WO2023104028A1 - 非编码rna介导的神经性疾病治疗 - Google Patents

非编码rna介导的神经性疾病治疗 Download PDF

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WO2023104028A1
WO2023104028A1 PCT/CN2022/136909 CN2022136909W WO2023104028A1 WO 2023104028 A1 WO2023104028 A1 WO 2023104028A1 CN 2022136909 W CN2022136909 W CN 2022136909W WO 2023104028 A1 WO2023104028 A1 WO 2023104028A1
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lncrna
mirna
cells
expression
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周海波
胡新德
苏锦霖
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上海鲸奇生物科技有限公司
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Definitions

  • the present disclosure relates to the field of biomedicine. More specifically, the present disclosure relates to the use of agents that increase or decrease the expression or activity of certain non-coding RNAs, such as certain miRNAs and lncRNAs, for the treatment of diseases associated with loss of neuronal function or death.
  • certain non-coding RNAs such as certain miRNAs and lncRNAs
  • Cell transdifferentiation refers to the process in which one type of differentiated cell transforms into another differentiated cell in structure and function through gene selective expression or gene reprogramming.
  • MicroRNA is a kind of highly conserved non-coding functional RNA molecule existing in animal or plant cells, and its length is about 17-27 nucleotides. miRNA inhibits the function of the target gene by binding to the 3'UTR region of the target mRNA sequence, resulting in translational repression or RNA degradation. When a miRNA is perfectly complementary to a target RNA it leads to the degradation of the mRNA, while when it is not fully complementary to the target RNA it prevents gene translation. In mammalian cells, miRNAs mainly prevent mRNA translation rather than affect its stability.
  • LncRNA Long non-coding RNA
  • RNA molecules with a length of more than 200 bases, which does not participate in protein coding, but can participate in the regulation of gene expression in the form of RNA.
  • LncRNA was regarded as the "noise" of gene transcription, a by-product of RNA transcription, and had no biological function.
  • studies in recent years have shown that lncRNAs are widely involved in a variety of biological processes, such as gene transcriptional regulation, genomic imprinting, chromatin silencing, chromatin modification, gene expression activation or inhibition, and many other biological processes.
  • LncRNAs participate in the regulation of gene expression from multiple levels such as epigenetics, gene transcription regulation, and post-transcriptional regulation, and some LncRNAs also participate in the regulation of miRNAs, thereby indirectly regulating gene expression.
  • astrocytes are a very abundant type of glial cells that exist around neurons and support and provide nutrition to neurons.
  • neuronal cells will die, resulting in some functional deficits, such as stroke, Alzheimer's disease and Parkinson's disease.
  • scientists can transdifferentiate glial cells into neurons by adding a variety of factors to astrocytes cultured in vitro, such as ASCL1, NeuroD1 and Ngn2.
  • ASCL1, NeuroD1 and Ngn2 factors that in a variety of factors to astrocytes cultured in vitro
  • ASCL1 a variety of factors to astrocytes cultured in vitro
  • it is difficult to reproduce the results of many studies in vitro in culture dishes. Therefore, it is of great scientific significance to study how to transdifferentiate astrocytes in situ in vivo to replenish lost neurons.
  • Parkinson's disease is a disorder associated with loss of neuronal function or death and is characterized by the loss of dopamine neurons in the substantia nigra of the midbrain.
  • the main treatment for Parkinson's disease is the medicine represented by levodopa preparation.
  • surgical treatment can also improve symptoms to a certain extent. It should be pointed out that all these means can only partially alleviate the disease, and cannot achieve the effect of preventing the disease from developing.
  • the present disclosure provides a method for producing neuronal cells from acellular cells, comprising: , miR-92b, miR-96, miR-106, miR-125a/b, miR-128, miR-134, miR-135, miR-137, miR-141, miR-143-3p, miR-184, miR -200, miR-218, miR-219, miR-228, miR-284, miR-429, miR-430 miRNA, or selected from utNgn1, RMST, Tuna, Linc-Brn1b, Dali, Miat/Gomafu, NBAT- 1.
  • lncRNA of Malat1, Dlx1as, Six3os, Evf2, LncKdm2b, lncRNA_N1, lncRNA_N2, lncRNA_N3, or any combination thereof is enhanced to make the non-neuronal cells transdifferentiate or reprogram into neuron cells.
  • the expression of said miRNA or lncRNA is enhanced or active.
  • the present disclosure provides a method for producing neuronal cells from non-neuronal cells comprising making a protein selected from the group consisting of miR-7a, miR-15, miR-23a/b, miR-25, miR- 29a, miR-129, miR-137, miR-138, miR-155, miR-195, miR-214, miR-222, miR-223, miR-132, miR-133 miRNA, or selected from Pnky, Paupar , HOTAIRM1, lncR492, TUG1 lncRNA, or the expression or activity reduction of any combination thereof to make the non-neuronal cells transdifferentiate or reprogram into neuronal cells.
  • a protein selected from the group consisting of miR-7a, miR-15, miR-23a/b, miR-25, miR- 29a, miR-129, miR-137, miR-138, miR-155, miR-195, miR-214, miR-222, miR-223, mi
  • DNA editing and RNA editing mediated by, for example, gene editing technology, RNA expression inhibitors, antisense oligonucleotides (ASO), small RNA interference, miRNA technology, small molecule compounds, gene suppression technology (dCas-Krab, etc.), and/or epigenetic regulation to reduce the expression or activity of the miRNA or lncRNA.
  • gene editing technology RNA expression inhibitors, antisense oligonucleotides (ASO), small RNA interference, miRNA technology, small molecule compounds, gene suppression technology (dCas-Krab, etc.), and/or epigenetic regulation to reduce the expression or activity of the miRNA or lncRNA.
  • RNA editing includes CRISPR-mediated RNA degradation and translation inhibition, RNA single base editing, insertion or deletion of bases on RNA, changes in RNA splicing, and RNA epigenetic modification.
  • the non-neuronal cells include, for example, glial cells, fibroblasts, stem cells, neural precursor cells, neural stem cells, wherein the glial cells are selected from the group consisting of astrocytes, microglia , oligodendrocytes, ependymal cells, Schwann cells, NG2 cells, satellite cells or combinations thereof, preferably astrocytes.
  • the glial cells are derived from the brain, spinal cord, eyes or ears, wherein the glial cells in the brain are derived from the striatum, substantia nigra, ventral tegmental area of the midbrain, spinal cord, and hypothalamus , dorsal midbrain or cerebral cortex, preferably striatum and substantia nigra.
  • the neuron cells are preferably dopamine neurons, GABA neurons, 5-HT neurons, glutamatergic neurons, ChAT neurons, NE neurons, motor neurons, spinal cord neurons , spinal motor neurons, spinal sensory neurons, pyramidal neurons, interneurons, medium spiny neurons (MSNs), Purkinje cells, granule cells, olfactory sensory neurons, periglobal cells, or combinations thereof, more Dopamine neurons are preferred.
  • said non-neuronal and/or neuronal cells are from eg humans, non-human primates, rats and mice, preferably humans.
  • the method is an in vivo method or an in vitro method.
  • the disclosure provides an agent selected from Let-7a, Let-7b, miR-18a/b, miR-24-3p, miR-34a, miR-92b, miR-96, miR-106 , miR-125a/b, miR-128, miR-134, miR-135, miR-137, miR-141, miR-143-3p, miR-184, miR-200, miR-218, miR-219, miR -228, miR-284, miR-429, miR-430 miRNA, or miRNA selected from utNgn1, RMST, Tuna, Linc-Brn1b, Dali, Miat/Gomafu, NBAT-1, Malat1, Dlx1as, Six3os, Evf2, LncKdm2b, The lncRNA of lncRNA_N1, lncRNA_N2, lncRNA_N3, or an agent for enhancing the expression or activity of any combination thereof is used in the
  • the disclosure provides an agent selected from the group consisting of miR-7a, miR-15, miR-23a/b, miR-25, miR-29a, miR-129, miR-137, miR-138, miR -155, miR-195, miR-214, miR-222, miR-223, miR-132, miR-133 miRNA, or lncRNA selected from Pnky, Paupar, HOTAIRM1, lncR492, TUG1, or any combination thereof or activity-reducing reagents for the preparation of medicines for the prevention and/or treatment of neuron function loss or death-related diseases, wherein the reagents reduce the expression or activity of the miRNA or the lncRNA.
  • the medicament is formulated for in vivo administration to the nervous system, e.g. striatum, substantia nigra, ventral tegmental area of midbrain, spinal cord, hypothalamus, dorsal midbrain, cerebral cortex , hippocampus, cerebellum, inner ear cochlea and vestibule, preferably striatum, substantia nigra, subretina, vitreous cavity and inner ear cochlea.
  • the nervous system e.g. striatum, substantia nigra, ventral tegmental area of midbrain, spinal cord, hypothalamus, dorsal midbrain, cerebral cortex , hippocampus, cerebellum, inner ear cochlea and vestibule, preferably striatum, substantia nigra, subretina, vitreous cavity and inner ear cochlea.
  • the diseases associated with neuronal function loss or death are selected from the group consisting of: Parkinson's disease, Alzheimer's disease, stroke (stroke), schizophrenia, Huntington's disease, depression, Motor neuron disease, cerebral ischemia, brain injury, amyotrophic lateral sclerosis (ALS), spinal muscular atrophy, Pick's disease, sleep disorders, epilepsy, ataxia, PloyQ disease, addiction, or combinations thereof, Parkinson's disease is preferred.
  • said enhancing the expression or activity of said miRNA or lncRNA or a combination thereof comprises:
  • the miRNA or lncRNA or a combination thereof exogenously, for example, through an expression vector comprising a promoter.
  • the expression or activity of said miRNA or lncRNA or a combination thereof is reduced by: antibody, small molecule compound, microRNA, siRNA, shRNA, antisense oligonucleotide, binding protein or protein domain , polypeptides, nucleic acid aptamers, gene editors, epigenetic regulatory elements, transcriptional repression elements, or combinations thereof.
  • the present disclosure provides a pharmaceutical composition or kit or kit comprising an agent selected from Let-7a, Let-7b, miR-18a/b, miR-24-3p, miR-34a , miR-92b, miR-96, miR-106, miR-125a/b, miR-128, miR-134, miR-135, miR-137, miR-141, miR-143-3p, miR-184, miR -200, miR-218, miR-219, miR-228, miR-284, miR-429, miR-430 miRNA, or selected from utNgn1, RMST, Tuna, Linc-Brn1b, Dali, Miat/Gomafu, NBAT- 1.
  • the agent for enhancing the expression or activity of the miRNA or lncRNA or its combination is selected from: an expression vector, the miRNA or lncRNA in the form of DNA or RNA or its combination, the miRNA or lncRNA or Endogenous activators thereof, analogs or agonists of said miRNA or lncRNA or combinations thereof.
  • the expression vector is a gene therapy vector, preferably a viral gene therapy vector, more preferably a viral vector selected from the following: adeno-associated virus (AAV) vector, recombinant adeno-associated virus vector (rAAV), auto Complementary AAV (scAAV), adenoviral vectors, lentiviral vectors, retroviral vectors, herpesviruses, SV40 vectors, poxvirus vectors, and combinations thereof, among which AAV and rAAV are preferred.
  • AAV adeno-associated virus
  • rAAV recombinant adeno-associated virus vector
  • scAAV auto Complementary AAV
  • adenoviral vectors lentiviral vectors
  • retroviral vectors lentiviral vectors
  • herpesviruses herpesviruses
  • SV40 vectors herpesviruses
  • poxvirus vectors poxvirus vectors
  • the agent that reduces the expression or activity of the miRNA or lncRNA or a combination thereof is selected from the group consisting of antibodies, small molecule compounds, microRNA, siRNA, shRNA, antisense oligonucleotides, binding proteins or proteins Domains, polypeptides, aptamers, gene editors, epigenetic regulatory elements, transcriptional repressor elements, or combinations thereof.
  • said pharmaceutical composition or kit or kit further comprises a carrier or vehicle for delivery of said agent.
  • the carrier or vehicle is a viral vector, liposome, nanoparticle, exosome, virus-like particle, preferably AAV.
  • the composition is administered topically to at least one of: i) glial cells in the striatum; ii) glial cells in the ventral tegmental area (VTA); iii) melanocytic cells. iv) glial cells in the hypothalamus; v) glial cells in the spinal cord; vi) glial cells in the prefrontal cortex; and vii) glial cells in the motor cortex.
