WO2024046393A1 - 非神经元细胞转分化为神经元的方法及应用 - Google Patents

非神经元细胞转分化为神经元的方法及应用 Download PDF

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WO2024046393A1
WO2024046393A1 PCT/CN2023/115930 CN2023115930W WO2024046393A1 WO 2024046393 A1 WO2024046393 A1 WO 2024046393A1 CN 2023115930 W CN2023115930 W CN 2023115930W WO 2024046393 A1 WO2024046393 A1 WO 2024046393A1
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cells
neurons
gene
aav
neuronal
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PCT/CN2023/115930
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English (en)
French (fr)
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周海波
胡新德
苏锦霖
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上海鲸奇生物科技有限公司
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    • 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
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • 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

Definitions

  • the present application relates to the field of translational medicine, and more specifically, to a method for transdifferentiating non-neuronal cells into neurons, and the use of this method for preparing drugs for the treatment or prevention of diseases related to neuronal damage or neuron death.
  • the main pathological changes caused by nervous system damage and various neurodegenerative diseases are neuronal degeneration, necrosis and neuronal damage. Since the self-repair ability of the nervous system (such as the brain, spinal cord, optic nerve, etc.) is very limited, it is difficult to repair neuron cells autonomously. Diseases related to neuronal function loss or neuron death have always been difficult to treat, such as Parkinson's. disease, schizophrenia, depression, Alzheimer's disease, Huntington's disease, sleep disorders, brain trauma, stroke, visual system diseases related to loss or death of RGC or photoreceptor cell function, blindness, deafness, etc., the current treatment methods are only It is to alleviate the progression of the disease, but there has been a lack of effective treatments.
  • Cell transdifferentiation refers to the process by which one type of differentiated cells can structurally and functionally transform into another type of differentiated cells under certain conditions. If some non-neuronal cells in the nervous system can be reprogrammed into neuronal cells, it will be expected to fundamentally treat diseases related to neuronal function loss or neuron death.
  • Another object of the present disclosure is to provide one or more inhibitors of the expression or activity of genes or their RNAs or their encoded proteins selected from the group consisting of for preventing and/or treating neuronal function loss or death associated with Disease uses: RCOR1, RCOR2, RCOR3, Sin3a, Sin3b, HDAC1, HDAC2, KDM1A, PHF21A, BAF53a, G9a, USP14, HuR, BrG1, EZH2, CDYL, HMG20B.
  • Another object of the present disclosure is to provide one or more enhancers of the expression or activity of genes or their RNAs or their encoded proteins selected from the group consisting of for preventing and/or treating neuronal function loss or death associated with Disease uses: DPYSL2, BAF45b, SCF, HuB, HuC, HuD, CYP1B1, BTRC.
  • the present disclosure provides a method of generating neuronal cells from glial cells, comprising using One or more inhibitors of the expression or activity of genes or their RNA or their encoded proteins transdifferentiate or reprogram the glial cells into neurons selected from: RCOR1, RCOR2, RCOR3, Sin3a, Sin3b , HDAC1, HDAC2, KDM1A, PHF21A, BAF53a, G9a, USP14, HuR, BrG1, EZH2, CDYL, HMG20B, wherein the inhibitor reduces the expression or activity of the gene or its RNA or its encoded protein, and the method includes :
  • the present disclosure provides a method of generating neuronal cells from glial cells, comprising using one or more enhancers of the expression or activity of a gene or its RNA or its encoded protein selected from: Transdifferentiating or reprogramming the glial cells into neurons: DPYSL2, BAF45b, SCF, HuB, HuC, HuD, CYP1B1, BTRC, wherein the enhancer increases the expression of the gene or its RNA or its encoded protein Or activity, the method includes:
  • the glial cells are mammalian glial cells, including glial cells of human, non-human primate, mouse, and rat species.
  • the mammalian glial cells include astrocytes, oligodendrocytes, microglia, NG2 cells, Müller glia, glioma cells or spiral nerves Glial cells.
  • the glial cells are astrocytes or Müller glia.
  • the astrocytes are derived from the brain, midbrain, cerebellum, brainstem, and spinal cord.
  • the astrocytes are derived from the striatum or substantia nigra.
  • the Müller glia cells are derived from the retina.
  • the spiral ganglion glial cells are derived from the inner ear or vestibule.
  • the neuronal cells are mammalian neurons, including neurons of humans, non-human primates, rats, and mice.
  • neuronal cells are selected from dopamine neurons, 5-HT neurons, NE neurons, ChAT neurons, motor neurons, GABA neurons, glutamatergic neurons, Spinal cord neurons, spinal motor neurons, spinal sensory neurons, photoreceptor cells (rods and cones), bipolar cells, amacrine cells, Retinal ganglion cells (RGC), cochlear nerve cells, pyramidal neurons, interneurons, medium spiny neurons (MSN), Purkinje cells, granule cells, olfactory sensory neurons, periglomerular cells or combinations thereof, more preferably dopamine neurons, retinal ganglion cells and photoreceptor cells.
  • dopamine neurons 5-HT neurons, NE neurons, ChAT neurons, motor neurons, GABA neurons, glutamatergic neurons, Spinal cord neurons, spinal motor neurons, spinal sensory neurons, photoreceptor cells (rods and cones), bipolar cells, amacrine cells, Retinal ganglion cells (RGC), cochlear nerve cells, pyramidal neurons, interneurons, medium spiny neurons (MSN
  • said glial cells are astrocytes; and said neuronal cells are dopamine neurons.
  • said glial cells are Müller glia; and said neuronal cells are RGCs or photoreceptor cells.
  • the present disclosure provides the use of one or more inhibitors of the expression or activity of genes or their RNA or their encoded proteins selected from the group consisting of: RCOR1, RCOR2, RCOR3, Sin3a, Sin3b, HDAC1, HDAC2, KDM1A, PHF21A, BAF53a, G9a, USP14, HuR, BrG1, EZH2, CDYL, HMG20B, the medicine is used to prevent and/or treat diseases related to neuronal function loss or death, wherein The inhibitor reduces the expression or activity of the gene or its RNA or its encoded protein.
  • the present disclosure provides the use of one or more enhancers of the expression or activity of genes or their RNA or their encoded proteins selected from the group consisting of: DPYSL2, BAF45b, SCF, HuB, HuC, HuD, CYP1B1, BTRC, the medicine is used to prevent and/or treat diseases related to neuronal function loss or death, wherein the enhancer increases the expression of the gene or its RNA or its encoded protein or active.
  • the medicament is formulated for in vivo administration to the nervous system, visual system, and auditory system, including the striatum, substantia nigra, midbrain ventral tegmental area, spinal cord, hypothalamus, dorsal Lateral midbrain, cerebral cortex, hippocampus, cerebellum, subretinal, vitreous cavity, inner ear cochlea and vestibule.
  • the medicament is formulated for administration to the striatum, substantia nigra, subretina and vitreous cavity.
  • the disease associated with neuronal function loss or death is a neurological disease, including Parkinson's disease, schizophrenia, depression, Alzheimer's disease, Huntington's disease, epilepsy, sleep disorders disorders, ataxia, PloyQ disease, cerebral ischemia, brain injury, addiction, motor neuron disease, amyotrophic lateral sclerosis, spinal muscular atrophy, Pick's disease, associated with loss of function or death of RGC or photoreceptor cells visual system diseases, blindness, and deafness.
  • a neurological disease including Parkinson's disease, schizophrenia, depression, Alzheimer's disease, Huntington's disease, epilepsy, sleep disorders disorders, ataxia, PloyQ disease, cerebral ischemia, brain injury, addiction, motor neuron disease, amyotrophic lateral sclerosis, spinal muscular atrophy, Pick's disease, associated with loss of function or death of RGC or photoreceptor cells visual system diseases, blindness, and deafness.
  • the diseases related to neuron function loss or death are Parkinson's disease and visual system diseases related to RGC or photoreceptor cell function loss or death.
  • the present disclosure provides a method of generating retinal ganglion cells (RGC) or photoreceptor cells from Müller glia, comprising using one or more genes or RNA thereof selected from or Inhibitors of the expression or activity of the encoded proteins enable the Müller glia to transdifferentiate or reprogram into RGCs or photoreceptor cells: RCOR1, RCOR2, RCOR3, Sin3a, Sin3b, HDAC1, HDAC2, KDM1A, PHF21A, BAF53a, G9a, USP14, HuR, BrG1, EZH2, CDYL, HMG20B, wherein the inhibitor reduces the gene or its RNA or its encoded protein White expression or activity.
  • RGC retinal ganglion cells
  • the present disclosure provides a method of generating retinal ganglion cells (RGC) or photoreceptor cells from Müller glia, comprising using one or more genes or RNA thereof selected from or The enhancer of the expression or activity of its encoded protein causes the Müller glial cells to transdifferentiate or reprogram into RGCs or photoreceptor cells: DPYSL2, BAF45b, SCF, HuB, HuC, HuD, CYP1B1, BTRC, wherein the enhancer The agent increases the expression or activity of the gene or its RNA or its encoded protein.
  • RGC retinal ganglion cells
  • the enhancer of the expression or activity of its encoded protein causes the Müller glial cells to transdifferentiate or reprogram into RGCs or photoreceptor cells: DPYSL2, BAF45b, SCF, HuB, HuC, HuD, CYP1B1, BTRC, wherein the enhancer The agent increases the expression or activity of the gene or its RNA or its encoded protein.
  • the Müller glia cells are derived from the retina.
  • the photoreceptor cells include rods and cones.
  • the present disclosure provides the use of one or more inhibitors of the expression or activity of genes or their RNA or their encoded proteins selected from the group consisting of: RCOR1, RCOR2, RCOR3, Sin3a in the preparation of a medicament.
  • the drug is used to prevent and/or treat the visual system related to RGC or photoreceptor cell function loss or death Diseases wherein the inhibitor reduces the expression or activity of the gene or its RNA or its encoded protein.
  • the present disclosure provides the use of one or more enhancers of the expression or activity of genes or their RNA or their encoded proteins selected from the group consisting of: DPYSL2, BAF45b, SCF, HuB, HuC, HuD, CYP1B1, BTRC, the drug is used to prevent and/or treat visual system diseases related to RGC or photoreceptor cell function loss or death, wherein the enhancer increases the gene or its RNA or its coding protein expression or activity.
  • the medicament is formulated for administration to the visual system.
  • the drug is formulated for subretinal or intravitreal use, wherein the drug acts by acting on Müller glia.
  • the neurological disease related to RGC function loss or death is selected from: vision impairment caused by RGC cell death, glaucoma, age-related RGC lesions, diabetes-related retinopathy, optic nerve damage, retinal defects Blood or hemorrhage, Leber hereditary optic neuropathy, or combinations thereof.
  • the visual system disease related to photoreceptor cell function loss or death is selected from: photoreceptor cell degeneration or death caused by injury or degenerative disease, macular degeneration, retinitis pigmentosa, diabetes-related blindness, Night blindness, color blindness, hereditary blindness, congenital amaurosis, or combinations thereof.
  • the inhibitor or agonist is selected from: antibodies, small molecule compounds, mRNA, microRNA, siRNA, shRNA, antisense oligonucleotides, binding proteins or protein domains, polypeptides, nucleic acid adapters Ligands, gene editors, PROTACs, expression vectors containing promoters, endogenous expression activators, protein analogs or enhancers, synthetic or modified inhibitors or enhancers mentioned above, or combinations thereof.
  • the present disclosure provides a pharmaceutical composition or kit or kit comprising one or more inhibitors of the expression or activity of a gene or its RNA or its encoded protein selected from : RCOR1, RCOR2, RCOR3, Sin3a, Sin3b, HDAC1, HDAC2, KDM1A, PHF21A, BAF53a, G9a, USP14, HuR, BrG1, EZH2, CDYL, HMG20B; and/or one or more genes selected from the following or their RNA or their encoding Enhancers of protein expression or activity: DPYSL2, BAF45b, SCF, HuB, HuC, HuD, CYP1B1, BTRC.
  • a gene or its RNA or its encoded protein selected from : RCOR1, RCOR2, RCOR3, Sin3a, Sin3b, HDAC1, HDAC2, KDM1A, PHF21A, BAF53a, G9a, USP14, HuR, BrG1, EZH2, CDYL, HMG20B
  • the inhibitor comprises:
  • gRNAs or expression vectors thereof wherein the gRNA is DNA or RNA that guides the gene editing protein to specifically bind to the gene.
  • the editing system includes: CRISPR system (including CRISPR/dCas system), ZFN system, TALEN system, RNA editing system, or a combination thereof.
  • the gene editing protein is an RNA-targeting gene editing protein.
  • the gRNA is an RNA-targeting gRNA.
  • said enhancer comprises: an expression vector containing a promoter, an endogenous expression activator, a protein analog or an enhancer.
  • the pharmaceutical composition or kit further comprises a vehicle for delivering the inhibitor.
  • the carrier is a viral vector, liposome, nanoparticle, exosome, virus-like particle, preferably AAV.
  • the RNA-targeting gene editing protein is selected from the group consisting of: Cas13d, Cas13e, Cas13a, Cas13b, Cas13c, Cas13f and their functional domains.
  • the RNA-targeting gene editing protein is selected from the group consisting of: CasRx, Cas13e, and Cas13f.
  • RNA-targeting gene editing protein is CasRx.
  • said pharmaceutical composition or kit or kit contains only a single type of gRNA targeting said mRNA sequence or 2, 3, 4, 5, 6, 7, 8, 9, 10 , 11, 12 different gRNAs.
  • the gRNA expression vector encodes a gRNA comprising only a single type of gRNA targeting the mRNA sequence or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 different gRNAs.
  • the expression vector contains:
  • nucleotide sequence encoding the gene editing protein operably linked to a promoter causing expression of the gene editing protein
  • the promoter is a broad spectrum promoter or a specific promoter.
  • the broad-spectrum promoter is selected from the group consisting of CMV, CBH, CAG, PGK, SV40, EFlA, EFS, and pGlobin promoters.
  • the specific promoter is a glial cell-specific promoter or a Müller glial (MG) cell-specific promoter.
  • the glial cell-specific promoter is selected from the group consisting of GFAP promoter, ALDH1L1 promoter, EAAT1/GLAST promoter, glutamine synthetase promoter, S100 ⁇ promoter and EAAT2/GLT- 1 promoter, NG2 promoter, CD68 promoter, F4/80 promoter, or the MG cell-specific promoter is selected from the group consisting of GFAP promoter, ALDH1L1 promoter, Glast (also known as Slc1a3) promoter and Rlbp1 promoter .
  • the expression vector is contained in a viral particle.
  • the expression vector is a gene therapy vector.
  • the gene therapy vector is a viral gene therapy vector.
  • the expression vector is a viral vector selected from the group consisting of: adeno-associated virus (AAV) vector, recombinant adeno-associated virus vector (rAAV), self-complementing adeno-associated virus vector (scAAV), adenovirus Vectors, lentiviral vectors, retroviral vectors, herpesvirus vectors, SV40 vectors, poxvirus vectors, and combinations thereof.
  • AAV adeno-associated virus
  • rAAV recombinant adeno-associated virus vector
  • scAAV self-complementing adeno-associated virus vector
  • adenovirus Vectors lentiviral vectors, retroviral vectors, herpesvirus vectors, SV40 vectors, poxvirus vectors, and combinations thereof.
  • the expression vector is an AAV vector or rAAV.
  • the composition is topically applied to at least one of: i) glial cells in the retina; ii) glial cells in the striatum, preferably in the putamen; iii ) Glial cells in the substantia nigra; iv) Glial cells in the inner ear; v) Glial cells in the spinal cord; vi) Glial cells in the prefrontal cortex; vii) Glial cells in the motor cortex; viii) Thalamus glial cells in; ix) glial cells in the ventral tegmental area (VTA); x) glial cells in the hippocampus; xi) glial cells in the cerebellum; and xii) glial cells in the brainstem Glial cells.
  • said composition further comprises i) one or more neuron-related factors, or ii) for expressing one or more neuron-related factors in said glial cells at least one expression vector.
  • the one or more neuron-related factors are selected from: AscL1, Mytl1, Ngn1, Ngn2, NeuroD1, NeuroD2, NeuroG1, Pax6, Ptbp1, P53, Zicl, Ctds1, miR- 9.miR-9-9*,miR-124,miR-124-124*,Let-7,Let-7b,miR-132,NeuroG2,Brn2,NeuroD4,Insm1,Prox1,FoxG1,Lhx6,Bcl2,Dlx1, Dlx2, Tlx3, Gata2, Gata3, Sox11, Lhx3, IsL1, etc.
  • said pharmaceutical composition or kit or kit further comprises i) one or more dopamine neuron-associated factors, or ii) for expressing in said glial cells a or at least one expression vector of multiple dopamine neuron-related factors.
  • the composition is further formulated for cell transfection, cell infection, endocytosis, Injection, intracranial administration, spinal administration, intraocular administration, intraaural administration, inhalation, parenteral administration, intravenous administration, intramuscular administration, intradermal administration, topical administration or oral administration, and ex vivo induction Transdifferentiate or reprogram and transplant the transdifferentiated or reprogrammed cells back into the body.
  • the AAV vector contains:
  • the promoter is a broad spectrum promoter or a specific promoter.
  • the broad-spectrum promoter is selected from the group consisting of CMV, CBH, CAG, PGK, SV40, EFlA, EFS, and pGlobin promoters.
  • the specific promoter is a glial cell-specific promoter or a Müller glial (MG) cell-specific promoter.
  • the glial cell-specific promoter is selected from the group consisting of GFAP promoter, ALDH1L1 promoter, EAAT1/GLAST promoter, glutamine synthetase promoter, S100 ⁇ promoter and EAAT2/GLT- 1 promoter, NG2 promoter, CD68 promoter, F4/80 promoter.
  • the MG cell-specific promoter is selected from the group consisting of GFAP promoter, ALDH1L1 promoter, Glast (also known as Slcla3) promoter and Rlbp1 promoter.
  • the transdifferentiation efficiency of glial cells is at least 0.1%, or at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26% ,27%,28%,29%,30%,31%,32%,33%,34%,35%,36%,37%,38%,39%,40%,41%,42%,43 %, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, or higher.
  • the disease associated with neuronal function loss or death is selected from: Parkinson's disease, schizophrenia, depression, vision impairment caused by RGC cell death, diabetes-related retinopathy, glaucoma, age Related RGC lesions, optic nerve damage, retinal ischemia or hemorrhage, Leber hereditary optic neuropathy, photoreceptor cell degeneration or death caused by injury or degeneration, macular degeneration, retinitis pigmentosa, diabetes-related blindness, night blindness, color blindness, genetics Sexual blindness, congenital amaurosis, or combinations thereof.
  • Parkinson's disease schizophrenia, depression, vision impairment caused by RGC cell death, diabetes-related retinopathy, glaucoma, age Related RGC lesions, optic nerve damage, retinal ischemia or hemorrhage, Leber hereditary optic neuropathy, photoreceptor cell degeneration or death caused by injury or degeneration, macular degeneration, retinitis pigmentosa, diabetes-related blindness, night blindness, color blindness, genetics
  • the RGCs can be integrated into the visual pathway and improve visual function.
  • the RGC can achieve functional projection to the central visual area and improve visual function.
  • said improving visual function is improving visual function in a mammal suffering from a retinal disease caused by neurodegeneration.
  • the MG cells are transdifferentiated into RGC cells, they are also differentiated into axonless cells.
  • a plurality of glial cells in the striatum are reprogrammed or transdifferentiated, and at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23% , 24%, 25%, 26%, 27%, 28%, 29%, or at least 30% of glial cells were converted into dopamine neurons.
  • the mammal includes a mammal suffering from a disease associated with loss of neuronal function or death.
  • the mammal includes a human or non-human mammal.
  • the non-human mammal includes rodents (such as mice, rats, or rabbits), primates (such as monkeys).
  • the gene editor is driven by a glial cell-specific promoter (eg, the GFAP promoter).
  • a glial cell-specific promoter eg, the GFAP promoter
  • the gene editor includes one or more gRNA and gene editing proteins.
  • the gRNA guides the gene editing protein to specifically bind to the RNA of the gene.
  • the gRNA guides the gene editing protein to specifically bind to the mRNA of the gene.
  • nucleotide sequence of the gRNA is shown in Table 1 below.
  • u is used interchangeably with the letter “t” in the context of RNA sequences to represent uridine or uridylic acid.
  • the source of the gene editing protein is selected from: Streptococcus pyogenes, Staphylococcus aureus, Acidaminococcus sp, Lachnospiraceae bacterium, Ruminococcus flavefaciens, or combinations thereof.
  • the gene or its RNA or its encoded protein is derived from a mammal; preferably, derived from a human, monkey, mouse, rat, or rabbit; more preferably, derived from a human.
  • the genes include wild-type genes and mutant genes.
  • the mutant type includes a mutant form in which the function of the encoded protein is not changed after mutation (ie, the function is the same or substantially the same as that of the wild-type encoded protein).
  • polypeptide encoded by the mutant gene is the same or substantially the same as the polypeptide encoded by the wild-type gene.
  • the mutant gene includes a homology of ⁇ 80% (preferably ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93% or ⁇ 94%, more Preferably ⁇ 95%, ⁇ 96% or ⁇ 97%, more preferably ⁇ 98% or 99%) polynucleotides.
  • the mutant gene includes truncating or adding 1-60 (preferably 1-30, more preferably 1-10) at the 5' end and/or 3' end of the wild-type gene. ) nucleotide polynucleotide.
  • the gene includes a cDNA sequence, a genomic sequence, or a combination thereof.
  • the protein includes active fragments or derivatives thereof.
  • the homology of the active fragment or derivative thereof to the gene or its RNA or its encoded protein is at least 90%, 91%, 92%, 93% or 94%, preferably 95%, 96% or 97%, more preferably 98%, 99%.
  • the active fragment or derivative thereof has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% activity.
  • amino acid sequence of the protein is selected from:
  • nucleotide sequence of said gene is selected from:
  • nucleotide sequence and SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 The homology of the nucleotide sequences shown in 40, 42, 44, 46, 48 and 50 is ⁇ 90%, ⁇ 91%, ⁇ 92%, ⁇ 93% or ⁇ 94% (preferably ⁇ 95%, ⁇ 96% or ⁇ 97%, more preferably ⁇ 98% or 99%) of the polynucleotide;
  • the protein is as shown in the above amino acid sequence.
  • nucleic acid encoding the protein is as shown in the above nucleotide sequence.
  • the region targeted by the inhibitor is positions 121931-121963 (RCOR1), 2364-2393 (RCOR2), and 53556-53585 of the gene sequence. bits (RCOR3), bits 59338-59367 (Sin3a), bits 12378-12406 (Sin3b), bits 24648-24678 (HDAC1), and bits 60126-60154 (KDM1A).
  • the inhibitor or enhancer inhibits or enhances the activity and/or expression of the gene or its RNA or its encoded protein.
  • the concentration of the inhibitor or enhancer is >1 ⁇ 10 12 .
  • the inhibition rate or enhancement rate of the inhibitor or enhancer on the activity and/or expression of the gene or its RNA or its encoded protein is greater than 90%, preferably, 90%-95 %.
  • the inhibitor or enhancer targets astrocytes of brain tissue.
  • the inhibitor or enhancer targets MG cells of the retina.
  • the gRNA guides the gene editing protein to specifically bind to the mRNA of the gene.
  • the composition includes a pharmaceutical composition.
  • the composition also includes other drugs for preventing and/or treating diseases associated with loss of neuronal function or death.
  • the composition further includes other drugs for the treatment of neurological diseases associated with neuronal death.
  • the composition also includes other drugs for the prevention and/or treatment of retinal diseases.
  • the expression vector of the gene editing protein includes a vector targeting glial cells.
  • the expression vector of the gene editing protein includes a vector targeting astrocytes in brain tissue.
  • the expression vector of the gene editing protein includes a vector targeting retinal MG cells.
  • the vector includes AAV2 or AAV9.
  • the gene encoding the gene editing protein and the gRNA are located in the same expression vector (such as an AAV vector).
  • the expression vector of the gene editing protein and the expression vector of gRNA are the same expression vector (such as AAV vector).
  • the expression vector further includes a glial cell-specific promoter (eg, GFAP promoter) for driving the expression of the gene editing protein.
  • a glial cell-specific promoter eg, GFAP promoter
  • the dosage form of the composition is selected from: lyophilized formulations, liquid formulations, or combinations thereof.
  • the dosage form of the composition is a liquid formulation.
  • the dosage form of the composition is an injectable dosage form.
  • other drugs for preventing and/or treating diseases associated with neuronal function loss or death are selected from: dopamine prodrugs, non-ergot dopamine receptor agonists, monoamine oxidase B inhibitors, or their combination.
  • the composition is a cellular preparation.
  • the expression vector of the gene editing protein and the expression vector of the gRNA are the same vector or different vectors.
  • the weight ratio of component (a) to component (b) is 100:1-0.01:1, preferably, 10:1-0.1:1, more preferably, 2: 1-0.5:1.
  • the content of component (a) in the composition is 0.001%-99%, preferably 0.1%-90%, more preferably 1%-70%.
  • the content of component (b) in the composition is 0.001%-99%, preferably, 0.1%-90%, more preferably, 1%-70%.
  • the content of component (c) in the composition is 1%-99%, preferably 10%-90%, more preferably 30%-70%.
  • the component (a) and component (b) and optional component (c) account for 0.01-99.99wt% of the total weight of the composition, Preferably 0.1-90wt%, more preferably 1-80wt%.
  • the present disclosure provides a kit comprising:
  • the kit further contains:
  • (c1) a third container, and other drugs located in said third container for preventing and/or diseases associated with neuronal loss or death, and/or containing other drugs for preventing and/or treating retinal diseases, and /or contains other drugs for the treatment of neurological diseases associated with neuronal death.
  • first container, the second container, and the third container are the same or different containers.
  • the drug in the first container is a single preparation containing a gene editing protein or its expression vector.
  • the drug in the second container is a single preparation containing gRNA or its expression vector.
  • the medicine in the third container is a single preparation containing other medicines pre-used to treat neurological diseases related to neuron death.
  • the dosage form of the drug is selected from: lyophilized preparations, liquid preparations, or combinations thereof.
  • the dosage form of the medicament is an oral dosage form or an injection dosage form.
  • the kit further contains instructions.
  • Item 1 Methods for transdifferentiating mammalian non-neuronal cells into neurons, including:
  • negative regulators that can reduce the expression of negative regulatory genes, including RCOR1, RCOR2, RCOR3, Sin3a, Sin3b, HDAC1, HDAC2, KDM1A, PHF21A, BAF53a, G9a, USP14, HuR, BrG1, EZH2, CDYL , or HMG20B, and/or
  • a positive regulator capable of increasing the expression of positively regulated genes, including DPYSL2, BAF45b, SCF, HuB, HuC, HuD, CYP1B1, or BTRC,
  • An effective amount of the negative regulator or the positive regulator contacts the non-neuronal cells to induce non-neuronal activity.
  • Cells are transdifferentiated into neurons.
  • the method is an in vitro, ex vivo or in vivo method.
  • RCOR1 is the amino acid sequence shown in SEQ ID NO: 1.
  • RCOR2 is the amino acid sequence shown in SEQ ID NO:3.
  • RCOR3 is the amino acid sequence shown in SEQ ID NO: 5.
  • Sin3a is the amino acid sequence shown in SEQ ID NO:7.
  • Sin3b is the amino acid sequence shown in SEQ ID NO: 9.
  • HDAC1 is the amino acid sequence shown in SEQ ID NO: 11.
  • HDAC2 is the amino acid sequence shown in SEQ ID NO: 13.
  • KDM1A is the amino acid sequence shown in SEQ ID NO: 15.
  • PHF21A is the amino acid sequence shown in SEQ ID NO: 17.
  • BAF53a is the amino acid sequence shown in SEQ ID NO: 19.
  • G9a is the amino acid sequence shown in SEQ ID NO: 21.
  • USP14 is the amino acid sequence shown in SEQ ID NO: 23.
  • HuR is the amino acid sequence shown in SEQ ID NO: 25.
  • BRG1 is the amino acid sequence shown in SEQ ID NO: 27.
  • EZH2 is the amino acid sequence shown in SEQ ID NO: 29.
  • CDYL is the amino acid sequence shown in SEQ ID NO: 31.
  • HMG20B is the amino acid sequence shown in SEQ ID NO: 33.
  • DPYSL2 is the amino acid sequence shown in SEQ ID NO: 35.
  • BAF45b is the amino acid sequence shown in SEQ ID NO: 37.
  • SCF is the amino acid sequence shown in SEQ ID NO: 39.
  • HuB is the amino acid sequence shown in SEQ ID NO: 41.
  • HuC is the amino acid sequence shown in SEQ ID NO: 43.
  • HuD is the amino acid sequence shown in SEQ ID NO: 45.
  • CYP1B1 is the amino acid sequence shown in SEQ ID NO: 47.
  • BTRC is the amino acid sequence shown in SEQ ID NO: 49.
  • Item 2 The method according to Item 1, wherein the non-neuronal cells are derived from humans.
  • Item 3 The method according to Item 1 or Item 2, wherein the non-neuronal cells are stem cells, progenitor cells or terminally differentiated cells; preferably, they are glial cells; more preferably, the glial cells are astrocytes. Glial cells, oligodendrocytes, microglia, NG2 cells, Müller glia, glioma cells or spiral ganglion glia, more preferably, the glial cells are astrocytes Plasmoblasts or Müller glia.
  • the non-neuronal cells are stem cells, progenitor cells or terminally differentiated cells; preferably, they are glial cells; more preferably, the glial cells are astrocytes. Glial cells, oligodendrocytes, microglia, NG2 cells, Müller glia, glioma cells or spiral ganglion glia, more preferably, the glial cells are astrocytes Plasmoblasts or Müller glia.
  • Item 4 The method according to Item 3, wherein the non-neuronal cells are derived from the brain.
  • the non-neuronal cells are from the brain, midbrain, cerebellum, brainstem, and spinal cord; more preferably, Comes from the striatum or substantia nigra in the brain.
  • Item 5 The method according to Item 4, wherein the non-neuronal cells are astrocytes derived from the brain, preferably astrocytes derived from the striatum or the substantia nigra.
  • Item 6 The method according to Item 4, wherein the non-neuronal cells are derived from the eye.
  • the non-neuronal cells are Müller glial cells derived from the eye.
  • Item 7 The method according to any one of Items 1 to 6, wherein the neurons are dopaminergic neurons, retinal ganglion cells, photoreceptor cells, 5-HT neurons, NE neurons, ChAT neurons, Motor neurons, GABA neurons, glutamatergic neurons, spinal cord neurons, spinal motor neurons, spinal sensory neurons, bipolar cells, amacrine cells, cochlear nerve cells, pyramidal neurons, interneurons , medium spiny neurons, Purkinje cells, granule cells, olfactory sensory neurons, or periglomerular cells, or combinations thereof.
  • the neurons are dopaminergic neurons, retinal ganglion cells, photoreceptor cells, 5-HT neurons, NE neurons, ChAT neurons, Motor neurons, GABA neurons, glutamatergic neurons, spinal cord neurons, spinal motor neurons, spinal sensory neurons, bipolar cells, amacrine cells, cochlear nerve cells, pyramidal neurons, interneurons , medium spiny neurons, Purkinje cells, granule cells, olfactory sensory neurons, or periglomerular cells,
  • Item 8 The method according to any one of Items 1 to 7, wherein the negative regulatory gene is RCOR1, RCOR2, RCOR3, Sin3a, Sin3b, KDM1A, BAF53a, G9a, HuR, BrG1, or EZH2, and the non- Neurons are animal cells derived from the brain or eyes.
  • the negative regulatory gene is RCOR1, RCOR2, RCOR3, Sin3a, Sin3b, KDM1A, BAF53a, G9a, HuR, BrG1, or EZH2
  • the non- Neurons are animal cells derived from the brain or eyes.
  • Item 9 The method according to Item 8, wherein the negative regulatory gene is RCOR1, RCOR2, RCOR3 or G9a, and the non-neuronal animal cells are derived from the brain; preferably, the non-neuronal cells are derived from Astrocytes of the brain; more preferably, the non-neuronal cells are astrocytes from the striatum or substantia nigra.
  • the negative regulatory gene is RCOR1, RCOR2, RCOR3 or G9a
  • the non-neuronal animal cells are derived from the brain; preferably, the non-neuronal cells are derived from Astrocytes of the brain; more preferably, the non-neuronal cells are astrocytes from the striatum or substantia nigra.
  • Item 10 The method according to Item 8, wherein the negative regulatory gene is Sin3a, Sin3b, KDM1A, BAF53a, G9a, HuR, BrG1, CDYL, or EZH2, and the non-neuronal cells are derived from the eye; preferably , the non-neuronal cells are Müller glial cells from the eye.
  • the negative regulatory gene is Sin3a, Sin3b, KDM1A, BAF53a, G9a, HuR, BrG1, CDYL, or EZH2
  • the non-neuronal cells are derived from the eye; preferably , the non-neuronal cells are Müller glial cells from the eye.
  • Item 11 The method according to item 10, wherein the negative regulatory gene is HuR and the neuron is an optic ganglion cell.
  • Item 12 The method according to Item 10, wherein the negative regulatory gene is Sin3a, KDM1A, BAF53a, G9a, HuR, BrG1, CDYL, or EZH2, and the neuron is a photoreceptor cell.
  • Item 13 The method according to any one of Items 1 to 7, wherein the positive regulatory gene is DPYSL2, BAF45b, SCF, HuB, HuC, HuD, CYP1B1, or BTRC, and the non-neuronal cells are from the brain ;
  • the non-neuronal cells come from the striatum, substantia nigra, ventral tegmental area of the midbrain, spinal cord, hypothalamus, dorsal midbrain, cerebral cortex, hippocampus, cerebellum; more preferably, from the striatum body.
  • Item 14 The method according to Item 13, wherein the positive regulatory gene is SCF or HuB.
  • Item 15 The method according to Item 13, wherein the positive regulatory gene is HuB or BTRC, and an effective amount of the positive regulator is brought into contact with the non-neuronal cells to induce non-neuronal non-neuronal cells. Transdifferentiate into dopaminergic neurons.
  • Item 16 The method according to any one of Items 13 to 15, wherein the non-neuronal cells are astrocytes; more preferably, the non-neuronal cells are astrocytes from the striatum or the substantia nigra. Glial cells.
  • Item 17 The method according to any one of Items 1 to 7, wherein the positive regulatory gene is DPYSL2, BAF45b, SCF, HuC, HuD, or CYP1B1, and the non-neuronal cells are from the eye; preferably, The non-neuronal cells are Müller glial cells from the eye.
  • the positive regulatory gene is DPYSL2, BAF45b, SCF, HuC, HuD, or CYP1B1
  • the non-neuronal cells are from the eye; preferably, The non-neuronal cells are Müller glial cells from the eye.
  • Item 18 The method according to Item 17, wherein the positive regulatory gene is SCF, HuD, or CYP1B1, and the neuron is an optic ganglion cell.
  • Item 19 The method according to Item 17, wherein the positive regulatory gene is DPYSL2 or BAF45b and the neuron is a photoreceptor cell.
  • Item 20 The method according to any one of Items 1 to 19, wherein said reducing the expression of a negative regulatory gene is capable of reducing the gene level of the negative regulatory gene, or reducing the mRNA level of the negative regulatory gene, or reducing the encoding of the negative regulatory gene. protein expression level;
  • Improving the expression of positively regulated genes can increase the gene level of positively regulated genes, or increase the mRNA level of positively regulated genes, or increase the expression level of encoded proteins of positively regulated genes.
  • Item 21 The method according to item 20, wherein the negative regulator is selected from the group consisting of gene editing tools or epigenetic regulation tools that reduce the expression of negatively regulated genes; inhibitors of negatively regulated genes, inhibitors of negatively regulated gene activity , or a degradation activator of the protein encoded by a negative regulatory gene.
  • the negative regulator is selected from the group consisting of gene editing tools or epigenetic regulation tools that reduce the expression of negatively regulated genes; inhibitors of negatively regulated genes, inhibitors of negatively regulated gene activity , or a degradation activator of the protein encoded by a negative regulatory gene.
  • Item 22 The method according to Item 21, wherein the inhibitor is: an inhibitory antibody of a negatively regulated gene; or a small molecule inhibitor of a negatively regulated gene; or an inhibitory mRNA, microRNA, siRNA, or shRNA, antisense oligonucleotide, binding protein or protein domain, polypeptide, nucleic acid aptamer, or PROTAC; or inhibitory binding protein or ligand that negatively regulates genes.
  • the inhibitor is: an inhibitory antibody of a negatively regulated gene; or a small molecule inhibitor of a negatively regulated gene; or an inhibitory mRNA, microRNA, siRNA, or shRNA, antisense oligonucleotide, binding protein or protein domain, polypeptide, nucleic acid aptamer, or PROTAC; or inhibitory binding protein or ligand that negatively regulates genes.
  • Item 23 The method according to item 21, wherein the negative regulator contains the gRNA and gene editing protein described in any one of SEQ ID NO: 51-67.
  • Item 24 The method according to item 20, wherein the positive regulator is selected from the group consisting of epigenetic regulatory tools capable of increasing expression of positively regulated genes, activators of positively regulated gene expression, and degradation inhibitors of proteins encoded by positively regulated genes. , a stabilizer of positive regulatory gene mRNA, or an exogenous positive regulatory gene or a functional fragment of a positive regulatory gene.
  • Item 25 The method according to item 24, wherein the activator is: an agonistic antibody that positively regulates a gene; or a small molecule agonist that positively regulates a gene; or an agonistic binding protein or ligand that positively regulates a gene; or inhibitors of competitive genes that positively regulate genes.
  • the activator is: an agonistic antibody that positively regulates a gene; or a small molecule agonist that positively regulates a gene; or an agonistic binding protein or ligand that positively regulates a gene; or inhibitors of competitive genes that positively regulate genes.
  • Item 26 The method of item 24, wherein the positive regulator contains a nucleic acid sequence as shown in SEQ ID NO: 36, 38, 40, 42, 44, 46, 48, or 50, or contains as SEQ ID NO: A functional fragment of the nucleic acid sequence shown in 36, 38, 40, 42, 44, 46, 48, or 50.
  • Item 27 The gene editing tool according to item 21, wherein the gene editing tool includes a gene editing system or an expression vector thereof, and the gene editing system is selected from: CRISPR system (including CRISPR/Cas system), ZFN system , TALEN system, or combination thereof.
  • CRISPR system including CRISPR/Cas system
  • ZFN system ZFN system
  • TALEN system TALEN system
  • a CRISPR system is used to reduce the expression or activity of a negative regulatory gene; preferably, the CRISPR system contains a nucleic acid encoding a Cas enzyme or a functional domain of a Cas enzyme and a nucleic acid targeting the gRNA of cell transdifferentiation factor; more preferably, the Cas enzyme is Cas13; more preferably, the Cas enzyme is Cas13d, Cas13X, Cas13a, Cas13b, Cas13c, or Cas13Y; more preferably, the Cas enzyme is CasRx .
  • Item 29 The method according to item 20, wherein the negative regulator or positive regulator is carried by a carrier; preferably, the carrier is a viral vector, lipid nanoparticle (LNP), lipid bodies, cationic polymers (such as PEI), nanoparticles, exosomes, or virus-like particles; more preferably, the carrier is an AAV vector or lipid nanoparticles.
  • the carrier is a viral vector, lipid nanoparticle (LNP), lipid bodies, cationic polymers (such as PEI), nanoparticles, exosomes, or virus-like particles; more preferably, the carrier is an AAV vector or lipid nanoparticles.
  • Item 30 The method according to any one of Items 1 to 29, wherein the effective amount of the negative regulator or the positive regulator is contacted with the non-neuronal cells in vitro to induce non-neuronal cells. Cells are transdifferentiated into neurons in vitro; or
  • the effective amount of the negative regulator or the positive regulator contacts the non-neuronal cells in vivo to induce transdifferentiation of the non-neuronal cells into neurons in vitro.
  • Item 31 Use of the negative regulator or positive regulator involved in any one of Items 1 to 30 for preparing a medicament for preventing or treating diseases related to neuron damage or neuron death.
  • Item 32 Use of the negative regulator or positive regulator involved in any one of Items 1 to 30 for preventing or treating diseases related to neuron damage or neuron death.
  • Item 33 The use according to Item 31 or 32, wherein the drug is formulated as a pharmaceutical agent for administration to the nervous system, visual system and auditory system in vivo, for example, administration to the striatum, substantia nigra, midbrain in vivo Ventral tegmental area, spinal cord, hypothalamus, dorsal midbrain, cerebral cortex, hippocampus, cerebellum, subretinal, vitreous cavity, inner ear cochlea and vestibule, preferably striatum, substantia nigra, subretinal and vitreous cavity.
  • Item 34 The use according to Item 31 or 32, wherein the disease related to neuron damage or neuron death is selected from the group consisting of Parkinson's disease, visual system diseases related to RGC or photoreceptor cell function loss or death, and Alzheimer's disease.
  • Alzheimer's disease brain injury, Huntington's disease, epilepsy, depression, sleep disorders, cerebral ischemia, motor neuron disease, amyotrophic lateral sclerosis, spinal muscular atrophy, ataxia, PloyQ disease, schizophrenia disease, addiction, Pick's disease, blindness, and deafness; preferably, it is Parkinson's disease and visual system diseases related to RGC or photoreceptor cell function loss or death.
  • Item 35 The use according to Item 34, wherein the visual system disease related to RGC or photoreceptor cell function loss or death is preferably from the group consisting of: visual impairment caused by RGC cell or photoreceptor cell death, glaucoma, and age-related RGC lesions.
