Direct transdifferentiation for the treatment of neurological diseases

Abstract

RE1 / NRSE blockers are used for treating nervous system diseases. This disclosure relates to the field of biomedicine. More specifically, this disclosure relates to the use of RE1 / NRSE elements as targets for transdifferentiating non-neuronal cells into neuronal cells; and the use of RE1 / NRSE blockers in preventing and / or treating diseases associated with neuronal loss or death. This disclosure provides methods for blocking RE1 / NRSE elements to regulate the expression of neuron-related genes in non-neuronal cells, which include reducing the binding of REST to RE1 / NRSE elements, or reducing the amount or activity of REST. This disclosure also provides RE1 / NRSE element blockers, especially the binding domains of endogenous RE1 / NRSE-binding proteins and their variants, which can be used for preventing and / or treating diseases associated with neuronal loss or death.

Description

Technical Field

[0001] The present disclosure relates to the field of biomedicine. More specifically, the present disclosure relates to the use of RE1 / NRSE elements as targets for transdifferentiating non-neuronal cells into neuronal cells; and the use of RE1 / NRSE blockers in the prevention and / or treatment of diseases associated with neuronal loss or death. Background Art

[0002] The repressor element 1 / neuron-restrictive silencer element (RE1 / NRSE) is a specific DNA sequence, about 21 bp in length (ranging from 20 to 23 bp), which mainly binds to REST (RE1-silencing transcription factor, which is also known as neuron-restrictive silencer factor, NRSF) and regulates the gene expression related to neuronal development and maturation. RE1 is a negative regulatory element related to neuronal maturation and was first discovered at the 5'-end of the promoters of NaV1.2 and SCG10 to regulate the expression of these genes. In non-neuronal cells, the RE1 locus is bound by a silencing complex composed of histone deacetylases and methylases, etc., which inhibits the expression of neuronal-related genes. However, there are approximately more than 1,800 RE1 elements in mice and humans, making it difficult to regulate them using some existing techniques. For example, CRISPR-mediated gene regulation and epigenetic modification techniques have very high precision and can precisely regulate the expression of specific genes, but it is difficult to regulate the expression of genes regulated by RE1 in this way.

[0003] Parkinson's disease (PD) is a disease associated with the loss or death of neuronal function, characterized by the loss of dopamine neurons in the substantia nigra of the midbrain. Currently, the main treatment methods for Parkinson's disease are small molecule drugs represented by dopamine analogs such as levodopa. The surgical treatments developed in recent years can also improve the symptoms of the disease to a certain extent, such as deep brain electrical stimulation. However, these methods can only partially relieve the condition, cannot prevent the development of the condition, and cannot slow down the death of dopamine neurons. The concept of transdifferentiation therapy has brought hope for the regeneration of dopamine neurons. By overexpressing some genes in glial cells or performing gene editing on glial cells, glial cells are transdifferentiated into dopamine neurons to supplement the missing or dead dopamine neurons. Müller glia (MG) are the main glial cells in the retinal tissue. Retinal ganglion cells (RGC) are nerve cells located in the innermost layer of the retina. Its dendrites mainly establish synaptic connections with bipolar cells. Its axons extend to the optic nerve papilla to form the optic nerve and extend to the brain. The damage or degenerative lesions of retinal ganglion cells (RGC) are the main causes of permanent blindness. RGC is the only output neuron in the retina. Therefore, the damage or degenerative lesions of RGC will lead to permanent blindness. Reconstructing functional RGC is currently the only method that can help blind patients restore their vision. However, RGC cells are very difficult to regenerate. Therefore, exploring how to regenerate RGC cells will bring hope for the blind patients caused by the death of RGC cells. Therefore, there is an urgent need in this field to develop methods that can regenerate dopamine neurons, retinal ganglion cells or other functional nerves. Summary of the Invention

[0004] In one embodiment, the present disclosure provides a method for blocking the RE1 / NRSE element to regulate the expression of neuron-related genes in non-neuronal cells, which includes reducing the binding of REST to the RE1 / NRSE element, or reducing the amount or activity of REST.

[0005] In a preferred embodiment, the amount of REST is reduced by methods such as gene editing, small RNA interference or accelerated protein degradation.

[0006] In a preferred embodiment, the amount of REST is reduced by methods such as gene editing, antisense oligonucleotides (ASO), small RNA interference, miRNA technology, small molecule compounds, or accelerated protein degradation.

[0007] In a preferred embodiment, the binding of REST to the RE1 / NRSE element is blocked by binding a REST binder to REST, and the REST binder is, for example, a REST antibody.

[0008] In a preferred embodiment, the binding of REST to the RE1 / NRSE element is blocked by binding of a RE1 / NRSE element blocker to the RE1 / NRSE element.

[0009] In a preferred embodiment, the RE1 / NRSE element blocker is a competitive binding protein, short peptide or gene editing protein of REST or its coding nucleic acid, or nucleic acid and nucleic acid analogs, or a small molecule RE1 / NRSE element blocker.

[0010] In a preferred embodiment, the RE1 / NRSE element blocker is a REST variant or its coding nucleic acid.

[0011] In a preferred embodiment, the REST variant is the DNA-binding domain of REST, which lacks the N-terminal and C-terminal inhibitory domains of REST, preferably amino acids 155-420 of REST.

[0012] In a preferred embodiment, the DNA-binding domain of REST is fused to an activation domain.

[0013] In a preferred embodiment, the activation domain is selected from: epigenetic modification proteins or gene activation regulatory elements, such as VP64, P65-HSF1, VP16, RTA, Suntag, P300, CBP or combinations thereof, preferably VP64 or P65-HSF1.

[0014] In a preferred embodiment, the REST variant has the amino acid sequences of SEQ ID NO: 1, 3, 5 and 9 or the nucleotide sequences of SEQ ID NO: 2, 4, 6 and 10, or sequences having at least 70%, 60%, 50% identity percentage thereto.

[0015] In a preferred embodiment, the non-neuronal cells include, for example, glial cells, fibroblasts, stem cells, neural progenitor cells, neural stem cells, wherein the glial cells are selected from astrocytes, oligodendrocytes, ependymal cells, Schwann cells, NG2 cells, satellite cells, Müller glial cells, inner ear glial cells or combinations thereof, preferably astrocytes, Müller glial cells and cochlear glial cells.

[0016] In a preferred embodiment, the glial cells are derived from the brain, spinal cord, eye or ear, wherein the brain glial cells are derived from the striatum, substantia nigra, ventral tegmental area of the midbrain, spinal cord, hypothalamus, dorsal midbrain or cerebral cortex, preferably the striatum and substantia nigra.

[0017] In a preferred embodiment, the expression of neuron-related genes in the non-neuronal cells is regulated to transdifferentiate the non-neuronal cells into neuronal cells, wherein the neuronal cells are mammalian neurons, preferably dopamine neurons, GABA neurons, 5-HT neurons, glutamatergic neurons, ChAT neurons, NE neurons, motor neurons, spinal cord neurons, spinal cord motor neurons, spinal cord sensory neurons, photoreceptor cells (rods and cones), bipolar cells, horizontal cells, amacrine cells, retinal ganglion cells (RGCs), cochlear nerve cells (cochlear spiral ganglion cells and vestibular neurons), pyramidal neurons, interneurons, medium spiny neurons (MSNs), Purkinje cells, granule cells, olfactory sensory neurons, periglomerular cells, or a combination thereof, more preferably dopamine neurons, retinal ganglion cells, photoreceptor cells, and cochlear spiral ganglion cells.

[0018] In a preferred embodiment, the non-neuronal cells and / or neuronal cells are from, for example, humans, non-human primates, rats, and mice, preferably humans.

[0019] In another embodiment, the present disclosure provides the use of an RE1 / NRSE element blocker for the preparation of a medicament for preventing and / or treating diseases associated with neuronal loss or death, wherein the RE1 / NRSE element blocker reduces the binding of an RE1 / NRSE endogenous binding factor to the RE1 / NRSE element, and the RE1 / NRSE endogenous binding factor includes zinc finger proteins such as REST.

[0020] In a preferred embodiment, the RE1 / NRSE element blocker binds to the RE1 / NRSE element to block the binding of the RE1 / NRSE endogenous binding factor to the RE1 / NRSE element.

[0021] In a preferred embodiment, the RE1 / NRSE element blocker is a competitive binding protein of REST, a short peptide, or a gene editing protein or its encoding nucleic acid, or a nucleic acid and nucleic acid analog, or a small molecule RE1 / NRSE element blocker.

[0022] In a preferred embodiment, the RE1 / NRSE element blocker is a REST variant or its encoding nucleic acid.

[0023] In a preferred embodiment, the REST variant is the DNA binding domain of REST, which lacks the N-terminal and C-terminal inhibitory domains of REST, preferably amino acids 155-420 of REST.

[0024] In a preferred embodiment, the DNA binding domain of REST is fused with an activation domain.

[0025] In a preferred embodiment, the activation domain is selected from: epigenetic modification proteins or gene activation regulatory elements, such as VP64, P65-HSF1, VP16, RTA, Suntag, P300, CBP, or a combination thereof, preferably VP64 or P65-HSF1.

[0026] In a preferred embodiment, the REST variant has the amino acid sequences of SEQ ID NO: 1, 3, 5, and 9 or the nucleotide sequences of SEQ ID NO: 2, 4, 6, and 10, or sequences having at least 70%, 60%, or 50% sequence identity therewith.

[0027] In a preferred embodiment, the diseases associated with neuronal loss or death are selected from: Parkinson's disease, Alzheimer's disease, stroke, schizophrenia, Huntington's disease, depression, motor neuron disease, amyotrophic lateral sclerosis, spinal muscular atrophy, Pick's disease, sleep disorders, epilepsy, ataxia, vision impairment due to RGC cell death, glaucoma, age-related RGC lesions, optic nerve injury, retinal ischemia or hemorrhage, Leber hereditary optic neuropathy, photoreceptor degeneration or death due to injury or degenerative lesions, macular degeneration, retinitis pigmentosa, diabetes-related blindness, night blindness, color blindness, hereditary blindness, congenital stationary night blindness, deafness or hearing loss due to spiral ganglion cell death, or a combination thereof.

[0028] In another embodiment, the present disclosure provides RE1 / NRSE element blockers, which are REST variants or their encoding nucleic acids, or artificially designed DNA binding domain analogs of REST.

[0029] In a preferred embodiment, the REST variant is the DNA binding domain of REST, which lacks the N-terminal and C-terminal inhibitory domains of REST, preferably amino acids 155-420 of REST.

