WO2022171167A1 - 胶质细胞向神经元转分化用于预防或治疗神经元功能缺失或死亡相关疾病 - Google Patents

胶质细胞向神经元转分化用于预防或治疗神经元功能缺失或死亡相关疾病 Download PDF

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WO2022171167A1
WO2022171167A1 PCT/CN2022/075823 CN2022075823W WO2022171167A1 WO 2022171167 A1 WO2022171167 A1 WO 2022171167A1 CN 2022075823 W CN2022075823 W CN 2022075823W WO 2022171167 A1 WO2022171167 A1 WO 2022171167A1
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rest
cells
promoter
kit
grna
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French (fr)
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周海波
胡新德
苏锦霖
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中国科学院脑科学与智能技术卓越创新中心
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Priority to CN202280014084.7A priority Critical patent/CN117279668A/zh
Publication of WO2022171167A1 publication Critical patent/WO2022171167A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • 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
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • 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 disclosure relates to the field of biomedicine. More specifically, the present disclosure relates to the use of REST (RE1-silencing transcription factor) inhibitors in the prevention and/or treatment of diseases associated with neuronal dysfunction or death.
  • REST RE1-silencing transcription factor
  • Parkinson's disease is a disorder associated with neuronal dysfunction or death, characterized by the loss of midbrain substantia nigra dopamine neurons.
  • the main treatment for Parkinson's disease is the drug represented by levodopa preparations.
  • surgical treatment can also improve symptoms to some extent. It should be pointed out that all these measures can only partially alleviate the disease, but can not achieve the effect of preventing the development of the disease.
  • Müller glia are the main glial cells in retinal tissue. In zebrafish, when retinal damage occurs, Müller glia can proliferate and differentiate into various retinal cells, including photoreceptor retinal ganglion cells (RGCs) and bipolar cells, to help repair the damage. But in higher mammals, MG loses the ability to differentiate into various functional cells of the retina after retinal maturation. Photoreceptors are a special type of nerve cell in the retina and the only cells in the visual system that will capture light signals. Retinitis pigmentosa, congenital amaurosis (LCA), age-related macular degeneration, diabetic retinopathy and other eye diseases can cause photoreceptor cell death, which can lead to blindness.
  • ROCs photoreceptor retinal ganglion cells
  • Photoreceptors are a special type of nerve cell in the retina and the only cells in the visual system that will capture light signals. Retinitis pigmentosa, congenital amaurosis (LCA), age-related ma
  • Retinal ganglion cells are nerve cells located in the innermost layer of the retina. Their dendrites mainly communicate with bipolar cells, and their axons extend to the optic nerve head to form the optic nerve. Retinal ganglion cell (RGC) degeneration is a major cause of permanent blindness. RGCs are the only output neurons of the retina, so RGC degeneration can lead to retinal diseases that cause permanent blindness. Therefore, reconstruction of functional photoreceptor cells or RGCs can help restore vision and is a potential therapeutic approach to restore visual function.
  • Another object of the present disclosure is to provide the use of REST inhibitors for the prevention and/or treatment of diseases associated with functional dopamine neuron dysfunction or death.
  • Another object of the present disclosure is to provide methods for generating functional retinal ganglion cells (RGCs) or photoreceptor cells from Mullerian glial cells.
  • Another object of the present disclosure is to provide the use of a REST inhibitor for preventing and/or treating diseases of the visual system associated with RGC or photoreceptor cell dysfunction or death.
  • the present disclosure provides a method of generating functional dopamine neurons from glial cells comprising transdifferentiating or reprogramming said glial cells into functional dopamine neurons using a REST inhibitor, wherein The REST inhibitor reduces the expression, content or activity of the REST gene or its RNA or its encoded protein.
  • the glial cells are selected from astrocytes, oligodendrocytes, ependymal cells, Schwann cells, NG2 cells, satellite cells, or a combination thereof.
  • the glial cells are astrocytes.
  • the astrocytes are derived from the central nervous system, including the striatum, substantia nigra, ventral tegmental area, spinal cord, hypothalamus, dorsal midbrain, or cerebral cortex.
  • the astrocytes are derived from the striatum and substantia nigra.
  • the present disclosure provides the use of a REST inhibitor in the manufacture of a medicament for the prevention and/or treatment of a disease associated with functional dopamine neuron dysfunction or death, wherein the REST Inhibitors reduce the expression, content or activity of a REST gene or its RNA or its encoded protein.
  • the medicament is formulated for administration to the central nervous system, including the striatum, substantia nigra, ventral tegmental area, spinal cord, hypothalamus, dorsal midbrain or cerebral cortex, etc. .
  • the medicament is formulated for administration to the striatum and substantia nigra.
  • the disease associated with the loss or death of functional dopamine neurons is a neurological disease, including stroke, Parkinson's disease, schizophrenia and depression.
  • the disease associated with the loss or death of functional dopamine neurons is Parkinson's disease.
  • the present disclosure provides a method of generating functional retinal ganglion cells (RGCs) or photoreceptor cells from Mullerian glial cells, comprising transdifferentiating the Mullerian glial cells with a REST inhibitor For or reprogramming into functional RGC or photoreceptor cells, wherein the REST inhibitor reduces the expression, content or activity of the REST gene or its RNA or its encoded protein.
  • RRCs retinal ganglion cells
  • photoreceptor cells For or reprogramming into functional RGC or photoreceptor cells
  • the Mullerian glial cells are derived from retina.
  • the photoreceptor cells include rod cells and cone cells.
  • the present disclosure provides the use of a REST inhibitor in the manufacture of a medicament for the prevention and/or treatment of diseases of the visual system associated with RGC or photoreceptor cell dysfunction or death, wherein the A REST inhibitor reduces the expression, content or activity of a REST gene or its RNA or its encoded protein.
  • the medicament is formulated for administration to the visual system.
  • the drug is formulated for use in the subretinal or vitreous cavity, wherein the drug acts by acting on Mullerian glial cells.
  • the neurological disease associated with RGC loss or death is selected from the group consisting of: visual impairment caused by RGC cell death, glaucoma, age-related RGC lesions, optic nerve damage, retinal ischemia or hemorrhage, Leber Inherited optic neuropathy, or a combination thereof.
  • the visual system disease associated with photoreceptor cell function loss or death is selected from the group consisting of photoreceptor cell degeneration or death caused by injury or degeneration, macular degeneration, retinitis pigmentosa, diabetes-related blindness, Night blindness, color blindness, hereditary blindness, amaurosis or a combination thereof.
  • the REST inhibitor is selected from the group consisting of: antibodies, small molecule compounds, microRNA, siRNA, shRNA, antisense oligonucleotides, REST binding proteins or protein domains, polypeptides, nucleic acid aptamers, Gene editors, PROTACs, epigenetic regulation, or a combination thereof.
  • the REST inhibitor comprises:
  • Gene editing protein or expression vector thereof editing system including: CRISPR system (including CRISPR/dCas system), ZFN system, TALEN system, RNA editing system, or a combination thereof, (b) one or more gRNAs or expression vectors thereof, all of which The gRNA is the DNA or RNA that guides the gene editing protein to specifically bind to the REST gene,
  • RNA-targeting gene editing proteins and RNA-targeting gRNAs are preferred.
  • the gRNA comprises a sequence complementary to the target sequence.
  • the gRNA directs the gene editing protein to specifically bind to nucleotides 867-1103 corresponding to the REST coding sequence (SEQ ID NO.: 3).
  • the gRNA comprises a sequence complementary to SEQ ID NO.:3.
  • the gRNA comprises a sequence selected from the group consisting of SEQ ID NO.: 4-20 and 83-118 or comprises a sequence encoded by the sequence of SEQ ID NO.: 55-62 and 71-76, preferably The gRNA comprises a sequence selected from SEQ ID NO.: 10 and 93-103.
  • the gRNA comprises a sequence that is completely complementary to the target sequence, or comprises a complementary sequence that has no more than 3 base mismatches with the target sequence.
  • the gRNA and the target sequence are from the same species or different species.
  • the gRNA and target sequence are from human, cynomolgus monkey or mouse.
  • the present disclosure provides a pharmaceutical composition or kit or kit comprising a REST inhibitor.
  • the REST inhibitor is selected from the group consisting of: antibodies, small molecule compounds, microRNA, siRNA, shRNA, antisense oligonucleotides, REST binding proteins or protein domains, polypeptides, nucleic acid aptamers, Gene editors, PROTACs, epigenetic regulation, or a combination thereof.
  • the REST inhibitor comprises:
  • the editing system includes: a CRISPR system (including a CRISPR/dCas system), a ZFN system, a TALEN system, an 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.
  • the gRNA comprises a sequence complementary to the target sequence.
  • the gRNA directs the gene editing protein to specifically bind to nucleotides 867-1103 corresponding to the REST coding sequence (SEQ ID NO.: 3).
  • the gRNA comprises a sequence complementary to SEQ ID NO.:3.
  • the gRNA comprises a sequence selected from the group consisting of SEQ ID NO.: 4-20 and 83-118 or comprises a sequence encoded by the sequence of SEQ ID NO.: 55-62 and 71-76, preferably The gRNA comprises a sequence selected from SEQ ID NO.: 10 and 93-103.
  • the gRNA comprises a sequence that is completely complementary to the target sequence, or comprises a complementary sequence that has no more than 3 base mismatches with the target sequence.
  • the gRNA and the target sequence are from the same species or different species.
  • the gRNA and target sequence are from human, cynomolgus monkey or mouse.
  • the pharmaceutical composition or kit or kit further comprises a carrier or vehicle for delivering the REST inhibitor.
  • the vector or vehicle is a viral vector, liposome, nanoparticle, exosome, viroid, preferably AAV.
  • the RNA-targeting gene editing protein is selected from the group consisting of Cas13d, CasRx, Cas13X, Cas13a, Cas13b, Cas13c, Cas13Y and functional domains thereof.
  • the RNA-targeting gene editing protein is selected from the group consisting of CasRx, Cas13X, and Cas13Y.
  • RNA-targeting gene editing protein is CasRx.
  • the pharmaceutical composition or kit or kit comprises only a single type of gRNA or 2, 3, 4, 5, 6 different gRNAs targeting the REST mRNA sequence.
  • the gRNA expression vector encodes only a single type of gRNA or 2, 3, 4, 5, 6 different gRNAs comprising the REST mRNA sequence targeting.
  • the expression vector comprises:
  • a promoter such as the U6 promoter to which the gRNA is expressed in mammalian cells is operably linked.
  • the promoter is a glial cell specific promoter or a Mueller 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 synthase promoter, S100 ⁇ promoter and EAAT2/GLT- 1 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 nanoparticles.
  • 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), adenoviral vector, lentiviral vector, retroviral vector , herpes virus, SV40 vector, poxvirus vector, and combinations thereof.
  • AAV adeno-associated virus
  • rAAV recombinant adeno-associated virus vector
  • adenoviral vector lentiviral vector
  • retroviral vector lentiviral vector
  • herpes virus SV40 vector
  • poxvirus vector poxvirus vector
  • the expression vector is an AAV vector or rAAV.
  • the composition is administered topically to at least one of: 1) 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; glial cells in the lateral tegmental area (VTA); and ix) glial cells in the hypothalamus.
  • 1) 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;
  • the pharmaceutical composition or kit or kit further comprises i) one or more dopamine neuron-related factors, or ii) for expressing in said glial cells a or at least one expression vector for more dopamine neuron-related factors.
  • the one or more dopamine neuron-related factors are selected from the group consisting of: Lmx1a, Lmx1b, FoxA2, Nurr1, Pitx3, Gata2, Gata3, FGF8, BMP, En1, En2, PET1, Pax family proteins, SHH, Wnt family proteins and TGF-beta family proteins.
  • the pharmaceutical composition or kit or kit further comprises i) one or more selected from the group consisting of ⁇ -catenin, Oct4, Sox2, Klf4, Crx, Brn3a, Brn3b, Math5, Factors for Nr2e3 and Nrl, and/or ii) for expression in glial cells of one or more factors selected from ⁇ -catenin, Oct4, Sox2, Klf4, Crx, Brn3a, Brn3b, Math5, Nr2e3 and Nrl at least one expression vector.
  • composition is further formulated for injection, intracranial, intraocular, inhalation, parenteral, intravenous, intramuscular, intradermal, topical, or Oral administration.
  • the AAV vector comprises:
  • nucleotide sequence encoding the gene editing protein operably linked to a promoter that causes expression of the gene editing protein in glial cells
  • the promoter is a glial cell specific promoter or a Mueller 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 synthase promoter, S100 ⁇ promoter and EAAT2/GLT- 1 promoter.
  • 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 transdifferentiation efficiency of the glial cells is at least 1%, or at least 10%, 20%, 30%, 40% or 50%.