  • the pharmaceutical composition or kit or kit is formulated for cell transfection, cell infection, endocytosis, injection, intracranial administration, inhalation, parenteral administration, intravenous administration , intramuscular, intradermal, topical or oral administration.
  • the non-neuronal cells are glial cells
  • the neuronal cells are dopamine neurons.
  • the non-neuronal cells have a transdifferentiation efficiency of at least 1%, or at least 10%, 20%, 30%, 40%, or 50%.
  • FIG. 1 Knockdown of Pnky-mediated transdifferentiation of glial cells into neurons.
  • FIG. 1 Schematic diagram of AAV vector design
  • Vector 1 is a vector diagram of GFAP driving mCherry expression
  • GFAP is a promoter specifically expressed in glial cells
  • mCherry is a red fluorescent protein, which is used to label glial cells.
  • Vector 2 is a schematic diagram of the GFAP-CasRx expression vector, and the expression of CasRx is driven by the astrocyte-specific promoter GFAP.
  • Vector3 is a schematic diagram of the U6-gRNA-GFAP-CasRx expression vector. The expression of gRNA is promoted by the U6 promoter, and CasRx is promoted by the GFAP promoter.
  • C Schematic diagram of AAV injection and transdifferentiation. Different AAVs were injected into the mouse brain to study the transdifferentiation of glial cells into neurons: the control group was injected with a mixed virus of GFAP-mCherry and GFAP-CasRx, and the experimental group was injected with GFAP-mCherry Mixed AAV with GFAP-CasRx-gRNA (Pnky), GFAP-mCherry labels astrocytes.
  • FIG. 1 MiRNA or LncRNA-mediated transdifferentiation of glial cells into neurons.
  • A Schematic diagram of the design of GFAP-mCherry and GFAP-miRNA/lncRNA expression vectors. Expression of mCherry, miRNA or LncRNA driven by the glial cell-specific promoter GFAP.
  • B Schematic diagram of AAV expression vectors for Tuna, Let-7b, and miR-137, where Tuna is LncRNA, and Let-7b and miRNA-137 are miRNAs, both of which are expressed by the GFAP promoter.
  • C Schematic diagram of AAV injection and miRNA/LncRNA transdifferentiation of glial cells into neurons.
  • E-G Representative graphs of the results of 1 month after injection of GFAP-Tuna, GFAP-Let-7b or miR-137 in the striatum of mice, the red fluorescent signal (mCherry) is the neurons labeled by GFAP-mCherry, The white signal is the neuron-specific marker NeuN, the yellow arrow indicates the cells co-labeled with mCherry and NeuN, and the scale bar is 50 ⁇ m.
  • mCherry is the neurons labeled by GFAP-mCherry
  • the white signal is the neuron-specific marker NeuN
  • the yellow arrow indicates the cells co-labeled with mCherry and NeuN
  • the scale bar is 50 ⁇ m.
  • FIG. 3 Overexpression of miRNAs induces transdifferentiation of astrocytes into neurons.
  • A Schematic diagram of AAV vector, the vector 1 is GFAP-EGFP-labeled AAV, which can specifically label astrocytes, and the vector 2 is GFAP-activated miRNA or LncRNA, which can specifically express miRNA or LncRNA in glial cells.
  • Control group injected with AAV-GFAP-EGFP, EGFP specifically labeled astrocytes C-G
  • C-G Injected corresponding AAV (GFAP-miR-18b, GFAP-miR-34a, GFAP-miR-34a, GFAP- miR-128, GFAP-miR-134, GFAP-miR-143) after transdifferentiation of astrocytes into neurons.
  • the green fluorescent signal is AAV-GFAP-EGFP labeled cells
  • NeuN is a neuron-specific protein marker.
  • White arrows point to cells co-labeled with green and white fluorescence, showing that these cells express the neuron-specific protein marker NeuN, and the bar is 50 ⁇ m.
  • FIG. 4 Overexpression of miRNAs induces transdifferentiation of astrocytes into dopamine neurons.
  • A One month after injection of AAV-GFAP-EGFP in the striatum of mice, the samples were analyzed.
  • B-F Overexpression of miRNAs (Let-7a, miR-92b, miR-96, miR-24, miR-106, -miR-125a) etc. Glial cells transdifferentiated into neurons, and some glial cells transdifferentiated into dopamine neurons.
  • the white arrow points to the green cells labeled with GFAP-EGFP, the white signal is the neuron-specific marker NeuN, and the red signal is the dopamine-specific protein marker TH.
  • the superimposed figure shows that there is partial overlap between different signals.
  • White arrows point to cells where the green signal is co-labeled with NeuN, yellow arrows point to TH-positive cells, and the bar is 50 ⁇ m.
  • FIG. 5 Overexpression of miRNAs transdifferentiates astrocytes into dopamine neurons.
  • A-F Overexpression of miR-135, miR-24, miR-141, miR-200, miR-218, miR-429 and other miRNAs in mouse striatal astrocytes transfected astrocytes into Differentiate into neurons, some of which transdifferentiate into dopamine neurons.
  • the white arrow points to the green cells labeled with GFAP-EGFP, the white signal is the neuron-specific marker NeuN, the red signal is the dopamine-specific protein marker TH, and the superimposed graph shows the overlap between different signals.
  • White arrows point to cells where the green signal is co-labeled with NeuN, yellow arrows point to TH-positive cells, and the bar is 50 ⁇ m.
  • FIG. 6 Overexpression of miRNAs transdifferentiates astrocytes into neurons and proportion of dopaminergic neurons.
  • A Overexpression of miR-18b, miR-24, miR-34a, miR-128, miR-134, miR-143, Let-7a, miR-92b, miR in mouse striatal astrocytes -96, miR-106, miR-125a, miR-135, miR-141, miR-200, miR-218, miR-429 and other miRNAs, the efficiency statistics of transdifferentiation of astrocytes into neurons.
  • miRNA and LncRNA are widely expressed in the nervous system and have important functions, and play an important role in the development of the nervous system and the occurrence and development of many neurological diseases.
  • miRNAs are widely expressed in a variety of nerve cells, and different types of miRNAs are expressed in different nerve cells. This suggests that different miRNAs may be involved in mediating different neuronal development.
  • miRNA-124 can promote the differentiation of neural precursor cells into neurons during the development of the nervous system, and miRNA-124 can inhibit the differentiation of neural precursor cells into glial cells by regulating the expression of Ptbp1 and other genes. differentiation.
  • Müller glia Müller glia, MG
  • overexpression of miRNA-9 and miRNA-124 can transdifferentiate Müller glia cells into retinal neurons
  • miRNA-9 and miRNA -124 can promote AscL1-mediated transdifferentiation of Müller glial cells into retinal neurons.
  • LncRNA is a kind of non-coding functional RNA that is very abundant in the nervous system, and about half of the lncRNA is specifically expressed in the nervous system. LncRNAs are not only involved in the development and functional maturation of the nervous system, but also in the regulation of synaptic connections, axon growth, and post-injury repair of the nervous system.
  • lncRNAs have important functions in regulating brain development, such as promoting the differentiation of neural stem cells into neurons or glial cells. LncRNA also has an important function in the process of disease damage repair. Studies have found that BACE1-AS is related to Alzheimer's disease, and MALAT1 is related to the occurrence and development of Parkinson's disease. However, so far there is no research on lncRNA and glial cell transdifferentiation.
  • neurodegenerative diseases have gradually become the second largest disease after cancer, and the number of patients is large.
  • drugs that can be used to treat neurodegenerative diseases, and most of them can only delay the disease.
  • the progress of the disease cannot achieve the effect of reversing the disease process.
  • nerve regeneration technology undoubtedly brings hope for the treatment of these major neurodegenerative diseases.
  • scientists have transdifferentiated glial cells into functional neurons, hoping to treat serious brain diseases such as neurodegenerative diseases or stroke. Therefore, it is particularly important to find high-efficiency neural transdifferentiation targets.
  • glial cells were transdifferentiated into neurons in the nervous system by AAV-mediated gene delivery technology.
  • the experimental results showed that miRNAs or LncRNAs such as Pnky, Tuna, Let-7b and miR137 could transdifferentiate astrocytes into neurons in vivo. These results indicate that miRNAs and LncRNAs can also serve as targets for the transdifferentiation of glial cells into neurons, and lay the foundation for the treatment of various neurodegenerative diseases.
  • diseases related to loss of function or death of neurons mainly include diseases related to loss of function or death of dopamine neurons, and visual impairment related to loss or death of optic ganglion or photoreceptor cells.
  • the diseases associated with neuronal function loss or death are selected from the group consisting of: Parkinson's disease, Alzheimer's disease, stroke (stroke), schizophrenia, Huntington's disease, depression, Motor neuron disease, cerebral ischemia, brain injury, amyotrophic lateral sclerosis (ALS), spinal muscular atrophy, Pick's disease, sleep disorders, epilepsy, ataxia, PloyQ disease, addiction, or combinations thereof, Parkinson's disease and disorders of the visual system resulting from loss of function or death of RGC or photoreceptor cells are preferred.
  • Parkinson's disease Alzheimer's disease, stroke (stroke), schizophrenia, Huntington's disease, depression, Motor neuron disease, cerebral ischemia, brain injury, amyotrophic lateral sclerosis (ALS), spinal muscular atrophy, Pick's disease, sleep disorders, epilepsy, ataxia, PloyQ disease, addiction, or combinations thereof.
  • Astrocytes are the most numerous type of cell in the mammalian brain. They perform many functions, including biochemical support (such as forming the blood-brain barrier), providing nutrition to neurons, maintaining extracellular ion balance, and participating in repair and scarring after brain and spinal cord injury. Astrocytes can be divided into two types according to the content of glial filaments and the shape of their processes: fibrous astrocytes are mostly distributed in the white matter of the brain and spinal cord, with slender processes and fewer branches , containing a large number of glial filaments in the cytoplasm; protoplasmic astrocytes (protoplasmic astrocytes), mostly distributed in gray matter, with thick and short cell processes and many branches.
  • biochemical support such as forming the blood-brain barrier
  • Astrocytes can be divided into two types according to the content of glial filaments and the shape of their processes: fibrous astrocytes are mostly distributed in the white matter of the brain and spinal cord, with slender processes and fewer branches ,
  • the astrocytes that can be used in the present disclosure are not particularly limited, and include various astrocytes derived from the central nervous system of mammals, such as striatum, ventral tegmental area of midbrain, hypothalamus, spinal cord, etc. , dorsal midbrain or cerebral cortex, preferably derived from striatum or substantia nigra.
  • a neuron may refer to a neuron capable of transmitting or receiving information through chemical or electrical signals.
  • neurons exhibit one or more functional properties of mature neurons present in the normal nervous system, including but not limited to: excitability (e.g., the ability to exhibit action potentials, such as rapid rise and Subsequent dips) (voltage or membrane potential across the cell membrane), formation of synaptic connections with other neurons, presynaptic neurotransmitter release, and postsynaptic responses (e.g., excitatory postsynaptic currents or inhibitory postsynaptic current).
  • excitability e.g., the ability to exhibit action potentials, such as rapid rise and Subsequent dips
  • postynaptic responses e.g., excitatory postsynaptic currents or inhibitory postsynaptic current.
  • neurons are characterized by the expression of one or more markers of functional neurons, including but not limited to synapsin, synaptophysin, glutamate decarboxylase 67 (GAD67), glutamate decarboxylate Enzyme 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 decarboxylate Enzyme 65 (GAD65), parvalbumin, dopamine- and cAMP-regulated neuro
  • Dopaminergic neurons contain and release dopamine (DA) as a neurotransmitter.
  • Dopamine is a catecholamine neurotransmitter, which 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) and ventral tegmentum of the midbrain. area (ventral tegmental area, VTA), hypothalamus and periventricular.
  • SNc substantria nigra pars compacta
  • VTA ventral tegmental area
  • Many experiments have confirmed that dopaminergic neurons are closely related to various diseases of the human body, the most typical being Parkinson's disease.
  • AAV adeno-associated virus
  • Adeno-associated virus also known as adeno-associated virus, belongs to the genus of Parvoviridae and is the simplest single-stranded DNA-deficient virus found so far. It needs helper virus (usually adenovirus) to participate in replication. It encodes cap and rep genes in two terminal inverted repeats (ITRs). ITRs play a decisive role in virus replication and packaging. The cap gene encodes the viral capsid protein, and the rep gene is involved in virus replication and integration. AAV can infect a variety of cells.
