Treatment of neurological disorders

Abstract

The present application provides methods and compositions for treating certain neurodegenerative diseases (e.g., RGC loss-associated degenerative diseases and Parkinson's disease) using CRISPR / Cas delivered via a viral vector (e.g., AAV vector) to convert glial cells into neurons in vivo by knocking down PTB and any nPTB.

Description

[Technical Field]

[0001] This application relates to the fields of regenerative medicine, neurology and biopharmaceuticals. In particular, this application relates to methods and compositions for differentiating non-neuronal cells into neuronal cells to treat neurodegenerative diseases and conditions. [Background technology]

[0002] Neurodegenerative diseases are destructive disorders associated with the progressive loss of neurons in various parts of the nervous system. Meanwhile, regenerative medicine holds promise in the treatment of neurodegenerative diseases that cause cell (e.g., neuron) loss. One approach employs cell replacement, while another employs cell transdifferentiation. Transdifferentiation exploits the existing cellular plasticity of endogenous cells to generate novel cell types. However, one challenge of this method is identifying effective strategies for converting specific target cells into desired cell types (e.g., neurons) not only in culture but also in their native environment in vivo, particularly in the desired location (e.g., tissue or organ type).

[0003] Recent studies have shown that downregulating a single gene, polypyrimidine tract-binding protein 1 (Ptbp1), in the striatum directly converts mouse astrocytes into dopaminergic neurons and alleviates symptoms in a mouse model of Parkinson's disease (Zhou et al., 2020, Cell 181:590-603). However, it remains unclear whether this strategy can be applied to nonhuman primates or humans. Many experimental therapies that have demonstrated efficacy in rodent models may be ineffective or impractical in nonhuman primates or humans. These strategies may fail for a variety of reasons, including inherent biological differences and lack of scalability. Nonhuman primate studies are an important step and offer significant value in resolving these issues before clinical trials.

[0004] It is therefore an object of the present application to provide methods and compositions for differentiating non-neuronal cells into neuronal cells for the treatment of neurodegenerative diseases and conditions in primates, i.e., human and non-human primates. Summary of the Invention

[0005] In a first aspect, the present application relates to a method of treating a neurological condition associated with degeneration of functional neurons in a region of the nervous system of a non-human primate or human.

[0006] In one embodiment, the method comprises administering to a non-neuronal cell in a region of the nervous system of a subject in need thereof an effective amount of a composition comprising a nucleic acid molecule that targets a target sequence in a primate PTB gene, transcript, or mRNA, or its complementary sequence, to inhibit expression or activity of a primate PTB protein in the non-neuronal cell and enable reprogramming of the non-neuronal cell into a functional neuron, wherein the target sequence is comprised of a sequence at least 95% identical to SEQ ID NO:87, and when the target sequence is targeted with a guide RNA (gRNA), the guide sequence has at least a 10-nucleotide overlap with at least one of SEQ ID NOs:1-86, and the gRNA is co-expressed with CasRx in at least one of Cos7 cells and 293T cells, and a relative expression of PTB mRNA of less than 0.5 is observed in the cells compared to corresponding control cells expressing CasRx alone.

[0007] SEQ ID NO:87: atggacggca ttgtcccaga tatagccgtt ggtacaaagc ggggatctga cgagcttttc 60 tctacttgtg tcactaacgg accgtttatc atgagcagca actcggcttc tgcagcaaac 120 ggaaatgaca gcaagaagtt caaaggtgac agccgaagtg caggcgtccc ctctagagtg 180 atccacatcc ggaagctccc catcgacgtc acggaggggg aagtcatctc cctggggctg 240 ccctttgggga aggtcaccaa cctcctgatg ctgaagggga aaaaccaggc cttcatcgag atgaacacgg aggaggctgc cacaccatg gtgaactact acacctcggt gacccctgtg 360 ctgcgcggcc agcccatcta catccagttc tccaaccaca aggagctgaa gaccgacagc tctcccaacc agggcggggc ccaggcggcc ctgcaggcgg tgaactcggt ccagtcgggg 480 aacctggcct tggctgcctc ggcggcggcc gtggacgcag ggatggcgat ggccgggcag 540 agccccgtgc tcaggatcat cgtggagaac ctcttctacc ctgtgaccct ggatgtgctg 600 caccagattt tctccaagtt cggcacagtg ttgaagatca tcaccttcac caagacaac cagttccagg ccctgctgca gtatgcggac cccgtgagcg cccagcacgc caagctgtcg 720 ctggacgggc agaacatcta caacgcctgc tgcacgctgc gcatcgactt ttccaagctc 780 accagcctca acgtcaagta caacaatgac aagagccgtg actacacacg cccagacctg ccttccgggg acagccagcc ctcgctggac cagaccatgg ccgcggcctt cggtgcacct 900 ggtataatct cagcctctcc gtagcagga gctggtttcc ctcccacctt tgccattcct 960 caagctgcag gcctttccgt tccgacgtc cacggcgccc tggccccct ggccatcccc 1020 tcggcggcgg cggcagctgc ggcggcaggt cggatcgcca tcccggcct ggcgggggca 1080 ggaaattctg tattgctggt cagcaacctc aacccagaga gagtcacacc ccaagccctc 1140 tttattcttt tcggcgtcta cggtgacgtg cagcgcgtga agatcctgtt caataagaag 1200 gagaacgccc tagtgcagat ggcggacggc aaccaggccc agctggccat gagccacctg 1260 aacggggcaca agctgcacgg gaagcccatc cgcatcacgc tctcgaagca ccagaacgtg 1320 cagctgcccc gcgaggggcca ggaggaccag ggcctgacca aggactacgg CAACTCACC 1380 ctgcaccgct tcagaagcc gggctccaag aacttccaga acatattccc gccctcggcc 1440 acgctgcacc tctccacat cccgccctca gtctccgagg aggatctca gttcctgttt 1500 tccagcaatg gggcgtcgt aaggattc aagttctcc agaggaccg caatggca 1560 ctgatccaga tggctccgt gggaggcg gtccaggccc tcattgacct gcacaccac 1620 gacctcgggg agaaccacca cctgcgggtc tccttctcca agtccaccat ctag 1674

[0008] In one embodiment, a method comprises administering to a non-neuronal cell in a region of the nervous system of a subject in need thereof an effective amount of a composition comprising a nucleic acid molecule that targets a target sequence in a primate PTB gene, transcript, or mRNA, or its complement, to inhibit expression or activity of PTB in the non-neuronal cell and enable reprogramming of the non-neuronal cell into a functional neuron, wherein the target sequence is comprised in a sequence that is at least 95% identical to positions 951-1487 of SEQ ID NO: 87. In some embodiments of the method, when the target sequence is targeted with a guide RNA (gRNA), the guide sequence has at least a 10-nucleotide overlap with at least one of SEQ ID NOs: 38-68, and the gRNA is co-expressed with CasRx in at least one of Cos7 cells and 293T cells, and a relative expression of PTB mRNA of less than 0.4 is observed compared to corresponding control cells expressing CasRx alone.

[0009] In one embodiment, the method is a method wherein the nucleic acid molecule is or encodes at least one of an antisense nucleic acid, an RNAi molecule, or a guide RNA (gRNA). In some embodiments, the method is a method wherein the composition comprises i) a Cas effector protein and at least one gRNA, or ii) at least one expression vector encoding a Cas effector protein and at least one gRNA, optionally wherein the Cas effector protein and at least one gRNA, or the at least one expression vector, are contained in a nanoparticle, preferably a liposome.

[0010] In one embodiment, the method is a method in which a) the Cas effector protein is an RNA-targeting Cas effector protein, and b) the gRNA comprises a guide sequence complementary to a contiguous sequence of 17 to 60 nucleotides in a sequence at least 95% identical to SEQ ID NO: 87. In some embodiments, the method is a method in which the RNA-targeting Cas effector protein is selected from the group consisting of Cas13d, CasRx, Cas13e, Cas13a, Cas13b, Cas13c, Cas13f, and functional domains thereof, preferably CasRx. In some embodiments, the method is a method in which the guide sequence in the gRNA comprises or consists of at least 10, 11, 12, 13, 14, 15, 16, 17, or all nucleotides of at least one of SEQ ID NOs: 1-86, preferably CasRx when the gRNA is co-expressed with CasRx in at least one of Cos7 cells and 293T cells. a relative expression of PTB mRNA of less than 0.5, 0.45, 0.4, 0.35, 0.3, 0.25, 0.24, 0.23, 0.22, 0.21, 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02 or 0.015 is observed compared to corresponding control cells expressing x alone. In some embodiments, the method is a method wherein the guide sequence in the gRNA comprises or consists of at least one sequence selected from the group consisting of SEQ ID NOs: 56, 60, 47, 48, 43, 45, 40, 61, 50, 55, 41, 44, 42, 66, 49, 51, and 46.

[0011] In one embodiment, the method is a method wherein the composition comprises only a single type of gRNA, or two, three, four, five, or six different gRNAs, that target a primate PTB mRNA sequence, or an expression vector for only a single type of gRNA, or two, three, four, five, or six different gRNAs that target a primate PTB mRNA sequence.

[0012] In one embodiment, the method comprises the step of: i) a nucleotide sequence encoding a Cas effector protein, operably linked to a promoter that drives expression of the Cas effector protein in a non-neuronal cell of a primate, preferably a glial cell-specific promoter or a Muller glial (MG) cell-specific promoter, wherein the glial cell-specific promoter is more preferably selected from the group consisting of GFAP promoter, ALDH1L1 promoter, EAAT1 / GLAST promoter, glutamine synthetase promoter, S100β promoter, and EAAT2 / GLT-1 promoter, or the MG cell-specific promoter is more preferably selected from the group consisting of GFAP promoter, ALDH1L1 promoter, GLAST (also known as Slc1a3) promoter, and Rlbp1 promoter; ii) at least one nucleotide sequence encoding a gRNA that targets a primate PTB mRNA sequence, wherein the nucleotide sequence is operably linked to a promoter, such as a U6 promoter, that drives expression of the gRNA in non-neuronal cells.

[0013] In one embodiment, the method is a method wherein the expression vector is contained in a nanoparticle, or wherein the expression vector is a gene therapy vector, preferably a viral gene therapy vector, more preferably a viral vector selected from the group consisting of an adeno-associated virus (AAV) vector, an adenovirus vector, a lentavirus vector, a retrovirus vector, a herpes virus, an SV40 vector, a poxvirus vector, and combinations thereof, and most preferably AAV.

[0014] In one embodiment, the method involves locally administering the composition to at least one of the following cells: i) non-neuronal cells of the mature retina; ii) non-neuronal cells of the striatum, preferably non-neuronal cells of the putamen; iii) non-neuronal cells of the substantia nigra; iv) non-neuronal cells of the inner ear; v) non-neuronal cells of the spinal cord; vi) non-neuronal cells of the prefrontal cortex; vii) non-neuronal cells of the motor cortex; and viii) non-neuronal cells of the ventral tegmental area (VTA). In some embodiments, the method involves administering the composition to non-neuronal cells of the striatum to generate functional dopaminergic neurons; preferably, the non-neuronal cells are glial cells; and preferably, the composition is administered to at least one of the putamen and the substantia nigra. In a preferred embodiment, the glial cells are astrocytes. In some embodiments, the method is a method in which the neurological condition is a condition associated with degeneration of functional neurons selected from the group consisting of Parkinson's disease, Alzheimer's disease, Huntington's disease, schizophrenia, depression, drug addiction, stroke, movement disorders such as chorea, spinal cord injury, choreoathetosis, and dyskinesia, bipolar disorder, autism spectrum disorder (ASD), and functional impairment. In some embodiments, the method is a method in which the composition further comprises i) one or more dopamine neuron-associated factors, or ii) at least one expression vector for expressing one or more dopamine neuron-associated factors in non-neuronal cells. In preferred embodiments, the one or more dopamine neuron-associated factors are selected from the group consisting of Lmx1a, Lmx1b, FoxA2, ​​Nurr1, Pitx3, Gata2, Gata3, FGF8, BMP, En1, En2, PET1, Pax family proteins, SHH, Wnt family proteins, and TGF-β family proteins.

[0015] In one embodiment, the method is a method in which the composition is administered to glial cells or Muller glial cells (MG) of the mature retina to generate functional retinal ganglion cell (RGC) neurons. In one embodiment, the method is a method in which the composition is administered to glial cells or Muller glial cells (MG) of the mature retina to generate functional retinal photoreceptors. In some embodiments, the method is a method in which the neurological condition is a condition associated with degeneration of functional neurons of the mature retina selected from the group consisting of glaucoma, age-related RGC loss, optic nerve injury, retinal ischemia, and Leber's hereditary optic neuropathy. In one embodiment, the method is a method in which the composition further comprises i) one or more factors selected from the group consisting of β-catenin, Oct4, Sox2, Klf4, Crx, Brn3a, Brn3b, Math5, Nr2e3, and Nrl, and / or ii) at least one expression vector for expressing one or more factors selected from the group consisting of β-catenin, Oct4, Sox2, Klf4, Crx, Brn3a, Brn3b, Math5, Nr2e3, and Nrl in a non-neuronal cell.

[0016] In one embodiment, the method further comprises administering an immunosuppressant before, simultaneously with, or after administering the cellular programming agent, more preferably wherein the immunosuppressant is selected from the group consisting of corticosteroids, calcineurin inhibitors, mTOR inhibitors, IMDH inhibitors, immunosuppressive antibodies, interferons, Janus kinase inhibitors, and biologics such as anakinra.

[0017] In a second aspect, the present application relates to a composition comprising a nucleic acid molecule that targets the above-defined target sequence or its complementary sequence in a primate PTB gene, transcript, or mRNA. In some embodiments, the composition comprises i) a Cas effector protein and at least one gRNA, or ii) an expression vector encoding a Cas effector protein and encoding at least one gRNA, optionally wherein the Cas effector protein and at least one gRNA or at least one expression vector are comprised in a nanoparticle, preferably a liposome. In a preferred embodiment, the Cas effector protein and at least one gRNA are as defined herein. In a preferred embodiment, the expression vector is an expression vector as defined herein.

[0018] In one embodiment, the composition for generating functional dopaminergic neurons further comprises i) one or more dopamine neuron-associated factors, or ii) at least one expression vector for expressing one or more dopamine neuron-associated factors in a non-neuronal cell; Preferably, the one or more dopamine neuron-related factors are selected from the group consisting of Lmx1a, Lmx1b, FoxA2, ​​Nurr1, Pitx3, Gata2, Gata3, FGF8, BMP, En1, En2, PET1, Pax family proteins, SHH, Wnt family proteins, and TGF-β family proteins.

[0019] In one embodiment, the composition for generating functional RGC neurons further comprises i) one or more factors selected from the group consisting of β-catenin, Oct4, Sox2, Klf4, Crx, Brn3a, Brn3b, Math5, Nr2e3, and Nrl, and / or ii) at least one expression vector for expressing one or more factors selected from the group consisting of β-catenin, Oct4, Sox2, Klf4, Crx, Brn3a, Brn3b, Math5, Nr2e3, and Nrl in non-neuronal cells.

[0020] In one embodiment, the composition is formulated for injection, inhalation, parenteral, intravenous, subcutaneous, intramuscular, intradermal, topical, or oral administration.

[0021] In a third aspect, the present application provides a method for producing a nucleic acid sequence comprising: (a) a coding sequence for a Cas effector protein that targets an RNA; and (b) at least one nucleotide sequence encoding a gRNA as defined herein. In some embodiments, the AAV vector is a vector in which the RNA-targeting Cas effector protein is selected from the group consisting of Cas13d, CasRx, Cas13e, Cas13a, Cas13b, Cas13c, Cas13f, and functional domains thereof, preferably CasRx. In some embodiments, the AAV vector comprises: i) a nucleotide sequence encoding a Cas effector protein operably linked to a promoter that drives expression of the Cas effector protein in a primate non-neuronal cell, preferably a glial cell-specific promoter or a Müller glial cell (MG) cell-specific promoter, wherein the glial cell-specific promoter is more preferably selected from the group consisting of the GFAP promoter, the ALDH1L1 promoter, the EAAT1 / GLAST promoter, the glutamine synthetase promoter, the S100β promoter, and the EAAT2 / GLT-1 promoter; or wherein the MG cell-specific promoter is more preferably selected from the group consisting of the GFAP promoter, the ALDH1L1 promoter, the GLAST (also known as Slc1a3) promoter, and the Rlbp1 promoter; and ii) A vector in which at least one nucleotide sequence encoding a gRNA is operably linked to a promoter, e.g., a U6 promoter, that drives expression of the gRNA in non-neuronal cells. [Brief explanation of the drawings]

[0022] [Figure 1]Screening for efficient gRNAs. (A) Two days after transient transfection of plasmids encoding CasRx and gRNAs in human 293T cells, Ptbp1 mRNA was downregulated with 86 gRNAs. Note that gRNA 60 was used in the following experiments. (B) Target sites and knockdown efficiencies of 86 gRNAs in the human Ptbp1 gene (dark: <0.1; light: >0.1). Key regions indicate target regions with high knockdown efficiency. The key region corresponds to positions 951 to 1487 of SEQ ID NO: 87, i.e., positions 1 to 536 of SEQ ID NO: 88. All values ​​are expressed as mean ± SEM. [Figure 2] gRNA 60 exhibits high knockdown efficiency in human 293T cells. All values ​​are expressed as mean ± SEM. Unpaired t-test: *p<0.05, **p<0.01, ***p<0.001. [Figure 3] Knockdown of Ptbp1 expression in monkey and mouse cells by gRNA 60. (A,B) gRNA 60 demonstrates high knockdown efficiency in monkey Cos7 cells and mouse N2a cells. All values ​​are expressed as mean ± SEM. Unpaired t-test: *p<0.05, **p<0.01, ***p<0.001. [Figure 4] Expression levels (y-axis) of all genes detected in RNA-sequencing (RNA-seq) libraries of CasRx-Ptbp1 relative to the CasRx control (x-axis) show that Ptbp1 was specifically downregulated in Cos7 cells, n = 3, two groups of independent duplicates. gRNA 60 was used. [Figure 5]Astrocytes are converted into dopamine neurons in vivo. (A) Schematic diagram of the AAV vectors and injection strategy. Vector 1 (AAV-GFAP-mCherry) encodes mCherry driven by the astrocyte-specific promoter GFAP, Vector 2 (AAV-GFAP-CasRx) encodes CasRx, and Vector 3 (AAV-GFAP-CasRx-Ptbp1) encodes CasRx and gRNA. AAV-GFAP-CasRx-Ptbp1 or the control vectors AAV-GFAP-CasRx and AAV-GFAP-mCherry are injected into the striatum. The conversion of astrocytes to dopamine neurons is assessed approximately 2–3 weeks after injection. ST: striatum. (B) Representative images show the specific expression of mCherry (Vector 1) and CasRx (Vector 3 fused with Flag) in mouse astrocytes. GFAP is an astrocyte-specific marker. (C) and (D) Confocal images show transduced mCherry+NeuN+ (white arrow) and mCherry+TH+ (white arrow) cells in mice 2 weeks after injection with Vector 1+3, but not in the striatum injected with control AAV. Note: NeuN is a neuron-specific marker, and TH is a dopamine neuron-specific marker. (E) Confocal images show transduced mCherry+NeuN+ (white arrow) and mCherry+TH+ (white arrow) cells in mice 1 month after injection with Vector 1+3, but not in the striatum injected with control AAV. Note: DAT is a mature dopamine neuron-specific marker. (F) Representative images show mCherry+TH+ cells in the putamen of a cynomolgus monkey injected with Vector 1 and 3 (yellow arrow). gRNA 60 was used. [Figure 6]Co-injection of dopamine neuron-related transcription factors improves the efficiency of astrocyte conversion to dopamine neurons. Confocal images show AAV-GFAP-mCherry + AAV-GFAP-CasRx-Ptbp1, AAV-GFAP-mCherry + AAV-GFAP-CasRx-Ptbp1 + AAV-GFAP-FoxA2, ​​AAV-GFAP-mCherry + AAV-GFAP-CasRx-Ptbp1 + AAV-GFAP-Lmx1a, and AAV-GFAP-mCherry + AAV-GFAP-CasRx-Ptbp1 + AAV-GFAP-FoxA2. Two to three weeks after injection of AAV-GFAP-Lmx1a and AAV-GFAP-mCherry, AAV-GFAP-CasRx-Ptbp1, AAV-GFAP-FoxA2, ​​AAV-GFAP-Lmx1a, and AAV-GFAP-Nurr1, converted mCherry+NeuN+TH+ (yellow arrows) cells were observed in mice, but not in control striatum injected with AAV-GFAP-mCherry+AAV-GFAP-CasRx. Note that NeuN is a neuron-specific marker, and TH is a dopamine neuron-specific marker. White arrows indicate mCherry+NeuN-TH- cells. gRNA 60 was used. [Figure 7]Co-injection of β-catenin into middle-aged mice improves the efficiency of MG-to-RGC conversion. (A) Schematic diagram of MG-to-RGC conversion. Vector I (AAV-GFAP-GFP-Cre) expresses Cre recombinase and GFP driven by the MG-specific promoter GFAP. Vector II (AAV-GFAP-CasRx-Ptbp1) expresses CasRx and gRNA. To induce MG-to-RGC conversion, AAV-GFAPCasRx-Ptbp1 or control AAV-GFAP-CasRx was injected into the retina together with AAV-GFAP-GFP-Cre (Ai9 mice, 4-5 months old). The induction of RGC conversion was verified 2-3 weeks after injection. ONL: outer nuclear layer; OPL: outer plexiform layer; INL: inner nuclear layer; IPL: inner plexiform layer; GCL: ganglion cell layer. (B-E) Representative images show the colocalization of RBPMS+tdTomato+ cells in the GCL and RBPMS+tdTomato+ optic nerves in the AAV-GFAP-GFP-Cre+AAV-GFAP-CasRx, AAV-GFAP-GFP-Cre+AAV-GFAP-β-catenin, AAV-GFAP-GFP-Cre+AAV-GFAP-CasRx-Ptbp1, and AAV-GFAP-GFP-Cre+AAV-GFAP-CasRx-Ptbp1+AAV-GFAP-β-catenin groups. Note that AAV-GFAP-GFP-Cre+AAV-GFAP-CasRx and AAV-GFAP-GFP-Cre+AAV-GFAP-β-catenin are control groups. Yellow arrows indicate colocalization of tdTomato and RBPMS in retinas injected with AAV-GFAP-GFP-Cre, AAV-GFAP-CasRx-Ptbp1, and AAV-GFAP-β-catenin. RBPMS is a specific marker for RGCs. gRNA 60 was used. DETAILED DESCRIPTION OF THE INVENTION

[0023] Description of the Invention definition Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Those skilled in the art will recognize that many methods and materials similar or equivalent to those described herein can be used to implement the present application. In fact, the present application is not limited to this method.

[0024] In this document and the claims, the verb "comprise" and its variations are used in an open-ended sense to mean the inclusion of the items following the word, but not the exclusion of items not specifically mentioned. Furthermore, the use of the indefinite article "a" or "an" to refer to an element does not exclude the possibility that one or more of the element are present, unless the context clearly requires only one element. Thus, the indefinite article "a" or "an" typically means "at least one."

[0025] As used herein, the term "and / or" means that one or more circumstances may occur alone or in combination with at least one circumstance, up to and including all circumstances.

[0026] As used herein, "at least" a particular value means the particular value or more. For example, "at least 2" should be understood to be the same as "2 or more," i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, etc.

[0027] When used in conjunction with a numerical value (e.g., about 10), the word "about" or "on the order of" preferably means that the value may be about 0.1% of the given value (of 10).

[0028] The term "astrocyte" generally refers to the star-shaped glial cells characteristic of the brain and spinal cord, characterized by one or more of the following: star shape; expression of markers such as glial fibrillary acidic protein (GFAP), aldehyde dehydrogenase 1 family member LI (ALDH1L1), excitatory amino acid transporter 1 / glutamate aspartate transporter (EAAT1 / GLAST), glutamine synthetase, S100β, or excitatory amino acid transporter 1 / GLAST1 (EAAT2 / GLT-1); association with endothelial cells in the blood-brain barrier; uptake and release of neurotransmitters; regulation of ion concentrations in the extracellular space; participation in response to neuronal injury and nervous system repair; and metabolic support of peripheral neurons.

[0029] In some embodiments, astrocytes refer to non-neuronal cells of the nervous system that express glial fibrillary acidic protein (GFAP), aldehyde dehydrogenase 1 family member L1 (ALDH1L1), or both.

[0030] In some embodiments, astrocytes are non-neuronal cells of the nervous system that express transgenes (e.g., red fluorescent protein (RFP), Cre recombinase) driven by the glial fibrillary acidic protein (GFAP) promoter.