[0030] In a preferred embodiment, the DNA binding domain of REST is fused with an activation domain.

[0031] In a preferred embodiment, the activation domain is selected from: epigenetic modification proteins or gene activation regulatory elements, such as VP64, P65-HSF1, VP16, RTA, Suntag, P300, CBP, or a combination thereof, preferably VP64 or P65-HSF1.

[0032] In a preferred embodiment, the REST variant has the amino acid sequences of SEQ ID NO: 1, 3, 5, and 9 or the nucleotide sequences of SEQ ID NO: 2, 4, 6, and 10, or a sequence having at least 70%, 60%, 50% identity thereto.

[0033] In a preferred embodiment, the REST variant or the DNA binding domain of REST is from, for example, human, non-human primate, rat, and mouse, preferably human.

[0034] In another embodiment, the present disclosure provides a pharmaceutical composition or a cartridge or a kit, which comprises the above blocker.

[0035] In a preferred embodiment, the pharmaceutical composition or the cartridge or the kit is formulated for injection, intracranial administration, intraocular administration, intra-aural administration, inhalation, parenteral administration, intravenous administration, intramuscular administration, intradermal administration, topical administration, or oral administration.

[0036] In a preferred embodiment, the pharmaceutical composition or the cartridge or the kit further comprises a carrier or vehicle for delivering the RE1 / NRSE element blocker, wherein the carrier or vehicle is a viral vector, liposome, nanoparticle, exosome, viroid particle, wherein the viral vector includes a recombinant adeno-associated virus vector (rAAV), an adeno-associated virus (AAV) vector, an adenovirus vector, a lentivirus vector, a retrovirus vector, a poxvirus vector, a herpesvirus, an SV40 virus vector, and combinations thereof, wherein AAV and rAAV are preferred.

[0037] In a preferred embodiment, the pharmaceutical composition or the cartridge or the kit comprises an expression vector for expressing a REST variant, wherein the expression vector comprises a nucleotide sequence encoding the REST variant, which is operably linked to a promoter that causes its expression.

[0038] In a preferred embodiment, the pharmaceutical composition or the cartridge or the kit is for local administration to at least one of the following: 1) glial cells in the striatum; ii) glial cells in the substantia nigra of the brain; iii) glial cells in the retina; iv) glial cells in the inner ear; v) glial cells in the spinal cord; vi) glial cells in the prefrontal cortex; vii) glial cells in the motor cortex; viii) glial cells in the hypothalamus; and ix) glial cells in the ventral tegmental area (VTA).

[0039] In a preferred embodiment, the pharmaceutical composition or the cartridge or the kit further comprises i) one or more dopamine neuron-related factors, or ii) for expressing one or more retinal ganglion cell-related factors in Müller glial cells,

[0040] 1) One or more of the dopamine neuron-related factors are selected from: FoxA2, Lmx1a, Lmx1b, Nurr1, Pbx1a, Pitx3, Gata2, Gata3, FGF8, BMP, En1, En2, PET1, Pax family proteins (such as Pax3, Pax6, etc.), SHH, Wnt family proteins, and TGF-β family proteins, or a combination thereof;

[0041] 2) One or more of the retinal ganglion cell-related factors include: β-catenin, Oct4, Sox2, Klf4, Crx, aCamKII, Brn3a, Brn3b, Brn3C, Math5, Otx2, Ngn2, Ngn1, AscL1, miRNA9, miRNA-124, Nr2e3, and Nrl and other factors.

[0042] In a preferred embodiment, the promoter is a glial cell-specific promoter or a Müller glial (MG) cell-specific promoter, and the glial cell-specific promoter is selected from the GFAP promoter, ALDH1L1 promoter, EAAT1 / GLAST promoter, glutamine synthetase promoter, S100β promoter, EAAT2 / GLT-1 promoter, and Rlbp1 promoter, preferably the GFAP promoter.

[0043] In a preferred embodiment, the transdifferentiation efficiency of glial cells is at least 1%, or at least 10%, 20%, 30%, 40%, or 50%. Brief Description of the Drawings

[0044] Figure 1. Schematic diagram of endogenous zinc finger protein design. (A) Schematic diagram of the REST protein structure. The REST protein contains an N-terminal inhibitory domain, a middle DNA-binding structure responsible for binding to RE1, and a C-terminal transcriptional inhibitory domain. RZFD-V1 represents the first design of RZFD, which contains 8 zinc finger domains (RZFD, REST Zinc Finger Domain) in the middle of the human REST protein responsible for binding to RE1. (B) Two designs of RZFD-V2 and RZFD-V3. To activate neuron-related genes inhibited by REST through binding to RE1 / NRSE, we fused RZFD with two different activators, named RZFD-V2 and RZFD-V3, respectively. RZFD-V2 is expressed by fusing RZFD with VP64, and RZFD-V3 is composed of the C-terminus of RZFD fused with a transcriptional activation domain of a P65 and an HSF1. (C) In glial cells, REST binds to RE1, and the C-terminus and N-terminus of REST recruit some transcriptional repressors respectively, so that neuron-related genes regulated by RE1 cannot be expressed. (D) We hypothesized that after expressing RZFD-V1 in glial cells, RZFD binds to RE1, preventing REST from binding to RE1, so that the REST silencing complex cannot inhibit the genes regulated by RE1, and the neuron-related genes regulated by RE1 are expressed. (E) RZFD-V2 binds to RE1 through the RZFD domain, preventing REST / NRSF from binding to RE1. At the same time, its VP64 domain can recruit transcriptional activators to enhance the expression of neuron-related genes regulated by RE1. (F) During the transdifferentiation of glial cells into neurons, the RZFD domain of RZFD-V3 binds to RE1, preventing REST from binding to RE1, relieving the expression of genes regulated by REST on RE1. At the same time, the P65-HSF1 activation domain recruits some transcriptional activators to promote the expression of neuron-related genes regulated by RE1.

[0045] Figure 2.RZFD-mediated glial cell-to-neuron transdifferentiation. (A) Schematic diagram of AAV vector design. Vector 1 is a schematic diagram of a vector for GFAP-driven mCherry expression. GFAP is a promoter specifically expressed in glial cells, and mCherry is a red fluorescent protein used to label glial cells. Vector 2 is a schematic diagram of a human RZFD expression vector, in which the expression of RZFD is driven by the astrocyte-specific promoter GFAP. (B) Schematic diagram of injection and sample collection for analysis. The day of AAV injection is designated as day 0. Samples are collected 2 weeks after injection to analyze the results of glial cell transdifferentiation into neurons, and 1.5 months after injection to analyze the results of glial cell transdifferentiation into neurons and dopamine neurons. (C) Schematic diagram of AAV virus injection and transdifferentiation. GFAP-mCherry is injected alone into the striatum or substantia nigra of mice, or a mixed AAV of GFAP-mCherry and GFAP-RZFD is injected. GFAP-mCherry labels glial cells red, while GFAP-RZFD transdifferentiates glial cells into neurons. (D) After injecting GFAP-mCherry alone into the striatum of wild-type C57 mice, astrocytes are labeled red. The mCherry channel shows astrocytes labeled with GFAP-mCherry, DAPI stains the cell nuclei, and NeuN is a neuron-specific marker. The Merge image shows that GFAP-mCherry specifically labels astrocytes but not neurons. (E) Samples are collected 2 weeks after injecting a mixed AAV of GFAP-mCherry and GFAP-RZFD into the striatum of mice for analysis. Most glial cells have started to deform, and a small number of cells have started to express NeuN, but no cells express TH. NeuN is a neuron-specific marker, and TH is a dopamine neuron-specific marker. (F) Samples are collected 1.5 months after injecting a mixed AAV of GFAP-mCherry and GFAP-RZFD into the striatum of mice for analysis. Most mCherry-positive cells express the neuron-specific marker, and a part of the cells express the dopamine-specific marker TH. The arrows indicate neurons that express both mCherry and TH.

[0046] Figure 3.Glial cell to neuron transdifferentiation mediated by RZFD-VP64 or RZFD-P65-HSF1. (A) Schematic diagram of the design of GFAP-RZFD-V2 and GFAP-RZFD-V3 expression vectors. RZFD-V2 is a fusion protein of RZFD and VP64, driven by the glial cell-specific promoter GFAP. RZFD-V3 consists of RZFD and the P65-HSF1 activation domain. (B) Schematic diagram of the injection of a mixture of GFAP-RZFD-V2 and GFAP-mCherry AAV into the striatum or substantia nigra of mice, and samples were taken for analysis 1.5 months after injection. (C) Representative diagram of the transdifferentiation of glial cells into neurons and dopamine neurons by GFAP-RZFD-V2 in the striatum. mCherry labels the cells marked by GFAP-mCherry, TH is a specific marker for dopamine neurons, and NeuN is a specific marker for neurons. The arrows indicate neurons that co-express mCherry and TH. (D) Schematic diagram of the injection of GFAP-RZFD-V3 into DAT-Cre:Ai9 mice. GFAP-RZFD-V3 was injected into the striatum or substantia nigra of DAT-Cre:Ai9 mice, and samples were taken for analysis 1.5 months later. Dat-Cre has Cre inserted behind the endogenous promoter of Dat, and Ai9 is a Rosa26-CAG-LSL-tdTomato-WPRE mouse. Only mature dopamine neurons in the brains of DAT-Cre:Ai9 mice can be labeled with the tdTomato red fluorescence signal. (E) Representative diagram of the results of sample analysis 1.5 months after the injection of GFAP-RZFD-V3 into the striatum. The red fluorescence signal (tdTomato) represents the transformed mature dopamine neurons, the green signal is the staining of the dopamine neuron-specific marker TH, and the white signal is the staining of the neuron-specific marker NeuN. The Merge diagram shows the coincidence of the tdTomato red signal and the TH staining green signal. The arrows indicate neurons that co-express tdTomato and TH Detailed implementation method

[0047] Although previous studies have shown that RE1 exists in the promoter regions of many neuron-related genes, it remains unknown whether glial cell-to-neuron transdifferentiation can be achieved by regulating RE1. In this study, by genetically engineering endogenous RE1-binding proteins, the zinc finger domain (ZFD) of the endogenous protein REST, which can bind to RE1, was used to regulate RE1 and achieve the regulation of the expression of its related genes. First, we analyzed the REST protein and found that its 159-412th positions are 8 zinc finger domains, which may be related to its binding to RE1. Its 1-83rd positions at the N-terminus are its N-terminal inhibitory region, which mainly binds to proteins such as Sin3a and Sin3b. Its 1008-1097th positions are its C-terminal inhibitory domain and a zinc finger domain, which mainly binds to proteins such as RCOR1. The middle 84-158th and 413-1007th positions have no obvious protein domains, and their functions are not yet clear, but they may be involved in regulating the binding of REST to RE1. Previous studies have shown that deleting the 1-83rd and 1008-1097th positions of the REST protein does not affect the binding of REST to RE1, but it cannot perform its normal function.