  • Recombinant adeno-associated virus vector is derived from non-pathogenic wild-type adeno-associated virus. Due to its good safety, wide range of host cells (dividing and non-dividing cells), and low immunogenicity, it is time-consuming to express foreign genes in vivo. Long and other characteristics, it is regarded as one of the most promising gene transfer vectors and has been widely used in gene therapy and vaccine research worldwide. After more than 10 years of research, the biological characteristics of recombinant adeno-associated virus have been deeply understood, especially a lot of information has been accumulated on its application effect in various cells, tissues and in vivo experiments.
  • rAAV is used in the research of gene therapy 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 genome of the cells they infect in a stable and site-specific manner. They are able to infect a large range of cells without any effect on cell growth, morphology or differentiation, and they do not appear to be involved in human pathology.
  • the AAV genome has been cloned, sequenced and characterized.
  • AAV contains an inverted terminal repeat (ITR) region of approximately 145 bases at each end, which serves as the viral origin of replication. The rest of the genome is divided into two important regions with encapsidation function: 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 Patent Nos. 6,566,118, 6,989,264, and 6,995,006, the disclosures of which are incorporated herein by reference in their entirety. The preparation of hybrid vectors is described, for example, in PCT Application No. PCT/US2005/027091, the disclosure of which is incorporated herein by reference in its entirety. The use of AAV-derived vectors for gene transfer in vitro and in vivo has been described (see, e.g., International Patent Application Publication Nos. WO91/18088 and WO93/09239; U.S. Patent Nos.
  • Replication-defective recombinant AAV can be prepared by co-transfecting into cell lines infected with a human helper virus (eg, adenovirus) a plasmid containing a nucleic acid sequence of interest flanked by two AAV inverted terminal repeats (ITRs) region, and a plasmid carrying the AAV encapsidation genes (rep and cap genes).
  • a human helper virus eg, adenovirus
  • ITRs AAV inverted terminal repeats
  • the recombinant vector is encapsidated into a virion (for example including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16 in AAV virions).
  • a virion for example including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16 in AAV virions.
  • the present disclosure includes recombinant virions (recombinant in that they comprise a recombinant polynucleotide) comprising any of the vectors described herein. Methods of producing such particles are known in the art and described in US Patent No. 6,596,535.
  • the plasmids in this study were all constructed by 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 is PCR with cell cDNA or genome as a template, and the PCR fragment is recovered after agarose gel electrophoresis.
  • the backbone vector was connected to the fragments according to the standard operating procedures of the ClonExpress MultiS One Step Cloning Kit (Vazyme, C113-02) of Novozyme Biotechnology Co., Ltd. Transformed into DH5a E. coli after ligation. Correct clones verified by sequencing were expanded and cultured, and plasmids were extracted.
  • the plasmids were constructed as follows: AAV-GFAP-mCherry, AAV-GFAP-CasRx, AAV-GFAP-CasRx-gRNA(Pnky), CAG-Pnky-WPRE; CAG-CasRx, CMV-mCherry-U6-gRNA(Pnky), AAV- GFAP-tuna, AAV-GFAP-Let-7b, AAV-GFAP-miRNA-137, AAV-GFAP-miR-18b, AAV-GFAP-miR-24-3p, AAV-GFAP-miR-34a, AAV-GFAP- miR-92b, AAV-GFAP-miR-96, AAV-GFAP-miR-106, AAV-GFAP-miR-125a, AAV-GFAP-miR-128, AAV-GFAP-miR-134AAV-GFAP-miR-135, AAV-GFAP-miR
  • 293T cells were cultured in DMEM + 10% fetal bovine serum + penicillin/streptomycin medium at 37°C in an incubator with 5% CO2 concentration. Transfection was performed when the cells grew to about 70% of the bottom of the culture dish. Cell transfection was carried out according to the standard operation procedure of EZ Trans cell transfection reagent (Shanghai Liji Biology, AC04L092).
  • the transfection plasmid in the experimental group was CAG-Pnky-WPRE+CAG-CasRx-EGFP+CMV-mCherry-U6-gRNA(Pnky), and the transfection plasmid in the control group was CAG-CasRx+CMV-mCherry-U6-gRNA(Pnky).
  • the qPCR primers were: 5'-aggcagtgtgcggaggacat-3' and 5'-gccattgtcctagcaagtgc-3'.
  • the injection was performed using the Ruiwode stereotaxic injection system.
  • the control group was injected with GFAP-mCherry+GFAP-CasRx, and the experimental group was injected with GFAP-mCherry+GFAP-CasRx-gRNA(Pnky).
  • the control group is injected with GFAP-mCherry, and the experimental group is injected with GFAP-mCherry+GFAP-miRNA/LncRNA.
  • the miRNA/LncRNA are GFAP-Tuna, GFAP-Let-7b and GFAP-miRNA-137 .
  • the titer of the AAV mixture in each group was greater than 5 ⁇ 10 12 vg/ml (1-3 ⁇ l per injection).
  • AAV was injected into striatum (AP+0.8mm, ML ⁇ 1.6mm and DV-2.8mm) or substantia nigra (AP-3.0mm, ML ⁇ 1.25mm and DV-4.5mm).
  • Antibody information used in this study is as follows:
  • 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) 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).
  • the core sequence of human and mouse miR-18a is 100% similar at the DNA level
  • the core sequence of human and mouse miR-24-3p is 100% similar at the DNA level
  • the core sequence of human and mouse miR-34a is 100% similar at the DNA level
  • the core sequence of human and mouse miR-92b has a similarity of 95.45% at the DNA level
  • the core sequence of human and mouse miR-96 is 100% similar at the DNA level
  • the core sequence of human and mouse miR-106 has a similarity of 91.3% at the DNA level;
  • the core sequence of human and mouse miR-128 is 100% similar at the DNA level
  • the core sequence of human and mouse miR-134 is 100% similar at the DNA level
  • the core sequence of human and mouse miR-135 is 100% similar at the DNA level
  • the core sequence of human and mouse miR-137 is 100% similar at the DNA level
  • the core sequence of human and mouse miR-141 has a similarity of 95.45% at the DNA level
  • the core sequence of human and mouse miR-143 is 100% similar at the DNA level
  • miR-200a 5'-catcttaccggacagtgctgga-3' (SEQ ID NO: 51)
  • miR-200b 5'-catcttactgggcagcattgga-3' (SEQ ID NO: 52)
  • miR-200c 5'-cgtcttacccagcagtgtttgg-3' (SEQ ID NO: 53)
  • miR-200a 5'-taacactgtctggtaacgatgt-3' (SEQ ID NO: 54)
  • miR-200b 5'-taatactgcctggtaatgatga-3' (SEQ ID NO: 55)
  • miR-200c 5'-taatactgccgggtaatgatgga-3' (SEQ ID NO: 56)
  • the core sequences of human and mouse miR-200a/b/c are 100% similar at the DNA level;
  • the core sequence of human and mouse miR-429 is 100% similar at the DNA level
  • the core sequence of human and mouse miR-25 is 100% similar at the DNA level
  • the core sequence of human and mouse miR-29a is 100% similar at the DNA level
  • the core sequence of human and mouse miR-137 is 100% similar at the DNA level
  • the core sequence of human and mouse miR-155 has a similarity of 95.8% at the DNA level;
  • the core sequence of human and mouse miR-195 is 100% similar at the DNA level
  • the core sequence of human and mouse miR-214 is 100% similar at the DNA level
  • the core sequence of human and mouse miR-223 is 100% similar at the DNA level
  • the core sequence of human and mouse miR-132 is 100% similar at the DNA level
  • the core sequence of human and mouse miR-133 has 100% similarity at the DNA level.
  • GFAP-mCherry+GFAP-CasRx Different AAV combinations were injected into the mouse brain, the control group was injected with GFAP-mCherry+GFAP-CasRx, and the experimental group was injected with GFAP-mCherry+GFAP-CasRx-gRNA(Pnky), in which GFAP-mCherry marked astrocytes. Samples were taken for analysis 1-2 months after injection (Fig. 1C).
  • Example 2 miRNA and lncRNA-mediated transdifferentiation of glial cells into neurons
  • AAV was injected into the striatum of the mouse brain, analyzed 1-2 months after injection, and immunofluorescent staining was performed with the neuron-specific marker NeuN, GFAP-mCherry labeled glial cells transdifferentiated into neurons Afterwards, it will be co-labeled with NeuN ( Figure 2C).
  • Figure 2C the results at 1 month after injection of GFAP-mCherry showed that glial cells still maintained the typical characteristics of glial cells and were not co-labeled with the neuron-specific marker NeuN (Fig. 2D).
  • the red fluorescently labeled cells already had the shape of neurons, the cell body was rounded, and there were some longer and thinner protrusions.
  • Example 3 Transdifferentiation of glial cells into neurons induced by overexpression of miRNA or lncRNA
  • miR-18b miR-24, miR-34a, miR-128, miR-134, miR-143, Let-7a, miR-92b, miR-96, miR-106, miR-125a, miR- 135.
  • miR-141, miR-200, miR-218, miR-429 and other miRNAs can also transdifferentiate astrocytes into neurons, we constructed the astrocyte-specific promoter GFAP miR-18b, miR-24, miR-34a, miR-128, miR-134, miR-143, Let-7a, miR-92b, miR-96, miR-106, miR-125a, miR-135, miR- 141, miR-200, miR-218, miR-429 expression vector, and packaging AAV. Each group of AAV was mixed with AAV-GFAP-EGFP and then injected into the striatum of mice, where AAV-GFAP-EGFP could specifically mark the astrocytes in the striatum as green.