  • the disease associated with neuronal dysfunction or death is selected from the group consisting of Parkinson's disease, schizophrenia, depression, vision impairment caused by RGC cell death, glaucoma, age-related RGC lesions, Optic nerve damage, retinal ischemia or hemorrhage, Leber hereditary optic neuropathy, photoreceptor cell degeneration or death due to injury or degeneration, macular degeneration, retinitis pigmentosa, diabetes-related blindness, night blindness, color blindness, hereditary blindness, congenital Amaurosis or a combination thereof.
  • 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.
  • the improving visual function is improving visual function in mammals suffering from neurodegenerative retinal diseases.
  • the MG cells are transdifferentiated into axon-free cells at the same time as the transdifferentiation into RGC cells.
  • the RGCs (1) express Brn3a, Rbpms, Foxp2, Brn3c and/or parvalbumin; (2) are F-RGCs, type 3 RGCs or PV-RGCs; (3) are integrated in all into existing retinal pathways in the subject (e.g., capable of projecting central information to the dLGN, and able to partially restore vision by relaying visual information to V1); and/or (4) capable of receiving visual information, which Information is characterized by the ability to establish action potentials upon light stimulation, synaptic connections (eg, with existing functional dLGN neurons in the brain), biogenesis of presynaptic neurotransmitters and/or subsequent synaptic responses.
  • the dopamine neurons (1) express tyrosine hydroxylase (TH), dopamine transporter (DAT), vesicular monoamine transporter 2 (VMAT2), corrugated homeobox 1 (Enl) ), FoxA2 and/or LIM homeobox transcription factor 1alpha (Lmxla); (2) exhibit synthesis and release of presynaptic neurotransmitters; (3) are integrated into the subject's brain with existing and/or (4) characterized by its ability to establish action potentials, synaptic connections, biogenesis of presynaptic neurotransmitters and/or postsynaptic responses.
  • TH tyrosine hydroxylase
  • DAT dopamine transporter
  • VMAT2 vesicular monoamine transporter 2
  • Enl corrugated homeobox 1
  • FoxA2 and/or LIM homeobox transcription factor 1alpha
  • a plurality of glial cells in the striatum are reprogrammed or transdifferentiated, and wherein at least 1% of the glial cells are converted into dopamine neurons.
  • the mammal includes a mammal suffering from a disease associated with neuronal dysfunction or death.
  • the mammal comprises a human or non-human mammal.
  • the non-human mammal includes rodents (eg, mice, rats, or rabbits), primates (eg, monkeys).
  • expression of 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 gNRAs and a gene editing protein.
  • the gRNA guides the gene editing protein to specifically bind to the RNA of the REST gene.
  • the gRNA-directed gene editing protein specifically binds to the mRNA of the REST gene.
  • nucleotide sequence of the gRNA is for example SEQ ID NO.: 4-20, preferably SEQ ID NO.: 10.
  • the source of the gene editing protein is selected from: Streptococcus pyogenes, Staphylococcus aureus, Acidaminococcus sp, Lachnospiraceae bacterium , Ruminococcus flavefaciens, or a combination thereof.
  • the REST is derived from a mammal; preferably, from a human, monkey, mouse, rat, or rabbit; more preferably, from a human.
  • the REST genes include wild-type REST genes and mutant REST genes.
  • the mutant form includes a mutant form in which the function of the encoded protein is unchanged after mutation (ie, the function is the same or substantially the same as the wild-type encoded protein).
  • polypeptide encoded by the mutant REST gene is the same or substantially the same as the polypeptide encoded by the wild-type REST gene.
  • the mutant REST gene comprises a homology of ⁇ 80% (preferably ⁇ 90%, more preferably ⁇ 95%, more preferably ⁇ 98% or 99%) of polynucleotides.
  • the mutant REST gene comprises truncation or addition of 1-60 (preferably 1-30, more preferably 1- 10) nucleotide polynucleotides.
  • the REST gene comprises a cDNA sequence, a genomic sequence, or a combination thereof.
  • the REST protein comprises an active fragment of REST or a derivative thereof.
  • the homology of the active fragment or its derivative to REST is at least 90%, preferably 95%, more preferably 98%, 99%.
  • the active fragment or derivative thereof has at least 80%, 85%, 90%, 95%, 100% REST activity.
  • amino acid sequence of the REST protein is selected from:
  • amino acid sequence shown above is formed by substitution, deletion or addition of one or several (such as 1-10) amino acid residues, and a polypeptide derived from (i) having the function of the protein; or
  • the homology of the amino acid sequence to the amino acid sequence shown above is ⁇ 90% (preferably ⁇ 95%, more preferably ⁇ 98% or 99%), and a polypeptide having the function of the protein.
  • nucleotide sequence of the REST gene is selected from:
  • 1-60 preferably 1-30, more preferably 1-10 nucleotides at the 5' end and/or 3' end of the polynucleotide having the nucleotide sequence shown above polynucleotide;
  • the REST protein is shown in the above amino acid sequence.
  • nucleic acid encoding the REST protein is shown in the above-mentioned nucleotide sequence.
  • the region targeted by the REST inhibitor is positions 15311-15338 of the REST gene sequence.
  • the REST inhibitor inhibits the activity and/or expression level of REST.
  • the concentration of the REST inhibitor is > 1 ⁇ 10 12 .
  • the inhibition rate of the REST inhibitor on the activity and/or expression of REST is greater than 90%, preferably, 90%-95%.
  • the inhibitor targets astrocytes of brain tissue.
  • the inhibitor targets retinal MG cells.
  • the gRNA-directed gene editing protein specifically binds to the mRNA of the REST gene.
  • the composition comprises a pharmaceutical composition.
  • the composition further includes other drugs for preventing and/or treating diseases associated with neuronal dysfunction or death.
  • composition further includes other drugs for the treatment of neurological diseases associated with functional neuronal death.
  • the composition further includes other drugs for preventing and/or treating retinal diseases.
  • the expression vector for the gene editing protein comprises a vector targeting glial cells.
  • the expression vector for the gene editing protein includes a vector targeting brain tissue astrocytes.
  • the expression vector for the gene editing protein comprises a vector targeting retinal MG cells.
  • the vector comprises AAV2, AAV8 or AAV9.
  • the gene encoding the gene editing protein and the gRNA are located in the same expression vector (eg, AAV vector).
  • the gene editing protein expression vector and the gRNA expression vector are the same expression vector (eg, AAV vector).
  • the expression vector further includes a glial cell-specific promoter (eg, a GFAP promoter) for driving the expression of the gene editing protein.
  • a glial cell-specific promoter eg, a GFAP promoter
  • the dosage form of the composition is selected from the group consisting of 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 related to neuronal dysfunction or death are selected from the group consisting of dopamine prodrugs, non-ergot dopamine receptor agonists, monoamine oxidase B inhibitors, or their combination.
  • the composition is a cellular preparation.
  • the gene editing protein expression vector and the gRNA expression vector are the same vector or different vectors.
  • the weight ratio of the component (a) to the component (b) is 100:1-0.01:1, preferably, 10:1-0.1:1, more preferably, 2: 1-0.5:1.
  • the content of the component (a) is 0.001%-99%, preferably 0.1%-90%, more preferably 1%-70%.
  • the content of the component (b) is 0.001%-99%, preferably 0.1%-90%, more preferably 1%-70%.
  • the content of the component (c) is 1%-99%, preferably 10%-90%, more preferably 30%-70%.
  • the component (a), the component (b) and the optional component (c) account for 0.01-99.99 wt% of the total weight of the composition, Preferably 0.1-90 wt%, more preferably 1-80 wt%.
  • a third aspect of the present disclosure provides a kit, comprising:
  • the gene editing protein is selected from: Cas13d, CasRx, Cas13X, Cas13a, Cas13b, Cas13c, Cas13Y, RNA-targeted gene editing proteins, or a combination thereof;
  • the gRNA guides the gene editing protein to specifically bind to the DNA or RNA of the REST gene.
  • the kit further comprises:
  • (c1) a third container, and other medicaments for preventing and/or diseases related to neuronal dysfunction or death, and/or containing other medicaments for preventing and/or treating retinal diseases, located in said third container, and and/or containing other drugs for the treatment of neurological disorders associated with functional neuronal death.
  • first container and the second and third containers are the same or different containers.
  • the drug in the first container is a single formulation containing the gene editing protein or its expression vector.
  • the medicine in the second container is a single formulation containing gRNA or its expression vector.
  • the medicament of the third container is a single formulation containing other medicaments that are pre-applied for the treatment of functional neuronal death-related neurological diseases.
  • the dosage form of the medicament is selected from: lyophilized formulation, liquid formulation, or a combination thereof.
  • the dosage form of the medicament is an oral dosage form or an injection dosage form.
  • the kit further contains instructions.
  • FIG. 1 Examine whether overexpression of miR124 can transdifferentiate glial cells into neurons in mice.
  • A Schematic diagram of overexpression of miR124 in mouse brain.
  • Vector-1 uses the glial cell-specific promoter GFAP to initiate mCherry red fluorescent protein expression, which is used to label glial cells
  • Vector-2 uses GFAP to initiate the expression of miR124. , can achieve the specific expression of miR124 in glial cells.
  • B After injection of GFAP-mCherry+GFAP-miR124 in mouse striatum, orange arrows point to the morphological changes in labeled glial cells, but not co-labeled with NeuN.
  • Tuj-1 is an early neuronal marker after injection of GFAP-mCherry+GFAP-miR124 in mouse striatum, orange arrows point to labeled glial cells, but these glial cells do not interact with Tuj-1 Co-scale, the scale bar is 40 ⁇ m.
  • D After infusion of GFAP-mCherry+GFAP-miR124 in mouse striatum, staining with neuron-specific marker NeuN and dopamine neuron-specific marker TH, white arrows point to labeled glial cells, these Glial cells were neither co-scaled with NeuN nor with the dopamine-specific marker TH, scale bar is 40 ⁇ m.
  • FIG. 1 Screening of gRNAs targeting mouse REST.
  • Vector-1 is a gRNA expression plasmid. The gRNA is driven by U6 and expresses red fluorescence to track transfection-positive cells.
  • Vector-2 is a CasRx expression plasmid. CasRx is driven by CAG and expresses green fluorescent tracer for transfection-positive cells.
  • B Schematic diagram of cell transfection and flow sorting. After transfecting cells, red and green double positive cells were sorted by flow sorting, and the amount of REST mRNA was detected by QCPR.
  • FIG. 3 Inhibition of REST transdifferentiates glial cells into neurons in the mouse brain.
  • A Schematic diagram of vector construction and transdifferentiation of glial cells in the brain.
  • the labeling system is GFAP-mCherry, and the expression of fluorescent protein mCherry is initiated by the astrocyte-specific promoter GFAP;
  • vector 2 is the control AAV plasmid, which is composed of astrocytes The glial cell-specific promoter GFAP initiates the expression of CasRx;
  • the vector 3 is an AAV plasmid targeting the REST group, and the gRNA expression is initiated by U6 (corresponding to gRNA-7 in Figure 2), and is initiated by the astrocyte-specific promoter GFAP at the same time CasRx expression; different AAV combinations were injected into mouse brains and samples were taken for analysis approximately 1 month after injection.
  • Control virus (GFAP-mCherry+GFAP-CasRx) was injected into mouse striatum, orange arrows point to labeled astrocytes, green is astrocyte-specific marker GFAP, white Nuclei were stained for mature neuron-specific marker NeuN, Dapi, and Merge images showed that mCherry signal co-labeled with GFAP signal, but not with NeuN.
  • FIG. 4 Epigenetic modulation techniques reduce REST gene expression.
  • DTM stands for DNA targeting protein or protein domain (such as: zinc finger proteins, TALEs, CRISPR-dCas, etc.)
  • DTM is linked to epigenetic regulatory proteins, including DNA epigenetic modification-related Enzymes and histone modification-related enzymes regulate the expression of downstream genes under the action of DTM-epigenetic modifier.
  • B Schematic diagram of the plasmid vector used in this study. The U6 promoter drives the expression of sgRNA, and CMV drives the expression of red fluorescent protein (mCherry).
  • vector 1 U6-sgRNA-CMV-mCherry
  • vector 2 dSpCas9-KRAB
  • vector 1 and vector 3 dSaCas9-KKH-KRAB
  • C After vector 1 and vector 2 were co-transfected into N2A cells, the inhibitory effect of epigenetic regulation on REST gene was detected by Q-PCR.
  • D After co-transfection of vector 1 and vector 3 in N2A cells, Q-PCR was used to detect the inhibitory effect of epigenetic regulation on REST gene.
  • FIG. 5 Screening of gRNAs in human cells (293T cells).
  • A The efficiency of each gRNA in knocking down REST expression in 293T cells, the red area indicates the gRNA region with high knockdown efficiency.
  • B Line graph of each gRNA knockdown REST expression, each gRNA corresponds to A.
  • C The distribution position of each gRNA on the REST gene, the gRNA marked in red is the gRNA with high inhibition efficiency, and the region marked in purple is the high-efficiency gRNA aggregation region.
  • FIG. 6 Efficient inhibition of REST in different species.
