 1 mm away for the contusion injury. The needle was then kept in place for three minutes after injection to prevent drawing out the virus during withdrawal.
  • IH- 0400 Infinite Horizon Impactor
  • mice were kept on a heating pad and treated with Carprofen for pain relieve via subcutaneous injection (5 mg/kg) on the day of surgery and drinking water (10 mg/kg) for three days after surgery and closely monitored for one week to ensure full recovery of health. Electrophysiology Mice were sacrificed at defined time points by anesthetization with 2.5% Avertin and decapitation.
  • the spinal cord segment was then removed from the spine into cutting solution (125 mM NaCl, 2.5 mM KCl, 1.3 mM MgSO 4 , 26 mM NaHCO 3 , 1.25 mM NaH 2 PO 4 , 2.0 mM CaCl2 and 10 mM glucose adjusted to pH 7.4 and 295 mOsm/L and bubbled for one hour with 95% O 2 /5% CO 2 ) cooled on ice, where it was encased in an agarose matrix (Sigma) and cut into 300 ⁇ m thickness slices using a VT3000 vibratome (Leica).
  • cutting solution 125 mM NaCl, 2.5 mM KCl, 1.3 mM MgSO 4 , 26 mM NaHCO 3 , 1.25 mM NaH 2 PO 4 , 2.0 mM CaCl2 and 10 mM glucose adjusted to pH 7.4 and 295 mOsm/L and bubbled for one hour with 95% O 2 /5% CO
  • Both native and converted cells were recorded by whole-cell recording using standard inner solution (135 mM K-gluconate, 10 mM KCl, 5 mM Naphosphocreatine, 10 mM HEPES, 2 mM EGTA, 4 mM MgATP, and 0.5 mM Na2GTP, adjusted to pH 7.4 and 295 mOsm/L) with the membrane potential held at -70 mV.
  • Typical values for the pipette and total series resistances were 2-10 M ⁇ and 20-60 M ⁇ , respectively.
  • Data were collected using the pClamp 9 software (Molecular Devices) by sampling at 10 kHz and filtering at 1 kHz. Data were then analyzed and plotted with the Clampfit 9.0 software (Molecular Devices).
  • the target region of the spinal cord ( ⁇ 0.5 cm in length) was surgically dissected, fixed in 4% paraformaldehyde (PFA) in PBS for one day, dehydrated in 30% sucrose solution for one day, and sectioned into 30 ⁇ m coronal or horizontal slices using a Leica CM1950 cryostat. The slices were collected serially in 24-well plates so that distance from the injury site could later be ascertained. The samples were then stored at 4°C in 0.02% sodium azide (NaN 3 ) in PBS to prevent bacterial degradation.
  • PFA paraformaldehyde
  • the samples were then incubated with primary antibodies diluted in the same blocking buffer at 4°C for two nights to allow thorough penetration of the antibodies.
  • the samples were recovered to room temperature, washed in PBS three times for five minutes per wash, and incubated with secondary antibodies diluted in blocking buffer for one hour.
  • the samples were washed in PBS three more times for ten minutes per wash and mounted on glass slides with coverslips using anti-fading mounting solution (Invitrogen).
  • the immunostained samples were examined and imaged using Olympus FV1200 and Zeiss LSM 800 laser confocal microscopes.
  • Strict background cutoffs for positive signals were calculated for each channel as three times the average background intensity for the relevant tissue and antibody.
  • Cells were binned by presence (i.e., above the background cutoff) or absence (i.e., below the background cutoff) for each marker in question, using the viral fluorophore (mCherry or GFP) to identify infected cells and DAPI to confirm each cell for counting.
  • mCherry or GFP the viral fluorophore
  • DAPI to confirm each cell for counting.
  • To estimate the total number of converted neurons per infection for our contusion experiments we multiplied the average number of NeuN+ infected cells per horizontal section, calculated from one dorsal, one central, and one ventral section, by the total number of horizontal sections per sample.
  • Example 1 NeuroD1 reprograms reactive astrocytes into neurons in the injured spinal cord SCI has been studied for decades but so far there is still limited therapy to treat SCI patients. Besides axonal degeneration, neuronal loss following SCI is a major obstacle for functional recovery.
  • expressing NeuroD1 in reactive astrocytes after brain injury can directly convert astrocytes into neurons (Guo et al., Cell Stem Cell, 14:188-202 (2014)). In this study, we investigated whether such in vivo direct conversion technology can be used to regenerate functional new neurons in injured spinal cord.
  • Cre-Flex gene expression system which contains two AAV vectors, with one encoding GFAP-Cre and the other encoding the transgene in reverse form flanked by double LoxP sites (FLEX vector)
  • FLEX vector double LoxP sites
  • Cre recombinase when the two AAVs are co-injected into the spinal cord, Cre recombinase will be expressed in the infected reactive astrocytes and turn on the transgene expression in FLEX vector by flipping the transgene sequence into the correct form for transcription (Figure 2B).
  • AAV GFAP::Cre and AAV FLEXCAG::mCherry (or ::GFP) into the stab-injured dorsal horn.
  • the control virus infected cells were mostly GFAP+, NeuN- astrocytes at 4 wpi ( Figure 2C).
  • NeuroD1 converts dorsal spinal astrocytes into Tlx3+ glutamatergic neurons After demonstrating astrocyte-to-neuron conversion in the spinal cord, we next investigated which subtypes of neurons were generated through NeuroD1-mediated conversion.
  • the dorsal horn of the spinal cord contains two main neuronal subtypes: glutamatergic and GABAergic neurons (Abraira and Ginty, Neuron, 79:618-639 (2013)).