  • the visual system disease related to photoreceptor cell function loss or death is preferably from : Photoreceptor cell degeneration or death caused by injury or degenerative disease, macular degeneration, retinitis pigmentosa, diabetes-related blindness, night blindness, color blindness, hereditary blindness, congenital amaurosis, or combinations thereof.
  • Item 36 The use according to Item 31 or 32, wherein the neurons are dopaminergic neurons, 5-HT neurons, NE neurons, ChAT neurons, GABA neurons, glutamatergic neurons, Motor neurons, photoreceptor cells (such as rods and cones), retinal ganglion cells (RGC), cochlear nerve cells (such as cochlear spiral ganglion cells and vestibular neurons), or medium spiny neurons (MSN) or combinations thereof, preferably dopaminergic neurons, retinal ganglion cells and photoreceptor cells.
  • the neurons are dopaminergic neurons, 5-HT neurons, NE neurons, ChAT neurons, GABA neurons, glutamatergic neurons, Motor neurons, photoreceptor cells (such as rods and cones), retinal ganglion cells (RGC), cochlear nerve cells (such as cochlear spiral ganglion cells and vestibular neurons), or medium spiny neurons (MSN) or combinations thereof, preferably dopaminergic neurons, retinal ganglion cells and photoreceptor cells.
  • Item 37 A method for preventing or treating diseases related to neuron damage or neuron death, comprising giving an effective amount of the negative regulator or positive regulator involved in any one of items 1 to 30 to a subject in need. Conditioner.
  • Item 38 The method according to Item 37, wherein the disease related to neuron damage or neuron death is selected from the group consisting of Parkinson's disease, visual system diseases related to RGC or photoreceptor cell function loss or death, Alzheimer's disease, Brain injury, Huntington's disease, epilepsy, depression, sleep disorders, cerebral ischemia, motor neuron disease, amyotrophic lateral sclerosis, spinal muscular atrophy, ataxia, PloyQ disease, schizophrenia, addiction , Pick's disease, blindness, deafness; preferably Parkinson's disease and visual system diseases related to RGC or photoreceptor cell function loss or death.
  • Parkinson's disease visual system diseases related to RGC or photoreceptor cell function loss or death
  • Alzheimer's disease Brain injury, Huntington's disease, epilepsy, depression, sleep disorders, cerebral ischemia, motor neuron disease, amyotrophic lateral sclerosis, spinal muscular atrophy, ataxia, PloyQ disease, schizophrenia, addiction , Pick's disease, blindness, de
  • Item 39 The method according to Item 38, wherein the visual system disease related to RGC function loss or death is preferably From: Vision impairment due to RGC cell death, glaucoma, age-related RGC pathology, optic nerve damage, age-related macular degeneration (AMD), diabetes-related retinopathy, retinal ischemia or hemorrhage, Leber hereditary optic neuropathy, or combinations thereof ;
  • the visual system diseases related to photoreceptor cell function loss or death are preferably from: photoreceptor cell degeneration or death caused by damage or degenerative disease, macular degeneration, retinitis pigmentosa, diabetes-related blindness, night blindness, color blindness, and hereditary blindness. , congenital amaurosis, or combinations thereof.
  • Item 40 The method according to Item 37, wherein the neurons are dopaminergic neurons, 5-HT neurons, NE neurons, ChAT neurons, GABA neurons, glutamatergic neurons, motor neurons cells, photoreceptor cells (such as rods and cones), retinal ganglion cells (RGC), cochlear nerve cells (such as cochlear spiral ganglion cells and vestibular neurons), or medium spiny neurons (MSN) or other
  • the neurons are dopaminergic neurons, 5-HT neurons, NE neurons, ChAT neurons, GABA neurons, glutamatergic neurons, motor neurons cells, photoreceptor cells (such as rods and cones), retinal ganglion cells (RGC), cochlear nerve cells (such as cochlear spiral ganglion cells and vestibular neurons), or medium spiny neurons (MSN) or other
  • dopaminergic neurons, retinal ganglion cells and photoreceptor cells are preferred.
  • the disease associated with optic neuron damage or optic neuron death may be retinitis pigmentosa (RP).
  • RP retinitis pigmentosa
  • Figure 1 Results of CasRx specifically knocking down each target gene in vitro.
  • expressing CasRx and gRNA targeting RCOR1, Sin3a, HDAC2, PHF21A, BAF53a, KDM1A, G9a, USP14, HuR, BrG1, EZH2, CDYL, and HMG20B can achieve RCOR1, Sin3a, HDAC2, KDM1A, Efficient knockdown of PHF21A, BAF53a, G9a, USP14, HuR, BrG1, EZH2, CDYL, and HMG20B.
  • Figure 2 Schematic diagram of transdifferentiating mouse astrocytes into neurons by knocking down or overexpressing different target genes.
  • Vector 1 AAV-GFAP-mCherry
  • Figure 2A drives the expression of fluorescent protein mCherry by the astrocyte-specific promoter GFAP
  • vector 2 AAV-GFAP-CasRx is driven by the astrocyte-specific promoter GFAP.
  • vector 3 encodes CasRx and gRNA targeting RCOR1, Sin3a, HDAC2, KDM1A, PHF21A, BAF53a, G9a, USP14, HuR, BrG1, EZH2, CDYL, or HMG20B ;
  • Vector 1 in Figure 2B is a schematic diagram of the vector for GFAP-driven mCherry
  • vector 4 is a schematic diagram of the vector for GFAP-driven expression of target genes.
  • the target genes are DPYSL2, BAF45b, SCF, HuB, HuC, HuD, CYP1B1, or BTRC respectively.
  • Figure 3 Conversion of astrocytes into neurons after knocking down different negative regulatory genes in the mouse striatum.
  • Figure 3A shows that mice were injected with control group AAV (AAV-GFAP-mCherry+AAV-GFAP- After CasRx), AAV-GFAP-mCherry can specifically label astrocytes without transdifferentiation into neurons.
  • the yellow arrow (the arrow in Figure 3A is a yellow arrow) indicates the astrocytes labeled by GFAP-mCherry.
  • Figure 3B to Figure 3M respectively show the use of CasRx to knock down RCOR1, HDAC2, PHF21A, BAF53a, G9a, USP14, HuR, The results of transdifferentiation of astrocytes into neurons by BrG1, EZH2, CDYL, HMG20B, and KDM1A.
  • the red signal is the cells labeled by AAV-GFAP-mCherry (the leftmost column of Figure 3A to Figure 3M is the result of mCherry labeling.
  • the bright part in the figure is red
  • NeuN is a neuron-specific marker
  • the white arrow indicates cells where the red mCherry signal is co-labeled with NeuN (the arrows in Figure 3B to Figure 3M are white arrows).
  • Scale bar is 50 microns.
  • Figure 4 Statistical diagram of astrocytes converted into neurons after knocking down different negative regulatory genes.
  • Figure 5 Overexpression of different positive regulatory genes converts astrocytes into neurons in the mouse striatum.
  • Figure 5A shows a control group AAV (AAV-GFAP-mCherry) specifically labeled astrocytes injected into the striatum of the mouse brain.
  • the yellow arrow indicates the astrocytes labeled by GFAP-mCherry.
  • Astrocytes are not co-labeled with NeuN, which is a neuron-specific marker;
  • Figure 5B to Figure 5I show the test group AAV (AAV-GFAP-mCherry+AAV-GFAP-positive) injected into the striatum of the mouse brain.
  • the results of regulatory genes among which the positively regulated genes are DPYSL2, BAF45b, SCF, HuB, HuC, HuD, CYP1B1, BTRC, and the white arrows indicate cells where the red mCherry signal is co-labeled with NeuN (the leftmost column of Figure 5A to Figure 5I is the result of mCherry labeling, the bright part in the figure is red, the arrows in Figure 5B to Figure 5I are white arrows), and NeuN is a neuron-specific marker. Scale bar is 50 microns.
  • Figure 6 Statistics of astrocytes converted into neurons after up-regulation of different positive regulatory genes.
  • Figure 7 Conversion of astrocytes into dopaminergic neurons after overexpression of BTRC or HuB in mouse striatum.
  • Figure 7A shows a control group AAV (AAV-GFAP-mCherry) injected into the striatum of mice.
  • the yellow arrow indicates that the labeled mCherry-positive cells do not co-label with TH, nor do they co-label with TH.
  • TH is a dopaminergic neuron-specific protein marker
  • NeuN is a neuron-specific marker
  • Figure 7B and Figure 7C respectively show the test group AAV (AAV-GFAP- mCherry+AAV-GFAP-positive regulatory genes), in which the positive regulatory genes are HuB and BTRC, and the white arrows (the arrows in Figure 7B and C are white arrows) indicate cells where the red mCherry signal is co-labeled with NeuN and TH.
  • Scale bar is 50 microns. Among them, the bright part in the mCherry column is red, the bright part in the TH column is green, the bright part in the NeuN column is white, and the bright part in the DAPI column is blue.
  • Figure 8 Schematic diagram of converting Müller glia cells into retinal ganglion cells or photoreceptor cells by overexpressing or knocking down the target gene in the retina.
  • Vector 1 in Figure 8A is a schematic diagram of the plasmid of AAV-GFAP-EGFP-2A-Cre. Cre expression is driven by the astrocyte-specific promoter GFAP.
  • Vector 2 is a schematic diagram of the plasmid of AAV-GFAP-CasRx, which is driven by astrocyte-specific promoter GFAP. Plasma cell-specific promoter GFAP drives the expression of RNA editing protein CasRx.
  • Vector 3 is a schematic diagram of the plasmid of AAV-GFAP-CasRx-gRNA, encoding CasRx and targeted negative regulatory genes (Sin3a, HDAC2, KDM1A, BAF53a, G9a, HuR, BrG1 , EZH2, CDYL) gRNA.
  • Figure 8B shows that overexpression of each positive regulatory gene in the retina transformed Müller glia cells. Schematic diagram of differentiation into retinal ganglion cells or photoreceptor cells.
  • Vector 1 is GFAP-driven mCherry
  • vector 4 is GFAP-driven expression of the target gene.
  • the target gene is DPYSL2, BAF45b, SCF, CYP1B1, or BTRC.
  • Figure 9 Knocking down negative regulatory genes in mouse eyes converts Müller glia into photoreceptor cells.
  • Figure 9A shows AAV (AAV-GFAP-EGFP-P2A-Cre) in the control group injected into the retina of Ai9 mice to specifically label Müller glia cells.
  • the yellow arrow indicates the Müller glial cells specifically labeled by GFAP-EGFP-P2A-Cre.
  • the Müller glial cell bodies are located in the INL layer, and no tdTomato-labeled cells were found in the ONL layer. red blood cells.
  • Figure 9B to Figure 9J respectively show the subretinal injection of different AAVs (AAV-GFAP-mCherry+AAV-GFAP-CasRx-gRNA) in the test group of mice, in which the gRNA targets Sin3a, HDAC2, BAF53a, G9a, HuR, and BrG1 respectively.
  • Figure 10A and Figure 10B respectively show the transformation of Müller glial cells into photoreceptor cells after subretinal injection of test group AAV (AAV-GFAP-EGFP-P2A-Cre+AAV-GFAP-positive regulatory gene) in mice.
  • the positive regulatory genes are DPYSL2 and BAF45b respectively
  • the white arrows indicate tdTomato-positive cells in the ONL layer
  • the yellow arrows indicate tdTomato-positive Müller glia cells in the INL layer.
  • the scale bar is 50 microns.
  • the leftmost column in the figure is the result of mCherry labeling. The bright part in the left column is red.
  • the arrow in the INL column in the figure is a yellow arrow
  • the arrow in the ONL column is a white arrow.
  • Figure 11 Cytogram of Müller glia reprogrammed into photoreceptor cells.
  • Figure 12 Knocking down Sin3a or KDM1A in mouse eyes converts Müller glia into rod photoreceptor cells.
  • Figure 12A shows the results of injecting AAV-GFAP-EGFP-P2A-Cre+AAV-GFAP-CasRx into the retina of Ai9 mice.
  • the white signal is staining of the rod-specific protein marker rhodopsin, and the yellow arrow (the arrow in Figure 12A (yellow arrow) indicates that tdTomato-labeled Müller glial cells are located in the INL layer, and there are no tdTomato-positive cells in the ONL layer.
  • Figure 12B and Figure 12C show the transdifferentiation results of the test group's AAV targeting Sin3a and KDM1A (AAV-GFAP-mCherry+AAV-GFAP-CasRx-gRNA) injected under the retina of Ai9 mice.
  • the white arrow indicates the tdTomato located in the ONL layer. Positive cells were co-labeled with rhodopsin. Scale bar is 50 microns.
  • the leftmost column of Figures 12A to 12C shows the results of mCherry labeling. The bright parts in the left column are red, and the arrows in Figures 12B and C are white arrows.
  • Figure 13 Knocking down Sin3a or EZH2 in mouse eyes converts Müller glia into cones.
  • Figure 13A shows the results of injecting AAV-GFAP-EGFP-P2A-Cre into the retina of Ai9 mice.
  • GFAP-EGFP-P2A-Cre can specifically label Müller glia.
  • the cell bodies of Müller glia are located in the INL layer. No red cells labeled by tdTomato were found in the ONL layer.
  • the green signal is the EGFP fluorescence expressed by GFAP-EGFP-P2A-Cre.
  • the yellow arrow (the arrow in Figure 13A is a yellow arrow) indicates the Muller's glue co-labeled with the green signal and the red signal. Plasma cells.
  • Figure 13B shows a control group AAV (AAV-GFAP-EGFP-P2A-Cre+AAV-GFAP-CasRx) injected into the retina of Ai9 mice.
  • the white signal is the cone-specific protein marker m-CAR and the cyan arrow ( Figure The arrow in 13B (cyan arrow) points to tdTomato-positive Müller glial cells, in No tdTomato-positive cells were found in the ONL layer.
  • Figure 13C and Figure 13D are the results of subretinal injection of test group AAV (AAV-GFAP-mCherry+AAV-GFAP-CasRx-gRNA) targeting Sin3a and EZH2 in mice, respectively.
  • white arrows in Figure 13C and Figure 13D Arrows (white arrows) indicate cells that are tdTomato-positive and co-labeled with mCAR in the ONL layer. Scale bar is 50 microns.
  • the bright part in the mCherry column is red
  • the bright part in the EGFP column is green
  • the bright part in the DAPI column is blue.
  • Figure 14 Knockdown of negative regulatory genes in mouse eyes converts Müller glia cells into retinal ganglion cells.
  • Figure 14A shows the results of injecting control group AAV (AAV-GFAP-EGFP-P2A-Cre) into the retina of Ai9 mice. There was almost no labeled optic nerve in the optic nerve.
  • Figures 14B to 14I respectively show the results of subretinal injection of test group AAV (AAV-GFAP-mCherry+AAV-GFAP-CasRx-gRNA) in Ai9 mice to inhibit different negative regulatory genes, in which the gRNA targets HDAC2, BAF53a, and G9a respectively.
  • HuR, BrG1, EZH2, CDYL, KDM1A HuR, BrG1, EZH2, CDYL, KDM1A.
  • Figure 15 Figures 15A to 15G show the transformation of Müller glial cells into retinal ganglion cells by overexpressing different positive regulatory genes in mouse eyes.
  • the positively regulated genes are DPYSL2, BAF45b, SCF, HuB, HuD, CYP1B1, and BTRC, and the scale bar is 150 ⁇ m.
  • Figure 16 Statistical diagram of Müller glial cells reprogramming into retinal ganglion cells.
  • Figure 17 Results of reprogramming Müller glia into photoreceptor cells in retinitis pigmentosa disease model mice.
  • Figure 17A is a schematic diagram of the degeneration and death of rod and cone photoreceptor cells and the time points of AAV injection in the retinitis pigmentosa disease model mouse (Pde6b-KO). AAV injection was performed after all photoreceptor cells in the retina of Pde6b-KO mice died (P50), and retinal tissue was harvested for analysis 50 days after injection;
  • Figure 17B and Figure 17C are retinal harvests from Pde6b-KO mice at 4 weeks and 6 weeks respectively. tissue and immunofluorescent staining with the rod-specific marker Rhodopsin, no rods were observed at either 4 or 6 weeks.
  • Figure 17D shows the results of immunofluorescence staining of retinal tissue sections injected with AAV (GFAP-CasRx+CBH-Pde6b) in the control group. There are no Rhodopsin-positive cells in the ONL layer.
  • Figure 17E to Figure 17F show the results of immunofluorescence staining of retinal tissue sections of AAV (GFAP-CasRx-gRNA+CBH-Pde6b) injection treatment group.
  • gRNA targeted knockdown of the expression of EZH2 or KDM1A, and Rhodopsin-positive cells reappeared in the ONL layer; where , the bright color in the Rhodopsin column is green, and the bright color in the DAPI column is blue.
  • Figure 17G is a statistical diagram of the number of retinal photoreceptor cells in different treatment groups of Pde6b-KO mice.
  • 4w and 6w are the number of photoreceptor cells in the retina of Pde6b-KO mice without AAV injection.
  • the number of photoreceptor cells in the EZH2 or KDM1A knockdown group increased significantly, and
  • Figure 17H shows the thickness of the retinal ONL layer in different treatment groups of Pde6b-KO mice.
  • 4w and 6w show the thickness of the retinal ONL layer of Pde6b-KO mice without AAV injection.
  • the EZH2 or KDM1A knockdown group was significantly thicker.
  • the inventor unexpectedly discovered for the first time that the expression or activity of one or more genes or RNAs or proteins encoding them selected from the group consisting of: Expression or activity of proteins: RCOR1, RCOR2, RCOR3, Sin3a, Sin3b, HDAC1, HDAC2, KDM1A, PHF21A, BAF53a, G9a, USP14, HuR, BrG1, EZH2, CDYL, HMG20B, and/or genes or RNAs that enhance the expression or activity of one or more genes or RNAs or proteins encoding the proteins in glial cells selected from the group consisting of: The expression or activity of its encoded proteins: DPYSL2, BAF45b, SCF, HuB, HuC, HuD, CYP1B1, BTRC, can effectively induce the differentiation of glial cells into neuronal cells, thereby treating neurological diseases related to neuronal function loss or death. . On this basis, the inventor completed the technical solution of the present application.
  • retinal ganglion cell (RGC) degeneration is the primary cause of permanent blindness.
  • the transdifferentiation of Müller glial cells (MG) into RGC can help restore vision.
  • MGs can be converted directly into RGCs by knocking down (e.g., knocking down using the RNA-targeted CRISPR system CasRx) the gene or its RNA or its encoded protein in mature mouse retinas.
  • NMDA N-methyl-D-aspartate
  • RGCs converted from MG achieved functional projections to central visual areas and resulted in improved visual function. . Therefore, reducing negative regulatory genes (e.g., CasRx-mediated knockdown) would be a promising therapy for the treatment of retinal diseases caused by neurodegeneration.
  • This application uses the recently characterized RNA-targeting CRISPR system CasRx to inhibit the gene or its RNA or its encoded protein.
  • Müller glia are the main glial cells in retinal tissue
  • retinal ganglion cells are nerve cells located in the innermost layer of the retina, and their dendrites mainly communicate with bipolar cells. , its axons extend to the optic nerve head to form the optic nerve.
  • differentiation has the same meaning herein and may refer to the generation of cells of a specific lineage from different types of non-neuronal cells, such as astrocytes (such as neuronal cells) without intermediate differentiation processes.
  • a negative regulator or a positive regulator is brought into contact with the non-neuronal cell
  • a negative regulator or a positive regulator is brought into contact with the non-neuronal cell
  • the positive modulator e.g., compound, nucleic acid, viral vector, etc.
  • contacting is by adding a negative regulator or a positive regulator to the cell culture.
  • Exposure may also be by injecting a negative or positive modulator, or a vehicle containing a negative or positive modulator, into a location in the body; This is accomplished by delivering the carrier of the positive regulator or positive regulator to a location in the body such that the negative regulator or positive regulator "contacts" the targeted cell type.
  • the effective amount of the negative regulator or the positive regulator is contacted with the non-neuronal cells in vivo to induce transdifferentiation of the non-neuronal cells into neurons in vitro.
  • Positive modulators or negative modulators are formulated for in vivo administration to the nervous system, visual system, and auditory system, for example, to the striatum, substantia nigra, ventral tegmental area of the midbrain, spinal cord, Hypothalamus, dorsal midbrain, cerebral cortex, hippocampus, cerebellum, subretinal, vitreous cavity, inner ear cochlea and vestibule, preferably striatum, substantia nigra, subretinal and vitreous cavity.
  • the effective amount of the negative regulator or the positive regulator is contacted with the non-neuronal cells in vitro to induce transdifferentiation of the non-neuronal cells into neurons in vitro.
  • neurons obtained through in vitro contact can be configured into appropriate cell therapy agents and administered to individuals in need through appropriate administration methods, such as intravenous infusion, in situ injection, etc.
  • NNL refers to the outer granule cell layer
  • INL refers to the inner granule cell layer
  • GCL refers to the retinal ganglion cell layer
  • non-neuronal cell may refer to any type of cell that is not a neuron.
  • the non-neuronal cells are stem cells, progenitor cells, or terminally differentiated cells.
  • non-neuronal cells are cells of cell lineages other than the neuronal lineage (eg, hematopoietic lineage).
  • the non-neuronal cells are cells of the neuronal lineage but are not neurons, such as glial cells.
  • the non-neuronal cells are non-neuronal terminally differentiated cells such as, but not limited to, glial cells, fibroblasts, embryonic fibroblasts, epithelial cells, melanocytes, keratinocytes, adipocytes , blood cells, bone marrow stromal cells, Langerhans cells, muscle cells, rectal cells or chondrocytes.
  • the non-neuronal cells are from a non-neuronal cell line, such as, but not limited to, glioblastoma cell line, HeLa cell line, NT2 cell line, ARPE19 cell line, or N2A cell line.
  • glioblastoma cell line such as, but not limited to, glioblastoma cell line, HeLa cell line, NT2 cell line, ARPE19 cell line, or N2A cell line.
  • Cell lineage or “lineage” may mean the developmental history of a tissue or organ from a fertilized embryo.
  • Neurogenesis may refer to the developmental history from neural stem cells to mature neurons, including various stages along this process (called neurogenesis), such as, but not limited to, neural stem cells (neuroepithelial cells, radial glia) , neural progenitor cells (e.g., interneuron precursors), neurons, astrocytes, oligodendrocytes, and microglia.
  • neural stem cells neuroepithelial cells, radial glia
  • neural progenitor cells e.g., interneuron precursors
  • neurons e.g., astrocytes, oligodendrocytes, and microglia.
  • the non-neuronal cells are stem cells, such as embryonic stem cells, neural stem cells, or induced pluripotent stem cells.
  • the non-neuronal cells are progenitor cells, such as neural grandmother cells or neural precursor cells (eg, dopamine neural precursor cells).
  • the non-neuronal cells are derived from the brain.
  • the non-neuronal cells are derived from the brain, midbrain, cerebellum, brainstem, and spinal cord; more preferably, they are derived from striae in the brain. body or substantia nigra.
  • the non-neuronal cells are astrocytes derived from the brain, preferably, astrocytes derived from the striatum or substantia nigra.
  • the non-neuronal cells are derived from the eye.
  • the non-neuronal cells are Müller glial cells from the eye.
  • progenitor cell refers to intermediate cells that exist in adult tissues before cells differentiate into terminally differentiated cells. The differentiation of progenitor cells is usually clear.
  • terminal differentiated cells also known as terminal cells, refers to cells of a specific type with specific functional nuclei that no longer undergo differentiation, division and proliferation. For example: glial cells, somatic cells, fibroblasts, red blood cells, mature epidermal cells, muscle cells, etc.
  • stem cells should be understood as undifferentiated cells that have differentiation potential and proliferation capacity (in particular self-renewal capacity) but retain differentiation potential. According to differentiation potential, stem cells include subpopulations such as pluripotent stem cells (PSC), multipotent stem cells, unipotent stem cells, embryonic stem cells, etc. In some implementations, the stem cells can be embryonic stem cells, neural stem cells, or induced pluripotent stem cells.
  • PSC pluripotent stem cells
  • multipotent stem cells multipotent stem cells
  • unipotent stem cells unipotent stem cells
  • embryonic stem cells etc.
  • the stem cells can be embryonic stem cells, neural stem cells, or induced pluripotent stem cells.
  • pluripotent stem cells refers to stem cells that can be cultured in vitro and have the ability to differentiate into any cell lineage belonging to the three germ layers (ectoderm, mesoderm, endoderm). PSCs can be induced from fertilized eggs, cloned embryos, germline stem cells, stem cells in tissues, somatic cells, etc. Examples of PSCs include embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs or ips), embryonic germ cells (EG cells), and the like.
  • ESCs embryonic stem cells
  • iPSCs or ips induced pluripotent stem cells
  • EG cells embryonic germ cells
  • induced pluripotent stem cells can be generated directly from adult cells by reprogramming.
  • Adult cells can be converted into PSCs by introducing the products of a specific set of pluripotency-related genes.
  • neural precursor cells refers to cells that have neuronal development potential and are in a precursor state of neuronal development.
  • neuron may have its general meaning as would be understood by those skilled in the art.
  • a neuron may refer to an electrically excitable cell that can receive, process, and transmit information through electrical signals (eg, membrane potential discharge) and chemical signals (eg, synaptic transmission of neurotransmitters).
  • electrical signals eg, membrane potential discharge
  • chemical signals eg, synaptic transmission of neurotransmitters.
  • chemical signals transduced between neurons can occur through specialized connections called synapses.
  • the neurons are dopaminergic neurons, retinal ganglion cells, photoreceptor cells, 5-HT neurons, NE neurons, ChAT neurons, motor neurons, GABA neurons, glutamate neurons Neurons, spinal neurons, spinal motor neurons, spinal sensory neurons, bipolar cells, amacrine cells, cochlear nerve cells, pyramidal neurons, interneurons, medium spiny neurons, Purkinje cells , granule cells, olfactory sensory neurons, or periglomerular cells, or combinations thereof.
  • protein As used herein, the terms “protein”, “peptide” and “polypeptide” may refer to an amino acid polymer or a group of two or more interacting or combined amino acid polymers, and have the same meaning.
  • nucleic acid and “polynucleotide” have the same meaning and may refer to a nucleic acid molecule containing one or more nucleotides.
  • target gene refers to a gene regulated by a "negative regulator” or a "positive regulator”.
  • photoreceptor refers to a type of neuroepithelial cell in the retina that is responsible for visual light transmission, including rods and cones. Some retinitis pigmentosa, macular degeneration, diabetes complications, etc. can cause the death of photoreceptor cells.
  • ganglion cells are neurons located in the final segment of the retina. Many eye diseases can lead to the death of optic ganglion cells, such as ischemic retinopathy, glaucoma, etc. The death of optic ganglion cells may also lead to permanent blindness.
  • Müller glia are the main glial cells in retinal tissue
  • retinal ganglion cells are nerve cells located in the innermost layer of the retina. Their dendrites mainly communicate with bipolar cells. , its axons extend to the optic nerve head to form the optic nerve.
  • vector refers to a tool that can transport exogenous nucleic acids into cells, which can be nucleic acids, proteins, etc.
  • the vector is capable of directing the synthesis of one or more proteins encoded by one or more genes carried by the vector or microRNA encoded by a polynucleotide carried by the vector.
  • vectors can be used to introduce the polynucleotides provided herein. In this article, the vector can be a variety of different forms of vectors.
  • the vector can be a viral vector, a plasmid vector, a minicircle vector, a linear DNA vector, a doggybone vector, a lipid vector, or a vector. Plastids, nanoparticles, exosomes, extracellular vesicles, cationic polymers (such as PEI) or virus-like particles, etc.
  • viral vector is a nucleic acid of viral origin that may be capable of transporting another nucleic acid into a cell. When a viral vector is present in an appropriate environment, it is capable of directing the expression of one or more proteins encoded by one or more genes carried by the vector or microRNA encoded by a polynucleotide carried by the vector.
  • viral vectors include, but are not limited to, retroviral vectors, adenoviral vectors, lentiviral vectors, poxviral vectors, herpesviral vectors, and adeno-associated viral vectors.
  • nanoparticle carriers may include polymer-based nanoparticles, aminolipid-based nanoparticles, and metal nanoparticles.
  • the vectors provided herein can be used to deliver the polynucleotide compositions provided herein.
  • a single vector is used to deliver at least about 2, 3, 4, or up to 5 polynucleotides.
  • a single vector is used to deliver at least about 2, 3, 4, or up to 5 different polynucleotides.
  • a single vector is used to deliver at least about 2, 3, 4, or up to 5 identical polynucleotides.
  • the vector can deliver DNA (eg, double-stranded DNA or single-stranded DNA), and can also deliver RNA.
  • RNA can include base modifications.
  • Vectors may include recombinant vectors.
  • the vector may be a vector modified from a naturally occurring vector.
  • the carrier may include at least a portion of a non-naturally occurring carrier. Any carrier can be utilized.
  • astrocytes may refer to the star-shaped glial cells that are characteristic of the brain and spinal cord. It will be clear to those skilled in the art that astrocytes can be characterized by being star-shaped, expressing markers such as glial fibrillary acidic protein (GFAP) and aldehyde dehydrogenase 1 family member L1 (ALDH1L1), excitatory Excitatory amino acid transporter 1/glutamate aspartate transporter (EAAT1/GLAST), glutamine synthetase, S100 ⁇ or excitatory amino acid transporter 1/glutamate transporter 1 (EAAT2/GLT-1), Involved with endothelial cells in the blood-brain barrier, transmitter uptake and release, regulation of ion concentrations in the extracellular space, response to neuronal injury and involvement in nervous system repair, and metabolic support of peripheral neurons.
  • markers such as glial fibrillary acidic protein (GFAP) and aldehyde dehydrogenase 1 family member L1 (ALDH1
  • astrocytes may refer to non-neuronal cells in the nervous system that express glial fibrillary acidic protein (GFAP), aldehyde dehydrogenase 1 family member L1 (ALDH1L1), or both. .
  • astrocytes may refer to non-neuronal cells in the nervous system that express glial fibrillary acidic protein (GFAP) promoter-driven transgenes (e.g., red fluorescent protein (RFP), Cre recombinase) .
  • GFAP glial fibrillary acidic protein
  • RFP red fluorescent protein
  • glial cells useful in the methods provided herein are glial cells isolated from the brain. In some embodiments, astrocytes useful in the methods provided herein are star-shaped glial cells in the brain or spinal cord.
  • an "effective amount" of the negative modulator or the positive modulator in contact with the non-neuronal cells refers to the amount of the negative modulator or positive modulator that is capable of converting the non-neuronal cells into neurons. , or the amount of carrier containing a negative regulator or a positive regulator.
  • active fragment As used herein, “active fragment”, “functional fragment”, and “functional fragment” have the same meaning and refer to a truncated fragment of a gene or protein that has the same or similar function as the full-length gene or protein.
  • “functional fragment of SEQ ID NO: 36” refers to a truncated fragment of SEQ ID NO: 36 that has a similar function to the sequence shown in SEQ ID NO: 36. This truncated fragment can perform the same function as SEQ ID NO: 36.
  • NO: 36 has the same or similar positive regulatory effect, promoting the transdifferentiation of non-neuronal cells into neurons.
  • an inhibitor of a negatively regulated gene may be an RNA interference agent that contains RNA and mediates targeted cleavage of RNA transcripts through the RNA-induced silencing complex (RISC) pathway.
  • RISC RNA-induced silencing complex
  • RNA interference agents direct the sequence-specific degradation of mRNA through the process of RNA interference (RNAi).
  • RNA interference agents can inhibit the expression of one or more negatively regulated genes in cells.
  • RNA interference agents include, but are not limited to, "small interfering RNA (siRNA)", “endoribonuclease-prepared siRNA (e-siRNA)", “short hairpin RNA (shRNA)” and “small time-regulated RNA” ( stRNA), “cleaved siRNA (d-siRNA),” and aptamers, oligonucleotides, and other synthetic nucleic acids containing at least one uracil base.
  • siRNA small interfering RNA
  • e-siRNA endoribonuclease-prepared siRNA
  • shRNA short hairpin RNA
  • stRNA small time-regulated RNA
  • d-siRNA cleaved siRNA
  • aptamers oligonucleotides, and other synthetic nucleic acids containing at least one uracil base.
  • RNA interfering agents are delivered by a carrier , such vectors include, but are not limited to, replication-deficient or replication-competent viral vectors (eg, adenovirus, lentivirus, gamma retrovirus, adeno-associated virus, etc.).
  • viral vectors eg, adenovirus, lentivirus, gamma retrovirus, adeno-associated virus, etc.
  • the invention provides methods of generating neurons in vivo.
  • Exemplary methods include administering a positive or negative modulator to an area of the subject's nervous system, such as the brain, eye, or spinal cord, and allowing non-neuronal cells to reprogram into functional neurons.
  • expression vectors refers to vectors that add expression elements (such as promoters, RBS, GOI, terminators, etc.) to the basic skeleton of the cloning vector to enable the expression of the target gene. Constructing an expression vector allows the target gene to be expressed and function in recipient cells.
  • expression elements such as promoters, RBS, GOI, terminators, etc.
  • a "negatively regulated gene” refers to a gene that, when the expression of the gene, or the expression of its mRNA, or the expression of the protein encoded by the gene is reduced or inhibited, can increase, promote or improve the transition of non-neuronal cells to neurons.
  • Differentiation genes such as: RCOR1, RCOR2, RCOR3, Sin3a, Sin3b, HDAC1, HDAC2, KDM1A, PHF21A, BAF53a, G9a, USP14, HuR, BrG1, EZH2, CDYL, HMG20B, etc.
  • a negative regulator refers to a regulator that is capable of reducing or inhibiting the gene expression, or the expression of mRNA, or the expression of the protein encoded by the "negatively regulated gene".
  • a negative regulator can be a gene editing tool or epigenetic regulation tool that can reduce the expression of a negatively regulated gene; it can also be an inhibitor of a negatively regulated gene, an inhibitor of a negatively regulated gene activity, or a negative regulator. Genes encoding proteins that are activators of degradation.
  • a "negative regulator” can be an inhibitory antibody of a negatively regulated gene; or a small molecule inhibitor of a negatively regulated gene; or an inhibitory mRNA, microRNA, siRNA, shRNA, or antisense of a negatively regulated gene. Oligonucleotides, binding proteins or protein domains, polypeptides, nucleic acid aptamers, or PROTACs; or inhibitory binding proteins or ligands that negatively regulate genes.
  • the gene editing tool used as a "negative regulator” can be a CRISPR gene editing tool, including a gene editing protein and a gRNA targeting a negative regulatory gene.
  • the gene editing protein can be a DNA editing protein, such as Cas9, or an RNA editing protein, such as Cas13.
  • the gene editing tool used as a "negative regulator” can be a gene editing tool containing a zinc finger nuclease, or it can be a TALENs (transcription activator-like (TAL) effector nucleases)) gene editing tool. .
  • TALENs transcription activator-like (TAL) effector nucleases
  • the “negative regulator” may be an epigenetic regulation tool.
  • Epigenetic regulation tools refer to the use of epigenetic modification methods (such as methylation, acetylation, phosphorylation, chromatin conformation changes, etc.) without changing the nucleotide sequence of the target gene.
  • Proteins, genes, small molecule compounds, etc. used to regulate the content and function of nucleic acids or proteins. For example: fusion of epigenetic regulatory proteins such as KRAB and DNMT3A on the mutated CRISPR/Cas system.
  • a "positively regulated gene” means that when the expression of the gene, or the expression of its mRNA, or the protein encoded by the gene When the expression of white is increased or activated, genes that can increase, promote or improve the transdifferentiation of non-neuronal cells into neurons, such as: DPYSL2, BAF45b, SCF, HuB, HuC, HuD, CYP1B1, or BTRC.
  • a “positive regulator” refers to a regulator that is capable of increasing or activating the gene expression of a “positive regulator gene", or the expression of mRNA, or the expression of the protein encoded by the gene.
  • the positive regulator can be an epigenetic regulation tool that can improve the expression of a positively regulated gene, an activator of positively regulated gene expression, a degradation inhibitor of the protein encoded by a positively regulated gene, or a stabilizer of the mRNA of a positively regulated gene. , or exogenous positive regulatory genes or functional fragments of positive regulatory genes.
  • a "positive regulator” can be: an agonistic antibody that positively regulates a gene; or a small molecule agonist that positively regulates a gene; or an agonistic binding protein or ligand that positively regulates a gene; or a positive regulator.
  • Competitive gene inhibitors of genes can be: an agonistic antibody that positively regulates a gene; or a small molecule agonist that positively regulates a gene; or an agonistic binding protein or ligand that positively regulates a gene; or a positive regulator.
  • diseases related to neuronal function loss or neuron death may be Parkinson's disease, visual system diseases related to RGC or photoreceptor cell function loss or death, Alzheimer's disease, brain injury, Huntington's disease , Epilepsy, Depression, Sleep Disorders, Cerebral Ischemia, Motor Neurone Disease, Amyotrophic Lateral Sclerosis, Spinal Muscular Atrophy, Ataxia, PloyQ Disease, Schizophrenia, Addiction, Pick's Disease, RGC or Blindness, macular degeneration, retinitis pigmentosa, deafness, night blindness, color blindness, hereditary blindness, congenital amaurosis, etc. caused by damage or death of photoreceptor cells.
  • the diseases related to neuron function loss or neuron death are preferably Parkinson's disease and visual system diseases related to RGC or photoreceptor cell function loss or death, for example: dopamine neuron function loss or death related diseases Disease, vision impairment associated with loss or death of optic ganglia or photoreceptor cells.
  • Visual system diseases related to RGC or photoreceptor cell function loss or death are preferably from: vision impairment caused by RGC cell or photoreceptor cell death, glaucoma, age-related RGC lesions, optic nerve damage, age-related macular degeneration (AMD), diabetes-related Retinopathy, retinal ischemia or hemorrhage, Leber's hereditary optic neuropathy, or a combination thereof;
  • the visual system disease related to photoreceptor cell function loss or death is preferably from: photoreceptor cell degeneration or death caused by injury or degenerative disease, macular degeneration, retinitis pigmentosa, diabetes-related blindness, night blindness, color blindness, hereditary blindness, congenital amaurosis, or combinations thereof.
  • Astrocytes are the most abundant type of cells in the mammalian brain. They perform many functions, including biochemical support (such as forming the blood-brain barrier), providing nutrients to neurons, maintaining extracellular ion balance, and participating in repair and scarring after brain and spinal cord injury. Astrocytes can be divided into two types based on the content of glial filaments and the shape of their processes: fibrous astrocytes are mostly distributed in the white matter of the brain and spinal cord, with slender processes and fewer branches. , the cytoplasm contains a large number of glial filaments; protoplasmic astrocytes are mostly distributed in the gray matter, with thick and short cell processes and many branches.
  • Astrocytes that can be used in the present disclosure are not particularly limited and include various astrocytes derived from the mammalian central nervous system, for example, from the striatum, substantia nigra, midbrain ventral tegmental area, inferior The thalamus, spinal cord, dorsal midbrain or cerebral cortex, preferably, originate from the striatum and substantia nigra.
  • functional neurons may refer to neurons capable of sending or receiving information through chemical or electrical signals.
  • functional neurons exhibit one or more functional properties of mature neurons present in the normal nervous system, including, but not limited to: excitability (e.g., the ability to exhibit action potentials, e.g., rapid rise and subsequent fall) (voltage or membrane potential across the cell membrane), formation of synaptic connections with other neurons, presynaptic neurotransmitter release, and postsynaptic responses (e.g., excitatory postsynaptic currents or inhibitory synaptic after-touch current).
  • excitability e.g., the ability to exhibit action potentials, e.g., rapid rise and subsequent fall
  • postynaptic responses e.g., excitatory postsynaptic currents or inhibitory synaptic after-touch current.
  • functional neurons are characterized by expressing one or more markers of functional neurons, including but not limited to synaptophysin, synaptophysin, glutamate decarboxylase 67 (GAD67), glutamine Acid decarboxylase 65 (GAD65), parvalbumin, dopamine- and cAMP-regulated neuronal phosphoprotein 32 (DARPP32), vesicular glutamate transporter 1 (vGLUT1), vesicular glutamate transporter 2 (vGLUT2) , acetylcholine, tyrosine hydroxylase (TH), dopamine, vesicular GABA transporter (VGAT) and gamma-aminobutyric acid (GABA).
  • GABA glutamate decarboxylase 67
  • GAD65 glutamine Acid decarboxylase 65
  • DARPP32 dopamine- and cAMP-regulated neuronal phosphoprotein 32
  • vGLUT1 vesicular glutamate transporter 1
  • functional neurons can be dopaminergic neurons, 5-HT neurons, NE neurons, ChAT neurons, GABA neurons, glutamatergic neurons, motor neurons, photoreceptor cells (e.g. rods and cones), retinal ganglion cells (RGC), cochlear nerve cells (such as cochlear spiral ganglion cells and vestibular neurons), or medium spiny neurons (MSN) or combinations thereof, preferably dopaminergic nerves cells, retinal ganglion cells, and photoreceptor cells.
  • photoreceptor cells e.g. rods and cones
  • RRC retinal ganglion cells
  • cochlear nerve cells such as cochlear spiral ganglion cells and vestibular neurons
  • MSN medium spiny neurons
  • the functional neurons are mammalian neurons, eg, human, non-human primate, rat, mouse neurons.
  • Dopaminergic neurons are neurons that contain and release dopamine (DA) as the neurotransmitter.