[0031] In some embodiments, Müller glia (MG) refer to non-neuronal glial cells found in the retina that express transgenes (e.g., red fluorescent protein (RFP)) driven by MG-specific promoters, including those from GFAP, GLAST (also known as Slc1a3), and Rlbp1.

[0032] "BRN2 transcription factor" or "Brain-2 transcription factor," also known as "POU domain, class 3 transcription factor 2" ("POU3F2") or "Oct-7," refers to a class III POU domain transcription factor that has a DNA-binding POU domain consisting of an N-terminal POU-specific domain of approximately 75 amino acids and a C-terminal POU-homology domain of approximately 60 amino acids, which are linked via a linker containing a short α-helical fold and can be expressed primarily in the central nervous system.

[0033] The term "cellular programming agent" generally refers to an agent that reprograms differentiated non-neuronal cells into neuronal cells by inhibiting the expression and / or function of PTB and / or nPTB. In certain embodiments, the cellular programming agent refers to a CRISPR / Cas effector protein (which may or may not include a variant, derivative, functional equivalent, or fragment thereof) with a guide RNA (gRNA) complementary to PTB mRNA or nPTB mRNA, and can knock down the expression and / or activity of PTB or nPTB to a sufficient extent to convert a non-neuronal cell into a neuronal cell, preferably in a local in vivo microenvironment in which the converted neuron is expected to be functional. The cellular programming agent may also refer to a polynucleotide encoding the CRISPR / Cas effector protein and / or guide RNA (gRNA) defined above. The polynucleotide may include mRNA of the Cas effector defined above. The polynucleotide may further include DNA encoding a gRNA complementary to the Cas effector and / or PTB / nPTB mRNA defined above. DNA encoding the above-defined Cas effector and / or gRNA can be part of a vector, including a viral vector (e.g., an AAV vector or a lentiviral vector, or any other viral vector described below). In the case of AAV, any AAV having tropism for glial or non-neuronal cells of the CNS and / or PNS can be used, including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, etc. Similarly, in the case of AAV, any of the above-defined Cas effectors can be used, as long as the coding sequence for the Cas effector is smaller than the packaging capacity of AAV, i.e., 4.7 kb, 4.5 kb, 4.0 kb, 3.5 kb, 3.0 kb, 2.5 kb, 2.0 kb, 1.5 kb, or less.Exemplary Cas effectors that can be used herein include Cas13a, Cas13b, Cas13c, Cas13d, CasRx, Cas13e, Cas13f, Cpf1, Cas9, and functional equivalents or fragments thereof. In the narrowest sense, the term "cellular programming agent" can be used interchangeably with a Cas effector having a gRNA or a polynucleotide (e.g., DNA or vector) encoding it.

[0034] Cas effector proteins that can be used in the applications described herein include CRISPR-Cas class 2 systems, which utilize a single large Cas protein to degrade target nucleic acids (e.g., mRNA). Suitable class 2 Cas effectors can include type II Cas effectors, such as Cas9 (e.g., Streptococcus pyogenes SpCas9 and Streptococcus thermophilus Cas9). Suitable Cas effectors can also be class 2 V-type Cas proteins, including Cas12a (formerly called Cpf1, e.g., Francisella novicida Cpf1 and Prevotella spp. Cpf1), C2c1, and C2c3, which lack HNH nuclease activity but possess RuvC nuclease activity. Particularly suitable Cas effector proteins can include class 2 type VI Cas proteins, including Cas13 (also called C2c2), Cas13a, Cas13b, Cas13c, Cas13d / CasRx, Cas13e, and Cas13f, each of which is an RNA-guided RNase (i.e., these Cas proteins recognize target RNA sequences using their crRNA, rather than target DNA sequences as in Cas9 and Cas12a). Generally, CRISPR / Cas13 systems achieve higher RNA digestion efficiency compared to traditional RNAi and CRISPRi technologies, while also producing less off-target cleavage compared to RNAi.

[0035] Thus, in certain embodiments, the cellular programming agent of the present application is or encodes a Cas effector protein that targets PTB or nPTB mRNA with its representative gRNA, while in other embodiments, the Cas effector targets PTB or nPTB DNA.

[0036] The phrase "contacting" a cell with a composition of the present disclosure means placing the composition (e.g., a compound, a nucleic acid, a viral vector, etc.) in a location that allows it to contact the cell to produce a "contacted" cell. Contacting can be done in any suitable manner. For example, in one embodiment, contacting is done by adding the compound to a cell culture. Contacting can also be achieved by injecting the composition or delivering it to a location in the body such that the composition "contacts" the target cell type.

[0037] The terms "differentiation" or "conversion" or "inducing differentiation" can be used interchangeably to mean changing a default cell type (genotype and / or phenotype) to a non-default cell type (genotype and / or phenotype). Thus, "inducing differentiation in astrocytes" refers to inducing a change in cell morphology (i.e., changes in gene expression as determined by genetic analysis such as microarrays) and / or phenotype (i.e., changes in protein expression) from an astrocyte morphology to a neuronal cell type morphology (i.e., changes in gene expression as determined by genetic analysis such as microarrays) and / or phenotype (i.e., changes in protein expression).

[0038] As used herein, "effective amount" refers to the amount of drug required to ameliorate disease symptoms in an untreated patient. For example, the effective amount of an active agent for practicing the present application, for the treatment of cancer, will vary depending on the method of administration and the subject's age, weight, and overall health. Ultimately, the appropriate amount and administration schedule will be determined by the attending physician or veterinarian. Such an amount is referred to as an "effective" amount, which can be determined as genome copies per kilogram (GC / kg). Thus, in the presently disclosed context, with respect to the administration of a drug that "effectively combats" a disease or condition, administration in a clinically relevant manner has been shown to produce a beneficial effect in at least a statistically significant portion of patients, such as symptomatic improvement, cure, alleviation of at least one disease sign or symptom, prolongation of life, improved quality of life, or other effect generally considered positive by a physician familiar with the treatment of a particular type of disease or condition.

[0039] The term "expression control sequence" is intended to include at least a sequence designed whose presence affects expression, and may also include additional advantageous components. For example, leader sequences and fusion partner sequences are expression control sequences. The term also includes designing a nucleic acid sequence to remove unwanted potential initiation codons, both in and out of frame, from the sequence. It may also include designing a nucleic acid sequence to remove unwanted potential splice sites. It includes sequences that direct the addition of a polyA tail or polyadenylation sequence (pA), which is a string of adenine residues at the 3' end of an mRNA, called a polyA sequence. It may also be designed to enhance mRNA stability. Expression control sequences, such as promoters that affect transcriptional and translational stability, and sequences such as Kozak sequences that affect translation, are known in the art. Expression control sequences have the property of regulating the nucleotide sequence operably linked to them, thereby achieving lower or higher expression levels.

[0040] The term "gene" refers to a DNA fragment comprising a region (transcribed region) that is operably linked to an appropriate regulatory region (e.g., a promoter) and that is transcribed into an RNA molecule (e.g., mRNA) in a cell. A gene generally comprises several operably linked fragments, such as a promoter, a 5' leader sequence, a coding region, and a 3'-untranslated sequence (3'-end) that includes a polyadenylation site. "Gene expression" refers to the process by which a DNA region operably linked to an appropriate regulatory region, particularly a promoter, is transcribed into biologically active RNA, which can then be translated into biologically active proteins or peptides.

[0041] The term "glial cells" may generally refer to a type of support cell in the central nervous system (eg, brain and spinal cord) and peripheral nervous system.

[0042] In some embodiments, glial cells do not conduct electrical pulses or exhibit action potentials. In some embodiments, glial cells do not communicate with each other or with neurons via synaptic connections or electrical signals. In the nervous system or in vitro culture systems, glial cells can surround neurons and provide support for and insulation between neurons. Non-limiting examples of glial cells include oligodendrocytes, astrocytes, Müller glial cells, ependymal cells, Schwann cells, microglial cells, spiral ganglion glial cells, and satellite cells.

[0043] As used herein, "guide sequence" refers to a sequence that guides an RNA- or DNA-guided endonuclease to a specific site in an RNA or DNA molecule. In the context of a guide RNA (gRNA)-CAS complex, "guide sequence" should also be understood herein as a part of the gRNA (or crRNA) that is required to target the gRNA-CAS complex to a specific site in a target RNA or DNA molecule. The "guide sequence" in a gRNA is complementary to a specific site, the "target sequence," in the target RNA or DNA molecule (see below).

[0044] The term "homologous," when used to describe the relationship between a given (recombinant) nucleic acid or polypeptide molecule and a given host organism or host cell, should be understood to mean that the nucleic acid or polypeptide molecule is produced by a host cell or organism of the same species, preferably the same variety or strain, in nature. When homologous to a host cell, the nucleic acid sequence encoding the polypeptide is usually (but not necessarily) operably linked to a different (heterologous) promoter sequence and, if applicable, to a different (heterologous) secretion signal sequence and / or terminator sequence in its natural environment. It should be understood that control sequences, signal sequences, terminator sequences, etc. may also be homologous to the host cell. In this context, the use of only "homologous" sequence elements allows the construction of "autocloned" transgenic organisms (GMOs) (the definition of autocloning in this application is the same as that in Annex II of Directive 98 / 81 / EC). When used to describe the relationship between two nucleic acid sequences, the term "homologous" means that one single-stranded nucleic acid sequence can hybridize with a complementary single-stranded nucleic acid sequence. The degree of hybridization can depend on many factors, including the degree of identity between the sequences and hybridization conditions such as temperature and salt concentration, which are discussed below.

[0045] The terms "heterologous" and "exogenous," when used with reference to a nucleic acid (DNA or RNA) or protein, refer to a nucleic acid or protein that is not naturally occurring as part of the organism, cell, genome, or DNA or RNA sequence in which the nucleic acid or protein is found, or that is found in one or more locations that are distinct from the cell, genome, or DNA or RNA sequence in which it is found in nature. Heterologous and exogenous nucleic acids or proteins are not endogenous to the cell into which they are introduced, but rather are obtained from another cell, or are synthetically or recombinantly produced. Typically, but not necessarily, such nucleic acids encode proteins that are not normally produced by the cell that transcribes or expresses the DNA, i.e., exogenous proteins. Similarly, exogenous RNA encodes proteins that are not normally expressed in the cell in which the exogenous RNA is present. Heterologous / exogenous nucleic acids and proteins are also referred to as foreign nucleic acids or proteins. Nucleic acids or proteins that one of skill in the art would consider to be foreign to the cell in which they are expressed are included in the term heterologous or exogenous nucleic acid or protein, as used herein. The terms heterologous and exogenous also apply to non-natural combinations of nucleic acid or amino acid sequences, ie, combinations in which at least two of the combined sequences are exogenous to each other.

[0046] "MicroRNA" or "miRNA" refers to a non-coding nucleic acid (RNA) sequence that binds to at least partially complementary nucleic acid sequences (mRNAs) and negatively regulates the expression of target mRNAs at the post-transcriptional level. MicroRNAs are typically processed into their "mature" form from "precursor" miRNAs that have a double-stranded hairpin loop structure. Mature microRNA sequences are typically approximately 19-25 nucleotides in length.

[0047] "miR-9" is a short non-coding RNA gene involved in gene regulation and highly conserved from Drosophila, mice, to humans. The mature ~21nt miRNA is derived from a hairpin precursor sequence processed by the Dicer enzyme. miR-9 may be one of the most expressed microRNAs in the developing and adult vertebrate brain. Key transcriptional regulators such as FoxG1, Hesl, and Tlx are direct targets of miR-9, placing it at the core of a gene network controlling the state of neuronal progenitor cells.

[0048] The terms "neuron" or "neuronal cell" as used herein may have the general meaning understood by those skilled in the art. In some embodiments, a neuron may refer to an electrically excitable cell that can receive, process, and transmit information through electrical signals (e.g., membrane potential discharge) and chemical signals (e.g., synaptic transmission of neurotransmitters). As understood by those skilled in the art, chemical signals transmitted between neurons (e.g., neurotransmitter-based release and discrimination) can occur through specialized connections called synapses.

[0049] The term "mature neuron" can refer to a differentiated neuron. In some embodiments, a neuron is a mature neuron if it expresses one or more markers of a mature neuron, such as, for example, microtubule-associated protein 2 (MAP2) and neuronal nuclear antigen (NeuN), neuron-specific enolase (NSE), 160 kDa neurofilament medium, 200 kDa neurofilament heavy chain, postsynaptic density protein 95 (PDS-95), synapsin I, synaptophysin, glutamic acid decarboxylase 67 (GAD67), glutamic acid decarboxylase 67 (GAD65), parvalbumin, dopamine- and cAMP-regulated neuronal phosphoprotein 32 (DARPP32), vesicular glutamate transporter 1 (vGLUT1), vesicular glutamate transporter 2 (vGLUT2), acetylcholine, or tyrosine hydroxylase (TH).

[0050] The term "functional neuron" may refer to a neuron that can send or receive information by chemical or electrical signals. In some embodiments, a functional neuron exhibits one or more functional characteristics of a mature neuron present in a normal nervous system, including, but not limited to, excitability (e.g., the ability to exhibit an action potential, such as a rapid rise and subsequent fall in voltage or membrane potential across a cell membrane), the formation of synaptic connections with other neurons, the release of presynaptic neurotransmitters, and postsynaptic responses (e.g., excitatory postsynaptic currents or inhibitory postsynaptic currents).

[0051] In some embodiments, functional neurons are characterized by expressing one or more markers of functional neurons, including, but not limited to, synapsin, synaptophysin, glutamic acid decarboxylase 67 (GAD67), glutamic acid decarboxylase 67 (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).

[0052] The term "non-neuronal cell" can refer to any type of cell that is not a neuron. Exemplary non-neuronal cells are cells with a cell lineage distinct from the neuronal lineage (e.g., hematopoietic lineage). In some embodiments, the non-neuronal cell is a cell of the neuronal lineage but is not a neuron, e.g., a glial cell. In some embodiments, the non-neuronal cell is a non-neuronal somatic cell such as, but not limited to, a glial cell, an adult primary fibroblast, an embryonic fibroblast, an epithelial cell, a melanocyte, a keratinocyte, an adipocyte, a blood cell, a bone marrow stromal cell, a Langerhans cell, a muscle cell, a colon cell, or a chondrocyte. In some embodiments, the non-neuronal cell is derived from a non-neuronal cell line, e.g., but not limited to, a glioblastoma cell line, a HeLa cell line, an NT2 cell line, an ARPE19 cell line, or an N2A cell line.

[0053] "Cell lineage" or "lineage" can refer to the developmental history of tissues or organs of a fertilized embryo.

[0054] "Neuronal lineage" may refer to, but is not limited to, the developmental history from neural stem cells to mature neurons, including various stages along the process (called neurogenesis), such as neural stem cells (neuroepithelial cells, radial glial cells), neural progenitor cells (e.g., intermediate neural precursors), neurons, astrocytes, oligodendrocytes, and microglial cells.

[0055] As used herein, the term "non-naturally occurring" when referring to an organism means that the organism has at least one genetic change that is not normally found in naturally occurring strains of the referenced species (including wild-type strains of the referenced species). Genetic changes include, for example, modifications that introduce expressible nucleic acids encoding proteins or enzymes, additions of other nucleic acids, deletions of nucleic acids, substitutions of nucleic acids, or other functional disruptions of the organism's genetic material. Such modifications include, for example, coding regions for heterologous or homologous polypeptides of the referenced species and functional fragments thereof. Additional modifications include, for example, non-coding regulatory regions that alter the expression of genes or operons. Genetic modifications of nucleic acid molecules encoding enzymes or functional fragments thereof can confer on a non-naturally occurring organism the capability of a biochemical reaction or metabolic pathway that is altered from its naturally occurring state.

[0056] The terms "nucleic acid" and "polynucleotide," used interchangeably herein, can refer to deoxyribonucleotides or ribonucleotides and polymers thereof in single- or double-stranded form. The terms can encompass synthetic, naturally occurring, and non-naturally occurring nucleic acids that contain known nucleotide analogs or modified backbone residues or linkers, have similar binding properties as the reference nucleic acid, and are metabolized in a manner similar to the reference nucleotide. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphates, chiral methyl phosphates, 2-O-methyl ribonucleotides, locked nucleic acids (LNAs), and peptide nucleic acids (PNAs).

[0057] As used herein, "nucleic acid construct" or "nucleic acid vector" refers to an artificial nucleic acid molecule produced using recombinant DNA technology. Therefore, the term "nucleic acid construct" does not include naturally occurring nucleic acid molecules, although a nucleic acid construct may (partially) contain naturally occurring nucleic acid molecules. A "vector" is a nucleic acid construct (usually DNA or RNA) for introducing an exogenous nucleic acid sequence (i.e., DNA or RNA) into a host cell. Preferably, the vector is maintained in the host by at least one of autonomous replication and integration into the host cell genome. The term "expression vector" or "expression construct" refers to a nucleotide sequence capable of affecting gene expression in a host cell or host organism compatible with such sequence. These expression vectors typically contain at least one "expression cassette," a functional unit capable of affecting the expression of a sequence encoding a product to be expressed, in which the coding sequence is operably linked to an appropriate expression control sequence, including at least an appropriate transcriptional regulatory sequence and, optionally, a 3' transcription termination signal. Other factors necessary or useful for affecting expression, such as expression enhancer elements, may also be present. The expression vector can be introduced into a suitable host cell to effect expression of the coding sequence in an in vitro cell culture of the host cell. Preferred expression vectors are suitable for expressing viral proteins and / or nucleic acids, particularly recombinant AAV proteins and / or nucleic acids.

[0058] "Oligodendrocyte" may refer to a type of glial cell that produces the myelin sheath that surrounds neuronal axons and supports and insulates axons in the central nervous system. Oligodendrocytes are also characterized by the expression of PDGF receptor alpha (PDGFR-α), SOXIO, neural / glial antigen 2 (NG2), Olig1, Olig2, and Olig3, oligodendrocyte-specific protein (OSP), myelin basic protein (MBP), or myelin oligodendrocyte glycoprotein (MOG).

[0059] "Polypyrimidine tract-binding protein" or "PTB" and its homolog, neuronal PTB (nPTB), are both commonly occurring RNA-binding proteins. Also known as polypyrimidine tract-binding protein 1, PTB is encoded by the PTBP1 gene in humans. The PTBP1 gene belongs to the commonly expressed heterogeneous nuclear ribonucleoprotein (hnRNP) subfamily.

[0060] hnRNPs are RNA-binding proteins that complex with heterogeneous nuclear RNA (hnRNA). These proteins associate with precursor mRNAs in the cell nucleus and are thought to affect precursor mRNA processing and other aspects of mRNA metabolism and transport. PTBs can contain four repeating quasi-RNA recognition motif (RRM) domains that bind RNA. Consistent with their widespread expression, PTBs can serve to inhibit many alternative splicing events. PTBs can recognize short RNA motifs, such as UCUU and UCUCU, located in pyrimidine-rich environments and are commonly associated with polypyrimidine tracts upstream of the 3' splice sites of constitutive and alternative exons.

[0061] In some cases, the binding site of PTB may also include exon sequence and intronic sequence downstream of the regulatory exon.

[0062] In most PTB-controlled alternative splicing systems, inhibition can be achieved by interaction of PTB with multiple PTB binding sites surrounding the alternative exons.

[0063] In some cases, inhibition may be associated with a single PTB binding site. PTB splicing inhibition can occur through direct competition between PTB and U2AF65, which in turn can prevent U2 snRNP assembly at the branch point. In some cases, PTB splicing inhibition may be associated with PTB binding sites located on either side of an alternative exon, or synergistic interactions between PTB molecules that loop out the RNA may prevent the splice site from entering the splicing machinery. PTB splicing inhibition may also be associated with multiple PTB binding sites from high-affinity binding sites, generating an inhibitory wave that covers the alternative exon and prevents its recognition.

[0064] While PTB is widely expressed in non-neuronal cells, nPTB may be restricted to neurons. PTB and nPTB can undergo programmed transformations during neuronal differentiation. For example, during neuronal differentiation, PTB is gradually downregulated during the neuronal induction stage, while, coincidentally or inevitably, nPTB levels gradually increase to a peak level. Subsequently, as neuronal differentiation enters the neuronal maturation stage, nPTB levels initially increase, then decline, and then recover to a relatively low level compared to the peak level during neuronal differentiation, at which point the cells develop into mature neurons.

[0065] Because the sequences of PTB and nPTB are known (see, for example, Romanelli et al. (2005) Gene 356:11-8; Robinson et al., PLoS One. (2008) 3(3):el801. doi:10.1371 / journal.pone.0001801; Makeyev et al., Mol. Cell (2007) 27(3):435-448), those skilled in the art can design and construct, for example, gRNA molecules that regulate the expression of PTB / nPTB so as to reduce or inhibit it, in order to carry out the methods of the present application.

[0066] The terms "protein," "peptide," and "polypeptide" can be used interchangeably and can refer to a polymer of amino acids or a set of two or more interacting or linked amino acid polymers, regardless of their particular pattern of action, size, three-dimensional structure, or origin.

[0067] As used herein, the term "promoter" or "transcriptional regulatory sequence" refers to a nucleic acid fragment that functions to control the transcription of one or more coding sequences, located upstream in the transcriptional direction of the coding sequence transcription initiation site and structurally identified by the presence of a DNA-dependent RNA polymerase binding site, a transcription initiation site, and any other DNA sequences, including, but not limited to, transcription factor binding sites, repressor and activator protein binding sites, enhancers, and any other nucleotide sequences known to those skilled in the art to directly or indirectly regulate the amount of transcription of a promoter. A "constitutive" promoter is a promoter that is active in most tissues under most environmental and developmental conditions. An "inducible" promoter is one that is environmentally or developmentally regulated, for example, by a chemical inducer or a biological entity.

[0068] As used herein, the term "operably linked" refers to the linkage of polynucleotide (or polypeptide) elements in a functional relationship. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a transcriptional regulatory sequence is operably linked to a coding sequence if it affects the transcription of the coding sequence. Operatively linked means that the DNA sequences being linked are usually contiguous, and, where necessary, linking two protein-coding regions, contiguous and in reading frame. An expression control sequence is "operably linked" to a nucleotide sequence if it controls and regulates the transcription and / or translation of the nucleotide sequence. Thus, expression control sequences can include promoters, enhancers, internal ribosome entry sites (IRES), transcription terminators, the start codon in front of a protein-encoding gene, splicing signals for introns, and termination codons.

[0069] The terms "reprogramming" or "transdifferentiation" can refer to an intermediate process that generates cells of a certain lineage (e.g., neuronal cells) from a different cell type (e.g., fibroblasts) without dedifferentiating the cells into cells that exhibit characteristics of pluripotent stem cells.

[0070] "Pluripotency" can refer to the ability of a cell to form all lineages of the body or somatic cells (i.e., the embryo itself). Exemplary "pluripotent stem cells" can include embryonic stem cells and induced pluripotent stem cells.

[0071] The terms "sequence identity," "homology," and the like may be used interchangeably herein. Sequence identity is defined herein as the relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences. In the art, "identity" refers to the degree of sequence relatedness between amino acid sequences or nucleic acid sequences, as the case may be, as determined by the match between strings of such sequences. "Similarity" between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide with the sequence of a second polypeptide. "Identity" and "similarity" can be readily calculated using known methods.

[0072] "Sequence identity" and "sequence similarity" can be determined by aligning two peptide or two nucleotide sequences using a global or local alignment algorithm, depending on the length of the two sequences. Sequences of similar length are preferably aligned using a global alignment algorithm (e.g., Needleman Wunsch) that optimally aligns sequences across their entire length, while sequences of disparate lengths are preferably aligned using a local alignment algorithm (e.g., Smith Waterman). Sequences may be referred to as "substantially the same" or "substantially similar" if they share at least a certain minimum percentage of sequence identity (as defined below) (e.g., when optimally aligned using default parameters with the programs GAP or BESTFIT). GAP uses the Needleman and Wunsch global alignment algorithm to align two sequences across their entire length (full length), maximizing the number of matches and minimizing the number of gaps. When two sequences have similar lengths, global alignment is appropriate for determining sequence identity. Typically, default GAP parameters are used, with a gap creation penalty of 50 (nucleotides) / 8 (proteins) and a gap extension penalty of 3 (nucleotides) / 2 (proteins). The default scoring matrices used are nwsgapdna for nucleotides and Blosum62 for proteins (Henikoff & Henikoff, 1992, PNAS 89, 915-919).Sequence alignments and percent sequence identity scores can be determined using computer programs such as the GCGWisconsin Package version 10.3, available from Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121-3752 USA, or open source software such as the programs "needle" (which uses the global Needleman-Wunsch algorithm) or "water" (which uses the local Smith-Waterman algorithm) in EmbossWIN version 2.10.0, using the same parameters as GAP described above, or default settings (for "needle" and "water" and for protein and DNA alignments, the default gap opening penalty is 10.0 and the default gap extension penalty is 0.5; the default scoring matrices are Blossum62 for proteins and DNAFull for DNA). When the overall lengths of the sequences differ substantially, local alignments are preferred, such as those using the Smith-Waterman algorithm.