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Abstract

提供了由非神经元细胞产生神经元细胞的方法，其包括通过增强或降低某些miRNA 和/或lncRNA在非神经元细胞中的表达或活性，使所述非神经元细胞转分化为或重编程为神经元细胞，还提供了使某些miRNA和/或IncRNA的表达或活性增强或降低的试剂用于预防和/或治疗与神经元功能缺失或死亡相关的疾病的用途。

Description

非编码RNA介导的神经性疾病治疗
本申请要求于2021年12月07日提交的发明名称为“非编码RNA介导的神经性疾病治疗”的申请号为202111488197.4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，本公开内容涉及使某些非编码RNA(例如某些miRNA和lncRNA)的表达或活性增强或降低的试剂用于治疗与神经元功能缺失或死亡相关的疾病的用途。
背景技术
细胞的转分化(cell transdifferentiation)是指一种类型的分化细胞通过基因选择性表达或基因的重编程使其在结构和功能上转变成另一种分化细胞的过程。
MicroRNA(miRNA)是一类存在于动物或植物细胞内的高度保守的非编码功能性RNA分子，其长度大约为17-27个核苷酸。miRNA通过与靶标mRNA序列的3’UTR区域结合，导致翻译抑制或者RNA降解，从而抑制靶标基因的功能。当miRNA与靶标RNA完全互补时导致mRNA的降解，而当与靶标RNA不完全互补时阻止基因翻译。在哺乳动物细胞中，miRNA主要以阻止mRNA翻译而不是影响其稳定性。长链非编码RNA(LncRNA)是一类长度超过200个碱基的RNA分子，并不参与蛋白编码，但能以RNA的形式参与调控基因的表达。以前LncRNA被当做是基因转录的“噪音”，是RNA转录的副产物，没有生物学功能。但近些年的研究表明，LncRNA广泛参与多种生物学过程，比如基因的转录调控、基因组印迹、染色质沉默、染色质修饰、基因表达激活或抑制等多种生物学过程。在基因组中大约有4-8％的序列用于转录LncRNA，但LncRNA的功能研究确比较少，而且受限于技术方法，LncRNA的研究相对滞后。LncRNA从表观遗传学、基因转录调控、转录后调控等多个层次参与基因的表达调控，而且有一些LncRNA还参与miRNA的调控，从而间接调控基因表达。
在中枢神经系统中，星形胶质细胞是一类非常丰富的胶质细胞，存在神经元的周围，对神经元有支持和提供营养等作用。在多种神经退行性疾病或神经外伤中，神经元细胞会发生死亡，从而导致一些功能缺陷，如中风、阿尔兹海默症和帕金森症等。科学家们在体外培养的星形胶质细胞中，通过添加多种因子，能实现将胶质细胞转分化为神经元，如ASCL1、NeuroD1和Ngn2等因子。但在体内，由于体内的环境复杂性和细胞之间的相互影响，导致很多在体外培养皿中研究的结果难以在体内重现。因此，研究如何在体内原位对星形胶质细胞进行转分化以补充丢失的神经元具有重要的科学意义。
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。
近几年，对于miRNA和LncRNA在神经系统中的功能探索逐渐增多，主要集中于研究不同的miRNA和LncRNA在神经系统中的分布，以及miRNA和LncRNA在神经系统生长发育中的功能，而有关其在神经疾病中的研究较少。其中研究最多的就是miR-9和miR-124在神经元发育和成熟中的功能研究。而利用miRNA和LncRNA的特性，调控基因表达网络，从而达到改变细胞的特性的研究非常的少。miRNA和LncRNA具有十分重要的功能，探究如何将miRNA和LncRNA应用于药物开发，更具体的，应用于神经再生药物中，具有十分重要的科学意义。
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
在一个实施方案中，本公开内容提供了由非细胞产生神经元细胞的方法，其包括通过使选自Let-7a、Let-7b、miR-18a/b、miR-24-3p、miR-34a、miR-92b、miR-96、miR-106、miR-125a/b、miR-128、miR-134、miR-135、miR-137、miR-141、miR-143-3p、miR-184、miR-200、miR-218、miR-219、miR-228、miR-284、miR-429、miR-430的miRNA、或者选自utNgn1、RMST、Tuna、Linc-Brn1b、Dali、Miat/Gomafu、NBAT-1、Malat1、Dlx1as、Six3os、Evf2、LncKdm2b、lncRNA_N1、lncRNA_N2、lncRNA_N3的lncRNA，或其任意组合的表达或活性增强来使所述非神经元细胞转分化为或重编程为神经元细胞。
在一个优选的实施方案中，通过例如过表达、基因激活剂、表观遗传修饰、miRNA模拟物、直接递送RNA、小分子化合物、和/或RNA稳定剂来增强所述miRNA或lncRNA的表达或活性。
在另一个实施方案中，本公开内容提供了由非神经元细胞产生神经元细胞的方法，其包括通过使选自miR-7a、miR-15、miR-23a/b、miR-25、miR-29a、miR-129、miR-137、miR-138、miR-155、miR-195、miR-214、miR-222、miR-223、miR-132、miR-133的miRNA、或者选自Pnky、Paupar、HOTAIRM1、lncR492、TUG1的lncRNA，或其任意组合的表达或活性降低来使所述非神经元细胞转分化为或重编程为神经元细胞。
在一个优选的实施方案中，通过例如基因编辑技术介导的DNA编辑和RNA编辑、RNA表达抑制剂、反义寡核苷酸(ASO)、小RNA干扰、miRNA技术、小分子化合物、基因抑制技术(dCas-Krab等)、和/或表观遗传调控来降低所述miRNA或lncRNA的表达或活性。
在一个优选的实施方案中，RNA编辑包括CRISPR介导的RNA降解和翻译抑制、RNA单碱基编辑、RNA上碱基的插入或缺失、RNA剪切的改变、RNA表观修饰。
在一个优选的实施方案中，所述选自Let-7a、Let-7b、miR-18a/b、miR-24-3p、miR-34a、 miR-92b、miR-96、miR-106、miR-125a/b、miR-128、miR-134、miR-135、miR-137、miR-141、miR-143-3p、miR-184、miR-200、miR-218、miR-219、miR-228、miR-284、miR-429、miR-430的miRNA、或选自miR-7a、miR-15、miR-23a/b、miR-25、miR-29a、miR-129、miR-137、miR-138、miR-155、miR-195、miR-214、miR-222、miR-223、miR-132、miR-133的miRNA、或者选自utNgn1、RMST、Tuna、Linc-Brn1b、Dali、Miat/Gomafu、NBAT-1、Malat1、Dlx1as、Six3os、Evf2、LncKdm2b、lncRNA_N1、lncRNA_N2、lncRNA_N3的lncRNA、或选自Pnky、Paupar、HOTAIRM1、lncR492、TUG1的lncRNA为来自不同物种的同源miRNA或同源lncRNA。
在一个优选的实施方案中，所述非神经元细胞包括例如胶质细胞、成纤维细胞、干细胞、神经前体细胞、神经干细胞，其中胶质细胞选自星形胶质细胞、小胶质细胞、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。
在一个优选的实施方案中，所述胶质细胞来源于脑、脊髓、眼或耳，其中脑部胶质细胞来源于纹状体、黑质、中脑腹侧被盖区、脊髓、下丘脑、背侧中脑或大脑皮层，优选纹状体和黑质。
在一个优选的实施方案中，所述神经元细胞优选多巴胺神经元、GABA神经元、5-HT神经元、谷氨酸能神经元、ChAT神经元、NE神经元、运动神经元、脊髓神经元、脊髓运动神经元、脊髓感觉神经元、锥体神经元、中间神经元、中型多棘神经元(MSN)、浦肯野细胞、颗粒细胞、嗅感觉神经元、球周细胞或其组合，更优选多巴胺神经元。
在一个优选的实施方案中，所述非神经元细胞和/或神经元细胞来自例如人、非人灵长类动物、大鼠和小鼠，优选人。
在一个优选的实施方案中，所述方法为体内方法或体外方法。
在另一个实施方案中，本公开内容提供了使选自Let-7a、Let-7b、miR-18a/b、miR-24-3p、miR-34a、miR-92b、miR-96、miR-106、miR-125a/b、miR-128、miR-134、miR-135、miR-137、miR-141、miR-143-3p、miR-184、miR-200、miR-218、miR-219、miR-228、miR-284、miR-429、miR-430的miRNA、或者选自utNgn1、RMST、Tuna、Linc-Brn1b、Dali、Miat/Gomafu、NBAT-1、Malat1、Dlx1as、Six3os、Evf2、LncKdm2b、lncRNA_N1、lncRNA_N2、lncRNA_N3的lncRNA，或其任意组合的表达或活性增强的试剂在制备用于预防和/或治疗与神经元功能缺失或死亡相关疾病的药物的用途，其中所述试剂增强所述miRNA或所述lncRNA的表达或活性。
在另一个实施方案中，本公开内容提供了使选自miR-7a、miR-15、miR-23a/b、miR-25、miR-29a、miR-129、miR-137、miR-138、miR-155、miR-195、miR-214、miR-222、miR-223、miR-132、miR-133的miRNA、或者选自Pnky、Paupar、HOTAIRM1、lncR492、TUG1的lncRNA，或其任意组合的表达或活性降低的试剂在制备用于预防和/或治疗与神经元功能缺失或死亡相关疾病的药物的用途，其中所述试剂降低所述miRNA或所述lncRNA的表达或活性。
在一个优选的实施方案中，所述药物配制成用于在体内施用于神经系统，例如纹状体、黑质、中脑腹侧被盖区、脊髓、下丘脑、背侧中脑、大脑皮层、海马、小脑、内耳耳蜗和前庭，优选纹状体、黑质、视网膜下、玻璃体腔和内耳耳蜗。
在一个优选的实施方案中，所述与神经元功能缺失或死亡相关的疾病选自：帕金森病、阿尔茨海默病、脑卒中(中风)、精神分裂症、亨廷顿舞蹈症、抑郁症、运动神经元病、脑缺血、脑损伤、肌萎缩性侧索硬化(ALS)、脊髓性肌萎缩症、Pick病、睡眠失调、癫痫、共济失调、PloyQ疾病、成瘾、或其组合，优选帕金森病。
在一个优选的实施方案中，所述使所述miRNA或lncRNA或其组合的表达或活性增强包括：
(a)外源表达所述miRNA或lncRNA或其组合，例如通过包含启动子的表达载体来实现；
(b)向细胞内递送DNA或RNA形式的所述miRNA或lncRNA或其组合；
(c)激活所述miRNA或lncRNA或其组合的内源表达，如基因表达激活剂和表观调控元件等；或者
(d)向细胞递送所述miRNA或lncRNA或其组合的类似物或激动剂；
其中优选外源表达所述miRNA或lncRNA或其组合，例如通过包含启动子的表达载体来实现。
在一个优选的实施方案中，通过以下使所述miRNA或lncRNA或其组合的表达或活性降低：抗体、小分子化合物、microRNA、siRNA、shRNA、反义寡核苷酸、结合蛋白或蛋白结构域、多肽、核酸适配体、基因编辑器、表观调控元件、转录抑制元件或其组合。
在另一个实施方案中，本公开内容提供了药物组合物或药盒或试剂盒，其包含使选自Let-7a、Let-7b、miR-18a/b、miR-24-3p、miR-34a、miR-92b、miR-96、miR-106、miR-125a/b、miR-128、miR-134、miR-135、miR-137、miR-141、miR-143-3p、miR-184、miR-200、miR-218、miR-219、miR-228、miR-284、miR-429、miR-430的miRNA、或者选自utNgn1、RMST、Tuna、Linc-Brn1b、Dali、Miat/Gomafu、NBAT-1、Malat1、Dlx1as、Six3os、Evf2、LncKdm2b、lncRNA_N1、lncRNA_N2、lncRNA_N3的lncRNA，或其任意组合的表达或活性增强的试剂；或者使选自miR-7a、miR-15、miR-23a/b、miR-25、miR-29a、miR-129、miR-137、miR-138、miR-155、miR-195、miR-214、miR-222、miR-223、miR-132、miR-133的miRNA、或者选自Pnky、Paupar、HOTAIRM1、lncR492、TUG1的lncRNA，或其任意组合的表达或活性降低的试剂。
在一个优选的实施方案中，使所述miRNA或lncRNA或其组合的表达或活性增强的试剂选自：表达载体，DNA或RNA形式的所述miRNA或lncRNA或其组合，所述miRNA或lncRNA或其组合的内源性激活剂，所述miRNA或lncRNA或其组合的类似物或激动剂。
在一个优选的实施方案中，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)、自互补AAV(scAAV)、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。
在一个优选的实施方案中，使所述miRNA或lncRNA或其组合的表达或活性降低的试剂选自：抗体、小分子化合物、microRNA、siRNA、shRNA、反义寡核苷酸、结合蛋白或蛋白结构域、多肽、核酸适配体、基因编辑器、表观调控元件、转录抑制元件，或其组合。
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试剂的载体或运载体。
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，所述组合物局部施用至以下至少一种：i)纹状体中的胶质细胞；ii)腹侧被盖区(VTA)中的胶质细胞；iii)黑质中的胶质细胞；iv)下丘脑中的胶质细胞；v)脊髓中的胶质细胞；vi)前额皮质中的胶质细胞；以及vii)运动皮质中的胶质细胞。
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配制成用于细胞转染、细胞感染、细胞内吞、注射、颅内给药、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，其中所述miRNA或lncRNA为Let-7a、miR-92b、miR-96、miR-106、miR-125a、miR-135、miR-141、miR-200、miR-218、miR-429，miR-24，所述非神经元细胞为胶质细胞，所述神经元细胞为多巴胺神经元。
在一个优选的实施方案中，非神经元细胞的转分化效率为至少1％，或至少10％、20％、30％、40％或50％。