  • glutamatergic and GABAergic neurons Abraira and Ginty, Neuron, 79:618-639 (2013).
  • Tlx3 and Pax2 appear to play roles in determining cell fate specification in the dorsal horn (Cheng et al., Nature Neurosci., 8:1510-1515 (2005); and Huang et al., Dev. Biol., 322:394-405 (2008)).
  • NeuroD1-converted neurons in the dorsal horn of the spinal cord are glutamatergic neurons, consistent with our finding in the mouse cortex (Chen et al., BioRxiv, Apr.4, 2018; doi: http://dx.doi.org/10.1101/294967).
  • Example 3 NeuroD1-converted neurons express region-specific neuronal subtype markers While NeuroD1-converted neurons appear to be mainly glutamatergic neurons in both the mouse cortex and spinal cord, we further investigated whether they are the same type of glutamatergic neurons or not.
  • the converted neurons in the brain resembled cortical pyramidal neurons with larger cell bodies (Figure 4A), while those in the spinal cord resembled dorsal horn interneurons with smaller cell bodies (Figure 4C).
  • the relative lower percentage of Tbr1+ cells among the converted neurons in the cortex suggest that the newly converted neurons may not be mature enough at 4 wpi and may take longer time to fully acquire their neuronal identity.
  • These distinct differences in the neuronal identity after conversion by the same transcription factor in the brain versus the spinal cord suggest that the glial cell lineage, here cortical lineage versus spinal lineage, as well as the local environment may exert an important influence on the resulting subtypes of converted neurons.
  • Example 4 NeuroD1-converted neurons are physiologically functional
  • the converted neurons could generate repetitive action potentials (Figure 5B) and displayed large Na+ and K+ currents (Figure 5C).
  • Figure 5D we detected robust spontaneous EPSCs from the NeuroD1-converted neurons ( Figure 5D).
  • Figure 5E we found that the NeuroD1-converted neurons showed similar levels of Na+ currents ( Figure 5E) and spontaneous EPSCs to their neighboring native neurons ( Figure 5F).
  • Example 5 NeuroD1-mediated cell conversion in the contusive SCI model
  • contusive injury e.g., a contusive injury created using a piston driven metal bar to strike the area
  • AAV GFAP a contusive injury created using a piston driven metal bar to strike the area
  • the advantage of the short-delay experiment is to maximize infection rate by taking advantage of the post- injury proliferation of reactive astrocytes, while the advantage of the long-delay experiment is to maximize the neuronal survival after conversion by allowing injury-induced neuroinflammation to taper down and minimize the secondary effects of the contusion injury.
  • viral injection was conducted at 10 days post-contusive injury, and tissues were collected at 6 weeks post-viral infection (Figure 6A). Viral injections were performed 1 mm away from the contusion site to avoid the injury core (Figure 6B). The injury core is apparent after contusion and is characterized by the loss of NeuN+ neuronal cell bodies ( Figure 6C, labeled by *).
  • NeuroD1-GFP infected cells were often colocalized with NeuN but rarely colocalized with GFAP (Figure 6C), indicating successful neuronal conversion.
  • Figure 6C the total number of converted neurons to be ⁇ 2,600 cells surrounding the lesion core areas.
  • Figure 6E The efficiency of NeuroD1-mediated neuronal conversion in the short-delay experiment as measured by NeuN immunoreactivity was ⁇ 55% (Figure 6E), while the remaining cells were mostly GFAP+ (Figure 6F).
  • the GFP-infected cells were mostly GFAP+ astrocytes and rarely NeuN+ neurons (only 3.9% NeuN+ in GFP group) ( Figures 6E and 6F).
  • Figure 7A illustrates the overall morphology of the spinal cord with immunostaining of GFP, NeuN, and GFAP.
  • the lesion core (labeled by *) also lacked NeuN+ neurons.
  • the viral infected cells were mainly S100b+ astrocytes ( Figure 7C), but rarely showed any NeuN+ signal ( Figure 7D, top row).
  • Example 6 Astrocyte to neuron conversion Nucleic acid driving expression of Mir124, NeuroD1, Isl1, Lhx3, Ngn2, or their combinations were introduced into the mouse ventral horn and found to convert astrocytes into neurons, with some of the converted neurons display motor neuron properties by immunostaining positive for ChAT, a typical motoneuron marker ( Figures 11-16).
  • Example 7 NeuroD1 and Dlx2-mediated cell conversion The following was performed to investigate whether it was possible to increase the proportion of GABAergic neurons by combining NeuroD1 with other transcription factors.
  • Example 8 Treating ALS
  • the mouse model for ALS (SOD1*G93A) was used to investigate a gene therapy treatment using in vivo astrocyte-to-neuron conversion technology to regenerate motor neurons in the spinal cord and restore motor functions in ALS mice.
  • AAV9-GFAP-Cre + AAV9-Flex-mCherry were used to infect astrocytes as control experiments.
  • AAV9-GFAP-Cre + AAV9-Flex-NeuroD1-mCherry, or AAV9-GFAP-Cre + AAV9-Flex-NeuroD1-GFP + AAV9-Flex-Isl1-mCherry, or AAV9-GFAP-Cre + AAV9- Flex-NeuroD1-GFP + AAV9-Flex-Lhx3-mCherry were employed to convert astrocytes into neurons in the spinal cord, through intra-spinal injection or intrathecal injection.
  • NeuroD1 + Isl1 resulted in a greater reduction of CD11b signal, compared to the NeuroD1 + Lhx3 group or GFP control group.
  • these results confirm that gene therapy treatment can significantly convert astrocytes into neurons and that the regenerated motor neurons can improve motor functions in ALS mice. See, also, Figures 17-25.
