NON-NEURONAL CELLS

CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority under 35 U.S.C. §119 to U.S. Provisional Application No. 62/913,104, filed October 9, 2019, the disclosures of which are incorporated herein by reference.

TECHNICAL FIELD

[0002] The disclosure relates to methods and composition for differentiating non-neuronal cells to neuronal cells and methods of treating neurodegenerative diseases and disorders.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING [0003] Accompanying this filing is a Sequence Listing entitled, "Sequence-Listing_ST25.txt" created on October 9, 2020 and having 21,814 bytes of data, machine formatted on IBM-PC, MS-Windows operating system. The sequence listing is hereby incorporated by reference in its entirety for all purposes.

BACKGROUND

[0004] Regenerative medicine has significant promise for addressing disorders of cell loss or degeneration. The primary approach for regenerative medicine strategies involves cell replacement by introducing pluripotent cells into a subject in order to treat a disease or disorder. This strategy has enjoyed produced successes in treating hematopoietic disorders but has demonstrated limited efficacy in neurodegenerative disorders and disease models. In contrast, strategies aimed at trans-differentiation can take advantage of the existing cellular plasticity of endogenous cells to generate new cell types.

SUMMARY

[0005] This disclosure relates to regenerative medicine for neurological diseases and therapeutic compositions and methods for transdifferentiating age-restricted cells to treat neurological degeneration, e.g., age-related neurodegenerative disorders, or neurological damage in elderly subjects. [0006] Provided herein are compositions for generating a functional neuron in vivo comprising: a PTB inhibition agent that suppresses PTB expression or activity; and a miR-9 agent that increases miR-9 expression or activity. In some embodiments the methods and compositions further include a miR-124. In some embodiments, the PTB inhibition agent and the miR-9 agent and the optional miR-124 each comprise an inhibitory nucleic acid molecule. In some embodiments, the inhibitory nucleic acid molecule is a ribonucleic acid polynucleotide. In some embodiments, the PTB inhibition agent and the miR-9 agent and the optional miR-124 are encoded by an expression vector. In some embodiments, the expression vector is a viral vector. In some embodiments, the viral vector is an AAV vector. In some embodiment, the vector comprises a PTB inhibitory agent. In some embodiments, the vector comprises more than one PTB inhibitory agent. In some embodiments, the PTB inhibition agent reduces or inhibits an activity of a PTB polypeptide. In some embodiments, the PTB inhibition agent reduces an amount of a PTB polypeptide within a non-neuronal cell. In some embodiments, the PTB inhibition agent is an anti-PTB inhibitor. In some embodiments, the anti-PTB inhibitor is an antisense oligonucleotide. In some embodiments, the anti-PTB inhibitor is selected from the group consisting of an anti-PTB shRNA, an anti-PTB antisense oligonucleotide, an anti-PTB antibody or fragment thereof, an anti- PTB nanobody, an anti-PTB affibody, an anti-PTB polypeptide, an anti-PTB small molecule, a dominant negative PTB mutant, and a sponge polyribonucleotide containing polypyrimidine. In some embodiments, the PTB inhibitor agent comprises an shPTB agent having a sequence of SEQ ID NO:2 or 3. In some embodiments, a vector of the disclosure comprises 1, 2 or 3 shPTB sequences. In some embodiments, the miR-9 agent increases an amount of a nucleic acid encoding a miR-9 molecule. In some embodiments, the miR-9 agent is a miR-9 ribonucleic acid molecule. In some embodiments, the miR-9 agent inhibits the expression or activity of an nPTB molecule. In some embodiments, the miR-9 agent is an anti-nPTB inhibitor. In some embodiments, the anti-nPTB inhibitor is selected from the group consisting of an anti-nPTB shRNA, an anti-nPTB antisense oligonucleotide, an anti-nPTB antibody or fragment thereof, an anti- nPTB nanobody, an anti-nPTB affibody, an anti-nPTB polypeptide, an anti-nPTB small molecule, a dominant negative nPTB mutant, and a sponge polyribonucleotide containing polypyrimidine. In some embodiment, the a miR-9 agent has a sequence of SEQ ID NO:l from nucleotide 3409-3469. In some embodiments, the viral vector comprises the PTB inhibition agent of SEQ ID NO:2 or 3 (shPTB), a miR-124 agent (SEQ ID NO:l from nucleotide 3069-3127, SEQ ID NO:6 or 7) and a miR-9 agent (SEQ ID NO:l from nucleotide 3409-3469, SEQ ID NO:4 or 5). In a specific embodiment, the vector comprises a sequence that is at least 85%, 90%, 92%, 95%, 97%, 98%, 99% or 100% (or any value between any two of the foregoing values) identical to SEQ ID NO:1.