[0048] In this study, a series of analyses and modifications were carried out on the endogenous zinc finger domain of REST by using protein structure prediction and combining genetic engineering techniques. First, through truncation experiments, we found that overexpressing the 155-420th amino acids of REST (only containing 8 zinc finger domains, named RZFD: REST Zinc Finger Domain) can block the binding of REST to RE1. By using AAV-mediated gene delivery technology, overexpressing RZFD in mouse striatal astrocytes can transdifferentiate glial cells into neurons. We further modified RZFD by fusing VP64 at the N-terminus or C-terminus of RZFD to form RZFD-VP64, and successfully achieved AAV-mediated in vivo glial cell-to-neuron transdifferentiation. We further fused the transcriptional activation domains of P65 and HSF1 to RZFD to form RZFD-P65-HSF1. By applying AAV-mediated in vivo transdifferentiation technology, we found that RZFD-P65-HSF1 can also transdifferentiate glial cells into neurons in the striatum.

[0049] Previous studies have shown that inhibiting REST can transdifferentiate glial cells into neurons, and REST binds to a DNA sequence called RE1 in the genome. RE1 is a type of sequence that cannot be targeted by CRISPR technology. In this study, a human endogenous zinc finger structure (REST zinc finger domain, RZFD) was used to ingeniously target the RE1 sequence, thereby blocking the binding of the REST silencing complex to RE1. In non-neuronal cells such as glial cells, the expression of RZFD (RZFD-V1) relieved the inhibition of the expression of neuron-related genes by the REST silencing complex. We further promoted the expression of neuron-related genes and the transdifferentiation of glial cells into neurons by fusing activation domains such as VP64 (RZFD-V2) or P65-HSF1 (RZFD-V3) to RZFD. Through immunofluorescence staining and the DAT-Cre:Ai9 labeling system, we also found that RZFD-V1, RZFD-V2, and RZFD-V3 could all transdifferentiate glial cells into dopamine neurons.

[0050] By injecting AAV into the subretinal space of the Ai9 retina, we found that RZFD, RZFD-VP64, and RZFD-P65-HSF1 could transdifferentiate Müller cells into retinal ganglion cells and observed some photoreceptor cells. Retinal ganglion cells are the only cells in the visual pathway that transmit visual signals to the brain, and their loss or death can lead to permanent blindness. The results in non-human primates were similar to those in mice. We found that in the brain, RZFD, RZFD-VP64, and RZFD-P65-HSF1 could transdifferentiate glial cells into dopamine neurons, and in the retina, RZFD, RZFD-VP64, and RZFD-P65-HSF1 could transdifferentiate Müller cells into retinal ganglion cells and photoreceptor cells.

[0051] Diseases associated with the loss or death of neuronal function

[0052] In this disclosure, diseases associated with the loss or death of neuronal function mainly include diseases associated with the loss or death of dopamine neurons, and vision disorders associated with the loss or death of optic ganglia or photoreceptor cells. Diseases associated with the loss or death of neuronal function include, but are not limited to: Parkinson's disease, schizophrenia, depression, vision impairment caused by RGC cell death, glaucoma, age-related RGC lesions, optic nerve injury, retinal ischemia or hemorrhage, Leber hereditary optic neuropathy, degeneration or death of photoreceptor cells caused by injury or degenerative lesions, macular degeneration, retinitis pigmentosa, diabetes-related blindness, night blindness, color blindness, hereditary blindness, congenital amaurosis, deafness or hearing loss caused by spiral ganglion cell death.

[0053] Dopamine neurons

[0054] Dopaminergic neurons are neurons that contain and release dopamine (DA) as a neurotransmitter. Dopamine belongs to the catecholamine neurotransmitters and plays an important biological role in the central nervous system. Dopaminergic neurons in the brain are mainly concentrated in the substantia nigra pars compacta (SNc), ventral tegmental area (VTA), hypothalamus, and periventricular region of the midbrain. Many experiments have confirmed that dopaminergic neurons are closely related to various human diseases, and the most typical one is Parkinson's disease.

[0055] General method

[0056] Animal ethics:

[0057] The breeding and use of animals in this study were completed under the guiding principles of the Biomedical Research Ethics Committee of the Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences.

[0058] Plasmid construction:

[0059] The plasmids in this study were all constructed in our laboratory. The AAV backbone vector was digested with restriction endonucleases, and agarose gel electrophoresis was performed to recover the backbone vector. The inserted DNA fragment was amplified by PCR using cellular cDNA as a template, and the PCR fragment was recovered after agarose gel electrophoresis. The ClonExpress MultiS One Step Cloning Kit (Vazyme, C113-02) from Novoprotein Scientific Inc. was used to ligate the backbone vector and the fragment. After ligation, the product was transformed into DH5α Escherichia coli and plated. Monoclonal colonies were picked the next day for identification, and positive clones were sequenced. Clones with completely correct sequencing were expanded and plasmid extraction was performed.

[0060] AAV injection into the mouse brain:

[0061] The AAV serotype used in this study was AAV8. A stereotaxic injection was performed using the RWD stereotaxic injection system (C57BL / 6 or Dat-Cre:Ai9 mice, aged over two months). The titers of AAV-GFAP-RZFD-V1, AAV-GFAP-RZFD-V2, and AAV-GFAP-RZFD-V3 were greater than 5×10 12vg / ml (1 - 3 μl per injection). AAV was injected into the striatum (AP +0.8 mm, ML ±1.6 mm, and DV -2.8 mm) or the substantia nigra (AP –3.0 mm, ML ±1.25 mm, and DV -4.5 mm).

[0062] Immunofluorescent staining of mouse tissues:

[0063] Brain tissues were collected, sectioned, and subjected to immunofluorescent staining at 2 weeks after injection and 1.5 - 2 months after injection. After perfusion with normal saline and 4% PFA, the brains were removed and fixed overnight with 4% paraformaldehyde (PFA), then dehydrated in 30% sucrose for at least 12 hours until the tissues sank to the bottom of the solution. After embedding with OCT, cryosections were prepared with a thickness of 30 μm or 40 μm. Before immunofluorescent staining, the brain sections were washed three times with 0.1 M phosphate - buffered saline (PBS) for 5 - 10 minutes each time. After incubation with the primary antibody overnight at 4°C, the sections were washed 3 - 4 times with PBS for 10 - 15 minutes each time. Then, the secondary antibody diluted with antibody diluent was added for incubation at room temperature for 2 - 3 hours. After incubation, the sections were washed 3 - 4 times with PBS for 10 - 15 minutes each time. Finally, the sections were mounted with an anti - fluorescence quenching mounting medium (Life Technology) for preservation.

[0064] Antibodies:

[0065] The primary antibodies used in this study included: guinea pig anti-NeuN (1:500, ABN90, Millipore), rabbit anti-TH (1:500, AB152, Millipore), rat anti-DAT (1:100, MAB369, Millipore), rabbit anti-RBPMS (Proteintech, Cat#15187-1-AP), and mouse anti-Flag (1:2000, F3165, Sigma). The secondary antibodies used in this study were: Cy5-AffiniPure Donkey Anti-Guinea Pig IgG(H+L) (1:500, 706-175-148, Jackson ImmunoResearch), Alexa Fluora-488 AffiniPure Donkey Anti-Rabbit IgG(H+L) (1:500, 711-545-152, Jackson ImmunoResearch), Alexa Fluora-488 AffiniPure Donkey Anti-Mouse IgG(H+L) (1:500, 715-545-150, Jackson ImmunoResearch), and Cy5 AffiniPure Donkey Anti-Rabbit IgG(H+L) (1:500, 711-175-152, Jackson ImmunoResearch).

[0066] Electrophysiological recording:

[0067] Electrophysiological recordings were performed 1 - 3 months after AAV injection. First, the mice were anesthetized and perfused with pre - cooled NMDA, then the brains were placed in N - methyl - D - glucamine (NMDG) artificial cerebrospinal fluid (ACSF) filled with carbon dioxide [NMDG ACSF (mM): NMDG 92, potassium chloride 2.5, sodium dihydrogen phosphate 1.25, sodium bicarbonate 30, HEPES 20, glucose 25, thiourea 2, sodium ascorbate 5, pyruvate 3, calcium chloride 0.5, magnesium sulfate 10] at room temperature. After perfusion, the brains were removed and placed in ice - cold NMDG ACSF solution for 30 seconds. Thick brain slices with a thickness of 250 - 350 μm were cut at a speed of 0.04 - 0.05 mm / s using a vibratome. The brain slices were transferred to a petri dish filled with carbon dioxide - containing NMDG ACSF and maintained at 32 - 34 °C for ≤12 minutes. At room temperature, the slices were transferred to a new petri dish filled with carbon dioxide - containing HEPES ACSF [HEPES ACSF containing (mM): sodium chloride 92, potassium chloride 2.5, sodium dihydrogen phosphate 1.25, sodium bicarbonate 30, HEPES 20, glucose 25, thiourea 2, sodium ascorbate 5, pyruvate 3, calcium chloride 2, magnesium sulfate 2]. After 1 hour, the slices were transferred to a recording dish containing a recording buffer [recording ACSF (mM): sodium chloride 119, potassium chloride 2.5, sodium dihydrogen phosphate 1.25, sodium bicarbonate 24, glucose 12.5, calcium chloride 2, magnesium sulfate 2]. Neuronal - like cells with red fluorescence signals were recorded under a microscope (Olympus BX51WI), and the electroactivity of trans - differentiated cells was captured using Clampex 10.

[0068] 6 - OHDA PD mouse model

[0069] The mice used in this experiment were adult C57BL / 6 mice (7 - 10 weeks old). Half an hour before anesthesia, 25 mg / kg desipramine hydrochloride (D3900, Sigma - Aldrich) was injected intraperitoneally. After anesthesia, 3 μg 6 - OHDA (H116, Sigma - Aldrich) or saline was injected into the right medial forebrain bundle of the mice: anteroposterior (A / P) = - 1.2 mm, mediolateral (M / L) = - 1.1 mm, dorsoventral (D / V) = - 5 mm. One hour after the surgery, 1 ml of 4% glucose - saline solution was injected subcutaneously into the mice.