  • Animal use In this example, B6.Cg-Tg (SOD1*G93A) dl1Gur/J mice (The Jackson Laboratory) were mated with C57BL/6J females (The Jackson Laboratory) to obtain mice on a pure background. Mice were genotyped by PCR against human SOD1 after weaning (P21-27), and the littermates without mutation were used as normal mice.
  • mice were housed in a 12 hour light/dark cycle and supplied with sufficient food and water.
  • Laminectomy, injury, and stereotaxic viral injection SOD1G93A mice were anesthetized by intraperitoneal injection of ketamine/xylazine (80-120 mg/kg ketamine; 10-16 mg/kg xylazine), followed by fur trimming on the back, and placement into a stereotaxic setup.
  • Artificial eye ointment was applied to cover the eye for protection purposes.
  • a laminectomy was then performed at the T11-L1 vertebrae to expose the spinal cord.
  • AAV9-GFAP-Cre + AAV9-Flex-NeuroD1-mCherry or AAV9- GFAP-Cre + AAV9-Flex-mCherry were injected into the ventral horn of spinal cord (0.45 mm lateral to the central artery with a depth of 0.9 mm), through a 50 ⁇ L Hamilton syringe with a 34-gauge injection needle at a rate of 0.05 ⁇ L/minute. After injection, the needle was kept in place for three minutes to prevent drawing out the virus and then slowly withdrawn. The surgical area was then treated with antibiotic ointment.
  • mice were kept on a heating pad and treated with Carprofen for pain relieve via subcutaneous injection (5 mg/kg) on the day of surgery and drinking water (10 mg/kg) for three days after surgery and closely monitored for one week to ensure full recovery of health.
  • Intrathecal injection For intrathecal injections, 9-week-old mice were anesthetized using isoflurane. Ten microliters of AAV-PHP.eB-GFAP-Cre + AAV-PHP.eB-Flex-TFs-GFP/mCherry or + AAV- PHP.eB-Flex-GFP/mCherry were injected into the mouse lumbar subarachnoid space using a 25 ⁇ L Hamilton syringe with a 31-gauge injection needle.
  • mice were anesthetized with 3% isoflurane and shaved 2*2 cm 2 of fur at the posterior end of the animal near the tail to facilitate a batter visualization during needle insertion.
  • Mice were placed in a nose cone for a continued isoflurane administration during the procedure in prone position, and the isoflurane was reduced to 1.5%.
  • An artificial eye ointment was applied to cover the eye for protection purposes.
  • the needle was carefully inserted between the groove of L5 and L6 vertebrae, and the mice were observe for a tail flick as a sign of a successful entry into the lumbar cistern. Once tail flick was observed, the syringe was stabilizes and the injection proceeded slowly.
  • This injection was repeated twice every 24 hours to achieve optimal amount of virus (totally 2x10 10 genome copies virus per mouse). All the injections were done with a timer to achieve a constant injection speed, inject 10 mL in 2 minutes. The syringe was removed after 1 minute to minimize CSF and vector leakage. Mice were maintained in the same position for 5 additional minutes. After surgery, animals were housed in cage with free access to food and water till end point. Body weight The body weight of each mouse was recorded every 8 days for 8 weeks from day 70 to day 126. Animal behavior Open field test Open field test was conducted in an opaque, open acrylic box (40 x 40 x 40 cm) in a brightly lit room. Mice were habituated to the testing environment for 1 hour in the testing room prior the test.
  • Each individual mouse was randomly placed at one corner of the box in a dark room with red light. Video camera recorded horizontal movement of the mouse for 10 minutes. Total distance traveled and duration of movement were measured by Noldus EthoVision XT software. The open field apparatus was cleaned with 70% ethanol between each trail. Catwalk test Mice were habituated at the testing room for 1 hour before the test. The catwalk was conducted in the Noldus CatWalk XT system. Each individual mouse was placed at the entrance of the walkway with a straight line to guild the movement. The mouse traveled freely on the walkway. Mouse traveling with a constant speed was as a successful trail. Three trails were recorded with the illuminated footprints technology. Paw print area, swing speed, and step cycle were measured by the CatWalk XT software.
  • SCI spinal cord injury
  • the spinal cord injury is due to a condition selected from the group consisting of: ischemic stroke; hemorrhagic stroke; physical injury; concussion; contusion; blast; penetration; tumor; inflammation; infection; traumatic spinal injury; ischemic or hemorrhagic myelopathy (spinal cord infarction); global ischemia as caused by cardiac arrest or severe hypotension (shock); hypoxic-ischemic encephalopathy as caused by hypoxia, hypoglycemia, or anemia; CNS embolism as caused by infective endocarditis or atrial myxoma; fibrocartilaginous embolic myelopathy; CNS thrombosis as caused by pediatric leukemia; cerebral venous sinus thrombosis as caused by nephrotic syndrome (kidney disease), chronic inflammatory disease, pregnancy, use of estrogen-based contraceptives, meningitis, dehydration; or a combination of any two or more thereof.
  • ischemic stroke hemorrhagic stroke
  • Embodiment 4 The method of embodiment 1, wherein said administering step comprises delivering an expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof to the spinal cord.
  • Embodiment 5. The method of embodiment 1, wherein said administering step comprises delivering a recombinant viral expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof to the spinal cord.
  • Embodiment 6. The method of embodiment 1, wherein said administering step comprises delivering a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof to the spinal cord.