  • Dopamine is a catecholamine neurotransmitter that plays an important biological role in the central nervous system.
  • Dopaminergic neurons in the brain are mainly concentrated in the substantia nigra pars compacta (SNc) of the midbrain and the ventral tegmentum. area (ventral tegmental area, VTA), hypothalamus and periventricular area.
  • SNc substantia nigra pars compacta
  • VTA ventral tegmental area
  • Many experiments have confirmed that dopaminergic neurons are closely related to various diseases in the human body, the most typical of which is Parkinson's disease.
  • Gene editing tools refer to the process of modifying target genes through gene editing technology, including the insertion, deletion or replacement of genes, thereby changing their genetic information and phenotypic characteristics.
  • available gene editing tools include, but are not limited to: CRIPSR gene editing tools (CRISPR/Cas system), zinc finger nuclease gene editing tools (ZFN system), TALENs (transcription activator-like (TAL) effector nucleases)) gene editing tools (TALEN system).
  • CRIPSR gene editing tools CRISPR/Cas system
  • ZFN system zinc finger nuclease gene editing tools
  • TALENs transcription activator-like (TAL) effector nucleases) gene editing tools
  • the gene editing tools include DNA gene editing tools or RNA gene editing tools.
  • the gene editing tool of the present disclosure is a CRIPSR gene editing tool.
  • the CRIPSR gene editing tool includes a nucleic acid encoding a Cas protein (also referred to as Cas enzyme herein) or a functional domain of the Cas protein, and a gRNA targeting a gene of interest. gRNA can guide the Cas protein to target the target gene and perform gene editing.
  • the Cas protein is Cas9, Cas12 (for example: Cas12a, Cas12b, Cas12c, Cas12d, etc.), Cas13, Cas 14, CasX or CasY family proteins or mutants thereof.
  • the Cas protein is Cas13a, Cas13d, Cas13X, Cas13a, Cas13b (for example: Cas13b-t1, Cas13b-t2, Cas13b-t3, etc.), Cas13c, or Cas13Y.
  • the Cas13d is CasRx.
  • the gene editing tool can reduce or silence the expression of the negative regulatory genes involved in this application.
  • gene editing tools can be delivered via vectors, for example, via adeno-associated viruses to deliver gRNAs and gene editing proteins (eg, CasRx) provided herein.
  • gRNAs and gene editing proteins eg, CasRx
  • the nucleotide of the Cas protein can be obtained through genetic engineering techniques, such as genome sequencing, polymerase chain reaction (PCR), etc., and its amino acid sequence can be derived from the nucleotide sequence.
  • the sources of the wild-type Cas protein include but are not limited to: Ruminococcus lavefaciens, Streptococcus pyogenes, Staphylococcus aureus, Acidaminococcus sp, Lachnospiraceae (Lachnospiraceaeacterium).
  • the Cas protein is capable of editing DNA.
  • the Cas protein is capable of editing RNA.
  • the Cas protein can be Cas13d, Cas13e, Cas13a, Cas13b, Cas13c, Cas13f and other RNA-targeting gene editing proteins.
  • protein of the present disclosure proteins
  • proteins proteins
  • polypeptide proteins
  • proteins are used interchangeably and may refer to RCOR1, RCOR2, RCOR3, Sin3a, Sin3b, HDAC1, HDAC2, KDM1A, Protein or polypeptide of the amino acid sequence of PHF21A, BAF53a, G9a, USP14, HuR, BrG1, EZH2, CDYL, HMG20B, BTRC, CYP1B1, DPYSL2, BAF45b, SCF, HuB, HuC, or HuD. They include the protein with or without the starting methionine. Furthermore, the term also includes the full length of said protein and fragments thereof.
  • the proteins referred to in this disclosure include their complete amino acid sequences, their secreted proteins, their mutants, and their functionally active fragments.
  • RCOR1, RCOR2 and RCOR3 are REST corepressor 1, REST corepressor 2 and REST corepressor 3.
  • the three have similar functions; the full name of HDAC1 is Histone deacetylase 1 (Histone deacetylase 1), HDAC2 stands for Histone deacetylase 2 (Histone deacetylase 2), both of which have similar functions; KDM1A is called Lysine-specific demethylase 1A (Lysine-specific demethylase 1A), also known as Lysine-specific histone demethylase (Lysine -specific histone demethylase 1A, LSD1).
  • Sin3a member A of the SIN3 transcriptional regulatory protein family
  • Sin3b member B of the SIN3 transcriptional regulatory protein family
  • the terms “gene”, “polynucleotide” and “nucleic acid” are used interchangeably and may all refer to genes having RCOR1, RCOR2, RCOR3, Sin3a, Sin3b, HDAC1, HDAC2, KDM1A, PHF21A, BAF53a, G9a , Nucleic acid sequence of the nucleotide sequence of USP14, HuR, BrG1, EZH2, CDYL, HMG20B, CYP1B1, BTRC, DPYSL2, BAF45b, SCF, HuB, HuC, or HuD.
  • the full genome length of the human RCOR1 gene is 173,980 bp (NCBI GenBank accession number is 23186).
  • the full genome length of mouse RCOR1 gene is 76536bp (NCBI GenBank accession number is 217864).
  • the full genome length of the human RCOR2 gene is 5935bp (NCBI GenBank accession number is 283248).
  • the full genome length of mouse RCOR2 gene is 7832bp (NCBI GenBank accession number is 104383).
  • the full genome length of the human RCOR3 gene is 57020bp (NCBI GenBank accession number is 55758).
  • the full genome length of mouse RCOR3 gene is 39526bp (NCBI GenBank accession number is 214742).
  • the full genome length of the human Sin3a gene is 86441bp (NCBI GenBank accession number is 25942).
  • the full genome length of the mouse Sin3a gene is 56382 bp (NCBI GenBank accession number is 20466).
  • the full genome length of the human Sin3b gene is 50958 bp (NCBI GenBank accession number is 23309).
  • the full genome length of mouse Sin3b gene is 34934bp (NCBI GenBank accession number is 20467).
  • the full genome length of the human HDAC1 gene is 41546bp (NCBI GenBank accession number is 3065).
  • the full genome length of mouse HDAC1 gene is 26543bp (NCBI GenBank accession number is 433759).
  • the full genome length of the human HDAC2 gene is 38121bp (NCBI GenBank accession number is 3066).
  • the full genome length of the mouse HDAC2 gene is 27593 bp (NCBI GenBank accession number is 15182).
  • the full genome length of the human KDM1A gene is 64249bp (NCBI GenBank accession number is 23028).
  • the full genome length of the mouse KDM1A gene is 52284 bp (NCBI GenBank accession number is 99982).
  • the full genome length of the human PHF21A gene is 192136bp (NCBI GenBank accession number is 51317).
  • the full genome length of mouse PHF21A gene is 180916bp (NCBI GenBank accession number is 192285).
  • the full genome length of human BAF53a gene is 25482bp (NCBI GenBank accession number is 86).
  • the full genome length of the mouse BAF53a gene is 18428 bp (NCBI GenBank accession number is 56456).
  • the full genome length of the human G9a gene is 17940 bp (NCBI GenBank accession number is 10919).
  • the full genome length of the mouse G9a gene is 15624 bp (NCBI GenBank accession number is 110147).
  • the full genome length of the human USP14 gene is 56073 bp (NCBI GenBank accession number is 9097).
  • the full genome length of mouse USP14 gene is 36535bp (NCBI GenBank accession number is 59025).
  • the full genome length of human HuR gene is 47069bp (NCBI GenBank accession number is 1994).
  • the full genome length of mouse HuR gene is 40324bp (NCBI GenBank accession number is 15568).
  • the full genome length of the human BrG1 gene is 101,279 bp (NCBI GenBank accession number is 6597).
  • the full genome length of the mouse BrG1 gene is 88150 bp (NCBI GenBank accession number is 20586).
  • the full genome length of the human EZH2 gene is 76971bp (NCBI GenBank accession number is 2146).
  • the full genome length of the mouse EZH2 gene is 65102 bp (NCBI GenBank accession number is 14056).
  • the full genome length of the mouse CDYL gene is 214247 bp (NCBI GenBank accession number is 12593).
  • the full genome length of the human HMG20B gene is 249407bp (NCBI GenBank accession number is 9425) and 6168bp (NCBI GenBank accession number is 10362).
  • the full genome length of the mouse HMG20B gene is 4898 bp (NCBI GenBank accession number is 15353).
  • the full genome length of the human DPYSL2 gene is 144145bp (NCBI GenBank accession number is 1808).
  • the full genome length of the mouse DPYSL2 gene is 108178 bp (NCBI GenBank accession number is 12934).
  • the full genome length of the human BAF45b gene is 18690 bp (NCBI GenBank accession number is 8193).
  • the full genome length of the mouse BAF45b gene is 13652 bp (NCBI GenBank accession number is 29861).
  • the full genome length of the human SCF gene is 87679 bp (NCBI GenBank accession number is 4254).
  • the full genome length of the mouse SCF gene is 84802bp (NCBI GenBank accession number is 17311).
  • the full genome length of the human HuB gene is 160503 bp (NCBI GenBank accession number is 1993).
  • the full genome length of the mouse HuB gene is 188638 bp (NCBI GenBank accession number is 15569).
  • the full genome length of the human HuC gene is 29721bp (NCBI GenBank accession number is 1995).
  • the full genome length of the mouse HuC gene is 37142 bp (NCBI GenBank accession number is 15571).
  • the full genome length of the human HuD gene is 155718 bp (NCBI GenBank accession number is 1996).
  • the full genome length of the mouse HuD gene is 148212 bp (NCBI GenBank accession number is 15572).
  • the full genome length of the human CYP1B1 gene is 8643bp (NCBI GenBank accession number is 1545).
  • the full genome length of mouse CYP1B1 gene is 8122bp (NCBI GenBank accession number is 13078).
  • the full genome length of the human BTRC gene is 203506bp (NCBI GenBank accession number is 8945).
  • the full genome length of the mouse BTRC gene is 169664 bp (NCBI GenBank accession number is 12234).
  • the similarity between human and mouse RCOR1 at the DNA level is 88.51%; the protein sequence similarity is 92.39%.
  • the similarity between human and mouse RCOR2 at the DNA level is 89.26%; the protein sequence similarity is 97.51%.
  • the similarity between human and mouse RCOR3 at the DNA level is 92.38%; the protein sequence similarity is 96.39%.
  • the similarity between human and mouse Sin3a at the DNA level is 90.91%; the protein sequence similarity is 98.04%.
  • the similarity between human and mouse Sin3b at the DNA level is 86.61%; the protein sequence similarity is 87.91%.
  • the similarity between human and mouse HDAC1 at the DNA level is 90.85%; the protein sequence similarity is 99.38%.
  • the similarity between human and mouse HDAC2 at the DNA level is 91.62%; the protein sequence similarity is 99.59%.
  • the similarity between human and mouse KDM1A at the DNA level is 89.75%; the protein sequence similarity is 97.72%.
  • the similarity between human and mouse PHF21A at the DNA level is 92.88%; the protein sequence similarity is 94.20%.
  • the similarity between human and mouse BAF53a at the DNA level is 88.60%; the protein sequence similarity is 98.84%.
  • the similarity between human and mouse G9a at the DNA level is 87.74%; the protein sequence similarity is 94.75%.
  • the similarity between human and mouse USP14 at the DNA level is 91.31%; the protein sequence similarity is 96.76%.
  • the similarity between human and mouse HuR at the DNA level is 91.03%; the protein sequence similarity is 98.47%.
  • the similarity between human and mouse BrG1 at the DNA level is 89.00%; the protein sequence similarity is 95.47%.
  • the similarity between human and mouse EZH2 at the DNA level is 91.98%; the protein sequence similarity is 97.60%.
  • the similarity between human and mouse CDYL at the DNA level is 86.77%; the protein sequence similarity is 93.12%.
  • the similarity between human and mouse HMG20B at the DNA level is 85.53%; the protein sequence similarity is 93.69%.
  • the similarity between human and mouse DPYSL2 at the DNA level is 91.05%; the protein sequence similarity is 97.64%.
  • the similarity between human and mouse BAF45b at the DNA level is 93.39%; the protein sequence similarity is 97.69%.
  • the similarity between human and mouse SCF at the DNA level is 89.44%; the protein sequence similarity is 82.78%.
  • Human and mouse HuB in DNA water The similarity is 95.46%; the protein sequence similarity is 99.72%.
  • the similarity between human and mouse HuC at the DNA level is 91.94%; the protein sequence similarity is 99.46%.
  • the similarity between human and mouse HuD at the DNA level is 94.64%; the protein sequence similarity is 99.21%.
  • the similarity between human and mouse CYP1B1 at the DNA level is 82.96%; the protein sequence similarity is 81.03%.
  • nucleic acid sequence encoding it can be constructed based on it, and a specific probe can be designed based on the nucleotide sequence.
  • the full-length nucleotide sequence or its fragments can usually be obtained by PCR amplification, recombination or artificial synthesis.
  • primers can be designed based on the nucleotide sequences disclosed in the present disclosure, especially the open reading frame sequence, and commercially available cDNA libraries or cDNA prepared by conventional methods known to those skilled in the art can be used
  • the library is used as a template to amplify the relevant sequence. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the amplified fragments together in the correct order.
  • recombination can be used to obtain the relevant sequence in large quantities. This is usually done by cloning it into a vector, transferring it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.
  • artificial synthesis methods can also be used to synthesize relevant sequences, especially when the fragment length is short. Often, fragments with long sequences are obtained by first synthesizing multiple small fragments and then ligating them.
  • DNA sequence encoding the protein of the present disclosure can be obtained entirely through chemical synthesis.
  • the DNA sequence can then be introduced into a variety of existing DNA molecules (eg, vectors) and cells known in the art.
  • polynucleotide sequences of the present disclosure may be used to express or produce recombinant polypeptides by conventional recombinant DNA techniques. Generally speaking there are the following steps:
  • polynucleotide sequences can be inserted into recombinant expression vectors.
  • any plasmid and vector can be used as long as it can replicate and be stable in the host body.
  • An important feature of expression vectors is that they usually contain an origin of replication, a promoter, a marker gene, and translation control elements.
  • the DNA sequence can be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis.
  • the expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green color for eukaryotic cell culture.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green color for eukaryotic cell culture.
  • GFP Fluorescent protein
  • tetracycline or ampicillin resistance in E. coli tetracycline or ampicillin resistance in E. coli.
  • Vectors containing appropriate DNA sequences as described above and appropriate promoter or control sequences can be used to transform appropriate host cells to enable expression of proteins.
  • the host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • a prokaryotic cell such as a bacterial cell
  • a lower eukaryotic cell such as a yeast cell
  • a higher eukaryotic cell such as a mammalian cell.
  • Representative examples include: Escherichia coli, bacterial cells of the genus Streptomyces; fungal cells such as yeast; plant cells; insect cells; animal cells, etc.
  • Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art.
  • competent cells capable of taking up DNA can be harvested after the exponential growth phase and treated with the CaCl2 method, using steps well known in the art. Another method is to use MgCl 2 .
  • transformation can also be performed by electroporation.
  • DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
  • the obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of the present disclosure.
  • the medium used in culture can be selected from various conventional media. Cultivate under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced using an appropriate method (such as temperature shift or chemical induction), and the cells are cultured for a further period of time.
  • the recombinant polypeptide in the above method can be expressed within the cell, or on the cell membrane, or secreted outside the cell.
  • the recombinant protein can be isolated and purified by various separation methods utilizing its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitating agents (salting out method), centrifugation, osmotic sterilization, ultratreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • Adeno-associated virus is smaller than other viral vectors, non-pathogenic, and can transfect both dividing and undividing cells, gene therapy methods for genetic diseases based on AAV vectors have received considerable attention. Widespread concern.
  • Adeno-associated virus also known as adeno-associated virus, belongs to the genus Parvoviridae and is a type of single-stranded DNA defective virus with the simplest structure currently discovered. It requires a helper virus (usually an adenovirus) to participate in replication. It encodes the cap and rep genes in two terminal inverted repeats (ITRs). ITRs play a decisive role in virus replication and packaging. The cap gene encodes the viral capsid protein, and the rep gene is involved in virus replication and integration. AAV can infect a variety of cells.
  • Recombinant adeno-associated virus vector is derived from non-pathogenic wild-type adeno-associated virus. Due to its good safety, wide range of host cells (dividing and non-dividing cells), and low immunogenicity, it can express foreign genes in vivo for a long time. With long and other characteristics, it is regarded as one of the most promising gene transfer vectors and has been widely used in gene therapy and vaccine research around the world. After more than 10 years of research, the biological properties of recombinant adeno-associated viruses have been deeply understood, especially their application effects in various cells, tissues and in vivo experiments. A lot of data has been accumulated.
  • rAAV is used in gene therapy research for a variety of diseases (including in vivo and in vitro experiments); at the same time, as a unique gene transfer vector, it is also widely used in gene function research, construction of disease models, and gene preparation. aspects such as knockout mice.
  • the vector is a recombinant AAV vector.
  • AAVs are relatively small DNA viruses that integrate into the genome of the cells they infect in a stable and site-specific manner. They are able to infect a large range of cells without any effect on cell growth, morphology or differentiation, and they do not appear to be involved in human pathology.
  • the AAV genome has been cloned, sequenced and characterized.
  • AAV contains an inverted terminal repeat (ITR) region of approximately 145 bases at each end, which serves as the virus's origin of replication. The remainder of the genome is divided into two important regions with encapsidation functions: the left portion of the genome containing the rep genes involved in viral replication and viral gene expression; and the right portion of the genome containing the cap gene encoding the viral capsid protein.
  • ITR inverted terminal repeat
  • AAV vectors can be prepared using standard methods in the art. Adeno-associated virus of any serotype is suitable. Methods for purifying vectors can be found, for example, in U.S. Patent Nos. 6,566,118, 6,989,264, and 6,995,006, the disclosures of which are incorporated herein by reference in their entirety. The preparation of hybrid vectors is described, for example, in PCT Application No. PCT/US2005/027091, the disclosure of which is incorporated herein by reference in its entirety. The use of AAV-derived vectors for gene transport in vitro and in vivo has been described (see, for example, International Patent Application Publication Nos. WO91/18088 and WO93/09239; U.S. Patent Nos.
  • Replication-deficient recombinant AAVs can be prepared by co-transfecting a plasmid containing a nucleic acid sequence of interest flanked by two AAV inverted terminal repeats (ITRs) into a cell line infected with a human helper virus (eg, adenovirus). region, and a plasmid carrying AAV encapsidation genes (rep and cap genes). The resulting AAV recombinants are then purified by standard techniques.
  • ITRs AAV inverted terminal repeats
  • the recombinant vector is encapsidated into viral particles (eg, including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16 AAV virions).
  • viral particles eg, including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16 AAV virions.
  • the present disclosure includes recombinant virions (recombinant because they contain recombinant polynucleotides) containing any of the vectors described herein. Methods of producing such particles are known in the art and are described in U.S. Patent No. 6,596,535.
  • the AAV vector may be modified to include a modified VP protein (e.g., modified to include a VP1 protein, AAV vector of VP2 protein or VP3 protein).
  • the AAV vector is a recombinant AAV (rAAV) vector.
  • rAAV may consist of a capsid sequence and structure that is substantially similar to that found in wild-type AAV (wtAAV).
  • wtAAV wild-type AAV
  • rAAV encapsulates a genome that is essentially devoid of AAV protein coding sequences and has a therapeutic gene expression cassette designed in its place, such as the subject polynucleotide.
  • virally derived sequences may be ITRs that may be required to direct genome replication and packaging during vector production.
  • Suitable AAV vectors may be selected from any AAV serotype or combination of serotypes.
  • the AAV vector can be any of the following: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV 12, AAV13, AAV 14, AAV 15, AAV 16, AAV .rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5 , AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HS
  • the vector is selected based on its natural orientation. In some cases, the vector serotype is selected based on its ability to cross the blood-brain barrier. AAV9 and AAV10 have been shown to cross the blood-brain barrier to transduce neurons and glial cells. In one aspect, the AAV vector is AAV2, AAV5, AAV6, AAV8 or AAV9. In some cases, the AAV vector is a chimera of at least two serotypes. In one aspect, the AAV vectors are serotypes AAV2 and AAV5. In some cases, chimeric AAV vectors include rep and ITR sequences from AAV2 and cap sequences from AAV5.
  • chimeric AAV vectors include rep and ITR sequences from AAV2 and cap sequences from any other AAV serotype.
  • AAV vectors may be self-complementary.
  • AAV vectors may include inverted terminal repeats.
  • AAV vectors may include inverted terminal repeat (scITR) sequences with mutated terminal melting sites.
  • rep, cap and ITR sequences from all the different AAV serotypes provided herein can be mixed and matched.
  • the AAV vector is from an adeno-associated virus with a serotype selected from: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV 12, AAV13, AAV 14 , AAV 15, AAV 16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV .PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV .HSC9, AAV.HSC10, AAV.HSC11
  • the vector can be a recombinant AAV (rAAV) vector, a hybrid AAV vector, a chimeric AAV vector, a self-complementing AAV (scAAV) vector, a single-stranded AAV, or any combination thereof.
  • the AAV vector includes a genome comprising replicated genes and inverted terminal repeats from a first AAV serotype and capsid proteins from a second AAV serotype.
  • the AAV vector may be chimeric and may be: an AAV2/5 vector, an AAV2/6 vector, an AAV2/7 vector, an AAV2/8 vector, or AAV2/9 vector.
  • the inverted terminal repeats of the AAV vector include 5' inverted terminal repeats, 3' inverted terminal repeats, and mutated inverted terminal repeats. In some cases, the mutated inverted terminal repeat lacks a terminal melting site.
  • suitable AAV vectors can be further modified to encompass modifications such as in the capsid or rep protein. Modifications may also include deletions, insertions, mutations, and combinations thereof.
  • the carrier is modified to reduce immunogenicity, allowing for repeated administration. In some cases, when repeated administration is performed to reduce and/or eliminate immunogenicity, the serotype of the vector used changes.
  • the inhibitor is one or several types of negative regulators, which inhibit the expression of negative regulatory genes, or inhibit the mRNA levels of negative regulatory genes, or inhibit the levels of encoded proteins of negative regulatory genes.
  • various conventional screening methods can be used to screen out gene expression levels, or mRNA levels, or protein expression levels, or proteins that can enhance DPYSL2, BAF45b, SCF, HuB, HuC, HuD, CYP1B1, or BTRC. Positive regulator of activity.
  • the enhancer agent is one or several types of positive regulators that enhance the expression of positively regulated genes, or enhance the mRNA levels of positively regulated genes, or enhance the levels of encoded proteins of positively regulated genes.
  • Negative or positive modulators useful in the present disclosure may be substances that reduce, eliminate, or eliminate the expression and/or activity of the gene, its RNA (eg, mRNA), or its encoded protein at the DNA, RNA, or protein level.
  • the negative regulator includes inhibitory antibodies of negatively regulated genes; or small molecule inhibitors of negatively regulated genes; or inhibitory mRNA, microRNA, siRNA, shRNA, antisense oligos of negatively regulated genes. Nucleotides, binding proteins or protein domains, polypeptides, nucleic acid aptamers, or PROTACs; or inhibitory binding proteins or ligands that negatively regulate genes.
  • negative modulators of RCOR1 of the present disclosure include inhibitors targeting a target sequence comprising SEQ ID NO: 68.
  • Negative modulators of RCOR2 of the present disclosure include inhibitors targeting a target sequence comprising SEQ ID NO:69.
  • Negative modulators of RCOR3 of the present disclosure include inhibitors targeting a target sequence comprising SEQ ID NO:70.
  • Negative modulators of Sin3a of the present disclosure include inhibitors targeting a target sequence comprising SEQ ID NO:71.
  • Negative modulators of Sin3b of the present disclosure include inhibitors targeting a target sequence comprising SEQ ID NO:72.
  • Negative modulators of HDAC1 of the present disclosure include inhibitors targeting a target sequence comprising SEQ ID NO:73.
  • Negative modulators of KDM1A of the present disclosure include inhibitors targeting a target sequence comprising SEQ ID NO:74.
  • Negative modulators of HDAC2 of the present disclosure include inhibitors targeting a target sequence comprising SEQ ID NO:75.
  • Negative modulators of PHF21A of the present disclosure include inhibitors targeting a target sequence comprising SEQ ID NO:76.
  • the present disclosure negatively regulates BAF53a
  • Modulating agents include inhibitors targeting a target sequence comprising SEQ ID NO:77.
  • Negative modulators of G9a of the present disclosure include inhibitors targeting a target sequence comprising SEQ ID NO:78.
  • Negative modulators of USP14 of the present disclosure include inhibitors targeting a target sequence comprising SEQ ID NO:79.
  • Negative modulators of HuR of the present disclosure include inhibitors targeting a target sequence comprising SEQ ID NO:80.
  • Negative modulators of BrG1 of the present disclosure include inhibitors targeting a target sequence comprising SEQ ID NO:81.
  • Negative modulators of EZH2 of the present disclosure include inhibitors targeting a target sequence comprising SEQ ID NO:82.
  • Negative modulators of CDYL of the present disclosure include inhibitors targeting a target sequence comprising SEQ ID NO:83.
  • Negative modulators of HMG20B of the present disclosure include inhibitors targeting a target sequence comprising SEQ ID NO:84.
  • the targets of the negative regulator or positive regulator of the present disclosure include astrocytes or MG cells.
  • the positive regulator can be a substance that increases the expression and/or activity of a positively regulated gene, or its RNA (such as mRNA), or its encoded protein at the DNA, RNA, or protein levels.
  • positive regulators include: expression vectors containing promoters, endogenous expression activators, protein analogs or enhancers, epigenetic regulatory tools that can improve the expression of positively regulated genes, positively regulated genes Expression activators, degradation inhibitors of proteins encoded by positive regulatory genes, stabilizers of positive regulatory gene mRNA, or exogenous positive regulatory genes or functional fragments of positive regulatory genes.
  • the methods and steps of negative regulator regulation include using antibodies to neutralize its proteins, using shRNA or siRNA carried by viruses (such as adeno-associated viruses), or using gene editing tools to silence genes.
  • the inhibition rate of negative regulators on negatively regulated genes is generally at least 50% or more, preferably 60%, 70%, 80%, 90%, or 95% or more.
  • the inhibition rate can be controlled and detected based on conventional techniques, such as flow cytometry, fluorescence quantitative PCR or Western blot.
  • Negative regulators including antibodies, antisense nucleic acids, gene editing tools and other inhibitors
  • positive regulators including: expression vectors containing promoters, endogenous expression activators, protein analogs or Enhancer
  • these materials when administered therapeutically, can inhibit or enhance the expression and/or activity of the gene or protein, thereby inducing the differentiation of glial cells into neuronal cells, thereby treating neuronal dysfunction. or death-related illness.
  • these materials may be formulated in a nontoxic, inert, and pharmaceutically acceptable aqueous medium, usually at a pH of about 5-8, preferably at a pH of about 6-8, although the pH may vary with the materials being formulated. It varies depending on the nature of the condition and the condition to be treated.
  • the prepared pharmaceutical composition can be administered by conventional routes, including (but not limited to): topical, intramuscular, intracranial, intraocular, intraperitoneal, intravenous, subcutaneous, intradermal, local administration, autologous Cells are extracted and cultured and then reinfused.
  • the present disclosure also provides a pharmaceutical composition, which contains a safe and effective amount of the negative modulator or positive modulator of the present disclosure and a pharmaceutically acceptable carrier or excipient.
  • Such carriers include, but are not limited to: saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof.
  • the drug formulation should match the mode of administration.
  • the pharmaceutical composition of the present disclosure can be formulated into an injection form, for example, using physiological saline or an aqueous solution containing glucose and other adjuvants by conventional methods. Make preparations.
  • Pharmaceutical compositions, such as tablets and capsules can be prepared by conventional methods.
  • Pharmaceutical compositions such as injections, solutions, tablets and capsules are preferably manufactured under sterile conditions.
  • the active ingredient is administered in a therapeutically effective amount, for example, about 1 microgram to 10 mg/kg of body weight per day.
  • This disclosure finds for the first time that RCOR1, RCOR2, RCOR3, Sin3a, Sin3b, HDAC1, HDAC2, KDM1A, PHF21A, BAF53a, G9a, USP14, HuR, BrG1,
  • EZH2, CDYL, or HMG20B genes or their encoded proteins and/or increasing the expression of DPYSL2, BAF45b, SCF, HuB, HuC, HuD, CYP1B1, or BTRC genes or their encoded proteins in glial cells or activity, which can induce the differentiation of glial cells into dopamine neuron cells, thereby preventing and/or treating diseases related to neuronal function loss or death.
  • DPYSL2, BAF45b, SCF, HuB, HuC, HuD, CYP1B1, or BTRC in glial cells, and directly transform Muller cells into RGC cells or photoreceptor cells.
  • Regenerated RGCs can be integrated into the visual pathway and improve visual function in RGC injury mouse models.
  • nSR100 In order to overexpress nSR100 in glial cells, the glial cell-specific promoter GFAP was selected to initiate the expression of nSR100.
  • Transient transfection of astrocytes and qPCR Isolate primary mouse astrocytes and seed astrocytes in 6-well plates. Transient transfection was performed with 3 ⁇ g of vector expressing gRNA-CasRx-GFP using Lipofectamine 3000 (Thermo Fisher Scientific) according to standard procedures. Control plasmid expresses non-targeting gRNA. 1-2 days after transient transfection, GFP-positive cells were collected by flow cytometric cell sorting (FACS) and lysed for qPCR analysis.
  • FACS flow cytometric cell sorting
  • AAV8 was used to carry the gene of interest.
  • the titer of AAV-CasRx, AAV-CasRx-gRNA, or AAV-GFAP-positive regulatory gene is greater than 5 ⁇ 10 12 vg/ml (1-3 ⁇ l per injection).
  • AAV was injected into the striatum (AP+0.8mm, ML ⁇ 1.6mm and DV-2.8mm).
  • Immunofluorescence staining Mice were perfused 6-12 weeks after injection, and brains were removed and fixed with 4% paraformaldehyde (PFA) overnight and dehydrated in 30% sucrose for at least 12 hours. After embedding, the sections were frozen and sectioned to a thickness of 30 ⁇ m. Before immunofluorescence staining, brain sections were rinsed thoroughly with 0.1 M phosphate buffered saline (PBS). Primary antibodies: rabbit polyclonal NeuN antibody (Brain, 1:500, #ABN78, Millipore), mouse TH antibody (1:300, MAB318, Millipore).
  • RNA-seq 293T or N2a cells were cultured in 15-cm dishes and transiently transfected with 70 ⁇ g of plasmid. The first 20% of GFP and mCherry double-positive N2a cells were collected by FACS, RNA was extracted, converted to cDNA, and then used for whole-transcriptome RNA-seq.
  • Intravitreal and subretinal injections were introduced via intravitreal and subretinal injections, respectively, as previously described.
  • Subretinal injection Inject into the eye using a Hamilton syringe (32G needle) under an Olympus microscope AAV (>1 ⁇ 10 13 vg/ml). To determine reprogramming in intact retinas, a total of 1 ⁇ l of GFAP-GFP-Cre (0.2 ⁇ l) and AAV-GFAP-nSR100, or GFAP-CasRx-gRNA (0.8 ⁇ l), or GFAP-GFP-Cre (0.2 ⁇ l) and GFAP-CasRx (0.8 ⁇ l) were delivered to the retina.
  • Immunofluorescence staining 1.5-3 months after AAV injection, the eyes, optic nerve and brain were removed and fixed with 4% paraformaldehyde (PFA) for 2 hours (eyes and optic nerve) or 24 hours (brain), and then incubated in 30% sucrose. Dehydrate in solution for 2 hours (eyes and optic nerves) or 24 hours (brain). After embedding and freezing, eyes and brains were sectioned at a thickness of 30 ⁇ m.
  • PFA paraformaldehyde
  • gRNAs targeting RCOR1, Sin3a, HDAC2, KDM1A, PHF21A, BAF53a, G9a, USP14, HuR, BrG1, EZH2, CDYL, and HMG20B were designed, and CasRx and gRNA were co-administered. Transfect N2A cells.
  • nucleotide sequence and target sequence of gRNA are shown in Table 1 above.
  • the transfection method is: transiently transfect 4 ⁇ g CAG-CasRx-P2A-GFP plasmid and 2 ⁇ g U6-gRNA-CMV-mCherry plasmid to determine the inhibitory effect of each gRNA in in vitro cell lines. Use CAG-CasRx-P2A-GFP plasmid alone. Transfection served as control. Lipofectamine 3000 (Thermo Fisher Scientific) was used according to standard procedures. Two days after transfection, 30,000 GFP and mCherry double-positive cells were collected from each sample by fluorescence-activated cell sorting (FACS), and lysed for qPCR analysis. See Table 2 for qPCR primers.
  • FACS fluorescence-activated cell sorting
  • Example 2 Study on the transdifferentiation of astrocytes into neurons by negative regulatory genes
  • AAV-GFAP was injected into the striatum of C57BL/6 mice older than eight weeks.
  • the plasmid design is shown in Figure 2A and B.
  • Figure 4 shows the statistics of astrocytes converted into neurons after knocking down different negative regulatory genes. Among them, RCOR1, G9a, BAF53A, etc. are more efficient in inducing glial cells to transdifferentiate into neurons.
  • Example 3 Study on the transdifferentiation of astrocytes into neurons by positive regulatory genes
  • AAV in the control or test group was injected into the striatum or substantia nigra of mice, in which vector 1 is AAV-GFAP-mCherry, and vector 2 (AAV-GFAP-Gene) is driven by the astrocyte-specific promoter GFAP.
  • Target gene expression Similar to the knockdown target gene expression group, samples were taken 1-3 months after AAV injection to analyze the efficiency of different targets in inducing glial cells to transdifferentiate into neurons. The results are shown in Figures 5 and 6.
  • Example 7 In order to further study whether the overexpression of positive regulatory genes and the inhibition of negative regulatory genes can promote the transdifferentiation of astrocytes into dopamine neurons, referring to the operations of Example 1 to Example 3, we injected AAV in the control group or test group was stained with the dopamine neuron-specific protein marker TH. The results are shown in Figure 7. No evidence was found in the control group. TH-positive cells ( Figure 7A), TH-positive dopamine neurons co-labeled with mCherry signals were found in the HuB and BTRC overexpression group, indicating that overexpression of HuB and BTRC can induce astrocytes to transform into dopamine neurons. differentiation.
  • Müller glia cells perform functions similar to glial cells, and the retina contains a variety of neurons with specialized functions, such as photoreceptor cells and bipolar cells.
  • GFAP-EGFP-2A-Cre system combined with Ai9 mice to achieve specific labeling of Müller glia.
  • the schematic diagram is shown in Figure 8. Referring to the operations of Examples 1 to 3, AAV-GFAP-CasRx-gRNA (or A mixed virus of control vector AAV-GFAP-CasRx) and AAV-GFAP-EGFP-2A-Cre.
  • AAV-GFAP-EGFP-2A-Cre is used to specifically label Müller glial cells or transformed from glial cells.
  • Neurons (retinal ganglion cells or photoreceptor cells), 1-3 months after injection, the retina is taken for slice analysis to study whether the corresponding negative regulatory genes are inhibited or the corresponding positive regulatory genes are upregulated.
  • Müller glia transdifferentiate into photoreceptor cells.
  • GFAP-EGFP-2A-Cre+GFAP-CasRx was used as the control group, and GFAP-EGFP-2A-Cre+GFAP-CasRx-gRNA was used as the test group.
  • the gRNA targeted Sin3a, HDAC2, BAF53a, G9a, HuR, BrG1, EZH2, CDYL, and KDM1A.
  • GFAP-EGFP-2A-Cre was used as the control group, and GFAP-EGFP-2A-Cre+GFAP-positive regulatory genes were used as the test group, among which the positive regulatory genes were DPYSL2, BAF45b, SCF, CYP1B1, and BTRC genes.
  • FIG. 9A The effects of negative regulatory genes on transdifferentiation are shown in Figure 9.
  • Figure 9A in the GFAP-EGFP-2A-Cre group, Müller glial cells showed a typical morphology, with the cell body located in the inner granular layer (INL) and processes distributed to the outer granular layer (ONL) and retinal ganglion cell layer. (GCL), and no red signal-labeled photoreceptor cells were observed in the ONL, indicating that there would be no transdifferentiation of Müller glia into photoreceptor cells in the control group.
  • CasRx knocks down the expression of negative regulatory genes or overexpresses positive regulatory genes to reprogram Müller glia into photoreceptor cells.
  • the quantitative statistics are shown in Figure 11, showing inhibition of Sin3a, KDM1A, BAF53a, G9a, HuR , BrG1, EZH2, and CDYL all show excellent photoreceptor cell regeneration ability.
  • Inhibiting HDAC2 also has photoreceptor cell regeneration ability, and overexpressing DPYSL2 and BAF45b also has photoreceptor cell regeneration ability.
  • Photoreceptor cells are divided into cones and rods.
  • Sin3a and KDM1A groups to use rod-specific Staining with sex protein markers, as shown in Figure 12, found that most of the photoreceptor cells transdifferentiated from Müller glia cells in the ONL layer were rod cells.
  • Müller glia can be transdifferentiated into retinal ganglion cells (RGC), subretinal injection of different AAV combinations into NMDA-modeled 6-8 week Ai9 (Rosa26-CAG-LSL-tdTomato-WPRE) mice .
  • RRC retinal ganglion cells
  • AAV-GFAP-CasRx-gRNA or control vector AAV-GFAP-CasRx
  • AAV-GFAP-EGFP-2A-Cre is used to specifically label Müller glia or neurons transformed from glial cells (optic ganglion cells).
  • AAV-GFAP-EGFP-2A-Cre was injected as the control group, and AAV-GFAP-EGFP-2A-Cre+AAV-GFAP-positive regulatory genes were injected (the positive regulatory genes were DPYSL2, BAF45b, SCF, HuB , HuC, HuD, CYP1B1, BTRC).
  • the number of cells and the statistical chart of the number of tdTomato-positive axons in the optic nerve are shown in Figure 16. More optic ganglion cells were found in the SCF, HuD, and CYP1B1 groups.
  • the Pde6b gene is a disease-causing gene related to retinitis pigmentosa (RP). Defects in the Pde6b gene can lead to the death of photoreceptor cells.
  • RP retinitis pigmentosa
  • Pde6b gene-deficient mice have been proven to be disease animal models with genetic characteristics of RP.
  • a Pde6b gene knockout mouse model constructed based on CRISPR-Cas9 editing was used to prove through immunohistochemical methods In Pde6b knockout mice, a large number of rod cells died at 4 weeks after birth, and all rod cells died at 6 weeks ( Figure 17A-C).
  • AAV AAV-U6-gRNA(KDM1A)-GFAP-CasRx and AAV-U6-gRNA(EZH2)-GFAP-CasRx
  • KDM1A and EZH2 transdifferentiation targets
  • EZH2 transdifferentiation targets
  • AAV-CBH-Pde6b AAV overexpressing Pde6b
  • the control group was simultaneously injected with control AAV (AAV-GFAP-CasRx) and AAV overexpressing Pde6b (AAV-CBH-Pde6b).
  • the injection volume of transdifferentiation target AAV The injection volume of Pde6b overexpressing AAV was 1x10 10 vg, and the injection volume was 5x10 9 vg, and the total volume of injected AAV was 1 ⁇ l.
  • retinal tissue was taken for analysis. It was found that there were no photoreceptor cells in the outer granular layer of the retina of mice injected with AVV (AAV-GFAP-CasRx+AAV-CBH-Pde6b) in the control group, using rod-specific markers. Rhodopsin was stained, and no Rhodopsin-positive cells were found (Figure 17D).
  • Example 3 and Example 4 the applicant described in detail the results of injecting the negative regulator or the positive regulator of the present application into the striatum.
  • Those skilled in the art can refer to the results of the present application.
  • Technical solutions and common knowledge in the field can choose to perform positioning injections in other parts of the brain, such as targeting the ventral tegmental area of the midbrain, spinal cord, hypothalamus, dorsal midbrain, cerebral cortex, hippocampus, cerebellum, or Injections into the substantia nigra, especially injections into the substantia nigra, also have the same or similar technical effects.
  • Example 5 the applicant described in detail the results of subretinal injection of the negative regulator or positive regulator of the present application.
  • Those skilled in the art can rely on the technical solutions of the present application and the According to conventional knowledge, you can choose to perform positioning injections in other parts of the eye, such as the vitreous cavity, which will also have the same or similar technical effects.