  • A Select 3 gRNA sequences targeting human REST and their mismatch sites in cynomolgus monkey and mouse, the bases marked in red are the difference sites in cynomolgus monkey or mouse and human REST sequences, gRNA-17, gRNA-18, gRNA-19 are the same numbered gRNAs targeting human REST in Figure 5.
  • B Schematic diagram of the construction of the vector, the gRNA in the expression vector is driven by U6, the CasRx is driven by CAG, and the green fluorescent protein gene is added to the vector to mark the transfection-positive cells.
  • C Schematic diagram of cell transfection and flow sorting.
  • gRNAs targeting human REST gRNA-17, gRNA-18 and gRNA-19
  • gRNAs targeting human REST gRNA-17, gRNA-18 and gRNA-19
  • FIG. 7 Human REST-targeting gRNAs transdifferentiate glial cells into neurons.
  • A Schematic diagram of AAV vector and transdifferentiation process, GFAP is astrocyte-specific promoter, mCherry is red fluorescent protein, CasRx is gene editing protein, U6-gRNA is U6-initiated REST-targeting gRNA expression cassette, The selected gRNA was gRNA-17 targeting human REST. Different AAV combinations were injected into mouse striatum and transdifferentiation effects were analyzed 1 month later.
  • the inventors After extensive and in-depth research, the inventors have unexpectedly discovered for the first time that inhibiting the expression, content or activity of the REST gene or RNA of glial cells or their encoded proteins can effectively induce the differentiation of glial cells into functional neurons, thereby effectively inducing the differentiation of glial cells into functional neurons. Treatment of neurological disorders associated with functional neuronal loss or death. On this basis, the present inventors have completed the present invention.
  • photoreceptor cell or retinal ganglion cell (RGC) degeneration is a major cause of permanent blindness.
  • the transdifferentiation of Mullerian glial cells (MG) into functional photoreceptor cells or RGCs can help restore vision.
  • the present application uses the recently characterized RNA-targeting CRISPR system CasRx for REST inhibition. Provides an excellent tool capable of treating a wide variety of diseases.
  • Mullerian glial cells are the primary glial cells in retinal tissue
  • retinal ganglion cells are nerve cells located in the innermost layer of the retina, whose dendrites are primarily associated with bipolar cells , its axons extend to the optic nerve head to form the optic nerve.
  • the gene editors include DNA gene editors, epigenetic regulatory editors, and RNA gene editors.
  • the gene editor of the present disclosure includes a gene editing protein and optionally a gRNA.
  • reprogramming or “transdifferentiation” can refer to the process of generating cells of a particular lineage (eg, neuronal cells) from different types of cells (eg, astrocytes).
  • diseases related to neuronal dysfunction or death mainly include diseases related to dopamine neuron dysfunction or death, and visual disturbances related to optic ganglion or photoreceptor cell loss or death.
  • diseases associated with neuronal dysfunction or death include, but are not limited to: Parkinson's disease, schizophrenia, depression, vision impairment due to RGC cell death, glaucoma, age-related RGC lesions , optic nerve damage, retinal ischemia or hemorrhage, Leber hereditary optic neuropathy, photoreceptor cell degeneration or death due to injury or degeneration, macular degeneration, retinitis pigmentosa, diabetes-related blindness, night blindness, color blindness, hereditary blindness, congenital Amaurosis, etc.
  • 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 homeostasis, and participating in repair and scarring following brain and spinal cord injury. According to the content of glial filaments and the shape of cell processes, astrocytes can be divided into two types: 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.
  • the astrocytes that can be used in the present disclosure are not particularly limited, and include various astrocytes derived from the mammalian central nervous system, such as those derived from the striatum, the ventral tegmental area of the midbrain, the hypothalamus, the spinal cord , dorsal midbrain or cerebral cortex, preferably, derived from the striatum.
  • a functional neuron may refer to a neuron 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 (eg, the ability to exhibit action potentials, such as rapid Rise and subsequent fall) (voltage or membrane potential across cell membranes), formation of synaptic connections with other neurons, presynaptic neurotransmitter release, and postsynaptic responses (eg, excitatory postsynaptic currents or inhibitory synapses) aftertouch current).
  • excitability eg, the ability to exhibit action potentials, such as rapid Rise and subsequent fall
  • postynaptic responses eg, excitatory postsynaptic currents or inhibitory synapses
  • functional neurons are characterized in that they express one or more markers of functional neurons, including but not limited to synapsin, synaptophysin, glutamate decarboxylase 67 (GAD67), glutamate 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).
  • markers of functional neurons including but not limited to synapsin, synaptophysin, glutamate decarboxylase 67 (GAD67), glutamate Acid decarboxylase 65 (GAD65), parvalbumin, dopamine- and cAMP-regulated neuronal
  • Dopaminergic neurons contain and release dopamine (DA) as a neurotransmitter.
  • Dopamine is a catecholamine neurotransmitter and plays an important biological role in the central nervous system.
  • the dopaminergic neurons in the brain are mainly concentrated in the substantria nigra pars compacta (SNc) of the midbrain and the ventral tegmentum.
  • SNc substantria nigra pars compacta
  • VTA Ventral tegmental area
  • Many experiments have confirmed that dopaminergic neurons are closely related to a variety of human diseases, the most typical of which is Parkinson's disease.
  • the gene editors include DNA gene editors and RNA gene editors.
  • the gene editor of the present disclosure includes a gene editing protein and optionally a gRNA.
  • the nucleotides of the gene editing protein can be obtained by genetic engineering techniques, such as genome sequencing, polymerase chain reaction (PCR), etc., and the amino acid sequence thereof can be deduced from the nucleotide sequence.
  • the source of the wild-type gene editing protein includes (but is not limited to): Ruminococcus lavefaciens, Streptococcus pyogenes, Staphylococcus aureus, Acidaminococcus sp , Lachnospiraceae acterium.
  • the gene editing proteins include, but are not limited to, Cas13d, CasRx, Cas13X, Cas13a, Cas13b, Cas13c, Cas13Y, and RNA targeting gene editing proteins.
  • protein of the present disclosure refers to a protein or polypeptide having a REST amino acid sequence. They include REST proteins with or without the starting methionine. In addition, the term also includes full-length REST and fragments thereof. REST proteins referred to in the present disclosure include their complete amino acid sequences, their secreted proteins, their mutants, and their functionally active fragments.
  • REST protein is a repressor element 1-silencing transcription factor (Repressor element 1-silencing transcription), also known as Neuron-Restrictive Silencer Factor (NRSF).
  • NRSF Neuron-Restrictive Silencer Factor
  • REST gene refers to a nucleic acid sequence having a REST nucleotide sequence.
  • the full-length genome of the human REST gene is 27948 bp (NCBI GenBank accession number is 5978).
  • the full-length genome of the mouse REST gene is 21007 bp (NCBI GenBank accession number is 19712).
  • nucleic acid sequence encoding it can be constructed from it, and a specific probe can be designed from the nucleotide sequence.
  • the full-length nucleotide sequence or its fragment can usually be obtained by PCR amplification method, recombinant method or artificial synthesis method.
  • primers can be designed based on the REST nucleotide sequences disclosed in the present disclosure, especially the open reading frame sequences, and prepared using commercially available cDNA libraries or conventional methods known to those skilled in the art
  • the cDNA library is used as a template to amplify the relevant sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splicing the amplified fragments together in the correct order.
  • recombinant methods can be used to obtain the relevant sequences in bulk. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.
  • synthetic methods can also be used to synthesize the relevant sequences, especially when the fragment length is short. Often, fragments of very long sequences are obtained by synthesizing multiple small fragments followed by ligation.
  • DNA sequences encoding proteins (or fragments, derivatives thereof) of the present disclosure can be obtained entirely by chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (eg, vectors) and cells known in the art.
  • polynucleotide sequences of the present disclosure can be used to express or produce recombinant REST polypeptides by conventional recombinant DNA techniques. Generally there are the following steps:
  • REST polynucleotide sequences can be inserted into recombinant expression vectors.
  • any plasmid and vector can be used as long as it is replicable and stable in the host.
  • An important feature of expression vectors is that they typically contain an origin of replication, a promoter, marker genes and translational control elements.
  • Expression vectors containing REST-encoding DNA sequences and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombinant technology, and the like.
  • the DNA sequence can be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis.
  • Expression vectors also include 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 for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or for tetracycline or ampicillin resistance in E. coli.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or for tetracycline or ampicillin resistance in E. coli.
  • Vectors comprising the appropriate DNA sequences described above, together with appropriate promoter or control sequences, can be used to transform appropriate host cells so that they can express the protein.
  • Host cells can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells.
  • prokaryotic cells such as bacterial cells
  • lower eukaryotic cells such as yeast cells
  • higher eukaryotic cells such as mammalian cells.
  • Representative examples are: Escherichia coli, bacterial cells of the genus Streptomyces; fungal cells such as yeast; plant cells; insect cells; animal cells and the like.
  • Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art.
  • the host is a prokaryotic organism such as E. coli
  • competent cells capable of uptake of DNA can be harvested after exponential growth phase and treated with the CaCl2 method using procedures well known in the art. Another method is to use MgCl 2 .
  • transformation can also be performed by electroporation.
  • the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging and the like.
  • the obtained transformants can be cultured by conventional methods to express the polypeptides encoded by the genes of the present disclosure.
  • the medium used in the culture can be selected from various conventional media depending on the host cells used. Cultivation is carried out under conditions suitable for growth of the host cells. After the host cells have grown to an appropriate cell density, the promoter of choice is induced by a suitable method (eg, temperature switching or chemical induction), and the cells are cultured for an additional period of time.
  • recombinant polypeptide in the above method can be expressed intracellularly, or on the cell membrane, or secreted outside the cell.
  • recombinant proteins can be isolated and purified by various isolation methods utilizing their 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 renaturation treatment, treatment with protein precipitants (salting-out method), centrifugation, osmotic disruption, ultratreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • AAV adeno-associated virus
  • Adeno-associated virus also known as adeno-associated virus, belongs to the genus Dependovirus of the family Parvoviridae. It is a class of single-stranded DNA-deficient viruses with the simplest structure found so far, and requires a helper virus (usually adenovirus) to participate in replication. It encodes the cap and rep genes in two terminal inverted repeats (ITRs). ITRs play a decisive role in viral replication and packaging. The cap gene encodes the viral capsid protein, and the rep gene is involved in the replication and integration of the virus. 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), low immunogenicity, and time to express foreign genes in vivo It is regarded as one of the most promising gene transfer vectors and has been widely used in gene therapy and vaccine research worldwide. After more than 10 years of research, the biological characteristics of recombinant adeno-associated virus have been deeply understood, especially its application effect in various cells, tissues and in vivo experiments has accumulated a lot of information.
  • rAAV is used in gene therapy research for various diseases (including in vivo and in vitro experiments); at the same time, as a characteristic gene transfer carrier, it is also widely used in gene function research, disease model construction, and gene preparation. Knockout mice, etc.
  • the vector is a recombinant AAV vector.
  • AAVs are relatively small DNA viruses that can integrate into the genomes of the cells they infect in a stable and site-specific manner. They are capable of infecting a wide 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 viral origin of replication. The rest of the genome is divided into two important regions with encapsidation functions: the left part of the genome containing the rep gene involved in viral replication and viral gene expression; and the right part 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. Any serotype of adeno-associated virus is suitable. Methods for purifying vectors can be found, for example, in US Pat. 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 transfer in vitro and in vivo has been described (see, eg, International Patent Application Publication Nos. WO 91/18088 and WO 93/09239; US Patent Nos.
  • Replication-deficient recombinant AAVs can be prepared by co-transfection of 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 plasmids carrying the 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 a virion (eg, including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV-DJ, AAV-rh10, PHP.S , PHP.B, PHP.eB, AAV virions of AAV2-7m8).
  • a virion eg, including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV-DJ, AAV-rh10, PHP.S , PHP.B, PHP.eB, AAV virions of AAV2-7m8.
  • the present disclosure includes recombinant virions (recombinant by virtue of their inclusion of recombinant polynucleotides) containing any of the vectors described herein. Methods of producing such particles are known in the art and described in US Patent No. 6,59
  • proteins of the present disclosure through various conventional screening methods, substances that interact with REST genes or proteins, especially inhibitors, can be screened.
  • REST inhibitors (or antagonists) useful in the present disclosure may reduce, eliminate the expression, content and/or activity of a REST gene, its RNA (eg, mRNA) or its encoded protein at the DNA, RNA, protein level.
  • RNA eg, mRNA
  • the REST inhibitor includes an antibody to REST, an antisense RNA to a REST nucleic acid, siRNA, shRNA, miRNA, a gene editor, or an inhibitor of REST activity.
  • a preferred REST inhibitor refers to a gene editor capable of inhibiting REST expression.
  • the REST inhibitors of the present disclosure include inhibitors targeting positions 15311-15338 of the REST gene sequence.
  • Objects to which the REST inhibitors of the present disclosure act include astrocytes or MG cells.
  • the methods and steps for inhibiting REST include neutralizing the protein of REST with an antibody, and silencing the REST gene with shRNA or siRNA or gene editor carried by a virus (eg, adeno-associated virus).