  • Embodiment 8 The method of any of embodiments 1-7, wherein said administering step comprises administering a recombinant expression vector comprising a nucleic acid sequence encoding NeuroD1 polypeptide, wherein the nucleic acid sequence encoding NeuroD1 polypeptide comprises a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence encoding SEQ ID NO:2 or a functional fragment thereof; a nucleic acid sequence encoding SEQ ID NO:4 or a functional fragment thereof; SEQ ID NO:1 or a functional fragment thereof; SEQ ID NO:3 or a functional fragment thereof; and a nucleic acid sequence encoding a protein which has 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%,
  • Embodiment 9 The method of any one of embodiments 1-8, wherein said administering step comprises a stereotactic injection to the spinal cord.
  • Embodiment 10. The method of any one of embodiments 1-8, wherein said administering step comprises an intravenous injection or intravenous infusion.
  • Embodiment 11. A method of treating a mammal having a spinal cord injury, wherein said method comprises administering a pharmaceutical composition comprising a pharmaceutically acceptable carrier containing adeno-associated virus particles comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof to the spinal cord of said mammal.
  • the pharmaceutical composition comprises about 1 ⁇ L to about 500 ⁇ L of a pharmaceutically acceptable carrier containing adeno-associated virus at a concentration of 10 10 -10 14 adeno-associated virus particles/mL of carrier comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof.
  • Embodiment 13 The method of embodiment 11 or 12, wherein the pharmaceutical composition is injected in the spinal cord of said mammal at a controlled flow rate of about 0.1 ⁇ L/minute to about 5 ⁇ L/minute.
  • Embodiment 14 is injected in the spinal cord of said mammal at a controlled flow rate of about 0.1 ⁇ L/minute to about 5 ⁇ L/minute.
  • a method for treating a mammal having spinal cord injury comprising administering a composition comprising exogenous nucleic acid encoding mir124, exogenous nucleic acid encoding a ISL LIM Homeobox 1 (Isl1) polypeptide or a biologically active fragment thereof, and exogenous nucleic acid encoding a LIM Homeobox 3 (Lhx3) polypeptide or biologically active fragment thereof to the spinal cord of said mammal.
  • Embodiment 15 The method of embodiment 14, wherein said mammal is a human.
  • said administering step comprises delivering (i) an expression vector comprising a nucleic acid encoding mir124, (ii) an expression vector comprising a nucleic acid encoding a Isl1 polypeptide or a biologically active fragment thereof, and (iii) an expression vector comprising a nucleic acid encoding a polypeptide or biologically active fragment thereof Lhx3 to the spinal cord of said mammal.
  • said administering step comprises delivering (i) a recombinant viral expression vector comprising a nucleic acid encoding mir124, (ii) a recombinant viral expression vector comprising a nucleic acid encoding a Isl1 polypeptide or biologically active fragment thereof, and (iii) a recombinant viral expression vector comprising a nucleic acid encoding a Lhx3 polypeptide or a biologically active fragment thereof to the spinal cord of said mammal.
  • a recombinant viral expression vector comprising a nucleic acid encoding mir124
  • a recombinant viral expression vector comprising a nucleic acid encoding a Isl1 polypeptide or biologically active fragment thereof
  • a recombinant viral expression vector comprising a nucleic acid encoding a Lhx3 polypeptide or a biologically active fragment thereof
  • said administering step comprises delivering (i) a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding mir124, (ii) a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding a Isl1 polypeptide or a biologically active fragment thereof, and (iii) a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding a Lhx3 polypeptide or biologically active fragment thereof to the spinal cord of said mammal.
  • a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding mir124
  • a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding a Isl1 polypeptide or a biologically active fragment thereof
  • a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding
  • said administering step comprises delivering an expression vector comprising a nucleic acid encoding mir124, a Isl1 polypeptide or a biologically active fragment thereof, and a Lhx3 polypeptide or a biologically active fragment thereof to the spinal cord of said mammal.
  • said administering step comprises delivering a recombinant viral expression vector comprising a nucleic acid encoding mir124, a Isl1 polypeptide or a biologically active fragment thereof, and a Lhx3 polypeptide or a biologically active fragment thereof to the spinal cord of said mammal.
  • Embodiment 21 Embodiment 21.
  • said administering step comprises delivering a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding mir124, a Isl1 polypeptide or biologically active fragment thereof, and a Lhx3 polypeptide or a biologically active fragment thereof to the spinal cord of said mammal.
  • a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding mir124, a Isl1 polypeptide or biologically active fragment thereof, and a Lhx3 polypeptide or a biologically active fragment thereof to the spinal cord of said mammal.
  • said administering step further comprises administering therapeutically effective doses of one or more of exogenous nucleic acid encoding a Neurogenin 2 (Ngn2) polypeptide or a biologically active fragment thereof, mir218, and a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof in combination with any combination of mir124, a Isl1 polypeptide or a biologically active fragment thereof, or a Lhx3 polypeptide or a biologically active fragment thereof to the spinal cord of said mammal.
  • Ngn2 Neurogenin 2
  • ALS Amyotrophic lateral sclerosis
  • Embodiment 25 The method of embodiment 24, wherein said mammal is a human.
  • said administering step comprises delivering an expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof to the brain.
  • Embodiment 27 Embodiment 27.
  • said administering step comprises delivering a recombinant viral expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof to the brain.
  • said administering step comprises delivering a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof to the brain.
  • Embodiment 29. The method of embodiment 28, wherein the adeno-associated virus is an AAV.PHP.eB.
  • Embodiment 30 is an AAV.PHP.eB.