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.敲低Pnky介导的胶质细胞向神经元转分化。(A)AAV载体设计示意图，Vector 1为GFAP驱动mCherry表达的载体示意图，GFAP是一种胶质细胞中特异性表达的启动子，mCherry是一种红色荧光蛋白，应用于标记胶质细胞。Vector 2是GFAP-CasRx表达载体示意图，由星形胶质细胞特异性启动子GFAP驱动CasRx的表达。Vector3是U6-gRNA-GFAP-CasRx表达载体示意图，gRNA由U6启动子启动表达，CasRx由GFAP启动子启动。(B)在293T细胞中利用CasRx敲低Pnky的表达，CasRx-gRNA(Pnky)高效敲低Pnky的mRNA。(C)AAV注射和转分化示意图，在小鼠脑中注射不同的AAV，研究胶质细胞向神经元转分化：对照组注射GFAP-mCherry与GFAP-CasRx的混合病毒，实验组注射 GFAP-mCherry与GFAP-CasRx-gRNA(Pnky)的混合AAV，GFAP-mCherry标记星形胶质细胞。
图2.miRNA或LncRNA介导的胶质细胞向神经元转分化。(A)GFAP-mCherry和GFAP-miRNA/lncRNA表达载体设计示意图。由胶质细胞特异性启动子GFAP驱动mCherry，miRNA或LncRNA表达。(B)Tuna，Let-7b，miR-137的AAV表达载体示意图，其中Tuna为LncRNA，Let-7b和miRNA-137为miRNA，都由GFAP启动子启动表达。(C)AAV注射和miRNA/LncRNA将胶质细胞转分化为神经元示意图。(D)在C57小鼠纹状体中注射对照组AAV结果代表图，在注射AAV后1个月取材分析，红色荧光信号为GFAP-mCherry所标记的细胞，白色信号为神经元特异性标志物NeuN，白色信号与红色信号不重合。(E-G)在小鼠纹状体中分别注射GFAP-Tuna，GFAP-Let-7b或miR-137后1个月取材分析结果代表图，红色荧光信号(mCherry)为GFAP-mCherry标记的神经元，白色信号为神经元特异性标志物NeuN，黄色箭头指示的为mCherry与NeuN共标的细胞，标尺为50微米。
图3.过表达miRNA诱导星形胶质细胞转分化为神经元。(A)AAV载体示意图，载体1位GFAP-EGFP标记AAV，能特异性标记星形胶质细胞，载体2为GFAP启动的miRNA或LncRNA，实现在胶质细胞中特异性表达miRNA或LncRNA。(B)注射对照组AAV-GFAP-EGFP，EGFP特异性标记星形胶质细胞(C-G)在小鼠纹状体中注射对应的AAV(GFAP-miR-18b，GFAP-miR-34a，GFAP-miR-128，GFAP-miR-134，GFAP-miR-143)后，星形胶质细胞转分化为神经元代表图。绿色荧光信号为AAV-GFAP-EGFP标记的细胞，NeuN为神经元特异性蛋白标志物。白色箭头指向绿色与白色荧光共标的细胞，显示这些细胞表达神经元的特异性蛋白标志物NeuN，标尺为50微米。
图4.过表达miRNA诱导星形胶质细胞转分化为多巴胺神经元。(A)在小鼠纹状体注射对照组AAV-GFAP-EGFP后1个月取材分析。(B-F)在小鼠纹状体星形胶质细胞中过表达miRNA(Let-7a，miR-92b，miR-96，miR-24，miR-106，-miR-125a)等，能将星形胶质细胞转分化为神经元，其中有部分胶质细胞转分化为多巴胺神经元。白色箭头指向GFAP-EGFP标记的绿色细胞，白色为神经元特异性标志物NeuN，红色信号为多巴胺特异性蛋白标志TH，叠合图显示不同信号之间有部分重叠。白色箭头指向绿色信号与NeuN共标的细胞，黄色箭头指向TH阳性的细胞，标尺为50微米。
图5.过表达miRNA将星形胶质细胞转分化为多巴胺神经元。(A-F)在小鼠纹状体星形胶质细胞中过表达miR-135，miR-24，miR-141，miR-200，miR-218，miR-429等miRNA，将星形胶质细胞转分化为神经元，其中部分转分化为多巴胺神经元。白色箭头指向GFAP-EGFP标记的绿色细胞，白色为神经元特异性标志物NeuN，红色信号为多巴胺特异性蛋白标志TH，叠合图显示不同信号之间的重叠情况。白色箭头指向绿色信号与NeuN共标的细胞，黄色箭头指向TH阳性的细胞，标尺为50微米。
图6.过表达miRNA将星形胶质细胞转分化为神经元和多巴胺神经元比例。(A)在小鼠纹状体星形胶质细胞中过表达miR-18b，miR-24，miR-34a，miR-128，miR-134，miR-143，Let-7a， miR-92b，miR-96，miR-106，miR-125a，miR-135，miR-141，miR-200，miR-218，miR-429等miRNA，将星形胶质细胞转分化为神经元效率统计。(B)在小鼠纹状体星形胶质细胞中过表达Let-7a，miR-106，miR-125，miR-134，miR-135，miR-141，miR-200，miR-429，星形胶质细胞转分化为多巴胺神经元效率统计结果。
具体实施方式
miRNA和LncRNA在神经系统中广泛表达并具有重要的功能，在神经系统的发育和多中神经疾病的发生发展中都扮演着重要的角色。神经系统发育过程中miRNA广泛表达于多种神经细胞，而且不同的神经细胞中表达的miRNA种类不同。这表明，不同的miRNA可能参与调解不同的神经细胞发育。之前的研究表明，在神经系统发育过程中miRNA-124具有促进神经前体细胞向神经元分化的作用，而且miRNA-124可以通过调控Ptbp1等基因的表达，抑制神经前体细胞向胶质细胞的分化。而且，在体外培养的穆勒胶质细胞(Müller glia，MG)中，过表达miRNA-9和miRNA-124可以将穆勒胶质细胞转分化为视网膜神经元，并且过表达miRNA-9和miRNA-124可以促进AscL1介导的穆勒胶质细胞向视网膜神经元转分化。LncRNA是一类在神经系统中非常丰富的非编码功能性RNA，大约有一半的LncRNA特异性表达于神经系统。LncRNA不但参与了神经系统的发育和功能的成熟，还参与调解突触连接，轴突生长以及神经系统的损伤后修复等过程。然而，对于LncRNA，由于技术方法所限而研究较少，许多LncRNA的功能尚不清楚。之前的研究表明，在LncRNA在调节大脑发育过程中有着重要的功能，比如促进神经干细胞向神经元或胶质细胞分化。在疾病损伤修复过程中LncRNA也具有重要的功能，有研究发现BACE1-AS与阿尔兹海默症有关，MALAT1与帕金森病的发生和发展有关。然而，目前为止没有有关LncRNA与胶质细胞转分化的研究。
随着老龄化进程的发展，神经退行性疾病逐渐成为除癌症以外的第二大疾病，病人数量众多，然而目前能用于治疗神经退行性疾病的药物非常少，而且大部分药物只能延缓疾病的进程，并不能达到逆转疾病进程的效果。而神经再生技术的出现，无疑为这些重大神经退行性疾病的治疗带来了希望。科学家们将胶质细胞转分化为功能性神经元，期待能治疗神经退行性疾病或者中风等重大脑疾病。因此，寻找高效的神经转分化靶点就尤为重要。
本研究通过AAV介导的基因递送技术，在神经系统中将胶质细胞转分化为神经元神经元。实验结果表明，Pnky、Tuna、Let-7b和miR137等miRNA或LncRNA可以在体内将星形胶质细胞转分化为神经元。这些结果说明，miRNA和LncRNA也可以作为胶质细胞向神经元转分化的靶点，而且为治疗多种神经退行性疾病奠定基础。
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。
在一个优选的实施方案中，所述与神经元功能缺失或死亡相关的疾病选自：帕金森病、阿尔茨海默病、脑卒中(中风)、精神分裂症、亨廷顿舞蹈症、抑郁症、运动神经元病、脑缺血、脑损伤、肌萎缩性侧索硬化(ALS)、脊髓性肌萎缩症、Pick病、睡眠失调、癫痫、共济失调、PloyQ疾病、成瘾、或其组合，优选帕金森病和RGC或感光细胞功能缺失或死亡导致的视觉系统疾病。
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，来源于纹状体或黑质。
神经元
在本公开内容中，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，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，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。
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、AAV10、AAV11、AAV12、AAV13、AAV14、AAV15和AAV16的AAV病毒粒子)中。因此，本公开包括含有本文所述任何载体的重组病毒粒子(因其包含重组多核苷酸而为重组的)。产生这样的粒子的方法是本领域己知的，并在美国专利No.6,596,535中有所描述。
通用方法
动物伦理：
本研究中动物的饲养和使用是在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指导下完成。
质粒构建：
本研究中的质粒均为本实验室自己构建，AAV骨架载体用限制性内切酶进行酶切，并进行琼脂糖凝胶电泳，回收骨架载体。插入DNA片段为以细胞cDNA或基因组为模板进行PCR，琼脂糖凝胶电泳后回收PCR片段。按照诺唯赞生物科技有限公司的ClonExpress MultiS One Step Cloning Kit(Vazyme，C113-02)标准操作步骤将骨架载体与片段连接。连接后转化到DH5a大肠杆菌中。经测序验证正确的克隆进行扩大培养，并提取质粒。构建质粒如下：AAV-GFAP-mCherry，AAV-GFAP-CasRx，AAV-GFAP-CasRx-gRNA(Pnky)，CAG-Pnky-WPRE；CAG-CasRx，CMV-mCherry-U6-gRNA(Pnky)，AAV-GFAP-tuna，AAV-GFAP-Let-7b，AAV-GFAP-miRNA-137，AAV-GFAP-miR-18b，AAV-GFAP-miR-24-3p，AAV-GFAP-miR-34a，AAV-GFAP-miR-92b，AAV-GFAP-miR-96，AAV-GFAP-miR-106，AAV-GFAP-miR-125a，AAV-GFAP-miR-128，AAV-GFAP-miR-134AAV-GFAP-miR-135，AAV-GFAP-miR-141，AAV-GFAP-miR-143，AAV-GFAP-miR-184，AAV-GFAP-miR-200，AAV-GFAP-miR-218，AAV-GFAP-miR-219，AAV-GFAP-miR-429，AAV-GFAP-RMST，AAV-GFAP-Brn1b，AAV-GFAP-Dali，AAV-GFAP-Dlxas。
细胞培养和转染
293T细胞培养于DMEM+10％胎牛血清+青/链霉素培养基中，在37℃，5％CO2浓度的的培养箱中培养。待细胞生长到铺满培养皿底70％左右进行转染。按照EZ Trans细胞转染试剂(上海李记生物，AC04L092)标准操作步骤进行细胞转染。实验组转染质粒为CAG-Pnky-WPRE+CAG-CasRx-EGFP+CMV-mCherry-U6-gRNA(Pnky)，对照组转染质粒为 CAG-CasRx+CMV-mCherry-U6-gRNA(Pnky)。转染后48h进行流式分选，每个样品收集30000个细胞，实验组收取GFP和mCherry双阳性细胞，对照组分选GFP阳性细胞。收集的细胞使用Trizol(Ambion)提取RNA，并使用逆转录试剂盒(用于qPCR的HiScript Q RT SuperMix，诺唯赞)进行其反转录。QPCR检测使用AceQ qPCR SYBR Green Master Mix(诺唯赞)。qPCR引物为：5’-aggcagtgtgcggaggacat-3’和5’-gccattgtcctagcaagtgc-3’。
小鼠脑部AAV注射：
利用瑞沃德立体定位注射系统进行注射，对于需要下调表达的RNA，对照组注GFAP-mCherry+GFAP-CasRx，实验组注射GFAP-mCherry+GFAP-CasRx-gRNA(Pnky)。对于需要上调表达的RNA，对照组注射GFAP-mCherry，实验组注射GFAP-mCherry+GFAP-miRNA/LncRNA，本实验中miRNA/LncRNA具体为GFAP-Tuna，GFAP-Let-7b和GFAP-miRNA-137。各组AAV混合液的滴度大于5×10 12vg/ml(每次注射1-3μl)。将AAV注入纹状体(AP+0.8mm，ML±1.6mm和DV-2.8mm)或黑质(AP-3.0mm，ML±1.25mm和DV-4.5mm)。
小鼠组织免疫荧光染色：
注射AAV后1-2个月取材、切片并进行免疫荧光染色。大概步骤为：先用生理盐水进行灌流，再用4％PFA灌流，取出目的组织并用4％多聚甲醛(PFA)固定过夜，然后在30％蔗糖中脱水至少12小时以上，待组织沉入蔗糖溶液底部再进行切片。用OCT进行包埋后进行冷冻切片，切片厚度为30μm或40μm。免疫荧光染色之时，用0.1M磷酸盐缓冲液(PBS)清洗脑片三次，每次5-10分钟。然后加上一抗4℃孵育过夜，用PBS清洗3-4次，每次10-15分钟。清洗结束后加入二抗稀释液稀释后的二抗进行孵育，室温孵育2-3个小时，孵育结束后再用PBS洗3-4次，每次10-15分钟。最后用抗荧光淬灭封片剂(Life Technology)进行封片。
本研究中使用的抗体信息如下：
本研究中所用一抗包括：荷兰猪抗NeuN(1∶500，ABN90，Millipore)，兔抗TH(1∶500，AB152，Millipore)，大鼠抗DAT(1∶100，MAB369，Millipore)和鼠抗Flag(1∶2000，F3165，Sigma)，。该研究中使用的二抗为：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)和Cy5 AffiniPure Donkey Anti-Rabbit IgG(H+L)(1∶500，711-175-152，Jackson ImmunoResearch)。
序列信息
miRNA的序列信息
Let-7a的核苷酸序列
人Let-7a编码序列：5’-tgaggtagtaggttgtatagtt-3’(SEQ ID NO：1)
人Let-7a＊编码序列：5’-ctatacaatctactgtctttc-3’(SEQ ID NO：2)
人Let-7a-2＊编码序列：5’-ctatacaatctactgtctttc-3’(SEQ ID NO：3)
人Let-7a Per-miRNA全长(人有3个Let-7a的Pre-miRNA)
Let-7a-1：
Figure PCTCN2022136909-appb-000001
Let-7a-2：
Figure PCTCN2022136909-appb-000002