  • a virus eg, adeno-associated virus
  • the inhibition rate of REST is generally at least 50% inhibition, preferably 60%, 70%, 80%, 90%, 95% inhibition, which can be based on conventional techniques, such as flow cytometry, fluorescence quantitative PCR or The inhibition rate of REST was controlled and detected by Western blot and other methods.
  • REST inhibitors of the present disclosure when administered (administered) therapeutically, inhibit the expression and/or activity of REST proteins, thereby inducing glia Cells differentiate into functional neurons, thereby treating diseases associated with neuronal loss of function or death.
  • these materials can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, usually at a pH of about 5-8, preferably at a pH of about 6-8, although the pH may vary depending on the formulation It varies depending on the nature of the substance and the condition being treated.
  • the formulated pharmaceutical compositions can be administered by conventional routes, including (but not limited to): topical, intramuscular, intracranial, intraocular, intraperitoneal, intravenous, subcutaneous, intradermal, topical, autologous Cells are extracted and cultured and then returned to infusion, etc.
  • the present disclosure also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a safe and effective amount of an inhibitor of the present disclosure (eg, an antibody, gene editor, antisense sequence (eg, siRNA), or inhibitor) and a pharmaceutically acceptable carrier or excipients.
  • Such carriers include, but are not limited to, saline, buffers, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the drug formulation should match the mode of administration.
  • the pharmaceutical compositions of the present disclosure can be prepared in the form of injections, for example, prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants.
  • 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, eg, about 1 microgram to 10 mg/kg body weight per day.
  • the present disclosure finds for the first time that reducing the expression, content or activity of the REST gene or its encoded protein in astrocytes can induce the differentiation of astrocytes into dopamine neurons, thereby preventing and/or treating Parkinson's Disease.
  • RNA-targeted CRISPR system CasRx can avoid the risk of permanent DNA changes caused by traditional CRISPR-Cas9 editing. Therefore, CasRx-mediated RNA editing provides an effective means for the treatment of various diseases.
  • the present disclosure directly converts MGs into functional photoreceptors and RGCs by inhibiting the expression of REST in the retina.
  • the present disclosure uses the RNA-targeted CRISPR system CasRx to knock down REST, providing an excellent tool capable of treating a variety of diseases.
  • Guide RNAs targeting mouse REST are for example SEQ ID NO.: 4-20, preferably SEQ ID NO.: 10.
  • the collected cells were extracted with Trizol (Ambion) for RNA and reverse transcribed into cDNA using a reverse transcription kit (HiScript Q RT SuperMix for qPCR, Vazyme, Biotech), and AceQ qPCR SYBR Green Master Mix (Vazyme) , Biotech) for QPCR analysis.
  • Trizol Ambion
  • HiScript Q RT SuperMix for qPCR, Vazyme, Biotech
  • AceQ qPCR SYBR Green Master Mix Vazyme
  • Targeted mouse REST qPCR primers are: upstream primer, 5'-ctggctcttccactgcagaa-3' (SEQ ID NO.:193); downstream primer, 5'-tggtgtttcaggtgtgctgt-3' (SEQ ID NO.:194);
  • the qPCR primers targeting mouse GAPDH are: upstream primer, 5'-ctacccccaatgtgtccgtc-3' (SEQ ID NO.:195); downstream primer, 5'-aagtcgcaggagacaacctg-3' (SEQ ID NO.:196);
  • Targeting human and cynomolgus REST qPCR primers are: upstream primer, 5'-gttagaactcatacaggaga-3' (SEQ ID NO.:197); downstream primer, 5'-gaggttttaggcccattgtga-3' (SEQ ID NO.:198);
  • Targeting human GAPDH qPCR primers are: upstream primer, 5'-gtctcctctgacttcaacagcg-3' (SEQ ID NO.:199); downstream primer, 5'-accaccctgttgctgtagccaa-3' (SEQ ID NO.:200);
  • Targeted cynomolgus GAPDH qPCR primers upstream primer, 5'-ggtcaccagggctgctttta-3' (SEQ ID NO.:201); downstream primer, 5'-ttcccgttctcagccttcac-3' (SEQ ID NO.:202).
  • the AAV serotype used in this study was AAV8, and the method of stereotaxic injection (C57BL/6, age approximately two months) was as described previously 2 .
  • the AAV mixture with a titer greater than 5 ⁇ 10 12 vg/ml was injected into the striatum (AP+0.8mm, ML ⁇ 1.6mm and DV-2.8mm) by a stereotaxic injector, and the injection volume was 1uL.
  • the injected AAV was AAV-GFAP-miR124 (approximately 1.7 ⁇ 10 13 vg/ml)
  • the control virus was GFAP-mcherry (approximately 5 ⁇ 10 11 vg) /ml)+AAV-GFAP-CasRx (titer is about 1.2 ⁇ 10 13 vg/ml, without gRNA targeting REST)
  • the AAV virus in the experimental group is GFAP-mcherry+AAV-GFAP-CasRx-REST (titer About 1.2 ⁇ 10 13 vg/ml, containing gRNA targeting REST)
  • 1-3 mice were injected in each group.
  • AAV8 was injected subretinal as previously described.
  • AAV was injected subretinal with a Hamilton syringe (32G needle) under an Olympus microscope (Olympus, Japan).
  • a total of 1 ⁇ l of GFAP-tdTomato 0.1 ⁇ l, approximately 1 ⁇ 10 12 vg/ml
  • GFAP-CasRx- REST 0.9 ⁇ l, about 1.2 ⁇ 10 13 vg/ml
  • GFAP-tdTomato 0.1 ⁇ l, about 1 ⁇ 10 12 vg/ml
  • GFAP-CasRx 0.9 ⁇ l, about 1.2 ⁇ 10 13 vg/ml
  • the primary antibodies used for immunofluorescence staining were: rabbit polyclonal NeuN antibody (1:500, #ABN78, Millipore), mouse TH antibody (1:300, MAB318, Millipore) and rat DAT antibody (1:100, MAB369 , Millipore).
  • Alexa 488 AffiniPure Donkey Anti-Mouse IgG (H+L) (1:500, 715-545-150, Jackson ImmunoResearch)
  • Alexa 488 AffiniPure Donkey Anti-Rabbit IgG(H+L) (1:500, 711-545-152, Jackson ImmunoResearch)
  • Primary antibodies for immunofluorescence staining rabbit anti-RBPMS (1:500, 15187-1-AP, Proteintech), mouse-anti-rhodopsin (1:2000, MAB5356, EMD Millipore) and secondary antibodies: Alexa 488 AffiniPure Donkey Anti-Rabbit IgG (H+L) (1:500, 715-545-150, Jackson ImmunoResearch), Alexa 488 AffiniPure Donkey Anti-Mouse IgG (H+L) (1:500, 711-545-152, Jackson ImmunoResearch). After application of the antibody, the slides were washed and mounted. Imaging was performed using an Olympus FV3000 microscope.
  • a high-efficiency REST-targeting gRNA (SEQ ID NO.: 4-54) screened in mouse NA2 cells
  • sgRNA guide sequences for epigenetic methods SEQ ID NO.: 55-82
  • gRNA SEQ ID NO. wizard sequence SEQ ID NO. DNA target sgRNA1 55 ggcgcagcagcagaagaccg 63 ggcgcagcagcagaagaccg sgRNA2 56 accgcagcgacggcagaacc 64 accgcagcgacggcagaacc sgRNA3 57 ccctggttctgccgtcgctg 65 ccctggttctgcgtcgctg sgRNA4 58 agcgacggcagaaccagggc 66 agcgacggcagaaccagggc sgRNA5 59 cgggatcagaccgccggccc 67 cgggatcagaccgccggccc sgRNA6 60 gatcgcacccgggatctcg 68 gatcgcaccccgggatctcg sgRNA7 61 gagt
  • Example 1 miR124 fails to transdifferentiate glial cells into neurons or dopamine neurons
  • Example 2 Screening of gRNAs targeting REST with high efficiency in N2A cells
  • glial cell-specific promoter GFAP to drive mCherry expression, and to specifically express CasRx in glial cells
  • glial cell-specific promoter GFAP to drive the expression of CasRx.
  • the virus injected in the control group was a mixed AAV of GFAP-mCherry and GFAP-CasRx, in which mCherry could label the infected glial cells
  • the AAV combination injected in the experimental group was GFAP-mCherry+GFAP-CasRx-REST (expressing gRNA-7) , of which GFAP-CasRx-REST could specifically target REST mRNA (Fig. 3A).
  • Epigenetic modification is also a common method for manipulating gene expression.
  • DNA-binding proteins such as Zinc fingers, TALEs, CRISPR-dCas, etc.
  • epigenetic regulation Elements eg, KRAB, Dnmt3a, Tet1, etc.
  • the DNA targeting proteins used in this study were two different CRISPR-dCas (dSpCas9, dSaCas9-KKH), which were fused with the Krab repression domain of the epigenetic modification protein, and the expression of EGFP protein was driven by SV40.
  • gRNA was independently driven by U6, and mCherry was expressed by CMV in the same plasmid vector as U6-gRNA for flow sorting of cells (Fig. 4B). 48 hours after transfection of N2A cells, it was found by Q-PCR that both dSpCas9-KRAB and dSaCas9-KKH-Krab could effectively reduce the expression of REST mRNA. The REST mRNA level was reduced to about half of the original level ( Figure 4C and 4D).
  • Example 6 High-efficiency human-targeting gRNAs enable efficient REST knockdown in non-human primates and mice
  • gRNA17, gRNA18 and gRNA19 3 gRNAs (gRNA17, gRNA18 and gRNA19).
  • the sequence of gRNA-17 is homologous in humans, non-human primates and mice, and the sequences are completely identical;
  • gRNA-18 has a 1 base mismatch in cynomolgus monkey and mouse, and
  • gRNA-19 There was a 2 base mismatch in cynomolgus monkey and mouse (FIG. 6A).
  • Figure 6B in this study, gRNA and CasRx were constructed into the same expression plasmid.
  • Example 7 CasRx-gRNA system targeting human REST can transdifferentiate glial cells into neurons in mice
  • human-targeting gRNAs can effectively transdifferentiate glial cells into neurons
  • this study constructed human-targeting gRNA-17 (gRNA(human)) and CasRx into AAV vectors and packaged AAVs.
  • GFAP-CasRx-REST and GFAP-mCherry were co-injected into the mouse brain, and the control group was injected with GFAP-CasRx+GFAP-mCherry, and the analysis was performed 1 month after injection (Fig. 7A).
  • the results showed that gRNA targeting human REST could transdifferentiate astrocytes into neurons, and the red fluorescently labeled cells were co-labeled with the neuron-specific protein marker NeuN (50.71% ⁇ 11.12%, SEM, 3 in each group).
  • mice while red fluorescently labeled cells in the brains of mice injected with control AAV still showed typical glial morphology and were not co-labeled with NeuN ( Figure 7B, 7C and 7D).
  • the above results indicate that the CasRx-gRNA system targeting human REST can efficiently transdifferentiate glial cells into neurons, and has the potential to treat neuron loss-related diseases.
  • knockdown of REST in the retina could transdifferentiate Mueller glia in the retina into functional neurons such as photoreceptors or retinal ganglion cells.