[0073] Alternatively, percent similarity or identity can be determined by searching common databases using algorithms such as FASTA or BLAST. Thus, the nucleic acid and protein sequences of the present application can further be used as "query sequences" to search common databases, for example, to identify other family members or related sequences. For this search, the BLASTn and BLASTx programs (version 2.0) of Altschul et al. (1990) J. Mol. Biol. 215:403-10 can be used. BLAST nucleotide searches can be performed using the NBLAST program (score = 100, word length = 12) to obtain nucleotide sequences homologous to the nucleic acid molecules of the present application. BLAST protein searches can be performed using the BLASTx program (score = 50, word length = 3) to obtain amino acid sequences homologous to the protein molecules of the present application. To obtain gapped alignments for comparison purposes, Gapped BLAST can be used as described in Altschul et al. (1997) Nucleic Acids Res. 25(17): 3389-3402. When using BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., BLASTx and BLASTn) can be used. See the homepage of the National Center for Biotechnology Information at http: / / www.ncbi.nlm.nih.gov / . Unless otherwise stated, the terms "subject" and "patient," which can be used interchangeably, can refer to mammals such as humans and non-human primates, as well as rabbits, rats, mice, goats, pigs, and other mammalian species. The term does not necessarily indicate that the subject has been diagnosed with a particular disease, but can refer to an individual under medical supervision.

[0074] "Target sequence" refers to the order of nucleotides within a nucleic acid that is targeted (e.g., where a modification is introduced or detected). In the context of a guide RNA (gRNA)-CAS complex, it is further understood herein that a "target sequence" is a portion within an RNA or DNA molecule that is targeted by the gRNA-CAS complex by virtue of its complementarity with a "guide sequence" in the gRNA (see also above). Similarly, an antisense oligonucleotide or miRNA is targeted by virtue of its complementarity with a "target sequence" within the targeted RNA or DNA molecule.

[0075] For example, a target sequence is the order of nucleotides contained in the first strand of a DNA duplex.

[0076] For example, mammalian species that may benefit from the disclosed methods and compositions include, but are not limited to, primates such as apes, chimpanzees, orangutans, and humans.

[0077] A "vector" is a nucleic acid that can carry another nucleic acid into a cell. When present in the appropriate environment, the vector can direct the expression of one or more proteins encoded by one or more genes or microRNAs encoded by polynucleotides carried by the vector.

[0078] The use of a substance described herein as a drug can also be interpreted as the use of that substance in the manufacture of a drug. Similarly, if a substance is used in therapy and as a drug, it can also be used in the manufacture of a therapeutic agent. The drug-use products described herein can be used in a method of treatment that includes administering the product.

[0079] A "viral vector" is a nucleic acid derived from a virus that can carry another nucleic acid into a cell. When present in the appropriate environment, the viral vector can direct the expression of one or more proteins encoded by one or more genes carried by the vector, or microRNAs encoded by polynucleotides carried by the vector. Examples of viral vectors include, but are not limited to, retroviral, adenoviral, lentiviral, and adeno-associated viral vectors.

[0080] Universal Methods and Compositions In one aspect, the present application relates to a method for treating a neurological condition associated with the degeneration of functional neurons in a region of the nervous system of a non-human primate or human. The method preferably comprises administering to a subject in need thereof an effective amount of a composition comprising a cellular programming agent. In one embodiment, the composition comprising a cellular programming agent comprises at least a nucleic acid molecule that targets a target sequence or its complementary sequence in a primate PTB gene, transcript, or mRNA to inhibit PTB expression or activity in the non-neuronal cell, thereby enabling the non-neuronal cell to be reprogrammed into a functional neuron. Thus, the nucleic acid molecule that targets a target sequence or its complementary sequence in a primate PTB gene, transcript, or mRNA may be or encode at least one of an antisense nucleic acid (e.g., for inducing exon skipping), an miRNA, or a guide RNA (gRNA for directing the nuclease activity of a CRISPR / Cas effector protein).

[0081] In one embodiment, the nucleic acid molecule in the composition comprising the cellular programming agent is a nucleic acid molecule that targets a target sequence in a primate PTB gene, transcript, or mRNA, or in the case of a double-stranded PTB DNA or RNA sequence, the nucleic acid molecule can target either of the two complementary strands. A nucleic acid molecule that targets a target sequence in a primate PTB gene, transcript, or mRNA (or its complementary sequence) is understood to mean that at least a portion of the nucleic acid molecule is substantially complementary to a target sequence in a primate PTB nucleic acid, such that it pairs with primate PTB nucleobases, preferably under physiological conditions, and exerts its biological effect, i.e., inhibition of PTB expression or activity in non-neuronal cells.

[0082] The inventors have identified target sequences among the effective target sequences of primate or human PTB genes, transcripts, or mRNAs, and targeting of the target sequences with the nucleic acid molecules of the present application ensures effective inhibition of PTB expression or activity in non-neuronal cells, as described in further detail below.

[0083] In one embodiment, the composition comprising a cellular programming agent comprises at least one expression vector encoding i) a CRISPR / Cas effector protein and a gRNA complementary to polypyrimidine tract-binding protein (PTB) mRNA, or ii) a guide RNA (gRNA) complementary to a CRISPR / Cas effector protein and a PTB mRNA.

[0084] Specifically, one aspect of the present application provides a method for reprogramming a non-neuronal cell into a mature neuron. An exemplary method includes providing a non-neuronal cell and reprogramming the non-neuronal cell into a mature neuron by contacting the non-neuronal cell with a composition comprising a cellular programming agent (e.g., a CRISPR / Cas effector with a guide RNA (gRNA) or a polynucleotide encoding the same) that inhibits expression and / or activity of PTB and / or nPTB in the non-neuronal cell. The method and composition not only convert cells in vitro, but also directly convert cells in vivo in the nervous system (e.g., the striatum, retina, inner ear, and spinal cord).

[0085] In some embodiments, the present application relates to methods for treating a neurological condition associated with degeneration of functional neurons in a region of the nervous system of a non-human primate or human, comprising administering to the non-neuronal cells in the region of the nervous system of a subject in need thereof an effective amount of a composition comprising: i) an RNA-targeting Cas effector protein and a guide RNA (gRNA) that targets a primate PTB mRNA sequence, or ii) at least one expression vector encoding an RNA-targeting Cas effector protein and encoding a gRNA that targets a primate PTB mRNA sequence, thereby inhibiting PTB expression or activity in the non-neuronal cells and allowing the non-neuronal cells to be reprogrammed into functional neurons.

[0086] According to some embodiments of the present disclosure, single-cell programming agents (e.g., Cas / gRNA) that inhibit the expression and / or activity of PTB / nPTB in non-human primate or human non-neuronal cells (e.g., MG cells in the mature retina or astrocytes in the striatum) can directly convert the non-neuronal cells into mature neurons (e.g., retinal ganglion cell (RGC) neurons, retinal photoreceptors, or dopamine neurons, respectively). In some embodiments, directly converting a non-neuronal cell into a neuron via a single-cell programming agent (e.g., Cas / gRNA) can mean converting a non-neuronal cell into a neuron simply by contacting it with a single-cell programming agent, without the need for any other intervention.

[0087] In another embodiment, the present disclosure provides a method for reprogramming astrocytes into mature neurons. An exemplary method includes providing astrocytes to be reprogrammed and contacting the astrocytes with a composition comprising a cell programming agent that inhibits PTB expression or activity in the astrocytes (e.g., a Cas with a gRNA targeting PTB / nPTB or a polynucleotide encoding the same) for at least one day, thereby reprogramming the astrocytes into mature neurons, such as dopamine neurons. In some embodiments, a single-cell programming agent that inhibits PTB expression or activity in astrocytes (e.g., a Cas with a gRNA targeting PTB) can directly convert astrocytes into neurons, such as dopamine neurons. In some examples, the astrocytes are in the striatum.

[0088] In another embodiment, the present application provides a method for reprogramming MG cells (e.g., cells in the mature retina) into RGC neurons. An exemplary method includes providing MG cells to be reprogrammed and reprogramming the MG cells into RGC neurons by contacting the MG cells with a composition comprising a cell programming agent that inhibits the expression or activity of PTB and / or nPTB in the MG cells (e.g., a Cas with a gRNA targeting PTB / nPTB or a polynucleotide encoding the same) for at least one day. In some embodiments, a single cell programming agent that inhibits the expression or activity of PTB in MG cells (e.g., a Cas with a gRNA targeting PTB) can directly convert MG cells into RGC neurons. In some embodiments, the MG cells are in the mature retina.

[0089] According to the present disclosure, in some cases, PTB reduction can induce the expression of many important neuronal differentiation factors. For example, without wishing to be bound by any theory, PTB and nPTB can each participate in two independent but intertwined loops, which may be important in neuronal differentiation. PTB can inhibit the neuronal induction loop, and the microRNA miR-124 can inhibit the transcriptional repressor RE1-silent transcription factor (REST), which can inhibit the induction of miR-124 and many neuron-specific genes (loop I). During normal neuronal differentiation, PTB can be gradually downregulated, and this downregulation can induce the expression of nPTB, which is part of the second loop of neuronal maturation involving the transcriptional activator Brn2 and miR-9 (loop II). In loop II, nPTB inhibits Brn2, thereby inhibiting miR-9, and conversely, miR-9 inhibits nPTB.

[0090] According to some embodiments, the expression levels of miR-9 or Brn2 in non-neuronal cells can be influenced by a cell programming agent that inhibits the expression or activity of PTB in the non-neuronal cells, thereby affecting the conversion of the non-neuronal cells into mature neurons. For example, adult fibroblasts may have low expression levels of miR-9 and Brn2. In some embodiments, a single agent that inhibits the expression or activity of PTB in adult fibroblasts can induce adult fibroblasts to differentiate into neuron-like cells (e.g., expression of Tuj1 protein), but not into mature neurons (e.g., expression of NeuN protein or other markers of mature neurons).

[0091] Without wishing to be bound by any particular theory, in some embodiments, the subject methods and compositions are particularly effective at generating a strengthened feedback loop in the molecular changes that induce non-neuronal cells to become neurons. Without wishing to be bound by any particular theory, when PTB expression or activity is first downregulated by an exogenous anti-PTB agent, REST levels can be downregulated, which in turn leads to upregulation of miR-124 levels.

[0092] Without wishing to be bound by any particular theory, in some cases, upregulated miR-124 can enhance the inhibition of PTB in cells because miR-124 can target and inhibit PTB expression. This positive enhancement can be persistent, even though anti-PTB agents, such as antisense oligonucleotides against PTB, may be present in the cells and only transiently active.

[0093] According to some embodiments of the present disclosure, single-cell programming agents that inhibit PTB / nPTB expression or activity in human non-neuronal cells (e.g., Cass with gRNAs targeting PTB / nPTB or polynucleotides encoding them) can directly convert the non-neuronal cells into mature neurons, optionally when the human non-neuronal cells express miR-9 or Brn2 at levels higher than those expressed in adult fibroblasts.

[0094] Exemplary human non-neuronal cells that can be used in the methods provided herein express miR-9 or Brn2 at levels at least 2-fold higher than those expressed in adult fibroblasts. In some embodiments, the human non-neuronal cells express miR-9 or Brn2 at levels at least about 1.2, 1.5, 2, 5, 10, 15, 20, or 50-fold higher than those expressed in adult fibroblasts.

[0095] In some embodiments, when human non-neuronal cells express miR-9 and Brn2 at levels higher than those expressed in adult fibroblasts, a single-cell programming agent that inhibits PTB / nPTB expression or activity in human non-neuronal cells (e.g., a Cas with a gRNA targeting PTB / nPTB) can directly convert the non-neuronal cells into mature neurons.

[0096] Exemplary human non-neuronal cells that can be used in the methods provided herein express miR-9 and Brn2 at levels at least 2-fold higher than those expressed in adult fibroblasts. In some embodiments, the human non-neuronal cells express miR-9 and Brn2 at levels at least about 1.2, 1.5, 2, 5, 10, 15, 20, or 50-fold higher than those expressed in adult fibroblasts.

[0097] In some embodiments, if a human non-neuronal cell expresses endogenous miR-9 or endogenous Brn2 at levels higher than those expressed in adult fibroblasts, a single-cell programming agent (e.g., a Cas with a gRNA targeting PTB / nPTB) that inhibits PTB / nPTB expression or activity in the human non-neuronal cell can directly convert the non-neuronal cell into a mature neuron. In some embodiments, the human non-neuronal cell has not been introduced with exogenous miR-9. In some embodiments, the human non-neuronal cell has not been introduced with exogenous Brn2. In some embodiments, the expression level of miR-9 or Brn2 in non-neuronal cells can be assessed by any technique understood by those skilled in the art. For example, the expression level of miR-9 in cells can be measured by reverse transcription (RT)-polymerase chain reaction (PCR), miRNA array, RNA sequencing (RNA-seq), and multiplex miRNA analysis. The expression level of miR-9 can also be measured by in situ methods such as in situ hybridization. The expression level of Brn2 as a protein can be measured by conventional techniques such as Western blot, enzyme-linked immunosorbent assay (ELISA), and immunostaining, or by other techniques, including, but not limited to, protein microarrays and spectroscopy (e.g., high-performance liquid chromatography (HPLC) and liquid chromatography mass spectrometry (LC / MS)). In some embodiments, information regarding the expression level of miR-9 in cells or certain tissues / cells can be obtained by referencing publicly available microRNA databases, such as, but not limited to, the Human MiRNA Expression Database (HMED), miRGator3.0, miRmine, and PhenomiR. In some embodiments, information regarding the expression level of miR-9 in a cell or a type of tissue / cell can be obtained by referencing publicly available protein expression databases, including, but not limited to, the Human Protein Atlas, GeMDBJ Proteomics, Human Proteinpedia, and the Kahn Dynamic Proteomics Database.

[0098] According to some embodiments, an exemplary method includes providing a human non-neuronal cell to be reprogrammed and contacting the human non-neuronal cell with a composition comprising a single-cell programming agent (e.g., a Cas with a gRNA targeting PTB / nPTB) that results in a decrease in PTB expression or activity in the human non-neuronal cell, and a decrease in nPTB expression or activity after the decrease in PTB expression or activity. In some embodiments, the cell programming agent can trigger sequential events regarding PTB and nPTB expression or activity levels in certain non-neuronal cells (e.g., human non-neuronal cells, e.g., human glial cells). In some embodiments, the direct effect of contact with the cell programming agent (e.g., a Cas with a gRNA targeting PTB) is to decrease PTB expression or activity in the non-neuronal cell. In some embodiments, the decrease in PTB expression or activity in the non-neuronal cell is accompanied by an initial increase in nPTB expression levels in the non-neuronal cell. In some embodiments, when PTB expression or activity is inhibited, the initial nPTB expression level increases to a high nPTB expression level. In some embodiments, nPTB expression decreases from a high nPTB expression level to a low nPTB expression level after an initial increase. In some embodiments, when PTB expression or activity is inhibited, the low nPTB expression level remains higher than the initial nPTB expression level. In some embodiments, in the absence of external intervention other than a cell programming agent that inhibits PTB expression or activity, the nPTB expression level decreases spontaneously after the initial increase. Without wishing to be bound by theory, the decrease in nPTB expression levels in non-neuronal cells after reduction of PTB expression or activity by a cell programming agent may be related to the direct conversion of the non-neuronal cell to a mature neuron by the cell programming agent. According to some embodiments, a single-cell programming agent that inhibits PTB expression or activity (e.g., a Cas with a gRNA targeting PTB) does not induce the above-described sequential events in adult fibroblasts (e.g., nPTB).For example, nPTB may undergo an initial increase in expression levels, but then fail to decline to some low level.

[0099] In some embodiments, in human astrocytes, a single-cell programming agent that inhibits PTB expression or activity in human astrocytes (e.g., a Cas with a gRNA targeting PTB) results in an immediate decrease in PTB expression or activity, an initial increase in nPTB expression levels, and a subsequent decrease in nPTB expression levels. In some embodiments, a single-cell programming agent that inhibits PTB expression or activity in human astrocytes (e.g., a Cas with a gRNA targeting PTB) directly converts human astrocytes into mature neurons. In some embodiments, the expression levels of miR-9 or Brn2 in non-neuronal cells may be related to whether or not the expression levels of nPTB in non-neuronal cells initially increase and then decrease after PTB expression or activity is inhibited by a cell programming agent. For example, in human astrocytes, the expression levels of miR-9 or Brn2 are higher than in adult fibroblasts, and the expression levels of nPTB in non-neuronal cells initially increase and then decrease after PTB expression or activity is inhibited by a cell programming agent, whereas in adult fibroblasts, as described above, the subsequent decrease in nPTB expression levels may not occur in some cases.

[0100] According to some embodiments, exemplary non-neuronal cells that can be reprogrammed into mature neurons in the methods provided herein can include, but are not limited to, glial cells such as astrocytes, oligodendrocytes, ependymal cells, microglia, Müller glial cells, spiral ganglion glial cells, Schwann cells, NG2 cells, and satellite cells. In some embodiments, the glial cells can be primate glial cells, e.g., human glial cells or non-human primate glial cells. Preferably, the glial cells are primate astrocytes, e.g., human astrocytes or non-human primate astrocytes.

[0101] In some embodiments, the glial cells that can be used in the methods provided herein are glial cells isolated from the brain. In some embodiments, the glial cells are glial cells in cell culture, for example, glial cells that have divided from parent glial cells. In some embodiments, the glial cells provided herein are glial cells differentiated from various cell types by external induction, for example, glial cells differentiated in vitro from neuronal stem cells in a medium containing a differentiation factor, or glial cells differentiated from induced pluripotent stem cells. In some other embodiments, the glial cells are glial cells in the nervous system, for example, MG cells in the mature retina, or astrocytes present in brain regions such as the striatum.

[0102] In some embodiments, astrocytes that can be used in the methods provided herein are astroglial cells in the brain or spinal cord. In some embodiments, the astrocytes express one or more known astrocyte markers, including, but not limited to, glial fibrillary acidic protein (GFAP) and aldehyde dehydrogenase 1 family member LI (ALDH1L1), excitatory amino acid transporter 1 / aspartate glutamate transporter (EAAT1 / GLAST), glutamine synthetase, S100β, or excitatory amino acid transporter 1 / glutamate transporter 1 (EAAT2 / GLT-1). In some embodiments, the astrocytes express glial fibrillary acidic protein (GFAP), aldehyde dehydrogenase 1 family member LI (ALDH1L1), or both. In some embodiments, the astrocytes are non-neuronal cells of the nervous system and express a transgene (e.g., red fluorescent protein (RFP), Cre recombinase) driven by the glial fibrillary acidic protein (GFAP) promoter. In some embodiments, the astrocytes described herein are not immunopositive for neuronal markers, such as Tuj1, NSE, NeuN, GAD67, VGluT1, or TH. In some embodiments, the astrocytes described herein are not immunopositive for oligodendrocyte markers, such as oligodendrocyte transcription factor 2, OLIG2. In some embodiments, the astrocytes described herein are not immunopositive for microglial cell markers, such as transmembrane protein 119 (TMEM119), CD45, ionized calcium-binding adaptor molecule 1 (Ibal), CD68, CD40, F4 / 80, or CD11 antigen-like family member B (CDllb). In some embodiments, the astrocytes described herein are not immunopositive for NG2 cell markers (e.g., neural / glial antigen 2, NG2).In some embodiments, the astrocytes described herein are not immunopositive for neural progenitor markers, such as nestin, CXCR4, Musashi, Notch-1, SRY-Box 1 (SOX1), SRY-Box 2 (SOX2), stage-specific embryonic antigen 1 (SSEA-1, also known as CD15), or vimentin. In some embodiments, the astrocytes described herein are not immunopositive for pluripotency markers, such as NANOG, octamer-binding transcription factor 4 (Oct-4), SOX2, Kruppel-like factor 4 (KLF4), SSEA-1, or stage-specific embryonic antigen 4 (SSEA-4). In some embodiments, the astrocytes described herein are not immunopositive for fibroblast markers (e.g., fibronectin).

[0103] Astrocytes may include various types or classes. The methods of the present application may be applied to various types of astrocytes. Non-limiting examples of various types of astrocytes include Ran2 + , GFAP + , fibroblast growth factor receptor 3 positive (FGFR3 + Type 1 astrocytes include type 1 astrocytes, which may be A2B5, A2B6, A2B7, A2B8, A2B9, A2B10, A2B11, A2B12, A2B13, A2B14, A2B15, A2B16, A2B17, A2B18, A2B19, A2B20, A2B210, A2B22, A2B23, A2B24, A2B25, A2B25, A2B30, A2B31, A2B32, A2B33, A2B34, A2B35, A2B36, A2B37, A2B38, A2B39, A2B40, A2B41, A2B42, A2B43, A2B44, A2B45, A2B56, A2B57, + , GFAP + , FGFR3 -Type 2 astrocytes include type 2 astrocytes, which may be GluT-, GluR-, and Ran2-. Type 2 astrocytes can be generated in vitro from 3-potential GRP or 2-potential O2A cells or in vivo when these progenitor cells are transplanted into an injury site. Astrocytes that can be used in the methods provided herein can be further classified according to their anatomical phenotype, such as protoplasmic astrocytes found in gray matter and having numerous branching processes whose endfeet enclose synapses, and fibrous astrocytes found in white matter and having long, slender, non-branching processes whose endfeet enclose nodes of Ranvier. Astrocytes that can be used in the methods provided herein also include GluT- and GluR-type astrocytes. GluT-type astrocytes express glutamate transporters (EAAT1 / SLC1A3 and EAAT2 / SLC1A2) and can respond to synaptic release of glutamate via transporter currents, whereas GluR-type astrocytes express glutamate receptors (mainly mGluR and AMPA types) and can respond to synaptic release of glutamate via channel-mediated currents and IP3-dependent Ca2+ + It responds instantaneously to synaptic release of glutamate via Cellular programming agents, components thereof and compositions containing same

[0104] As provided herein, compositions comprising a cell programming agent include a nucleic acid molecule that targets a target sequence in a primate PTB gene, transcript, or mRNA, or its complementary sequence, to inhibit PTB expression or activity in the non-neuronal cell, thereby enabling the reprogramming of the non-neuronal cell into a functional neuron. In one embodiment, the nucleic acid molecule is or encodes at least one antisense nucleic acid, RNAi, or guide RNA (gRNA) that targets a target sequence in a primate PTB gene, transcript, or mRNA, or its complementary sequence.

[0105] In some embodiments, the nucleic acid molecule (e.g., gRNA) targets a target sequence in a non-human primate PTB gene sequence, preferably a primate PTB gene sequence, such as a human PTB gene sequence. Examples of targeted primate PTB gene sequences include, for example, the human PTBP1 gene sequence (e.g., GenBank ID 5725).

[0106] The sequences of primate and human PTB and nPTB are known (see, e.g., Romanelli et al. (2005) Gene, August 15:356:11-8; Robinson et al., PLoS One. 2008 Mar. 12; 3(3):e1801. doi: 10.1371 / journal.pone.0001801; Makeyev et al., Mol. Cell (2007) August 3; 27(3):435-48), and therefore, one of skill in the art can design and construct antisense, miRNA, siRNA guide RNA molecules, etc. to modulate, e.g., reduce or inhibit, expression of primate PTB and / or nPTB to practice the methods of the present application.

[0107] In some embodiments, the nucleic acid molecule (e.g., gRNA) targets a target sequence in a primate PTB gene, transcript, or mRNA sequence that is conserved between humans and non-human primates.

[0108] In some embodiments, the nucleic acid molecule (e.g., gRNA) targets a target sequence in a primate PTB mRNA sequence, such as a non-human primate PTB mRNA sequence, preferably a human PTB mRNA sequence. In some embodiments, the nucleic acid molecule (e.g., gRNA) targets a target sequence in a protein-coding sequence in a primate mRNA sequence.