[0007] Also provided in some embodiments are methods of reprogramming a non-neuronal cell into a neuron, the method comprising contacting a composition comprising: a PTB inhibition agent that suppresses PTB expression or activity and a miR-9 agent that increases miR-9 expression or activity and an optional miR-124 with the non-neuronal cell, thereby reprogramming the non-neuronal cell into the neuron. In some embodiments, the non-neuronal cell is a glial cell that expresses miR-9 at a reduced level as compared to a non-neuronal cell from the same brain region of a younger subject. In some embodiments, the glial cell is selected from the group consisting of an astrocyte, an oligodendrocyte, an ependymal cell, a Schwan cell, a NG2 cell, and a satellite cell. In some embodiments, the non-neuronal cell and the neuron are located within a brain of a subject. In some embodiments, the subject has a neurodegenerative disorder. In some embodiments, the neurodegenerative disorder is an age-related neurodegenerative disorder. In some embodiments, the age-related neurodegenerative disorder is selected from the group consisting of Alzheimer 's disease, Parkinson's Disease, dementia, stroke, and a disease associated with a loss of functional neurons within the brain of a subject. In some embodiments, the subject is 30 years or older, 40 years or older, 50 years or older, 55 years or older, 60 years or older, 65 years or older, 70 years or older, 75 years or older, or 80 years or older. [0008] Also provided herein are methods of reprogramming a non neuronal cell into a neuron, the method comprising: contacting the non-neuronal cell with a PTB inhibition agent that suppresses PTB expression or activity and a miR-9 agent that increases miR-9 expression or activity and an optional miR-124 in the non-neuronal cell, thereby reprogramming the non-neuronal cell into the neuron.

In some embodiments, the non-neuronal cell is a glial cell that expresses miR-9 at a reduced level as compared to a non-neuronal cell from the same brain region of a younger subject. In some embodiments, the glial cell is selected from the group consisting of an astrocyte, an oligodendrocyte, an ependymal cell, a Schwan cell, a NG2 cell, and a satellite cell. In some embodiments, the PTB inhibition agent reduces or inhibits an expression level of a nucleic acid encoding a PTB polypeptide. In some embodiments, the PTB inhibition agent reduces or inhibits an activity of a PTB polypeptide. In some embodiments, the PTB inhibition agent reduces an amount of a PTB polypeptide within the non-neuronal cell. In some embodiments, the PTB inhibition agent is an anti-PTB inhibitor. In some embodiments, the anti-PTB inhibitor is an antisense oligonucleotide. In some embodiments, the anti-PTB inhibitor is selected from the group consisting of an anti-PTB shRNA, an anti-PTB miRNA, an anti-PTB antisense oligonucleotide, an anti-PTB antibody or fragment thereof, an anti-PTB nanobody, an anti-PTB affibody, an anti-PTB polypeptide, an anti-PTB small molecule, a dominant negative PTB mutant, and a sponge polyribonucleotide containing polypyrimidine. In some embodiments, the miR-9 agent increases an amount of a nucleic acid encoding for a miR-9 molecule. In some embodiments, the miR-9 agent is a miR-9 ribonucleic acid molecule.