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Abstract

提供了将哺乳动物的非神经元细胞转分化为神经元的方法，其中，包括：提供能够降低负调控基因表达的负向调节剂，负调控基因包括RCOR1、RCOR2、RCOR3、Sin3a、Sin3b、HDAC1、HDAC2、KDM1A、PHF21A、BAF53a、G9a、USP14、HuR、BrG1、EZH2、CDYL、或HMG20B，和/或提供能够提高正调控基因表达的正向调节剂，正调整基因包括DPYSL2、BAF45b、SCF、HuB、HuC、HuD、CYP1B1、或BTRC，有效量的所述负向调节剂或所述正向调节剂与所述非神经元细胞接触，能够诱导非神经元动物细胞转分化为神经元。

Description

非神经元细胞转分化为神经元的方法及应用
本申请要求2022年8月30日递交的申请号为PCT/CN2022/115708的专利的优先权。
技术领域
本申请涉及转化医学领域，更具体的，涉及将非神经元细胞转分化为神经元的方法，以及该方法用于制备治疗或预防神经元损伤或神经元死亡相关的疾病的药物的用途。
背景技术
神经系统损伤和多种神经退行性疾病引起的主要病理变化是神经元变性坏死和神经元损伤。由于神经系统(例如：大脑、脊髓、视神经等)的自我修复的能力非常有限，很难自主修复神经元细胞，与神经元功能缺失或神经元死亡相关的疾病一直是治疗的难点，如帕金森病、精神分裂症、抑郁症、老年痴呆、亨廷顿舞蹈症、睡眠失调、脑外伤、脑卒中、与RGC或感光细胞功能缺失或死亡有关的视觉系统疾病、失明、耳聋等，目前的治疗手段仅仅是缓解病情进展，一直缺乏有效的治疗手段。
细胞的转分化(cell transdifferentiation)是指一种类型的分化细胞在一定的条件下，能够在结构和功能上转变成另一种分化细胞的过程。如果能将神经系统的一些非神经元细胞重编程为神经元细胞，将有望从根本上治疗与神经元功能缺失或神经元死亡相关的疾病。
最近的研究表明，调控神经系统的某些基因的表达，可以引导非神经元细胞转化为神经元，例如抑制PTBP1，能够诱导胶质细胞转分化为神经元。然而，目前这些研究还都处于早期研发阶段，本领域依然缺乏有效的将非神经元细胞转分化为神经元的手段。
发明内容
本公开内容的一个目的在于提供在体内由胶质细胞产生神经元细胞的方法。
本公开内容的另一目的在于提供一种或更多种选自以下的基因或其RNA或其编码蛋白的表达或活性的抑制剂用于预防和/或治疗与神经元功能缺失或死亡相关的疾病的用途：RCOR1、RCOR2、RCOR3、Sin3a、Sin3b、HDAC1、HDAC2、KDM1A、PHF21A、BAF53a、G9a、USP14、HuR、BrG1、EZH2、CDYL、HMG20B。
本公开内容的另一目的在于提供一种或更多种选自以下的基因或其RNA或其编码蛋白的表达或活性的增强剂用于预防和/或治疗与神经元功能缺失或死亡相关的疾病的用途：DPYSL2、BAF45b、SCF、HuB、HuC、HuD、CYP1B1、BTRC。
在一个实施方案中，本公开内容提供了由胶质细胞产生神经元细胞的方法，其包括使用 一种或更多种选自以下的基因或其RNA或其编码蛋白的表达或活性的抑制剂使所述胶质细胞转分化为或重编程为神经元：RCOR1、RCOR2、RCOR3、Sin3a、Sin3b、HDAC1、HDAC2、KDM1A、PHF21A、BAF53a、G9a、USP14、HuR、BrG1、EZH2、CDYL、HMG20B，其中所述抑制剂降低所述基因或其RNA或其编码蛋白的表达或活性，所述方法包括：
(i)提供所述抑制剂；
(ii)提供所述胶质细胞；
(iii)使所述抑制剂以有效地引起胶质细胞转分化或重编程为神经元细胞的量与所述胶质细胞接触；以及
(iv)任选地，鉴定和/或分离经转分化或重编程的细胞。
在一个实施方案中，本公开内容提供了由胶质细胞产生神经元细胞的方法，其包括使用一种或更多种选自以下的基因或其RNA或其编码蛋白的表达或活性的增强剂使所述胶质细胞转分化为或重编程为神经元：DPYSL2、BAF45b、SCF、HuB、HuC、HuD、CYP1B1、BTRC，其中所述增强剂提高所述基因或其RNA或其编码蛋白的表达或活性，所述方法包括：
(i)提供所述增强剂；
(ii)提供所述胶质细胞；
(iii)使所述增强剂以有效地引起胶质细胞转分化或重编程为神经元细胞的量与所述胶质细胞接触；以及
(iv)任选地，鉴定和/或分离经转分化或重编程的细胞。
在一个优选的实施方案中，所述胶质细胞为哺乳动物胶质细胞，包括人、非人灵长类动物、小鼠、大鼠物种的胶质细胞。
在一个更优选的实施方案中，所述哺乳动物胶质细胞包括星形胶质细胞、少突胶质细胞、小胶质细胞、NG2细胞、穆勒胶质细胞、胶质瘤细胞或螺旋神经节胶质细胞。
在一个更优选的实施方案中，所述胶质细胞是星形胶质细胞或穆勒胶质细胞。
在一个优选的实施方案中，所述星形胶质细胞来源于大脑、中脑、小脑、脑干、脊髓。
在一个更优选的实施方案中，所述星形胶质细胞来源于纹状体或黑质。
在一个更优选的实施方案中，所述穆勒胶质细胞来源于视网膜。
在一个更优选的实施方案中，所述螺旋神经节胶质细胞来源于内耳或前庭。
在一个优选的实施方案中，所述神经元细胞为哺乳动物神经元，包括人、非人灵长类动物、大鼠、小鼠的神经元。
在一个更优选的实施方案中，其中所述神经元细胞选自多巴胺神经元、5-HT神经元、NE神经元、ChAT神经元、运动神经元、GABA神经元、谷氨酸能神经元、脊髓神经元，脊髓运动神经元、脊髓感觉神经元、感光细胞(视杆细胞和视锥细胞)、双极细胞、无长突细胞、 视网膜神经节细胞(RGC)、耳蜗神经细胞、锥体神经元、中间神经元、中型多棘神经元(medium spiny neuron，MSN)、浦肯野细胞、颗粒细胞、嗅感觉神经元、球周细胞或其组合，更优选多巴胺神经元，视网膜神经节细胞和感光细胞。
在一个更优选的实施方案中，其中所述胶质细胞是星形胶质细胞；并且所述神经元细胞是多巴胺神经元。
在一个更优选的实施方案中，其中所述胶质细胞是穆勒胶质细胞；并且所述神经元细胞是RGC或感光细胞。
在另一个实施方案中，本公开内容提供了一种或更多种选自以下的基因或其RNA或其编码蛋白的表达或活性的抑制剂在制备药物中的用途：RCOR1、RCOR2、RCOR3、Sin3a、Sin3b、HDAC1、HDAC2、KDM1A、PHF21A、BAF53a、G9a、USP14、HuR、BrG1、EZH2、CDYL、HMG20B，所述药物用于预防和/或治疗与神经元功能缺失或死亡相关的疾病，其中所述抑制剂降低所述基因或其RNA或其编码蛋白的表达或活性。
在另一个实施方案中，本公开内容提供了一种或更多种选自以下的基因或其RNA或其编码蛋白的表达或活性的增强剂在制备药物中的用途：DPYSL2、BAF45b、SCF、HuB、HuC、HuD、CYP1B1、BTRC，所述药物用于预防和/或治疗与神经元功能缺失或死亡相关的疾病，其中所述增强剂提高所述基因或其RNA或其编码蛋白的表达或活性。
在一个优选的实施方案中，所述药物配制成用于在体内施用于神经系统、视觉系统和听觉系统，包括纹状体、黑质、中脑腹侧被盖区、脊髓、下丘脑、背侧中脑、大脑皮层、海马、小脑、视网膜下、玻璃体腔、内耳耳蜗和前庭。
在一个更优选的实施方案中，所述药物配制成用于施用于纹状体、黑质、视网膜下和玻璃体腔。
在一个优选的实施方案中，所述与神经元功能缺失或死亡相关的疾病为神经系统疾病，包括帕金森病、精神分裂症、抑郁症、阿尔茨海默病、亨廷顿舞蹈症、癫痫、睡眠失调、共济失调、PloyQ疾病、脑缺血、脑损伤、成瘾、运动神经元病、肌萎缩性侧索硬化、脊髓性肌萎缩症、Pick病、与RGC或感光细胞功能缺失或死亡有关的视觉系统疾病、失明、耳聋。
在一个更优选的实施方案中，所述与神经元功能缺失或死亡相关的疾病为帕金森病和与RGC或感光细胞功能缺失或死亡有关的视觉系统疾病。
在另一个实施方案中，本公开内容提供了由穆勒胶质细胞产生视网膜神经节细胞(RGC)或感光细胞的方法，其包括用一种或更多种选自以下的基因或其RNA或其编码蛋白的表达或活性的抑制剂使所述穆勒胶质细胞转分化为或重编程为RGC或感光细胞：RCOR1、RCOR2、RCOR3、Sin3a、Sin3b、HDAC1、HDAC2、KDM1A、PHF21A、BAF53a、G9a、USP14、HuR、BrG1、EZH2、CDYL、HMG20B，其中所述抑制剂降低所述基因或其RNA或其编码蛋 白的表达或活性。
在另一个实施方案中，本公开内容提供了由穆勒胶质细胞产生视网膜神经节细胞(RGC)或感光细胞的方法，其包括用一种或更多种选自以下的基因或其RNA或其编码蛋白的表达或活性的增强剂使所述穆勒胶质细胞转分化为或重编程为RGC或感光细胞：DPYSL2、BAF45b、SCF、HuB、HuC、HuD、CYP1B1、BTRC，其中所述增强剂提高所述基因或其RNA或其编码蛋白的表达或活性。
在一个优选的实施方案中，所述穆勒胶质细胞来源于视网膜。
在一个优选的实施方案中，所述感光细胞包括视杆细胞和视锥细胞。
另一个实施方案中，本公开内容提供了一种或更多种选自以下的基因或其RNA或其编码蛋白的表达或活性的抑制剂在制备药物中的用途：RCOR1、RCOR2、RCOR3、Sin3a、Sin3b、HDAC1、HDAC2、KDM1A、PHF21A、BAF53a、G9a、USP14、HuR、BrG1、EZH2、CDYL、HMG20B，所述药物用于预防和/或治疗与RGC或感光细胞功能缺失或死亡有关的视觉系统疾病，其中所述抑制剂降低所述基因或其RNA或其编码蛋白的表达或活性。
在另一个实施方案中，本公开内容提供了一种或更多种选自以下的基因或其RNA或其编码蛋白的表达或活性的增强剂在制备药物中的用途：DPYSL2、BAF45b、SCF、HuB、HuC、HuD、CYP1B1、BTRC，所述药物用于预防和/或治疗与RGC或感光细胞功能缺失或死亡有关的视觉系统疾病，其中所述增强剂提高所述基因或其RNA或其编码蛋白的表达或活性。
在一个优选的实施方案中，所述药物配制成用于施用于视觉系统。
在一个更优选的实施方案中，所述药物配制成用于视网膜下或玻璃体腔，其中所述药物通过作用于穆勒胶质细胞来发挥作用。
在一个优选的实施方案中，所述与RGC功能缺失或死亡有关的神经系统疾病选自：RGC细胞死亡导致的视力损伤、青光眼、年龄相关的RGC病变、糖尿病相关视网膜病变、视神经损伤、视网膜缺血或出血、Leber遗传性视神经病变、或其组合。
在一个优选的实施方案中，所述与感光细胞功能缺失或死亡有关的视觉系统疾病选自：损伤或退行性病变导致的感光细胞变性或死亡、黄斑变性、视网膜色素变性、糖尿病有关的失明、夜盲症、色盲、遗传性失明、先天性黑蒙症或其组合。
在一个优选的实施方案中，所述抑制剂或激动剂选自：抗体、小分子化合物、mRNA,microRNA、siRNA、shRNA、反义寡核苷酸、结合蛋白或蛋白结构域、多肽、核酸适配体、基因编辑器、PROTAC、包含启动子的表达载体、内源性表达激活剂、蛋白质类似物或增强剂，人工合成或修饰的以上所述抑制剂或增强剂或其组合。
在另一个的实施方案中，本公开内容提供了药物组合物或药盒或试剂盒，其包含一种或更多种选自以下的基因或其RNA或其编码蛋白的表达或活性的抑制剂：RCOR1、RCOR2、 RCOR3、Sin3a、Sin3b、HDAC1、HDAC2、KDM1A、PHF21A、BAF53a、G9a、USP14、HuR、BrG1、EZH2、CDYL、HMG20B；和/或一种或更多种选自以下的基因或其RNA或其编码蛋白的表达或活性的增强剂：DPYSL2、BAF45b、SCF、HuB、HuC、HuD、CYP1B1、BTRC。
在一个优选的实施方案中，所述抑制剂包含：
(a)基因编辑蛋白或其表达载体，以及
(b)一个或多个gRNA或其表达载体，其中所述gRNA是引导基因编辑蛋白特异性结合所述基因的DNA或RNA。
在一个优选的实施方案中，编辑系统包括：CRISPR系统(包括CRISPR/dCas系统)、ZFN系统、TALEN系统、RNA编辑系统，或其组合。
在一个优选的实施方案中，所述基因编辑蛋白为靶向RNA的基因编辑蛋白。
在一个优选的实施方案中，所述gRNA为靶向RNA的gRNA。
在一个优选的实施方案中，其中所述增强剂包含：包含启动子的表达载体、内源性表达激活剂、蛋白质类似物或增强剂。
在一个优选的实施方案中，所述药物组合物或药盒或试剂盒还包含用于递送所述抑制剂的运载体。
在一个优选的实施方案中，所述运载体为病毒载体、脂质体、纳米颗粒、外泌体、类病毒颗粒，优选AAV。
在一个优选的实施方案中，所述靶向RNA的基因编辑蛋白选自：Cas13d、Cas13e、Cas13a、Cas13b、Cas13c、Cas13f及其功能结构域。
在一个更优选的实施方案中，所述靶向RNA的基因编辑蛋白选自：CasRx、Cas13e、Cas13f。
在一个更优选的实施方案中，所述靶向RNA的基因编辑蛋白为CasRx。
在一个优选的实施方案中，所述药物组合物或药盒或试剂盒包含靶向所述mRNA序列的仅单一类型的gRNA或2、3、4、5、6、7、8、9、10、11、12种不同的gRNA。
在另一个优选的实施方案中，所述gRNA表达载体编码包含靶向所述mRNA序列的仅单一类型的gRNA或2、3、4、5、6、7、8、9、10、11、12种不同的gRNA。
在一个优选的实施方案中，所述表达载体包含：
i)编码所述基因编辑蛋白的核苷酸序列，其与引起所述基因编辑蛋白表达的启动子可操作地连接，以及
ii)至少一种编码靶向所述mRNA或DNA序列的gRNA的核苷酸序列，所述核苷酸序列与引起所述gRNA在哺乳动物细胞中表达的启动子例如U6启动子可操作地连接。
在一个优选的实施方案中，所述启动子是广谱启动子或特异性启动子。
在一个更优选的实施方案中，其中所述广谱启动子选自CMV、CBH、CAG、PGK、SV40、EF1A、EFS、pGlobin启动子。
在一个更优选的实施方案中，所述特异性启动子是胶质细胞特异性启动子或穆勒胶质细胞(MG)细胞特异性启动子。
在一个更优选的实施方案中，所述胶质细胞特异性启动子选自GFAP启动子、ALDH1L1启动子、EAAT1/GLAST启动子、谷氨酰胺合成酶启动子、S100β启动子和EAAT2/GLT-1启动子、NG2启动子、CD68启动子、F4/80启动子，或者所述MG细胞特异性启动子选自GFAP启动子、ALDH1L1启动子、Glast(也称为Slc1a3)启动子和Rlbp1启动子。
在一个优选的实施方案中，所述表达载体包含在病毒颗粒中。
在一个优选的实施方案中，所述表达载体是基因治疗载体。
在一个更优选的实施方案中，所述基因治疗载体是病毒基因治疗载体。
在一个更优选的实施方案中，所述表达载体是选自以下的病毒载体：腺相关病毒(AAV)载体、重组腺相关病毒载体(rAAV)、自互补腺相关病毒载体(scAAV)、腺病毒载体、慢病毒载体、逆转录病毒载体、疱疹病毒载体、SV40载体、痘病毒载体、及其组合。
在一个更优选的实施方案中，所述表达载体是AAV载体或rAAV。
在一个优选的实施方案中，所述组合物局部施用至以下至少一种：i)视网膜中的胶质细胞；ii)纹状体中的胶质细胞，优选壳核中的胶质细胞；iii)黑质中的胶质细胞；iv)内耳中的胶质细胞；v)脊髓中的胶质细胞；vi)前额皮质中的胶质细胞；vii)运动皮质中的胶质细胞；viii)丘脑中的胶质细胞；ix)腹侧被盖区(VTA)中的胶质细胞；x)海马(Hippocampus)中的胶质细胞；xi)小脑中的胶质细胞；以及xii)脑干中的胶质细胞。
在一个优选的实施方案中，所述组合物还包含i)一种或更多种神经元相关因子，或ii)用于在所述胶质细胞中表达一种或更多种神经元相关因子的至少一种表达载体。
在一个更优选的实施方案中，所述一种或更多种神经元相关因子选自：AscL1、Mytl1、Ngn1、Ngn2、NeuroD1、NeuroD2、NeuroG1、Pax6、Ptbp1、P53、Zic1、Ctdsp1、miR-9、miR-9-9*、miR-124、miR-124-124*、Let-7、Let-7b、miR-132、NeuroG2、Brn2、NeuroD4、Insm1、Prox1、FoxG1、Lhx6、Bcl2、Dlx1、Dlx2、Tlx3、Gata2、Gata3、Sox11、Lhx3、IsL1等。
在一个优选的实施方案中，所述药物组合物或药盒或试剂盒还包含i)一种或更多种多巴胺神经元相关因子，或ii)用于在所述胶质细胞中表达一种或更多种多巴胺神经元相关因子的至少一种表达载体。
在一个优选的实施方案中，所述组合物还配制成用于细胞转染、细胞感染、细胞内吞、 注射、颅内给药、脊髓给药、眼内给药、耳内给药、吸入、肠胃外施用、静脉内施用、肌内施用、皮内施用、表面施用或经口施用，以及离体诱导转分化或重编程并将经转分化或重编程的细胞移植回体内。
在一个优选的实施方案中，所述AAV载体包含：
i)编码所述基因编辑蛋白的核苷酸序列，其与引起所述基因编辑蛋白在胶质细胞中表达的启动子可操作地连接；以及
ii)至少一种编码靶向所述mRNA或DNA序列的gRNA的核苷酸序列，其与引起所述gRNA在哺乳动物细胞中表达的启动子例如U6启动子可操作地连接。
在一个优选的实施方案中，所述启动子是广谱启动子或特异性启动子。
在一个更优选的实施方案中，其中所述广谱启动子选自CMV、CBH、CAG、PGK、SV40、EF1A、EFS、pGlobin启动子。
在一个更优选的实施方案中，所述特异性启动子是胶质细胞特异性启动子或穆勒胶质细胞(MG)细胞特异性启动子。
在一个更优选的实施方案中，所述胶质细胞特异性启动子选自GFAP启动子、ALDH1L1启动子、EAAT1/GLAST启动子、谷氨酰胺合成酶启动子、S100β启动子和EAAT2/GLT-1启动子、NG2启动子、CD68启动子、F4/80启动子。
在一个更优选的实施方案中，所述MG细胞特异性启动子选自GFAP启动子、ALDH1L1启动子、Glast(也称为Slc1a3)启动子和Rlbp1启动子。
在一个优选的实施方案中，胶质细胞的转分化效率为至少0.1％，或至少1％、2％、3％、4％、5^％、6％、7％、8％、9％、10％、11％、12％、13％、14％、15％、16％、17％、18％、19％、20％、21％、22％、23％、24％、25％、26％、27％、28％、29％、30％、31％、32％、33％、34％、35％、36％、37％、38％、39％、40％、41％、42％、43％、44％、45％、46％、47％、48％、49％、50％、51％、52％、53％、54％、55％、56％、57％、58％、59％、60％、或更高。
在另一个优选实施方案中，所述与神经元功能缺失或死亡相关的疾病选自：帕金森病、精神分裂症、抑郁症、RGC细胞死亡导致的视力损伤、糖尿病相关视网膜病变、青光眼、年龄相关的RGC病变、视神经损伤、视网膜缺血或出血、Leber遗传性视神经病变、损伤或退行性病变导致的感光细胞变性或死亡、黄斑变性、视网膜色素变性、糖尿病有关的失明、夜盲症、色盲、遗传性失明、先天性黑蒙症或其组合。
在另一个优选实施方案中，所述RGC可整合到视觉通路中，并改善视觉功能。
在另一个优选实施方案中，所述RGC可以实现对中央视觉区域的功能性投射，并改善视觉功能。
在另一个优选实施方案中，所述改善视觉功能是改善患有神经变性引起的视网膜疾病的哺乳动物的视觉功能。
在另一个优选实施方案中，所述MG细胞转分化为RGC细胞的同时，还分化为无轴突细胞。
在另一个优选实施方案中，对所述纹状体中的多个神经胶质细胞进行重编程或转分化，并且其中至少1％、2％、3％、4％、5％、6％、7％、8％、9％、10％、11％、12％、13％、14％、15％、16％、17％、18％、19％、20％、21％、22％、23％、24％、25％、26％、27％、28％、29％或至少30％的胶质细胞被转化为多巴胺神经元。
在另一个优选实施方案中，所述哺乳动物包括患有与神经元功能缺失或死亡相关的疾病的哺乳动物。
在另一个优选实施方案中，所述哺乳动物包括人或非人哺乳动物。
在另一个优选实施方案中，所述非人哺乳动物包括啮齿动物(如小鼠、大鼠、或兔)、灵长类动物(如猴)。
在另一个优选实施方案中，所述基因编辑器由神经胶质细胞特异性启动子(例如GFAP启动子)驱动表达。
在另一个优选实施方案中，所述基因编辑器包括1个或多个gRNA和基因编辑蛋白。
在另一个优选实施方案中，所述gRNA引导基因编辑蛋白特异性结合所述基因的RNA。
在另一个优选实施方案中，所述gRNA引导基因编辑蛋白特异性结合所述基因的mRNA。
在另一个优选实施方案中，所述gRNA的核苷酸序列参见如下表1。
表1：gRNA的核苷酸序列及靶序列