[0109] In some embodiments, the target sequence in the PTB gene, transcript, or mRNA is contained in a PTB sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 87. Preferred target sequences are those targeted to effectively inhibit expression or activity of primate PTB protein in non-neuronal cells. Thus, in one embodiment, the target sequence in the PTB gene, transcript, or mRNA is contained in a PTB sequence that is at least 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 87, and when the target sequence is targeted with a guide RNA (gRNA), the guide sequence of the gRNA has at least 10 nucleotides overlap with at least one sequence of SEQ ID NOs: 1-86, and the gRNA is targeted to at least one of Cos7 cells and 293T cells. and a relative expression level of PTB mRNA of less than 0.5, 0.45, 0.4, 0.35, 0.3, 0.25, 0.24, 0.23, 0.22, 0.21, 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, or 0.015 is observed in these cells compared to corresponding control cells expressing CasRx alone. More preferably, the target sequence in the PTB gene, transcript, or mRNA is contained in a PTB sequence that is at least 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO: 87, and when the target sequence is targeted with a guide RNA (gRNA), the guide sequence of said gRNA comprises or consists of at least one of SEQ ID NOs: 1 to 86, and said gRNA is expressed in at least one of Cos7 cells and 293T cells. On the other hand, relative expression levels of PTB mRNA co-expressed with CasRx of less than 0.5, 0.45, 0.4, 0.35, 0.3, 0.25, 0.24, 0.23, 0.22, 0.21, 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, or 0.015 are observed in these cells compared to corresponding control cells expressing CasRx alone.

[0110] In one embodiment, the target sequence in the PTB gene, transcript, or mRNA comprises or consists of a contiguous sequence of at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides and / or no more than 60, 55, 50, 45, 40, 35, 34, 33, 32, 31, 30, 29, 28, 27, or 26 nucleotides in the PTB sequence, which PTB sequence is at least 95, 96, 97, 98, 99, or 100% identical to SEQ ID NO:87.

[0111] In one embodiment, the target sequence in the PTB gene, transcript, or mRNA comprises or consists of a contiguous sequence of at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides and / or no more than 60, 55, 50, 45, 40, 35, 34, 33, 32, 31, 30, 29, 28, 27, or 26 nucleotides in the PTB sequence, which PTB sequence is at least 95, 96, 97, 98, 99, or 100% identical to positions 951 to 1487 of SEQ ID NO: 87, and preferably when the target sequence is targeted with a guide RNA (gRNA), The guide sequence of the gRNA comprises or consists of at least one of SEQ ID NOs: 38-68, and the gRNA is co-expressed with CasRx in at least one of Cos7 cells and 293T cells, and a relative expression level of PTB mRNA of less than 0.50, 0.45, 0.40, 0.35, 0.3, 0.25, 0.24, 0.23, 0.22, 0.21, 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, or 0.015 is observed in the cells compared to corresponding control cells expressing CasRx alone.

[0112] In one embodiment, the target sequence in the PTB gene, transcript, or mRNA comprises or consists of a contiguous sequence of at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides and / or no more than 60, 55, 50, 45, 40, 35, 34, 33, 32, 31, 30, 29, 28, 27, or 26 nucleotides in the PTB sequence, the PTB sequence being selected from the group consisting of positions 1000 to 1487 of SEQ ID NO: 87; positions 1050 to 1487 of SEQ ID NO: 87; positions 1100 to 1487 of SEQ ID NO: 7; positions 1150 to 1487 of SEQ ID NO: 87; positions 1200 to 1287 of SEQ ID NO: 87; positions 1250 to 1487 of SEQ ID NO: 87; positions 1300 to 1487 of SEQ ID NO: 87; positions 1350 to 1487 of SEQ ID NO: 7; positions 1400 to 1487 of SEQ ID NO: 87; positions 951 to 1400 of SEQ ID NO: 87; positions 951 to 1350 of SEQ ID NO: 87; positions 951 to 1300 of SEQ ID NO: 87; positions 951 to 1250 of SEQ ID NO: 87; positions 951 to 1200 of SEQ ID NO: 87; positions 951 to 1150 of SEQ ID NO: 87; positions 951 to 1100 of SEQ ID NO: 87; positions 951 to 1050 of SEQ ID NO: 87; and positions 951 to 1000 of SEQ ID NO: 87. Also, preferably, when the target sequence is targeted by a guide RNA (gRNA), the guide sequence of the gRNA comprises or consists of at least one of SEQ ID NOs: 38 to 68, and the gRNA is co-expressed with CasRx in at least one of Cos7 cells and 293T cells, and a relative expression level of PTB mRNA of less than 0.50, 0.45, 0.40, 0.35, 0.3, 0.25, 0.24, 0.23, 0.22, 0.21, 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, or 0.015 is observed in the cells compared to corresponding control cells expressing CasRx alone.

[0113] According to the present application, the nucleic acid molecule or nucleic acid sequence used to target a primate PTB target sequence may be an oligonucleotide, nucleotide, polynucleotide, or any fragment thereof, DNA or RNA of genomic or synthetic origin (which may be single-stranded or double-stranded and may represent the sense or antisense strand), peptide nucleic acid (PNA), or any DNA- or RNA-like substance of natural or synthetic origin. Nucleic acid molecules for practicing this application include "nucleic acids" or "nucleic acid sequences," which include oligonucleotides, nucleotides, polynucleotides, or any fragment thereof; DNA or RNA (e.g., mRNA, rRNA, tRNA, iRNA), or peptide nucleic acids (PNAs), of genomic or synthetic origin (which may be single-stranded or double-stranded and may represent the sense or antisense strand); or any DNA- or RNA-like entity, of natural or synthetic origin, such as iRNA, ribonucleoproteins (e.g., double-stranded iRNA such as iRNPs). Nucleic acid molecules for practicing this application include nucleic acids, i.e., oligonucleotides containing known analogs of natural nucleotides. Nucleic acid molecules for practicing this application can be any of the nucleic acid sequences described in, for example, Mata (1997) Toxicol. Appl. Pharmacol. 144:189-197; Strauss-Soukup (1997) Biochemistry 36:8692-8698; Samstag (1996) Antisense Nucleic Acid Drug Development 6:153-156, including nucleic acid-like structures with synthetic backbones. Nucleic acid molecules for carrying out the present application include "oligonucleotides" comprising a single-stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands, which can be chemically synthesized. Nucleic acid molecules for carrying out the present application include synthetic oligonucleotides that lack a 5' phosphate and therefore will not ligate to other oligonucleotides without the addition of a phosphate with ATP in the presence of a kinase. Synthetic oligonucleotides may be ligated to non-dephosphorylated fragments.

[0114] In one embodiment, the present application provides a nucleic acid molecule used to target a primate PTB target sequence as an antisense inhibitory nucleic acid molecule. Thus, in some embodiments, the present application provides antisense or other inhibitory nucleic acid molecules capable of reducing or inhibiting expression of the PTB and / or nPTB gene or protein. In one embodiment, the antisense inhibitory nucleic acid molecule can reduce or inhibit expression of the PTB and / or nPTB gene or protein by inducing exon skipping of the PTB pre-mRNA. In some embodiments, the present application's methods involve the use of molecules that can generate PTB and nPTB knockdown or eliminate or significantly reduce PTB and nPTB expression. In some embodiments, the present application's methods involve the use of these molecules to efficiently convert primate non-neuronal cells into functional neuronal cells with mature neuronal markers by sequentially knocking down first PTB and then nPTB.

[0115] Naturally occurring or synthetic nucleic acids are useful as antisense oligonucleotides. Antisense oligonucleotides can be of any length; for example, in alternative embodiments, the antisense oligonucleotides are between about 5 and 100, about 10 and 80, about 15 and 60, or about 18 and 40. The optimal length can be determined by routine screening. Antisense oligonucleotides can be present at any concentration. The optimal concentration can be determined by routine screening. Many synthetic, non-naturally occurring nucleotide and nucleic acid analogs are known to solve this potential problem. For example, peptide nucleic acids (PNAs) containing non-ionic backbones (e.g., N-(2-aminoethyl)glycine units) can be used. Antisense oligonucleotides having phosphorothioate linkages may also be used, as described in WO 97 / 03211; WO 96 / 39154; Mata (1997) Toxicol Appl Pharmacol 144:189-197; Antisense Therapeutics, ed. Agrawal (Humana Press, Totowa, NJ, 1996). Antisense oligonucleotides having synthetic DNA backbone analogs provided herein can further include phosphorodithioate, methylphosphonate, phosphoramidate, alkylphosphotriester, sulfamate, 3'-thioacetal, methylene (methylimino), 3'-N-carbamate, and morpholinocarbamate nucleic acids.

[0116] In some embodiments, the present application uses RNAi inhibitory nucleic acid molecules that can reduce or inhibit expression of a primate PTB or nPTB gene, gene, or protein.

[0117] In one embodiment, the RNAi molecule comprises a double-stranded RNA (dsRNA) molecule. The RNAi molecule can comprise a double-stranded RNA (dsRNA) molecule, such as an siRNA, miRNA (microRNA), and / or a short hairpin RNA (shRNA) molecule. For example, in one embodiment, the present application uses inhibitory nucleic acids, such as siRNA, miRNA, or shRNA, that inhibit or block the expression and / or activity of primate PTB and / or nPTB.

[0118] In some embodiments, the length of RNAi is about 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more double-stranded nucleotides. Although the present application is not limited to a particular mechanism of action, RNAi is introduced into cells and causes the degradation of single-stranded RNA (ssRNA) of similar or identical sequences, including endogenous mRNA. When cells are exposed to double-stranded RNA (dsRNA), mRNA from homologous genes is selectively degraded by a process called RNA interference (RNAi). The basic mechanism behind RNAi (e.g., siRNA to inhibit transcription and / or miRNA to inhibit translation) may be that double-stranded RNA (dsRNA) that matches a specific gene sequence is cleaved into short fragments called short interfering RNAs, thereby triggering the degradation of mRNA that matches the sequence. In one aspect, the RNAi of the present application is used for gene silencing therapy, such as silencing one or a set of transcription factors involved in maintaining the differentiated phenotype of differentiated cells; see, e.g., Shuey (2002) Drug Discov. Today 7:1040-1046. In one aspect, the present application provides a method for selectively degrading RNA using the RNAi of the present application. In one aspect, the RNAi molecules of the present application are useful for generating loss-of-function mutations in cells. These processes can be carried out in vitro, ex vivo, or in vivo.

[0119] In one embodiment, RNAi (e.g., miRNA or siRNA) is introduced into cells via the internalization of a target cell-specific ligand that binds to an RNA-binding protein containing adsorbed RNAi (e.g., microRNA). The ligand may be specific for a specific target cell surface antigen. The ligand can spontaneously internalize after binding to the cell surface antigen. If a specific cell surface antigen is not spontaneously internalized after binding to its ligand, internalization can be promoted by incorporating an arginine-rich peptide or other membrane-permeable peptide into the structure of the ligand or RNA-binding protein, or by linking such a peptide to the ligand or binding protein. See, e.g., U.S. Patent Application Publications 20060030003, 20060025361, 20060019286, and 20060019258. In one aspect, the present application provides lipid-based formulations for delivery of the present nucleic acids into cells as nucleic acid-lipid particles containing RNAi molecules, see, e.g., U.S. Patent Application Publication 20060008910.

[0120] Methods for producing and using RNAi molecules (e.g., siRNA and / or miRNA) that selectively degrade RNA are well known in the art; see, for example, U.S. Patent Nos. 6,506,559, 6,511,824, 6,515,109, and 6,489,127. Methods for producing expression constructs (e.g., vectors or plasmids) that transcribe inhibitory polynucleotides (e.g., the double-stranded siRNAs of the present application) are well known and conventional. Regulatory regions (e.g., promoters, enhancers, silencers, splice donors, acceptors, etc.) can be used to transcribe one or more RNA strands of the inhibitory polynucleotide from the expression construct. When preparing double-stranded siRNA inhibitory molecules, the sense and antisense strands targeting the target portion of the IRES can be transcribed as two separate RNA strands that are annealed together, or as a single RNA strand that anneals to itself to form a hairpin loop. For example, a construct targeting a portion of a gene can be inserted between two promoters so that transcription occurs in both directions, producing complementary RNA strands, which can then be annealed to form the inhibitory siRNA of the present application.

[0121] Alternatively, the target portion of a gene, coding sequence, promoter, or transcript can be designed as a first and second antisense binding region together on a single expression vector, for example, comprising a first coding region of the target gene in the sense orientation relative to a regulated promoter, and a second coding region of the gene in the antisense orientation relative to the regulated promoter. When transcription of the sense and antisense coding regions of the target portion of the target gene occurs from two independent promoters, the result is two independent RNA strands that can then be annealed to form a gene-inhibiting siRNA for carrying out the present invention.

[0122] In another embodiment, transcription of the sense and antisense target portions of the target gene is controlled by a single promoter, and the resulting transcript is a self-complementary single hairpin RNA strand, i.e., a double-stranded RNA that self-folds to form a duplex to generate a gene-inhibiting siRNA molecule. In this configuration, a spacer, e.g., a nucleotide spacer, between the sense and antisense coding regions of the target gene can improve the ability of the single-stranded RNA to form a hairpin loop, and the hairpin loop comprises the spacer. In one embodiment, the spacer comprises nucleotides having a length of between about 5 and 50 nucleotides. In one aspect, the sense and antisense coding regions of the siRNA can be placed on separate expression vectors and controlled by their own promoters.

[0123] In one embodiment, the nucleic acid molecule is an antisense oligonucleotide that targets the PTB pre-mRNA to induce exon hopping, which results in the inhibition of expression or activity of the primate PTB protein in non-neuronal cells. Exon skipping, as used herein, refers to the induction of a mature mRNA in a cell that does not contain a particular exon that is normally present therein. Exon skipping is achieved by providing a cell expressing the pre-mRNA of the mRNA with a molecule capable of interfering with a sequence, such as a splicing donor or splicing acceptor sequence required for the enzymatic process that allows splicing, or an exon inclusion signal required to recognize a portion of nucleotides as an exon to be included in the mRNA. The term pre-mRNA refers to unprocessed or partially processed precursor mRNA, synthesized by transcription from a DNA template in the cell nucleus. In the context of this application, induction and / or promotion of exon skipping as referred to herein means that at least 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the PTB mRNA in non-neuronal cells does not contain said exon, preferably as assessed by PCR as described in the Examples.

[0124] In one preferred embodiment, the nucleic acid molecule targeting a target sequence or its complementary sequence in a primate PTB gene, transcript, or mRNA is a guide RNA (gRNA) for a CRISPR / Cas family effector protein (Cas effector protein). Thus, in one embodiment, a composition comprises a cellular programming agent comprising: i) a Cas effector protein and at least one gRNA that targets a target sequence or its complementary sequence in a primate PTB gene, transcript, or mRNA, or ii) at least one expression vector encoding a Cas effector protein and encoding at least one gRNA. Similarly, the present application provides compositions comprising such cellular programming agents.

[0125] In some embodiments, the cell programming agent (e.g., comprising a Cas effector protein and at least one gRNA, or at least one expression vector) is contained in a nanoparticle, such as a liposome.

[0126] In one embodiment, a cell programming agent is provided that comprises a CRISPR / Cas family effector protein (Cas effector protein) and a guide RNA (gRNA) that targets a PTB gene sequence, or at least one expression vector that encodes a Cas effector protein and a gRNA that targets a PTB gene sequence.

[0127] In some embodiments, the Cas effector protein in the cell programming agent is selected from the group consisting of Cas13d, CasRx, Cas13e, CRISPR / Cas9, Cpf1, Cas9, Cas13a, Cas13b, Cas13c, Cas13f, and functional domains thereof. In some embodiments, the Cas effector protein is encoded by an ORF (start codon to stop codon) of 4.5 kb or less, 4 kb or less, 3.5 kb or less, 3 kb or less, 2.5 kb or less, 2.1 kb or less, or 1.5 kb or less. In some embodiments, the Cas effector protein is modified to include a nuclear localization signal. In some embodiments, the Cas effector protein is a DNA-targeting Cas effector protein. In some embodiments, the Cas effector protein is a DNA-targeting Cas effector protein selected from the group consisting of spCas9 or a variant thereof, SaCas9 or a variant thereof, Cpf1 or a variant thereof, and combinations thereof.

[0128] In some embodiments, the Cas effector protein in the cell programming agent is an RNA-targeting Cas effector protein. As used herein, an RNA-targeting Cas effector protein is understood to refer to a Cas effector protein that uses the crRNA / gRNA of the Cas effector protein to identify and degrade a target RNA sequence instead of the Cas effector protein of the target DNA sequence. In some embodiments, the Cas effector protein is an effector protein of a type VI CRISPR-CAS system, preferably a type VI-D CRISPR-CAS system. In some embodiments, the Cas effector protein contains two HEPN ribonuclease motifs and includes an RXXXXH motif (see Anantharaman et al., 2013, Biol Direct. 2013; 8: 15). In some embodiments, the RNA-targeting Cas effector protein is selected from the group consisting of Cas13a, Cas13b, Cas13c, Cas13d / CasRx, CRISPR / Cas9, Cpf1, Cas13e, and Cas13f, and functional domains thereof. In some embodiments, the RNA-targeting Cas effector protein is encoded by an ORF (start codon to stop codon) of 4.5 kb or less, 4 kb or less, 3.5 kb or less, 3 kb or less, 2.5 kb or less, 2.1 kb or less, or 1.5 kb or less. In some embodiments, the RNA-targeting Cas effector protein is modified to include a nuclear localization signal.

[0129] In a preferred embodiment, the RNA-targeting Cas effector protein is Cas13d or its orthologue, most preferably CasRx (described in Konermann et al., 2018, Cell 173, 665-676). CasRx is the orthologue of CRISPR-Cas13d, and is the first choice for in vivo therapy due to its smallest size and high target specificity and efficiency. Preferably, gRNA that targets primate PTB mRNA sequence is used in the cell programming agent of the present application in combination with the CRISPR / Cas family effector protein that targets RNA.Therefore, in some embodiments, gRNA is the sequence that is complementary to the target sequence in primate PTB mRNA, for example, human PTBP1 coding sequence (NM_002819; SEQ ID NO: 87), and preferably, gRNA comprises the sequence that is complementary to the target sequence as defined herein above.

[0130] In one embodiment, the gRNA comprises a guide sequence complementary to a contiguous portion of 14 to 60 nucleotides in a primate PTB mRNA sequence (e.g., SEQ ID NO: 87). Preferably, the gRNA comprises a guide sequence complementary to a contiguous portion of at least 17 but not more than 60 nucleotides in a primate PTB mRNA sequence (e.g., SEQ ID NO: 87). In some specific embodiments, the gRNA comprises a guide sequence complementary to a contiguous portion of at least 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in a primate PTB mRNA sequence (e.g., SEQ ID NO: 87), and / or a guide sequence complementary to a contiguous portion of no more than 60, 55, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, or 40 nucleotides in a primate PTB sequence (e.g., SEQ ID NO: 87). In some embodiments, the gRNA comprises a guide sequence complementary to a contiguous portion of 25-45 nucleotides in a primate PTB mRNA sequence (e.g., SEQ ID NO: 87).

[0131] In one embodiment, the guide sequence in the gRNA comprises or consists of at least 10, 11, 12, 13, 14, 15, 16, 17 consecutive nucleotides, or all nucleotides, of at least one of SEQ ID NOs: 1-86.

[0132] In one embodiment, the guide sequence in the gRNA comprises or consists of at least 10, 11, 12, 13, 14, 15, 16, 17 contiguous nucleotides or all nucleotides of at least one of SEQ ID NOs: 1-86, and when the gRNA is co-expressed with CasRx in at least one of Cos7 and / or 293T cells, it results in a PTB of less than 0.5, 0.45, 0.4, 0.35, 0.3, 0.25, 0.24, 0.23, 0.22, 0.21, 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02 or 0.015 compared to corresponding control cells expressing CasRx alone. The relative expression of mRNA is observed.

[0133] In one embodiment, the guide sequence in the gRNA comprises or consists of at least one of SEQ ID NOs: 1-86. In one embodiment, the guide sequence in the gRNA comprises or consists of at least one of SEQ ID NOs: 1-86, preferably generating a relative expression of less than 0.5, 0.45, 0.4, 0.35, 0.3, 0.25, 0.24, 0.23, 0.22, 0.21, 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, or 0.015 as determined in Table 1.

[0134] In one embodiment, the guide sequence in the gRNA comprises or consists of at least one sequence selected from the group consisting of SEQ ID NOs: 56, 60, 47, 48, 43, 45, 40, 61, 50, 55, 41, 44, 42, 66, 49, 51, 46, 79, 07, 58, 53, 38, 64, 63, 59, 54, 62, 81, 83, 57, 05, 08, 70, 73, 76, 10, 04, 36, 52, 26, 67, 25, 39, 34, 80, 06, 23, 85, 24, 30, 02, 13, 03, 77, 31, 21, 69, 16, 75, 12, 78, 20, 74, 71, and 37. In one embodiment, the guide sequence in the gRNA comprises or consists of at least one sequence selected from the group consisting of SEQ ID NOs: 56, 60, 47, 48, 43, 45, 40, 61, 50, 55, 41, 44, 42, 66, 49, 51, 46, 79, 07, 58, 53, 38, 64, 63, 59, 54, 62, 81, 83, 57, 05, 08, 70, 73, 76, 10, 04, 36, 52, 26, 67, 25, 39, 34, 80, 06, 23, 85, 24, 30, and 02. In one embodiment, the guide sequence in the gRNA comprises or consists of at least one sequence selected from the group consisting of SEQ ID NOs: 56, 60, 47, 48, 43, 45, 40, 61, 50, 55, 41, 44, 42, 66, 49, 51, 46, 79, 07, 58, 53, 38, 64, 63, 59, 54, 62, 81, 83, 57, 05, 08, 70, 73, 76, 10, 04, 36, 52, 26, 67, and 25. In one embodiment, the guide sequence in the gRNA comprises or consists of at least one sequence selected from the group consisting of SEQ ID NOs: 56, 60, 47, 48, 43, 45, 40, 61, 50, 55, 41, 44, 42, 66, 49, 51, 46, 79, 07, 58, 53, 38, 64, 63, 59, 54, 62, 81, 83, 57, 05, 08, 70, 73, and 76. In one embodiment, the guide sequence in the gRNA comprises or consists of at least one sequence selected from the group consisting of SEQ ID NOs: 56, 60, 47, 48, 43, 45, 40, 61, 50, 55, 41, 44, 42, 66, 49, 51, 46, 79, 07, 58, 53, 38, 64, 63, and 59.In one embodiment, the guide sequence in the gRNA comprises or consists of at least one sequence selected from the group consisting of SEQ ID NOs: 56, 60, 47, 48, 43, 45, 40, 61, 50, 55, 41, 44, 42, 66, 49, 51, and 46. In one embodiment, the guide sequence in the gRNA comprises or consists of at least one sequence selected from the group consisting of SEQ ID NOs: 56, 60, 47, 48, 43, 45, 40, 61, 50, 55, 41, 44, 42, 66, and 49. In one embodiment, the guide sequence in the gRNA comprises or consists of at least one sequence selected from the group consisting of SEQ ID NOs: 56, 60, 47, 48, 43, 45, 40, and 61. In one embodiment, the guide sequence in the gRNA comprises or consists of at least one sequence selected from the group consisting of SEQ ID NOs: 56, 60, 47, 48, 43, 45, and 40. In one embodiment, the guide sequence in the gRNA comprises or consists of at least one sequence selected from the group consisting of SEQ ID NOs: 56, 60, 47, 48, 43, and 45. In one embodiment, the guide sequence in the gRNA comprises or consists of at least one sequence selected from the group consisting of SEQ ID NOs: 56, 60, 47, 48, and 43. In one embodiment, the guide sequence in the gRNA comprises or consists of at least one sequence selected from the group consisting of SEQ ID NOs: 56, 60, 47, and 48. In one embodiment, the guide sequence in the gRNA comprises or consists of at least one sequence selected from the group consisting of SEQ ID NOs: 56, 60, and 47. In one embodiment, the guide sequence in the gRNA comprises or consists of at least one sequence selected from the group consisting of SEQ ID NOs: 56 and 60.

[0135] In a preferred embodiment, the guide sequence in the gRNA comprises or consists of SEQ ID NO: 60. In another preferred embodiment, the guide sequence in the gRNA comprises or consists of SEQ ID NO: 56.

[0136] In some embodiments, the gRNA targets a sequence in primate PTB mRNA, the sequence being: a) positions 59-91 of SEQ ID NO:87; b) positions 303-349 of SEQ ID NO:87; c) positions 422-451 of SEQ ID NO:87; d) positions 460-489 of SEQ ID NO:87; e) positions 542-576 of SEQ ID NO:87; f) positions 646-681 of SEQ ID NO:87; g) positions 706-769 of SEQ ID NO:87; h) positions 773-806 of SEQ ID NO:87; i) positions 1079-1139 of SEQ ID NO:87; j) positions 1152-1184 of SEQ ID NO:87; k) positions 1191-1254 of SEQ ID NO:87; l) positions 1374-1434 of SEQ ID NO:87; m) positions 951-1487 of SEQ ID NO:87; and o) positions 1502 to 1575 of SEQ ID NO: 87; and o) positions 1626 to 1669 of SEQ ID NO: 87. As understood herein, sequences in a primate PTB mRNA that correspond to sequences relative to positions in SEQ ID NO: 87 (excluding SEQ ID NO: 87) are sequences that correspond to these positions in SEQ ID NO: 87 in a nucleotide sequence alignment, preferably using the global Needleman-Wunsch algorithm with default settings (default gap opening penalty 10.0, default gap extension penalty 0.5, default scoring matrix DNAFull).