In some embodiments, the miR-9 agent inhibits the expression or activity of an nPTB molecule. In some embodiments, the miR-9 agent is an anti-nPTB inhibitor. In some embodiments, the anti-nPTB inhibitor is selected from the group consisting of an anti-nPTB shRNA, an anti-nPTB miRNA, an anti-nPTB antisense oligonucleotide, an anti-nPTB antibody or fragment thereof, an anti-nPTB nanobody, an anti-nPTB affibody, an anti-nPTB polypeptide, an anti-nPTB small molecule, a dominant negative nPTB mutant, and a sponge polyribonucleotide containing polypyrimidine. In some embodiments, the PTB inhibition agent suppresses the level of PTB expression or activity for at least 12 hours, at least 24 hours, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 10 days, or at least 15 days In some embodiments, the miR-9 agent increases the level of miR-9 expression or activity for at least 12 hours, at least 24 hours, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 10 days, or at least 15 days. In some embodiments, the non-neuron cell is located within a brain region.

In some embodiments, the neuron is a dopaminergic neuron. In some embodiments, the neuron is a cholinergic neuron.

[0009] The disclosure also provides methods of generating a neuron within a subject, the method comprising: administering to a brain region comprising a non-neuronal cell that expresses miR-9 at a reduced level as compared to a young non-neuronal cell of the subject a PTB inhibition agent that suppresses PTB expression or activity and a miR-9 agent that increases miR-9 expression or activity in the non-neuronal cell, thereby reprogramming the non neuronal cell into the neuron. In some embodiments, the method can further comprise delivery a miR-124 to the non-neuronal cell. In some embodiments, the non-neuronal cell is a glial cell. In some embodiments, the glial cell is selected from the group consisting of an astrocyte, an oligodendrocyte, an ependymal cell, a Schwan cell, and a satellite cell. In some embodiments, the PTB inhibition agent reduces or inhibits an expression level of a nucleic acid encoding a PTB polypeptide. In some embodiments, the PTB inhibition agent reduces or inhibits an activity of a PTB polypeptide. In some embodiments, the PTB inhibition agent reduces an amount of a PTB polypeptide within the non-neuronal cell. In some embodiments, the PTB inhibition agent is an anti-PTB inhibitor. In some embodiments, the anti-PTB inhibitor is an antisense oligonucleotide. In some embodiments, the anti-PTB inhibitor is selected from the group consisting of an anti-PTB shRNA, an anti-PTB miRNA, an anti-PTB antisense oligonucleotide, an anti-PTB antibody or fragment thereof, an anti-PTB nanobody, an anti-PTB affibody, an anti-PTB polypeptide, an anti-PTB small molecule, a dominant negative PTB mutant, and a sponge polyribonucleotide containing polypyrimidine In some embodiments, the miR-9 agent increases an amount of a nucleic acid encoding for a miR-9 molecule. In some embodiments, the miR-9 agent is a miR-9 ribonucleic acid molecule. In some embodiments, the miR- 9 agent inhibits the expression or activity of an nPTB molecule. In some embodiments, the miR-9 agent is an anti-nPTB inhibitor. In some embodiments, the anti-nPTB inhibitor is selected from the group consisting of an anti-nPTB shRNA, an anti-nPTB miRNA, an anti-nPTB antisense oligonucleotide, an anti-nPTB antibody or fragment thereof, an anti-nPTB nanobody, an anti-nPTB affibody, an anti-nPTB polypeptide, an anti-nPTB small molecule, a dominant negative nPTB mutant, and a sponge polyribonucleotide containing polypyrimidine.

In some embodiments, the functional neuron is a dopaminergic neuron

In some embodiments, the functional neuron is a cholinergic neuron.

In some embodiments, the administering of the PTB inhibition agent and the miR-9 agent comprises administering a viral vector comprising a nucleic acid encoding the PTB inhibition agent and the miR-9 agent. In some embodiments, the administering the PTB inhibition agent and the miR-9 agent comprises administering a viral vector comprising a nucleic acid encoding the PTB inhibition agent and a viral vector comprising a nucleic acid encoding the miR-9 agent. In some embodiments, the administering comprises contacting a non-neuronal cell with the PTB inhibition agent and the miR-9 agent. In some embodiments, the subject comprises a phenotype wherein contacting the non-neuronal cell within the brain of the subject with the PTB inhibition agent alone does not reprogram the non-neuronal cell into a functional neuron. In some embodiments, the subject is an elderly individual with a brain injury or an individual with an age-related neurodegenerative disorder. In some embodiments, the age-related neurodegenerative disorder is selected from the group consisting of Alzheimer's Disease, Parkinson's Disease, dementia, stroke, and a disease associated with a loss of functional neurons within the brain of a subject.