  • said administering step comprises administering a recombinant expression vector comprising a nucleic acid sequence encoding NeuroD1 protein
  • the nucleic acid sequence encoding NeuroD1 protein comprises a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence encoding SEQ ID NO:2 or a functional fragment thereof; a nucleic acid sequence encoding SEQ ID NO:4 or a functional fragment thereof; SEQ ID NO:1 or a functional fragment thereof; SEQ ID NO:3 or a functional fragment thereof; and a nucleic acid sequence encoding a protein which has 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater, identity to
  • Embodiment 31 The method of any one of embodiments 24-30, wherein said administering step comprises a stereotactic intracranial injection.
  • Embodiment 32 The method of embodiment 31, wherein said administering step comprises two or more stereotactic intracranial injections.
  • Embodiment 33 The method of any one of embodiments 24-30, wherein said administering step comprises a retro-orbital injection.
  • Embodiment 34 A method of treating a mammal having ALS, wherein said method comprises administering a pharmaceutical composition comprising a pharmaceutically acceptable carrier containing adeno-associated virus particles comprising a nucleic acid encoding NeuroD1 to the central nervous system of said mammal.
  • Embodiment 35 A method of treating a mammal having ALS, wherein said method comprises administering a pharmaceutical composition comprising a pharmaceutically acceptable carrier containing adeno-associated virus particles comprising a nucleic acid encoding NeuroD1 to the central nervous system of said mammal.
  • the pharmaceutical composition comprises about 1 ⁇ L to about 500 ⁇ L of a pharmaceutically acceptable carrier containing adeno-associated virus at a concentration of 10 10 -10 14 adeno-associated virus particles/ml of carrier comprising a nucleic acid encoding a NeuroD1 polypeptide.
  • Embodiment 36 The method of embodiment 34 or 35, wherein the pharmaceutical composition is injected in the central nervous system of said mammal at a controlled flow rate of about 0.1 ⁇ L/minute to about 5 ⁇ L/minute.
  • Embodiment 37 Embodiment 37.
  • a method for treating a mammal having Amyotrophic lateral sclerosis comprising administering a composition comprising exogenous nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof, exogenous nucleic acid encoding an Isl1 polypeptide or a biologically active fragment thereof, and exogenous nucleic acid encoding a Lhx3 polypeptide or a biologically active fragment thereof to the central nervous system of said mammal.
  • ALS Amyotrophic lateral sclerosis
  • said administering step comprises delivering (i) an expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof, (ii) an expression vector comprising a nucleic acid encoding a Isl1 polypeptide or a biologically active fragment thereof, and (iii) an expression vector comprising a nucleic acid encoding a Lhx3 polypeptide or a biologically active fragment thereof to the central nervous system of said mammal.
  • Embodiment 40 comprises delivering (i) an expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof, (ii) an expression vector comprising a nucleic acid encoding a Isl1 polypeptide or a biologically active fragment thereof, and (iii) an expression vector comprising a nucleic acid encoding a Lhx3 polypeptide or a biologically active fragment thereof to the central nervous system of said mammal.
  • said administering step comprises delivering (i) a recombinant viral expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof, (ii) a recombinant viral expression vector comprising a nucleic acid encoding a Isl1 polypeptide or a biologically active fragment thereof, and (iii) a recombinant viral expression vector comprising a nucleic acid encoding a Lhx3 polypeptide or a biologically active fragment thereof to the central nervous system of said mammal.
  • a recombinant viral expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof
  • a recombinant viral expression vector comprising a nucleic acid encoding a Isl1 polypeptide or a biologically active fragment thereof
  • a recombinant viral expression vector comprising a nucleic acid encoding a Lhx3
  • said administering step comprises delivering (i) a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof, (ii) a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding a Isl1 polypeptide or a biologically active fragment thereof, and (iii) a recombinant adeno- associated virus expression vector comprising a nucleic acid encoding a Lhx3 polypeptide or a biologically active fragment thereof to the central nervous system of said mammal.
  • a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof
  • a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding a Isl1 polypeptide or a biologically active fragment
  • said administering step comprises delivering an expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof, a Isl1 polypeptide or a biologically active fragment thereof and a Lhx3 polypeptide or a biologically active fragment thereof to the central nervous system of said mammal.
  • an expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof, a Isl1 polypeptide or a biologically active fragment thereof and a Lhx3 polypeptide or a biologically active fragment thereof to the central nervous system of said mammal.
  • said administering step comprises delivering a recombinant viral expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof, a Isl1 polypeptide or a biologically active fragment thereof, and a Lhx3 polypeptide or a biologically active fragment thereof to the central nervous system of said mammal.
  • a recombinant viral expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof, a Isl1 polypeptide or a biologically active fragment thereof, and a Lhx3 polypeptide or a biologically active fragment thereof to the central nervous system of said mammal.
  • said administering step comprises delivering a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof, a Isl1 polypeptide or a biologically active fragment thereof, and a Lhx3 polypeptide or a biologically active fragment thereof to the central nervous system of said mammal.
  • a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof, a Isl1 polypeptide or a biologically active fragment thereof, and a Lhx3 polypeptide or a biologically active fragment thereof to the central nervous system of said mammal.
  • said administering step further comprises administering therapeutically effective doses of one or more of exogenous nucleic acid encoding Ngn2, mir218, and mir124 in combination with any combination of a NeuroD1 polypeptide or a biologically active fragment thereof, a Isl1 polypeptide or a biologically active fragment thereof, and a Lhx3 polypeptide or a biologically active fragment thereof to the central nervous system of said mammal.
  • a NeuroD1 polypeptide or a biologically active fragment thereof a Isl1 polypeptide or a biologically active fragment thereof, and a Lhx3 polypeptide or a biologically active fragment thereof to the central nervous system of said mammal.