[0137] In some specific embodiments, the gRNA comprises or consists of at least 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides, and / or no more than 60, 55, 50, 49, 48, 47, 46, 44, 43, 42, 41, or 40 nucleotides of a primate PTB mRNA sequence, wherein the primate PTB mRNA sequence is at least 95, 96, 97, 98, 99%, or 100% identical to positions 951-1487 of SEQ ID NO: 87, and preferably, when the target sequence is targeted with a guide RNA (gRNA), the guide sequence of the gRNA comprises or consists of at least one of SEQ ID NOs: 38-68, and wherein the gRNA is co-expressed with CasRx in at least one of Cos7 cells and 293T cells ... A relative expression of PTB mRNA of less than 0.50, 0.45, 0.40, 0.35, 0.3, 0.25, 0.24, 0.23, 0.22, 0.21, 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02 or 0.015 is observed in these cells compared to corresponding control cells expressing sRx only.

[0138] In one embodiment, the gRNA comprises or consists of a guide sequence complementary to a contiguous portion of at least 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides, and / or no more than 60, 55, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, or 40 nucleotides, of a primate PTB mRNA sequence, The mRNA sequence is as follows: positions 1000 to 1487 of SEQ ID NO: 87; positions 1050 to 1487 of SEQ ID NO: 87; positions 1100 to 1487 of SEQ ID NO: 87; positions 1150 to 1487 of SEQ ID NO: 87; positions 1200 to 1487 of SEQ ID NO: 87; positions 1250 to 1487 of SEQ ID NO: 87; positions 1300 to 1487 of SEQ ID NO: 87; positions 1350 to 1487 of SEQ ID NO: 87; positions 1400 to 1487 of SEQ ID NO: 87; positions 1400 to 1487 of SEQ ID NO: 87; positions 1410 to 1411 of SEQ ID NO: 87; positions 1420 to 1421 of SEQ ID NO: 87; positions 1430 to 1431 of SEQ ID NO: 87; positions 1440 to 1441 of SEQ ID NO: 87; positions 1450 to 1451 of SEQ ID NO: 87; positions 1460 to 1461 of SEQ ID NO: 87; positions 1470 to 1471 of SEQ ID NO: 87; positions 1480 to 1487 of SEQ ID NO: 87; positions 1490 to 1491 of SEQ ID NO: 87; positions 1500 to 1500 of SEQ ID NO: 87; positions 1510 to 1511 of SEQ ID NO: 87; positions 1520 to 1521 of SEQ ID NO: 87; positions 1530 to 1531 of SEQ ID NO: 8 positions 951 to 1400 of SEQ ID NO: 87; positions 951 to 1350 of SEQ ID NO: 87; positions 951 to 1300 of SEQ ID NO: 87; positions 951 to 1250 of SEQ ID NO: 87; positions 951 to 1200 of SEQ ID NO: 7; positions 951 to 1150 of SEQ ID NO: 7; positions 951 to 1100 of SEQ ID NO: 87; positions 951 to 1050 of SEQ ID NO: 87; and positions 951 to 1000 of SEQ ID NO: 87. , 97%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the target sequence. Preferably, when the target sequence is targeted by a guide RNA (gRNA), the guide sequence of the gRNA comprises or consists of at least one of SEQ ID NOs: 38 to 68, and the gRNA is co-expressed with CasRx in at least one of Cos7 cells and 293T cells, and expresses only CasRx. A relative expression of PTB mRNA of less than 0.50, 0.45, 0.40, 0.35, 0.3, 0.25, 0.24, 0.23, 0.22, 0.21, 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02 or 0.015 is observed in the cells compared to corresponding control cells.

[0139] In some embodiments, co-expression of a gRNA of the present application with CasRx in human or non-human primate cells (e.g., Cos7 cells and / or 293T cells) results in a PTB of less than 0.9, 0.85, 0.8, 0.75, 0.7, 0.65, 0.6, 0.55, 0.5, 0.45, 0.4, 0.35, 0.3, 0.25, 0.24, 0.23, 0.22, 0.21, 0.20, 0.19, 0.18, 0.17, 0.16, 0.15, 0.14, 0.13, 0.12, 0.11, 0.10, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, or 0.01 compared to corresponding control cells expressing CasRx alone. Relative mRNA expression can be measured as described in the Examples section of this specification. For example, instantaneous cotransfection can be performed using Lipofectamine 3000 (or a similar transfection reagent) with 4 μg of a vector expressing CAG-CasRx-P2A-GFP and 2 μg of the U6-gRNA-CMV-mCherry plasmid. Cells transfected with the CAG-CasRx-P2A-GFP plasmid alone can be used as a control. Two days after transient transfection, approximately 30K GFP and mCherry double-positive cells (top 20% of GFP) are harvested by fluorescence-activated cell sorting (FACS), digested, and subjected to qPCR analysis to determine the relative expression of PTB mRNA compared to a corresponding amount of control cells (sorted only to the top 20% of GFP).

[0140] As provided herein, a cellular programming agent (e.g., a Cas with a gRNA targeting PTB or a polynucleotide encoding it) inhibits PTB expression or activity by at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of endogenous or natural levels. As provided herein, a cellular programming agent (e.g., a Cas having a gRNA targeting nPTB or a polynucleotide encoding the same) inhibits nPTB expression or activity by about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of endogenous or natural levels.

[0141] In some embodiments, the cellular programming agents provided herein (e.g., Cass having gRNAs targeting PTB / nPTB or polynucleotides encoding them) directly inhibit the expression levels of PTB / nPTB, e.g., transcription, translation, or protein stability of PTB and / or nPTB.

[0142] In some embodiments, the cellular programming agents provided herein (e.g., Cass with gRNAs targeting PTB / nPTB or polynucleotides encoding them) directly affect the expression or activity of PTB / nPTB and do not affect other cellular signaling pathways.

[0143] In some embodiments, the composition comprising a cellular programming agent comprises at least one gRNA targeting a primate PTB mRNA sequence or at least one expression vector encoding at least one gRNA targeting a primate PTB mRNA sequence. In other embodiments, the composition comprising a cellular programming agent comprises no more than one type of gRNA targeting a primate PTB mRNA sequence or at least one expression vector encoding no more than one type of gRNA targeting a primate PTB mRNA sequence. In yet other embodiments, the composition comprising a cellular programming agent comprises two, three, four, five, or six different types of gRNAs targeting a primate PTB mRNA sequence or at least one expression vector encoding two, three, four, five, or six different types of gRNAs targeting a primate PTB mRNA sequence.

[0144] As provided herein, contacting a non-neuronal cell with a composition comprising a cell programming agent provided herein can be done in any suitable manner, depending on the type of non-neuronal cell to be reprogrammed, the environment in which the non-neuronal cell is placed, and the desired outcome of cell reprogramming.

[0145] In some embodiments, non-neuronal cells are contacted with a composition comprising a cellular programming agent provided herein in the form of a polynucleotide encoding a Cas having a gRNA that targets PTB / nPTB. Thus, in one embodiment, non-neuronal cells are contacted with a cellular programming agent provided herein in the form of an expression vector that encodes a Cas effector protein and encodes at least one gRNA that targets a primate PTB mRNA sequence.

[0146] In some embodiments, at least one expression vector comprises a nucleotide sequence encoding a Cas effector protein, the nucleotide sequence operably linked to a promoter that drives expression of the Cas effector protein in a non-neuronal cell. In some embodiments, the promoter that drives expression in a non-neuronal cell is a glial cell-specific promoter or a Müller glial cell (MG) cell-specific promoter. In some embodiments, the glial cell-specific promoter is selected from the group consisting of a GFAP promoter, an ALDH1L1 promoter, an EAAT1 / GLAST promoter, a glutamine synthetase promoter, an S100β promoter, and an EAAT2 / GLT-1 promoter. In some embodiments, the MG cell-specific promoter is selected from the group consisting of a GFAP promoter, an ALDH1L1 promoter, a GLAST (also known as Slc1a3) promoter, and an Rlbp1 promoter. In some embodiments, the glial cell-specific promoter or the Müller glial cell (MG) cell-specific promoter is a primate, human, or non-human primate promoter.

[0147] In some embodiments, at least one expression vector comprises at least one nucleotide sequence encoding a gRNA that targets a primate PTB mRNA sequence, the nucleotide sequence being operably linked to a promoter that drives expression of the gRNA in non-neuronal cells. In some examples, the promoter operably linked to the gRNA coding sequence (and that drives expression of the gRNA in non-neuronal cells) is a promoter derived from the U6 snRNA gene, preferably a primate, human, or non-human primate U6 promoter.

[0148] In some embodiments, nucleotide sequences encoding Cas effector proteins are suitable for optimizing their codon usage to match the codon usage of primate, human, or non-human primate (non-neuronal) host cells. The adaptability of a nucleotide sequence encoding a polypeptide to host cell codon usage can be expressed as a codon adaptation index (CAI). The codon adaptation index is defined herein as a metric of the relative adaptability of a gene's codon usage relative to the codon usage of highly expressed genes in a particular host cell or organism. The relative adaptability (w) of each codon is the ratio of each codon's usage to the usage of the most abundant codon for the same amino acid. The CAI index is defined as the geometric mean of these relative adaptability values. Nonsynonymous codons and stop codons (according to the genetic code) are excluded. CAI values ​​range from 0 to 1, with higher values ​​indicating a higher proportion of the most abundant codons (see Sharp and Li, 1987, Nucleic Acids Research 15: 1281-1295; and Jansen et al., 2003, Nucleic Acids Res. 31(8):2242-51). Suitable nucleotide sequences encoding Cas effector proteins preferably have a CAI of 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9 or greater. Methods of codon optimization for optimal gene expression in heterologous organisms are known in the art and have been previously described (see, e.g., Welch et al., 2009, PLoS One 4:e7002; Gustafsson et al., 2004, Trends Biotechnol. 22:346-353; Wu et al., 2007, Nucl. Acids Res. 35:D76-79; Villalobos et al., 2006, BMC Bioinformatics 7:285; U.S. Patent Publication No. 2011 / 0111413; and U.S. Patent Publication No. 2008 / 0292918).

[0149] In these configurations, the cell programming agent is introduced using a non-viral transfection or viral transduction method. Non-viral transfection can refer to any cell transfection method that does not involve a virus. Non-limiting examples of non-viral transfection include electroporation, microinjection, calcium phosphate precipitation, DEAE-dextran followed by cationic polymers such as polyethylene glycol, dendrimer-mediated transfection, liposome-mediated transfection ("lipid transfection"), microparticle bombardment ("gene gun"), fugene, direct sonic loading, cell squeezing, optical transfection, protoplast fusion, needle transfection, magnetic transfection, nuclear transfection, and any combination thereof. In some embodiments, the methods provided herein utilize gene therapy vectors, e.g., viral vectors, as a suitable vehicle for delivery of cell programming agents to non-neuronal cells. As provided herein, viral vector methods can include the use of DNA or RNA viral vectors. Examples of suitable viral vectors include adenovirus, lentavirus, adeno-associated virus (AAV), poliovirus, herpes simplex virus (HSV), or murine Maloney-based viral vectors.

[0150] In some examples, the vector is an AAV vector. In some embodiments, the cellular programming agent is administered in the form of an AAV vector. In some embodiments, the cellular programming agent is administered in the form of a lentiviral vector. For example, the cellular programming agent can be delivered to non-neuronal cells using lentivirus or adenovirus-associated virus (AAV) to express a Cas effector protein with a gRNA against PTB / nPTB.

[0151] According to some embodiments of the present disclosure, the methods provided herein include inhibiting PTB / nPTB expression or activity in a non-neuronal cell (e.g., a glial cell or an astrocyte) with a cell programming agent in an amount sufficient to reprogram the non-neuronal cell into a mature neuron. As one of ordinary skill in the art can readily appreciate, a sufficient amount of a cell programming agent can be empirically determined. In some embodiments, the amount of a cell programming agent can be determined by any type of assay that examines the activity of the cell programming agent in a non-neuronal cell.

[0152] For example, if a cell programming agent is configured to inhibit expression of PTB / nPTB in non-neuronal cells, a sufficient amount of the cell programming agent can be determined by assessing the expression level of PTB / nPTB in exemplary non-neuronal cells after administration, e.g., by Western blot. In some embodiments, after delivery of a cell programming agent to exemplary non-neuronal cells, the activity of PTB / nPTB is assessed in a functional assay. In some embodiments, other functional assays of downstream neuronal properties, such as immunostaining for neuronal markers or electrical recordings of neuronal functional properties, are examined to determine a sufficient amount of the cell programming agent.

[0153] In some embodiments, the cell programming agent is delivered in the form of a viral vector, which can include one or more copies of an expression sequence encoding the cell programming agent, e.g., a Cas effector protein with one or more copies, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 copies, of a gRNA coding sequence for PTB / nPTB.

[0154] As can be determined by one of skill in the art, the viral vector can be titrated to any suitable dosage. For example, the titer, as determined by PCR, RT-PCR, or other methods, is at least about 10 5 Virus particles / mL, 10 6 particles / mL, 107 particles / mL, 10 8 particles / mL, 10 9 particles / mL, 10 10 particles / mL, 10 11 particles / mL, 10 12 particles / mL, 10 13 particles / mL, 10 14 particles / mL or 10 15 It can be particles / mL.

[0155] In some embodiments, the titer of the administered viral vector is at least about 10 10 particles / mL, 10 11 particles / mL, 10 12 particles / mL, 10 13 particles / mL or 10 14 particles / mL. In other embodiments of the present application, the composition comprising a cellular programming agent that targets PTB, as defined above, comprises additional components, such as one or more cellular programming agents that do not target PTB, to further improve neuronal conversion efficiency.

[0156] Combining a cell programming agent that targets PTB with one or more cell programming agents that do not target PTB can act synergistically to improve neuronal conversion efficiency.

[0157] Therefore, in one embodiment, the composition comprising a cell programming agent targeting PTB as defined above further comprises i) one or more dopamine neuron-associated factors, and / or ii) at least one expression vector for expressing one or more dopamine neuron-associated factors, preferably in non-neuronal cells. Preferably, such a composition comprising a cell programming agent targeting PTB and a dopamine neuron-associated factor is used for local administration to non-neuronal cells in the striatum. More preferably, the composition is administered to non-neuronal cells in the striatum to generate functional dopaminergic neurons, and even more preferably, to glial cells in the striatum to generate functional dopaminergic neurons.

[0158] In one embodiment, the one or more dopamine neuron-associated factors are selected from the group consisting of Lmx1a, Lmx1b, FoxA2, ​​Nurr1, Pitx3, Gata2, Gata3, FGF8, BMP, En1, En2, PET1, Pax family proteins, SHH, Wnt family proteins, and TGF-β family proteins. In one embodiment, the one or more dopamine neuron-associated factors are selected from the group consisting of FoxA2, ​​Lmx1a, and Nurr1. In a preferred embodiment, the one or more dopamine neuron-associated factors are FoxA2 alone, Lmx1a alone, Nurr1 alone, a combination of FoxA2 and Lmx1a, a combination of FoxA2 and Nurr1, a combination of Nurr1 and Lmx1a, or a combination of FoxA2, ​​Lmx1a, and Nurr1. Suitable primate and human amino acid and / or nucleotide sequences for these dopamine neuron-associated factors / genes are known to those of skill in the art and can be accessed in public databases. Construction and delivery of expression vectors for expressing one or more dopamine neuron-associated factors in non-neuronal cells can be as described above for expression vectors for PTB-targeted cell programming.

[0159] In another embodiment, the composition comprising a cell programming agent targeting PTB as defined above comprises at least one expression vector for expressing, preferably in non-neuronal cells, i) one or more factors selected from the group consisting of β-catenin, OSK factors (also referred to as Oct4, Sox2, Klf4, and other factors involved in cell recycling or epigenetic remodeling), Crx, Brn3a, Brn3b, Math5, Nr2e3, and Nrl, and / or ii) one or more factors selected from the group consisting of β-catenin, OSK factors (also referred to as Oct4, Sox2, Klf4, and other factors involved in cell recycling or epigenetic remodeling), Crx, Brn3a, Brn3b, Math5, Nr2e3, and Nrl. In one embodiment, the one or more factors are selected from the group consisting of β-catenin, OSK factors, Oct4, Sox2, and Klf4. In preferred embodiments, the one or more factors are a combination of β-catenin and Oct4, a combination of β-catenin and Sox2, a combination of β-catenin and Klf4, a combination of β-catenin, Oct4 and Sox2, a combination of β-catenin, Oct4 and Klf4, a combination of β-catenin, Sox2 and Klf4, a combination of β-catenin and all three of the OSK factors Oct4, Sox2 and Klf4, or a combination of β-catenin and all three of the OSK factors Oct4, Sox2 and Klf4 (without β-catenin). Preferably, such compositions comprising cell programming agents and factors that target PTB are used for local administration to non-neuronal cells in the mature retina. More preferably, the composition is applied to non-neuronal cells in the mature retina to generate functional retinal ganglion cell (RGC) neurons and / or functional retinal photoreceptors, and even more preferably, the composition is applied to glial cells or Muller glia (MG) cells in the mature retina to generate functional retinal ganglion cell (RGC) neurons and / or functional retinal photoreceptors. In a preferred embodiment, the factor is β-catenin.Suitable primate and human amino acid and / or nucleotide sequences for β-catenin, Oct4, Sox2, Klf4, Crx, Brn3a, Brn3b, Math5, Nr2e3, and Nrl, or genes thereof, are known to those skilled in the art and can be accessed in public databases. Construction and delivery of expression vectors for expressing one or more factors selected from β-catenin, Oct4, Sox2, Klf4, Crx, Brn3a, Brn3b, Math5, Nr2e3, and Nrl in non-neuronal cells may be as described above for the expression vectors for cell programming agents targeting PTB. Dosing and Treatment Regimen

[0160] The methods provided herein can include inhibiting expression or activity of PTB / nPTB in a non-neuronal cell for a period of time sufficient to reprogram the non-neuronal cell into a mature neuron.

[0161] In some embodiments, exemplary methods include reprogramming a non-neuronal cell into a mature neuron by contacting the non-neuronal cell with a cell programming agent that inhibits PTB / nPTB expression or activity in the non-neuronal cell for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 2 months, at least 3 months, at least 4 months, at least 5 months, or at least 1 year.

[0162] In some embodiments, the inhibition of PTB and nPTB expression or activity is sequential, e.g., PTB expression or activity is inhibited first, e.g., by any of the time periods described above, before nPTB expression or activity is inhibited.

[0163] In some embodiments, the inhibition of PTB and nPTB expression or activity occurs simultaneously.

[0164] In some embodiments, an exemplary method includes reprogramming a non-neuronal cell into a mature neuron by contacting the non-neuronal cell with a cell programming agent that inhibits expression or activity of PTB in the non-neuronal cell for about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 3 weeks, 4 weeks, 5 weeks, 2 months, 3 months, 4 months, or 5 months.

[0165] In some configurations, the methods provided herein comprise a one-time administration of a cellular programming agent, e.g., adding the cellular programming agent to a cell culture comprising non-neuronal cells, or a one-time delivery of the cellular programming agent to a brain region (e.g., the striatum) comprising non-neuronal cells, wherein the cellular programming agent can remain active for the period of time required to inhibit PTB / nPTB expression or activity in the non-neuronal cells (e.g., at least 1 day, at least 2 days, at least 4 days, or the last 10 days). For example, when the cellular programming agent comprises an AAV vector expressing coding sequences for a Cas effector and an anti-PTB gRNA, the AAV vector can be designed to maintain transcriptional activity over a long period of time.

[0166] In some embodiments, the methods provided herein include administering the cell programming agent multiple times, such as at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 12 times, at least 15 times, at least 20 times or more.

[0167] In some embodiments, the methods provided herein further comprise administering at least one immunosuppressant. In one embodiment, for example, when the cellular programming agent comprises an AAV vector expressing coding sequences for a Cas effector and an anti-PTB gRNA, the immunosuppressant is administered before, simultaneously with, and / or after administration of the cellular programming agent. Suitable immunosuppressants include, for example, corticosteroids (e.g., prednisone, prednisolone, dexamethasone, etc.), calcineurin inhibitors (e.g., cyclosporine, tacrolimus, etc.), mTOR inhibitors (e.g., sirolimus, everimus, etc.), IMDH inhibitors (e.g., azathioprine, mycopherol acid, etc.), antibodies (e.g., basiliximab, rituximab, alemtuzumab, etc.), interferons (e.g., IFN-β, IFN-γ, etc.), Janus kinase inhibitors (e.g., tofacitinib, etc.), and biologics (e.g., anakinra, etc.). In one embodiment, the immunosuppressant is administered about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 3 weeks, 4 weeks, 5 weeks, 2 months, 3 months, 4 months, or 5 months before and / or after administration of the cellular programming agent. In one embodiment, the immunosuppressant is administered approximately simultaneously with administration of the cellular programming agent. marker

[0168] According to some embodiments of the present disclosure, the methods provided herein include efficiently reprogramming a plurality of non-neuronal cells into mature neurons.

[0169] In some embodiments, the method comprises reprogramming MG cells or astrocytes into mature neurons, converting at least 60% of the MG cells / astrocytes into Map2 or NeuN positive mature neurons.

[0170] In some embodiments, at least 40% of the astrocytes are converted into mature Map2-positive neurons, hi some embodiments, at least about 20%, 25%, 30%, 35%, 38%, 40%, 42%, 44%, 46%, 48%, 50%, 52%, 54%, 56%, 58%, 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, or 100% of the MG cells / astrocytes are converted into mature NeuN- or Map2-positive neurons.

[0171] In some embodiments, at least 10 MG cells in a 10 mm x 50 μm retinal ganglion cell layer (GCL) are converted to RGCs expressing Brn3a or Rbpms. In some embodiments, at least about 1, 2, 4, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, or 60 MG cells in a 10 mm x 50 μm retinal ganglion cell layer (GCL) are converted to RGCs expressing Brn3a or Rbpms.

[0172] In some embodiments, the method comprises reprogramming human astrocytes into mature neurons, converting at least 40%, at least 60%, or at least 80% of the human astrocytes into Map2- or NeuN-positive mature neurons, hi some embodiments, at least 20%, at least 40%, or at least 60% of the human astrocytes are converted into Map2- or NeuN-positive mature neurons. In some embodiments, at least about 20%, 25%, 30%, 35%, 38%, 40%, 42%, 44%, 46%, 48%, 50%, 52%, 54%, 56%, 58%, 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99% or 100% of human astrocytes are converted into Map2 or NeuN positive mature neurons. In some embodiments, the methods provided herein include reprogramming a plurality of non-neuronal cells, e.g., human non-neuronal cells, e.g., human glial cells or astrocytes, and about 20%, 25%, 30%, 35%, 38%, 40%, 42%, 44%, 46%, 48%, 50%, 52%, 54%, 56%, 58%, 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, or 99% of the non-neuronal cells, e.g., human non-neuronal cells, e.g., human glial cells or astrocytes, into mature neurons. In some embodiments, the methods provided herein reprogram about 20%, 25%, 30%, 35%, 38%, 40%, 42%, 44%, 46%, 48%, 50%, 52%, 54%, 56%, 58%, 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, or 100% of non-neuronal cells, e.g., human non-neuronal cells, e.g., human glial cells, MG cells, or astrocytes, into mature neurons.

[0173] In some embodiments, mature neurons are characterized by the expression of NeuN (neuronal nuclear antigen), Map2 (microtubule-associated protein 2), NSE (neuron-specific enolase), 160 kDa neurofilament medium, 200 kDa neurofilament heavy chain, and the like. The non-neuronal cells are characterized by expressing one or more neuronal markers selected from the group consisting of: PDS-95 (postsynaptic density protein 95), synapsin I, synaptophysin, GAD67 (glutamic acid decarboxylase 67), GAD65 (glutamic acid decarboxylase 65), parvalbumin, DARPP32 (dopamine- and cAMP-regulated neuronal phosphoprotein 32), vGLUT1 (vesicular glutamate transporter 1), vGLUT2 (vesicular glutamate transporter, acetylcholine, vesicular GABA transporter (VGAT), gamma-aminobutyric acid (GABA), and TH (tyrosine hydroxylase). In some embodiments, at least 40% of the non-neuronal cells, e.g., human non-neuronal cells, e.g., human glial cells, or astrocytes, are reprogrammed into mature neurons.