[0070] Apomorphine - induced rotation test

[0071] Mice were intraperitoneally injected with 0.5 mg / kg of apomorphine (A4393, Sigma-Aldrich) 10 minutes before the test. During the test, each mouse was placed individually in an opaque cylinder (30 cm in diameter), and its behavior was recorded from above by a camera for 20 minutes. Rotation was defined as a full-body turn with one hind paw as the center and no change in head orientation. The number of rotations on the injected side and the contralateral side of the injection were counted, and the data were quantified as the number of contralateral rotations within 20 minutes.

[0072] Cylinder test

[0073] Mice were gently placed into a glass beaker (1000 ml), and their behavior was recorded with a video camera in front of them for 10 minutes. The number of times the forelimbs touched the wall on the injected side and the contralateral side of the injection were calculated separately, and the data were quantified as the ratio of the number of ipsilateral wall touches to the total number of wall touches.

[0074] Rotarod test

[0075] Mice were first trained for 2 days and then subjected to behavioral testing on the third day. On the first day, the rotarod was set at a fixed speed of 4 revolutions per minute to train the mice 4 times for 300 seconds each time. On the second day, the mice were trained 4 times in an accelerating manner from 4 to 40 revolutions per minute. On the third day, behavioral testing was performed at a speed of 4 - 40 revolutions per minute, 4 times. The time the mice stayed on the rod before falling off was recorded as the retention period, and the average of the 3 longest retention periods was used for analysis.

[0076] NMDA model establishment:

[0077] To study whether RGCs can be regenerated in the damaged retina, an NMDA solution of 200 mM was prepared in PBS and then, through intravitreal injection, 1.5 μl of the NMDA solution was injected into the eyes of 4 - 8-week-old Ai9 mice or 5 - 6-week-old C57BL / 6 mice (for VEP and light-dark scene preference tests). Two to three weeks after NMDA injection, GFAP-GFP-Cre was co-delivered to the retina by subretinal injection together with GFAP-CasRx-REST or GFAP-CasRx. To evaluate the functional rescue of the damaged retina (VEP and light-dark box shuttle experiments), 5-week-old to 12-month-old mice (C57BL / 6) were injected with NMDA to induce retinal damage, and 2 - 3 weeks after injection, GFAP-mCherry (0.1 μl) was subretinally injected together with PBS (0.9 μl) or a mixture of GFAP-RZFD-V1 (0.9 μl) / GFAP-RZFD-V2 (0.9 μl) / GFAP-RZFD-V3 (0.9 μl).

[0078] Subretinal AAV injection

[0079] Inject high-titer (>1×10 13 vg / ml) AAV into the eye using a Hamilton syringe (32G needle) under an Olympus microscope (Olympus, Tokyo, Japan). To determine reprogramming in the intact retina, a total of 1 μl of GFAP-GFP-Cre (0.1 ul) + PBS (0.9 ul), or GFAP-GFP-Cre (0.1 μl) and GFAP-RZFD-V1 (0.9 μl) / GFAP-RZFD-V2 (0.9 μl) / GFAP-RZFD-V3 (0.9 μl) was injected subretinally (Ai9 and C57BL / 6 mice, 4 to 12 months old).

[0080] Retinal tissue collection and sectioning:

[0081] One to three months after AAV injection, the eyes, optic nerves, and brain tissues were harvested, fixed with 4% paraformaldehyde (PFA) for 2 hours (eyes and optic nerves) or 24 hours (brain), then dehydrated in 30% sucrose solution for 2 h (eyes) or 24 h (brain), and the optic nerves were directly observed after being sealed with PBS. Then, they were embedded with OCT, and the eyes and brains were sectioned with a thickness of 30 μm.

[0082] Retinal immunofluorescence staining:

[0083] Primary antibodies for immunofluorescence staining: Rabbit anti-RBPMS (1:500, 15187-1-AP, Proteintech), mouse anti-Brn3a (1:100, MAB1585, Millipore), rabbit anti-Sox9 (1:500, AB5535, Millipore), rabbit anti-Prox1 (1:500, AB5475, Millipore), rabbit anti-Pax6 (1:500, 901301, Biolegent). Secondary antibodies were: Cy TM 5 AffiniPure Donkey anti-mouse IgG (H+L) (1:500, 715-175-150, Jackson ImmunoResearch) and Cy TM 5 AffiniPure Donkey anti-rabbit IgG (H+L) (1:500, 711-175-152, Jackson ImmunoResearch). The primary antibodies were incubated overnight at 4°C, then washed 3 times with PBS for 10 minutes each time. The secondary antibodies were incubated at room temperature for 2 - 3 h, then washed 3 times with PBS for 10 minutes each time. Finally, the slides were sealed with an anti-fluorescence quencher (Life technology), and imaging was performed using an Olympus FV3000 microscope.

[0084] Retinal electrophysiological recording

[0085] Mice were placed in a dark room one day in advance for dark adaptation overnight. Under an infrared microscope, retinal dissection was performed in oxygenated (95% O2 / 5% CO2) artificial cerebrospinal fluid (ACSF) containing 126 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 2 mM CaCl2, 2 mM NaHCO3, and 10 mM glucose. On the stage of an upright microscope, the retina was placed with the RGC facing the cell recording chamber. Two-photon (λ = 1030 nm) microscopy was used to identify tdTomato-positive cells in the ganglion cell layer, and cell-attached recordings were made of them under infrared light. The recording electrode (4 - 7 MΩ) was filled with ACSF and 0.25 mM Alexa488 fluorescent dye. Recordings were made using a Multiclamp 700A amplifier and pClamp10 software suite (Molecular Devices). The signal was low-pass filtered at 1 kHz and digitized at 10 kHz. Full-field light stimulation was delivered using a white LED light source. After the recording, a current pulse was used to inject the fluorescent dye into the cells to label the recorded cells.

[0086] Mouse visual evoked potential

[0087] Mice were anesthetized by intraperitoneal injection with a mixture of fentanyl (0.05 mg / kg), midazolam (5 mg / kg), and medetomidine (0.5 mg / kg). The mouse head was fixed with a stereotaxic apparatus, and its body temperature was maintained at 37 °C using a heating pad. Craniotomies (about 1 mm in diameter) were made above both sides of the visual cortex (V1) (AP - 3.6 to - 3.9 mm, ML 2.2 mm), and the dura mater was carefully removed. Visual stimuli were emitted from a 17-inch liquid crystal display (Dell P170S, maximum brightness 69 cd / m2), which was 8 cm away from the eye at the recording end, while the side of the eye ipsilateral to the recording end was occluded from visual stimulation. We performed 100 repeated flash stimuli (full field, 100% contrast) for 2 seconds each, with an interval of 2 seconds. Recordings were made in V1 (AP - 3.6 to - 3.9 mm, ML 2.2 mm) using a multi-site silicon probe (A1×16 - 5 mm - 50 - 177, NeuroNexus Technologies), and the cortical depth reached by the electrode tip for each recording was approximately 900 μm. The reference line and ground line were both placed at least 3 mm away from the recording point. The Cerebus 32-channel system (Blackrock microsystems) was used to amplify the signals and filter the neural responses. The local field potential (LFP) signals were sampled at 2 kHz or 10 kHz using a broadband front-end filter (0.3 - 500 Hz). The LFP responses to full-screen flash stimuli were used for current source density (CSD) analysis to determine the location of cortical layer 43. To generate the CSD distribution profile, we calculated the second-order spatial derivative of the LFP using the following equation:

[0088]

[0089] where is the LFP, z is the coordinate of the recording end, Δz is the distance between adjacent recording ends, and nΔz is the differentiation grid (n = 2). Layer 4 (granular layer) was defined as the recording locations at the initial current sinks. We used the layer 4 channels showing the maximum average amplitude to analyze the visually evoked responses of each mouse.

[0090] Black and white box preference test

[0091] The box used for the black and white preference test consisted of two parts, a one-third dark box part and a two-thirds light box part (550 - 600 lumens). Mice could move freely between the two compartments, and after placing the mice, they were recorded for 10 minutes using a camera. The time the mice spent in the light and dark compartments was counted and analyzed using Ethovision XT software. After each trial, the box was wiped with 70% ethanol to avoid olfactory interference.

[0092] Production of PD model monkeys

[0093] The cynomolgus monkeys used in this study were adult male cynomolgus monkeys (7 - 10 years old). In order to obtain a stable animal model of Parkinson's disease, MPTP (1 - methyl - 4 - phenyl - 1,2,3,6 - tetrahydropyridine) was administered by intravenous injection for model establishment. After continuous injection for 1 week, the animals began to show phenotypes, and the injection dose was adjusted according to the animal phenotypes. As a result, a large number of dopamine neurons in the substantia nigra region of their brains underwent apoptosis, and the administration of the drug was stopped after continuous emergence of disease phenotypes such as tremors, bradykinesia, and impaired balance ability. The pathological phenotypes were continuously monitored, and AAV injection was performed after five weeks of stable manifestation of the pathological phenotypes.

[0094] Positron emission tomography experiment

[0095] In order to monitor the characteristics and functions of newly transformed dopamine neurons in the monkey brain, we performed PET imaging (radionuclides including 18F - FP - CIT, 18F - DTBZ, and 18F - DOPA) one month before AAV administration and 1 - 6 months after AAV administration, respectively. Before scanning, the cynomolgus monkeys were first induced and normally anesthetized, and then the animals were placed in a PET scanner for scanning, and all physiological parameters were monitored. The original data of the scans were analyzed using standard analysis methods.

[0096] Cynomolgus monkeys with NMDA - induced retinal damage

[0097] The cynomolgus monkeys were anesthetized, and then a 200 mM NMDA solution was injected into the vitreous body with a 1 ml syringe to eliminate most of the RGCs. Within 1 week after injection, an appropriate amount of Diclofenac Sodium Eye Ointment was applied to the monkeys every day to prevent infection or eye redness and swelling.

[0098] Subretinal injection in cynomolgus monkeys:

[0099] First, the modeled monkeys 2 - 3 weeks after NMDA modeling were anesthetized and dilated, and then AAV injection was performed. A 1 ml syringe was used to make a hole at the edge of the junction of the sclera and the cornea first, and then a 100 μl Hamilton flat - tip syringe was used for AAV injection. The virus mixture (100 μl) of the experimental group or the control group was slowly injected subretinally, and the needle was withdrawn and eye ointment was applied after injection. Diclofenac Sodium Eye Ointment was applied to the animals continuously for 7 days after injection to prevent eye infection or redness and swelling.