Let-7a-3：
Figure PCTCN2022136909-appb-000003
人和鼠Let-7a(Let-7a-1/2)核心序列，在DNA水平的相似性为100％。
Let-7b的核苷酸序列
人Let-7b编码序列：5’-tgaggtagtaggttgtgtggtt-3’(SEQ ID NO：7)
人Let-7b＊编码序列：5’-ctatacaacctactgccttccc-3’(SEQ ID NO：8)
人Let-7b Per-miRNA全长
Figure PCTCN2022136909-appb-000004
人和鼠Let-7b核心序列，在DNA水平的相似性为100％。
miR-18的核苷酸序列
人miR-18a编码序列：5’-taaggtgcatctagtgcagatag-3’(SEQ ID NO：10)
人miR-18a＊编码序列：5’-actgccctaagtgctccttctgg-3’(SEQ ID NO：11)
人miR-18b＊编码序列：5’-taaggtgcatctagtgcagttag-3’(SEQ ID NO：12)
人miR-18 Per-miRNA全长
miR-18a
Figure PCTCN2022136909-appb-000005
miR-18b
Figure PCTCN2022136909-appb-000006
人和鼠miR-18a核心序列，在DNA水平的相似性为100％；
miR-24-3p的核苷酸序列
人miR-24-3p编码序列：5’-tggctcagttcagcaggaacag-3’(SEQ ID NO：15)
人miR-24-3p Per-miRNA全长(人有2个编码序列)
miR-24-1
Figure PCTCN2022136909-appb-000007
miR-24-2
Figure PCTCN2022136909-appb-000008
人和鼠miR-24-3p核心序列，在DNA水平的相似性为100％；
miR-34a的核苷酸序列
人miR-34a编码序列：5’-tggcagtgtcttagctggttgt-3’(SEQ ID NO：18)
人miR-34a＊编码序列：5’-caatcagcaagtatactgccct-3’(SEQ ID NO：19)
人miR-34a Per-miRNA全长
Figure PCTCN2022136909-appb-000009
人和鼠miR-34a核心序列，在DNA水平的相似性为100％；
miR-92b的核苷酸序列
人miR-92b编码序列：5’-agggacgggacgcggtgcagtg-3’(SEQ ID NO：21)
人miR-92b＊编码序列：5’-tattgcactcgtcccggcctcc-3’(SEQ ID NO：22)
人miR-92b Per-miRNA全长
Figure PCTCN2022136909-appb-000010
人和鼠miR-92b核心序列，在DNA水平的相似性为95.45％；
miR-96的核苷酸序列
人miR-96编码序列：5’-tttggcactagcacatttttgct-3’(SEQ ID NO：24)
人miR-96＊编码序列：5’-aatcatgtgcagtgccaatatg-3’(SEQ ID NO：25)
人miR-96 Per-miRNA全长
Figure PCTCN2022136909-appb-000011
人和鼠miR-96核心序列，在DNA水平的相似性为100％；
miR-106的核苷酸序列
人miR-106编码序列：5’-aaaagtgcttacagtgcaggtag-3’(SEQ ID NO：27)
人miR-106＊编码序列：5’-ctgcaatgtaagcacttcttac-3’(SEQ ID NO：28)
人miR-106 Per-miRNA全长
Figure PCTCN2022136909-appb-000012
人和鼠miR-106核心序列，在DNA水平的相似性为91.3％；
miR-128的核苷酸序列
人miR-128编码序列：5’-cggggccgtagcactgtctgaga-3’(SEQ ID NO：30)
人miR-128＊编码序列：5’-tcacagtgaaccggtctcttt-3’(SEQ ID NO：31)
人miR-128 Per-miRNA全长
Figure PCTCN2022136909-appb-000013
人和鼠miR-128核心序列，在DNA水平的相似性为100％；
miR-134的核苷酸序列
人miR-134编码序列：5’-tgtgactggttgaccagagggg-3’(SEQ ID NO：33)
人miR-134＊编码序列：5’-cctgtgggccacctagtcaccaa-3’(SEQ ID NO：34)
人miR-134 Per-miRNA全长
Figure PCTCN2022136909-appb-000014
人和鼠miR-134核心序列，在DNA水平的相似性为100％；
miR-135的核苷酸序列
人miR-135编码序列：5’-tatggctttttattcctatgtga-3’(SEQ ID NO：36)
人miR-135＊编码序列：5’-tatagggattggagccgtggcg-3’(SEQ ID NO：37)
人miR-135 Per-miRNA全长
Figure PCTCN2022136909-appb-000015
人和鼠miR-135核心序列，在DNA水平的相似性为100％；
miR-137的核苷酸序列
人miR-137编码序列：5’-acgggtattcttgggtggataat-3’(SEQ ID NO：39)
人miR-137＊编码序列：5’-ttattgcttaagaatacgcgtag-3’(SEQ ID NO：40)
人miR-137 Per-miRNA全长
Figure PCTCN2022136909-appb-000016
人和鼠miR-137核心序列，在DNA水平的相似性为100％；
miR-141的核苷酸序列
人miR-141编码序列：5’-catcttccagtacagtgttgga-3’(SEQ ID NO：42)
人miR-141＊编码序列：5’-taacactgtctggtaaagatgg-3’(SEQ ID NO：43)
人miR-141 Per-miRNA全长
Figure PCTCN2022136909-appb-000017
人和鼠miR-141核心序列，在DNA水平的相似性为95.45％；
miR-143的核苷酸序列
人miR-143编码序列：5’-ggtgcagtgctgcatctctggt-3’(SEQ ID NO：45)
人miR-143＊编码序列：5’-tgagatgaagcactgtagctc-3’(SEQ ID NO：46)
人miR-143 Per-miRNA全长
Figure PCTCN2022136909-appb-000018
人和鼠miR-143核心序列，在DNA水平的相似性为100％；
miR-184的核苷酸序列
人miR-184编码序列：5’-ccttatcacttttccagcccagc-3’(SEQ ID NO：48)
人miR-184＊编码序列：5’-tggacggagaactgataagggt-3’(SEQ ID NO：49)
人miR-184 Per-miRNA全长
Figure PCTCN2022136909-appb-000019
人和鼠miR-184核心序列，在DNA水平的相似性为95.45％；
miR-200的核苷酸序列
人miR-200编码序列
miR-200a：5’-catcttaccggacagtgctgga-3’(SEQ ID NO：51)
miR-200b：5’-catcttactgggcagcattgga-3’(SEQ ID NO：52)
miR-200c：5’-cgtcttacccagcagtgtttgg-3’(SEQ ID NO：53)
人miR-200编码序列
miR-200a：5’-taacactgtctggtaacgatgt-3’(SEQ ID NO：54)
miR-200b：5’-taatactgcctggtaatgatga-3’(SEQ ID NO：55)
miR-200c：5’-taatactgccgggtaatgatgga-3’(SEQ ID NO：56)
人miR-200 Per-miRNA全长
miR-200a：
Figure PCTCN2022136909-appb-000020
miR-200b：
Figure PCTCN2022136909-appb-000021
miR-200c：
Figure PCTCN2022136909-appb-000022
人和鼠miR-200a/b/c核心序列，在DNA水平的相似性为100％；
miR-218的核苷酸序列
人miR-218-1编码序列：5’-ttgtgcttgatctaaccatgt-3’(SEQ ID NO：60)
人miR-218-1＊编码序列：5’-atggttccgtcaagcaccatgg-3’(SEQ ID NO：61)
人miR-218-2＊编码序列：5’-catggttctgtcaagcaccgcg-3’(SEQ ID NO：62)
人miR-218 Per-miRNA全长
miR-218-1：
Figure PCTCN2022136909-appb-000023
miR-218-2：
Figure PCTCN2022136909-appb-000024
人和鼠miR-218-1/2核心序列，在DNA水平的相似性为100％；
miR-219的核苷酸序列
人miR-219-1编码序列：5’-tgattgtccaaacgcaattct-3’(SEQ ID NO：65)
人miR-219-1＊编码序列：5’-agagttgagtctggacgtcccg-3’(SEQ ID NO：66)
人miR-219-2编码序列：5’-tgattgtccaaacgcaattct-3’(SEQ ID NO：67)
人miR-219-2＊编码序列：5’-agaattgtggctggacatctgt-3’(SEQ ID NO：68)
人miR-219 Per-miRNA全长
miR-219-1：
Figure PCTCN2022136909-appb-000025
miR-219-2：
Figure PCTCN2022136909-appb-000026
人和鼠miR-219-1/2核心序列，在DNA水平的相似性为100％；
miR-429的核苷酸序列
人miR-429编码序列：5’-gtcttaccagacatggttaga-3’(SEQ ID NO：71)
人miR-429＊编码序列：5’-taatactgtctggtaaaaccgt-3’(SEQ ID NO：72)
人miR-429 Per-miRNA全长
Figure PCTCN2022136909-appb-000027
人和鼠miR-429核心序列，在DNA水平的相似性为100％；
miR-430的核苷酸序列
人miR-430编码序列：5’-acttaaacgtggatgtacttgct-3’(SEQ ID NO：74)
人miR-430＊编码序列：5’-taagtgcttccatgttttggtga-3’(SEQ ID NO：75)
人miR-430 Per-miRNA全长
Figure PCTCN2022136909-appb-000028
miR-7a的核苷酸序列
人miR-7a编码序列：5’-tggaagactagtgattttgttgtt-3’(SEQ ID NO：77)
人miR-7a＊编码序列：5’-caacaaatcacagtctgccata-3’(SEQ ID NO：78)
人miR-7a Per-miRNA全长
Figure PCTCN2022136909-appb-000029
3.人和鼠miR-7a核心序列，在DNA水平的相似性为100％；
miR-15的核苷酸序列
人miR-15-1编码序列：5’-tagcagcacataatggtttgtg-3’(SEQ ID NO：80)
人miR-15-1＊编码序列：5’-caggccatattgtgctgcctca-3’(SEQ ID NO：81)
人miR-15-2编码序列：5’-tagcagcacatcatggtttaca-3’(SEQ ID NO：82)
人miR-15-2＊编码序列：5’-cgaatcattatttgctgctcta-3’(SEQ ID NO：83)
人miR-15 Per-miRNA全长
miR-15-1：
Figure PCTCN2022136909-appb-000030
miR-15-2：
Figure PCTCN2022136909-appb-000031
人和鼠miR-15核心序列，在DNA水平的相似性为100％；
miR-23的核苷酸序列
人miR-23编码序列：5’-ggggttcctggggatgggattt-3’(SEQ ID NO：86)
人miR-23＊编码序列：5’-atcacattgccagggatttcc-3’(SEQ ID NO：87)
人miR-23 Per-miRNA全长
Figure PCTCN2022136909-appb-000032
人和鼠miR-23核心序列，在DNA水平的相似性为100％；
miR-25的核苷酸序列
人miR-25编码序列：5’-aggcggagacttgggcaattg-3’(SEQ ID NO：89)
人miR-25＊编码序列：5’-cattgcacttgtctcggtctga-3’(SEQ ID NO：90)
人miR-25 Per-miRNA全长
Figure PCTCN2022136909-appb-000033
人和鼠miR-25核心序列，在DNA水平的相似性为100％；
miR-29a的核苷酸序列
人miR-29a编码序列：5’-actgatttcttttggtgttcag-3’(SEQ ID NO：92)
人miR-29a＊编码序列：5’-tagcaccatctgaaatcggtta-3’(SEQ ID NO：93)
人miR-29a Per-miRNA全长
Figure PCTCN2022136909-appb-000034
人和鼠miR-29a核心序列，在DNA水平的相似性为100％；
miR-129的核苷酸序列
人miR-129-1编码序列：5’-ctttttgcggtctgggcttgc-3’(SEQ ID NO：95)
人miR-129-1＊编码序列：5’-aagcccttaccccaaaaagtat-3’(SEQ ID NO：96)
人miR-129-2编码序列：5’-ctttttgcggtctgggcttgc-3’(SEQ ID NO：97)
人miR-129-2＊编码序列：5’-aagcccttaccccaaaaagcat-3’(SEQ ID NO：98)
人miR-129 Per-miRNA全长
miR-129-1：
Figure PCTCN2022136909-appb-000035
miR-129-2：
Figure PCTCN2022136909-appb-000036