[0174] In some embodiments, the MG cells are converted into retinal photoreceptors characterized by expressing one or more rod cell markers selected from rhodopsin and GNAT1, and / or characterized by expressing one or more cone cell markers selected from S-opsin, M-opsin, and mCAR.

[0175] As those skilled in the art will readily appreciate, the expression of all of the above markers can be assessed by any conventional technique. For example, immunostaining using antibodies against specific cell-type markers described herein can reveal whether target cells express the corresponding cell-type markers. Immunostaining under certain conditions can also reveal the subcellular distribution of cell-type markers, which is important for determining the developmental stage of target cells. For example, expression of Map2 is found in various neurites (e.g., dendrites) of postmitotic mature neurons but is absent from neuronal axons. Another example is the expression of voltage-gated sodium ion channels (e.g., the a-subunit Navi.1-1.9 and b-subunit Navi.1-1.9), which can be clustered in mature neurons at the initial axonal segment (where action potentials can be initiated) and at the node of Ranvier. In some embodiments, other techniques, such as flow cytometry, mass spectrometry, in situ hybridization, RT-PCR, and microarrays, can also be used to assess the expression of specific cell-type markers described herein. Conversion efficiency

[0176] Methods provided by certain aspects of the present disclosure include reprogramming a plurality of non-neuronal cells, and at least about 20%, 25%, 30%, 35%, 38%, 40%, 42%, 44%, 46%, 48%, 50%, 52%, 54%, 56%, 58%, 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, or 99% of the non-neuronal cells, e.g., human non-neuronal cells, e.g., human glial cells, MG cells, or astrocytes, into functional neurons. In some embodiments, the methods provided herein reprogram at least 20% of non-neuronal cells, e.g., human non-neuronal cells, e.g., human glial cells, MG cells, or astrocytes, into functional neurons. In some embodiments, the methods provided herein reprogram at least 20%, 25%, 30%, 35%, 38%, 40%, 42%, 44%, 46%, 48%, 50%, 52%, 54%, 56%, 58%, 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 99%, or 100% of the non-neuronal cells, e.g., human non-neuronal cells, e.g., human glial cells, MG cells, or astrocytes, into functional neurons.

[0177] In some embodiments, the methods provided herein comprise the use of a combination of i) a cellular programming agent that targets PTB as defined herein, and ii) one or more cellular programming agents that do not target PTB as defined herein, wherein the combination improves neuronal conversion efficiency by at least 1.1-fold, 1.2-fold, 1.5-fold, 2.0-fold, 3.0-fold, 4.0-fold, 5.0-fold, 8.0-fold, 10-fold, 15-fold, or 20-fold. Functional evaluation

[0178] In some embodiments, a functional neuron is characterized by the ability to form neuronal networks, the ability to send and receive neuronal signals, or both. In some embodiments, a functional neuron fires action potentials. In some embodiments, a functional neuron establishes synaptic connections with other neurons. For example, a functional neuron can be a postsynaptic neuron at a synapse, e.g., having its dendritic terminals, e.g., dendritic spines, forming a postsynaptic compartment at a synapse with another neuron. For example, a functional neuron can be a presynaptic neuron at a synapse, e.g., having an axon terminal that forms a presynaptic terminal at a synapse with another neuron.

[0179] Synapses that functional neurons can form with other neurons include, but are not limited to, axon-axon synapses, axon-dendritic synapses, and axon-somatic synapses. Synapses that functional neurons can form with other neurons can be excitatory (e.g., glutamatergic), inhibitory (e.g., γ-aminobutyric acidergic), modulatory, or any combination thereof. In some embodiments, the synapses that functional neurons form with other neurons are glutamatergic, γ-aminobutyric acidergic, cholinergic, adrenergic, dopaminergic, or any other suitable type. As a presynaptic neuron, a functional neuron can release neurotransmitters, including, but not limited to, glutamate, GABA, acetylcholine, aspartate, D-serine, glycine, nitric oxide (NO), carbon monoxide (CO), hydrogen sulfide (HS), dopamine, norepinephrine (also called noradrenaline), epinephrine (adrenaline), histamine, serotonin, phenethylamine, N-methylphenethylamine, tyramine, 3-iodothyronamine, octopamine, tryptamine, somatostatin, substance P, opioid peptides, adenosine triphosphate (ATP), adenosine, and anandamide. As a postsynaptic neuron, a functional neuron can elicit a postsynaptic response in response to neurotransmitters released into the synaptic cleft from the presynaptic neuron. The postsynaptic responses of functional neurons generated in the methods provided herein can be excitatory, inhibitory, or any combination thereof, depending on the type of neurotransmitter receptors expressed by the functional neurons. In some embodiments, the functional neurons express ionotropic neurotransmitter receptors, such as ionotropic glutamate receptors and ionotropic GABA receptors.Ionotropic glutamate receptors can include, but are not limited to, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors (e.g., GluAl / GRIA1, GluA2 / GRIA2, GluA3 / GRIA3, GluA4 / GRIA4), δ receptors (e.g., GluDl / GRIDl, GluD2 / GRID2), kinin receptors (e.g., GluKl / GRIKl, GluK2 / GRIK2, GluK3 / GRIK3, GluK4 / GRIK4, GluK5 / GRIK5), and N-methyl-D-aspartate (NMDA) receptors (e.g., GluNl / GRINl, GluN2A / GRIN2A, GluN2B / GRIN2B, GluN2C / GRIN2C, GluN2D / GRIN2D, GluN3A / GRIN3A, GluN3B / GRIN3B). Ionotropic GABA receptors can include, but are not limited to, GABA receptors. In some embodiments, the functional neuron expresses metabotropic neurotransmitter receptors, such as metabotropic glutamate receptors (e.g., mGluRi, mGluRs, mGluR, mGluRi, mGluRe, mGluR, mGluRs) and metabotropic GABA receptors (e.g., GABA receptors). In some embodiments, the functional neuron expresses one type of dopamine receptor, for example, a dopamine receptor of the D1-like family, such as D1 and D5 receptors (D1R and D5R), or a dopamine receptor of the D2-like family, such as D2, D3, and D4 receptors (D2R, D3R, and D4R). In some embodiments, the functional neuron provided herein forms an electrical synapse (e.g., a gap junction) with another neuron. In some embodiments, the functional neuron provided herein forms a chemical or electrical synapse with itself, known as an autapse.

[0180] Characteristics of functional neurons can be evaluated using conventional techniques available to those skilled in the art. For example, electrical properties of functional neurons, such as postsynaptic responses to action potential excitation and neurotransmitter release, can be examined using techniques such as patch clamp recording (e.g., current clamp recording and voltage clamp recording), intracellular recording, and extracellular recording (e.g., tetrode recording, single-wire recording, and field potential recording). Specific properties of functional neurons (e.g., ion channel expression and resting membrane potential) can also be examined using patch clamp recording, where various variations of patch clamp recording can be applied for various purposes, such as cell-attached patch clamp, inside-out patch clamp, outside-out patch clamp, whole-cell recording, perforated patch clamp, and loose patch clamp. Evaluation of postsynaptic responses by electrical methods can be combined with either electrical stimulation of the presynaptic neuron or application of neurotransmitter or receptor agonists or antagonists. In some cases, AMPA-type glutamate receptor-mediated postsynaptic currents can be assessed by AMPA receptor agonists (e.g., AMPA) or antagonists (e.g., 2,3-hydroxy-6-nitro-7-sulfonylamino-benzoquinoxaline (NBQX) or 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX)). In some cases, NMDA-type glutamate receptor-mediated postsynaptic currents can be assessed by NMDA receptor agonists (e.g., NMDA and glycine) or antagonists (e.g., AP5 and ketamine).

[0181] In some embodiments, functional neurons are examined by techniques other than electrical methods. For example, electrical signals transmitted or delivered by functional neurons can be monitored using a variety of recently developed fluorescent dyes or genetically encoded fluorescent proteins and imaging techniques. In this case, calcium-dependent fluorescent dyes (e.g., calcium indicators) such as fura-2, indo-1, fluo-3, fluo-4, and calcium green-1, and calcium-dependent fluorescent proteins such as Cameleons, FIP-CBSM, Pericams, GCaMP, TN-L15, TN-humTnC, TN-XL, TNXXL, and Twitch's are useful for tracking calcium influx and efflux as an indicator of neuronal membrane potential. Alternatively, or in addition, voltage-sensitive dyes that can change their spectral properties in response to voltage changes can also be used to monitor neuronal activity.

[0182] Neurotransmitter release can be an important aspect of functional neurons. The methods provided herein can include reprogramming non-neuronal cells into functional neurons that release certain neurotransmitters. In some embodiments, the functional neurons release neurotransmitters such as glutamate, GABA, acetylcholine, aspartate, D-serine, glycine, nitric oxide (NO), carbon monoxide (CO), hydrogen sulfide (HS), dopamine, norepinephrine (also called noradrenaline), epinephrine (adrenaline), histamine, serotonin, phenethylamine, N-methylphenethylamine, tyramine, 3-iodothyronamine, octopamine, tryptamine, somatostatin, substance P, opioid peptides, adenosine triphosphate (ATP), adenosine, and anandamide.

[0183] In some embodiments, the functional neuron releases dopamine as its primary neurotransmitter. In some embodiments, the functional neuron releases multiple types of neurotransmitters. In some embodiments, the functional neuron releases neurotransmitters in response to an action potential. In some embodiments, the functional neuron releases neurotransmitters in response to graded potentials (e.g., changes in membrane potential that do not exceed the threshold for eliciting an action potential). In some embodiments, the functional neuron exhibits neurotransmitter release at a basal level (e.g., spontaneous neurotransmitter release). Neurotransmitters released by the functional neurons described herein can be assessed by various techniques available to those skilled in the art. In some embodiments, imaging methods can be used to characterize neurotransmitter release in functional neurons, for example, by imaging genetically encoded fluorescent fusion molecules containing vesicle proteins to monitor the process of synaptic vesicle fusion to the presynaptic membrane.

[0184] Alternatively, or in addition, other methods can be applied to directly monitor the levels of specific neurotransmitters. For example, HPLC probes can be used to measure dopamine content in a dish or in brain regions to which functional neurons project their axons. Dopamine levels detected by HPLC can indicate presynaptic activity of functional neurons. In some embodiments, such assessments can be combined with stimulation of functional neurons to change their membrane potential, e.g., initiate action potentials.

[0185] In one aspect, the present disclosure provides a method for generating functional neurons in vivo. An exemplary method includes administering a composition comprising a cellular programming agent (e.g., a Cas effector protein and a gRNA targeting / complementary to PTB and / or nPTB, or a polynucleotide encoding the same) to a subject's nervous system region, such as the mature retina or inner ear, or a region of the brain or spinal cord (e.g., the striatum), to non-neuronal cells (e.g., glial cells or astrocytes) in that region, thereby reprogramming the non-neuronal cells into functional neurons. In some embodiments, the cellular programming agent inhibits expression or activity of PTB and / or nPTB. Administration route

[0186] According to some embodiments of the present disclosure, the methods provided herein include directly administering a cellular programming agent (e.g., a Cas effector protein and a gRNA targeting / complementary to PTB and / or nPTB, or a polynucleotide encoding the same) to a region of a subject's nervous system (e.g., the mature retina or inner ear, or a region within the brain or spinal cord (e.g., the striatum)). In some embodiments, the cellular programming agent (e.g., a Cas effector protein and a gRNA targeting / complementary to PTB and / or nPTB, or a polynucleotide encoding the same) is delivered locally to a region of the nervous system (e.g., the mature retina, a region within the brain, or spinal cord (e.g., the striatum)). In one embodiment, a composition comprising a cellular programming agent, such as a viral vector (e.g., an AAV vector), is administered to a subject or organism by stereotaxic or convection-enhanced delivery to a brain region (e.g., the striatum).

[0187] One skilled in the art can use a stereotactic positioning system to locate a specific brain region (e.g., the striatum) to which a composition containing a cellular programming agent will be administered. Such methods and devices can readily be used to deliver the compositions provided herein to a subject or living organism. In another embodiment, the compositions provided herein are delivered systemically to a subject or a region of the subject's nervous system, such as the brain (e.g., the striatum) or spinal cord, e.g., the cerebrospinal fluid or ventricles, and the composition comprises one or more agents configured to relocate the cellular programming agent to a specific region of the subject's nervous system (e.g., the striatum) or to a specific type of cell in the subject's nervous system.

[0188] In some embodiments, the cellular programming agent used in the methods provided herein comprises a virus expressing a Cas effector and an anti-PTB or anti-nPTB gRNA, and the methods include stereotactically injecting the virus into a desired brain region. In some embodiments, the virus comprises an adenovirus, a lentivirus, an adeno-associated virus (AAV), a poliovirus, a herpes simplex virus (HSV), or a mouse Moloney-based virus. AAV that can be used in the methods provided herein can be any suitable AAV serotype, such as, but not limited to, AAV2, AAV5, AAV6, AAV7, AAV8, and AAV9. In some embodiments, the methods include delivering an AAV2- or AAV9-based viral vector, which expresses an agent that inhibits PTB and / or nPTB expression or activity in non-neuronal cells in a nervous system region (e.g., the brain (e.g., the striatum) or the spinal cord).

[0189] In some embodiments, as described above, the methods provided herein include reprogramming a plurality of non-neuronal cells into mature neurons. In some embodiments, the methods provided herein include administering a composition comprising a cell programming agent to a region of the nervous system of a subject (e.g., the brain (e.g., the striatum) or spinal cord), wherein the cell programming agent inhibits PTB and / or nPTB expression or activity in a number of non-neuronal cells, e.g., but not limited to, glial cells such as astrocytes, oligodendrocytes, NG2 cells, satellite cells, or ependymal cells of the nervous system, thereby enabling the non-neuronal cells to be reprogrammed into functional neurons. In some embodiments, the methods provided herein include reprogramming astrocytes in a region of the nervous system of a subject (e.g., the brain (e.g., the striatum) or spinal cord) into functional neurons.

[0190] As described above, the methods provided herein can include reprogramming non-neuronal cells in a particular brain region (e.g., the striatum) into functional neurons. Exemplary brain regions that can be used in the methods provided herein can be any of the hindbrain, midbrain, or forebrain. In some embodiments, the methods provided herein include administering to the midbrain, striatum, or cortex of a subject a composition comprising a cellular programming agent that inhibits PTB expression or activity in non-neuronal cells in the mature retina or striatum, thereby enabling reprogramming of the non-neuronal cells into functional neurons. In some embodiments, the methods provided herein include administering to the mature retina or striatum of a subject a composition comprising a cellular programming agent that inhibits PTB / nPTB expression or activity in non-neuronal cells in the mature retina or striatum, thereby enabling reprogramming of the non-neuronal cells into functional neurons.

[0191] In some embodiments, the methods provided herein comprise reprogramming non-neuronal cells into functional neurons in a brain region, such as, for example, the medulla oblongata, pyramidal medulla, olivores, inferior olivary nucleus, rostral ventrolateral medulla, caudal ventrolateral medulla, solitary nucleus, respiratory center-respiratory group, dorsal respiratory group, ventral respiratory group or apneustic center, anterior Butzinger complex, Botzinger complex, posterior trapezoid nucleus, posterior facial nucleus, nucleus ambiguus retronucleus, subnucleus ambiguus, paramedian reticular nucleus, gigantocellular reticular nucleus, parafacial nucleus, zone), cuneiform nucleus, gracilis nucleus, hypoglossal nucleus, interneuron, prepositus nucleus, hypoglossal nucleus, area posterior, medullary cranial nucleus, inferior salivary nucleus, nucleus ambiguus, dorsal nucleus of the vagus, hypoglossal nucleus, hindbrain, pons, pontine nuclei, pontine cranial nerve nuclei, principal nucleus of the trigeminal sensory nucleus or pontine nucleus, trigeminal motor nucleus (v), abducens nucleus (vi), facial nucleus (vii), vestibulocochlear nucleus (vestibular nucleus and cochlear nucleus) (viii), superior salivary nucleus, pontine tegmentum, pontine micturition center (Barrington's nucleus), locus coeruleus, pedunculopontine nucleus, posterodorsal tegmental nucleus, reticular nucleus of the pontine tegmentum, parabrachial area, parabrachial nucleus, lateral parabrachial nucleus, parabrachial nucleus (Kdlliker-Fuse nucleus), pontine respiratory complex, superior olivary complex, medial superior olive olive), lateral superior olive, medial nucleus of the trapezoid body, paramedian pontine reticular formation, parvocellular reticular nucleus, caudal pontine reticular nucleus, cerebellar peduncle, superior cerebellar peduncle, middle cerebellar peduncle, inferior cerebellar peduncle, fourth ventricle, cerebellum, cerebellar vermis, cerebellar hemispheres, anterior lobe, posterior lobe, flocculus tuberosus, cerebellar nuclei, nucleus osseostaticus, internodal nucleus, nucleus globus, nucleus emboli, dentate nucleus, midbrain (mesencephalon), tectum, tectum tetragiom, inferior colliculus, superior colliculus, pretectum, tegmentum, periaqueductal gray, rostral interstitial nucleus of the medial longitudinal fasciculus, midbrain reticular formation, dorsal raphe nucleus, red nucleus, ventral tegmental area, parabrachial pigmented nucleus, paranigral nucleus, rostromedial tegmental nucleus, caudal linear nucleus, rostral linear raphe nucleus nucleus of the raphe, interfascicular nucleus, substantia nigra, pars compacta, pars reticularis, interpeduncular nucleus, cerebral peduncle (crus cerebri), midbrain cranial nerve nuclei, oculomotor nucleus (III), oculomotor accessory nucleus (Edinger-Westphalnucleus), trochlear nucleus (IV), mesencephalic tract (cerebral aqueduct, aqueduct of Sylvius), forebrain (forebrain / prosencephalon), diencephalon, epithalamus, pineal gland, habenular nucleus, medullary stria oblongata, stria striata thalamus (taenia thalami), third ventricle, subcommissural organ, thalamus, anterior nucleus, anterior ventral nucleus (also known as ventral anterior nucleus), anterior dorsal nucleus, anterior medial nucleus, medial nucleus, median nucleus, parathenia nucleus, nucleus of reuniens nucleus), rhomboid nucleus, intralaminar nucleus group, centromedian nucleus, parafascicular nucleus, paracentral nucleus, lateral nucleus group, lateral dorsal nucleus, lateral posterior nucleus, pulvinar, ventral nucleus group, ventral anterior nucleus, ventral posterior nucleus, ventroventral nucleus, ventroposterior nucleus, ventroposterior nucleus, posterior thalamus, medial geniculate body, lateral geniculate body, reticular nucleus of the thalamus, hypothalamus (limbic system) (hpa axis), anterior, medial region, preoptic area, medial preoptic nucleus, suprachiasmatic nucleus, paraventricular nucleus, supraoptic nucleus (mainly), anterior nucleus of the hypothalamus, lateral region, preoptic area, lateral preoptic nucleus, anterior part of the lateral nucleus, part of the supraoptic nucleus, other nuclei of the preoptic area, median optic tract Anterior nucleus, periventricular preoptic nucleus, tubercle, medial region, dorsomedial nucleus of the hypothalamus, ventromedial nucleus, arcuate nucleus, lateral region, lateral tubercular nucleus, lateral tubercular nucleus, posterior, medial region, mammillary nucleus, posterior nucleus, lateral region, posterior lateral nucleus, optic chiasm, subround organ, periventricular nucleus, pituitary stalk, gray matter tubercle, tubercular nucleus, tuberomammillary nucleus, tubercular region, mammillary body, mammillary nucleus, subthalamus, subthalamic nucleus, zona incerta, pituitary gland, pituitary nerve, pars intermedia (intermediate lobe), pituitary gland, frontal lobe, parietal lobe, occipital lobe, temporal lobe, cerebellum, brainstem, centrum semiovale, corona radialis, internal capsule, external capsule, extreme capsule capsule), subcortical area, hippocampus, dentate gyrus, cornu ammonis (CA area), cornu ammonis area 1 (CA1), cornu ammonis area 2 (CA2), cornu ammonis area 3 (CA3), cornu ammonis area 4 (CA4), amygdala, central nucleus of the amygdala, medial nucleus of the amygdala, cortical and basomedial nuclei of the amygdala, lateral and basolateral nuclei of the amygdala, extended amygdalaamygdala, stria terminalis, bed nucleus of stria terminalis, anterior claustrum, basal ganglia, striatum, dorsal striatum, putamen, caudate nucleus, ventral striatum, nucleus accumbens, olfactory tubercle, globus pallidus, ventral globus pallidus, subthalamic nucleus, basal forebrain, anterior perforated substance, substantia innominata, basal ganglia, Broca's diagonal band, septal nucleus, medial septal nucleus, lamina terminalis, organus vasculosus of lamina terminalis, rhinencephalon (paleocortex), olfactory bulb, olfactory tract, anterior olfactory nucleus, piriform cortex, anterior commissure, uncus, peri-amygdaloid cortex, cerebral cortex, frontal lobe, cortex, primary motor cortex (precentral gyrus, Ml), supplementary motor area, premotor cortex, prefrontal cortex, orbitofrontal cortex, dorsolateral prefrontal cortex, gyrus, superior frontal gyrus, middle frontal gyrus, inferior frontal gyrus, Brodmann's areas 4, 6, 8, 9, 10, 11, 12, 2 4, 25, 32, 33, 44, 45, 46, 47, parietal lobe, cortex, primary somatosensory cortex (SI), secondary somatosensory cortex (S2), posterior parietal cortex, gyrus, postcentral gyrus (primary sensory cortex), precuneus, Brodmann areas 1, 2, 3, 5, 7, 23, 26, 29, 31, 39, and 40, occipital lobe, cortex, primary visual cortex (VI), v2, v3, v4, v5 / mt, gyrus, lateral occipital gyrus, cuneus, Brodmann areas 17 (VI, primary visual cortex) 18 and 19, temporal lobe, cortex, primary auditory cortex (A1), secondary auditory cortex (A2), inferior temporal cortex, posterior inferior temporal cortex, gyrus, superior temporal gyrus, middle temporal gyrus, inferior temporal gyrus, entorhinal cortex, perirhinal cortex cortex), parahippocampal gyrus, fusiform gyrus, Brodmann's area 20, 21, 22, 27, 34, 35, 36, 37, 38, 41 and 42, medial superior temporal area (MST), insular cortex, cingulate cortex, anterior cingulate cortex, posterior cingulate cortex, retrosplenial cortex, indusium griseum, subgenual area 25, and Brodmann's area 23, 24; 26, 29, 30 (retrosplenial area) 31, 32.

[0192] In one aspect, the present application provides a method for generating dopaminergic neurons in vivo. An exemplary method includes administering to the striatum of a subject's brain a composition comprising a cellular programming agent (e.g., a Cas effector protein and a coding sequence for a gRNA for PTB and / or nPTB) that inhibits expression or activity of PTB and / or nPTB in non-neuronal cells (e.g., glial cells or astrocytes) of the brain, thereby enabling reprogramming of the non-neuronal cells into dopaminergic neurons. In some embodiments, the method includes administering to the putamen of the subject a composition comprising a cellular programming agent (e.g., a Cas effector protein and a coding sequence for a gRNA for PTB and / or nPTB) that inhibits expression or activity of PTB and / or nPTB in non-neuronal cells (e.g., glial cells or astrocytes) of the brain, thereby enabling reprogramming of the non-neuronal cells into dopaminergic neurons. In some embodiments, the method includes administering specifically to the putamen of the subject (e.g., avoiding administration to other parts of the caudate nucleus and / or striatum) a composition comprising a cellular programming agent (e.g., a Cas effector protein and a coding sequence for a gRNA against PTB and / or nPTB) that inhibits expression or activity of PTB and / or nPTB in non-neuronal cells of the brain (e.g., glial cells or astrocytes) and enables reprogramming of the non-neuronal cells into dopaminergic neurons.

[0193] In another aspect, the present invention provides a method for generating RGC neurons in vivo. An exemplary method includes administering to a subject's mature retina a composition comprising a cell programming agent (e.g., a Cas effector protein and a gRNA coding sequence for PTB and / or nPTB) that inhibits the expression or activity of PTB and / or nPTB in non-neuronal cells (e.g., glial cells or MG cells) of the mature retina, allowing the non-neuronal cells to be reprogrammed into RGC neurons.

[0194] In some embodiments, the methods provided herein include administering to a region of the nervous system (e.g., the brain or spinal cord) of a subject a composition comprising a cellular programming agent (e.g., a Cas effector protein and a coding sequence for a gRNA against PTB and / or nPTB) that inhibits expression or activity of PTB and / or nPTB in non-neuronal cells within the region and enables reprogramming of the non-neuronal cells into functional neurons of the subtype that predominates within the region.