[0100] Behavioral experiments and video analysis in cynomolgus monkeys

[0101] For video analysis, behavioral videos of the monkeys were recorded before drug administration and 1 to 12 months after drug administration, and the videos were analyzed after collection. The PD symptoms of the monkeys were analyzed in the following sub-items: head movement, facial expression, voluntary movement, movement after stimulation, tremor, body posture, and gait.

[0102] Tissue Sampling and Sectioning of Cynomolgus Monkeys

[0103] For cynomolgus monkey brain tissue sectioning and staining, the monkeys were euthanized 1 - 6 months after AAV injection, then perfused with normal saline and 4% PFA, and the brain tissue was taken and placed in 4% PFA for continued fixation for 1 week. After fixation, the tissue was taken out and placed in 30% sucrose solution for dehydration for 1 - 2 weeks. Then sectioning was performed. The brain tissue was cut into appropriate sizes, embedded with OCT embedding agent, and sectioned on a cryostat, with a section thickness of 30 - 40 μm. For retina sampling, similar to taking brain tissue, the monkeys were euthanized about 1 - 6 months after AAV injection, then perfused with normal saline and 4% PFA, the eyes, optic nerves, and brains were taken out and fixed with 4% paraformaldehyde (PFA) for 1 - 2 weeks, and then placed in 30% sucrose solution for dehydration. After embedding, the eyes and brains were cut into thin slices of 30 - 40 μm.

[0104] Immunofluorescence Staining of Cynomolgus Monkey Tissues:

[0105] The staining steps of cynomolgus monkey brain tissue were similar to those of mice. After adding the primary antibody, it was incubated at 4°C for 24 hours, washed 3 - 4 times with PBS, 15 - 20 minutes each time. After washing, the secondary antibody was added for incubation, incubated at room temperature for 6 - 8 hours, washed 3 - 4 times with PBS, 15 - 20 minutes each time, and then sealed with an anti-fluorescence quenching mounting medium and stored. The staining steps of cynomolgus monkey retina tissue were similar to those of mouse retina tissue. First, it was washed 3 times with PBS, 10 - 15 minutes each time, and then stained in a wet box. First, the primary antibody was diluted with an antibody diluent and added to the sections, completely covering the sections, stained at 4°C for 12 - 24 hours, and then washed 3 - 4 times with PBS, 10 - 15 minutes each time. Then secondary antibody staining was performed, the antibody completely covered the tissue samples, incubated at room temperature for 6 - 8 hours. Then washed 3 - 4 times with PBS, 10 - 15 minutes each time, and finally sealed with an anti-fluorescence quenching agent.

[0106] Statistical Analysis:

[0107] Error bars were set by s.e.m., and statistical significance (p < 0.05) was calculated by unpaired two-tailed t-test or one-way ANOVA. All experiments were randomly assigned, and no statistical method was used to pre-determine the sample size. It was assumed that the data were normally distributed but not formally tested. Data collection and analysis were not performed under blinded experimental conditions.

[0108] Example

[0109] Example 1. Construction of REST variants

[0110] To achieve the regulation of RE1, taking advantage of the fact that REST can bind to RE1, we explored the RE1-binding domain of its endogenous zinc finger protein (ZF) to regulate RE1, which is called RZFD (REST Zinc Finger Domain). Through domain prediction and protein structure modeling, we found that the 159-412th positions of the human REST protein contain 8 zinc finger protein domains( Figure 1 A). To further regulate the expression of neuron-related proteins controlled by RE1, we fused a gene expression regulator VP64 and P65-HSF1 at the C-terminus of RZFD to construct RZFD-VP64 and RZFD-P65-HSF( Figure 1 B). VP64 and P65-HSF can recruit proteins such as transcription factors and histone acetylation to the vicinity of RE1, regulate the chromosomal structure near RE1, and enable the genes it controls to be expressed. In non-neurons (such as glial cells), the REST complex binds to RE1, and under the action of histone deacetylase and methylase in the complex, the chromatin near RE1 transforms into a dense state, and the expression of neuron-related genes is turned off( Figure 1 C). We expressed RZFD, RZFD-VP64 or RZFD-P65-HSF1 in non-neuronal cells (such as glial cells). RZFD, RZFD-VP64 or RZFD-P65-HSF1 competitively binds to RE1, preventing the binding of the REST silencer to RE1( Figure 1 D, E and F). The mechanism of RZFD is through competitive binding to RE1, while RZFD-VP64 and RZFD-P65-HSF1 can not only competitively bind to RE1, but also further change the chromosomal state near RE1, promoting the expression of neuron-related genes it regulates.

[0111] Example 2. RZFD transdifferentiates astrocytes into neurons

[0112] To achieve the transdifferentiation of glial cells into neuronal cells in mice, we constructed an AAV expression vector. To specifically label astrocytes, we used the astrocyte-specific promoter GFAP to drive the expression of mCherry, and the human RZFD was also driven by the GFAP promoter and specifically expressed in astrocytes( Figure 2A). To detect whether the time 2 weeks or more after injection can induce the transdifferentiation of glial cells into neurons or dopamine neurons, we collected samples 2 weeks or 1.5 months after injecting AAV, and performed NeuN and TH staining. Figure 2 B). GFAP-mCherry or GFAP-mCherry + GFAP-RZFD was injected into the striatum or substantia nigra of 8-week-old C57 mice. The injection of GFAP-mCherry alone served as the control group. Figure 2 C). Our previous studies have shown that no neurons are generated in the control group from 1 week to 3 months after injection. Consistent with the previous research results, we did not observe the generation of neurons 2 weeks after injecting the control group AAV. Figure 2 D). In the experimental group, we found that 2 weeks after injection, some cells had begun to deform, and a small number of cells had begun to express the neuron-specific marker NeuN. Figure 2 E). This indicates that the expression of RZFD in astrocytes can transdifferentiate astrocytes into neurons. After analyzing 1.5 months after injecting the mixed AAV virus of GFAP-mCherry + GFAP-RZFD, we found a large number of mCherry + NeuN + double-positive cells, indicating that RZFD can efficiently transdifferentiate astrocytes into neurons. At the same time, we found that some mCherry-positive cells expressed TH, indicating that RZFD can transdifferentiate astrocytes into dopamine neurons. Figure 2 F).

[0113] Example 3. RZFD-VP64 and RZFD-P65-HSF1 transdifferentiate astrocytes into dopamine neurons

[0114] To further study the regulation of the expression of RE1 neuron-related genes, we constructed AAV expression vectors of RZFD-VP64 and RZFD-P65-HSF1. Figure 3 A). Under the drive of the glial cell-specific promoter GFAP, RZFD-VP64 and RZFD-P65-HSF1 were expressed. We injected the mixed AAV of GFAP-mCherry + GFAP-RZFD-VP64 into the striatum or substantia nigra of C57 mice and collected samples for analysis 1.5 months after injection. Figure 3 B). We found that most of the red fluorescence-labeled cells in the GFAP-RZFD-VP64 injection group expressed the neuron-specific marker NeuN, and at the same time, some cells expressed the dopamine neuron-specific marker TH. Figure 3C). We performed similar AAV injections in the striatum or substantia nigra of DAT-Cre:Ai9 mice, injecting GFAP-RZFD-P65-HSF1 into the striatum or substantia nigra of the mice, and analyzing the samples 1.5 months later ( Figure 3 D). In DAT-Cre:Ai9 mice, only dopaminergic neurons in Chengdu could be labeled, and our study found that there were no cells with red fluorescence labeling in the striatum of control mice. However, red cells appeared in the striatum of DAT-Cre:Ai9 mice injected with GFAP-RZFD-P65-HSF1. After NeuN and TH staining, we found that these cells with red fluorescence signals expressed the neuron-specific marker NeuN and also expressed the dopaminergic neuron-specific marker TH. Figure 3 E). This indicates that the expression of RZFD-P65-HSF1 in glial cells can transdifferentiate astrocytes into dopaminergic neurons.

[0115] Sequence information

[0116] Amino acid sequence of human RZFD (SEQ ID NO.:1)

[0117]

[0118]

[0119] The similarity between the amino acid sequences of human and mouse RZFD is 95.49%. Coding sequence of human RZFD (SEQ ID NO.:2)

[0120]

[0121] Amino acid sequence of NLS-RZFD-V1 (SEQ ID NO.:3)

[0122]

[0123] Nucleotide sequence of NLS-RZFD-V1 (SEQ ID NO.:4)

[0124]

[0125] Amino acid sequence of NLS-RZFD-V2 (SEQ ID NO.:5)

[0126]

[0127] Nucleotide sequence of NLS-RZFD-V2 (SEQ ID NO.:6)

[0128]

[0129]

[0130] VP64 amino acid sequence (SEQ ID NO.: 7)

[0131]

[0132] VP64 nucleotide sequence (SEQ ID NO.: 8)

[0133]

[0134] NLS-RZFD-V3 amino acid sequence (SEQ ID NO.: 9)

[0135]

[0136] NLS-RZFD-V3 nucleotide sequence (SEQ ID NO.: 10)

[0137]

[0138]

[0139] P65-HSF1 amino acid sequence (SEQ ID NO.: 11)

[0140]

[0141] P65-HSF1 nucleotide sequence (SEQ ID NO.: 12)