人和鼠miR-129-1/2核心序列，在DNA水平的相似性为100％；
miR-137的核苷酸序列
人miR-137编码序列：5’-acgggtattcttgggtggataat-3’(SEQ ID NO：101)
人miR-137＊编码序列：5’-ttattgcttaagaatacgcgtag-3’(SEQ ID NO：102)
人miR-137 Per-miRNA全长
Figure PCTCN2022136909-appb-000037
人和鼠miR-137核心序列，在DNA水平的相似性为100％；
miR-138的核苷酸序列
人miR-138-1编码序列：5’-agctggtgttgtgaatcaggccg-3’(SEQ ID NO：104)
人miR-138-1＊编码序列：5’-gctacttcacaacaccagggcc-3’(SEQ ID NO：105)
人miR-138-2编码序列：5’-agctggtgttgtgaatcaggccg-3’(SEQ ID NO：106)
人miR-138-2＊编码序列：5’-gctatttcacgacaccagggtt-3’(SEQ ID NO：107)
人miR-138 Per-miRNA全长
miR-138-1：
Figure PCTCN2022136909-appb-000038
miR-138-2：
Figure PCTCN2022136909-appb-000039
人和鼠miR-138-1/2核心序列，在DNA水平的相似性为100％；
miR-155的核苷酸序列
人miR-155编码序列：5’-ttaatgctaatcgtgataggggtt-3’(SEQ ID NO：110)
人miR-155＊编码序列：5’-ctcctacatattagcattaaca-3’(SEQ ID NO：111)
人miR-155 Per-miRNA全长
Figure PCTCN2022136909-appb-000040
人和鼠miR-155核心序列，在DNA水平的相似性为95.8％；
miR-195的核苷酸序列
人miR-195编码序列：5’-tagcagcacagaaatattggc-3’(SEQ ID NO：113)
人miR-195＊编码序列：5’-ccaatattggctgtgctgctcc-3’(SEQ ID NO：114)
人miR-195Per-miRNA全长
Figure PCTCN2022136909-appb-000041
人和鼠miR-195核心序列，在DNA水平的相似性为100％；
miR-214的核苷酸序列
人miR-214编码序列：5’-tgcctgtctacacttgctgtgc-3’(SEQ ID NO：116)
人miR-214＊编码序列：5’-acagcaggcacagacaggcagt-3’(SEQ ID NO：117)
人miR-214Per-miRNA全长
Figure PCTCN2022136909-appb-000042
人和鼠miR-214核心序列，在DNA水平的相似性为100％；
miR-222的核苷酸序列
人miR-222编码序列：5’-ctcagtagccagtgtagatcct-3’(SEQ ID NO：119)
人miR-222＊编码序列：5’-agctacatctggctactgggt-3’(SEQ ID NO：120)
人miR-222 Per-miRNA全长
Figure PCTCN2022136909-appb-000043
人和鼠miR-222核心序列，在DNA水平的相似性为100％；
miR-223的核苷酸序列
人miR-223编码序列：5’-cgtgtatttgacaagctgagtt-3’(SEQ ID NO：122)
人miR-223＊编码序列：5’-tgtcagtttgtcaaatacccca-3’(SEQ ID NO：123)
人miR-223 Per-miRNA全长
Figure PCTCN2022136909-appb-000044
人和鼠miR-223核心序列，在DNA水平的相似性为100％；
miR-132的核苷酸序列
人miR-132编码序列：5’-accgtggctttcgattgttact-3’(SEQ ID NO：125)
人miR-132＊编码序列：5’-taacagtctacagccatggtcg-3’(SEQ ID NO：126)
人miR-132 Per-miRNA全长
Figure PCTCN2022136909-appb-000045
人和鼠miR-132核心序列，在DNA水平的相似性为100％；
miR-133的核苷酸序列
人miR-133编码序列：5’-agctggtaaaatggaaccaaat-3’(SEQ ID NO：128)
人miR-133＊编码序列：5’-tttggtccccttcaaccagctg-3’(SEQ ID NO：129)
人miR-133 Per-miRNA全长
Figure PCTCN2022136909-appb-000046
人和鼠miR-133核心序列，在DNA水平的相似性为100％。
lncRNA的序列信息
Human-Pnky
Figure PCTCN2022136909-appb-000047
Human-Paupar
Figure PCTCN2022136909-appb-000048
Figure PCTCN2022136909-appb-000049
human-HOTAIRM1
Figure PCTCN2022136909-appb-000050
human-TUG1
Figure PCTCN2022136909-appb-000051
Figure PCTCN2022136909-appb-000052
Human-RMST
Figure PCTCN2022136909-appb-000053
Mouse-RMST
Figure PCTCN2022136909-appb-000054
human-tuna
Figure PCTCN2022136909-appb-000055
Figure PCTCN2022136909-appb-000056
mouse-tuna
Figure PCTCN2022136909-appb-000057
human-Linc-Brn1b
Figure PCTCN2022136909-appb-000058
Figure PCTCN2022136909-appb-000059
mouse-Linc-Brn1b
Figure PCTCN2022136909-appb-000060
Mouse-Dali
Figure PCTCN2022136909-appb-000061
human-miat
Figure PCTCN2022136909-appb-000062
Figure PCTCN2022136909-appb-000063
Figure PCTCN2022136909-appb-000064
mouse-miat
Figure PCTCN2022136909-appb-000065
human-NBAT-1
Figure PCTCN2022136909-appb-000066
Figure PCTCN2022136909-appb-000067
human-Malat1
Figure PCTCN2022136909-appb-000068
Figure PCTCN2022136909-appb-000069
mouse-Malat1
Figure PCTCN2022136909-appb-000070
Figure PCTCN2022136909-appb-000071
mouse-Dlx1as
Figure PCTCN2022136909-appb-000072
human-Six3os
Figure PCTCN2022136909-appb-000073
mouse-Six3os
Figure PCTCN2022136909-appb-000074
Figure PCTCN2022136909-appb-000075
human-Evf2
Figure PCTCN2022136909-appb-000076
Figure PCTCN2022136909-appb-000077
mouse-Evf2
Figure PCTCN2022136909-appb-000078
human-lncRNA N1
Figure PCTCN2022136909-appb-000079
human-lncRNA N2
Figure PCTCN2022136909-appb-000080
Figure PCTCN2022136909-appb-000081
实施例
下面结合具体实施例，进一步阐述本公开内容。应理解，这些实施例仅用于举例说明本公开内容而不用于限制本公开内容的范围。
实施例1.敲低Pnky介导的胶质细胞向神经元转分化
为了研究CasRx敲低Pnky的效率，我们在293T细胞中分别转染对照组质粒和实验组质粒，Q-PCR结果表明在人类细胞中可以利用CasRx高效敲低Pnky的表达，Pnky敲低实验组的Pnky表达只有对照组的0.5％(图1A)。为了进一步在体内利用CasRx对Pnky进行编辑，我们构建了一套AAV表达系统，用胶质细胞特异性启动子GFAP来启动mCherry的表达，以特异性标记星形胶质细胞，同时用GFAP启动CasRx的表达，实现特异性在星形胶质细胞中表达CasRX(图1B)。在小鼠大脑中注射不同的AAV组合，对照组注射GFAP-mCherry+GFAP-CasRx，实验组注射GFAP-mCherry+GFAP-CasRx-gRNA(Pnky)，其中GFAP-mCherry标记星形胶质细胞，在注射后1-2个月进行取材分析(图1C)。
实施例2.miRNA和lncRNA介导的胶质细胞向神经元转分化
为了在体内探究miRNA和lncRNA是否可以将胶质细胞转分化为神经元，我们构建了AAV系统，利用AAV-GFAP-mCherry标记胶质细胞，并利用胶质细胞特异性启动子GFAP启动miRNA/LncRNA的在胶质细胞中表达(图2A)。为了探究Tuna，Let-7b和miRNA-137的功能，我们分别构建了AAV-GFAP-Tuna，AAV-GFAP-Let-7b和AAV-GFAP-miRNA-137的AAV表达载体(图2B)。将AAV注射到小鼠脑部纹状体中，在注射后的1-2个月分析，并用神经元特异性标志物NeuN进行免疫荧光染色，GFAP-mCherry标记的胶质细胞转分化为神经元后会与NeuN共标(图2C)。在对照组中，注射了GFAP-mCherry后1个月的结果显示，胶质细胞依然保持着胶质细胞的典型特征，并且不与神经元特异性标志物NeuN共标(图2D)。而在GFAP-Tuna组，红色荧光标记的细胞已经明显具有神经元的形态，胞体变圆，同时具 有一些较长的而且较细的突起，进一步用神经元特异性标志物NeuN进行染色，发现红色细胞与NeuN重叠，这表明这些红色的细胞已经从胶质细胞转分化为神经元(图2E)。与此类似，在GFAP-Let-7b组和GFAP-miRNA-137组有一部分红色细胞具有典型神经元形态，而且与神经元特异性标志物NeuN共标。与此同时我们在GFAP-Let-7b组和GFAP-miRNA-137组也观察到有一部分红色细胞依然保持胶质细胞形态，这表明有一部分胶质细胞转分化为神经元，有一部分细胞并未发生转分化，依然是星形胶质细胞(图2F和G)。
实施例3.过表达miRNA或lncRNA诱导的胶质细胞向神经元转分化
为了进一步研究在miR-18b，miR-24，miR-34a，miR-128，miR-134，miR-143，Let-7a，miR-92b，miR-96，miR-106，miR-125a，miR-135，miR-141，miR-200，miR-218，miR-429等miRNA是否也能将星形胶质细胞转分化为神经元，我们构建了由星形胶质细胞特异性启动子GFAP启动的miR-18b，miR-24，miR-34a，miR-128，miR-134，miR-143，Let-7a，miR-92b，miR-96，miR-106，miR-125a，miR-135，miR-141，miR-200，miR-218，miR-429表达载体，并包装AAV。各组AAV与AAV-GFAP-EGFP进行混合后注射到小鼠纹状体中，其中AAV-GFAP-EGFP可以特异性将纹状体中的星形胶质细胞标记为绿色。在注射后1-2个月进行取材分析，发现在对照组，绿色荧光信号标记的细胞仍然保持着典型的星形胶质细胞形态，而miR-18b，miR-24，miR-34a，miR-128，miR-134，miR-143，Let-7a，miR-92b，miR-96，miR-106，miR-125a，miR-135，miR-141，miR-200，miR-218，miR-429等miRNA过表达组中发现有大量AAV-GFAP-EGFP标记的星形胶质细胞已经转变为神经元形态(图3B-G，图4A-F和图5A-F)。进一步通过神经元特异性蛋白标志物进行染色，发现虽然有些细胞表达NeuN还比较弱，但大部分具有典型神经元形态的细胞已经表达NeuN(图3B-G，图4A-F和图5A-F)。为了进一步研究是否能转分化产生多巴胺神经元，我们用多巴胺神经元特异性蛋白标志物TH进行免疫荧光染色，发现在Let-7a，miR-92b，miR-96，miR-106，miR-125a，miR-135，miR-141，miR-200，miR-218，miR-429等miRNA过表达组发现有TH阳性细胞，其中miR-106，Let-7a，miR-141，miR-200这几个组的TH阳性细胞较多，而且TH阳性细胞比例较高(图6)。这表明，过表达miR-18b，miR-24，miR-34a，miR-128，miR-134，miR-143，Let-7a，miR-92b，miR-96，miR-106，miR-125a，miR-135，miR-141，miR-200，miR-218，miR-429等miRNA能将星形胶质细胞转分化为神经元。而且，过表达Let-7a，miR-92b，miR-96，miR-106，miR-125a，miR-135，miR-141，miR-200，miR-218，miR-429，miR-24等miRNA不仅能将星形胶质细胞转分化为神经元，而且能将其转分化为多巴胺神经元。
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 (29)

  1. 由非神经元细胞产生神经元细胞的方法，其包括通过使选自Let-7a、Let-7b、miR-18a/b、miR-24-3p、miR-34a、miR-92b、miR-96、miR-106、miR-125a/b、miR-128、miR-134、miR-135、miR-137、miR-141、miR-143-3p、miR-184、miR-200、miR-218、miR-219、miR-228、miR-284、miR-429、miR-430的miRNA、或者选自utNgn1、RMST、Tuna、Linc-Brn1b、Dali、Miat/Gomafu、NBAT-1、Malat1、Dlx1as、Six3os、Evf2、LncKdm2b、lncRNA_N1、lncRNA_N2、lncRNA_N3的lncRNA，或其任意组合的表达或活性增强来使所述非神经元细胞转分化为或重编程为神经元细胞。