如本申请所用，在RNA序列的背景下字母“u”可与字母“t”互换使用，以代表尿苷或尿苷酸。
在另一个优选实施方案中，所述基因编辑蛋白的来源选自：酿脓链球菌(Streptococcus pyogenes)、葡萄球菌(Staphylococcus aureus)、氨基酸球菌属(Acidaminococcussp)、毛螺科菌(Lachnospiraceae bacterium)、黄化瘤胃球菌(Ruminococcusflavefaciens)、或其组合。
在另一个优选实施方案中，所述基因或其RNA或其编码蛋白来源于哺乳动物；优选地，来源于人、猴、小鼠、大鼠、或兔；更优选地，来源于人。
在另一个优选实施方案中，所述基因包括野生型基因和突变型基因。
在另一个优选实施方案中，所述突变型包括突变后编码蛋白的功能未发生改变的突变形式(即功能与野生型编码蛋白相同或基本相同)。
在另一个优选实施方案中，所述突变型基因编码的多肽与野生基因所编码的多肽相同或基本相同。
在另一个优选实施方案中，所述突变型基因包括与野生基因相比，同源性≥80％(优选地≥90％，≥91％，≥92％，≥93％或≥94％，更优选地≥95％，≥96％或≥97％，更优选地，≥98％或99％)的多核苷酸。
在另一个优选实施方案中，所述突变型基因包括在野生型基因的5＇端和/或3＇端截短或添加1-60个(优选地1-30，更优选地1-10个)核苷酸的多核苷酸。
在另一个优选实施方案中，所述基因包括cDNA序列、基因组序列、或其组合。
在另一个优选实施方案中，所述蛋白包括活性片段或其衍生物。
在另一个优选实施方案中，所述活性片段或其衍生物与所述基因或其RNA或其编码蛋白的同源性至少为90％，91％，92％，93％或94％，优选为95％，96％或97％，更优选为98％、99％。
在另一个优选实施方案中，所述活性片段或其衍生物至少具有80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的活性。
在另一个优选实施方案中，所述蛋白的氨基酸序列选自：
(i)具有SEQ ID NO:1、3、5、7、9、11、13、15、17、19、21、23、25、27、29、31、 33、35、37、39、41、43、45、47、49所示氨基酸序列的多肽；
(ii)将SEQ ID NO:1、3、5、7、9、11、13、15、17、19、21、23、25、27、29、31、33、35、37、39、41、43、45、47、49所示氨基酸序列经过一个或几个(如1-10个)氨基酸残基的取代、缺失或添加而形成的，具有所述蛋白功能的、由(i)衍生的多肽；或
(iii)氨基酸序列与SEQ ID NO:1、3、5、7、9、11、13、15、17、19、21、23、25、27、29、31、33、35、37、39、41、43、45、47、49所示氨基酸序列的同源性≥90％、≥91％、≥92％、≥93％或≥94％(优选地≥95％，≥96％或≥97％，更优选地≥98％或99％)，具有所述蛋白功能的多肽。
在另一个优选实施方案中，所述基因的核苷酸序列选自：
(a)编码具有如上所示氨基酸序列的多肽的多核苷酸；
(b)SEQ ID NO:2、4、6、8、10、12、14、16、18、20、22、24、26、28、30、32、34、36、38、40、42、44、46、48、50所示序列的多核苷酸；
(c)核苷酸序列与SEQ ID NO:2、4、6、8、10、12、14、16、18、20、22、24、26、28、30、32、34、36、38、40、42、44、46、48、50所示核苷酸序列的同源性≥90％、≥91％、≥92％、≥93％或≥94％(优选地≥95％，≥96％或≥97％，更优选地≥98％或99％)的多核苷酸；
(d)在具有SEQ ID NO:2、4、6、8、10、12、14、16、18、20、22、24、26、28、30、32、34、36、38、40、42、44、46、48、50所示核苷酸序列的多核苷酸的5’端和/或3’端截短或添加1-60个(优选地1-30，更优选地1-10个)核苷酸的多核苷酸；
(e)与(a)-(d)任一所述多核苷酸互补的多核苷酸。
在另一个优选实施方案中，所述蛋白如上述氨基酸序列所示。
在另一个优选实施方案中，所述蛋白的编码核酸如上述核苷酸序列所示。
在另一个优选实施方案中，所述抑制剂(如基因编辑蛋白)靶向的区域为所述基因序列的第121931-121963位(RCOR1)、第2364-2393位(RCOR2)、第53556-53585位(RCOR3)、第59338-59367位(Sin3a)、第12378-12406位(Sin3b)、第24648-24678位(HDAC1)和第60126-60154位(KDM1A)。
在另一个优选实施方案中，所述抑制剂或增强剂抑制或增强所述基因或其RNA或其编码蛋白的活性和/或表达量。
在另一个优选实施方案中，所述抑制剂或增强剂的浓度(病毒的滴度)＞1×1012。
在另一个优选实施方案中，所述抑制剂或增强剂对所述基因或其RNA或其编码蛋白的活性和/或表达量的抑制率或增强率大于90％，优选地，90％-95％。
在另一个优选实施方案中，所述抑制剂或增强剂靶向脑组织的星形胶质细胞。
在另一个优选实施方案中，所述抑制剂或增强剂靶向视网膜的MG细胞。
在另一个优选实施方案中，所述gRNA引导基因编辑蛋白特异性结合所述基因的mRNA。
在另一个优选实施方案中，所述组合物包括药物组合物。
在另一个优选实施方案中，所述组合物还包括其他预防和/或治疗与神经元功能缺失或死亡相关的疾病的药物。
在另一个优选实施方案中，所述组合物还包括其他用于治疗神经元死亡相关的神经系统疾病的药物。
在另一个优选实施方案中，所述组合物还包括其他预防和/或治疗视网膜疾病的药物。
在另一个优选实施方案中，所述基因编辑蛋白的表达载体包括靶向胶质细胞的载体。
在另一个优选实施方案中，所述基因编辑蛋白的表达载体包括靶向脑组织星形胶质细胞的载体。
在另一个优选实施方案中，所述基因编辑蛋白的表达载体包括靶向视网膜MG细胞的载体。
在另一个优选实施方案中，所述载体包括AAV2或AAV9。
在另一个优选实施方案中，所述基因编辑蛋白的编码基因与gRNA位于同一表达载体(如AAV载体)。
在另一个优选实施方案中，所述基因编辑蛋白的表达载体与gRNA的表达载体为同一表达载体(如AAV载体)。
在另一个优选实施方案中，所述表达载体还包括神经胶质细胞特异性启动子(例如GFAP启动子)，用于驱动所述基因编辑蛋白的表达。
在另一个优选实施方案中，所述组合物的剂型选自：冻干制剂、液体制剂、或其组合。
在另一个优选实施方案中，所述组合物的剂型为液体制剂。
在另一个优选实施方案中，所述组合物的剂型为注射剂型。
在另一个优选实施方案中，其他预防和/或治疗与神经元功能缺失或死亡相关的疾病的药物选自：多巴胺前体药物、非麦角类多巴胺受体激动剂、单胺氧化酶B抑制剂、或其组合。
在另一个优选实施方案中，所述组合物为细胞制剂。
在另一个优选实施方案中，所述基因编辑蛋白的表达载体和gRNA的表达载体为同一载体或不同载体。
在另一个优选实施方案中，所述组分(a)与组分(b)的重量比为100:1-0.01:1，优选地，10:1-0.1:1，更优选地，2:1-0.5:1。
在另一个优选实施方案中，所述组合物中，所述组分(a)的含量为0.001％-99％，优选地，0.1％-90％，更优选地，1％-70％。
在另一个优选实施方案中，所述组合物中，所述组分(b)的含量为0.001％-99％，优选地， 0.1％-90％，更优选地，1％-70％。
在另一个优选实施方案中，所述组合物中，所述组分(c)的含量为1％-99％，优选地，10％-90％，更优选地，30％-70％。
在另一个优选实施方案中，所述组合物中，所述组分(a)和组分(b)和任选的组分(c)占所述组合物总重的0.01-99.99wt％，优选地0.1-90wt％，更优选地1-80wt％。
在另一个实施方案中，本公开内容提供了一种药盒，其包含：
(a1)第一容器，以及位于所述第一容器中的基因编辑蛋白或其表达载体，或含有基因编辑蛋白或其表达载体的药物，所述基因编辑蛋白选自：Cas13d、Cas13e、Cas13a、Cas13b、Cas13c、Cas13f、RNA靶向基因编辑蛋白、或其组合；
(b1)第二容器，以及位于所述第二容器中的gRNA或其表达载体，或含有gRNA或其表达载体的药物，所述gRNA引导基因编辑蛋白特异性结合所述基因的DNA或RNA。
在另一个优选实施方案中，所述药盒还包含：
(c1)第三容器，以及位于所述第三容器中的其他预防和/或与神经元功能缺失或死亡相关的疾病的药物，和/或含有其他预防和/或治疗视网膜疾病的药物，和/或含有其他治疗神经元死亡相关的神经系统疾病的药物。
在另一个优选实施方案中，所述第一容器和第二容器、第三容器是相同或不同的容器。
在另一个优选实施方案中，所述第一容器的药物是含基因编辑蛋白或其表达载体的单方制剂。
在另一个优选实施方案中，所述第二容器的药物是含gRNA或其表达载体的单方制剂。
在另一个优选实施方案中，所述第三容器的药物是含其他预用于治疗神经元死亡相关的神经系统疾病的药物的单方制剂。
在另一个优选实施方案中，所述药物的剂型选自：冻干制剂、液体制剂、或其组合。
在另一个优选实施方案中，所述药物的剂型为口服剂型或注射剂型。在另一个优选实施方案中，所述试剂盒还含有说明书。
具体来说，本申请还涉及如下方案：
项1.将哺乳动物的非神经元细胞转分化为神经元的方法，其中，包括：
提供能够降低负调控基因表达的负向调节剂，所述负调控基因包括RCOR1、RCOR2、RCOR3、Sin3a、Sin3b、HDAC1、HDAC2、KDM1A、PHF21A、BAF53a、G9a、USP14、HuR、BrG1、EZH2、CDYL、或HMG20B，和/或
提供能够提高正调控基因表达的正向调节剂，所述正调整基因包括DPYSL2、BAF45b、SCF、HuB、HuC、HuD、CYP1B1、或BTRC，
有效量的所述负向调节剂或所述正向调节剂与所述非神经元细胞接触，诱导非神经元动 物细胞转分化为神经元。
在一个具体的方式中，所述方法为体外、离体或体内的方法。
在一个具体的方式中，负调控基因涉及的蛋白的序列如下：RCOR1为SEQ ID NO：1所示的氨基酸序列。RCOR2为SEQ ID NO：3所示的氨基酸序列。RCOR3为SEQ ID NO：5所示的氨基酸序列。Sin3a为SEQ ID NO：7所示的氨基酸序列。Sin3b为SEQ ID NO：9所示的氨基酸序列。HDAC1为SEQ ID NO：11所示的氨基酸序列。HDAC2为SEQ ID NO：13所示的氨基酸序列。KDM1A为SEQ ID NO：15所示的氨基酸序列。PHF21A为SEQ ID NO：17所示的氨基酸序列。BAF53a为SEQ ID NO：19所示的氨基酸序列。G9a为SEQ ID NO：21所示的氨基酸序列。USP14为SEQ ID NO：23所示的氨基酸序列。HuR为SEQ ID NO：25所示的氨基酸序列。BRG1为SEQ ID NO：27所示的氨基酸序列。EZH2为SEQ ID NO：29所示的氨基酸序列。CDYL为SEQ ID NO：31所示的氨基酸序列。HMG20B为SEQ ID NO：33所示的氨基酸序列。
在一个具体的方式中，正调控基因涉及的蛋白的序列如下：DPYSL2为SEQ ID NO：35所示的氨基酸序列。BAF45b为SEQ ID NO：37所示的氨基酸序列。SCF为SEQ ID NO：39所示的氨基酸序列。HuB为SEQ ID NO：41所示的氨基酸序列。HuC为SEQ ID NO：43所示的氨基酸序列。HuD为SEQ ID NO：45所示的氨基酸序列。CYP1B1为SEQ ID NO：47所示的氨基酸序列。BTRC为SEQ ID NO：49所示的氨基酸序列。
项2.如项1所述的方法，其中，所述非神经元细胞来源于人。
项3.如项1或项2所述的方法，其中，所述非神经元细胞为干细胞、祖细胞或终末分化细胞；优选为胶质细胞；更优选的，所述胶质细胞为星形胶质细胞、少突胶质细胞、小胶质细胞、NG2细胞、穆勒胶质细胞、胶质瘤细胞或螺旋神经节胶质细胞，更优选的，所述胶质细胞为星形胶质细胞或穆勒胶质细胞。
项4.如项3所述的方法，其中，所述非神经元细胞来源于脑部，优选的，所述非神经元细胞来自大脑、中脑、小脑、脑干、脊髓；更优选的，来源于大脑中的纹状体或黑质。
项5.如项4所述的方法，其中，所述非神经元细胞为来源于脑部的星形胶质细胞，优选的，为来源于纹状体或黑质的星形胶质细胞。
项6.如项4所述的方法，其中，所述非神经元细胞来源于眼部，优选的，所述非神经元细胞为来自眼部的穆勒胶质细胞。
项7.如项1至项6任一所述的方法，其中，所述神经元为多巴胺能神经元、视网膜神经节细胞、感光细胞、5-HT神经元、NE神经元、ChAT神经元、运动神经元、GABA神经元、谷氨酸能神经元、脊髓神经元，脊髓运动神经元、脊髓感觉神经元、双极细胞、无长突细胞、耳蜗神经细胞、锥体神经元、中间神经元、中型多棘神经元、浦肯野细胞、颗粒细胞、嗅感觉神经元、或球周细胞、或其组合。
项8.如项1至项7任一所述的方法，其中，所述负调控基因为RCOR1、RCOR2、RCOR3、Sin3a、Sin3b、KDM1A、BAF53a、G9a、HuR、BrG1、或EZH2，所述非神经元动物细胞来源于脑部或眼部。
项9.如项8所述的方法，其中，所述负调控基因为RCOR1、RCOR2、RCOR3或G9a，所述非神经元动物细胞来源于脑部；优选的，所述非神经元细胞为来自脑部的星形胶质细胞；更优选的，所述非神经元细胞为来自纹状体或黑质的星形胶质细胞。
项10.如项8所述的方法，其中，所述负调控基因为Sin3a、Sin3b、KDM1A、BAF53a、G9a、HuR、BrG1、CDYL、或EZH2，所述非神经元细胞来源于眼部；优选的，所述非神经元细胞为来自眼部穆勒胶质细胞。
项11.如项10所述的方法，其中，所述负调控基因为HuR，所述神经元为视神经节细胞。
项12.如项10所述的方法，其中，所述负调控基因为Sin3a、KDM1A、BAF53a、G9a、HuR、BrG1、CDYL、或EZH2，所述神经元为感光细胞。
项13.如项1至项7任一所述的方法，其中，所述正调控基因为DPYSL2、BAF45b、SCF、HuB、HuC、HuD、CYP1B1、或BTRC，所述非神经元细胞来自脑部；优选的，所述非神经元细胞来自纹状体、黑质、中脑腹侧被盖区、脊髓、下丘脑、背侧中脑、大脑皮层、海马、小脑；更优选的，来自纹状体。
项14.如项13所述的方法，其中，所述正调控基因为SCF或HuB。
项15.如项13所述的方法，其中，所述正调控基因为HuB或BTRC，有效量的所述正向调节剂与所述非神经元细胞接触，诱导非神经元类非神经元细胞转分化为多巴胺能神经元。
项16.如项13-15任一所述的方法，其中，所述非神经元细胞为星形胶质细胞；更优选的，所述非神经元细胞为来自纹状体或黑质的星形胶质细胞。
项17.如项1至项7任一所述的方法，其中，所述正调控基因为DPYSL2、BAF45b、SCF、HuC、HuD、或CYP1B1，所述非神经元细胞来自眼部；优选的，所述非神经元细胞为来自眼部穆勒胶质细胞。
项18.如项17所述的方法，其中，所述正调控基因为SCF、HuD、或CYP1B1，所述神经元为视神经节细胞。
项19.如项17所述的方法，其中，所述正调控基因为DPYSL2或BAF45b，所述神经元为感光细胞。
项20.如项1-19任一所述的方法，其中，所述降低负调控基因表达是能够降低负调控基因的基因水平、或降低负调控基因的mRNA水平、或降低负调控基因的编码蛋白表达水平；
所述提高正调控基因表达是能够提高正调控基因的基因水平、或提高正调控基因的mRNA水平、或提高正调控基因的编码蛋白表达水平。
项21.如项20所述的方法，其中，所述负向调节剂选自降低负调控基因表达的基因编辑工具或表观遗传调控工具；负调控基因的抑制剂、负调控基因活性抑制剂、或负调控基因的编码蛋白的降解激活剂。
项22.如项21所述的方法，其中，所述抑制剂为：负调控基因的抑制性抗体；或负调控基因的小分子抑制剂；或负调控基因的抑制性mRNA、microRNA、siRNA、shRNA、反义寡核苷酸、结合蛋白或蛋白结构域、多肽、核酸适配体、或PROTAC；或负调控基因的抑制性结合蛋白或配体。
项23.如项21所述的方法，其中，所述的负向调节剂含有如SEQ ID NO：51-67中任一所述的gRNA及基因编辑蛋白。
项24.如项20所述的方法，其中，所述正向调节剂选自能够提高正调控基因表达的表观遗传调控工具、正调控基因表达激活剂、正调控基因编码蛋白的降解抑制剂、正调控基因mRNA的稳定剂、或外源性的正调控基因或正调控基因的功能性片段。
项25.如项24所述的方法，其中，所述激活剂为：正调控基因的激动性抗体；或正调控基因的小分子激动剂；或正调控基因的激动性结合蛋白或配体；或正调控基因的竞争性基因的抑制剂。
项26.如项24所述的方法，其中，所述正向调节剂含有如SEQ ID NO：36、38、40、42、44、46、48、或50所示的核酸序列，或含有如SEQ ID NO：36、38、40、42、44、46、48、或50所示的核酸序列的功能性片段。
项27.如项21所述的基因编辑工具，其中，所述基因编辑工具包括基因编辑系统或其表达载体，所述基因编辑系统有选自：CRISPR系统(包括CRISPR/Cas系统)、ZFN系统、TALEN系统、或其组合。
项28.根据项27所述的方法，其中，采用CRISPR系统降低负调控基因的表达或活性；优选的，所述CRISPR系统含有Cas酶或Cas酶的功能结构域的编码核酸以及靶向所述细胞转分化因子的gRNA；更优选的，所述Cas酶为Cas13；更优选的，所述Cas酶为Cas13d、Cas13X、Cas13a、Cas13b、Cas13c、或Cas13Y；更优选的，所述Cas酶为CasRx。
项29.根据项20所述的方法，其中，所述负向调节剂或正向调节剂采用运载体携带；优选的，所述运载体为病毒载体、脂质纳米颗粒(LNP)、脂质体、阳离子聚合物(如PEI)、纳米颗粒、外泌体、或类病毒颗粒；更优选的，所述载体为AAV载体或脂质纳米颗粒。
项30.如项1-29任一所述的方法，其中，所述有效量的所述负向调节剂或所述正向调节剂与所述非神经元细胞在体外接触，诱导非神经元细胞在体外转分化为神经元；或
所述有效量的所述负向调节剂或所述正向调节剂与所述非神经元细胞在体内接触，诱导非神经元细胞在体外转分化为神经元。
项31.项1-30任一所涉及的负向调节剂或正向调节剂用于制备预防或治疗与神经元损伤或神经元死亡相关的疾病的药物的用途。
项32.项1-30任一所涉及的负向调节剂或正向调节剂用于预防或治疗与神经元损伤或神经元死亡相关的疾病的用途。
项33.如项31或32所述的用途，其中，所述药物配制成用于在体内施用于神经系统、视觉系统和听觉系统的药剂，例如体内施用于纹状体、黑质、中脑腹侧被盖区、脊髓、下丘脑、背侧中脑、大脑皮层、海马、小脑、视网膜下、玻璃体腔、内耳耳蜗和前庭，优选纹状体、黑质、视网膜下和玻璃体腔。
项34.如项31或32所述的用途，其中，所述与神经元损伤或神经元死亡相关的疾病选自帕金森病、RGC或感光细胞功能缺失或死亡有关的视觉系统疾病、阿尔茨海默病、脑损伤、亨廷顿舞蹈症、癫痫、抑郁症、睡眠失调、脑缺血、运动神经元病、肌萎缩性侧索硬化、脊髓性肌萎缩症、共济失调、PloyQ疾病、精神分裂症、成瘾、Pick病、失明、耳聋；优选为帕金森病和RGC或感光细胞功能缺失或死亡有关的视觉系统疾病。
项35.如项34所述的用途，其中，所述与RGC或感光细胞功能缺失或死亡有关的视觉系统疾病优选自：RGC细胞或感光细胞死亡导致的视力损伤、青光眼、年龄相关的RGC病变、视神经损伤、年龄相关性黄斑变性(AMD)、糖尿病相关视网膜病变、视网膜缺血或出血、Leber遗传性视神经病变、或其组合；所述与感光细胞功能缺失或死亡有关的视觉系统疾病优选自：损伤或退行性病变导致的感光细胞变性或死亡、黄斑变性、视网膜色素变性、糖尿病有关的失明、夜盲症、色盲、遗传性失明、先天性黑蒙症、或其组合。
项36.根据项31或32所述的用途，其中，所述神经元为多巴胺能神经元、5-HT神经元、NE神经元、ChAT神经元、GABA神经元、谷氨酸能神经元、运动神经元、感光细胞(如视杆细胞和视锥细胞)、视网膜神经节细胞(RGC)、耳蜗神经细胞(如耳蜗螺旋神经节细胞和前庭神经元)、或中型多棘神经元(MSN)或其组合，优选多巴胺能神经元、视网膜神经节细胞和感光细胞。
项37.一种预防或治疗与神经元损伤或神经元死亡相关的疾病的方法，包括给有此需要的受试者有效量的项1-30任一所涉及的负向调节剂或正向调节剂。
项38.根据项37的方法，其中，所述与神经元损伤或神经元死亡相关的疾病选自帕金森病、RGC或感光细胞功能缺失或死亡有关的视觉系统疾病、阿尔茨海默病、脑损伤、亨廷顿舞蹈症、癫痫、抑郁症、睡眠失调、脑缺血、运动神经元病、肌萎缩性侧索硬化、脊髓性肌萎缩症、共济失调、PloyQ疾病、精神分裂症、成瘾、Pick病、失明、耳聋；优选为帕金森病和RGC或感光细胞功能缺失或死亡有关的视觉系统疾病。
项39.如项38所述的方法，其中，所述与RGC功能缺失或死亡有关的视觉系统疾病优选 自：RGC细胞死亡导致的视力损伤、青光眼、年龄相关的RGC病变、视神经损伤、年龄相关性黄斑变性(AMD)、糖尿病相关视网膜病变、视网膜缺血或出血、Leber遗传性视神经病变、或其组合；所述与感光细胞功能缺失或死亡有关的视觉系统疾病优选自：损伤或退行性病变导致的感光细胞变性或死亡、黄斑变性、视网膜色素变性、糖尿病有关的失明、夜盲症、色盲、遗传性失明、先天性黑蒙症、或其组合。
项40.根据项37所述的方法，其中，所述神经元为多巴胺能神经元、5-HT神经元、NE神经元、ChAT神经元、GABA神经元、谷氨酸能神经元、运动神经元、感光细胞(如视杆细胞和视锥细胞)、视网膜神经节细胞(RGC)、耳蜗神经细胞(如耳蜗螺旋神经节细胞和前庭神经元)、或中型多棘神经元(MSN)或其组合，优选多巴胺能神经元、视网膜神经节细胞和感光细胞。
本申请所涉及的技术方案还包括：
项1-项30任一所涉及的KDM1A或EZH2的负向调节剂或正向调节剂用于制备预防或治疗与视神经元损伤或视神经元死亡相关的疾病的药物的用途。
在一些具体实施方式中，视神经元损伤或视神经元死亡相关的疾病可以是视网膜色素变性(RP)。
应理解，在本公开内容范围内中，本公开内容的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1：CasRx在体外特异性敲低各目的基因的结果。在体外N2A细胞系中，表达CasRx和靶向RCOR1、Sin3a、HDAC2、PHF21A、BAF53a、KDM1A、G9a、USP14、HuR、BrG1、EZH2、CDYL、HMG20B的gRNA可以实现对RCOR1、Sin3a、HDAC2、KDM1A、PHF21A、BAF53a、G9a、USP14、HuR、BrG1、EZH2、CDYL、HMG20B的高效敲低。
图2：敲低或过表达不同的目的基因将小鼠星形胶质细胞转分化为神经元示意图。图2A的载体1(AAV-GFAP-mCherry)由星形胶质细胞特异性启动子GFAP驱动荧光蛋白mCherry表达，载体2(AAV-GFAP-CasRx)由星形胶质细胞特异性启动子GFAP驱动RNA编辑蛋白CasRx表达，载体3(AAV-GFAP-CasRx-sg)编码CasRx和靶向RCOR1、Sin3a、HDAC2、KDM1A、PHF21A、BAF53a、G9a、USP14、HuR、BrG1、EZH2、CDYL、或HMG20B的gRNA；图2B的载体1为GFAP驱动的mCherry的载体示意图，载体4为GFAP驱动的目的基因表达的载体示意图，目的基因分别为DPYSL2、BAF45b、SCF、HuB、HuC、HuD、CYP1B1、或BTRC。
图3：在小鼠纹状体中，敲低不同的负调控基因后，将星形胶质细胞转化为神经元的情况。图3A显示，在小鼠的纹状体中注射对照组AAV(AAV-GFAP-mCherry+AAV-GFAP- CasRx)后，AAV-GFAP-mCherry能够特异性标记星形胶质细胞而且不会向神经元转分化，黄色箭头(图3A中的箭头为黄色箭头)指示被GFAP-mCherry标记的星形胶质细胞，不与NeuN共标，NeuN为神经元特异性标志物；图3B至图3M分别为在小鼠纹状体中，利用CasRx敲低RCOR1、HDAC2、PHF21A、BAF53a、G9a、USP14、HuR、BrG1、EZH2、CDYL、HMG20B、KDM1A将星形胶质细胞转分化为神经元的结果，红色信号为AAV-GFAP-mCherry标记的细胞(图3A至图3M的最左栏为mCherry标记的结果，图中亮色的部分为红色)，NeuN为神经元特异性标志物，白色箭头指示红色mCherry信号与NeuN共标的细胞(图3B至图3M中的箭头为白色箭头)。标尺为50微米。
图4：敲低不同的负调控基因后将星形胶质细胞转化为神经元的统计图。
图5：在小鼠纹状体中，过表达不同的正调控基因将星形胶质细胞转化为神经元的情况。图5A是在小鼠大脑纹状体中注射对照组AAV(AAV-GFAP-mCherry)特异性标记星形胶质细胞，黄色箭头(图5A中的箭头为黄色箭头)指示被GFAP-mCherry标记的星形胶质细胞不与NeuN共标，NeuN为神经元特异性标志物；图5B至图5I为在小鼠大脑纹状体中注射测试组AAV(AAV-GFAP-mCherry+AAV-GFAP-正调控基因)的结果，其中，正调控基因分别为DPYSL2、BAF45b、SCF、HuB、HuC、HuD、CYP1B1、BTRC，白色箭头指示红色mCherry信号与NeuN共标的细胞(图5A至图5I的最左栏为mCherry标记的结果，图中亮色的部分为红色，图5B至图5I中的箭头为白色箭头)，NeuN为神经元特异性标志物。标尺为50微米。
图6：上调不同的正调控基因后将星形胶质细胞转化为神经元的统计情况。
图7：在小鼠纹状体中过表达BTRC或HuB后，星形胶质细胞转化为多巴胺能神经元的情况。图7A为在小鼠纹状体中注射对照组AAV(AAV-GFAP-mCherry)，黄色箭头(图7A中的箭头为黄色箭头)指示被标记的mCherry阳性细胞不与TH共标，也不与NeuN和共标，TH是多巴胺能神经元特异性蛋白标志物，NeuN为神经元特异性标志物；图7B和图7C分别为在小鼠大脑纹状体中注射测试组AAV(AAV-GFAP-mCherry+AAV-GFAP-正调控基因)的结果，其中正调控基因为HuB和BTRC，白色箭头(图7B和C中的箭头为白色箭头)指示红色mCherry信号与NeuN和TH共标的细胞。标尺为50微米。其中在mCherry栏中亮色部分为红色，在TH栏中亮色部分为绿色，在NeuN栏中亮色部分为白色，在DAPI中亮色部分为蓝色。
图8：在视网膜中过表达或敲低目的基因将穆勒胶质细胞转化为视网膜神经节细胞或感光细胞示意图。图8A的载体1为AAV-GFAP-EGFP-2A-Cre的质粒示意图，由星形胶质细胞特异性启动子GFAP驱动Cre表达，载体2为AAV-GFAP-CasRx的质粒示意图，由星形胶质细胞特异性启动子GFAP驱动RNA编辑蛋白CasRx表达，载体3为AAV-GFAP-CasRx-gRNA的质粒示意图，编码CasRx和靶向负调控基因(Sin3a、HDAC2、KDM1A、BAF53a、G9a、HuR、BrG1、EZH2、CDYL)的gRNA。图8B为在视网膜中过表达各正调控基因将穆勒胶质细胞转 分化为视网膜神经节细胞或感光细胞的示意图，载体1为GFAP驱动的mCherry，载体4为GFAP驱动的目的基因表达，目的基因为DPYSL2、BAF45b、SCF、CYP1B1、或BTRC。
图9：在小鼠眼内敲低负调控基因将穆勒胶质细胞转化为感光细胞的情况。图9A为在Ai9小鼠视网膜中注射对照组AAV(AAV-GFAP-EGFP-P2A-Cre)特异性标记穆勒胶质细胞。黄色箭头(图9A中的箭头为黄色箭头)指示被GFAP-EGFP-P2A-Cre特异性标记的穆勒胶质细胞，穆勒胶质细胞胞体位于INL层，在ONL层未发现被tdTomato标记的红色细胞。图9B-图9J分别为在小鼠视网膜下注射测试组不同的AAV(AAV-GFAP-mCherry+AAV-GFAP-CasRx-gRNA)，其中gRNA分别靶向Sin3a、HDAC2、BAF53a、G9a、HuR、BrG1、EZH2、CDYL、KDM1A等基因，白色箭头指示在ONL层tdTomato阳性细胞，黄色箭头指示INL层tdTomato阳性穆勒胶质细胞，图9A至图9J的最左栏为mCherry标记的结果，图中亮色的部分为红色，图9A至图9J中INL一栏的箭头为黄色箭头，ONL一栏的箭头为白色箭头。
图10：图10A和图10B分别为在小鼠视网膜下注射测试组AAV(AAV-GFAP-EGFP-P2A-Cre+AAV-GFAP-正调控基因)后穆勒胶质细胞转化为感光细胞的情况，正调控基因分别为DPYSL2和BAF45b，白色箭头指示在ONL层tdTomato阳性细胞，黄色箭头指示INL层tdTomato阳性穆勒胶质细胞。标尺为50微米，图中的最左栏为mCherry标记的结果，左栏中亮色的部分为红色，图中INL一栏的箭头为黄色箭头，ONL一栏的箭头为白色箭头。
图11：穆勒胶质细胞重编程为感光细胞的细胞统计图。
图12：在小鼠眼内敲低Sin3a或KDM1A，将穆勒胶质细胞转化为视杆细胞的情况。图12A为在Ai9小鼠视网膜中注射AAV-GFAP-EGFP-P2A-Cre+AAV-GFAP-CasRx的结果，白色信号为视杆细胞特异性蛋白标志物rhodopsin染色，黄色箭头(图12A中的箭头为黄色箭头)指示tdTomato标记的穆勒胶质细胞位于INL层，在ONL层没有tdTomato阳性细胞。图12B和图12C为在Ai9小鼠视网膜下注射测试组靶向Sin3a和KDM1A的AAV(AAV-GFAP-mCherry+AAV-GFAP-CasRx-gRNA)的转分化结果，白色箭头指示位于ONL层的tdTomato阳性细胞与rhodopsin共标。标尺为50微米。图12A至图12C的最左栏为mCherry标记的结果，左栏中亮色的部分为红色，图12B和C中的箭头为白色箭头。
图13：在小鼠眼内敲低Sin3a或EZH2，将穆勒胶质细胞转化为视锥细胞的情况。图13A为在Ai9小鼠视网膜中注射AAV-GFAP-EGFP-P2A-Cre的结果，GFAP-EGFP-P2A-Cre能特异性标记穆勒胶质细胞，穆勒胶质细胞胞体位于INL层，在ONL层未发现被tdTomato标记的红色细胞，绿色信号为GFAP-EGFP-P2A-Cre表达出的EGFP荧光，黄色箭头(图13A中的箭头为黄色箭头)指示绿色信号与红色信号共标的穆勒胶质细胞。图13B为在Ai9小鼠视网膜中注射对照组AAV(AAV-GFAP-EGFP-P2A-Cre+AAV-GFAP-CasRx)，白色信号为视锥细胞特异性蛋白标志物m-CAR，青色箭头(图13B中的箭头为青色箭头)指向tdTomato阳性的穆勒胶质细胞，在 ONL层未发现tdTomato阳性细胞。图13C和图13D分别为在小鼠视网膜下注射靶向Sin3a和EZH2的测试组AAV(AAV-GFAP-mCherry+AAV-GFAP-CasRx-gRNA)的结果，白色箭头(图13C和图13D中的箭头为白色箭头)指示在ONL层tdTomato阳性且与mCAR共标的细胞。标尺为50微米。其中在mCherry栏中亮色部分为红色，在EGFP栏中亮色部分为绿色，在DAPI中亮色部分为蓝色。
图14：在小鼠眼内敲低负调控基因将穆勒胶质细胞转化为视网膜神经节细胞的情况。图14A为在Ai9小鼠视网膜中注射对照组AAV(AAV-GFAP-EGFP-P2A-Cre)的结果，在视神经中几乎没有被标记的视神经。图14B至图14I分别为在Ai9小鼠视网膜下注射测试组AAV(AAV-GFAP-mCherry+AAV-GFAP-CasRx-gRNA)抑制不同负调控基因的结果，其中gRNA分别靶向HDAC2、BAF53a、G9a、HuR、BrG1、EZH2、CDYL、KDM1A。
图15：图15A至图15G为在小鼠眼内分别过表达不同的正调控基因基因将穆勒胶质细胞转化为视网膜神经节细胞的情况。其中正调控基因分别为DPYSL2、BAF45b、SCF、HuB、HuD、CYP1B1、BTRC，标尺为150微米。
图16：穆勒胶质细胞重编程为视网膜神经节细胞统计图。
图17：在视网膜色素变性疾病模型小鼠中将穆勒胶质细胞重编程为感光细胞的结果。图17A是视网膜色素变性疾病模型小鼠(Pde6b-KO)中视杆细胞和视锥细胞退化死亡和AAV注射时间点示意图。在Pde6b-KO小鼠视网膜感光细胞全部死亡后(P50)进行AAV注射，注射后50天取视网膜组织进行分析；图17B和图17C是Pde6b-KO小鼠4周龄和6周龄分别取视网膜组织，并用视杆细胞特异性标志物Rhodopsin进行免疫荧光染色，在4周和6周都没有观察到视杆细胞。图17D是注射对照组AAV(GFAP-CasRx+CBH-Pde6b)视网膜组织切片免疫荧光染色结果，在ONL层没有Rhodopsin阳性细胞。图17E至图17F显示注射治疗组AAV(GFAP-CasRx-gRNA+CBH-Pde6b)视网膜组织切片免疫荧光染色结果，gRNA靶向敲低EZH2或KDM1A的表达，在ONL层重新出现Rhodopsin阳性细胞；其中，Rhodopsin一栏中亮色为绿色，DAPI一栏亮色为蓝色。图17G为Pde6b-KO小鼠不同处理组视网膜感光细胞数量统计图，4w和6w为未注射AAV的Pde6b-KO小鼠视网膜中感光细胞数量，敲低EZH2或KDM1A组感光细胞数量明显增多，和图17H显示Pde6b-KO小鼠不同处理组视网膜ONL层厚度，4w和6w为未注射AAV的Pde6b-KO小鼠视网膜ONL层厚度，敲低EZH2或KDM1A组明显增厚。
具体实施方式
本发明人经过广泛而深入的研究，首次意外地发现，抑制胶质细胞的一种或更多种选自以下的基因或其RNA或其编码蛋白的表达或活性的基因或其RNA或其编码蛋白的表达或活性：RCOR1、RCOR2、RCOR3、Sin3a、Sin3b、HDAC1、HDAC2、KDM1A、PHF21A、BAF53a、 G9a、USP14、HuR、BrG1、EZH2、CDYL、HMG20B，和/或增强胶质细胞的一种或更多种选自以下的基因或其RNA或其编码蛋白的表达或活性的基因或其RNA或其编码蛋白的表达或活性：DPYSL2、BAF45b、SCF、HuB、HuC、HuD、CYP1B1、BTRC，可有效诱导胶质细胞向神经元细胞的分化，从而治疗神经元功能缺失或死亡相关的神经系统疾病。在此基础上，本发明人完成了本申请的技术方案。
除非特别说明，本文所使用的单数形式的“一个”，“一种”和“该”也可以包括复数对象。例如，对“一种抑制剂”的引用可以包括多种抑制剂，对“该试剂”的引用包括对一种或多种试剂以及本领域技术人员所知的其等同物的引用。
除非另有说明，否则本文中使用的所有技术和科学术语具有与本发明所属的技术领域的普通技术人员通常理解的含义相同的含义。尽管与本文描述的那些类似或等同的任何方法和试剂都可以用于所公开的方法和组合物的实践中，但现在描述示例性的方法和材料。
除非另有说明，在本文中，“和”的使用也包括“和/或”，相似地，“包含”和“包括”是可互换的，且不旨在加以限制。
应当进一步理解的是，在各个实施方案的描述使用术语“包含”的情况下，本领域技术人员将理解，在某些特定的实例中，实施方案可以替代地使用语言“基本上由...组成”或“由...组成”进行描述。