[0142] Sequence Listing <110> Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences <120> Direct Transdifferentiation for the Treatment of Neurological Diseases <160> 12 <170> SIPOSequenceListing 1.0 <210> 1 <211> 266 <212> PRT <213> Homo sapiens <400> 1 Lys Thr Lys Pro Phe Arg Cys Lys Pro Cys Gln Tyr Glu Ala Glu Ser 1 5 10 15 Glu Glu Gln Phe Val His His Ile Arg Val His Ser Ala Lys Lys Phe 20 25 30 Phe Val Glu Glu Ser Ala Glu Lys Gln Ala Lys Ala Arg Glu Ser Gly 35 40 45 Ser Ser Thr Ala Glu Glu Gly Asp Phe Ser Lys Gly Pro Ile Arg Cys 50 55 60 Asp Arg Cys Gly Tyr Asn Thr Asn Arg Tyr Asp His Tyr Thr Ala His 65 70 75 80 Leu Lys His His Thr Arg Ala Gly Asp Asn Glu Arg Val Tyr Lys Cys 85 90 95 Ile Ile Cys Thr Tyr Thr Thr Val Ser Glu Tyr His Trp Arg Lys His 100 105 110 Leu Arg Asn His Phe Pro Arg Lys Val Tyr Thr Cys Gly Lys Cys Asn 115 120 125 Tyr Phe Ser Asp Arg Lys Asn Asn Tyr Val Gln His Val Arg Thr His 130 135 140 Thr Gly Glu Arg Pro Tyr Lys Cys Glu Leu Cys Pro Tyr Ser Ser Ser 145 150 155 160 Gln Lys Thr His Leu Thr Arg His Met Arg Thr His Ser Gly Glu Lys 165 170 175 Pro Phe Lys Cys Asp Gln Cys Ser Tyr Val Ala Ser Asn Gln His Glu 180 185 190 Val Thr Arg His Ala Arg Gln Val His Asn Gly Pro Lys Pro Leu Asn 195 200 205 Cys Pro His Cys Asp Tyr Lys Thr Ala Asp Arg Ser Asn Phe Lys Lys 210 215 220 His Val Glu Leu His Val Asn Pro Arg Gln Phe Asn Cys Pro Val Cys 225 230 235 240 Asp Tyr Ala Ala Ser Lys Lys Cys Asn Leu Gln Tyr His Phe Lys Ser 245 250 255 Lys His Pro Thr Cys Pro Asn Lys Thr Met 260 265 <210> 2 <211> 798 <212> DNA <213> Homo sapiens <400> 2 aagaccaaac cctttcgctg taagccatgc caatatgaag cagaatctga agaacagttt 60 gtgcatcaca tcagagttca cagtgctaag aaattttttg tggaagagag tgcagagaag 120 caggcaaaag ccagggaatc tggctcttcc actgcagaag agggagattt ctccaagggc 180 cccattcgct gtgaccgctg cggctacaat actaatcgat atgatcacta tacagcacac 240 ctgaaacacc acaccagagc tggggataat gagcgagtct acaagtgtat catttgcaca 300 tacacaacag tgagcgagta tcactggagg aaacatttaa gaaaccattt tccaaggaaa 360 gtatacacat gtggaaaatg caactatttt tcagacagaa aaaacaatta tgttcagcat 420 gttagaactc atacaggaga acgcccatat aaatgtgaac tttgtcctta ctcaagttct 480 cagaagactc atctaactag acatatgcgt actcattcag gtgagaagcc atttaaatgt 540 gatcagtgca gttatgtggc ctctaatcaa catgaagtaa cccgccatgc aagacaggtt 600 cacaatgggc ctaaacctct taattgccca cactgtgatt acaaaacagc agatagaagc 660 aacttcaaaa aacatgtaga gctacatgtg aacccacggc agttcaattg ccctgtatgt 720 gactatgcag cttccaagaa gtgtaatcta cagtatcact tcaaatctaa gcatcctact 780 tgtcctaata aaacaatg 798 <210> 3 <211> 277 <212> PRT <213> Artificial Sequence <400> 3 Met Pro Lys Lys Lys Arg Lys Val Gly Ser Gly Lys Thr Lys Pro Phe 1 5 10 15 Arg Cys Lys Pro Cys Gln Tyr Glu Ala Glu Ser Glu Glu Gln Phe Val 20 25 30 His His Ile Arg Val His Ser Ala Lys Lys Phe Phe Val Glu Glu Ser 35 40 45 Ala Glu Lys Gln Ala Lys Ala Arg Glu Ser Gly Ser Ser Thr Ala Glu 50 55 60 Glu Gly Asp Phe Ser Lys Gly Pro Ile Arg Cys Asp Arg Cys Gly Tyr 65 70 75 80 Asn Thr Asn Arg Tyr Asp His Tyr Thr Ala His Leu Lys His His Thr 85 90 95 Arg Ala Gly Asp Asn Glu Arg Val Tyr Lys Cys Ile Ile Cys Thr Tyr 100 105 110 Thr Thr Val Ser Glu Tyr His Trp Arg Lys His Leu Arg Asn His Phe 115 120 125 Pro Arg Lys Val Tyr Thr Cys Gly Lys Cys Asn Tyr Phe Ser Asp Arg 130 135 140 Lys Asn Asn Tyr Val Gln His Val Arg Thr His Thr Gly Glu Arg Pro 145 150 155 160 Tyr Lys Cys Glu Leu Cys Pro Tyr Ser Ser Ser Gln Lys Thr His Leu 165 170 175 Thr Arg His Met Arg Thr His Ser Gly Glu Lys Pro Phe Lys Cys Asp 180 185 190 Gln Cys Ser Tyr Val Ala Ser Asn Gln His Glu Val Thr Arg His Ala 195 200 205 Arg Gln Val His Asn Gly Pro Lys Pro Leu Asn Cys Pro His Cys Asp 210 215 220 Tyr Lys Thr Ala Asp Arg Ser Asn Phe Lys Lys His Val Glu Leu His 225 230 235 240 Val Asn Pro Arg Gln Phe Asn Cys Pro Val Cys Asp Tyr Ala Ala Ser 245 250 255 Lys Lys Cys Asn Leu Gln Tyr His Phe Lys Ser Lys His Pro Thr Cys 260 265 270 Pro Asn Lys Thr Met 275 <210> 4 <211> 831 <212> DNA <213> Artificial Sequence <400> 4 atgcctaaaa agaaaagaaa ggtgggttct ggtaagacca aaccctttcg ctgtaagcca 60 tgccaatatg aagcagaatc tgaagaacag tttgtgcatc acatcagagt tcacagtgct 120 tgccaatatg aagcagaatc tgaagaacag tttgtgcatc acatcagagt tcacagtgct 120 aagaaatttt ttgtggaaga gagtgcagag aagcaggcaa aagccaggga atctggctct 180 aagaaatttt ttgtggaaga gagtgcagag aagcaggcaa aagccaggga atctggctct 180 tccactgcag aagagggaga tttctccaag ggccccattc gctgtgaccg ctgcggctac 240 tccactgcag aagagggaga tttctccaag ggccccattc gctgtgaccg ctgcggctac 240 aatactaatc gatatgatca ctatacagca cacctgaaac accacaccag agctggggat 300 aatactaatc gatatgatca ctatacagca cacctgaaac accacaccag agctggggat 300 aatgagcgag tctacaagtg tatcatttgc acatacacaa cagtgagcga gtatcactgg 360 aatgagcgag tctacaagtg tatcatttgc acatacacaa cagtgagcga gtatcactgg 360 aggaaacatt taagaaacca ttttccaagg aaagtataca catgtggaaa atgcaactat 420 aggaaacatt taagaaacca ttttccaagg aaagtataca catgtggaaa atgcaactat 420 ttttcagaca gaaaaaacaa ttatgttcag catgttagaa ctcatacagg agaacgccca 480 ttttcagaca gaaaaaacaa ttatgttcag catgttagaa ctcatacagg agaacgccca 480 tataaatgtg aactttgtcc ttactcaagt tctcagaaga ctcatctaac tagacatatg 540 tataaatgtg aactttgtcc ttactcaagt tctcagaaga ctcatctaac tagacatatg 540 cgtactcatt caggtgagaa gccatttaaa tgtgatcagt gcagttatgt ggcctctaat 600 cgtactcatt caggtgagaa gccatttaaa tgtgatcagt gcagttatgt ggcctctaat 600 caacatgaag taacccgcca tgcaagacag gttcacaatg ggcctaaacc tcttaattgc 660 caacatgaag taacccgcca tgcaagacag gttcacaatg ggcctaaacc tcttaattgc 660 ccacactgtg attacaaaac agcagataga agcaacttca aaaaacatgt agagctacat 720 ccacactgtg attacaaaac agcagataga agcaacttca aaaaacatgt agagctacat 720 gtgaacccac ggcagttcaa ttgccctgta tgtgactatg cagcttccaa gaagtgtaat 780 gtgaacccac ggcagttcaa ttgccctgta tgtgactatg cagcttccaa gaagtgtaat 780 ctacagtatc acttcaaatc taagcatcct acttgtccta ataaaacaat g 831 <210> 5 <211> 344 <212> PRT <213> Artificial Sequence <400> 5 Met Pro Lys Lys Lys Arg Lys Val Gly Ser Gly Lys Thr Lys Pro Phe 1 5 10 15 Arg Cys Lys Pro Cys Gln Tyr Glu Ala Glu Ser Glu Glu Gln Phe Val 20 25 30 His His Ile Arg Val His Ser Ala Lys Lys Phe Phe Val Glu Glu Ser 35 40 45 Ala Glu Lys Gln Ala Lys Ala Arg Glu Ser Gly Ser Ser Thr Ala Glu 50 55 60 Glu Gly Asp Phe Ser Lys Gly Pro Ile Arg Cys Asp Arg Cys Gly Tyr 65 70 75 80 Asn Thr Asn Arg Tyr Asp His Tyr Thr Ala His Leu Lys His His Thr 85 90 95 Arg Ala Gly Asp Asn Glu Arg Val Tyr Lys Cys Ile Ile Cys Thr Tyr 100 105 110 Thr Thr Val Ser Glu Tyr His Trp Arg Lys His Leu Arg Asn His Phe 115 120 125 Pro Arg Lys Val Tyr Thr Cys Gly Lys Cys Asn Tyr Phe Ser Asp Arg 130 135 140 Lys Asn Asn Tyr Val Gln His Val Arg Thr His Thr Gly Glu Arg Pro 145 150 155 160 Tyr Lys Cys Glu Leu Cys Pro Tyr Ser Ser Ser Gln Lys Thr His Leu 165 170 175 Thr Arg His Met Arg Thr His Ser Gly Glu Lys Pro Phe Lys Cys Asp 180 185 190 Gln Cys Ser Tyr Val Ala Ser Asn Gln His Glu Val Thr Arg His Ala 195 200 205 Arg Gln Val His Asn Gly Pro Lys Pro Leu Asn Cys Pro His Cys Asp 210 215 220 Tyr Lys Thr Ala Asp Arg Ser Asn Phe Lys Lys His Val Glu Leu His 225 230 235 240 Val Asn Pro Arg Gln Phe Asn Cys Pro Val Cys Asp Tyr Ala Ala Ser 245 250 255 Lys Lys Cys Asn Leu Gln Tyr His Phe Lys Ser Lys His Pro Thr Cys 260 265 270 Pro Asn Lys Thr Met Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 275 280 285 Ser Arg Ala Asp Ala Leu Asp Asp Phe Asp Leu Asp Met Leu Gly Ser 290 295 300 Asp Ala Leu Asp Asp Phe Asp Leu Asp Met Leu Gly Ser Asp Ala Leu 305 310 315 320 Asp Asp Phe Asp Leu Asp Met Leu Gly Ser Asp Ala Leu Asp Asp Phe 325 330 335 Asp Leu Asp Met Leu Tyr Ile Asp 340 <210> 6 <211> 1032 <212> DNA <213> Artificial Sequence <400> 6 atgcctaaaa agaaaagaaa ggtgggttct ggtaagacca aaccctttcg ctgtaagcca 60 tgccaatatg aagcagaatc tgaagaacag tttgtgcatc acatcagagt tcacagtgct 120 aagaaatttt ttgtggaaga gagtgcagag aagcaggcaa aagccaggga atctggctct 180 tccactgcag aagagggaga tttctccaag ggccccattc gctgtgaccg ctgcggctac 240 aatactaatc gatatgatca ctatacagca cacctgaaac accacaccag agctggggat 300 aatgagcgag tctacaagtg tatcatttgc acatacacaa cagtgagcga gtatcactgg 360 aggaaacatt taagaaacca ttttccaagg aaagtataca catgtggaaa atgcaactat 420 ttttcagaca gaaaaaacaa ttatgttcag catgttagaa ctcatacagg agaacgccca 480 tataaatgtg aactttgtcc ttactcaagt tctcagaaga ctcatctaac tagacatatg 540 cgtactcatt caggtgagaa gccatttaaa tgtgatcagt gcagttatgt ggcctctaat 600 caacatgaag taacccgcca tgcaagacag gttcacaatg ggcctaaacc tcttaattgc 660 ccacactgtg attacaaaac agcagataga agcaacttca aaaaacatgt agagctacat 720 gtgaacccac ggcagttcaa ttgccctgta tgtgactatg cagcttccaa gaagtgtaat 780 ctacagtatc acttcaaatc taagcatcct acttgtccta ataaaacaat gggtagtggc 840 ggtggtggtt caggaggcgg cggaagccgc gccgacgcgc tggacgattt cgatctcgac 900 atgctgggtt ctgatgccct cgatgacttt gacctggata tgttgggaag cgacgcattg 960 gatgactttg atctggacat gctcggctcc gatgctctgg acgatttcga tctcgatatg 1020 ttatatatcg at 1032 <210> 7 <211> 55 <212> PRT <213> Artificial Sequence <400> 7 Arg Ala Asp Ala Leu Asp Asp Phe Asp Leu Asp Met Leu Gly Ser Asp 1 5 10 15 Ala Leu Asp Asp Phe Asp Leu Asp