  2. 根据权利要求1所述的方法，其中通过例如过表达、基因激活剂、表观遗传修饰、miRNA模拟物、直接递送RNA、小分子化合物、和/或RNA稳定剂来增强所述miRNA或lncRNA的表达或活性。
  3. 由非神经元细胞产生神经元细胞的方法，其包括通过使选自miR-7a、miR-15、miR-23a/b、miR-25、miR-29a、miR-129、miR-137、miR-138、miR-155、miR-195、miR-214、miR-222、miR-223、miR-132、miR-133的miRNA、或者选自Pnky、Paupar、HOTAIRM1、lncR492、TUG1的lncRNA，或其任意组合的表达或活性降低来使所述非神经元细胞转分化为或重编程为神经元细胞。
  4. 根据权利要求3所述的方法，其中通过例如基因编辑技术介导的DNA编辑和RNA编辑、RNA表达抑制剂、反义寡核苷酸、小RNA干扰、miRNA技术、小分子化合物、基因抑制技术、和/或表观遗传调控来降低所述miRNA或lncRNA的表达或活性。
  5. 根据权利要求4所述的方法，其中RNA编辑包括CRISPR介导的RNA降解和RNA翻译抑制、RNA单碱基编辑、RNA上碱基的插入或缺失、RNA剪切的改变、RNA表观修饰。
  6. 根据权利要求1或权利要求3所述的方法，其中所述选自Let-7a、Let-7b、miR-18a/b、miR-24-3p、miR-34a、miR-92b、miR-96、miR-106、miR-125a/b、miR-128、miR-134、miR-135、miR-137、miR-141、miR-143-3p、miR-184、miR-200、miR-218、miR-219、miR-228、miR-284、miR-429、miR-430的miRNA、或选自miR-7a、miR-15、miR-23a/b、miR-25、miR-29a、miR-129、miR-137、miR-138、miR-155、miR-195、miR-214、miR-222、miR-223、miR-132、miR-133的miRNA、或者选自utNgn1、RMST、Tuna、Linc-Brn1b、Dali、Miat/Gomafu、NBAT-1、Malat1、Dlx1as、Six3os、Evf2、LncKdm2b、lncRNA_N1、lncRNA_N2、lncRNA_N3的lncRNA、或选自Pnky、Paupar、HOTAIRM1、lncR492、TUG1的lncRNA，为来自不同物种的同源miRNA或同源lncRNA。
  7. 根据前述权利要求中任一项所述的方法，其中所述非神经元细胞包括例如胶质细胞、成纤维细胞、干细胞、神经前体细胞、神经干细胞，其中胶质细胞选自星形胶质细胞、小胶质细胞、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。
  8. 根据权利要求7所述的方法，其中所述胶质细胞来源于脑、脊髓、眼或耳，其中脑部胶质细胞来源于纹状体、黑质、中脑腹侧被盖区、脊髓、下丘脑、背侧中脑或大脑皮层，优选纹状体和黑质。
  9. 根据前述权利要求中任一项所述的方法，其中所述神经元细胞优选多巴胺神经元、GABA神经元、5-HT神经元、谷氨酸能神经元、ChAT神经元、NE神经元、运动神经元、脊髓神经元、脊髓运动神经元、脊髓感觉神经元、锥体神经元、中间神经元、中型多棘神经元、浦肯野细胞、颗粒细胞、嗅感觉神经元、球周细胞或其组合，更优选多巴胺神经元。
  10. 根据前述权利要求中任一项所述的方法，其中所述非神经元细胞和/或神经元细胞来自例如人、非人灵长类动物、大鼠和小鼠，优选人。
  11. 根据前述权利要求中任一项所述的方法，其中所述方法为体内方法或体外方法。
  12. 根据权利要求1至2和6至11中任一项所述的方法，其中所述miRNA或lncRNA为Let-7a、miR-92b、miR-96、miR-106、miR-125a、miR-135、miR-141、miR-200、miR-218、miR-429，miR-24，所述非神经元细胞为胶质细胞，所述神经元细胞为多巴胺神经元。
  13. 使选自Let-7a、Let-7b、miR-18a/b、miR-24-3p、miR-34a、miR-92b、miR-96、miR-106、miR-125a/b、miR-128、miR-134、miR-135、miR-137、miR-141、miR-143-3p、miR-184、miR-200、miR-218、miR-219、miR-228、miR-284、miR-429、miR-430的miRNA、或者选自utNgn1、RMST、Tuna、Linc-Brn1b、Dali、Miat/Gomafu、NBAT-1、Malat1、Dlx1as、Six3os、Evf2、LncKdm2b、lncRNA_N1、lncRNA_N2、lncRNA_N3的lncRNA，或其任意组合的表达或活性增强的试剂在制备用于预防和/或治疗与神经元功能缺失或死亡相关疾病的药物的用途，其中所述试剂增强所述miRNA或所述lncRNA的表达或活性。
  14. 使选自miR-7a、miR-15、miR-23a/b、miR-25、miR-29a、miR-129、miR-137、miR-138、miR-155、miR-195、miR-214、miR-222、miR-223、miR-132、miR-133的miRNA、或者选自Pnky、Paupar、HOTAIRM1、lncR492、TUG1的lncRNA，或其任意组合的表达或活性降低的试剂在制备用于预防和/或治疗与神经元功能缺失或死亡相关疾病的药物的用途，其中所述试剂降低所述miRNA或所述lncRNA的表达或活性。
  15. 根据权利要求13或14所述的用途，其中所述药物配制成用于在体内施用于神经系统，例如纹状体、黑质、中脑腹侧被盖区、脊髓、下丘脑、背侧中脑、大脑皮层、海马、小脑，优选纹状体和黑质。
  16. 根据权利要求13至15中任一项所述的用途，其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、阿尔茨海默病、脑卒中、精神分裂症、亨廷顿舞蹈症、抑郁症、运动神经元病、脑缺血、脑损伤、肌萎缩性侧索硬化、脊髓性肌萎缩症、Pick病、睡眠失调、癫痫、共济失调、PloyQ疾病、成瘾，优选帕金森病。
  17. 根据前述权利要求中任一项所述的方法或用途，其中所述使所述miRNA或lncRNA 或其组合的表达或活性增强包括：
    (a)外源表达所述miRNA或lncRNA或其组合，例如通过包含启动子的表达载体来实现；
    (b)向细胞内递送DNA或RNA形式的所述miRNA或lncRNA或其组合；
    (c)激活所述miRNA或lncRNA或其组合的内源表达，如基因表达激活剂和表观调控元件等；或者
    (d)向细胞递送所述miRNA或lncRNA或其组合的类似物或激动剂；
    其中优选外源表达所述miRNA或lncRNA或其组合，例如通过包含启动子的表达载体来实现。
  18. 根据前述权利要求中任一项所述的方法或用途，其中通过以下使所述miRNA或lncRNA或其组合的表达或活性降低：抗体、小分子化合物、microRNA、siRNA、shRNA、反义寡核苷酸、结合蛋白或蛋白结构域、多肽、核酸适配体、基因编辑器、表观调控元件、转录抑制元件或其组合。
  19. 根据权利要求13和15至18中任一项所述的方法，其中所述miRNA或lncRNA为Let-7a、miR-92b、miR-96、miR-106、miR-125a、miR-135、miR-141、miR-200、miR-218、miR-429，miR-24，所述非神经元细胞为胶质细胞，所述神经元细胞为多巴胺神经元。
  20. 药物组合物或药盒或试剂盒，其包含使选自Let-7a、Let-7b、miR-18a/b、miR-24-3p、miR-34a、miR-92b、miR-96、miR-106、miR-125a/b、miR-128、miR-134、miR-135、miR-137、miR-141、miR-143-3p、miR-184、miR-200、miR-218、miR-219、miR-228、miR-284、miR-429、miR-430的miRNA、或者选自utNgn1、RMST、Tuna、Linc-Brn1b、Dali、Miat/Gomafu、NBAT-1、Malat1、Dlx1as、Six3os、Evf2、LncKdm2b、lncRNA_N1、lncRNA_N2、lncRNA_N3的lncRNA，或其任意组合的表达或活性增强的试剂；或者使选自miR-7a、miR-15、miR-23a/b、miR-25、miR-29a、miR-129、miR-137、miR-138、miR-155、miR-195、miR-214、miR-222、miR-223、miR-132、miR-133的miRNA、或者选自Pnky、Paupar、HOTAIRM1、lncR492、TUG1的lncRNA，或其任意组合的表达或活性降低的试剂。
  21. 根据权利要求20的药物组合物或药盒或试剂盒，其中使所述miRNA或lncRNA或其组合的表达或活性增强的试剂选自：表达载体，DNA或RNA形式的所述miRNA或lncRNA或其组合，所述miRNA或lncRNA或其组合的内源性激活剂，所述miRNA或lncRNA或其组合的类似物或激动剂。
  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：腺相关病毒载体、重组腺相关病毒载体、自互补AAV、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。
  23. 根据权利要求20的药物组合物或药盒或试剂盒，其中使所述miRNA或lncRNA或其组合的表达或活性降低的试剂选自：抗体、小分子化合物、microRNA、siRNA、shRNA、 反义寡核苷酸、结合蛋白或蛋白结构域、多肽、核酸适配体、基因编辑器、表观调控元件、转录抑制元件，或其组合。
  24. 根据权利要求20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试剂的载体或运载体。
  25. 根据权利要求24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。
  26. 根据权利要求20至25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：i)纹状体中的胶质细胞；ii)腹侧被盖区中的胶质细胞；iii)黑质中的胶质细胞；iv)下丘脑中的胶质细胞；v)脊髓中的胶质细胞；vi)前额皮质中的胶质细胞；以及vii)运动皮质中的胶质细胞。
  27. 根据权利要求20至26中任一项的药物组合物或药盒或试剂盒，其配制成用于细胞转染、细胞感染、细胞内吞、注射、颅内给药、眼内给药、内耳注射、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。
  28. 根据权利要求20至27中任一项所述的方法，其中所述miRNA或lncRNA为Let-7a、miR-92b、miR-96、miR-106、miR-125a、miR-135、miR-141、miR-200、miR-218、miR-429，miR-24，所述非神经元细胞为胶质细胞，所述神经元细胞为多巴胺神经元。
  29. 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，其中非神经元细胞的转分化效率为至少1％，或至少10％、20％、30％、40％或50％。
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150037299A1 (en) * 2012-02-22 2015-02-05 Brainstem Biotec Ltd. Generation of neural stem cells and motor neurons
US20150037298A1 (en) * 2012-02-22 2015-02-05 Brainstem Biotec Ltd. MicroRNAS FOR THE GENERATION OF ASTROCYTES
US20190015453A1 (en) * 2012-02-22 2019-01-17 Exostem Biotec Ltd. MicroRNAS FOR THE GENERATION OF ASTROCYTES
CN109674809A (zh) * 2018-12-27 2019-04-26 吉林大学 一种包括miR-124-3P的组合物及其在诱导神经元形成的药物中的应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150037299A1 (en) * 2012-02-22 2015-02-05 Brainstem Biotec Ltd. Generation of neural stem cells and motor neurons
US20150037298A1 (en) * 2012-02-22 2015-02-05 Brainstem Biotec Ltd. MicroRNAS FOR THE GENERATION OF ASTROCYTES
US20190015453A1 (en) * 2012-02-22 2019-01-17 Exostem Biotec Ltd. MicroRNAS FOR THE GENERATION OF ASTROCYTES
CN109674809A (zh) * 2018-12-27 2019-04-26 吉林大学 一种包括miR-124-3P的组合物及其在诱导神经元形成的药物中的应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
YANG HUI, WAN GUANG, GAO XUAN-ZHAO, LIU YI, WANG SHOU-CHUN: "let-7a inhibits MAP4K3/MKK4/JNK signaling pathway to decrease neuronal apoptosis in rats with intracerebral hemorrhage", CHINESE JOURNAL OF PATHOPHYSIOLOGY, vol. 36, no. 6, 25 June 2022 (2022-06-25), pages 1055 - 1062, XP093068818, DOI: 1000-4718(2020)06-1055-08 *

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