[0010] The disclosure also provides methods of treating a neurological condition associated with the degeneration of functional neurons within a brain of a subject, the method comprising: contacting a non-neuronal cell with a PTB inhibition agent that suppresses PTB expression or activity and a miR-9 agent that increases miR-9 expression or activity in the non-neuronal cell, thereby reprogramming the non-neuronal cell into the neuron and treating the neurological condition. In some embodiments, the contacting with the PTB inhibition agent and the miR-9 agent is performed simultaneously. In some embodiments, the contacting comprises co-administering the PTB inhibition agent and the miR-9 agent to a region of the brain comprising the non-neuronal cell of the subject. In some embodiments, the co-administering the PTB inhibition agent and the miR-9 agent comprises administering a single viral vector comprising a nucleic acid encoding the PTB inhibition agent and the miR-9 agent. In some embodiments, the neurological disorder is a neurodegenerative disorder. In some embodiments, the neurodegenerative disorder is an age-related neurodegenerative disorder. In some embodiments, the subject comprises a phenotype wherein contacting the non-neuronal cell within the brain of the subject with the PTB inhibition agent alone does not reprogram the non-neuronal cell into a functional neuron.

In some embodiments, the non-neuronal cell is a glial cell. In some embodiments, the glial cell is selected from the group consisting of an astrocyte, an oligodendrocyte, an ependymal cell, a Schwan cell, a NG2 cell, and a satellite cell.

[0011] The disclosure also provides methods of generating a neuron within a brain of a subject, the method comprising: contacting a non-neuronal cell that is located within a region of the brain that the functional neuron originates from with a PTB inhibition agent that suppresses PTB expression or activity, thereby reprogramming the non-neuronal cell into the neuron. In some embodiments, the method further comprises: contacting a non-neuronal cell that is located within a region of the brain that the functional neuron originates with a miR-9 agent that increases miR-9 expression or activity in the non-neuronal cell and optionally a miR-124. In some embodiments, the functional neuron is a dopaminergic neuron and the non-neuronal cell that is located within a mesencephalon region of the brain. In some embodiments, the functional neuron is a cholinergic neuron and the non-neuronal cell that is located within a basal forebrain region of the brain. In some embodiments, the functional neuron comprises a transcriptional phenotype similar to the non-neuronal cell that is located within a region of the brain. In some embodiments, the non-neuronal cell is a glial cell. In some embodiments, the non-neuronal cell is an astrocyte.

[0012] The disclosure provides methods of generating a functional neuron within a brain of a subject with Alzheimer's Disease, the method comprising: contacting a non-neuronal cell with a PTB inhibition agent that suppresses PTB expression or activity and a miR-9 agent that increases miR-9 expression or activity, thereby reprogramming the non-neuronal cell into a functional neuron, wherein the non-neuronal cell is within a region of the brain from which the functional neuron originates.

[0013] The disclosure also provides methods of generating a functional neuron within a brain of a subject with Parkinson's Disease, the method comprising: contacting a non-neuronal cell with a PTB inhibition agent that suppresses PTB expression or activity and a miR-9 agent that increases miR-9 expression or activity, thereby reprogramming the non-neuronal cell into a functional neuron wherein the non-neuronal cell is within a region of the brain from which the functional neuron originates.

[0014] The disclosure provides kits for generating a functional neuron within a brain of a subject comprising: a PTB inhibition agent that suppresses PTB expression or activity; a miR-9 agent that increases miR-9 expression or activity; and a set of instructions for administering the PTB inhibition agent and the miR-9 agent to the brain of the subject.