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Abstract

涉及通过抑制脑中的星形胶质细胞的REST基因或RNA或其编码蛋白的表达、含量或活性，可有效诱导星形胶质细胞向多巴胺神经元的转分化，并且通过抑制视网膜中的穆勒胶质细胞中的REST基因或RNA或其编码蛋白的表达、含量或活性，可有效诱导穆勒胶质细胞向视网膜神经节细胞(RGC)或感光细胞的转分化，从而预防和/或治疗与神经元功能缺失或死亡相关的疾病。

Description

胶质细胞向神经元转分化用于预防或治疗神经元功能缺失或死亡相关疾病
相关申请
本申请要求于2021年2月10日提交的标题为“胶质细胞向神经元转分化用于预防或治疗神经元功能缺失或死亡相关疾病”的申请号为202110185323.2的申请的优先权，其全部内容通过引用并入本文。
技术领域
本公开内容涉及生物医药领域。更具体地，本公开内容涉及REST(RE1-silencing transcription factor)抑制剂在预防和/或治疗与神经元功能缺失或死亡相关的疾病中的用途。
背景技术
帕金森病(PD)是一种与神经元功能缺失或死亡相关的疾病，其特征是中脑黑质多巴胺神经元的丧失。目前对帕金森病的主要治疗手段是以左旋多巴制剂为代表的药物。同时手术治疗也能在一定程度上改善症状。需要指出的是所有这些手段只能部分的缓解病情，还达不到阻止病情发展的效果。
穆勒胶质细胞(Müller glia，MG)是视网膜组织中的主要神经胶质细胞。在斑马鱼中，视网膜发生损伤时，穆勒胶质细胞能增殖并分化为感光细胞视网膜神经节细胞(retinal ganglion cell，RGC)和双极细胞等多种视网膜细胞，从而帮助修复损伤。但在高等哺乳动物中，在视网膜成熟后MG失去了分化为视网膜各种功能性细胞的能力。感光细胞是视网膜中一种特殊的神经细胞，也是视觉系统中唯一将捕捉光信号的细胞。视网膜色素变性，先天性黑蒙(LCA),老年黄斑变性，糖年病性视网膜病等多种眼科疾病都会导致感光细胞死亡，从而导致失明。视网膜神经节细胞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。视网膜神经节细胞(RGC)退化是造成永久性失明的主要缘由。RGC是视网膜惟一的输出神经元，因此RGC退化可导致造成永久性失明的视网膜疾病。因此，重建功能性感光细胞或RGC有助于恢复视力，是恢复视觉功能的潜在治疗方法。
本领域仍然迫切需要开发能够有效治疗与神经元功能缺失或死亡相关的疾病的新靶点和新疗法。
发明内容
本公开内容的一个目的在于提供由胶质细胞产生功能性多巴胺神经元的方法。
本公开内容的另一目的在于提供REST抑制剂用于预防和/或治疗与功能性多巴胺神经元功能缺失或死亡相关的疾病的用途。
本公开内容的另一目的在于提供由穆勒胶质细胞产生功能性视网膜神经节细胞(RGC)或感光细胞的方法。
本公开内容的另一目的在于提供REST抑制剂用于预防和/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的用途。
在一个实施方案中，本公开内容提供了由胶质细胞产生功能性多巴胺神经元的方法，其包括 使用REST抑制剂使所述胶质细胞转分化为或重编程为功能性多巴胺神经元，其中所述REST抑制剂降低REST基因或其RNA或其编码蛋白的表达、含量或活性。
在一个优选的实施方案中，所述胶质细胞选自星形胶质细胞、少突胶质细胞、室管膜细胞、施万细胞(Schwann cell)、NG2细胞、卫星细胞、或其组合。
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。
在一个优选的实施方案中，所述星形胶质细胞来源于中枢神经系统，包括纹状体、黑质、中脑腹侧被盖区、脊髓、下丘脑、背侧中脑或大脑皮层。
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和黑质。
在另一个实施方案中，本公开内容提供了REST抑制剂在制备药物中的用途，所述药物用于预防和/或治疗与功能性多巴胺神经元功能缺失或死亡相关的疾病，其中所述REST抑制剂降低REST基因或其RNA或其编码蛋白的表达、含量或活性。
在一个优选的实施方案中，所述药物配制成用于施用于中枢神经系统，包括纹状体、黑质、中脑腹侧被盖区、脊髓、下丘脑、背侧中脑或大脑皮层等。
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和黑质。
在一个优选的实施方案中，所述与功能性多巴胺神经元功能缺失或死亡相关的疾病为神经系统疾病，包括中风、帕金森病、精神分裂症和抑郁症。
在一个更优选的实施方案中，所述与功能性多巴胺神经元功能缺失或死亡相关的疾病为帕金森病。
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功能性视网膜神经节细胞(RGC)或感光细胞的方法，其包括用REST抑制剂使所述穆勒胶质细胞转分化为或重编程为功能性RGC或感光细胞，其中所述REST抑制剂降低REST基因或其RNA或其编码蛋白的表达、含量或活性。
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。
在另一个实施方案中，本公开内容提供了REST抑制剂在制备药物中的用途，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REST抑制剂降低REST基因或其RNA或其编码蛋白的表达、含量或活性。
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、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REST抑制剂选自：抗体、小分子化合物、microRNA、siRNA、shRNA、反义寡核苷酸、REST结合蛋白或蛋白结构域、多肽、核酸适配体、基因编辑器、PROTAC、 表观遗传调控或其组合。
在一个优选的实施方案中，所述REST抑制剂包含：
基因编辑蛋白或其表达载体，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，(b)一个或多个gRNA或其表达载体，所述gRNA是引导基因编辑蛋白特异性结合REST基因的DNA或RNA，
其中优选靶向RNA的基因编辑蛋白和靶向RNA的gRNA。
在一个优选的实施方案中，所述gRNA包含与目标序列互补的序列。
在一个优选的实施方案中，所述gRNA引导所述基因编辑蛋白特异性结合于对应于REST编码序列的第867-1103位核苷酸(SEQ ID NO.:3)。
在一个优选的实施方案中，所述gRNA包含与SEQ ID NO.:3互补的序列。
在一个优选的实施方案中，所述gRNA包含选自SEQ ID NO.:4-20和83-118的序列或者包含由SEQ ID NO.:55-62和71-76的序列编码的序列，优选所述gRNA包含选自SEQ ID NO.:10和93-103的序列。
在一个优选的实施方案中，所述gRNA包含与目标序列完全互补的序列，或者包含与目标序列具有不多于3个碱基错配的互补序列。
在一个优选的实施方案中，所述gRNA与目标序列来自相同物种或不同物种。
在一个优选的实施方案中，所述gRNA与目标序列来自人、食蟹猴或小鼠。
在另一个的实施方案中，本公开内容提供了包含REST抑制剂的药物组合物或药盒或试剂盒。
在一个优选的实施方案中，所述REST抑制剂选自：抗体、小分子化合物、microRNA、siRNA、shRNA、反义寡核苷酸、REST结合蛋白或蛋白结构域、多肽、核酸适配体、基因编辑器、PROTAC、表观遗传调控或其组合。
在一个优选的实施方案中，所述REST抑制剂包含：
基因编辑蛋白或其表达载体，以及
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REST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，所述gRNA包含与目标序列互补的序列。
在一个优选的实施方案中，所述gRNA引导所述基因编辑蛋白特异性结合于对应于REST编码序列的第867-1103位核苷酸(SEQ ID NO.:3)。
在一个优选的实施方案中，所述gRNA包含与SEQ ID NO.:3互补的序列。
在一个优选的实施方案中，所述gRNA包含选自SEQ ID NO.:4-20和83-118的序列或者包含由SEQ ID NO.:55-62和71-76的序列编码的序列，优选所述gRNA包含选自SEQ ID NO.:10和93-103的序列。
在一个优选的实施方案中，所述gRNA包含与目标序列完全互补的序列，或者包含与目标序 列具有不多于3个碱基错配的互补序列。
在一个优选的实施方案中，所述gRNA与目标序列来自相同物种或不同物种。
在一个优选的实施方案中，所述gRNA与目标序列来自人、食蟹猴或小鼠。
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REST抑制剂的载体或运载体。
在一个优选的实施方案中，所述载体或运载体为病毒载体、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、CasRx、Cas13X、Cas13a、Cas13b、Cas13c、Cas13Y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、Cas13X、Cas13Y。
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REST mRNA序列的仅单一类型的gRNA或2、3、4、5、6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REST mRNA序列的仅单一类型的gRNA或2、3、4、5、6种不同的gRNA。
在一个优选的实施方案中，所述表达载体包含：
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
至少一种编码靶向REST mRNA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。
在一个更优选的实施方案中，所述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，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纳米颗粒中。
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)、腺病毒载体、慢病毒载体、逆转录病毒载体、疱疹病毒、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：1)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)腹侧被盖区(VTA)中的胶质细胞；以及ix)下丘脑中的胶质细胞。
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，其中所述一种或更多种多巴胺神经元相关因子选自：Lmx1a、Lmx1b、FoxA2、Nurr1、Pitx3、Gata2、Gata3、FGF8、BMP、En1、En2、PET1、Pax家族蛋白、SHH、Wnt家族蛋白和TGF-β家族蛋白。
在一个优选的实施方案中，其中所述药物组合物或药盒或试剂盒还包含i)一种或更多种选自β-catenin、Oct4、Sox2、Klf4、Crx、Brn3a、Brn3b、Math5、Nr2e3和Nrl的因子，和/或ii)用于在胶质细胞中表达选自β-catenin、Oct4、Sox2、Klf4、Crx、Brn3a、Brn3b、Math5、Nr2e3和Nrl的一种或更多种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注射、颅内给药、眼内给药、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。
在一个优选的实施方案中，所述AAV载体包含：
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
至少一种编码靶向REST mRNA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。
在一个更优选的实施方案中，所述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。
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1％，或至少10％、20％、30％、40％或50％。
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、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，RGC(1)表达Brn3a、Rbpms、Foxp2、Brn3c和/或小清蛋白；(2)是 F-RGC，3型RGC或PV-RGC；(3)被整合在所述受试者中现有的视网膜通路中(例如，能够将中心信息投影到dLGN，并且通过将视觉信息中继到V1而能够部分恢复视觉)；和/或(4)能够接收视觉信息，这些信息的特征在于能够在光刺激，突触连接(例如与大脑中现有的功能性dLGN神经元)，突触前神经递质的生物发生和/或后继作用建立动作电位的能力突触反应。
在另一个优选实施方案中，多巴胺神经元(1)表达酪氨酸羟化酶(TH)，多巴胺转运蛋白(DAT)，囊泡单胺转运蛋白2(VMAT2)，波纹同源盒1(Enl)，FoxA2和/或LIM同源盒转录因子1alpha(Lmxla)；(2)表现出突触前神经递质的合成和释放；(3)被整合到所述受试者的大脑中现有的神经元回路中；和/或(4)其特征在于，其建立动作电位，突触连接，突触前神经递质的生物发生和/或突触后反应的能力。
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％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gNRA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REST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REST基因的mRNA。
在另一个优选实施方案中，所述gRNA的核苷酸序列例如SEQ ID NO.:4-20，优选为SEQ ID NO.:10。
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(Acidaminococcus sp)、毛螺科菌(Lachnospiraceae bacterium)、黄化瘤胃球菌(Ruminococcus flavefaciens)、或其组合。
在另一个优选实施方案中，所述REST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REST基因包括野生型REST基因和突变型REST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REST基因编码的多肽与野生REST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REST基因包括与野生REST基因相比，同源性≥80％(优选地≥90％，更优选地≥95％，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REST基因包括在野生型REST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REST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REST蛋白包括REST的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REST的同源性至少为90％，优选为95％，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％、85％、90％、95％、100％的REST活性。