[0195] Without being bound by any particular theory, the methods provided herein can utilize local inductive signals in a region (e.g., a specific brain region) when non-neuronal cells are reprogrammed into functional neurons in vivo. For example, local signals in the striatum may induce PTB / nPTB-inhibited non-neuronal cells to convert into dopamine neurons. Local neurons, non-neuronal cells (e.g., astrocytes, microglial cells, or both), or other local components of the striatum can contribute to the subtype specificity of neurons induced by cell programming agents and generated by non-neuronal cells.

[0196] In some embodiments, the methods provided herein include administering to a brain region (e.g., the striatum) of a subject a composition comprising a cellular programming agent (e.g., a coding sequence for a Cas effector protein and a gRNA for PTB and / or nPTB) that inhibits PTB / nPTB expression or activity in a plurality of non-neuronal cells in the brain region, the method further comprising administering to the ... , 25%, 30%, 35%, 38%, 40%, 42%, 44%, 46%, 48%, 50%, 52%, 54%, 56%, 58%, 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, or 99% of the non-neuronal cells into dopaminergic neurons.

[0197] In some embodiments, the methods provided herein include administering to a mature retina or brain region (e.g., striatum) of a subject a composition comprising a cellular programming agent (e.g., a coding sequence for a Cas effector protein and a gRNA for PTB and / or nPTB) that inhibits expression or activity of PTB / nPTB in a plurality of non-neuronal cells in the brain region, and wherein at least about 5% of the cells produced by this method are cytoplasmic or cytoplasmic. 10%, 20%, 25%, 30%, 35%, 38%, 40%, 42%, 44%, 46%, 48%, 50%, 52%, 54%, 56%, 58%, 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, or 99% of the functional neurons are RGC neurons or dopaminergic neurons, respectively.

[0198] In some embodiments, the dopaminergic neurons generated by the methods provided herein express one or more markers of dopaminergic neurons, including, but not limited to, dopamine, tyrosine hydroxylase (TH), dopamine transporter (DAT), vesicular monoamine transporter 2 (VMAT2), Engrailed homeobox 1 (Enl), nuclear receptor related-1 (Nurrl), G protein-regulated inwardly rectifying potassium channel 2 (Girk2), forkhead box A2 (FoxA2), orthodenticle homeobox 2 (OTX2), and / or LIM homeobox transcription factor 1 alpha (Lmx1a).

[0199] In some embodiments, dopamine neurons generated using the methods provided herein exhibit Ih currents that can be mediated by hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. Ih currents are characterized as slowly activated inward currents and can be activated by hyperpolarization steps. For example, when voltage clamp and holding potential Vh are at -40 mV, dopamine neurons trigger a slowly activating inward current with a reversal potential close to -30 mV. The activation curve of the Ih current characteristics of dopamine neurons generated using the methods provided herein can be in the range of -50 mV to -120 mV, with a midpoint activation point between -84 mV and 1 mV.

[0200] In some embodiments, the dopaminergic neurons produced by the methods provided herein have a gene expression profile similar to that of naturally occurring dopaminergic neurons. In some embodiments, the dopaminergic neurons generated by the methods provided herein release dopamine as a neurotransmitter.

[0201] The dopaminergic neurons generated by the methods provided herein may be any subtype of dopaminergic neurons, including, but not limited to, A9 (e.g., immunopositive for Girk2), A10 (e.g., immunopositive for calcium binding protein-D28k), A11, A12, A13, A16, Aaq, and telencephalic dopamine neurons.

[0202] According to some embodiments of the present disclosure, the methods provided herein include reprogramming non-neuronal cells in a region of a subject's nervous system, such as the mature retina, brain, or spinal cord region (e.g., the striatum), into functional neurons. In some embodiments, the functional neurons described herein are integrated into neural networks within the nervous system. As described herein, the reprogrammed functional neurons can form synaptic connections with local neurons (e.g., neurons adjacent to the reprogrammed functional neurons). For example, as the reprogrammed neurons mature in vivo, synaptic connections can be formed between the reprogrammed neurons and adjacent primary neurons (e.g., glutamatergic neurons), GABAergic intermediary neurons, or other adjacent neurons (e.g., dopaminergic neurons, adrenergic neurons, or cholinergic neurons). In these synaptic connections to local neurons, the reprogrammed functional neurons can be presynaptic neurons, postsynaptic neurons, or both.

[0203] In some embodiments, the reprogrammed functional neurons send axonal projections to remote brain regions.

[0204] In some embodiments, the reprogrammed functional neurons can integrate themselves into one or more existing neural pathways in the brain or spinal cord, such as the superior longitudinal fasciculus, arcuate fasciculus, uncinate fasciculus, perforant pathway, thalamocortical radiation, corpus callosum, anterior commissure, ventral amygdala efferent pathway, interthalamic adhesions, posterior commissure, habenular commissure, fornix, mammillary tegmental bundle, interthalamic pathway, cerebral peduncle, medial forebrain bundle, medial longitudinal fasciculus, myoclonic triangular, mesocortical pathway, mesolimbic pathway, substantia nigra-liatallar pathway, tuberoinfundibular pathway, and the like. pathway), extrapyramidal system, pyramidal tract, corticospinal tract or cerebrospinal fibers, lateral corticospinal tract, anterior corticospinal tract, corticopontine fibers, frontopontine fibers, temporopontine fibers, corticobulbar tract, corticomesencephalic tract, tectumspinal tract, interstitial spinal tract, rubrospinal tract, rubro-olivary canal, olivocerebellar tract, olivospinal tract, vestibulospinal tract, lateral vestibulospinal tract, medial vestibulospinal tract, reticulospinal tract, lateral suturespinal tract, dorsal column medial lemniscus pathway, fasciculus gracilis, fasciculus cuneate, medial lemniscus, spinothalamic tract, lateral spinothalamic tract, anterior spinothalamic tract, spinomesencephalic tract, spinocerebellar tract, spinolivary tract, and spinal reticular tract.

[0205] Without being bound by any particular theory, the local cellular environment may be related to the projection of functional neurons generated according to some embodiments of the present disclosure. For example, functional neurons generated in the striatum according to some embodiments of the methods provided herein may be influenced by other cells in the local environment of the striatum. Treatable conditions / diseases

[0206] In one aspect, the present disclosure provides a method for treating a neurological condition associated with the degeneration of functional neurons in a nervous system region. An exemplary method includes administering to a nervous system region of a subject in need thereof, such as the mature retina, brain or spinal region (e.g., the striatum) or inner ear, a composition comprising a cell programming agent that inhibits PTB / nPTB expression or activity in non-neuronal cells in the region, allowing the non-neuronal cells to reprogram into functional neurons (e.g., RGCs or dopaminergic neurons), thereby replacing degenerated functional neurons in the region.

[0207] According to some embodiments of the present disclosure, the methods provided herein include treating neurological conditions, including, but not limited to, Parkinson's disease, blindness, hearing loss, spinal cord injury, Alzheimer's disease, Huntington's disease, schizophrenia, depression, and drug addiction.

[0208] Applicable neurological conditions can also include, but are not limited to, diseases associated with neuronal loss in the spinal cord, such as amyotrophic lateral sclerosis (ALS) and motor neuron disease. The methods provided herein are also useful for treating or ameliorating one or more symptoms of neurodegenerative diseases, including autosomal dominant cerebellar ataxia, autosomal recessive spastic ataxia of Charlevoix-Saguenay, corticobasal degeneration, corticobasal syndrome, Creutzfeldt-Jakob disease, fragile X-associated tremor / ataxia syndrome, frontotemporal dementia with parkinsonism linked to chromosome 17, Kufour-Rakeb syndrome, Lyme disease, Machado-Joseph disease, Niemann-Pick disease, pontocerebellar hypoplasia, Refsum disease, pyruvate dehydrogenase complex deficiency, Sandhoff disease, Shy-Drager syndrome, Tay-Sachs disease, and Wobbly hedgehog syndrome. These include, but are not limited to, encephalopathy syndrome.

[0209] As provided herein, "neurodegeneration" or its grammatical equivalents can refer to the progressive loss of neuronal structure, function, or both, including neuronal death.

[0210] Neurodegeneration can be caused by any type of mechanism. The neurological conditions to which the methods provided herein are applicable can be of any etiology. Neurological conditions can be genetic or accidental and can result from genetic mutations, protein misfolding, oxidative stress, or environmental exposure (e.g., toxins or drug abuse).

[0211] In some embodiments, the methods provided herein treat neurological conditions associated with degeneration of dopaminergic neurons in brain regions. In some embodiments, the methods provided herein treat neurological conditions associated with degeneration of RGC neurons in the mature retina. In other embodiments, the methods provided herein treat a neurological condition related to the degeneration of any type of neuron, including glutamatergic neurons, GABAergic neurons, cholinergic neurons, adrenergic neurons, dopaminergic neurons, or any other suitable type of neuron, which releases aspartate, D-serine, glycine, nitric oxide (NO), carbon monoxide (CO), hydrogen sulfide (HS), norepinephrine (also called noradrenaline), histamine, serotonin, phenethylamine, N-methylphenethylamine, tyramine, 3-iodothyronamine, octopamine, tryptamine, somatostatin, substance P, opioid peptides, adenosine triphosphate (ATP), adenosine, or anandamide as a neurotransmitter. The methods provided herein can be used to treat neurological conditions associated with neuronal degeneration in any region, such as, but not limited to, the midbrain region (e.g., the substantia nigra or the ventral tegmental area), the forebrain region, the hindbrain region, or the spinal cord. The methods provided herein can include reprogramming non-neuronal cells in any suitable region of the nervous system into functional neurons to treat neurological conditions associated with neuronal degeneration.

[0212] The methods provided herein are useful for treating or ameliorating one or more symptoms associated with Parkinson's disease. Parkinson's disease is a neurodegenerative disorder characterized by early and significant functional impairment and death of dopaminergic neurons in the substantia nigra pars compacta (SNpc). The resulting dopamine deficiency in the basal ganglia can lead to movement disorders characterized by typical parkinsonian motor symptoms. Parkinson's disease may also be associated with many non-motor symptoms. One criterion for diagnosing Parkinson's disease is the presence or absence of SNpc degeneration and Lewy pathology in postmortem pathology examination. Lewy pathology may include abnormal aggregations of acinuclein protein called Lewy bodies and Lewy neurites. Patients with Parkinson's disease may exhibit a variety of symptoms, including motor and non-motor symptoms. The methods provided herein can treat or ameliorate one or more of these motor or non-motor symptoms associated with Parkinson's disease. Motor symptoms of Parkinson's disease (symptoms of parkinsonism) include motor slowness (bradykinesia), rigidity, balance problems, a shuffling gait, and unstable posture. The motor characteristics of Parkinson's disease patients can be heterogeneous, leading to attempts to categorize the disease into subtypes, such as tremor-dominant Parkinson's disease (relative absence of other motor symptoms), non-tremor-dominant Parkinson's disease (which may include phenotypes described as akinetic-rigid syndrome and postural instability gait disorder), and additional subgroups with mixed or indeterminate phenotypes with several motor symptoms of comparable severity. Non-motor symptoms of Parkinson's disease can include olfactory dysfunction, cognitive impairment, psychiatric symptoms (e.g., depression), sleep disorders, autonomic dysfunction, pain, and fatigue. These symptoms are often seen early in Parkinson's disease. Non-motor features of Parkinson's disease can often appear before the onset of typical motor symptoms. The premotor or prodromal stage of the disease may be characterized by anosmia, constipation, depression, excessive daytime sleepiness, and rapid eye movement sleep behavior disorder.

[0213] In some embodiments, the methods provided herein reduce or slow the progression of Parkinson's disease, which can be characterized by a deterioration in motor function. As the disease progresses, complications associated with long-term symptomatic treatment can include motor and non-motor fluctuations, movement disorders, and psychosis.

[0214] One of the pathological hallmarks of Parkinson's disease can be the loss of dopaminergic neurons in the substantia nigra, e.g., the substantia nigra pars compacta (SNpc). According to some embodiments, the methods provided herein replenish dopamine (secreted from dopamine neurons converted from the striatum) lost due to the loss of dopamine neurons in the substantia nigra (e.g., SNpc) of a patient. Neuronal loss in Parkinson's disease can also occur in many other brain regions, such as the locus coeruleus, nucleus basalis of Meynert, pedunculopontine nucleus, raphe nucleus, dorsal motor nucleus of the vagus nerve, amygdala, and hypothalamus. In some embodiments, as provided herein, a method of treating or ameliorating one or more symptoms of Parkinson's disease in a subject includes reprogramming non-neuronal cells in a brain region experiencing neuronal loss in a Parkinson's disease patient into functional neurons.

[0215] The methods provided herein are useful for treating Parkinson's disease of various etiologies. For example, Parkinson's disease may be caused by one or more genetic mutations, such as, but not limited to, mutations in the genes SNCA, LRRK2, VPS35, EIF4G1, DNAJC13, CHCHD2, Parkin, PINK1, DJ-1, ATP13A2, C9ORF72, FBX07, PLA2G6, POLG1, SCA2, SCA3, SYNJ1, and RAB39B, and mutations in one or more genes that may affect microdeletion syndrome in 22qll2. Alternatively, Parkinson's disease may have no known genetic characteristics.

[0216] As provided herein, one or more symptoms of Parkinson's disease that can be improved by the methods provided herein can include not only the motor and non-motor symptoms described above, but also pathological features at other levels. For example, the reduction in dopamine signaling in the brain of a Parkinson's disease patient can be reversed or alleviated by the methods provided herein by recruiting functional dopamine neurons that can be integrated into neural circuits and reconstruct the projection of dopamine neurons to appropriate brain regions.

[0217] In one aspect, the present disclosure also provides methods for restoring dopamine release in a subject experiencing a decrease in the biological production of dopamine compared to normal levels. An exemplary method includes reprogramming non-neuronal cells in a brain region (e.g., the striatum) of the subject, allowing the non-neuronal cells to reprogram into dopaminergic neurons, thereby restoring at least 50% of the loss of dopamine. In some embodiments, the reprogramming is achieved by administering to the brain region (e.g., the striatum) of the subject a composition comprising a cellular programming agent that inhibits PTB / nPTB expression or activity in non-neuronal cells (e.g., astrocytes) in the brain region. In some embodiments, the methods provided herein restore at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% of the loss of dopamine. In some embodiments, the methods provided herein reverse about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% of the loss in dopamine. In some embodiments, the methods provided herein reverse at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% of the loss in dopamine. In some embodiments, the methods provided herein reverse at least about 50% of the loss in dopamine. Pharmaceutical Composition

[0218] In one aspect, the present disclosure provides pharmaceutical compositions comprising a cell programming agent in an amount effective to reprogram a mammalian non-neuronal cell into a mature neuron by inhibiting the expression or activity of PTB / nPTB in the non-neuronal cell. Exemplary pharmaceutical compositions can further comprise a pharmaceutically acceptable carrier or excipient. As described above, the cell programming agent provided herein can be a coding sequence for a Cas effector protein and a gRNA for PTB / nPTB.

[0219] The pharmaceutical compositions provided herein can include one or more carriers and excipients (including, but not limited to, buffers, sugars, mannitol, proteins, peptides, or amino acids such as glycine, antioxidants, bacteriostats, chelating agents, suspending agents, thickening agents, and / or preservatives), water, oils (including those of petroleum, animal, vegetable, or synthetic origin) such as peanut oil, soybean oil, mineral oil, and sesame oil, saline solutions, aqueous glucose and glycerin solutions, flavorings, coloring agents, anti-adherents, and other acceptable additives, or other pharmaceutically acceptable auxiliary substances required to approximate physiological conditions, such as binders, pH buffers, tonicity adjusters, emulsifiers, wetting agents, etc. Examples of excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, nonfat dry milk, glycerin, propylene, ethylene glycol, water, ethanol, etc. In other cases, the composition is substantially free of preservatives. In other embodiments, the composition contains at least one preservative. General methods for drug dosage forms are described in Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems (Lippencott Williams & Wilkins, Baltimore, Md. (1999)). Any suitable carrier known to those skilled in the art can be used to administer the pharmaceutical compositions described herein, but those skilled in the art should recognize that the type of carrier can vary depending on the method of administration. Suitable formulations and additional carriers are described in Remington "The Science and Practice of Pharmacy" (20th Ed., Lippincott Williams & Wilkins, Baltimore, Md.), the teachings of which are incorporated herein by reference in their entirety.

[0220] Exemplary pharmaceutical compositions may be formulated for injection, inhalation, parenteral, intravenous, subcutaneous, intramuscular, intradermal, topical, or oral administration.

[0221] In some embodiments, a pharmaceutical composition comprising an AAV vector encoding a Cas effector and a coding sequence for a gRNA against PTB / nPTB can be injected into the mature retina or striatum of a subject's brain.

[0222] As will be appreciated by those skilled in the art, pharmaceutical compositions can contain any suitable carrier or excipient, depending on the type of cellular programming agent and the intended route of administration of the composition. For example, compositions containing the cellular programming agents provided herein can be formulated for parenteral administration and can be presented in unit dose form in ampoules, prefilled syringes, small injectable syringes, or multi-dose containers with added preservatives. The compositions can take the form of a suspension, solution, or emulsion in an oily or aqueous vehicle, such as a solution in aqueous polyethylene glycol. For example, in the case of injectable formulations, the vehicle can be selected from aqueous solutions or oil suspensions, including sesame oil, corn oil, cottonseed oil, or peanut oil, or emulsions and elixirs, mannitol, glucose, or sterile aqueous solutions, and similar pharmaceutical vehicles known to those skilled in the art.

[0223] The formulations may also include biocompatible, biodegradable polymer compositions, such as polylactic acid-co-hydroxyacetic acid. These materials can be loaded with drugs and further coated or derivatized into microspheres or nanospheres to provide superior sustained release. Vehicles suitable for periocular or intraocular injection include, for example, suspensions of the active agent in injection-grade water, liposomes, and vehicles suitable for lipophilic substances, as well as those known in the art. The compositions provided herein can include additional agents in addition to a cell programming agent and a pharmaceutically acceptable carrier or excipient. For example, additional reagents can be provided for the purpose of promoting neuronal survival. Alternatively, or in addition, additional reagents can be provided for pharmacodynamic monitoring purposes. In some embodiments, the compositions include additional reagents as penetration enhancers or for sustained or controlled release of the active ingredient (e.g., a cell programming agent).

[0224] The compositions provided herein can be administered to a subject in doses of about 0.0005, 0.001, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.45, 0.5, 0.55, 0.6, 0.7, 0.8, 0.9, 1.0 mL or more. The compositions can be administered in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more dose-treatment regimens. In some cases, the compositions can be administered in 2, 3, or 4 dose-treatment regimens. In some cases, the compositions can be administered in a single dose-treatment regimen.

[0225] The administration of the first agent (e.g., an AAV vector encoding a gRNA for a Cas effector and a PTB) and the second agent (e.g., an AAV vector encoding a gRNA for a Cas effector and a nPTB) of a two-dose treatment regimen can be separated by about 0, 1, 2, 5, 7, 14, 21, 30 days, 2 months, 4 months, 6 months, 9 months, 1 year, 1.5 years, 2 years, 3 years, 4 years, 5 years, 10 years, 20 years, or more. Compositions of the invention can be administered to a subject once daily, once weekly, once every two weeks, once monthly, once yearly, twice yearly, three times yearly, or once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more years.

[0226] The composition may be administered to the subject once every 2, 3, 4, 5, 6, 7 or more years. The composition may be administered to the subject once. Other Aspects

[0227] Some embodiments of the present disclosure provide methods and compositions for cell or tissue transplantation. Exemplary methods can include reprogramming non-neuronal cells into neurons in vitro and transplanting the reprogrammed neurons into a brain region of a subject. In some embodiments, the in vitro reprogramming can be performed according to the methods provided herein. Exemplary compositions can include neurons reprogrammed according to any embodiment of the methods provided herein.

[0228] In other embodiments, the methods provided herein include reprogramming non-neuronal cells into neurons in vivo and explanting the reprogrammed neurons. In some embodiments, the explant comprises brain tissue containing the reprogrammed neurons. In some embodiments, the explant is transplanted into a brain region of a subject. As provided herein, transplantation of neurons reprogrammed according to the methods provided herein can be used to replenish degenerated neurons in subjects suffering from conditions associated with neuronal loss. Some other aspects of the present disclosure relate to animals comprising neurons reprogrammed according to any of the embodiments of the methods provided herein.

[0229] As provided herein, the animal may be any mammal. The animal may be a human. The animal may be, for example, but not limited to, a non-human primate, such as a rhesus monkey, a cynomolgus monkey, a stamp-tailed monkey, a pig-tailed monkey, a squirrel monkey, an owl monkey, a baboon, a chimpanzee, a marmoset, and a spider monkey. The animal may be a research animal, a transgenic animal, or any other suitable type of animal.

[0230] Also provided herein is animal brain tissue (e.g., explants) containing one or more neurons reprogrammed according to any embodiment of the present disclosure. Such brain tissue may be viable. In some embodiments, the brain tissue may be fixed with any suitable fixative. The brain tissue may be used for transplantation, medical research, basic research, or any other purpose.

[0231] This disclosure demonstrates that this method is applicable to neurodegenerative disease models. For example, this disclosure demonstrates that the astrocyte-to-neuron conversion strategy can function in a chemically induced Parkinson's disease model. The method and composition can convert astrocytes into neurons, including dopaminergic, glutamatergic, and GABAergic neurons, which can form synapses in the brain. Furthermore, the converted neurons can efficiently reconstruct damaged nigrostriatal pathways to correct measurable Parkinson's phenotypes. The effectiveness of this method was confirmed in cultured astrocytes (human and mouse) and in mouse Parkinson's disease models. Therefore, this strategy has the potential to cure Parkinson's disease and can also be applied to a wide range of neurodegenerative diseases, such as other neurological disorders associated with neuronal dysfunction.

[0232] In some embodiments, the disclosed methods exploit the genetic basis of a pre-existing but latent neuronal maturation program in two mammalian astrocytes, which, when reprogrammed by PTB inhibition, gradually generate mature neurons. These findings provide a clinically viable method for generating neurons from local astrocytes in the mammalian brain using a single dose of a vector containing sequences encoding a Cas effector and a gRNA against PTB / nPTB. The neuronal phenotypes induced by PTB / nPTB knockdown may be related to the environment in which they are generated and / or the astrocytes from which they are derived. The present disclosure demonstrates the effective conversion of astrocytes into neurons (e.g., dopamine neurons in the striatum). More specifically, the present disclosure demonstrates that this strategy is effective in converting astrocytes into neurons in a primate model, thereby satisfying all five factors for in vivo reprogramming. The data provided herein suggest that reducing PTB in the primate brain converts astrocytes into dopamine neurons (e.g., dopaminergic neurons).

[0233] A "therapeutically effective amount" of a composition of the present disclosure varies depending on factors such as the individual's disease state, age, sex, and weight, and the ability of the composition to elicit a desired response in the individual. A therapeutically effective amount may also be an amount in which any toxic or adverse effects of the composition outweigh the therapeutically beneficial effects. While not wishing to be bound by theory, it is expected that a therapeutically effective amount of a cellular programming agent provided herein may, in some cases, be an amount of cellular programming agent that converts a predetermined percentage of astrocytes in a brain region (experiencing neuronal loss), such that the conversion of such percentage of astrocytes in the brain region to functional neurons is sufficient to ameliorate or treat a disease or condition associated with neuronal loss, while not exceeding a threshold level that may result in adverse effects that outweigh the beneficial effects of neuronal conversion, such as, for example, an excessive reduction in the number of astrocytes in the brain region due to a direct result of neuronal conversion. The following examples are intended to illustrate, but not limit, the present disclosure. They are typical of those that may be used, although other procedures known to those skilled in the art may alternatively be used. [Example]

[0234] Example 1 Methods and Materials Mice and cell lines C57BL / 6 mice were purchased from the Shanghai SLAC Laboratory. Mice were kept in a light / dark circulating chamber with water and food. All animal experiments were performed and approved by the Animal Care and Use Committee of CEBSIT, Chinese Academy of Sciences, Shanghai, China. Cos7, 293T, and N2a cell lines were obtained from the cell bank of the Shanghai Institute of Biochemistry and Cell Biology (SIBCB), Chinese Academy of Sciences, and cultured in DMEM containing 10% fetal bovine serum and 1% penicillin / streptomycin in a 37°C incubator with 5% CO2.