Met Leu Gly Ser Asp Ala Leu Asp 20 25 30 Asp Phe Asp Leu Asp Met Leu Gly Ser Asp Ala Leu Asp Asp Phe Asp 35 40 45 Leu Asp Met Leu Tyr Ile Asp 50 55 <210> 8 <211> 165 <212> DNA <213> Artificial Sequence <400> 8 cgcgccgacg cgctggacga tttcgatctc gacatgctgg gttctgatgc cctcgatgac 60 tttgacctgg atatgttggg aagcgacgca ttggatgact ttgatctgga catgctcggc 120 tccgatgctc tggacgattt cgatctcgat atgttatata tcgat 165 <210> 9 <211> 680 <212> PRT <213> Artificial Sequence <400> 9 Met Pro Lys Lys Lys Arg Lys Val Gly Ser Gly Lys Thr Lys Pro Phe 1 5 10 15 Arg Cys Lys Pro Cys Gln Tyr Glu Ala Glu Ser Glu Glu Gln Phe Val 20 25 30 His His Ile Arg Val His Ser Ala Lys Lys Phe Phe Val Glu Glu Ser 35 40 45 Ala Glu Lys Gln Ala Lys Ala Arg Glu Ser Gly Ser Ser Thr Ala Glu 50 55 60 Glu Gly Asp Phe Ser Lys Gly Pro Ile Arg Cys Asp Arg Cys Gly Tyr 65 70 75 80 Asn Thr Asn Arg Tyr Asp His Tyr Thr Ala His Leu Lys His His Thr 85 90 95 Arg Ala Gly Asp Asn Glu Arg Val Tyr Lys Cys Ile Ile Cys Thr Tyr 100 105 110 Thr Thr Val Ser Glu Tyr His Trp Arg Lys His Leu Arg Asn His Phe 115 120 125 Pro Arg Lys Val Tyr Thr Cys Gly Lys Cys Asn Tyr Phe Ser Asp Arg 130 135 140 Lys Asn Asn Tyr Val Gln His Val Arg Thr His Thr Gly Glu Arg Pro 145 150 155 160 Tyr Lys Cys Glu Leu Cys Pro Tyr Ser Ser Ser Gln Lys Thr His Leu 165 170 175 Thr Arg His Met Arg Thr His Ser Gly Glu Lys Pro Phe Lys Cys Asp 180 185 190 Gln Cys Ser Tyr Val Ala Ser Asn Gln His Glu Val Thr Arg His Ala 195 200 205 Arg Gln Val His Asn Gly Pro Lys Pro Leu Asn Cys Pro His Cys Asp 210 215 220 Tyr Lys Thr Ala Asp Arg Ser Asn Phe Lys Lys His Val Glu Leu His 225 230 235 240 Val Asn Pro Arg Gln Phe Asn Cys Pro Val Cys Asp Tyr Ala Ala Ser 245 250 255 Lys Lys Cys Asn Leu Gln Tyr His Phe Lys Ser Lys His Pro Thr Cys 260 265 270 Pro Asn Lys Thr Met Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 275 280 285 Ser Arg Ala Asp Ala Leu Asp Asp Phe Asp Leu Asp Met Leu Gly Ser 290 295 300 Asp Ala Leu Asp Asp Phe Asp Leu Asp Met Leu Gly Ser Asp Ala Leu 305 310 315 320 Asp Asp Phe Asp Leu Asp Met Leu Gly Ser Asp Ala Leu Asp Asp Phe 325 330 335 Asp Leu Asp Met Leu Tyr Ile Asp Asp Tyr Lys Leu Gly Ser Gly Gly 340 345 350 Gly Gly Ser Gly Gly Gly Gly Gly Ser Pro Ser Gly Gln Ile Ser Asn Gln 355 360 365 Ala Leu Ala Leu Ala Pro Ser Ser Ala Pro Val Leu Ala Gln Thr Met 370 375 380 Val Pro Ser Ser Ala Met Val Pro Leu Ala Gln Pro Pro Ala Pro Ala 385 390 395 400 Pro Val Leu Thr Pro Gly Pro Pro Gln Ser Leu Ser Ala Pro Val Pro 405 410 415 Lys Ser Thr Gln Ala Gly Glu Gly Thr Leu Ser Glu Ala Leu Leu His 420 425 430 Leu Gln Phe Asp Ala Asp Glu Asp Leu Gly Ala Leu Leu Gly Asn Ser 435 440 445 Thr Asp Pro Gly Val Phe Thr Asp Leu Ala Ser Val Asp Asn Ser Glu 450 455 460 Phe Gln Gln Leu Leu Asn Gln Gly Val Ser Met Ser His Ser Thr Ala 465 470 475 480 Glu Pro Met Leu Met Glu Tyr Pro Glu Ala Ile Thr Arg Leu Val Thr 485 490 495 Gly Ser Gln Arg Pro Pro Asp Pro Ala Pro Thr Pro Leu Gly Thr Ser 500 505 510 Gly Leu Pro Asn Gly Leu Ser Gly Asp Glu Asp Phe Ser Ser Ile Ala 515 520 525 Asp Met Asp Phe Ser Ala Leu Leu Ser Gln Ile Ser Ser Ser Gly Gln 530 535 540 Gly Gly Gly Gly Ser Gly Phe Ser Val Asp Thr Ser Ala Leu Leu Asp 545 550 555 560 Leu Phe Ser Pro Ser Val Thr Val Pro Asp Met Ser Leu Pro Asp Leu 565 570 575 Asp Ser Ser Leu Ala Ser Ile Gln Glu Leu Leu Ser Pro Gln Glu Pro 580 585 590 Pro Arg Pro Pro Glu Ala Glu Asn Ser Ser Pro Asp Ser Gly Lys Gln 595 600 605 Leu Val His Tyr Thr Ala Gln Pro Leu Phe Leu Leu Asp Pro Gly Ser 610 615 620 Val Asp Thr Gly Ser Asn Asp Leu Pro Val Leu Phe Glu Leu Gly Glu 625 630 635 640 Gly Ser Tyr Phe Ser Glu Gly Asp Gly Phe Ala Glu Asp Pro Thr Ile 645 650 655 Ser Leu Leu Thr Gly Ser Glu Pro Pro Lys Ala Lys Asp Pro Thr Val 660 665 670 Ser Pro Lys Lys Lys Arg Lys Val 675 680 <210> 10 <211> 2040 <212> DNA <213> Artificial Sequence <400> 10 atgcctaaaa agaaaagaaa ggtgggttct ggtaagacca aaccctttcg ctgtaagcca 60 tgccaatatg aagcagaatc tgaagaacag tttgtgcatc acatcagagt tcacagtgct 120 aagaaatttt ttgtggaaga gagtgcagag aagcaggcaa aagccaggga atctggctct 180 tccactgcag aagagggaga tttctccaag ggccccattc gctgtgaccg ctgcggctac 240 aatactaatc gatatgatca ctatacagca cacctgaaac accacaccag agctggggat 300 aatgagcgag tctacaagtg tatcatttgc acatacacaa cagtgagcga gtatcactgg 360 aggaaacatt taagaaacca ttttccaagg aaagtataca catgtggaaa atgcaactat 420 ttttcagaca gaaaaaacaa ttatgttcag catgttagaa ctcatacagg agaacgccca 480 tataaatgtg aactttgtcc ttactcaagt tctcagaaga ctcatctaac tagacatatg 540 cgtactcatt caggtgagaa gccatttaaa tgtgatcagt gcagttatgt ggcctctaat 600 caacatgaag taacccgcca tgcaagacag gttcacaatg ggcctaaacc tcttaattgc 660 ccacactgtg attacaaaac agcagataga agcaacttca aaaaacatgt agagctacat 720 gtgaacccac ggcagttcaa ttgccctgta tgtgactatg cagcttccaa gaagtgtaat 780 ctacagtatc acttcaaatc taagcatcct acttgtccta ataaaacaat gggtagtggc 840 ggtggtggtt caggaggcgg cggaagccgc gccgacgcgc tggacgattt cgatctcgac 900 atgctgggtt ctgatgccct cgatgacttt gacctggata tgttgggaag cgacgcattg 960 gatgactttg atctggacat gctcggctcc gatgctctgg acgatttcga tctcgatatg 1020 ttatatatcg atgattacaa gcttggtagt ggcggtggtg gttcaggagg cggcggaagc 1080 ccttcagggc agatcagcaa ccaggccctg gctctggccc ctagctccgc tccagtgctg 1140 gcccagacta tggtgccctc tagtgctatg gtgcctctgg cccagccacc tgctccagcc 1200 cctgtgctga ccccaggacc accccagtca ctgagcgctc cagtgcccaa gtctacacag 1260 gccggcgagg ggactctgag tgaagctctg ctgcacctgc agttcgacgc tgatgaggac 1320 ctgggagctc tgctggggaa cagcaccgat cccggagtgt tcacagatct ggcctccgtg 1380 gacaactctg agtttcagca gctgctgaat cagggcgtgt ccatgtctca tagtacagcc 1440 gaaccaatgc tgatggagta ccccgaagcc attacccggc tggtgaccgg cagccagcgg 1500 ccccccgacc ccgctccaac tcccctggga accagcggcc tgcctaatgg gctgtccgga 1560 gatgaagact tctcaagcat cgctgatatg gactttagtg ccctgctgtc acagatttcc 1620 tctagtgggc agggaggagg tggaagcggc ttcagcgtgg acaccagtgc cctgctggac 1680 ctgttcagcc cctcggtgac cgtgcccgac atgagcctgc ctgaccttga cagcagcctg 1740 gccagtatcc aagagctcct gtctccccag gagcccccca ggcctcccga ggcagagaac 1800 agcagcccgg attcagggaa gcagctggtg cactacacag cgcagccgct gttcctgctg 1860 gaccccggct ccgtggacac cgggagcaac gacctgccgg tgctgtttga gctgggagag 1920 ggctcctact tctccgaagg ggacggcttc gccgaggacc ccaccatctc cctgctgaca 1980 ggctcggagc ctcccaaagc caaggacccc actgtctccc ccaagaagaa gcgcaaggtg 2040 <210> 11 <211> 313 <212> PRT <213> Artificial Sequence <400> 11 Pro Ser Gly Gln Ile Ser Asn Gln Ala Leu Ala Leu Ala Pro Ser Ser 1 5 10 15 Ala Pro Val Leu Ala Gln Thr Met Val Pro Ser Ser Ala Met Val Pro 20 25 30 Leu Ala Gln Pro Pro Ala Pro Ala Pro Val Leu Thr Pro Gly Pro Pro 35 40 45 Gln Ser Leu Ser Ala Pro Val Pro Lys Ser Thr Gln Ala Gly Glu Gly 50 55 60 Thr Leu Ser Glu Ala Leu Leu His Leu Gln Phe Asp Ala Asp Glu Asp 65 70 75 80 Leu Gly Ala Leu Leu Gly Asn Ser Thr Asp Pro Gly Val Phe Thr Asp 85 90 95 Leu Ala Ser Val Asp Asn Ser Glu Phe Gln Gln Leu Leu Asn Gln Gly 100 105 110 Val Ser Met Ser His Ser Thr Ala Glu Pro Met Leu Met Glu Tyr Pro 115 120 125 Glu Ala Ile Thr Arg Leu Val Thr Gly Ser Gln Arg Pro Pro Asp Pro 130 135 140 Ala Pro Thr Pro Leu Gly Thr Ser Gly Leu Pro Asn Gly Leu Ser Gly 145 150 155 160 Asp Glu Asp Phe Ser Ser Ile Ala Asp Met Asp Phe Ser Ala Leu Leu 165 170 175 Ser Gln Ile Ser Ser Ser Gly Gln Gly Gly Gly Gly Ser Gly Phe Ser 180 185 190 Val Asp Thr Ser Ala Leu Leu Asp Leu Phe Ser Pro Ser Val Thr Val 195 200 205 Pro Asp Met Ser Leu Pro Asp Leu Asp Ser Ser Leu Ala Ser Ile Gln 210 215 220 Glu Leu Leu Ser Pro Gln Glu Pro Pro Arg Pro Pro Glu Ala Glu Asn 225 230 235 240 Ser Ser Pro Asp Ser Gly Lys Gln Leu Val His Tyr Thr Ala Gln Pro 245 250 255 Leu Phe Leu Leu Asp Pro Gly Ser Val Asp Thr Gly Ser Asn Asp Leu 260 265 270 Pro Val Leu Phe Glu Leu Gly Glu Gly Ser Tyr Phe Ser Glu Gly Asp 275 280 285 Gly Phe Ala Glu Asp Pro Thr Ile Ser Leu Leu Thr Gly Ser Glu Pro 290 295 300 Pro Lys Ala Lys Asp Pro Thr Val Ser 305 310 <210> 12 <211> 939 <212> DNA <213> Artificial Sequence <400> 12 ccttcagggc agatcagcaa ccaggccctg gctctggccc ctagctccgc tccagtgctg 60 gcccagacta tggtgccctc tagtgctatg gtgcctctgg cccagccacc tgctccagcc 120 cctgtgctga ccccaggacc accccagtca ctgagcgctc cagtgcccaa gtctacacag 180 gccggcgagg ggactctgag tgaagctctg ctgcacctgc agttcgacgc tgatgaggac 240 ctgggagctc tgctggggaa cagcaccgat cccggagtgt tcacagatct ggcctccgtg 300 gacaactctg agtttcagca gctgctgaat cagggcgtgt ccatgtctca tagtacagcc 360 gaaccaatgc tgatggagta ccccgaagcc attacccggc tggtgaccgg cagccagcgg 420 ccccccgacc ccgctccaac tcccctggga accagcggcc tgcctaatgg gctgtccgga 480 gatgaagact tctcaagcat cgctgatatg gactttagtg ccctgctgtc acagatttcc 540 tctagtgggc agggaggagg tggaagcggc ttcagcgtgg acaccagtgc cctgctggac 600 ctgttcagcc cctcggtgac cgtgcccgac atgagcctgc ctgaccttga cagcagcctg 660 gccagtatcc aagagctcct gtctccccag gagcccccca ggcctcccga ggcagagaac 720 agcagcccgg attcagggaa gcagctggtg cactacacag cgcagccgct gttcctgctg 780 gaccccggct ccgtggacac cgggagcaac gacctgccgg tgctgtttga gctgggagag 840 ggctcctact tctccgaagg ggacggcttc gccgaggacc ccaccatctc cctgctgaca 900 ggctcggagc ctcccaaagc caaggacccc actgtctcc 939