在本公开内容中，视网膜神经节细胞(RGC)退化是造成永久性失明的主要缘由。而穆勒胶质细胞(MG)转分化为RGC可有助于恢复视力。发明人发现，通过在成熟小鼠视网膜中降低(例如：使用RNA靶向的CRISPR系统CasRx来敲低)所述基因或其RNA或其编码蛋白，可将MG直接转变为RGC。此外，在由NMDA(N-甲基-D-天冬氨酸)诱导的视网膜损伤的小鼠模型中，从MG转变的RGC实现了对中央视觉区域的功能性投射，并使视觉功能得到改善。因此，由降低负调控基因(例如：CasRx介导的敲低)会是一种很有前景的治疗由神经变性引起的视网膜疾病的疗法。
本申请使用最近表征的RNA靶向CRISPR系统CasRx对所述基因或其RNA或其编码蛋白进行抑制。
如本文所用，穆勒胶质细胞(MG)是视网膜组织中的主要神经胶质细胞，视网膜神经节细胞(RGC)是位于视网膜最内层的神经细胞，它的树突主要与双极细胞联系，它的轴突延伸至视神经乳头处，形成视神经。
除非另有说明，术语“分化”、“重编程”或“转分化”在本文中具有相同的含义，可以指从不同类型的非神经细胞(例如星形胶质细胞)产生特定谱系的细胞(例如神经元细胞)而没有中间分化的过程。
如本文所用，“负向调节剂或正向调节剂与所述非神经元细胞接触”是指将负向调节剂或 正向调节剂(例如化合物、核酸、病毒载体等)放置在允许其接触细胞以产生“接触的”细胞的位置。可以使用任何合适的方法来完成接触。例如，在一个实施方案中，接触是通过将负向调节剂或正向调节剂添加至细胞培养物中。接触也可以通过将负向调节剂或正向调节剂或者含有负向调节剂或正向调节剂的载体注射至体内的位置；或将负向调节剂或正向调节剂或者含有负向调节剂或正向调节剂的运载体递送至体内的位置以使负向调节剂或正向调节剂“接触”靶向的细胞类型来完成。
在一些具体实施方式中，所述有效量的所述负向调节剂或所述正向调节剂与所述非神经元细胞在体内接触，诱导非神经元细胞在体外转分化为神经元。将正向调节剂或负向调节剂配制成用于在体内施用于神经系统、视觉系统和听觉系统的药剂，例如体内施用于纹状体、黑质、中脑腹侧被盖区、脊髓、下丘脑、背侧中脑、大脑皮层、海马、小脑、视网膜下、玻璃体腔、内耳耳蜗和前庭，优选纹状体、黑质、视网膜下和玻璃体腔。
在一些具体实施方式中，所述有效量的所述负向调节剂或所述正向调节剂与所述非神经元细胞在体外接触，诱导非神经元细胞在体外转分化为神经元。在一些具体实施方式中，经体外接触所获得的神经元，可以配置成合适的细胞治疗药剂，以合适的给药方式，如静脉输注、原位注射等，给予有需要的个体。
术语“ONL”指外颗粒细胞层，术语“INL”指内颗粒细胞层，术语“GCL”指视网膜神经节细胞层。
如本文所用，术语“非神经元细胞”可以指不是神经元的任何类型的细胞。在一些具体实施方式中，非神经元细胞为干细胞、祖细胞或终末分化细胞。
示例性非神经元细胞是除神经元谱系以外的细胞谱系(例如，造血谱系)的细胞。在一些实施方案中，非神经元细胞是神经元谱系但不是神经元的细胞，例如神经胶质细胞。在一些实施方案中，非神经元细胞是非神经元的终末分化细胞，例如但不限于神经胶质细胞、成纤维细胞、胚胎成纤维细胞、上皮细胞、黑素细胞、角化细胞、脂肪细胞、血细胞、骨髓基质细胞、朗格汉斯细胞、肌肉细胞、直肠细胞或软骨细胞。在一些实施方案中，非神经元细胞来自非神经元细胞系，例如但不限于胶质母细胞瘤细胞系、Hela细胞系、NT2细胞系、ARPE19细胞系或N2A细胞系。“细胞谱系”或“谱系”可以表示来自受精胚胎的组织或器官的发育史。“神经元谱系”可以指从神经干细胞到成熟神经元的发育史，包括沿着该过程(称为神经发生)的各个阶段，例如但不限于神经干细胞(神经上皮细胞、放射状神经胶质细胞)、神经祖细胞(例如，中间神经元前体)、神经元、星形胶质细胞、少突细胞和小神经胶质。
在一些实施方案中，非神经元细胞是干细胞，例如为胚胎干细胞、神经干细胞、或诱导多能干细胞。在一些实施方案中，非神经元细胞是祖细胞，例如神经祖母细胞或神经前体细胞(如多巴胺神经前体细胞)。
在一些具体实施方式中，所述非神经元细胞来源于脑部，优选的，所述非神经元细胞来自大脑、中脑、小脑、脑干、脊髓；更优选的，来源于大脑中的纹状体或黑质。优选的，所述非神经元细胞为来源于脑部的星形胶质细胞，优选的，为来源于纹状体或黑质的星形胶质细胞。在一些具体实施方式中，所述非神经元细胞来源于眼部，优选的，所述非神经元细胞为来自眼部的穆勒胶质细胞。
术语“祖细胞”(Progenitor cell)，指存在于成体组织中的，细胞在分化为终末分化细胞前的中间态细胞(intermediate cell)，祖细胞的分化通常具有明确性。
术语“终末分化细胞”也称终末细胞，是指具有特定功能核特定类型，且不再进行分化、分裂和增殖的细胞。例如：胶质细胞、体细胞、纤维细胞、红细胞、成熟表皮细胞、肌肉细胞等。
术语“干细胞”应被理解为具有分化潜能和增殖能力(特别是自我更新能力)但保持分化潜能的未分化细胞。根据分化潜能，干细胞包括诸如多能干细胞(PSC)、专能(multipotent)干细胞、单能干细胞、胚胎干细胞等亚群。在一些具体实施中，所述干细胞可以为胚胎干细胞、神经干细胞、或诱导多能干细胞。
在本文中，术语“多能干细胞”(PSC)是指能够在体外培养并具有分化成属于三个胚层(外胚层、中胚层、内胚层)的任何细胞谱系的能力的干细胞。PSC可以从受精卵、克隆胚胎、生殖干细胞、组织中的干细胞、体细胞等诱导。PSC的实例包括胚胎干细胞(ESC)、诱导多能干细胞(iPSC或ips)、胚胎生殖细胞(EG细胞)等。
在本文中，术语“诱导多能干细胞”(iPS或iPSC)可以通过重编程直接从成体细胞生成。通过引入特定组的多能性相关基因的产物，可以将成体细胞转化为PSC。
在本文中，术语“神经前体细胞”是具有神经元发育潜能，处于神经元发育前体状态的细胞。
术语“神经元”可以具有本领域技术人员将理解的一般含义。在一些实施方案中，神经元可以指可以通过电信号(例如，膜电位放电)和化学信号(例如，神经递质的突触传递)接收、处理和传输信息的电可兴奋细胞。如本领域技术人员所理解的，在神经元之间转导的化学信号(例如，基于神经递质的释放和识别)可以通过称为突触的专门连接发生。在一些具体实施方式中，所述神经元为多巴胺能神经元、视网膜神经节细胞、感光细胞、5-HT神经元、NE神经元、ChAT神经元、运动神经元、GABA神经元、谷氨酸能神经元、脊髓神经元，脊髓运动神经元、脊髓感觉神经元、双极细胞、无长突细胞、耳蜗神经细胞、锥体神经元、中间神经元、中型多棘神经元、浦肯野细胞、颗粒细胞、嗅感觉神经元、或球周细胞、或其组合。
在本文中，术语“蛋白质”、“肽”和“多肽”可以指氨基酸聚合物或一组两个或更多个相互作用或结合的氨基酸聚合物，具有相同的含义。
在本文中，术语“核酸”和“多核苷酸”具有相同的含义，可以指含有一个或多个核苷酸的核酸分子。
在本文中，术语“多巴胺能神经元”和“多巴胺神经元”具有相同的含义。
在本文中，术语“目的基因”是指被“负向调节剂”或“正向调节剂”调控的基因。例如：RCOR1、RCOR2、RCOR3、Sin3a、Sin3b、HDAC1、HDAC2、KDM1A、PHF21A、BAF53a、G9a、USP14、HuR、BrG1、EZH2、CDYL、HMG20B、DPYSL2、BAF45b、SCF、HuB、HuC、HuD、CYP1B1、或BTRC。
术语“感光细胞”是视网膜中的一种的神经上皮细胞，能够进行视觉光传导，包括视杆细胞和视锥细胞。一些色素性视网膜炎、黄斑变性、糖尿病并发症等均能造成感光细胞的死亡。
术语“神经节细胞”(ganglion cell)是位于视网膜最终段的神经元。很多眼病都可以导致视神经节细胞的死亡，如缺血性视网膜病、青光眼等，视神经节细胞的死亡也可能会导致永久性失明。
如本文所用，穆勒胶质细胞(MGcell)是视网膜组织中的主要神经胶质细胞，视网膜神经节细胞(RGC)是位于视网膜最内层的神经细胞，它的树突主要与双极细胞联系，它的轴突延伸至视神经乳头处，形成视神经。
术语“运载体”是指能够将外源核酸转运到细胞中的工具，可以为核酸、蛋白等。当运载体存在于适当的环境中时，运载体能够指导由该运载体携带的一种或多种基因编码的一种或多种蛋白质或由该运载体携带的多核苷酸编码的微小RNA的表达。在一些具体实施方式中，运载体可以用于引入本文所提供的多核苷酸。在本文中，运载体可以是多种不同的形式的载体，在一些具体实施方式中，运载体可以是病毒载体、质粒载体、微环载体、线性DNA载体、狗骨载体(doggybone vectors)、脂质体、纳米颗粒、外泌体、细胞外囊泡、阳离子聚合物(如PEI)或类病毒颗粒等。
术语“病毒载体”是可以能够将另一种核酸转运到细胞中的病毒来源的核酸。当病毒载体存在于适当的环境中时，它能够指导由该载体携带的一种或多种基因编码的一种或多种蛋白质或由该载体携带的多核苷酸编码的微小RNA的表达。病毒载体的实例包括但不限于逆转录病毒载体、腺病毒载体、慢病毒载体、痘病毒载体、疱疹病毒载体、和腺相关病毒载体。
在一些实施例中，纳米颗粒载体可以包括基于聚合物的纳米颗粒、基于氨基脂的纳米颗粒、金属纳米颗粒。
本文所提供的载体可以用于递送本文所提供的多核苷酸组合物。在一些情况下，使用单个载体递送至少约2个、3个、4个或至多5个多核苷酸。在一些情况下，使用单个载体递送至少约2个、3个、4个或至多5个不同的多核苷酸。在一些情况下，使用单个载体递送至少约2个、3个、4个或至多5个相同的多核苷酸。
在一些实施例中，载体可以递送DNA(如双链DNA或单链DNA)，还可以递送RNA。在一些情况下，RNA可以包括碱基修饰。载体可以包括重组载体。载体可以是从天然存在的载体修饰的载体。载体可以包括非天然存在的载体的至少一部分。可以利用任何载体。
在本文中，“星形胶质细胞”可以指脑和脊髓中特征性的星形的胶质细胞。对本领域技术人员来说清楚的是，星形胶质细胞的特征可以是星形的，表达标志物如胶质原纤维酸性蛋白(GFAP)和醛脱氢酶1家族成员L1(ALDH1L1)、兴奋性氨基酸转运体1/谷氨酸天门冬氨酸转运体(EAAT1/GLAST)、谷氨酰胺合成酶、S100β或兴奋性氨基酸转运体1/谷氨酸转运体1(EAAT2/GLT-1)，与内皮细胞一起参与血-脑屏障、递质摄取和释放、调节细胞外空间中的离子浓度、对神经元损伤的反应和参与神经系统修复以及代谢支持周围神经元。在本发明的某些实施方案中，星形胶质细胞可以指神经系统中表达胶质原纤维酸性蛋白(GFAP)、醛脱氢酶1家族成员L1(ALDH1L1)或两者的非神经元细胞。在某些实施方案中，星形胶质细胞可以指神经系统中表达胶质原纤维酸性蛋白(GFAP)启动子驱动的转基因(例如红色荧光蛋白(RFP)、Cre重组酶)的非神经元细胞。
在一些实施方案中，可用于本文提供的方法中的神经胶质细胞是从脑分离的神经胶质细胞。在一些实施方案中，可用于本文提供的方法中的星形胶质细胞是脑或脊髓中星形的神经胶质细胞。
“有效量”的所述负向调节剂或所述正向调节剂与所述非神经元细胞接触是指能够使得非神经元细胞转化为神经元的负向调节剂或正向调节剂的量，或含有负向调节剂或正向调节剂的运载体的量。
在本文中，“活性片段”、“功能性片段”、和“功能片段”具有相同含义，指与全长的基因或蛋白具有相同或相似的功能的基因或蛋白的截短片段。例如“SEQ ID NO：36的功能性片段”，是指与SEQ ID NO：36所示的序列具有相类似的功能的SEQ ID NO：36的截短片段，该截短片段能够发挥与SEQ ID NO：36相同或相似的正向调节作用，促进非神经元细胞转分化为神经元。
如本文所用，负调控基因的抑制剂可以是含有RNA且通过RNA诱导的沉默复合物(RISC)通路介导RNA转录物的靶向裂解的RNA干扰剂。RNA干扰剂通过RNA干扰(RNAi)的过程指导mRNA的序列特异性降解。RNA干扰剂可以抑制细胞中一种或多种负调控基因的表达。RNA干扰剂包括但不限于“小干扰RNA(siRNA)”、“内切核糖核酸酶制备的siRNA(e-siRNA)”、“短发夹RNA(shRNA)”和“小时间调控的RNA”(stRNA)”、“切割的siRNA(d-siRNA)”以及适体、寡核苷酸和包含至少一个尿嘧啶碱基的其他合成核酸。在一些实施方案中，此类RNA干扰剂由运载体递送，所述运载体例如但不限于复制缺陷或有复制能力的病毒载体(例如，腺病毒、慢病毒、γ逆转录病毒、腺相关病毒等)。
在一些具体实施方式中，本发明提供了在体内产生神经元的方法。示例性方法包括向受试者的神经系统例如脑、眼、或脊髓中的区域施用正向调节剂或负向调节剂，并允许非神经元细胞重编程为功能性神经元。
术语“表达载体”(Expression vectors)指在克隆载体基本骨架的基础上增加表达元件(如启动子、RBS、GOI、终止子等)，使目的基因能够表达的载体。构建表达载体可以使目的基因能在受体细胞中表达并发挥作用。
“负调控基因”和“负向调节剂”
如本文所用，“负调控基因”是指当该基因的表达、或其mRNA的表达、或基因编码的蛋白的表达降低或被抑制时，能够提高、促进或改善非神经元细胞向神经元转分化的基因，例如：RCOR1、RCOR2、RCOR3、Sin3a、Sin3b、HDAC1、HDAC2、KDM1A、PHF21A、BAF53a、G9a、USP14、HuR、BrG1、EZH2、CDYL、HMG20B等。
如本文所用，“负向调节剂”是指能够降低或抑制“负调控基因”的基因表达、或mRNA的表达、或基因编码的蛋白的表达的调节剂。在一些具体实施方式中，负向调节剂可以是能够降低负调控基因表达的基因编辑工具或表观遗传调控工具；也可以是负调控基因的抑制剂、负调控基因活性抑制剂、或负调控基因的编码蛋白的降解激活剂。
在一些具体实施方式中，“负向调节剂”可以是负调控基因的抑制性抗体；或负调控基因的小分子抑制剂；或负调控基因的抑制性mRNA、microRNA、siRNA、shRNA、反义寡核苷酸、结合蛋白或蛋白结构域、多肽、核酸适配体、或PROTAC；或负调控基因的抑制性结合蛋白或配体。
在一些具体实施方式中，作为“负向调节剂”的基因编辑工具，可以是CRISPR基因编辑工具，包含基因编辑蛋白和靶向负调控基因的gRNA。在具体实施例中，基因编辑蛋白可以为DNA编辑蛋白，如Cas9，也可以为RNA编辑蛋白，如Cas13。
在一些具体实施方式中，作为“负向调节剂”的基因编辑工具，可以为包含锌指核酸酶的基因编辑工具，也可以是TALENs(transcription activator-like(TAL)effector nucleases))基因编辑工具。
在一些具体实施方式中，作为“负向调节剂”的可以为表观遗传调控工具。表观遗传调控工具是指在目的基因的核苷酸序列不发生改变的情况下，通过表观遗传修饰方式(如甲基化、乙酰化、磷酸化、染色质构象变化等)，对细胞内核酸或蛋白质的含量与功能进行调节所使用的蛋白、基因、小分子化合物等。例如：在突变的CRISPR/Cas系统上融合KRAB，DNMT3A等表观调控蛋白。
“正调控基因”和“正向调节剂”
如本文所用，“正调控基因”是指当该基因的表达、或其mRNA的表达、或基因编码的蛋 白的表达升高或被激活时，能够提高、促进或改善非神经元细胞向神经元转分化的基因，例如：DPYSL2、BAF45b、SCF、HuB、HuC、HuD、CYP1B1、或BTRC。
如本文所用，“正向调节剂”是指能够升高或被激活“正调控基因”的基因表达、或mRNA的表达、或基因编码的蛋白的表达的调节剂。在一些具体实施方式中，正向调节剂可以是能够提高正调控基因表达的表观遗传调控工具、正调控基因表达激活剂、正调控基因编码蛋白的降解抑制剂、正调控基因mRNA的稳定剂、或外源性的正调控基因或正调控基因的功能性片段。
在一些具体实施方式中，“正向调节剂”可以为：正调控基因的激动性抗体；或正调控基因的小分子激动剂；或正调控基因的激动性结合蛋白或配体；或正调控基因的竞争性基因的抑制剂。
与神经元功能缺失或神经元死亡相关的疾病
在本公开内容中，与神经元功能缺失或神经元死亡相关的疾病可以是帕金森病、RGC或感光细胞功能缺失或死亡有关的视觉系统疾病、阿尔茨海默病、脑损伤、亨廷顿舞蹈症、癫痫、抑郁症、睡眠失调、脑缺血、运动神经元病、肌萎缩性侧索硬化、脊髓性肌萎缩症、共济失调、PloyQ疾病、精神分裂症、成瘾、Pick病、RGC或感光细胞损伤或死亡导致的失明、黄斑变性、视网膜色素变性、耳聋、夜盲症、色盲、遗传性失明、先天性黑蒙症等。在一些具体实施方式中，与神经元功能缺失或神经元死亡相关的疾病优选为帕金森病和RGC或感光细胞功能缺失或死亡有关的视觉系统疾病，例如：多巴胺神经元功能缺失或死亡相关的疾病，与视神经节或感光细胞缺失或死亡相关的视力障碍。
与RGC或感光细胞功能缺失或死亡有关的视觉系统疾病优选自：RGC细胞或感光细胞死亡导致的视力损伤、青光眼、年龄相关的RGC病变、视神经损伤、年龄相关性黄斑变性(AMD)、糖尿病相关视网膜病变、视网膜缺血或出血、Leber遗传性视神经病变、或其组合；所述与感光细胞功能缺失或死亡有关的视觉系统疾病优选自：损伤或退行性病变导致的感光细胞变性或死亡、黄斑变性、视网膜色素变性、糖尿病有关的失明、夜盲症、色盲、遗传性失明、先天性黑蒙症、或其组合。
星形胶质细胞
星形胶质细胞，是哺乳动物脑内数量最多的一类细胞。它们执行许多功能，包括生化支撑(例如形成血-脑屏障)，为神经元提供营养，维持细胞外离子平衡，并参与脑和脊髓损伤后的修复和瘢痕形成。根据胶质丝的含量以及胞突的形状可将星形胶质细胞分为两种：纤维性星形胶质细胞(fibrous astrocyte)多分布在脑和脊髓的白质，突起细长，分支较少，胞质中含大量胶质丝；原浆性星形胶质细胞(protoplasmic astrocyte)，多分布在灰质，细胞突起粗短，分支多。
可用于本公开内容的星形胶质细胞没有特别限制，包括哺乳动物中枢神经系统来源的各种星形胶质细胞，例如来源于纹状体、黑质、中脑腹侧被盖区、下丘脑、脊髓、背侧中脑或大脑皮层，优选地，来源于纹状体和黑质。
神经元
在本公开内容中，功能性神经元可以指能够通过化学或电信号发送或接收信息的神经元。在一些实施方案中，功能性神经元展现出存在于正常神经系统中的成熟神经元的一种或多种功能特性，包括但不限于：兴奋性(例如，表现出动作电位的能力，例如快速上升和随后的下降)(跨细胞膜的电压或膜电位)，与其他神经元形成突触连接，突触前神经递质释放和突触后反应(例如，兴奋性突触后电流或抑制性突触后电流)。
在一些实施方案中，功能性神经元的特征在于其表达功能神经元的一种或多种标记，包括但不限于突触蛋白，突触素，谷氨酸脱羧酶67(GAD67)，谷氨酸脱羧酶65(GAD65)，小白蛋白，多巴胺-和cAMP调节的神经元磷蛋白32(DARPP32)，囊泡谷氨酸转运蛋白1(vGLUT1)，囊泡谷氨酸转运蛋白2(vGLUT2)，乙酰胆碱，酪氨酸羟化酶(TH)，多巴胺，囊泡GABA转运蛋白(VGAT)和γ-氨基丁酸(GABA)。
在一些实施方案中，功能性神经元可以为多巴胺能神经元、5-HT神经元、NE神经元、ChAT神经元、GABA神经元、谷氨酸能神经元、运动神经元、感光细胞(如视杆细胞和视锥细胞)、视网膜神经节细胞(RGC)、耳蜗神经细胞(如耳蜗螺旋神经节细胞和前庭神经元)、或中型多棘神经元(MSN)或其组合，优选多巴胺能神经元、视网膜神经节细胞和感光细胞。
在一些实施方案中，功能性神经元为哺乳动物神经元，例如为人、非人灵长类动物、大鼠、小鼠的神经元。
多巴胺能神经元或多巴胺神经元
多巴胺能神经元(dopaminergic neuron)含有并释放多巴胺(dopamine，DA)作为神经递质的神经元。多巴胺属于儿茶酚胺类神经递质，在中枢神经系统中发挥重要的生物学作用，大脑内的多巴胺能神经元主要集中在中脑的黑质致密区(substantria nigra pars compacta,SNc)、腹侧被盖区(ventral tegmental area,VTA)、下丘脑和脑室周围。很多实验证实多巴胺能神经元与人体的多种疾病密切相关，最典型的就是帕金森病。
基因编辑工具
基因编辑工具是指通过基因编辑技术对目的基因进行修饰的过程，包括基因的插入、缺失或替换，从而改变其遗传信息和表现型特征。
在本公开内容中，可用的基因编辑工具包括但不限于：CRIPSR基因编辑工具(CRISPR/Cas系统)、锌指核酸酶的基因编辑工具(ZFN系统)、TALENs(transcription activator-like(TAL)effector nucleases))基因编辑工具(TALEN系统)。
在一些具体实施方式中，所述基因编辑工具包括DNA基因编辑工具或RNA基因编辑工具。
在一优选实施方式中，本公开内容的基因编辑工具为CRIPSR基因编辑工具。在一些具体实施方式中，CRIPSR基因编辑工具包括Cas蛋白(在本文中也称作Cas酶)或Cas蛋白的功能结构域的编码核酸，和靶向目的基因的gRNA。gRNA可以引导Cas蛋白靶向目的基因，并进行基因编辑。
在一些具体实施方式中，Cas蛋白为Cas9，Cas12(例如：Cas12a、Cas12b、Cas12c、Cas12d等)，Cas13，Cas 14、CasX或CasY等家族的蛋白或其突变体。在一些具体实施方式中，所述Cas蛋白为Cas13a、Cas13d、Cas13X、Cas13a、Cas13b(例如：Cas13b-t1、Cas13b-t2、Cas13b-t3等)、Cas13c、或Cas13Y。在一些具体实施方式中，所述Cas13d为CasRx。
在一优选实施方式中，基因编辑工具能够使得本申请涉及的负调控基因表达降低、或沉默。
在一些情况下，基因编辑工具可以通过运载体进行递送，例如，通过腺相关病毒递送本文所提供的gRNA及基因编辑蛋白(例如CasRx)。
Cas蛋白
在本公开内容中，Cas蛋白的核苷酸可以通过基因工程技术来获得，如基因组测序、聚合酶链式反应(PCR)等，其氨基酸序列可由核苷酸序列推导而得到。所述野生型的Cas蛋白的来源包括但并不限于：黄化瘤胃球菌(Ruminococcuslavefaciens)、酿脓链球菌(Streptococcus pyogenes)、葡萄球菌(Staphylococcus aureus)、氨基酸球菌属(Acidaminococcussp)、毛螺科菌(Lachnospiraceaeacterium)。
在本公开内容的一个优选实施方案中，所述Cas蛋白能够编辑DNA。
在本公开内容的一个优选实施方案中，所述Cas蛋白能够编辑RNA，例如，Cas蛋白可以是Cas13d、Cas13e、Cas13a、Cas13b、Cas13c、Cas13f等RNA靶向基因编辑蛋白。
多肽、蛋白、蛋白质、和多核苷酸
在本公开内容中，术语“本公开内容蛋白”、“蛋白”、“多肽”、“蛋白质”可互换使用，都可指具有RCOR1、RCOR2、RCOR3、Sin3a、Sin3b、HDAC1、HDAC2、KDM1A、PHF21A、BAF53a、G9a、USP14、HuR、BrG1、EZH2、CDYL、HMG20B、BTRC、CYP1B1、DPYSL2、BAF45b、SCF、HuB、HuC、或HuD的氨基酸序列的蛋白或多肽。它们包括含有或不含起始甲硫氨酸的所述蛋白。此外，该术语还包括全长的所述蛋白及其片段。本公开内容所指的蛋白包括其完整的氨基酸序列、其分泌蛋白、其突变体以及其功能上活性的片段。
RCOR1、RCOR2和RCOR3全称为REST辅阻遏物1(REST corepressor 1)、REST辅阻遏物2(REST corepressor 2)和REST辅阻遏物3(REST corepressor 3)，三者具有相似的功能；HDAC1全称为组蛋白脱乙酰酶1(Histone deacetylase 1)，HDAC2全称为组蛋白脱乙酰酶2(Histone  deacetylase 2)，二者具有相似的功能；KDM1A全称为赖氨酸特异性去甲基化酶1A(Lysine-specific demethylase 1A)，也称为赖氨酸特异性组蛋白去甲基化酶(Lysine-specific histone demethylase 1A，LSD1)。Sin3a(SIN3转录调控蛋白家族成员A)与Sin3b(SIN3转录调控蛋白家族成员B)具有相似的功能。
在本公开内容中，术语“基因”、“多核苷酸”、“核酸”可互换使用，都可以指具有RCOR1、RCOR2、RCOR3、Sin3a、Sin3b、HDAC1、HDAC2、KDM1A、PHF21A、BAF53a、G9a、USP14、HuR、BrG1、EZH2、CDYL、HMG20B、CYP1B1、BTRC、DPYSL2、BAF45b、SCF、HuB、HuC、或HuD的核苷酸序列的核酸序列。人RCOR1基因的基因组全长173980bp(NCBI GenBank登录号为23186)。鼠RCOR1基因的基因组全长76536bp(NCBI GenBank登录号为217864)。人RCOR2基因的基因组全长5935bp(NCBI GenBank登录号为283248)。鼠RCOR2基因的基因组全长7832bp(NCBI GenBank登录号为104383)。人RCOR3基因的基因组全长57020bp(NCBI GenBank登录号为55758)。鼠RCOR3基因的基因组全长39526bp(NCBI GenBank登录号为214742)。人Sin3a基因的基因组全长86441bp(NCBI GenBank登录号为25942)。鼠Sin3a基因的基因组全长56382bp(NCBI GenBank登录号为20466)。人Sin3b基因的基因组全长50958bp(NCBI GenBank登录号为23309)。鼠Sin3b基因的基因组全长34934bp(NCBI GenBank登录号为20467)。人HDAC1基因的基因组全长41546bp(NCBI GenBank登录号为3065)。鼠HDAC1基因的基因组全长26543bp(NCBI GenBank登录号为433759)。人HDAC2基因的基因组全长38121bp(NCBI GenBank登录号为3066)。鼠HDAC2基因的基因组全长27593bp(NCBI GenBank登录号为15182)。人KDM1A基因的基因组全长64249bp(NCBI GenBank登录号为23028)。鼠KDM1A基因的基因组全长52284bp(NCBI GenBank登录号为99982)。人PHF21A基因的基因组全长192136bp(NCBI GenBank登录号为51317)。鼠PHF21A基因的基因组全长180916bp(NCBI GenBank登录号为192285)。人BAF53a基因的基因组全长25482bp(NCBI GenBank登录号为86)。鼠BAF53a基因的基因组全长18428bp(NCBI GenBank登录号为56456)。人G9a基因的基因组全长17940bp(NCBI GenBank登录号为10919)。鼠G9a基因的基因组全长15624bp(NCBI GenBank登录号为110147)。人USP14基因的基因组全长56073bp(NCBI GenBank登录号为9097)。鼠USP14基因的基因组全长36535bp(NCBI GenBank登录号为59025)。人HuR基因的基因组全长47069bp(NCBI GenBank登录号为1994)。鼠HuR基因的基因组全长40324bp(NCBI GenBank登录号为15568)。人BrG1基因的基因组全长101279bp(NCBI GenBank登录号为6597)。鼠BrG1基因的基因组全长88150bp(NCBI GenBank登录号为20586)。人EZH2基因的基因组全长76971bp(NCBI GenBank登录号为2146)。鼠EZH2基因的基因组全长65102bp(NCBI GenBank登录号为14056)。鼠CDYL基因的基因组全长214247bp(NCBI GenBank登录号为12593)。人CDYL基因的基因组全长 249407bp(NCBI GenBank登录号为9425)人HMG20B基因的基因组全长6168bp(NCBI GenBank登录号为10362)。鼠HMG20B基因的基因组全长4898bp(NCBI GenBank登录号为15353)。人DPYSL2基因的基因组全长144145bp(NCBI GenBank登录号为1808)。鼠DPYSL2基因的基因组全长108178bp(NCBI GenBank登录号为12934)。人BAF45b基因的基因组全长18690bp(NCBI GenBank登录号为8193)。鼠BAF45b基因的基因组全长13652bp(NCBI GenBank登录号为29861)。人SCF基因的基因组全长87679bp(NCBI GenBank登录号为4254)。鼠SCF基因的基因组全长84802bp(NCBI GenBank登录号为17311)。人HuB基因的基因组全长160503bp(NCBI GenBank登录号为1993)。鼠HuB基因的基因组全长188638bp(NCBI GenBank登录号为15569)。人HuC基因的基因组全长29721bp(NCBI GenBank登录号为1995)。鼠HuC基因的基因组全长37142bp(NCBI GenBank登录号为15571)。人HuD基因的基因组全长155718bp(NCBI GenBank登录号为1996)。鼠HuD基因的基因组全长148212bp(NCBI GenBank登录号为15572)。人CYP1B1基因的基因组全长8643bp(NCBI GenBank登录号为1545)。鼠CYP1B1基因的基因组全长8122bp(NCBI GenBank登录号为13078)。人BTRC基因的基因组全长203506bp(NCBI GenBank登录号为8945)。鼠BTRC基因的基因组全长169664bp(NCBI GenBank登录号为12234)。
人和鼠RCOR1，在DNA水平的相似性为88.51％；蛋白序列相似性为92.39％。人和鼠RCOR2，在DNA水平的相似性为89.26％；蛋白序列相似性为97.51％。人和鼠RCOR3，在DNA水平的相似性为92.38％；蛋白序列相似性为96.39％。人和鼠Sin3a，在DNA水平的相似性为90.91％；蛋白序列相似性为98.04％。人和鼠Sin3b，在DNA水平的相似性为86.61％；蛋白序列相似性为87.91％。人和鼠HDAC1，在DNA水平的相似性为90.85％；蛋白序列相似性为99.38％。人和鼠HDAC2，在DNA水平的相似性为91.62％；蛋白序列相似性为99.59％。人和鼠KDM1A，在DNA水平的相似性为89.75％；蛋白序列相似性为97.72％。人和鼠PHF21A，在DNA水平的相似性为92.88％；蛋白序列相似性为94.20％。人和鼠BAF53a，在DNA水平的相似性为88.60％；蛋白序列相似性为98.84％。人和鼠G9a，在DNA水平的相似性为87.74％；蛋白序列相似性为94.75％。人和鼠USP14，在DNA水平的相似性为91.31％；蛋白序列相似性为96.76％。人和鼠HuR，在DNA水平的相似性为91.03％；蛋白序列相似性为98.47％。人和鼠BrG1，在DNA水平的相似性为89.00％；蛋白序列相似性为95.47％。人和鼠EZH2，在DNA水平的相似性为91.98％；蛋白序列相似性为97.60％。人和鼠CDYL，在DNA水平的相似性为86.77％；蛋白序列相似性为93.12％。人和鼠HMG20B，在DNA水平的相似性为85.53％；蛋白序列相似性为93.69％。人和鼠DPYSL2，在DNA水平的相似性为91.05％；蛋白序列相似性为97.64％。人和鼠BAF45b，在DNA水平的相似性为93.39％；蛋白序列相似性为97.69％。人和鼠SCF，在DNA水平的相似性为89.44％；蛋白序列相似性为82.78％。人和鼠HuB，在DNA水 平的相似性为95.46％；蛋白序列相似性为99.72％。人和鼠HuC，在DNA水平的相似性为91.94％；蛋白序列相似性为99.46％。人和鼠HuD，在DNA水平的相似性为94.64％；蛋白序列相似性为99.21％。人和鼠CYP1B1，在DNA水平的相似性为82.96％；蛋白序列相似性为81.03％。人和鼠BTRC，在DNA水平的相似性为90.87％；蛋白序列相似性为98.68％。需理解的是，当编码相同的氨基酸时，密码子中核苷酸的取代是可接受的。另外需理解的是，由核苷酸取代而产生保守的氨基酸取代时，核苷酸的变换也是可被接受的。
在得到了氨基酸片段的情况下，可根据其构建出编码它的核酸序列，并且根据核苷酸序列来设计特异性探针。核苷酸全长序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。对于PCR扩增法，可根据本公开内容所公开的核苷酸序列，尤其是开放阅读框序列来设计引物，并用市售的cDNA库或按本领域技术人员已知的常规方法所制备的cDNA库作为模板，扩增而得有关序列。当序列较长时，常常需要进行两次或多次PCR扩增，然后再将各次扩增出的片段按正确次序拼接在一起。
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入运载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。
此外，还可用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。
目前，已经可以完全通过化学合成来得到编码本公开内容蛋白(或其片段，衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(如运载体)和细胞中。
通过常规的重组DNA技术，可利用本公开内容的多核苷酸序列可用来表达或生产重组的多肽。一般来说有以下步骤：
(1).用本公开内容的编码多肽的多核苷酸(或变异体)，或用含有该多核苷酸的重组表达载体转化或转导合适的宿主细胞；
(2).在合适的培养基中培养的宿主细胞；
(3).从培养基或细胞中分离、纯化蛋白质。
本公开内容中，多核苷酸序列可插入到重组表达载体中。总之，只要能在宿主体内复制和稳定，任何质粒和运载体都可以用。表达载体的一个重要特征是通常含有复制起点、启动子、标记基因和翻译控制元件。
本领域的技术人员熟知的方法能用于构建含RCOR1、RCOR2、RCOR3、Sin3a、Sin3b、HDAC1、HDAC2、KDM1A、PHF21A、BAF53a、G9a、USP14、HuR、BrG1、EZH2、CDYL、CYP1B1、BTRC、HMG20B、DPYSL2、BAF45b、SCF、HuB、HuC、或HuD的编码DNA序列和合适的转录/翻译控制信号的表达载体。这些方法包括体外重组DNA技术、DNA 合成技术、体内重组技术等。所述DNA序列可有效连接到表达载体中的适当启动子上，以指导mRNA合成。表达载体还包括翻译起始用的核糖体结合位点和转录终止子。
此外，表达载体优选地包含一个或多个选择性标记基因，以提供用于选择转化的宿主细胞的表型性状，如真核细胞培养用的二氢叶酸还原酶、新霉素抗性以及绿色荧光蛋白(GFP)，或用于大肠杆菌的四环素或氨苄青霉素抗性。
包含上述的适当DNA序列以及适当启动子或者控制序列的载体，可以用于转化适当的宿主细胞，以使其能够表达蛋白质。
宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。代表性例子有：大肠杆菌，链霉菌属的细菌细胞；真菌细胞如酵母；植物细胞；昆虫细胞；动物细胞等。