  • Embodiment 46 The method of embodiment 41 or embodiment 44, wherein the adeno- associated virus is an AAV.PHP.eB.
  • Embodiment 47 Embodiment 47.
  • said administering step comprises delivering an expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof to the spinal cord.
  • Embodiment 50 The method of embodiment 47, wherein said administering step comprises delivering a recombinant viral expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof to the spinal cord.
  • Embodiment 51 The method of embodiment 47, wherein said administering step comprises delivering a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof to the spinal cord.
  • Embodiment 52 Embodiment 52.
  • adeno-associated virus is an AAV.PHP.eB.
  • Embodiment 53 The method of any one of embodiments 47-52, wherein said administering step comprises administering a recombinant expression vector comprising a nucleic acid sequence encoding NeuroD1 polypeptide, wherein the nucleic acid sequence encoding NeuroD1 polypeptide comprises a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence encoding SEQ ID NO:2 or a functional fragment thereof; a nucleic acid sequence encoding SEQ ID NO:4 or a functional fragment thereof; SEQ ID NO:1 or a functional fragment thereof; SEQ ID NO:3 or a functional fragment thereof; and a nucleic acid sequence encoding a protein which has 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%
  • Embodiment 54 The method of any one of embodiments 47-53, wherein said administering step comprises a stereotactic injection to the spinal cord.
  • Embodiment 55 The method of any one of embodiments 47-54, wherein said administering step comprises an intravenous injection or intravenous infusion.
  • Embodiment 56 The method of any one of embodiments 47-53, wherein said administering step comprises a stereotactic injection to the spinal cord.
  • Embodiment 57 The method of embodiment 56, wherein said mammal is a human.
  • said administering step comprises delivering an expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof to the spinal cord.
  • said administering step comprises delivering a recombinant viral expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof to the spinal cord.
  • said administering step comprises delivering a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof to the spinal cord.
  • Embodiment 61 Embodiment 61.
  • adeno-associated virus is an AAV.PHP.eB.
  • Embodiment 62 The method of any one of embodiments 56-61, wherein said administering step comprises administering a recombinant expression vector comprising a nucleic acid sequence encoding NeuroD1 polypeptide, wherein the nucleic acid sequence encoding NeuroD1 polypeptide comprises a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence encoding SEQ ID NO:2 or a functional fragment thereof; a nucleic acid sequence encoding SEQ ID NO:4 or a functional fragment thereof; SEQ ID NO:1 or a functional fragment thereof; SEQ ID NO:3 or a functional fragment thereof; and a nucleic acid sequence encoding a protein which has 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 8
  • Embodiment 63 The method of any one of embodiments 56-62, wherein said administering step comprises a stereotactic injection to the spinal cord.
  • Embodiment 64 The method of any one of embodiments 56-63, wherein said administering step comprises an intravenous injection or intravenous infusion.
  • Embodiment 65 The method of any one of embodiments 56-62, wherein said administering step comprises a stereotactic injection to the spinal cord.
  • Embodiment 64 The method of any one of embodiments 56-63, wherein said administering step comprises an intravenous injection or intravenous infusion.
  • Embodiment 66 The method of embodiment 65, wherein said mammal is a human.
  • Embodiment 67 The method of embodiment 66, wherein said administering step comprises delivering (i) an expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof, (ii) an expression vector comprising a nucleic acid encoding a Isl1 polypeptide or a biologically active fragment thereof, or (iii) an expression vector comprising a nucleic acid encoding a Lhx3 polypeptide or a biologically active fragment thereof to the central nervous system of said mammal.
  • Embodiment 68 Embodiment 68.
  • said administering step comprises delivering (i) a recombinant viral expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof, (ii) a recombinant viral expression vector comprising a nucleic acid encoding a Isl1 polypeptide or a biologically active fragment thereof, or (iii) a recombinant viral expression vector comprising a nucleic acid encoding a Lhx3 polypeptide or a biologically active fragment thereof to the central nervous system of said mammal.
  • a recombinant viral expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof
  • a recombinant viral expression vector comprising a nucleic acid encoding a Isl1 polypeptide or a biologically active fragment thereof
  • a recombinant viral expression vector comprising a nucleic acid encoding a Lhx3
  • invention 66 wherein said administering step comprises delivering (i) a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof, (ii) a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding a Isl1 polypeptide or a biologically active fragment thereof, or (iii) a recombinant adeno- associated virus expression vector comprising a nucleic acid encoding a Lhx3 polypeptide or a biologically active fragment thereof to the central nervous system of said mammal.
  • Embodiment 70 Embodiment 70.
  • adeno-associated virus is an AAV.PHP.eB.
  • Embodiment 71 The method of any one of embodiments 65-70, wherein said administering step comprises a stereotactic injection to the spinal cord.
  • Embodiment 72 The method of any one of embodiments 65-71, wherein said administering step comprises an intravenous injection or intravenous infusion.
  • Embodiment 73 The method of any one of embodiments 65-71, wherein said administering step comprises an intravenous injection or intravenous infusion.
  • a method for treating a mammal having spinal cord injury comprises administering a composition comprising (a) exogenous nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof and (b) exogenous nucleic acid encoding a Distal-Less Homeobox 2 (Dlx2) polypeptide or biologically active fragment thereof to the spinal cord of said mammal.
  • a composition comprising (a) exogenous nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof and (b) exogenous nucleic acid encoding a Distal-Less Homeobox 2 (Dlx2) polypeptide or biologically active fragment thereof to the spinal cord of said mammal.
  • Dlx2 Distal-Less Homeobox 2
  • said administering step comprises delivering (i) an expression vector comprising a nucleic acid a NeuroD1 polypeptide and (ii) an expression vector comprising a nucleic acid encoding a Dlx2 polypeptide or a biologically active fragment thereof to the spinal cord of said mammal.