在另一个优选实施方案中，所述REST蛋白的氨基酸序列选自：
具有SEQ ID NO.:1所示氨基酸序列的多肽；
将如上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
氨基酸序列与如上所示氨基酸序列的同源性≥90％(优选地≥95％，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REST基因的核苷酸序列选自：
编码具有如上所示氨基酸序列的多肽的多核苷酸；
SEQ ID NO.:2所示序列的多核苷酸；
核苷酸序列与如上所示核苷酸序列的同源性≥95％(优选地≥98％，更优选地≥99％)的多核苷酸；
在具有如上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；
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REST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REST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REST抑制剂(如基因编辑蛋白)靶向的区域为REST基因序列的第15311-15338位。
在另一个优选实施方案中，所述REST抑制剂抑制REST的活性和/或表达量。
在另一个优选实施方案中，所述REST抑制剂的浓度(病毒的滴度)＞1×10 12。
在另一个优选实施方案中，所述REST抑制剂对REST的活性和/或表达量的抑制率大于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REST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功能性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、AAV8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％。
本公开内容第三方面提供了一种药盒，包括：
(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、CasRx、Cas13X、Cas13a、Cas13b、Cas13c、Cas13Y、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REST基因的DNA或RNA。
在另一个优选实施方案中，所述药盒还包括：
(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/或含有其他治疗功能性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功能性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。
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.在小鼠体内探究过表达miR124是否能将胶质细胞转分化为神经元。(A)在小鼠脑内过表达miR124示意图，Vector-1为利用胶质细胞特异性启动子GFAP启动mCherry红色荧光蛋白表达，用于标记胶质细胞，Vector-2是由GFAP启动miR124的表达，可以实现在胶质细胞中特异性表达miR124。(B)在小鼠纹状体中注入GFAP-mCherry+GFAP-miR124后，橙色箭头指向被标记生的胶质细胞发生了形态改变，但不与NeuN共标。(C)在小鼠纹状体中注入GFAP-mCherry+GFAP-miR124后，Tuj-1是神经元早期标志物，橙色箭头指向被标记的胶质细胞，但这些胶质细胞不与Tuj-1共标，标尺为40微米。(D)在小鼠纹状体中注入GFAP-mCherry+GFAP-miR124后，用神经元特异性标志物NeuN和多巴胺神经元特异性标志物TH染色，白色箭头指向被标记的胶质细胞，这些胶质细胞既不与NeuN共标，也不与多巴胺特异性标志物TH共标，标尺为40微米。
图2.靶向小鼠REST的gRNA筛选。(A)质粒构建示意图，Vector-1是gRNA表达质粒，gRNA由U6驱动，同时表达红色荧光示踪转染阳性的细胞。Vector-2是CasRx表达质粒，CasRx由CAG驱动，同时表达绿色荧光示踪转染阳性的细胞。(B)细胞转染和流式分选示意图，在转染细胞后，通过流式分选红绿双阳的细胞，并通过QCPR检测REST mRNA的量。(C)Cas13d与不同的gRNA共转N2A细胞后，流式分选阳性细胞并通过QPCR检测不同的gRNA转染N2A细胞后REST的mRNA含量，残余REST mRNA量越低表明gRNA敲低效率越高。红色标注的gRNA-7在所筛选的gRNA中效率最高。
图3.在小鼠大脑中抑制REST将胶质细胞转分化为神经元。(A)载体构建及脑内胶质细胞转分化示意图，标记系统是GFAP-mCherry，由星形胶质细胞特异性启动子GFAP启动荧光蛋白mCherry表达；载体2为对照组AAV质粒，由星形胶质细胞特异性启动子GFAP启动CasRx表达；载体3为靶向REST组AAV质粒，由U6启动gRNA表达(对应图2的gRNA-7)，同时由星形胶质细胞特异性启动子GFAP启动CasRx表达；将不同的AAV组合注射到小鼠脑中，注射后大约1个月取材分析。(B)在小鼠纹状体中注射对照组病毒(GFAP-mCherry+GFAP-CasRx),橙色箭头指向被标记的星形胶质细胞，绿色为星形胶质细胞特异性标志物GFAP，白色为成熟神经元特异性标志物NeuN，Dapi染细胞核，Merge图像显示mCherry信号与GFAP 信号共标，但不与NeuN共标。(C)载体1和载体3共注后，在小鼠纹状体中敲低REST，将胶质细胞转分化为神经元，白色为成熟神经元特异性标志物NeuN，白色箭头指向mCherry信号与NeuN信号共标的神经元，标尺为20微米。(D-E)在注射可敲低REST的AAV组(GFAP-mCherry+GFAP-CasRx-REST)中，有一小部分红色荧光标记的细胞表达多巴胺神经元特异性细胞标志物TH和DAT。
图4.表观遗传调控技术降低REST基因表达。(A)表观遗传调控原理示意图，DTM代表DNA靶向蛋白或蛋白结构域(如：锌指蛋白，TALEs，CRISPR-dCas等)，DTM与表观调控蛋白相连，包括DNA表观修饰相关的酶和组蛋白修饰相关酶，在DTM-epigenetic modifier作用下，调控下游基因的表达。(B)本研究中所用质粒载体示意图，由U6启动子驱动sgRNA的表达，CMV驱动红色荧光蛋白(mCherry)表达；另一个载体是由SV40启动子启动绿色荧光蛋白表达，由EF1A驱动dCas9(dSpCas9或dSaCas9-KKH)表达，载体1(U6-sgRNA-CMV-mCherry)和载体2(dSpCas9-KRAB)或载体1和载体3(dSaCas9-KKH-KRAB)共转N2A细胞，进行研究分析。(C)载体1和载体2共转N2A细胞后，Q-PCR检测表观调控对REST基因的抑制效果。(D)载体1和载体3共转N2A细胞后，Q-PCR检测表观调控对REST基因的抑制效果。
图5.在人类细胞(293T细胞)中筛选gRNA。(A)各gRNA在293T细胞中敲低REST表达的效率，红色区域表示敲低效率高的gRNA区域。(B)各gRNA敲低REST表达的折线图，各gRNA与A图对应。(C)各gRNA在REST基因上的分布位置，红色标记的gRNA为抑制效率高的gRNA，紫红色标记的区域为高效gRNA聚集区域。
图6.在不同物种中都能对REST进行高效抑制。(A)选取3个靶向人REST的gRNA序列及其在食蟹猴和小鼠中错配位点，红色标记的碱基为食蟹猴或小鼠与人REST序列中的差异位点，gRNA-17,gRNA-18,gRNA-19为图5中的靶向人REST的相同编号gRNAs。(B)载体构建示意图，表达载体中gRNA由U6驱动，CasRx由CAG驱动，并在载体中加上绿色荧光蛋白基因，以标记转染阳性的细胞。(C)细胞转染和流式分选示意图，转染不同细胞后，通过流式细胞分选EGFP阳性细胞并通过QPCR分析。(D)QPCR分析REST mRNA表达量，靶向人类REST的gRNA(gRNA-17,gRNA-18和gRNA-19)也能高效敲低非人灵长类(食蟹猴)和小鼠的REST的mRNA表达量。
图7.靶向人类REST的gRNA能将胶质细胞转分化为神经元。(A)AAV载体及转分化过程示意图，GFAP是星形胶质细胞特异性启动子，mCherry为红色荧光蛋白，CasRx为基因编辑蛋白，U6-gRNA为U6启动的靶向REST的gRNA表达框，选用的gRNA为靶向人类REST的gRNA-17。将不同的AAV组合注射到小鼠纹状体中，并在1个月后分析转分化效果。(B)在小鼠纹状体中注射对照组病毒GFAP-mCherry+GFAP-CasRx，红色荧光信号为GFAP-mCherry，白色荧光信号为成熟神经元特异性标志物NeuN染色，图中显示mCherry与NeuN不共标。(C)在小鼠纹状体中注射GFAP-mCherry+GFAP-CasRx-REST病毒组合，NeuN是成熟神经元特异性标志物，橙黄色箭头指向mCherry与NeuN共标的神经元。标尺为40微米。(D)统计分析，mCherry和NeuN双阳细胞在mCherry阳性细胞中的比例(SEM，每组3只小鼠)。(E)在视网膜中尝试将穆勒胶质细胞转分化为感光细胞，在视网膜中GFAP是穆勒胶质细胞的启动子，视网膜下注射GFAP-tdTomato+GFAP-CasRx-REST的病毒后，其中GFAP-tdTomato用来标记视网膜穆勒胶质细胞，GFAP-CasRx-REST用来在穆勒胶质细胞中敲低REST，Rhodopsin是视网膜感光细胞中视杆细胞的特异性蛋白标志物，白色箭头指示的细胞同时表达tdTomato和Rhodopsin。(F)在视网膜中尝试将穆勒胶质细胞转分化为视网膜神经节细胞，视网膜下注射GFAP-tdTomato+GFAP-CasRx-REST的病毒后，红色细胞为GFAP-tdTomato标记的细胞， 绿色为视网膜神经节细胞特异性蛋白标志物Rbpms染色，白色箭头指示的细胞同时表达tdTomato和Rbpms，标尺为20微米。
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REST基因或RNA或其编码蛋白的表达、含量或活性，可有效诱导胶质细胞向功能性神经元的分化，从而治疗功能性神经元功能缺失或死亡相关的神经系统疾病。在此基础上，本发明人完成了本发明。
在本公开内容中，感光细胞或视网膜神经节细胞(RGC)退化是造成永久性失明的主要缘由。而穆勒胶质细胞(MG)转分化为功能性感光细胞或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使用RNA靶向的CRISPR系统CasRx来敲低REST，可将MG直接转变为功能性感光细胞或RGC。因此，由CasRx介导的REST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的疗法。
本申请使用最近表征的RNA靶向CRISPR系统CasRx对REST进行抑制。提供了一种能够治疗多种疾病的卓越工具。
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。
在本公开内容中，所述基因编辑器包括DNA基因编辑器、表观遗传调控编辑和RNA基因编辑器。在一优选实施方式中，本公开内容的基因编辑器包括基因编辑蛋白和任选的gRNA。
术语“重编程”或“转分化”可以指从不同类型的细胞(例如星形胶质细胞)产生特定谱系的细胞(例如神经元细胞)的过程。
与神经元功能缺失或死亡相关的疾病
在本公开内容中，与神经元功能缺失或死亡相关的疾病主要包括与多巴胺神经元功能缺失或死亡相关的疾病，以及与视神经节或感光细胞缺失或死亡相关的视力障碍。
在一优选实施方式中，与神经元功能缺失或死亡相关的疾病包括，但并不限于：帕金森病、精神分裂症、抑郁症、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、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、先天性黑蒙症等。
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、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。
功能性神经元
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)。
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。
基因编辑器
在本公开内容中，所述基因编辑器包括DNA基因编辑器和RNA基因编辑器。在一优选实施方式中，本公开内容的基因编辑器包括基因编辑蛋白和任选的gRNA。
基因编辑蛋白
在本公开内容中，基因编辑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。所述野生型的基因编辑蛋白的来源包括(但并不限于)：黄化瘤胃球菌(Ruminococcus lavefaciens)、酿脓链球菌(Streptococcus pyogenes)、葡萄球菌(Staphylococcus aureus)、氨基酸球菌属(Acidaminococcus sp)、毛螺科菌(Lachnospiraceae acterium)。
在本公开内容的一个优选实施方案中，所述基因编辑蛋白包括，但并不限于Cas13d、CasRx、Cas13X、Cas13a、Cas13b、Cas13c、Cas13Y、RNA靶向基因编辑蛋白。
REST蛋白和多核苷酸
在本公开内容中，术语“本公开内容蛋白”、“REST蛋白”、“REST多肽”、“REST”可互换使 用，都指具有REST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REST蛋白。此外，该术语还包括全长的REST及其片段。本公开内容所指的REST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。
REST蛋白是抑制元件1沉默转录因子(Repressor element 1-silencing transcription)，也称为神经元限制性沉默因子(Neuron-Restrictive Silencer Factor，NRSF)。
在本公开内容中，术语“REST基因”、“REST多核苷酸”、“REST基因”可互换使用，都指具有REST核苷酸序列的核酸序列。
人REST基因的基因组全长27948bp(NCBI GenBank登录号为5978)。鼠REST基因的基因组全长21007bp(NCBI GenBank登录号为19712)。
人和鼠REST，在DNA水平的相似性为72％，蛋白序列相似性为62％。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REST的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REST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REST多肽。一般来说有以下步骤：
(1).用本公开内容的编码人REST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，REST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REST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CaCl 2法处理，所用的步骤在本领域众所周知。另一种方法是使用MgCl 2。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，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、AAV-DJ、AAV-rh10、PHP.S、PHP.B、PHP.eB、AAV2-7m8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。