用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核生物如大肠杆菌时，能吸收DNA的感受态细胞可在指数生长期后收获，用CaCl2法处理，所用的步骤在本领域众所周知。另一种方法是使用MgCl2。如果需要，转化也可用电穿孔的方法进行。当宿主是真核生物，可选用如下的DNA转染方法：磷酸钙共沉淀法，常规机械方法如显微注射、电穿孔、脂质体包装等。
获得的转化子可以用常规方法培养，表达本公开内容的基因所编码的多肽。根据所用的宿主细胞，培养中所用的培养基可选自各种常规培养基。在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后，用合适的方法(如温度转换或化学诱导)诱导选择的启动子，将细胞再培养一段时间。
在上面的方法中的重组多肽可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
腺相关病毒
因腺相关病毒(Adeno-associated virus，AAV)较其他病毒载体小，无致病性，可转染正在分裂和未分裂的细胞等特性，基于AAV载体的针对遗传性疾病的基因治疗方法受到了广泛的关注。
腺相关病毒(AAV),也称腺伴随病毒，属于微小病毒科依赖病毒属,是目前发现的一类结构最简单的单链DNA缺陷型病毒，需要辅助病毒(通常为腺病毒)参与复制。它编码两个末端的反向重复序列(ITR)中的cap和rep基因。ITRs对于病毒的复制和包装具有决定性作用。cap基因编码病毒衣壳蛋白，rep基因参与病毒的复制和整合。AAV能感染多种细胞。
重组腺相关病毒载体(rAAV)源于非致病的野生型腺相关病毒，由于其安全性好、宿主细胞范围广(分裂和非分裂细胞)、免疫源性低，在体内表达外源基因时间长等特点，被视为最有前途的基因转移载体之一，在世界范围内的基因治疗和疫苗研究中得到广泛应用。经过10余年的研究，重组腺相关病毒的生物学特性己被深入了解，尤其是其在各种细胞、组织和体内实验中的应用效果方面已经积累了许多资料。在医学研究中，rAAV被用于多种疾病的基因治疗的研究(包括体内、体外实验)；同时作为一种有特点的基因转移载体，还广泛用于基因功能研究、构建疾病模型、制备基因敲除鼠等方面。
在本公开内容一个优选的实施例中，运载体为重组AAV载体。AAV是相对较小的DNA病毒，其可以稳定和位点特异性方式整合到它们所感染的细胞的基因组中。它们能够感染一大系列的细胞而不对细胞生长、形态或分化产生任何影响，并且它们似乎并不涉及人体病理学。AAV基因组己被克隆、测序及表征。AAV在每个末端包含约145个碱基的反向末端重复序列(ITR)区域，其作为病毒的复制起点。该基因组的其余被分成两个带有衣壳化功能的重要区域：包含涉及病毒复制和病毒基因表达的rep基因的基因组左边部分；以及包含编码病毒衣壳蛋白的cap基因的基因组右边部分。
AAV载体可采用本领域的标准方法制备。任何血清型的腺相关病毒均是合适的。用于纯化载体的方法可见于例如美国专利No.6566118、6989264和6995006，它们的公开内容整体以引用方式并入本文。杂合载体的制备在例如PCT申请No.PCT/US2005/027091中有所描述，该申请的公开内容整体以引用方式并入本文。用于体外和体内转运基因的衍生自AAV的载体的使用己有描述(参见例如国际专利申请公布No.WO91/18088和WO93/09239；美国专利No.4,797,368、6,596,535和5,139,941，以及欧洲专利No.0488528，它们均整体以引用方式并入本文)。这些专利公布描述了其中rep和/或cap基因缺失并被所关注的基因替换的各种来源于AAV的构建体，以及这些构建体在体外(进入培养的细胞中)或体内(直接进入生物体)转运所关注的基因的用途。复制缺陷重组AAV可通过将以下质粒共转染进被人类辅助病毒(例如腺病毒)感染的细胞系而制备：所含的所关注核酸序列的侧翼为两个AAV反向末端重复序列(ITR)区域的质粒，和携带AAV衣壳化基因(rep和cap基因)的质粒。然后通过标准技术纯化所产生的AAV重组体。
在一些实施方案中，重组载体被衣壳化到病毒粒子(例如包括但不限于AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV10、AAV11、AAV12、AAV13、AAV14、AAV15和AAV16的AAV病毒粒子)中。因此，本公开包括含有本文所述任何载体的重组病毒粒子(因其包含重组多核苷酸而为重组的)。产生这样的粒子的方法是本领域己知的，并在美国专利No.6,596,535中有所描述。
在一些情况下，AAV载体可以经修饰以包含经修饰的VP蛋白(如经修饰以包含VP1蛋白、 VP2蛋白或VP3蛋白的AAV载体)。一方面，AAV载体是重组AAV(rAAV)载体。rAAV可以由与野生型AAV(wtAAV)中发现的基本上相似的衣壳序列和结构组成。然而，rAAV包封了基本上没有AAV蛋白编码序列的基因组，并在其位置设计了治疗基因表达盒，如主题多核苷酸。在一些情况下，病毒来源的序列可以是ITR，在载体生产期间可能需要所述ITR来指导基因组复制和包装。合适的AAV载体可以选自任何AAV血清型或血清型的组合。例如，AAV载体可以是以下中的任一个：AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV 10、AAV11、AAV 12、AAV13、AAV 14、AAV 15、AAV 16、AAV.rh8、AAV.rh10、AAV.rh20、AAV.rh39、AAV.Rh74、AAV.RHM4-1、AAV.hu37、AAV.Anc80、AAV.Anc80L65、AAV.7m8、AAV.PHP.B、AAV2.5、AAV2tYF、AAV3B、AAV.LK03、AAV.HSC1、AAV.HSC2、AAV.HSC3、AAV.HSC4、AAV.HSC5、AAV.HSC6、AAV.HSC7、AAV.HSC8、AAV.HSC9、AAV.HSC10、AAV.HSC11、AAV.HSC12、AAV.HSC13、AAV.HSC14、AAV.HSC15、AAV.HSC16和AAVhu68、或其任何组合。在一些情况下，基于其自然取向选择载体。在一些情况下，基于其跨血脑屏障的能力选择载体血清型。AAV9和AAV10已被证明能够跨血脑屏障以转导神经元和胶质细胞。一方面，AAV载体是AAV2、AAV5、AAV6、AAV8或AAV9。在一些情况下，AAV载体是至少两种血清型的嵌合体。一方面，AAV载体是血清型AAV2和AAV5。在一些情况下，嵌合AAV载体包括来自AAV2的rep和ITR序列以及来自AAV5的帽序列。在一些情况下，嵌合AAV载体包括来自AAV2的rep和ITR序列以及来自任何其它AAV血清型的帽序列。在一些实施例中，AAV载体可以是自互补的。在一些情况下，AAV载体可以包括反向末端重复序列。在其它情况下，AAV载体可以包括具有突变的末端解链位点的反向末端重复(scITR)序列。在一些情况下，可以将来自本文所提供的所有不同AAV血清型的rep、帽和ITR序列混合并匹配。在一些情况下，AAV载体来自具有选自以下的血清型的腺相关病毒：AAV1、AAV2、AAV3、AAV4、AAV5、AAV6、AAV7、AAV8、AAV9、AAV 10、AAV11、AAV 12、AAV13、AAV 14、AAV 15、AAV 16、AAV.rh8、AAV.rh10、AAV.rh20、AAV.rh39、AAV.Rh74、AAV.RHM4-1、AAV.hu37、AAV.Anc80、AAV.Anc80L65、AAV.7m8、AAV.PHP.B、AAV2.5、AAV2tYF、AAV3B、AAV.LK03、AAV.HSC1、AAV.HSC2、AAV.HSC3、AAV.HSC4、AAV.HSC5、AAV.HSC6、AAV.HSC7、AAV.HSC8、AAV.HSC9、AAV.HSC10、AAV.HSC11、AAV.HSC12、AAV.HSC13、AAV.HSC14、AAV.HSC15、AAV.HSC16和AAVhu68。在一些情况下，载体可以是重组AAV(rAAV)载体、杂交AAV载体、嵌合AAV载体、自互补AAV(scAAV)载体、单链AAV或其任何组合。在一些情况下，AAV载体包括包含来自第一AAV血清型的复制基因和反向末端重复序列以及来自第二AAV血清型的衣壳蛋白的基因组。在一些情况下，AAV载体可以是嵌合的，并且可以是：AAV2/5载体、AAV2/6载体、AAV2/7载体、AAV2/8载体或 AAV2/9载体。在一些情况下，AAV载体的反向末端重复序列包括5'反向末端重复序列、3'反向末端重复序列和突变的反向末端重复序列。在一些情况下，突变的反向末端重复序列缺少末端解链位点。在一些情况下，合适的AAV载体可以被进一步修饰以涵盖如在衣壳或rep蛋白中的修饰。修饰还可以包含缺失、插入、突变和其组合。在一些情况下，对载体进行修饰以降低免疫原性，从而允许重复给药。在一些情况下，当进行重复给药以降低和/或消除免疫原性时，所使用的载体的血清型发生变化。
药物组合物
利用本公开的内容，通过各种常规筛选方法，可筛选出对RCOR1、RCOR2、RCOR3、Sin3a、Sin3b、HDAC1、HDAC2、KDM1A、PHF21A、BAF53a、G9a、USP14、HuR、BrG1、EZH2、CDYL、或HMG20B的基因或蛋白发生抑制作用的负向调节剂。
在一些具体实施方式中，抑制剂是负向调节剂的一种或几种，抑制负调控基因的表达、或抑制负调控基因的mRNA水平，或抑制负调控基因的编码蛋白水平。
利用本公开的内容，通过各种常规筛选方法，可筛选出能够增强DPYSL2、BAF45b、SCF、HuB、HuC、HuD、CYP1B1、或BTRC的基因表达水平、或mRNA水平、或蛋白表达水平、或蛋白活动的正向调节剂。
在一些具体实施方式中，增强剂剂是正向调节剂的一种或几种，增强正调控基因的表达、或增强正调控基因的mRNA水平，或增强正调控基因的编码蛋白水平。
可用于本公开内容的负向调节剂或正向调节剂可以在DNA、RNA、蛋白质水平降低、消除所述基因、其RNA(如mRNA)或其编码蛋白的表达和/或活性的物质。
在一些具体实施方式中，所述负向调节剂包括负调控基因的抑制性抗体；或负调控基因的小分子抑制剂；或负调控基因的抑制性mRNA、microRNA、siRNA、shRNA、反义寡核苷酸、结合蛋白或蛋白结构域、多肽、核酸适配体、或PROTAC；或负调控基因的抑制性结合蛋白或配体。
在一些具体实施方式中，本公开内容的RCOR1的负向调节剂包括靶向包含SEQ ID NO：68的靶序列的抑制剂。本公开内容的RCOR2的负向调节剂包括靶向包含SEQ ID NO：69的靶序列的抑制剂。本公开内容的RCOR3的负向调节剂包括靶向包含SEQ ID NO：70的靶序列的抑制剂。本公开内容的Sin3a的负向调节剂包括靶向包含SEQ ID NO：71的靶序列的抑制剂。本公开内容的Sin3b的负向调节剂包括靶向包含SEQ ID NO：72的靶序列的抑制剂。本公开内容的HDAC1的负向调节剂包括靶向包含SEQ ID NO：73的靶序列的抑制剂。本公开内容的KDM1A的负向调节剂包括靶向包含SEQ ID NO：74的靶序列的抑制剂。本公开内容的HDAC2的负向调节剂包括靶向包含SEQ ID NO：75的靶序列抑制剂。本公开内容的PHF21A的负向调节剂包括靶向包含SEQ ID NO：76的靶序列抑制剂。本公开内容的BAF53a的负向调 节剂包括靶向包含SEQ ID NO：77的靶序列的抑制剂。本公开内容的G9a的负向调节剂包括靶向包含SEQ ID NO：78的靶序列的抑制剂。本公开内容的USP14的负向调节剂包括靶向包含SEQ ID NO：79的靶序列的抑制剂。本公开内容的HuR的负向调节剂包括靶向包含SEQ ID NO：80的靶序列的抑制剂。本公开内容的BrG1的负向调节剂包括靶向包含SEQ ID NO：81的靶序列的抑制剂。本公开内容的EZH2的负向调节剂包括靶向包含SEQ ID NO：82的靶序列的抑制剂。本公开内容的CDYL的负向调节剂包括靶向包含SEQ ID NO：83的靶序列的抑制剂。本公开内容的HMG20B的负向调节剂包括靶向包含SEQ ID NO：84的靶序列的抑制剂。
在一些具体实施方式中，本公开内容的负向调节剂或正向调节剂的作用对象包括星形胶质细胞或MG细胞。
在一些具体实施方式中，所述正向调节剂可以在DNA、RNA、蛋白质水平提高、增强正调控基因、或其RNA(如mRNA)、或其编码蛋白的表达和/或活性的物质。
在一些具体实施方式中，正向调节剂包括：包含启动子的表达载体、内源性表达激活剂、蛋白质类似物或增强剂，能够提高正调控基因表达的表观遗传调控工具、正调控基因表达激活剂、正调控基因编码蛋白的降解抑制剂、正调控基因mRNA的稳定剂、或外源性的正调控基因或正调控基因的功能性片段。
在一种优选的实施方式中，负向调节剂调控的方法和步骤包括利用抗体中和其蛋白，利用病毒(如腺相关病毒)携带的shRNA或siRNA、或利用基因编辑工具进行基因的沉默。
负向调节剂对负调控基因的抑制率一般为达到至少50％以上，优选为60％、70％、80％、90％、95％以上。可以基于常规技术，例如流式细胞术、荧光定量PCR或Western blot等方法对抑制率进行控制和检测。
本公开内容的负向调节剂(包括抗体、反义核酸、基因编辑工具以及其他抑制剂)或正向调节剂(包括：包含启动子的表达载体、内源性表达激活剂、蛋白质类似物或增强剂)，当在治疗上进行施用(给药)时，可抑制或增强所述基因或蛋白的表达和/或活性，进而诱导胶质细胞分化为神经元细胞，从而治疗与神经元功能缺失或死亡相关的疾病。通常，可将这些物质配制于无毒的、惰性的和药学上可接受的水性介质中，其中pH通常约为5-8，优选地pH约为6-8，尽管pH值可随被配制物质的性质以及待治疗的病症而有所变化。配制好的药物组合物可以通过常规途径进行给药，其中包括(但并不限于)：局部、肌内、颅内、眼内、腹膜内、静脉内、皮下、皮内、局部给药、自体细胞提取培养后回输等。
本公开内容还提供了一种药物组合物，它含有安全有效量的本公开内容的负向调节剂或正向调节剂以及药学上可接受的载体或赋形剂。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。药物制剂应与给药方式相匹配。本公开内容的药物组合物可以被制成针剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法 进行制备。诸如片剂和胶囊之类的药物组合物，可通过常规方法进行制备。药物组合物如针剂、溶液、片剂和胶囊宜在无菌条件下制造。活性成分的给药量是治疗有效量，例如每天约1微克-10毫克/千克体重。
本公开内容的主要优点包括：
(1)本公开内容首次发现，降低非神经元细胞，特别是胶质细胞中的RCOR1、RCOR2、RCOR3、Sin3a、Sin3b、HDAC1、HDAC2、KDM1A、PHF21A、BAF53a、G9a、USP14、HuR、BrG1、EZH2、CDYL、或HMG20B的基因或其编码蛋白的表达或活性，和/或提高胶质细胞中的DPYSL2、BAF45b、SCF、HuB、HuC、HuD、CYP1B1、或BTRC的基因或其编码蛋白的表达或活性，可诱导胶质细胞向多巴胺神经元细胞的分化，从而预防和/或治疗神经元功能缺失或死亡相关的疾病。
(2)采用本发明提供的负向调节剂或正向调节剂，能够诱导多巴胺神经元的产生，从而具有潜在的治疗帕金森疾病的作用。
(3)本公开内容通过抑制视网膜中RCOR1、RCOR2、RCOR3、Sin3a、Sin3b、HDAC1、HDAC2、KDM1A、PHF21A、BAF53a、G9a、USP14、HuR、BrG1、EZH2、CDYL、或HMG20B的表达，和/或增强胶质细胞中的DPYSL2、BAF45b、SCF、HuB、HuC、HuD、CYP1B1、或BTRC、的表达，将穆勒细胞直接转变为RGC细胞或感光细胞。
(4)再生的RGC可被整合到视觉通路中，并改善RGC损伤小鼠模型的视觉功能。
下面结合具体实施例，进一步阐述本公开内容。应理解，这些实施例仅用于说明本公开内容而不用于限制本公开内容的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
除非特别说明，否则本公开内容实施例中所用材料和试剂均为市售产品。
通用方法
动物伦理：动物的使用和饲养符合中国科学院脑科学与智能技术卓越创新中心生物医学研究伦理委员会的指导原则。
向导RNA序列和AAV表达质粒构建：为了在胶质细胞中过表达nSR100，选用胶质细胞特异性启动子GFAP来启动nSR100的表达。
瞬时转染星形胶质细胞和qPCR：分离小鼠原代星形胶质细胞，将星形胶质细胞接种在6孔板中。按照标准程序使用Lipofectamine 3000(Thermo Fisher Scientific)，用3μg表达gRNA-CasRx-GFP的载体瞬时转染。对照质粒表达非靶向gRNA。瞬时转染后1-2天，通过流式荧光细胞分选(FACS)收集GFP阳性细胞并裂解作qPCR分析。qPCR分析方法为：使用 Trizol(Ambion)提取RNA，然后使用逆转录试剂盒(用于qPCR的HiScript Q RT SuperMix，Vazyme，Biotech)将RNA逆转录为cDNA。通过AceQ qPCR SYBR Green Master Mix(Vazyme，Biotech)追踪扩增。qPCR引物见下表2：
表2：qPCR引物
定位注射：在该研究中使用到了AAV8携带目的基因。AAV-CasRx,AAV-CasRx-gRNA，或AAV-GFAP-正调控基因的滴度为大于5×1012vg/ml(每次注射1-3μl)。将AAV注入纹状体(AP+0.8mm，ML±1.6mm和DV-2.8mm)。
免疫荧光染色：小鼠注射后6-12周，灌注，取脑并用4％多聚甲醛(PFA)固定过夜，并在30％蔗糖中脱水至少12小时。在包埋后冷冻切片，切片厚度为30μm。免疫荧光染色之前，用0.1M磷酸盐缓冲液(PBS)彻底冲洗脑切片。一抗：兔多克隆NeuN抗体(Brain，1：500，#ABN78，Millipore)，小鼠TH抗体(1：300，MAB318，Millipore)。二抗：在该研究中使用驴抗小鼠(#715-545-150，Jackson ImmunoResearch)，驴抗兔(#711-545-152，Jackson ImmunoResearch)。在抗体孵育后，洗涤切片并用封片剂(Life Technology)覆盖。
统计分析：由s.e.m.设置误差线，以非成对双尾t检验或单因素方差分析计算统计学显着性(p<0.05)，所有实验均随机制定，未使用统计学方法预确定样本量。
对于RNA-seq，在15-cm培养皿中培养293T或者N2a细胞，并用70μg质粒进行瞬时转染。通过FACS收集前20％GFP和mCherry双阳性的N2a细胞，提取RNA，转变为cDNA，然后用于全转录组RNA-seq。
玻璃体内注射和视网膜下注射：如前所述，分别通过玻璃体内和视网膜下注射引入NMDA和AAV8。对于
视网膜下注射：在Olympus显微镜下用Hamilton注射器(32G针)向眼睛注射 AAV(>1×1013vg/ml)。为确定完整视网膜中的重编程，通过视网膜下注射(Ai9和C57BL/6小鼠，5周到12个月年龄)将共计1μl的GFAP-GFP-Cre(0.2μl)和AAV-GFAP-nSR100，或GFAP-CasRx-gRNA(0.8μl)，或GFAP-GFP-Cre(0.2μl)和GFAP-CasRx(0.8μl)传送至视网膜。为确定受损视网膜中的重编程，在PBS中溶解NMDA至浓度200mM，然后通过玻璃体内注射将1.5μl NMDA溶液注入4-8周龄的Ai9小鼠或5-6周龄的C57BL/6小鼠的眼睛(用于VEP和黑白场景偏好测试)。NMDA注射2-3周后，各病毒组合通过视网膜下注射共同递送至视网膜。
免疫荧光染色：AAV注射1.5-3个月后，取眼睛，视神经和脑，用4％多聚甲醛(PFA)固定2小时(眼睛和视神经)或24小时(脑部)，然后在30％蔗糖溶液中脱水2小时(眼睛和视神经)或24小时(脑)。嵌入和冷冻后，将眼睛和大脑以30μm的厚度切片。用于免疫荧光染色的一抗：兔抗RBPMS(1：500,15187-1-AP，Proteintech)，以及二抗：Jackson ImmunoResearch)，Cy TM 5AffiniPure Donkey兔抗IgG(H+L)(1：500,711-175-152，Jackson ImmunoResearch)。孵育抗体后，用PBS进行洗片，并用抗荧光淬灭剂进行封片。使用Olympus FV3000显微镜进行成像。
实施例
实施例1、使用CasRx的体外特异性敲低
为了评估对负调控基因的敲低情况，分别设计了靶向RCOR1、Sin3a、HDAC2、KDM1A、PHF21A、BAF53a、G9a、USP14、HuR、BrG1、EZH2、CDYL、HMG20B的gRNA，并将CasRx和gRNA共转染N2A细胞。
其中，gRNA的核苷酸序列及靶序列如上表1所示。
转染方法为：瞬时转染4μg CAG-CasRx-P2A-GFP质粒和2μg U6-gRNA-CMV-mCherry质粒来确定各gRNA在体外细胞系里的抑制效果，使用CAG-CasRx-P2A-GFP质粒单转染作为对照组。依标准程序使用Lipofectamine 3000(Thermo Fisher Scientific)。转染两天后，通过荧光激活细胞分选(FACS)对每个样品收集30000个GFP和mCherry双阳性细胞，裂解后进行qPCR分析，qPCR引物参见表2。
结果如图1所示，靶向负调控基因的mRNA的gRNA和CasRx共转染，可以在小鼠N2A细胞中对目的基因实现显著的敲低。
实施例2、负调控基因对星形胶质细胞向神经元的换分化研究
为了研究负调控基因的敲低是否能诱导星形胶质细胞向神经元转分化或向多巴胺能神经元转分化，在年龄大于八周的C57BL/6小鼠纹状体中，注射AAV-GFAP-CasRx-gRNA或不含gRNA的对照载体AAV-GFAP-CasRx以及AAV-GFAP-mCherry。质粒设计如图2A和B所示，注射载体1+载体2或注射载体1+载体3，其中，GFAP是星形胶质细胞特异性启动子，AAV- GFAP-mCherry用于特异性的标记星形胶质细胞，AAV-GFAP-CasRx为对照组AAV，gRNA分别靶向负调控基因RCOR1、HDAC2、PHF21A、BAF53a、G9a、USP14、HuR、BrG1、EZH2、CDYL、HMG20B、KDM1A。
注射后1-3个月取材分析。结果如图3所示。如图3A，在注射了GFAP-mCherry+AAV-GFAP-CasRx的对照组中，mCherry阳性的红色细胞呈现典型的胶质细胞形态，未发现明显神经元形态的红色细胞，进一步用神经元特异性标志蛋白NeuN进行免疫荧光染色发现对照组几乎无红色细胞与NeuN共标，这表明GFAP-mCherry能特异性标记星形胶质细胞，且注射了GFAP-mCherry+AAV-GFAP-CasRx的对照组不能诱导星形胶质细胞向神经元转分化。如图3B至3M，在注射了GFAP-mCherry+AAV-GFAP-CasRx-gRNA的实验组中，均有不同数量的mCherry阳性细胞与NeuN共标，表明RCOR1、HDAC2、PHF21A、BAF53a、G9a、USP14、HuR、BrG1、EZH2、CDYL、HMG20B、KDM1A均能不同程度的诱导胶质细胞向神经元转分化。
如图4显示的敲低不同的负调控基因后将星形胶质细胞转化为神经元的统计情况，其中RCOR1、G9a、BAF53A等诱导胶质细胞向神经元转分化的效率较高。
实施例3、正调控基因对星形胶质细胞向神经元的转分化研究
为了研究正调控基因的过表达对转分化的影响，尝试在小鼠纹状体过表达DPYSL2、BAF45b、SCF、HuB、HuC、HuD、CYP1B1、BTRC。
在小鼠纹状体或黑质中注射对照组或测试组AAV，其中载体1为AAV-GFAP-mCherry，载体2(AAV-GFAP-Gene)是由星形胶质细胞特异性启动子GFAP驱动靶点基因表达。与敲低目的基因表达组类似，在注射AAV后1-3个月进行取材分析各不同靶点诱导胶质细胞向神经元转分化的效率。结果如图5和图6所示。
如图5A，在注射有AAV-GFAP-mCherry对照组AAV的小鼠脑切片中，红色荧光标记的细胞为典型星形胶质细胞形态，几乎无mCherry与NeuN共标的细胞。而在注射测试组AAV(AAV-GFAP-mCherry+AAV-GFAP-正调控基因)，其中正调控基因为DPYSL2、BAF45b、SCF、HuB、HuC、HuD、CYP1B1、BTRC的各组中有不同比例的mCherry与NeuN共标的细胞(图5B至5I)。这些结果表明过表达DPYSL2、BAF45b、SCF、HuB、HuC、HuD、CYP1B1、BTRC等基因能不同程度的诱导星形胶质细胞向神经元转分化。如图6，SCF、HuB、HuC、和CYP1B1均显示出优异的转分化效率。
实施例4、星形胶质细胞向多巴胺神经元转分化
为了进一步研究正调控基因的过表达和负调控基因的抑制能否促进星形胶质细胞向多巴胺神经元转分化，参照实施例1至实施例3的操作，我们在小鼠纹状体中注射对照组或测试组的AAV，用多巴胺神经元特异性蛋白标志物TH进行染色，结果如图7所示，对照组中未发现 TH阳性的细胞(图7A)，在HuB和BTRC过表达组发现有TH阳性的，且与mCherry信号共标的多巴胺神经元，表明过表达HuB和BTRC能诱导星形胶质细胞向多巴胺神经元转分化。
实施例5、穆勒胶质细胞向感光细胞转分化
在视网膜中，穆勒胶质细胞发挥着类似于胶质细胞的功能，而且视网膜中包含多种特异功能的神经元，如感光细胞和双极细胞等。为了在视网膜系统中探究各基因是否能将穆勒胶质细胞转分化为感光细胞，我们利用GFAP-EGFP-2A-Cre系统结合Ai9小鼠，实现对穆勒胶质细胞的特异性标记，操作示意图如图8所示，参照实施例1至实施例3的操作，向6-10周的Ai9(Rosa26-CAG-LSL-tdTomato-WPRE)小鼠视网膜下注射AAV-GFAP-CasRx-gRNA(或对照运载体AAV-GFAP-CasRx)与AAV-GFAP-EGFP-2A-Cre的混合病毒，AAV-GFAP-EGFP-2A-Cre用来特异性的标记穆勒胶质细胞或者由胶质细胞转化来的神经元(视网膜神经节细胞或感光细胞)，注射后1-3个月后取视网膜进行切片分析，研究抑制相应的负调控基因，或上调相应的正调控基因，在视网膜系统中是否具有将穆勒胶质细胞转分化为感光细胞。
以GFAP-EGFP-2A-Cre+GFAP-CasRx为对照组，以表达GFAP-EGFP-2A-Cre+GFAP-CasRx-gRNA为测试组，其中gRNA分别靶向Sin3a、HDAC2、BAF53a、G9a、HuR、BrG1、EZH2、CDYL、和KDM1A。
以GFAP-EGFP-2A-Cre为对照组，以GFAP-EGFP-2A-Cre+GFAP-正调控基因为测试组，其中正调控基因为DPYSL2、BAF45b、SCF、CYP1B1、BTRC基因。
将不同组的AAV注射到NMDA造模的Ai9小鼠视网膜下，注射后1-3个月进行分析。
负调控基因组对转分化的影响如图9所示。图9A中，在GFAP-EGFP-2A-Cre组中，穆勒胶质细胞呈现典型的形态，胞体位于内颗粒层(INL)，突起分别分布至外颗粒层(ONL)和视网膜神经节细胞层(GCL)，并且在ONL中未观测到红色信号标记的感光细胞，表明对照组不会有穆勒胶质细胞转分化为感光细胞。如图9B-图9J所示，在负调控基因敲低的实验组，Sin3a、BAF53a、G9a、HuR、BrG1、EZH2、和KDM1A组发现大量红色荧光标记的感光细胞，这些结果表明敲低Sin3a、HDAC2、BAF53a、G9a、HuR、BrG1、EZH2、CDYL、和KDM1A基因能将穆勒胶质细胞转分化为感光细胞。
正调控基因组对转分化的影响如图10所示，在BAF45b和DPYSL2的ONL层发现了红色荧光标记的感光细胞，显示过表达DPYSL2和BAF45b能穆勒胶质细胞转分化为感光细胞。
在视网膜中，CasRx敲低负调控基因基因的表达或过表达正调控基因将穆勒胶质细胞重编程为感光细胞的数量统计如图11所示，显示抑制Sin3a、KDM1A、BAF53a、G9a、HuR、BrG1、EZH2、CDYL均显示出优异的感光细胞再生能力，抑制HDAC2也具有感光细胞再生能力，过表达DPYSL2和BAF45b，也具有感光细胞再生能力。
感光细胞分为视锥细胞和视杆细胞，我们选取其中的Sin3a和KDM1A组用视杆细胞特异 性蛋白标志物进行染色，如图12所示，发现在ONL层的由穆勒胶质细胞转分化为感光细胞大部分都是视杆细胞。
我们用视锥细胞特异性蛋白标志物m-CAR对部分组别进行染色，并在共聚焦显微镜下进行成像分析，结果如图13所示，发现有视锥细胞。
实施例6、穆勒胶质细胞向视神经节细胞转分化
为了研究敲低RCOR1、Sin3a、HDAC2、KDM1A、PHF21A、BAF53a、G9a、USP14、HuR、BrG1、EZH2、CDYL、HMG20B或过表达DPYSL2、BAF53b、BAF45b、SCF、HuB、HuC、HuD、CYP1B1、或BTRC是否可以将穆勒胶质细胞转分化为视网膜神经节细胞(RGC)，向NMDA造模的6-8周的Ai9(Rosa26-CAG-LSL-tdTomato-WPRE)小鼠视网膜下注射不同的AAV组合。在基因表达敲低组，我们注射AAV-GFAP-CasRx-gRNA(或对照载体AAV-GFAP-CasRx)与AAV-GFAP-EGFP-2A-Cre的混合病毒。AAV-GFAP-EGFP-2A-Cre用来特异性的标记穆勒胶质细胞或者由胶质细胞转化来的神经元(视神经节细胞)。在过表达组，注射AAV-GFAP-EGFP-2A-Cre作为对照组，注射AAV-GFAP-EGFP-2A-Cre+AAV-GFAP-正调控基因(其中正调控基因为DPYSL2、BAF45b、SCF、HuB、HuC、HuD、CYP1B1、BTRC)。由于穆勒胶质细胞在GCL层的伪足对观测转分化的RGC细胞有比较大的干扰，而每一个RGC细胞都会向视神经发出一条轴突，我们采用视神经成像的分析方法，在注射后1-3个月取视神经进行结果分析，结果如图14和图15所示。如图14A，在对照组中，我们发现非常少量的视神经，平均每个视神经中只有少于5条红色荧光信号标记的轴突被发现，如图14B至图14I，在敲低HuR组，观测到大量的红色荧光信号标记的轴突，而KDM1A、Sin3a、HDAC2这些组别中几乎没有观测到明显的红色荧光信号标记的轴突信号。如图15所示，在过表达SCF、HuD和CYP1B1组，观测到大量的红色荧光信号标记的轴突，SCF组红色荧光信号标记的轴突最多。这些结果表明敲低HuR的表达，或者过表达SCF、HuD和CYP1B1能促进穆勒胶质细胞向RGC的转分化，而KDM1A、Sin3a、HDAC2等基因不能发挥促进穆勒胶质细胞向RGC的转分化的功能。
在视网膜中敲低KDM1A、HDAC2、BAF53a、G9a、HuR、BrG1、EZH2、CDYL的表达或过表达DPYSL2、BAF45b、SCF、HuB、HuD、CYP1B1、BTRC将穆勒胶质细胞重编程为视网膜神经节细胞的数量，在视神经中统计tdTomato阳性的轴突数量的统计图如图16所示，在SCF、HuD、CYP1B1组均发现较多的视神经节细胞。
实施例7：RP疾病模型小鼠中发现感光细胞再生
方法：Pde6b基因是一种和视网膜色素变性(RP)相关的致病基因，Pde6b基因的缺陷会导致感光细胞的死亡。目前Pde6b基因缺陷小鼠已经被证明具有RP遗传特征的疾病动物模型。本专利中利用基于CRISPR-Cas9编辑构建的Pde6b基因敲除小鼠模型，通过免疫组化方法证明 Pde6b基因敲除小鼠在生后4周rod细胞发生大量死亡，6周rod细胞全部死完(图17A-C)。在该疾病模型小鼠7周龄时同时进行视网膜下注射转分化靶点KDM1A和EZH2的AAV(AAV-U6-gRNA(KDM1A)-GFAP-CasRx和AAV-U6-gRNA(EZH2)-GFAP-CasRx)以及过表达Pde6b的AAV(AAV-CBH-Pde6b)，对照组同时注射对照AAV(AAV-GFAP-CasRx)和过表达Pde6b的AAV(AAV-CBH-Pde6b)，转分化靶点AAV的注射量为1x1010vg，Pde6b过表达AAV注射量为5x109vg，注射AAV总体积为1μl。注射后2个月，取视网膜组织进行分析，发现在注射对照组AVV(AAV-GFAP-CasRx+AAV-CBH-Pde6b)的小鼠视网膜外颗粒层并没有感光细胞，用视杆细胞特异性标志物Rhodopsin进行染色，也没有发现有Rhodopsin阳性的细胞(图17D)。而在注射了转分化靶点KDM1A和EZH2的AAV(AAV-U6-gRNA(KDM1A)-GFAP-CasRx和AAV-U6-gRNA(EZH2)-GFAP-CasRx)组，在小鼠视网膜外颗粒层发现了一层细胞，对其进行免疫荧光染色发现这些细胞是Rhodopsin阳性的感光细胞(图17E-F)。对各组进行统计分析，发现在敲低KDM1A和EZH2组中，在200um中大约能产生20多个Rhodopsin阳性的感光细胞(图17G)。对外颗粒层的厚度进行分析，发现敲低KDM1A和EZH2组的外颗粒层明显增厚(图17H)。
在上述的实施例中(例如实施例3和实施例4)，申请人详细描述了针对纹状体注射本申请的负向调节剂或正想调节剂的结果，本领域技术人员根据本申请的技术方案及本领域的常规认知，可以选择在脑部的其他部位进行定位注射，例如针对中脑腹侧被盖区、脊髓、下丘脑、背侧中脑、大脑皮层、海马、小脑、或黑质等进行注射，特别是在黑质中进行注射，也具有相同或相似的技术效果。
在上述的实施例中(例如实施例5)，申请人详细描述了在视网膜下注射本申请的负向调节剂或正想调节剂的结果，本领域技术人员根据本申请的技术方案及本领域的常规认知，可以选择在眼部的其他部位进行定位注射，例如玻璃体腔，也具有相同或相似的技术效果。
应知晓，上述实施例仅仅是作为示例性的说明，而非具体限定，例如，在上述实施例中，申请人详细描述了负向调控RCOR1基因的结果，本领域技术人员根据本申请的技术方案及本领域的常规认知，可以选择与RCOR1相似的基因(例如RCOR2或RCOR3)进行调控，也具相同或相似的技术效果；申请人详细描述了负向调控Sin3a基因的结果，本领域技术人员根据本申请的技术方案及本领域的常规认知，可以选择与Sin3a相似的基因(例如Sin3b)进行调控，也具相同或相似的技术效果；申请人详细描述了负向调控HDAC2基因的结果，本领域技术人员根据本申请的技术方案及本领域的常规认知，可以选择与HDAC2相似的基因(例如HDAC1)进行调控，也具相同或相似的技术效果。
在本公开内容提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本公开内容的上述讲授内容之后，本领域技术人员可以对本公开内容作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定 的范围。
以下为本申请涉及到正调控基因和负调控基因的序列信息：
人RCOR1蛋白序列(SEQ ID NO:1)
人RCOR1编码序列(SEQ ID NO:2)
人RCOR2蛋白序列(SEQ ID NO:3)
人RCOR2编码序列(SEQ ID NO:4)
人RCOR3蛋白序列(SEQ ID NO:5)
人RCOR3编码序列(SEQ ID NO:6)