  • said administering step comprises delivering (i) a recombinant viral expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide and (ii) a recombinant viral expression vector comprising a nucleic acid encoding a Dlx2 polypeptide or biologically active fragment thereof to the spinal cord of said mammal.
  • Embodiment 77 The method of embodiment 73, wherein said administering step comprises delivering (i) a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide and (ii) a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding a Dlx2 polypeptide or a biologically active fragment thereof to the spinal cord of said mammal.
  • Embodiment 78 Embodiment 78.
  • said administering step comprises delivering an expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof and a Dlx2 polypeptide or a biologically active fragment thereof to the spinal cord of said mammal.
  • said administering step comprises delivering a recombinant viral expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof and a Dlx2 polypeptide or a biologically active fragment thereof to the spinal cord of said mammal.
  • Embodiment 80 Embodiment 80.
  • said administering step comprises delivering a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or biologically active fragment thereof and a Dlx2 polypeptide or a biologically active fragment thereof to the spinal cord of said mammal.
  • adeno-associated virus is an AAV.PHP.eB.
  • Embodiment 82 The method of embodiments 77 or 80, wherein the adeno-associated virus is an AAV.PHP.eB.
  • said administering step comprises administering a recombinant expression vector comprising a nucleic acid sequence encoding NeuroD1 polypeptide
  • the nucleic acid sequence encoding NeuroD1 polypeptide comprises a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence encoding SEQ ID NO:2 or a functional fragment thereof; a nucleic acid sequence encoding SEQ ID NO:4 or a functional fragment thereof; SEQ ID NO:1 or a functional fragment thereof; SEQ ID NO:3 or a functional fragment thereof; and a nucleic acid sequence encoding a protein which has 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%
  • Embodiment 83 The method of any one of embodiments 73-82, wherein said administering step comprises administering a recombinant expression vector comprising a nucleic acid sequence encoding Dlx2 polypeptide, wherein the nucleic acid sequence encoding Dlx2 polypeptide comprises a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence encoding SEQ ID NO: 11 or a functional fragment thereof; a nucleic acid sequence encoding SEQ ID NO:13 or a functional fragment thereof; SEQ ID NO:10 or a functional fragment thereof; SEQ ID NO:12 or a functional fragment thereof; and a nucleic acid sequence encoding a protein which has 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%
  • Embodiment 84 A method for (1) regenerating dorsal spinal cord neurons, (2) generating new neurons, or (3) increasing circulation in the spinal cord within a mammal having a SCI and in need of said (1), (2), or (3), wherein said method comprises administering a composition comprising (i) exogenous nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof and (ii) exogenous nucleic acid encoding a Dlx2 polypeptide or a biologically active fragment thereof, wherein (a) said spinal cord neurons are regenerated, (b) new neurons are generated, or (c) spinal cord circulation is increased.
  • Embodiment 85 The method of embodiment 84, wherein said mammal is a human.
  • Embodiment 86 The method of embodiment 84, wherein said administering step comprises delivering (i) an expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof and (ii) an expression vector comprising a nucleic acid encoding a Dlx2 polypeptide or a biologically active fragment thereof to the central nervous system of said mammal.
  • Embodiment 87 Embodiment 87.
  • invention 84 wherein said administering step comprises delivering (i) a recombinant viral expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof and (ii) a recombinant viral expression vector comprising a nucleic acid encoding a Dlx2 polypeptide or a biologically active fragment thereof to the central nervous system of said mammal.
  • a recombinant viral expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof
  • a recombinant viral expression vector comprising a nucleic acid encoding a Dlx2 polypeptide or a biologically active fragment thereof
  • invention 84 wherein said administering step comprises delivering (i) a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof and (ii) a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding a Dlx2 polypeptide or a biologically active fragment thereof to the central nervous system of said mammal.
  • a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof
  • a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding a Dlx2 polypeptide or a biologically active fragment thereof
  • said administering step comprises delivering an expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof and a Dlx2 polypeptide or a biologically active fragment thereof to the spinal cord of said mammal.
  • said administering step comprises delivering a recombinant viral expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof and a Dlx2 polypeptide or a biologically active fragment thereof to the spinal cord of said mammal.
  • Embodiment 91 Embodiment 91.
  • said administering step comprises delivering a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or biologically active fragment thereof and a Dlx2 polypeptide or a biologically active fragment thereof to the spinal cord of said mammal.
  • adeno-associated virus is an AAV.PHP.eB.
  • Embodiment 93 The method of any one of embodiments 73-92, wherein said administering step comprises a stereotactic injection to the spinal cord.
  • Embodiment 95 The method of embodiment 84, wherein said new neurons are selected from the group consisting of glutamatergic neurons and GABAergic neurons.
  • Embodiment 99 The method of embodiments 88 or 91, wherein the adeno-associated virus is an AAV serotype 5.
  • Embodiment 100 A method for treating a mammal having ALS, wherein said method comprises administering a composition comprising (a) exogenous nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof and (b) exogenous nucleic acid encoding an Isl1 polypeptide or biologically active fragment thereof to said mammal.
  • Embodiment 101 The method of embodiment 100, wherein said mammal is a human.
  • Embodiment 102 The method of embodiment 100, wherein said mammal is a human.
  • said administering step comprises delivering (i) an expression vector comprising a nucleic acid a NeuroD1 polypeptide and (ii) an expression vector comprising a nucleic acid encoding an Isl1 polypeptide or a biologically active fragment thereof to said mammal.