REST抑制剂和药物组合物
利用本公开内容蛋白，通过各种常规筛选方法，可筛选出与REST基因或蛋白发生相互作用的物质，尤其是抑制剂等。
可用于本公开内容的REST抑制剂(或拮抗剂)可以在DNA、RNA、蛋白质水平降低、消除REST基因、其RNA(如mRNA)或其编码蛋白的表达、含量和/或活性的物质。
例如，所述REST抑制剂包括REST的抗体、REST核酸的反义RNA、siRNA、shRNA、miRNA、基因编辑器、或REST的活性抑制剂。一种优选的REST抑制剂指的是能够抑制REST表达的基因编辑器。
优选的，本公开内容的REST抑制剂包括靶向REST基因序列的第15311-15338位抑制剂。本公开内容REST抑制剂所作用的对象包括星形胶质细胞或MG细胞。
在一种优选的实施方式中，抑制REST的方法和步骤包括利用REST的抗体中和其蛋白，利用病毒(如腺相关病毒)携带的shRNA或siRNA或基因编辑器进行REST基因的沉默。
对REST的抑制率一般为达到至少50％以上的抑制，优选为60％、70％、80％、90％、95％以上的抑制，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REST的 抑制率进行控制和检测。
本公开内容REST抑制剂(包括抗体、反义核酸、基因编辑器以及其他抑制剂)，当在治疗上进行施用(给药)时，可抑制REST蛋白的表达和/或活性，进而诱导胶质细胞分化为功能性神经元，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载体介质中，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抑制剂(如抗体、基因编辑器、反义序列(如siRNA)、或抑制剂)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，降低星形胶质细胞中的REST基因或其编码蛋白的表达、含量或活性，可诱导星形胶质细胞向多巴胺神经元的分化，从而预防和/或治疗帕金森病。
(2)本公开内容首次发现，用基因编辑器(包括基因编辑蛋白和gRNA)抑制星形胶质细胞中的REST的表达，可以使星形胶质细胞转分化为多巴胺神经元，进而为帕金森治疗提供了一种潜在的途径。
(3)本公开内容首次发现，RNA靶向的CRISPR系统CasRx可避免传统的CRISPR-Cas9编辑引起的永久性DNA改变的风险。因此，CasRx介导的RNA编辑为治疗各种疾病提供了一种有效的手段。
(4)本公开内容通过抑制视网膜中REST的表达，将MG直接转变为功能性感光细胞和RGC。
(5)本公开内容使用RNA靶向的CRISPR系统CasRx来敲低REST，提供了一种能够治疗多种疾病的卓越工具。
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
靶向小鼠REST的向导RNA例如SEQ ID NO.:4-20，优选SEQ ID NO.:10。
Cos7细胞，293T或者N2a细胞的瞬时转染和qPCR分析
细胞系瞬时转染4μg CAG-CasRx-P2A-GFP质粒和2μg U6-gRNA-CMV-mCherry质粒来确定REST在体外细胞系里的抑制效果。同时使用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(对照组收集EGFP阳性细胞)。收集好的细胞用Trizol(Ambion)提取RNA，并使用逆转录试剂盒(用于qPCR的HiScript Q RT SuperMix，Vazyme，Biotech)将其反转录为cDNA，并使用AceQ qPCR SYBR Green Master Mix(Vazyme，Biotech)进行QPCR分析。
靶向小鼠REST qPCR引物是：上游引物，5’-ctggctcttccactgcagaa-3’(SEQ ID NO.:193)；下游引物，5’-tggtgtttcaggtgtgctgt-3’(SEQ ID NO.:194)；
靶向小鼠GAPDH qPCR引物是：上游引物，5’-ctacccccaatgtgtccgtc-3’(SEQ ID NO.:195)；下游引物，5’-aagtcgcaggagacaacctg-3’(SEQ ID NO.:196)；
靶向人和食蟹猴REST qPCR引物是：上游引物，5’-gttagaactcatacaggaga-3’(SEQ ID NO.:197)；下游引物，5’-gaggtttaggcccattgtga-3’(SEQ ID NO.:198)；
靶向人GAPDH qPCR引物是：上游引物，5’-gtctcctctgacttcaacagcg-3’(SEQ ID NO.:199)；下游引物，5’-accaccctgttgctgtagccaa-3’(SEQ ID NO.:200)；
靶向食蟹猴GAPDH qPCR引物：上游引物，5’-ggtcaccagggctgctttta-3’(SEQ ID NO.:201)；下游引物，5’-ttcccgttctcagccttcac-3’(SEQ ID NO.:202)。
立体定位注射
在该研究中使用的AAV血清型为AAV8，立体定位注射(C57BL/6,年龄大约两个月)的方法如前所述 2。将滴度为大于5×10 12vg/ml的AAV混合液，通过立体定位注射仪注入纹状体(AP+0.8mm，ML±1.6mm和DV-2.8mm)中，注射量为1uL。在miR124过表达实验中，注射的AAV为AAV-GFAP-miR124(约为1.7×10 13vg/ml)，在REST敲低实验中，对照组病毒为GFAP-mcherry(约为5×10 11vg/ml)+AAV-GFAP-CasRx(滴度约为1.2×10 13vg/ml,不含靶向REST的gRNA),实验组AAV病毒为GFAP-mcherry+AAV-GFAP-CasRx-REST(滴度约为1.2×10 13vg/ml,含靶向REST的gRNA)，每组注射1-3只小鼠。
视网膜下注射
如前所述，通过视网膜下注射AAV8。对于视网膜下注射，在Olympus显微镜(Olympus，日本)下用Hamilton注射器(32G针)向视网膜下注射AAV。为确定完整视网膜中的重编程，通过视网膜下注 射(C57BL/6小鼠，大约5周龄)将共计1μl的GFAP-tdTomato(0.1μl,约1×10 12vg/ml)和GFAP-CasRx-REST(0.9μl，约1.2×10 13vg/ml)，或GFAP-tdTomato(0.1μl,约1×10 12vg/ml)和GFAP-CasRx(0.9μl，约1.2×10 13vg/ml)注射至视网膜下。
免疫荧光染色
对于脑部免疫荧光染色：在注射后约1个月灌注小鼠后取脑，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(PB)彻底冲洗脑切片。用于免疫荧光染色的一抗为：兔多克隆NeuN抗体(1：500，#ABN78，Millipore)，小鼠TH抗体(1：300，MAB318，Millipore)以及大鼠DAT抗体(1：100，MAB369，Millipore)。二抗为：Alexa
Figure PCTCN2022075823-appb-000001
488 AffiniPure Donkey Anti-Mouse IgG(H+L)(1:500，715-545-150，Jackson ImmunoResearch)，Alexa
Figure PCTCN2022075823-appb-000002
488 AffiniPure Donkey Anti-Rabbit IgG(H+L)(1:500,711-545-152，Jackson ImmunoResearch)；Alexa
Figure PCTCN2022075823-appb-000003
488 AffiniPure Donkey Anti-Rat IgG(H+L)
(1:500，712-545-153，Jackson ImmunoResearch)；Cy TM5 AffiniPure Donkey Anti-Rabbit IgG(H+L)(1:500，711-175-152,Jackson ImmunoResearch)。在抗体孵育后，洗涤切片并用封片剂(Life Technology)封片。对于视网膜切片，在AAV注射后大约1个月，取眼睛，用4％多聚甲醛(PFA)固定2小时(眼睛)，然后在30％蔗糖溶液进行脱水然后将组织进行包埋盒切片，厚度为30μm。用于免疫荧光染色的一抗：兔抗RBPMS(1:500,15187-1-AP，Proteintech)，mouse-anti-rhodopsin(1:2000,MAB5356，EMD Millipore)以及二抗：Alexa
Figure PCTCN2022075823-appb-000004
488 AffiniPure Donkey Anti-Rabbit IgG(H+L)(1：500,715-545-150，Jackson ImmunoResearch)，Alexa
Figure PCTCN2022075823-appb-000005
488AffiniPure Donkey Anti-Mouse IgG(H+L)(1：500,711-545-152，Jackson ImmunoResearch)。敷完抗体后，进行洗片并封片。使用Olympus FV3000显微镜进行成像。
序列信息
人REST蛋白的氨基酸序列(SEQ ID NO.:1)：
Figure PCTCN2022075823-appb-000006
人REST编码序列(SEQ ID NO.:2)：
Figure PCTCN2022075823-appb-000007
Figure PCTCN2022075823-appb-000008
人REST编码序列的第867-1103位核苷酸序列(SEQ ID NO.:3)：
Figure PCTCN2022075823-appb-000009
在小鼠NA2细胞中筛选到的高效靶向REST的gRNA(SEQ ID NO.:4-54)
Figure PCTCN2022075823-appb-000010
Figure PCTCN2022075823-appb-000011
用于表观遗传学方法的sgRNA向导序列(SEQ ID NO.:55-82)
dSpCas9-KRAB的sgRNA1-8
gRNA SEQ ID NO. 向导序列 SEQ ID NO. DNA靶标
sgRNA1 55 ggcgcagcagcagaagaccg 63 ggcgcagcagcagaagaccg
sgRNA2 56 accgcagcgacggcagaacc 64 accgcagcgacggcagaacc
sgRNA3 57 ccctggttctgccgtcgctg 65 ccctggttctgccgtcgctg
sgRNA4 58 agcgacggcagaaccagggc 66 agcgacggcagaaccagggc
sgRNA5 59 cgggatcagaccgccggccc 67 cgggatcagaccgccggccc
sgRNA6 60 gatcgcaccccgggatctcg 68 gatcgcaccccgggatctcg
sgRNA7 61 gagttggagcggcggcgacg 69 gagttggagcggcggcgacg
sgRNA8 62 atactgtggctcgggcggcg 70 atactgtggctcgggcggcg
dSaCas9-KKH-Krab的sgRNA1-6
Figure PCTCN2022075823-appb-000012
所构建的一系列靶向人类RESTmRNA的gRNA(SEQ ID NO.:83-190)
Figure PCTCN2022075823-appb-000013
Figure PCTCN2022075823-appb-000014
Figure PCTCN2022075823-appb-000015
小鼠REST氨基酸序列(SEQ ID NO.:191)：
Figure PCTCN2022075823-appb-000016
小鼠REST编码序列(SEQ ID NO.:192)：
Figure PCTCN2022075823-appb-000017
实施例
实施例1：miR124不能将胶质细胞转分化为神经元或多巴胺神经元
之前的研究表明，过表达miR124可以将干细胞分化为神经元，还有研究表明Ptbp1通过miR124介导的转分化能将胶质细胞转分化为神经元。但这些都是在体外细胞中的研究，为了研究在动物体内miR124是否能将胶质细胞转分化为神经元，本研究构建了能在胶质细胞中特异性表达miR124的AAV载体，通过在小鼠脑中注射miR124过表达的AAV，探究miR124是否能在体内将胶质细胞转分化为神经元(图1A)。在注射后大约1个月取材分析，发现与体外研究结果不同，红色荧光蛋白标记的细胞并不与神经元特异性标志物NeuN共标(图1B)。这表明在小鼠体内，过表达miR124不能将胶质细胞转分化为神经元。为了进一步研究过表达miR124的细胞是否处于新生神经元阶段，我们通过新生神经元特异性蛋白标志物Tuj-1进行染色，发现这些红色荧光标记的细胞并与Tuj-1共标(图1C)，表明过表达miR124并不能将星形胶质细胞直接转分化为神经元。通过用多巴胺特异性蛋白标志物TH进行染色分析发现，miR124过表达的红色细胞也不表达TH，这表明过表达miR124不能将胶质细胞转分化为多巴胺神经元(图1D)。综合以上结果表明，在体内过表达miR124不能将胶质细胞转分化为神经元或多巴胺神经元。
实施例2：在N2A细胞中筛选高效靶向REST的gRNA
为了筛选到CasRx高效靶向REST的gRNA，首先我们设计了17个靶向REST的gRNA(参见SEQ ID NO.:4-54)，并构建到U6-gRNA-CMV-mCherry载体上，不同的gRNA分别与CAG-CasRx-P2A-EGFP质粒共转N2A细胞(图2A和2B)。转染细胞48小时后通过流式分选转染的GFP和mCherry双阳性细胞，并通过Q-PCR检测REST mRNA的表达量，从而筛选出靶向REST效率最高的gRNA。QPCR结果表明，大部分gRNA都能有效敲低REST mRNA水平，其中gRNA-7效率最高，能敲低大约94％的REST mRNA表达水平(图2C)。
实施例3：在体内实现星形胶质细胞向神经元细胞转分化
之前的研究表明，在体外敲低REST的表达，能将成纤维细胞转分化为神经元，虽然转分化效率只有5％左右。但在体内的复杂环境下，敲低REST的表达，是否可以实现转分化的过程呢。为了进一步探究CasRx介导的REST敲低技术是否能在体内实现胶质细胞向神经元的转分化，我们构建了的AAV表达载体并包装AAV，将AAV注射到小鼠大脑纹状体中(图3A)。为了标记胶质细胞，我们用胶质细胞特异性启动子GFAP驱动mCherry表达，为了在胶质细胞中特异性表达CasRx，我们同样利用胶质细胞特异性启动子GFAP驱动CasRx的表达。对照组注射的病毒为GFAP-mCherry与GFAP-CasRx的混合AAV，其中mCherry能标记被感染的胶质细胞，实验组注射的AAV组合为GFAP-mCherry+GFAP-CasRx-REST(表达gRNA-7)，其中GFAP-CasRx-REST能特异性靶向REST mRNA(图3A)。在注射AAV后大约1个月进行分析，发现对照组的红色荧光标记的细胞依然与胶质细胞特异性蛋白标志物GFAP共标，而不与神经元特异性标志物NeuN共标(图3B)。而在注射GFAP-mCherry+GFAP-CasRx-REST组，发现有大量mCherry阳性细胞与NeuN共标，而不与GFAP共标(图3C)。这些结果表明，在小鼠体内，靶向敲低REST的表达能高效将星形胶质细胞转分化为 神经元细胞。为了进一步研究敲低REST能否将胶质细胞转分化为多巴胺神经元，本研究利用多巴胺神经元特异性细胞标志物TH和DAT进行染色。在GFAP-mCherry+GFAP-CasRx对照组，红色荧光标记的细胞既不表达TH也不表达DAT，而在注射可敲低REST的AAV组(GFAP-mCherry+GFAP-CasRx-REST)中，有一小部分红色荧光标记的细胞表达多巴胺神经元特异性细胞标志物TH和DAT(图3D和3E)。以上结果表明，在纹状体中敲低REST的表达，能将星形胶质细胞转分化为多巴胺神经元。