人Sin3a蛋白序列(SEQ ID NO:7)
人Sin3a编码序列(SEQ ID NO:8)

人Sin3b蛋白序列(SEQ ID NO:9)
人Sin3b编码序列(SEQ ID NO:10)
人HDAC1蛋白序列(SEQ ID NO:11)

人HDAC1编码序列(SEQ ID NO:12)
人HDAC2蛋白序列(SEQ ID NO:13)
人HDAC2编码序列(SEQ ID NO:14)
人Kdm1a蛋白序列(SEQ ID NO:15)
人Kdm1a编码序列(SEQ ID NO:16)

人PHF21A蛋白序列(SEQ ID NO:17)
人PHF21A编码序列(SEQ ID NO:18)
人BAF53a蛋白序列(SEQ ID NO:19)
人BAF53a编码序列(SEQ ID NO:20)

人G9a蛋白序列(SEQ ID NO:21)
人G9a编码序列(SEQ ID NO:22)
人USP14蛋白序列(SEQ ID NO:23)

人USP14编码序列(SEQ ID NO:24)
人HuR蛋白序列(SEQ ID NO:25)
人HuR编码序列(SEQ ID NO:26)
人BrG1蛋白序列(SEQ ID NO:27)
人BrG1编码序列(SEQ ID NO:28)

人EZH2蛋白序列(SEQ ID NO:29)
人EZH2编码序列(SEQ ID NO:30)

人CDYL蛋白序列(SEQ ID NO:31)
人CDYL编码序列(SEQ ID NO:32)
人HMG20B蛋白序列(SEQ ID NO:33)
人HMG20B编码序列(SEQ ID NO:34)
人DPYSL2蛋白序列(SEQ ID NO:35)

人DPYSL2编码序列(SEQ ID NO:36)
人BAF45b蛋白序列(SEQ ID NO:37)
人BAF45b编码序列(SEQ ID NO:38)
人SCF蛋白序列(SEQ ID NO:39)
人SCF编码序列(SEQ ID NO:40)
人HuB蛋白序列(SEQ ID NO:41)
人HuB编码序列(SEQ ID NO:42)
人HuC蛋白序列(SEQ ID NO:43)
人HuC编码序列(SEQ ID NO:44)
人HuD蛋白序列(SEQ ID NO:45)
人HuD编码序列(SEQ ID NO:46)
人CYP1B1蛋白序列(SEQ ID NO:47)
人CYP1B1编码序列(SEQ ID NO:48)

人BTRC蛋白序列(SEQ ID NO:49)
人BTRC编码序列(SEQ ID NO:50)

Claims (40)

  1. 将哺乳动物的非神经元细胞转分化为神经元的方法，其中，包括：
    提供能够降低负调控基因表达的负向调节剂，所述负调控基因包括RCOR1、RCOR2、RCOR3、Sin3a、Sin3b、HDAC1、HDAC2、KDM1A、PHF21A、BAF53a、G9a、USP14、HuR、BrG1、EZH2、CDYL、或HMG20B，和/或
    提供能够提高正调控基因表达的正向调节剂，所述正调整基因包括DPYSL2、BAF45b、SCF、HuB、HuC、HuD、CYP1B1、或BTRC，
    有效量的所述负向调节剂或所述正向调节剂与所述非神经元细胞接触，诱导非神经元动物细胞转分化为神经元。
  2. 如权利要求1所述的方法，其中，所述非神经元细胞来源于人。
  3. 如权利要求1或2所述的方法，其中，所述非神经元细胞为干细胞、祖细胞或终末分化细胞；优选为胶质细胞；更优选的，所述胶质细胞为星形胶质细胞、少突胶质细胞、小胶质细胞、NG2细胞、穆勒胶质细胞、胶质瘤细胞或螺旋神经节胶质细胞，更优选的，所述胶质细胞为星形胶质细胞或穆勒胶质细胞。
  4. 如权利要求3所述的方法，其中，所述非神经元细胞来源于脑部，优选的，所述非神经元细胞来自大脑、中脑、小脑、脑干、脊髓；更优选的，来源于大脑中的纹状体或黑质。
  5. 如权利要求4所述的方法，其中，所述非神经元细胞为来源于脑部的星形胶质细胞，优选的，为来源于纹状体或黑质的星形胶质细胞。
  6. 如权利要求4所述的方法，其中，所述非神经元细胞来源于眼部，优选的，所述非神经元细胞为来自眼部的穆勒胶质细胞。
  7. 如权利要求1-6任一所述的方法，其中，所述神经元为多巴胺能神经元、视网膜神经节细胞、感光细胞、5-HT神经元、NE神经元、ChAT神经元、运动神经元、GABA神经元、谷氨酸能神经元、脊髓神经元，脊髓运动神经元、脊髓感觉神经元、双极细胞、无长突细胞、耳蜗神经细胞、锥体神经元、中间神经元、中型多棘神经元、浦肯野细胞、颗粒细胞、嗅感觉神经元、或球周细胞、或其组合。
  8. 如权利要求1-7任一所述的方法，其中，所述负调控基因为RCOR1、RCOR2、RCOR3、Sin3a、Sin3b、KDM1A、BAF53a、G9a、HuR、BrG1、或EZH2，所述非神经元动物细胞来源于脑部或眼部。
  9. 如权利要求8所述的方法，其中，所述负调控基因为RCOR1、RCOR2、RCOR3或G9a，所述非神经元动物细胞来源于脑部；优选的，所述非神经元细胞为来自脑部的星形胶质细胞； 更优选的，所述非神经元细胞为来自纹状体或黑质的星形胶质细胞。
  10. 如权利要求8所述的方法，其中，所述负调控基因为Sin3a、Sin3b、KDM1A、BAF53a、G9a、HuR、BrG1、CDYL、或EZH2，所述非神经元细胞来源于眼部；优选的，所述非神经元细胞为来自眼部穆勒胶质细胞。
  11. 如权利要求10所述的方法，其中，所述负调控基因为HuR，所述神经元为视神经节细胞。
  12. 如权利要求10所述的方法，其中，所述负调控基因为Sin3a、KDM1A、BAF53a、G9a、HuR、BrG1、CDYL、或EZH2，所述神经元为感光细胞。-
  13. 如权利要求1-7任一所述的方法，其中，所述正调控基因为DPYSL2、BAF45b、SCF、HuB、HuC、HuD、CYP1B1、或BTRC，所述非神经元细胞来自脑部；优选的，所述非神经元细胞来自纹状体、黑质、中脑腹侧被盖区、脊髓、下丘脑、背侧中脑、大脑皮层、海马、小脑；更优选的，来自纹状体。
  14. 如权利要求13所述的方法，其中，所述正调控基因为SCF或HuB。
  15. 如权利要求13所述的方法，其中，所述正调控基因为HuB或BTRC，有效量的所述正向调节剂与所述非神经元细胞接触，诱导非神经元类非神经元细胞转分化为多巴胺能神经元。
  16. 如权利要求13-15任一所述的方法，其中，所述非神经元细胞为星形胶质细胞；更优选的，所述非神经元细胞为来自纹状体或黑质的星形胶质细胞。
  17. 如权利要求1-7任一所述的方法，其中，所述正调控基因为DPYSL2、BAF45b、SCF、HuC、HuD、或CYP1B1，所述非神经元细胞来自眼部；优选的，所述非神经元细胞为来自眼部穆勒胶质细胞。
  18. 如权利要求17所述的方法，其中，所述正调控基因为SCF、HuD、或CYP1B1，所述神经元为视神经节细胞。
  19. 如权利要求17所述的方法，其中，所述正调控基因为DPYSL2或BAF45b，所述神经元为感光细胞。
  20. 如权利要求1-19任一所述的方法，其中，所述降低负调控基因表达是能够降低负调控基因的基因水平、或降低负调控基因的mRNA水平、或降低负调控基因的编码蛋白表达水平；
    所述提高正调控基因表达是能够提高正调控基因的基因水平、或提高正调控基因的mRNA水平、或提高正调控基因的编码蛋白表达水平。
  21. 如权利要求20所述的方法，其中，所述负向调节剂选自降低负调控基因表达的基因编 辑工具或表观遗传调控工具；负调控基因的抑制剂、负调控基因活性抑制剂、或负调控基因的编码蛋白的降解激活剂。
  22. 如权利要求21所述的方法，其中，所述抑制剂为：负调控基因的抑制性抗体；或负调控基因的小分子抑制剂；或负调控基因的抑制性mRNA、microRNA、siRNA、shRNA、反义寡核苷酸、结合蛋白或蛋白结构域、多肽、核酸适配体、或PROTAC；或负调控基因的抑制性结合蛋白或配体。
  23. 如权利要求21所述的方法，其中，所述的负向调节剂含有如SEQ ID NO：51-67中任一所述的gRNA及基因编辑蛋白。
  24. 如权利要求20所述的方法，其中，所述正向调节剂选自能够提高正调控基因表达的表观遗传调控工具、正调控基因表达激活剂、正调控基因编码蛋白的降解抑制剂、正调控基因mRNA的稳定剂、或外源性的正调控基因或正调控基因的功能性片段。
  25. 如权利要求24所述的方法，其中，所述激活剂为：正调控基因的激动性抗体；或正调控基因的小分子激动剂；或正调控基因的激动性结合蛋白或配体；或正调控基因的竞争性基因的抑制剂。
  26. 如权利要求24所述的方法，其中，所述正向调节剂含有如SEQ ID NO：36、38、40、42、44、46、48、或50所示的核酸序列，或含有如SEQ ID NO：36、38、40、42、44、46、48、或50所示的核酸序列的功能性片段。
  27. 如权利要求21所述的基因编辑工具，其特征在于，所述基因编辑工具包括基因编辑系统或其表达载体，所述基因编辑系统有选自：CRISPR系统(包括CRISPR/Cas系统)、ZFN系统、TALEN系统，或其组合
  28. 根据权利要求27所述的方法，其中，采用CRISPR系统降低负调控基因的表达或活性；优选的，所述CRISPR系统含有Cas酶或Cas酶的功能结构域的编码核酸以及靶向所述细胞转分化因子的gRNA；更优选的，所述Cas酶为Cas13；更优选的，所述Cas酶为Cas13d、Cas13X、Cas13a、Cas13b、Cas13c、或Cas13Y；更优选的，所述Cas酶为CasRx。
  29. 根据权利要求20所述的方法，其中，所述负向调节剂或正向调节剂采用运载体携带；优选的，所述运载体为病毒载体、脂质纳米颗粒(LNP)、脂质体、阳离子聚合物(如PEI)、纳米颗粒、外泌体、或类病毒颗粒；更优选的，所述载体为AAV载体或脂质纳米颗粒。
  30. 如权利要求1-29任一所述的方法，其中，所述有效量的所述负向调节剂或所述正向调节剂与所述非神经元细胞在体外接触，诱导非神经元细胞在体外转分化为神经元；或
    所述有效量的所述负向调节剂或所述正向调节剂与所述非神经元细胞在体内接触，诱导非神经元细胞在体外转分化为神经元。
  31. 权利要求1-30任一所涉及的负向调节剂或正向调节剂用于制备预防或治疗与神经元损伤或神经元死亡相关的疾病的药物的用途。
  32. 权利要求1-30任一所涉及的负向调节剂或正向调节剂用于预防或治疗与神经元损伤或神经元死亡相关的疾病的用途。
  33. 如权利要求31或32所述的用途，其中，所述药物配制成用于在体内施用于神经系统、视觉系统和听觉系统的药剂。
  34. 如权利要求31或32所述的用途，其中，所述与神经元损伤或神经元死亡相关的疾病选自帕金森病、RGC或感光细胞功能缺失或死亡有关的视觉系统疾病、阿尔茨海默病、脑损伤、亨廷顿舞蹈症、癫痫、抑郁症、睡眠失调、脑缺血、运动神经元病、肌萎缩性侧索硬化、脊髓性肌萎缩症、共济失调、PloyQ疾病、精神分裂症、成瘾、Pick病、失明、耳聋；优选为帕金森病和RGC或感光细胞功能缺失或死亡有关的视觉系统疾病。
  35. 如权利要求34所述的用途，其中，所述与RGC或感光细胞功能缺失或死亡有关的视觉系统疾病优选自：RGC细胞或感光细胞死亡导致的视力损伤、青光眼、年龄相关的RGC病变、视神经损伤、年龄相关性黄斑变性(AMD)、糖尿病相关视网膜病变、视网膜缺血或出血、Leber遗传性视神经病变、或其组合；所述与感光细胞功能缺失或死亡有关的视觉系统疾病优选自：损伤或退行性病变导致的感光细胞变性或死亡、黄斑变性、视网膜色素变性、糖尿病有关的失明、夜盲症、色盲、遗传性失明、先天性黑蒙症、或其组合。
  36. 根据权利要求31或32所述的用途，其中，所述神经元为多巴胺能神经元、5-HT神经元、NE神经元、ChAT神经元、GABA神经元、谷氨酸能神经元、运动神经元、感光细胞(如视杆细胞和视锥细胞)、视网膜神经节细胞(RGC)、耳蜗神经细胞(如耳蜗螺旋神经节细胞和前庭神经元)、或中型多棘神经元(MSN)或其组合，优选多巴胺能神经元、视网膜神经节细胞和感光细胞。
  37. 一种预防或治疗与神经元损伤或神经元死亡相关的疾病的方法，包括给有此需要的受试者有效量的权利要求1-30任一所涉及的负向调节剂或正向调节剂。
  38. 根据权利要求37的方法，其中，所述与神经元损伤或神经元死亡相关的疾病选自帕金森病、RGC或感光细胞功能缺失或死亡有关的视觉系统疾病、阿尔茨海默病、脑损伤、亨廷顿舞蹈症、癫痫、抑郁症、睡眠失调、脑缺血、运动神经元病、肌萎缩性侧索硬化、脊髓性 肌萎缩症、共济失调、PloyQ疾病、精神分裂症、成瘾、Pick病、失明、耳聋；优选为帕金森病和RGC或感光细胞功能缺失或死亡有关的视觉系统疾病。
  39. 如权利要求38所述的方法，其特征在于，所述与RGC功能缺失或死亡有关的视觉系统疾病优选自：RGC细胞死亡导致的视力损伤、青光眼、年龄相关的RGC病变、视神经损伤、年龄相关性黄斑变性(AMD)、糖尿病相关视网膜病变、视网膜缺血或出血、Leber遗传性视神经病变、或其组合；所述与感光细胞功能缺失或死亡有关的视觉系统疾病优选自：损伤或退行性病变导致的感光细胞变性或死亡、黄斑变性、视网膜色素变性、糖尿病有关的失明、夜盲症、色盲、遗传性失明、先天性黑蒙症、或其组合。
  40. 根据权利要求37所述的方法，其特征在于，所述神经元为多巴胺能神经元、5-HT神经元、NE神经元、ChAT神经元、GABA神经元、谷氨酸能神经元、运动神经元、感光细胞(如视杆细胞和视锥细胞)、视网膜神经节细胞(RGC)、耳蜗神经细胞(如耳蜗螺旋神经节细胞和前庭神经元)、或中型多棘神经元(MSN)或其组合，优选多巴胺能神经元、视网膜神经节细胞和感光细胞。
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