  • said administering step comprises delivering (i) a recombinant viral expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide and (ii) a recombinant viral expression vector comprising a nucleic acid encoding an Isl1 polypeptide or biologically active fragment thereof to said mammal.
  • Embodiment 104 Embodiment 104.
  • said administering step comprises delivering (i) a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide and (ii) a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding an Isl1 polypeptide or a biologically active fragment thereof to the spinal cord of said mammal.
  • said administering step comprises delivering an expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof and an Isl1 polypeptide or a biologically active fragment thereof to said mammal.
  • Embodiment 106 The method of embodiment 100, wherein said administering step comprises delivering a recombinant viral expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof and an Isl1 polypeptide or a biologically active fragment thereof to said mammal.
  • Embodiment 107 The method of embodiment 100, wherein said administering step comprises delivering a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or biologically active fragment thereof and an Isl1 polypeptide or a biologically active fragment thereof to said mammal.
  • Embodiment 108 Embodiment 108.
  • adeno- associated virus is an AAV.PHP.eB.
  • Embodiment 109 The method of any one of embodiments 100-108, wherein said administering step comprises administering a recombinant expression vector comprising a nucleic acid sequence encoding NeuroD1 polypeptide, wherein the nucleic acid sequence encoding NeuroD1 polypeptide comprises a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence encoding SEQ ID NO:2 or a functional fragment thereof; a nucleic acid sequence encoding SEQ ID NO:4 or a functional fragment thereof; SEQ ID NO:1 or a functional fragment thereof; SEQ ID NO:3 or a functional fragment thereof; and a nucleic acid sequence encoding a protein which has 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 81%, 82%, 83%, 84%, 85%, 86%
  • Embodiment 110 The method of any one of embodiments 100-109, wherein said administering step comprises administering a recombinant expression vector comprising a nucleic acid sequence encoding an Isl1 polypeptide, wherein the nucleic acid sequence encoding an Isl1 polypeptide comprises a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence encoding SEQ ID NO: 15 or a functional fragment thereof; a nucleic acid sequence encoding SEQ ID NO:17 or a functional fragment thereof; SEQ ID NO:14 or a functional fragment thereof; SEQ ID NO:16 or a functional fragment thereof; and a nucleic acid sequence encoding a protein which has 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%
  • Embodiment 111 A method for treating a mammal having spinal cord injury, wherein said method comprises administering a composition comprising (a) exogenous nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof and (b) exogenous nucleic acid encoding an Isl1 polypeptide or biologically active fragment thereof to the spinal cord of said mammal.
  • Embodiment 112. The method of embodiment 111, wherein said mammal is a human.
  • Embodiment 113 The method of embodiment 111, wherein said mammal is a human.
  • said administering step comprises delivering (i) an expression vector comprising a nucleic acid a NeuroD1 polypeptide and (ii) an expression vector comprising a nucleic acid encoding an Isl1 polypeptide or a biologically active fragment thereof to the spinal cord of said mammal.
  • said administering step comprises delivering (i) a recombinant viral expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide and (ii) a recombinant viral expression vector comprising a nucleic acid encoding an Isl1 polypeptide or biologically active fragment thereof to the spinal cord of said mammal.
  • Embodiment 115 The method of embodiment 111, wherein said administering step comprises delivering (i) a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide and (ii) a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding an Isl1 polypeptide or a biologically active fragment thereof to the spinal cord of said mammal.
  • Embodiment 116 Embodiment 116.
  • said administering step comprises delivering an expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof and an Isl1 polypeptide or a biologically active fragment thereof to the spinal cord of said mammal.
  • said administering step comprises delivering a recombinant viral expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or a biologically active fragment thereof and an Isl1 polypeptide or a biologically active fragment thereof to the spinal cord of said mammal.
  • Embodiment 118 Embodiment 118.
  • invention 111 wherein said administering step comprises delivering a recombinant adeno-associated virus expression vector comprising a nucleic acid encoding a NeuroD1 polypeptide or biologically active fragment thereof and an Isl1 polypeptide or a biologically active fragment thereof to the spinal cord of said mammal.
  • adeno-associated virus is an AAV.PHP.eB.
  • Embodiment 120 The method of embodiments 115 or 118, wherein the adeno-associated virus is an AAV.PHP.eB.
  • said administering step comprises administering a recombinant expression vector comprising a nucleic acid sequence encoding NeuroD1 polypeptide
  • the nucleic acid sequence encoding NeuroD1 polypeptide comprises a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence encoding SEQ ID NO:2 or a functional fragment thereof; a nucleic acid sequence encoding SEQ ID NO:4 or a functional fragment thereof; SEQ ID NO:1 or a functional fragment thereof; SEQ ID NO:3 or a functional fragment thereof; and a nucleic acid sequence encoding a protein which has 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%
  • Embodiment 121 The method of any one of embodiments 111-120, wherein said administering step comprises administering a recombinant expression vector comprising a nucleic acid sequence encoding an Isl1 polypeptide, wherein the nucleic acid sequence encoding an Isl1 polypeptide comprises a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence encoding SEQ ID NO: 15 or a functional fragment thereof; a nucleic acid sequence encoding SEQ ID NO:17 or a functional fragment thereof; SEQ ID NO:14 or a functional fragment thereof; SEQ ID NO:16 or a functional fragment thereof; and a nucleic acid sequence encoding a protein which has 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,

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EP20875773.2A EP4045148A4 (en) 2019-10-17 2020-10-16 REGENERATION OF FUNCTIONAL NEURONS FOR THE TREATMENT OF SPINAL CORD INJURY AND ALS
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