实施例4：表观遗传方法抑制REST基因的表达
表观遗传修饰也是操控基因表达的一个常用方法，为了探究表观遗传的方法是否可以有效抑制REST mRNA的表达，我们将DNA结合蛋白(如Zinc fingers,TALEs,CRISPR-dCas等)与表观调控元件(如：KRAB，Dnmt3a,Tet1等)通过柔性连接氨基酸进行融合表达(图4A)。本研究中所使用的DNA靶向蛋白为两种不用的CRISPR-dCas(dSpCas9，dSaCas9-KKH)，并与表观修饰蛋白Krab抑制结构域进行融合表达，并同时由SV40驱动EGFP蛋白的表达用于流式分选，gRNA由U6独立驱动表达，并在U6-gRNA同一质粒载体中由CMV驱动mCherry荧光表达，用于细胞流式分选(图4B)。转染N2A细胞48小时后，通过Q-PCR检测发现dSpCas9-KRAB和dSaCas9-KKH-Krab都能有效降低REST mRNA的表达，大部分sgRNA(参见SEQ ID NO.:55-82)靶向REST后能将REST mRNA水平降低至原来的一半左右(图4C和4D)。
实施例5：在人类细胞中CasRx介导的高效REST敲低
为了进一步探究在人类细胞中是否可以高效敲低REST的表达，我们构建了一系列靶向人类REST mRNA的gRNA(参见SEQ ID NO.:83-190)。通过Q-PCR的方法检测不同gRNA敲低效率，发现大部分gRNA都能有效敲低REST mRNA(图5A)。对这些gRNA位置进行分析，发现敲低效率很高的这些gRNA集中分布于REST mRNA的一小段区域(图5B和5C)。结果表明，该区域是设计gRNA靶向的优选位置。
实施例6：高效靶向人的gRNA可以在非人灵长类和小鼠中实现REST高效敲低
为了研究高效靶向人的gRNA是否同样可以高效靶向非人灵长类或者小鼠，本研究从已经筛选的高效靶向人类REST基因的gRNA中选取了3个gRNA进行测试(gRNA17、gRNA18和gRNA19)。其中gRNA-17序列在人类、非人灵长类和小鼠中都是同源的，序列完全一致；gRNA-18在食蟹猴和小鼠中有1个碱基的错配，gRNA-19在食蟹猴和小鼠中有2个碱基的错配(图6A)。如图6B所示，本研究将gRNA与CasRx构建到同一个表达质粒中，将质粒转染293T、Cos-7和N2A细胞后，通过流式分选转染的阳性细胞，并通过QPCR检测REST mRNA表达量的差异(图6C)。结果表明，靶向人的3个gRNA都可以高效靶向非人灵长类和小鼠的REST，也能有效敲低非人灵长类和小鼠的REST mRNA的表达量(图6D)。以上结果表明，本发明的gRNA可以应用于不同物种，而同样实现本发明的技术效果。
实施例7：靶向人REST的CasRx-gRNA系统可以在小鼠中将胶质细胞转分化为神经元
为了研究靶向人的gRNA是否可以有效将胶质细胞转分化为神经元，本研究将靶向人的gRNA-17(gRNA(human))和CasRx构建到AAV载体中并包装AAV。将GFAP-CasRx-REST与GFAP-mCherry共注到小鼠脑内，对照组注射GFAP-CasRx+GFAP-mCherry，注射后1个月进行分析(图7A)。结果显示，靶向人类REST的gRNA可以将星形胶质细胞转分化为神经元，红色荧光标记的细胞与神经元特异性蛋白标志物NeuN共标(50.71％±11.12％，SEM,每组3只小鼠)，而注射对照组AAV的小鼠脑中红色荧光标记的细胞依然表现出典型的胶质细胞形态，并且不与NeuN共标(图7B,7C和7D)。以上结果表明，靶向人类REST的CasRx-gRNA系统可以高效将胶质细胞转分化为神经元，具有治疗神经元缺失相关疾病的潜力。为了进一步研究在视网膜中敲低REST是否可以将视网膜中的穆勒胶质细胞转分化为功能性神经元，如感光细胞或视网膜神经节细胞。我们在大约5周的C57小鼠视网膜下注射GFAP-tdTomato和GFAP-CasRx-REST的AAV(之前的文献显示GFAP可以作为穆勒胶质细胞的特异启动子，GFAP-tdTomato用来标记穆勒胶质细胞)，在视网膜穆勒胶质细胞中敲低REST的表达，发现视网膜外颗粒层中有细胞同时表达Rhodopsin和tdTomato,而在视网膜神经节细胞层中有细胞同时表达Rbpms和tdTomato，这些数据暗示着在视网膜中敲低REST，能将穆勒胶质细胞分别转分化为感光细胞或视网膜神经节细胞(图7E和7F)。
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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。

Claims (39)

  1. 由胶质细胞产生功能性多巴胺神经元的方法，其包括使用REST抑制剂使所述胶质细胞转分化为或重编程为功能性多巴胺神经元，其中所述REST抑制剂降低REST基因或其RNA或其编码蛋白的表达、含量或活性。
  2. 根据权利要求1所述的方法，其中所述胶质细胞选自星形胶质细胞、少突胶质细胞、室管膜细胞、施万细胞、NG2细胞、卫星细胞、或其组合，优选星形胶质细胞。
  3. 根据权利要求2所述的方法，其中所述星形胶质细胞来源于中枢神经系统，包括纹状体、黑质、中脑腹侧被盖区、脊髓、下丘脑、背侧中脑或大脑皮层，优选来源于纹状体和黑质。
  4. REST抑制剂在制备药物中的用途，所述药物用于预防和/或治疗与功能性多巴胺神经元功能缺失或死亡相关的疾病，其中所述REST抑制剂降低REST基因或其RNA或其编码蛋白的表达、含量或活性。
  5. 根据权利要求4所述的用途，其中所述药物配制成用于施用于中枢神经系统，包括纹状体、黑质、中脑腹侧被盖区、脊髓、下丘脑、背侧中脑或大脑皮层等，优选纹状体和黑质。
  6. 根据权利要求4所述的用途，其中所述与功能性多巴胺神经元功能缺失或死亡相关的疾病为神经系统疾病，包括中风、帕金森病、精神分裂症和抑郁症，优选帕金森病。
  7. 由穆勒胶质细胞产生功能性视网膜神经节细胞(RGC)或感光细胞的方法，其包括用REST抑制剂使所述穆勒胶质细胞转分化为或重编程为功能性RGC或感光细胞，其中所述REST抑制剂降低REST基因或其RNA或其编码蛋白的表达、含量或活性。
  8. 根据权利要求7所述的方法，其中所述穆勒胶质细胞来源于视网膜，并且其中所述感光细胞包括视杆细胞和视锥细胞。
  9. REST抑制剂在制备药物中的用途，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REST抑制剂降低REST基因或其RNA或其编码蛋白的表达、含量或活性。
  10. 根据权利要求9所述的用途，其中所述药物配制成用于施用于视觉系统，优选视网膜下或玻璃体腔。
  11. 根据权利要求9所述的用途，其中所述与RGC功能缺失或死亡有关的视觉系统疾病选自：RGC细胞死亡导致的视力损伤、青光眼、年龄相关的RGC病变、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；并且其中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。
  12. 根据前述权利要求中任一项所述的方法或用途，其中所述REST抑制剂选自：抗体、小分子化合物、microRNA、siRNA、shRNA、反义寡核苷酸、REST结合蛋白或蛋白结构域、多肽、核酸适配体、基因编辑器、PROTAC、表观遗传调控或其组合。
  13. 根据权利要求12所述的方法或用途，其中所述REST抑制剂包含：
    (a)基因编辑蛋白或其表达载体，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，(b)一个或多个gRNA或其表达载体，所述gRNA是引导基因编辑蛋白特异性结合REST基因的DNA或RNA，
    其中优选靶向RNA的基因编辑蛋白和靶向RNA的gRNA，优选地所述gRNA包含与REST基因互补的序列。
  14. 根据权利要求13所述的方法或用途，其中所述gRNA引导所述基因编辑蛋白特异性结合于对应于REST编码序列的第867-1103位核苷酸(SEQ ID NO.:3)，优选地所述gRNA包含与SEQ ID NO.:3互补的序列。
  15. 根据权利要求13或14所述的方法或用途，其中所述gRNA包含选自SEQ ID NO.:4-20和83-118的序列或者包含由SEQ ID NO.:55-62和71-76的序列编码的序列，优选所述gRNA包含选自SEQ ID NO.:10和93-103的序列。
  16. 根据权利要求13-15中任一项所述的方法或用途，其中所述gRNA包含与目标序列完全互补的序列，或者包含与目标序列具有不多于3个碱基错配的互补序列。
  17. 根据权利要求13-15中任一项所述的方法或用途，其中所述gRNA与目标序列来自相同物种或不同物种。
  18. 根据权利要求17中任一项所述的方法或用途，其中所述gRNA与目标序列来自人、食蟹猴或小鼠。
  19. 包含REST抑制剂的药物组合物或药盒或试剂盒。
  20. 根据权利要求19所述的药物组合物或药盒或试剂盒，其中所述REST抑制剂选自：抗体、小分子化合物、microRNA、siRNA、shRNA、反义寡核苷酸、REST结合蛋白或蛋白结构域、多肽、核酸适配体、基因编辑器、PROTAC、表观遗传调控或其组合。
  21. 根据权利要求19或20的药物组合物或药盒或试剂盒，其中所述REST抑制剂包含：
    (a)基因编辑蛋白或其表达载体，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，(b)一个或多个gRNA或其表达载体，所述gRNA是引导基因编辑蛋白特异性结合REST基因的DNA或RNA，
    其中优选靶向RNA的基因编辑蛋白和靶向RNA的gRNA，优选地所述gRNA包含与REST基因互补的序列。
  22. 根据权利要求21所述的药物组合物或药盒或试剂盒，其中所述gRNA引导所述基因编辑蛋白特异性结合于对应于REST编码序列的第867-1103位核苷酸(SEQ ID NO.:3)，优选地所述gRNA包含与SEQ ID NO.:3互补的序列。
  23. 根据权利要求21或22所述的药物组合物或药盒或试剂盒，其中所述gRNA包含选自SEQ ID NO.:4-20和83-118的序列或者包含由SEQ ID NO.:55-62和71-76的序列编码的序列，优选所述gRNA包含选自SEQ ID NO.:10和93-103的序列。
  24. 根据权利要求21-23中任一项所述的药物组合物或药盒或试剂盒，其中所述gRNA包含与 目标序列完全互补的序列，或者包含与目标序列具有不多于3个碱基错配的互补序列。
  25. 根据权利要求21-23中任一项所述的药物组合物或药盒或试剂盒，其中所述gRNA与目标序列来自相同物种或不同物种。
  26. 根据权利要求25中任一项所述的药物组合物或药盒或试剂盒，其中所述gRNA与目标序列来自人、食蟹猴或小鼠。
  27. 根据权利要求19-26中任一项所述的药物组合物或药盒或试剂盒，其还包含用于递送所述REST抑制剂的载体或运载体。
  28. 根据权利要求27的药物组合物或药盒或试剂盒，其中所述载体或运载体为病毒载体、脂质体、纳米颗粒、外泌体、类病毒颗粒，优选AAV。
  29. 根据权利要求21-28中任一项的药物组合物或药盒或试剂盒，其中所述靶向RNA的基因编辑蛋白选自：Cas13d、CasRx、Cas13X、Cas13a、Cas13b、Cas13c、Cas13Y及其功能结构域，其中优选CasRx、Cas13X、Cas13Y，更优选CasRx。
  30. 根据权利要求21-28中任一项的药物组合物或药盒或试剂盒，其中所述药物组合物或药盒或试剂盒包含靶向REST mRNA序列的仅单一类型的gRNA或2、3、4、5、6种不同的gRNA，或者所述gRNA表达载体编码包含靶向REST mRNA序列的仅单一类型的gRNA或2、3、4、5、6种不同的gRNA。
  31. 根据权利要求21-28中任一项的药物组合物或药盒或试剂盒，其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，其中优选地所述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，其中更优选地，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，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；以及
    ii)至少一种编码靶向REST mRNA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。
  32. 根据权利要求21-28中任一项的药物组合物或药盒或试剂盒，其中所述表达载体包含在纳米颗粒中，或者其中所述表达载体是基因治疗载体，优选病毒基因治疗载体，更优选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)、腺病毒载体、慢病毒载体、逆转录病毒载体、疱疹病毒、SV40载体、痘病毒载体、及其组合，其中优选AAV和rAAV。
  33. 根据权利要求19-28中任一项的药物组合物或药盒或试剂盒，其中所述组合物局部施用至以下至少一种：1)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)下丘脑中的胶质细胞；以及ix)腹侧被盖区(VTA)中的胶质细胞。
  34. 根据权利要求21-28中任一项的药物组合物或药盒或试剂盒，其中所述组合物还包含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。
  35. 根据权利要求34的药物组合物或药盒或试剂盒，其中所述一种或更多种多巴胺神经元相关因子选自：Lmx1a、Lmx1b、FoxA2、Nurr1、Pitx3、Gata2、Gata3、FGF8、BMP、En1、En2、PET1、Pax家族蛋白、SHH、Wnt家族蛋白和TGF-β家族蛋白。
  36. 根据权利要求21-28中任一项的药物组合物或药盒或试剂盒，其中所述组合物还包含i)一种或更多种选自β-catenin、Oct4、Sox2、Klf4、Crx、Brn3a、Brn3b、Math5、Nr2e3和Nrl的因子，和/或ii)用于在胶质细胞中表达选自β-catenin、Oct4、Sox2、Klf4、Crx、Brn3a、Brn3b、Math5、Nr2e3和Nrl的一种或更多种因子的至少一种表达载体。
  37. 根据权利要求19-28中任一项的药物组合物或药盒或试剂盒，其配制成用于注射、颅内给药、眼内给药、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。
  38. 根据权利要求28的药物组合物或药盒或试剂盒，其中所述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，其中优选地所述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，其中更优选地，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，或者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；以及
    ii)至少一种编码靶向REST mRNA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。
  39. 根据前述权利要求中任一项的方法、用途、或者药物组合物或药盒或试剂盒，其中胶质细胞的转分化效率为至少1％，或至少10％、20％、30％、40％或50％。
PCT/CN2022/075823 2021-02-10 2022-02-10 胶质细胞向神经元转分化用于预防或治疗神经元功能缺失或死亡相关疾病 WO2022171167A1 (zh)

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