[0235] Transfection, qPCR and RNA-seq Transient transfection was performed using Liposome 3000 with 4 μg of the CAG-CasRx-P2A-GFP expression vector and 2 μg of the U6-gRNA-CMV-mCherry plasmid. The CAG-CasRx-P2A-GFP plasmid served as a control. Two days after transient transfection, approximately 30K GFP and mCherry double-positive cells (top 20% of GFP) were harvested by fluorescence-activated cell sorting (FACS) and digested for qPCR analysis. RNA was extracted using Trizol (Ambion) and then converted to cDNA using a reverse transcription kit (HiScript Q RT SuperMix for qPCR, Vazyme, Biotech). Amplification was monitored using AceQ qPCR SYBR Green Master Mix (Vazyme, Biotech). For RNA-seq, Cos7 cells were cultured in 10-cm Petri dishes. Approximately 100k positive cells (top 20% of GFP cells) were isolated by FACS, RNA was extracted, reverse transcribed to cDNA, and used for RNA-seq. RNA-seq data analysis was performed as previously described (Zhou et al., 2018, Cell 181: 590-603) and is presented as the average of all duplicates.

[0236] Guide RNA sequence The guide sequences of the guide RNAs used (i.e., sequences complementary to the targeted PTB mRNA sequence) are shown in Table 1. Table 1. Guide RNA guide sequences and the relative expression levels obtained with each guide RNA (compared to the no-gRNA control). The highly efficient gRNAs are all located in the critical region (positions 951 to 1487 of SEQ ID NO: 87, i.e., SEQ ID NO: 88, see Figure 1), which is shown in bold.

[0237] [Table 1-1] [Table 1-2]

[0238] Stereotactic injection and immunofluorescence staining In this study, we used AAV8. Stereotaxic injections were performed on mice as previously described (Zhou et al., 2014, ELife 3, e02536, doi:10.7554 / eLife.02536). Mice were placed in a stereotaxic frame. Next, the skin over the skull was shaved with a razor, and the skull was opened. A craniotomy was performed at coordinates (AP +0.8 mm, ML ±1.6 mm) above the border between the frontal and parietal bones, allowing for placement of an injection micropipette (tip outer diameter approximately 20 μm). A virus solution containing either AAV-GFAP-CasRx-Ptbp1 + AAV-GFAP-mCherry or AAV-GFAP-CasRx + AAV-GFAP-mCherry was injected slowly (approximately 0.3 μl / min). AAV was injected into the striatum of mice (AP +0.8 mm, ML ±1.6 mm, and DV 2.6 mm) (>1 × 10 12 (vg / ml, 1 μL, 8-10 week old mice). Cynomolgus monkeys were given the immunosuppressant dexamethasone (1.5 mg / kg) intramuscularly approximately 14 hours before AAV injection. For AAV injection, 80 μL (>1 × 10 12AAV-GFAP-CasRx-Ptbp1 + AAV-GFAP-mCherry and AAV-GFAP-CasRx + AAV-GFAP-mCherry were injected at a volume ratio of 1:20 (vg / ml). For immunofluorescence staining, brains were perfused with 4% paraformaldehyde (PFA) overnight and then fixed in 30% sucrose for at least 12 hours (mice) or 2 weeks (cynomolgus monkeys). For embedded and frozen brain sections, 30 μm thick sections from mice and 30 μm thick sections from cynomolgus monkeys were used for immunofluorescence staining. Brain sections were thoroughly washed with 0.1 M phosphate buffer (PB). Primary antibodies: rabbit anti-NeuN (1:500, 24307S, Cell Signaling Technology), guinea pig anti-NeuN (1:500, ABN90, Millipore), mouse anti-Flag (1:2000, F3165, Sigma), rabbit anti-TH (1:500, AB152, Millipore), rat anti-DAT (1:100, MAB369, Millipore), rabbit anti-RBPMS (Proteintech, Cat#15187-1-AP). In this study, we used Alexa Fluora 488 AffiniPure donkey anti-rabbit IgG (H+L) (1:500, 711-545-152, Jackson ImmunoResearch), Alexa Fluora 488 AffiniPure donkey anti-rat IgG (H+L) (1:500, 715-545-150, Jackson ImmunoResearch), Cy5-AffiniPure donkey anti-guinea pig IgG (H+L) (1:500, 706-175-148, Jackson ImmunoResearch), and Cy5 AffiniPure donkey anti-rabbit IgG (H+L) (1:500, 711-175-152, Jackson ImmunoResearch). After antibody incubation, sections were washed and covered with mounting medium (Life Technology). Images were collected under an Olympus FV3000 microscope.

[0239] Subretinal injection and immunofluorescence staining NMDA and AAV (AAV8) were transduced by subretinal injection. For subretinal injection, mice were anesthetized and AAV was slowly injected into the subretinal space. To determine complete retinal transduction, a total of 1 μl of AAV-GFAP-GFP-Cre (0.2 μl) and AAV-GFAP-CasRx-Ptbp1 (0.4 μl), or AAV-GFAP-GFP-Cre (0.2 μl) and AAV-GFAP-CasRx (0.4 μl) and PBS (0.4 μl), or AAV-GFAP-GFP-Cre (0.2 μl) and AAV-GFAP-CasRx-Ptbp1 (0.4 μl) and AAV-GFAP-β-catenin (0.4 μl) was delivered to the retina by subretinal injection (Ai9 and C57BL / 6 mice, 4 months old). Two to three weeks after AAV injection, eyes and optic nerves were harvested and fixed in 4% paraformaldehyde (PFA) and then preserved in a solution containing 30% sucrose. After embedding, eyes were sliced, washed, and covered with mounting medium (Life Technology). Images were collected using an Olympus FV3000 microscope.

[0240] Results Example 1 In this study, we designed a system that can specifically downregulate Ptbp1 expression in mouse, cynomolgus monkey, and human cells, and further demonstrated that CasRx-mediated downregulation of Ptbp1 can efficiently and directly convert astrocytes into dopamine neurons in the striatum of cynomolgus monkeys. In summary, our study provides preclinical evidence that Ptbp1-mediated dopamine neuron conversion is also possible in primates (i.e., humans and non-human primates), paving the way for clinical trials.

[0241] To knock down Ptbp1 expression, we used CasRx, an RNA-guided and RNA-targeting CRISPR protein, which is an effective and specific method for downregulating RNA. To examine the efficiency of CasRx-mediated Ptbp1 knockdown, we designed 86 gRNAs targeting the human Ptbp1 gene. Two days after transfection, co-transfection of a vector containing the CasRx gene with a gRNA targeting Ptbp1 resulted in a decrease in Ptbp1 mRNA in human 293T cells (Figure 1A; see Table 1 for gRNA sequences). Specifically, the critical region targeting the human Ptbp1 gene (positions 951–1487 of SEQ ID NO: 87, corresponding to positions 1–536 of SEQ ID NO: 88) often induced effective downregulation (Figure 1B). In this experiment, gRNAs targeting this critical region achieved less than 40% knockdown, and only in this critical region was effective knockdown of less than 10% observed.

[0242] SEQ ID NO:88: gccattcctc aagctgcagg cctttccgtt ccgaacgtcc acggcgccct ggcccccctg 60 gccatcccct cggcggcggc ggcagctgcg gcggcaggtc ggatcgccat cccgggcctg 120 gcggggggcag gaaattctgt attgctggtc agcaacctca acccagagag agtcacaccc 180 caaagcctct ttattctttt cggcgtctac ggtgacgtgc agcgcgtgaa gatcctgttc 240 aataagaagg agaacgccct agtgcagatg gcggacggca accaggccca gctggccatg 300 agccacctga acgggcacaa gctgcacggg aagcccatcc gcatcacgct ctcgaagcac 360 cagaacgtgc agctgccccg cgagggccag gaggaccagg gcctgaccaa ggactacggc 420 aactcacccc tgcaccgctt caagaagccg ggctccaaga acttccagaa catattcccg 480 ccctcggcca cgctgcacct ctccaacatc ccgccctcag tctccgagga ggatct 536

[0243] The target site of gRNA 60 is conserved in the cynomolgus monkey, human Ptbp1, and mouse genes, and it effectively downregulated the Ptbp1 gene in human 293T, monkey Cos7 cells, and mouse N2a cells. Therefore, we used it in the following experiments (Figures 2 and 3). To determine the target specificity of this strategy, we performed RNA-seq and found that Ptbp1 was specifically downregulated in Cos7 cells (Figure 4). Recent studies have shown that Ptbp1 knockdown can convert striatal astrocytes into dopamine neurons. Next, we examined whether knockdown of Ptbp1 in striatal astrocytes of non-human primates can locally convert astrocytes into dopamine neurons in vivo. To validate the AAV vector in vivo, we first injected wild-type mice with AAV-GFAP-CasRx-Ptbp1 and gRNA 60, which expresses CasRx, and AAV-GFAP-mCherry, which fluorescently labels astrocytes. We also constructed a control vector, AAV-GFAP-CasRx, which does not express Ptbp1 gRNA (Figure 5A). One week after injection, both mCherry and CasRx were specifically expressed in astrocytes and showed high colocalization efficiency in the striatum. Furthermore, mCherry + A high proportion of cells expressed the mature neuronal marker NeuN, but not in control striatum injected with AAV-GFAP-mCherry and AAV-GFAP-CasRx (Figures 5B and 5C). +The majority of cells expressed the dopamine neuron marker TH 2 weeks after AAV injection and DAT 1 month after AAV injection (Figures 5D and 5E). To explore whether knocking down Ptbp1 in the striatum of cynomolgus monkeys could convert astrocytes into dopamine neurons, we injected AAV-GFAP-CasRx-Ptbp1 and AAV-GFAP-mCherry into the right hemisphere putamen of 8-year-old cynomolgus monkeys, and AAV-GFAP-CasRx and AAV-GFAP-mCherry into the left hemisphere putamen. One month after AAV injection, mCherry was expressed in the right putamen. + TH + cells were observed in the control putamen but not in the control putamen ( Fig. 5<em>F ), suggesting that knocking down Ptbp1 can also convert astrocytes into dopamine neurons in nonhuman primates.

[0244] We further explored the possibility of improving the efficiency of dopamine neuron conversion by combining other transcription factors involved in dopamine neuron conversion, such as FoxA2, ​​Lmx1a, and Nurr1. We hypothesized that overexpression of these transcription factors might enhance dopamine neuron conversion. Indeed, our results showed that coexpression of AAV-GFAP-CasRx-Ptbp1 with AAV-GFAP-FoxA2, ​​AAV-GFAP-Lmx1a, AAV-GFAP-FoxA2 + AAV-GFAP-Lmx1a, or AAV-GFAP-FoxA2 + AAV-GFAP-Lmx1a + AAV-GFAP-NurR1 enhanced the efficiency of dopamine neuron conversion in the mouse striatum compared with expression of AAV-GFAP-CasRx-Ptbp1 alone (see Figure 6).

[0245] In addition to the conversion of astrocytic dopamine neurons, our previous results showed that knockdown of Ptbp1 can convert Müller glia (MG) into retinal ganglion cells (RGCs) in the mature retina at 4 to 8 weeks of age. However, it is unclear whether this strategy can be applied to middle-aged and older mice. Therefore, we subretinal injected AAV-GFAP-CasRx-Ptbp1 and AAV-GFAP-GFP-Cre into the eyes of 4- to 5-month-old ai9 mice (CAG-LSL-tdTomato), and tdTomato was detected in the optic nerve 3 weeks after injection. + A small number of axons were observed. To explore whether stimulating MG recirculation could improve MG-to-RGC conversion, we injected AAV-GFAP-CasRx-Ptbp1, AAV-GFAP-GFP-Cre, and AAV-GFAP-β-catenin into the retinas of 4- to 5-month-old mice. Approximately 3 weeks after injection, we confirmed that injection of AAV-GFAP-CasRx-ptbp1 + AAV-GFAP-β-catenin + AAV-GFAP-GFP-Cre increased the amount of converted RGCs compared with injection of AAV-GFAP-CasRx-Ptbp1 and AAV-GFAP-GFP-Cre (see Figure 7). In summary, our results provide a novel strategy for improving RGC conversion in middle-aged or aged mammals.

[0246] Example 2 Symptom reduction in MD model monkeys To determine whether astrocytes could be converted to dopamine neurons in the putamen of a monkey PD model, histological observations revealed that the majority of putamen injected with AAV-GFAP-mCherry and AAV-GFAP-CasRx-Ptbp1 expressed mCherry. +The cells expressed the dopamine neuron marker TH, but not the putamen injected with control AAVs (AAV-GFAP-mCherry and AAV-GFAP-CasRx). Furthermore, we found that a high percentage of mCherry cells also expressed the dopamine transporter (DAT), a marker for substantia nigra A9 dopaminergic neurons. These results suggest that downregulating Ptbp1 expression in putamen astrocytes successfully induced dopamine neurons. Next, we used video-based motor analysis of monkeys to assess whether the induced dopamine neurons could alleviate Parkinson's disease symptoms. Approximately one month after AAV injection, PD monkeys injected with AAV-GFAP-mCherry+AAV-GFAP-CasRx-Ptbp1 showed a significant reduction (approximately 15%) in PD symptoms. However, control monkeys injected with AAV-GFAP-mCherry and AAV-GFAP-CasRx encoding gRNA targeting Ptbp1 did not show significant reduction in PD symptoms. To determine whether the induced dopamine neurons functioned as dopaminergic neurons in vivo, we performed 18F-Dopa PET, 18F-DTBZ PET, and 18F-FP-CIT PET to detect presynaptic dopaminergic function, monitor vesicular monoamine transporter type 2 (VMAT2) activity, and accurately quantify DAT density. Enhanced signals were observed in the putamen injected with AAV-GFAP-mCherry and AAV-GFAP-CasRx-Ptbp1, but not in the control AAV. These results demonstrate functional dopamine neurons induced in intact monkey putamen.

[0247] Conversion of MG to RGC (RGC) in a monkey model of NMDA-induced retinal injury To test whether the induced RGCs could replenish RGCs in a monkey model of retinal injury, adult monkeys were injected with N-methyl-D-aspartate (NMDA), which resulted in the loss of most RGCs. Two to three weeks after NMDA injection, the eyes were injected with AAV-GFAP-tdTomato + AAV-GFAP-CasRx-Ptbp1 + AAV-GFAP-β-catenin, AAV-GFAP-tdTomato + AAV-GFAP-CasRx-Ptbp1, or control AAVs not encoding gRNAs targeting Ptbp1 (AAV-GFAP-tdTomato + AAV-GFAP-CasRx + AAV-GFAP-β-catenin and AAV-GFAP-tdTomato + AAV-GFAP-GFAP-CasRx). Furthermore, we hypothesized that coexpression of OSK (Oct4 (also known as Pou5f1), Sox2, and Klf4 genes) with CasRx-Ptbp1 in middle-aged or aged retinal MG might promote RGC conversion. Therefore, we also injected AAV-GFAP-tdTomato + AAV-GFAP-CasRx-Ptbp1 + AAV-GFAP-OSK into the retina. One to two months after AAV injection, tdTomato in the GCL was significantly reduced in retinas injected with AAV-GFAP-tdTomato + AAV-GFAP-CasRx-ptbp1 + AAV-GFAP-β-catenin, AAV-GFAP-tdTomato + AAV-GFAP-CasRx-ptbp1 + AAV-GFAP-OSK, or AAV-GFAP-tdTomato + AAV-GFAP-CasRx-ptbp1. + Rbpms + Although the number of MG cells increased, there were very few such cells in the control AAV-injected retinas, suggesting that the NMDA-induced retinal injury monkey model successfully induced the conversion of MGs to RGCs. + Rbpms +The cells were more frequently observed in retinas injected with AAV-GFAP-tdTomato+AAV-GFAP-CasRx-Ptbp1+AAV-GFAP-β-catenin or AAV-GFAP-tdTomato+AAV-GFAP-CasRx-Ptbp1+AAV-GFAP-OSK than in retinas injected with AAV-GFAP-tdTomato+AAV-GFAP-CasRx-Ptbp1.In the visual system, RGCs send axons to the brain via the optic nerve and form central projections with the dorsal lateral geniculate nucleus (dLGN) and superior colliculus (SC) in the monkey brain. To explore whether MG-derived RGCs are integrated into the visual system, monkey brain slices were prepared. Optic nerves injected with AAV-GFAP-tdTomato + AAV-GFAP-CasRx-Ptbp1 + AAV-GFAP-β-catenin or AAV-GFAP-tdTomato + AAV-GFAP-CasRx-Ptbp1 + AAV-GFAP-tdTomato contained more tdTomato than those injected with AAV-GFAP-tdTomato + AAV-GFAP-CasRx-Ptbp1. + In the dLGN and SC, tdTomato axons were observed, whereas in the control group, almost no such axons were observed. + Detect axons and tdTomato + Axons were more abundant in the contralateral brain than in the ipsilateral brain, suggesting that the induced RGCs formed connections with the right side of the brain. To examine whether MGs could convert to retinal photoreceptors, immunostaining was performed. In the retinas injected with AAAV-GFAP-tdTomato + AAV-GFAP-CasRx-Ptbp1 + AAV-GFAP-β-catenin, AAV-GFAP-tdTomato + AAV-GFAP-CasRx-Ptbp1 + AAV-GFAP-OSK, and AAV-GFAP-tdTomato + AAV-GFAP-CasRx-Ptbp1, tdTomato was significantly increased. + A portion of the cells expressed rod cell markers rhodopsin and GNAT1, and cone cell markers S-opsin, M-opsin, and mCAR, indicating that cone and rod cells can also be induced by MG.

[0248] method PD model monkeys This study used adult (7-10 years old) male cynomolgus monkeys. To create a non-human primate model of Parkinson's disease, animals were intravenously injected with MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) to deplete dopamine neurons in the substantia nigra. Persistent symptoms, including tremors, motor slowing, and impaired balance, were observed. Stable symptoms were observed for at least 5 weeks before AAV injection.

[0249] Positron Emission Tomography (PET) Study (PD Part) To monitor the dopamine function of the converted dopamine neurons in intact monkey brains, PET imaging (18F-DOPA, 18F-DTBZ, and 18F-FP-CIT) was performed before and 1 month after AAV injection. Before scanning, the monkeys were anesthetized, and then the animals were placed in a PET scanner to monitor all physiological parameters. After scanning, the PET data were analyzed using standard protocols.

[0250] Video analysis of behavioral recovery (PD part) For video analysis, monkey movements were recorded before AAV injection and at 1 and 2 months after injection. The recorded videos were analyzed after collection. The monkeys' PD symptoms were assessed using items such as head movement, facial expression, spontaneous activity, movement in response to stimuli, tremor, posture, and gait.

[0251] Preparation of a monkey model of NMDA-induced retinal injury and AAV injection (RGC region) NMDA was injected intravitreally, and AAV was injected subretinal. For intravitreal injection, monkeys were anesthetized and alcaine was instilled into the eyes. NMDA solution was injected into the vitreous to deplete the majority of RGCs, and then o-ofloxacin eye ointment was applied to the eyes to prevent infection. For subretinal injection, monkeys were anesthetized 2–3 weeks after NMDA injection, and their pupils were dilated. AAV was slowly injected into the subretinal space. After injection, the needle was removed and eye ointment was administered. To determine the conversion, the following three strategies were designed to induce the conversion of MGs to RGCs: We injected AAV-GFAP-tdTomato + AAV-GFAP-CasRx-Ptbp1 + AAV-GFAP-β-catenin, AAV-GFAP-tdTomato + AAV-GFAP-CasRx-Ptbp1 + AAV-GFAP-OSK, and AAV-GFAP-tdTomato + AAV-GFAP-CasRx-Ptbp1. Additionally, we injected control AAVs that do not encode a gRNA targeting Ptbp1: AAV-GFAP-tdTomato + AAV-GFAP-CasRx + AAV-GFAP-β-catenin, AAV-GFAP-tdTomato + AAV-GFAP-CasRx + AAV-GFAP-OSK, or AAV-GFAP-tdTomato + AAV-GFAP-CasRx. Note that gRNA transcription is driven by the U6 promoter. The AAVs were delivered to the retina through the subretinal route. Approximately 1–2 months after AAV injection, the eyes, optic nerves, and brains were harvested and fixed in 4% paraformaldehyde (PFA) and then stored in 30% sucrose solution. After embedding, the eyes and brains were sectioned, incubated with antibodies, and examined under a microscope.

Claims

1. 1. A pharmaceutical composition for treating a neurological condition associated with degeneration of functional neurons in a region of the nervous system of a non-human primate or a human, the composition comprising: i) a Cas effector protein and at least one gRNA; or ii) at least one expression vector encoding a Cas effector protein and encoding said at least one gRNA; wherein the guide sequence of the gRNA is SEQ ID NO: 4, 5, 7, 8, 10, 26, 36, 38, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 66, 70, 73, 76, 79, 81 or 83.

2. 2. The pharmaceutical composition of claim 1, wherein the Cas effector protein and the at least one gRNA or at least one expression vector are contained in nanoparticles.

3. 2. The pharmaceutical composition of claim 1, wherein the Cas effector protein is an RNA-targeting Cas effector protein.

4. The pharmaceutical composition of claim 3, wherein the RNA-targeting Cas effector protein is selected from the group consisting of Cas13d, CasRx, Cas13e, Cas13a, Cas13b, Cas13c, Cas13f and functional domains thereof.

5. 2. The pharmaceutical composition of claim 1, wherein the composition comprises only a single type of gRNA, or two, three, four, five, or six different gRNAs, that target a primate PTB mRNA sequence, or comprises an expression vector encoding only a single type of gRNA, or two, three, four, five, or six different gRNAs that target a primate PTB mRNA sequence.

6. The at least one expression vector i) a nucleotide sequence encoding said Cas effector protein, wherein said nucleotide sequence is operably linked to a promoter that drives expression of said Cas effector protein in a non-neuronal cell of a primate; ii) at least one nucleotide sequence encoding a gRNA that targets a primate PTB mRNA sequence, the nucleotide sequence being operably linked to a promoter that drives expression of the gRNA in the non-neuronal cell.

7. 2. The pharmaceutical composition of claim 1, wherein the composition is administered locally to at least one of the following cells: i) non-neuronal cells of the mature retina, ii) non-neuronal cells of the striatum, iii) non-neuronal cells of the substantia nigra, iv) non-neuronal cells of the inner ear, v) non-neuronal cells of the spinal cord, vi) non-neuronal cells of the prefrontal cortex, vii) non-neuronal cells of the motor cortex, and viii) non-neuronal cells of the ventral tegmental area (VTA) of the midbrain.

8. 8. The pharmaceutical composition of claim 7, wherein the neurological condition is a condition associated with degeneration of functional neurons selected from the group consisting of Parkinson's disease; Alzheimer's disease; Huntington's disease; schizophrenia; depression; drug addiction; stroke; movement disorder; bipolar disorder; autism spectrum disorder (ASD); and functional impairment.

9. The composition further comprises i) one or more dopamine neuron-associated factors, or ii) at least one expression vector for expressing one or more dopamine neuron-associated factors in a non-neuronal cell. The pharmaceutical composition of claim 7.

10. 8. The pharmaceutical composition of claim 7, wherein the composition is administered to mature retinal glial cells or Müller glial cells (MG) to generate functional retinal ganglion cell (RGC) neurons and / or functional retinal photoreceptors.

11. 11. The pharmaceutical composition of claim 7 or 10, wherein the neurological condition is a condition associated with degeneration of functional neurons in the mature retina selected from the group consisting of glaucoma, age-related RGC loss, optic nerve damage, retinal ischemia, and Leber's hereditary optic neuropathy.

12. 12. The pharmaceutical composition of claim 10 or 11, wherein the composition further comprises i) one or more factors selected from the group consisting of β-catenin, Oct4, Sox2, Klf4, Crx, Brn3a, Brn3b, Math5, Nr2e3 and Nrl, and / or ii) at least one expression vector for expressing one or more factors selected from the group consisting of β-catenin, Oct4, Sox2, Klf4, Crx, Brn3a, Brn3b, Math5, Nr2e3 and Nrl in non-neuronal cells.

13. The pharmaceutical composition of claim 1 , wherein at least one immunosuppressant is administered before, simultaneously with, or after administration of the composition.

14. (a) a coding sequence for an RNA-targeting Cas effector protein; (b) at least one nucleotide sequence encoding a gRNA, wherein the guide sequence of the gRNA is SEQ ID NO: 4, 5, 7, 8, 10, 26, 36, 38, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 66, 70, 73, 76, 79, 81, or 83.

15. The AAV vector of claim 14, wherein the RNA-targeting Cas effector protein is selected from the group consisting of Cas13d, CasRx, Cas13e, Cas13a, Cas13b, Cas13c, Cas13f, and functional domains thereof.

16. i) the nucleotide sequence encoding the Cas effector protein is operably linked to a promoter that drives expression of the Cas effector protein in a primate non-neuronal cell; and ii) the at least one nucleotide sequence encoding a gRNA is operably linked to a promoter that causes expression of the gRNA in the non-neuronal cell.

Images