Claims

1. RE1 / NRSE element blocker, characterized in that, The RE1 / NRSE element blocker is a REST variant or its encoding nucleotide, and the amino acid sequence of the REST variant is as shown in SEQ ID NO: 1, 3, 5 or 9, and the nucleotide sequence of the REST variant is as shown in SEQ ID NO: 2, 4, 6 or 10.

2. A pharmaceutical composition comprising the blocker according to claim 1.

3. A medicine box comprising the blocker according to claim 1.

4. A kit comprising the blocker according to claim 1.

5. The pharmaceutical composition according to claim 2, formulated for injection or oral administration.

6. The pharmaceutical composition according to claim 2, formulated for intracranial administration, intraocular administration, intra-aural administration, intravenous administration, intramuscular administration or intradermal administration.

7. The pharmaceutical composition according to claim 2, formulated for inhalation.

8. The pharmaceutical composition according to claim 2, or the medicine box according to claim 3, or the kit according to claim 4, further comprising a carrier for delivering the RE1 / NRSE element blocker, wherein the carrier is a viral vector, liposome, nanoparticle, exosome, and the viral vector includes a recombinant adeno-associated virus vector rAAV, an adeno-associated virus AAV vector, an adenovirus vector, a lentivirus vector, a retrovirus vector, a poxvirus vector, a herpes virus, an SV40 virus vector, and combinations thereof.

9. The pharmaceutical composition, medicine box or kit according to claim 8, wherein the carrier is AAV and rAAV.

10. The pharmaceutical composition according to claim 2, or the medicine box according to claim 3, or the kit according to claim 4, comprising an expression vector for expressing the REST variant, wherein the expression vector comprises a nucleotide sequence encoding the REST variant, which is operably linked to a promoter that causes its expression.

11. The pharmaceutical composition according to claim 2, for topical administration to at least one of the following: i) glial cells in the striatum; ii) glial cells in the substantia nigra of the brain; iii) glial cells in the retina; iv) glial cells in the inner ear; v) Glial cells in the spinal cord; vi) Glial cells in the prefrontal cortex; vii) Glial cells in the motor cortex; viii) Glial cells in the hypothalamus; and ix) Glial cells in the ventral tegmental area.

12. The pharmaceutical composition according to claim 2, or the medicine box according to claim 3, or the kit according to claim 4, further comprising i) one or more dopamine neuron-related factors, or ii) for expressing one or more retinal ganglion cell-related factors in Müller glial cells, 1) One or more of the dopamine neuron-related factors are selected from: FoxA2, Lmx1a, Lmx1b, Nurr1, Pbx1a, Pitx3, Gata2, Gata3, FGF8, BMP, En1, En2, PET1, Pax family proteins, SHH, Wnt family proteins, and TGF-β family proteins, or a combination thereof; 2) One or more of the retinal ganglion cell-related factors are selected from: β-catenin, Oct4, Sox2, Klf4, Crx, aCamKII, Brn3a, Brn3b, Brn3C, Math5, Otx2, Ngn2, Ngn1, AscL1, miRNA9, miRNA-124, Nr2e3, and Nrl factors.

13. The pharmaceutical composition or cartridge or kit according to claim 12, wherein the Pax family protein is Pax3 or Pax6.

14. The pharmaceutical composition or cartridge or kit according to claim 10, wherein the promoter is a glial cell-specific promoter, and the glial cell-specific promoter is selected from the GFAP promoter, ALDH1L1 promoter, EAAT1 / GLAST promoter, glutamine synthetase promoter, S100β promoter, EAAT2 / GLT-1 promoter, and Rlbp1 promoter.

15. The pharmaceutical composition or cartridge or kit according to claim 10, wherein the promoter is a Müller glial cell-specific promoter.

16. The pharmaceutical composition or cartridge or kit according to claim 14, wherein the promoter is the GFAP promoter.

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