BRIEF DESCRIPTION OF THE DRAWINGS [0015] An understanding of the features and advantages of the invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which: [0016] FIG. 1 shows exemplary data from quantitative PCR analysis of key microRNAs in astrocytes derived from mice of different ages. Young astrocytes were isolated from the brain of postnatal day 5 mice. Old astrocytes were isolated from the brain of postnatal day 180 mice. miR-124 and miR-9 expression levels are shown.

[0017] FIGs 2A-E show exemplary data for age-restriction to neuronal reprogramming due to reduced miR-9. Astrocyte reprogramming to neurons induced by shPTB alone or in combination with miR-9 is shown in FIG. 2A. FIG. 2B shows quantitation of cell survival (left) and converted Tuj1-positive neurons (right) in response to shPTB treatment alone or in combination with miR-9 overexpression. FIG. 2C shows western blotting analysis of Tau and Tuj1 expression in response to shPTB treatment alone or in combination with miR-9 overexpression. FIG. 2D shows the expression of the constructs of FIG. 2E measured by RT-PCR. FIG. 2E provides exemplary constructs of the disclosure.

[0018] FIG. 3 shows exemplary data for expression of neuronal specific transcription factors in different astrocytes from different regions of the brain. The selective subset of TFs was analyzed by real time RT-PCR. The data were normalized against actin.

[0019] FIG. 4 shows exemplary data demonstrating the region specific conversion of astrocytes to neurons. Targeting astrocytes within the basal forebrain results in the generation of cholinergic neurons.

[0020] FIG. 5 shows a schematic of a construct encoding an anti-PTB shRNA and miR-9. The construct also includes inverted terminal repeats (L-ITR and R-ITR), a CMV promoter, LoxP, stop codon, LoxP, and Turbo-RFP.

[0021] FIG. 6 provides a map of a construct of the disclosure.

[0022] FIG. 7 provides an exemplary sequence of a construct of the disclosure (SEQ ID NO:l) annotated to show various domain/coding sequences. SEQ ID NO:8 is similar containing human sequences. DETAILED DESCRIPTION

[0023] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a sample" includes a plurality of such samples and reference to "the cell" includes reference to one or more cells, and so forth. [0024] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs.

Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices and materials are described herein.

[0025] Also, the use of "or" means "and/or" unless stated otherwise. Similarly, "comprise," "comprises," "comprising" "include," "includes," and "including" are interchangeable and not intended to be limiting.

[0026] It is to be further understood that where descriptions of various embodiments use the term "comprising, " those skilled in the art would understand that in some specific instances, an embodiment can be alternatively described using language "consisting essentially of" or "consisting of."

[0027] The publications discussed above and throughout the text are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior disclosure. Moreover, with respect to any term that is presented in one or more publications that is similar to, or identical with, a term that has been expressly defined in this disclosure, the definition of the term as expressly provided in this disclosure will control in all respects.

[0028] It is to be understood that this disclosure is not limited to particular embodiments described, as such may, of course, vary.

It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0029] Throughout this application, the term "about" is used to indicate that a value that can include similar values or that a value can include the standard deviation of error for the device or method being employed to determine the value. The term "about" when used before a numerical designation, e.g., temperature, time, amount, and concentration, including range, indicates approximations which may vary by (+) or (-) 20%, 10%, 5%, or 1%.

[0030] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

[0031] Throughout this application, various embodiments may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0032] "Age" can refer to the age of a subject or the age of cell within the subject. Non-limiting examples of age-related neurodegenerative disorders include Alzheimer's disease, Parkinson's disease, dementia, Huntington's disease, Downs syndrome (DS), DS- linked early onset of Alzheimer's (DS-AD), stroke, and Amyotrophic lateral sclerosis. In some embodiments, the age-related neurodegenerative disorders is Alzheimer's disease, Parkinson's disease, dementia, or Huntington's disease. In some embodiments, the age-related neurodegenerative disorders is Alzheimer's disease. In some embodiments, the age-related neurodegenerative disorders is Parkinson's disease. In some embodiments, the age-related neurodegenerative disorders is dementia. In some embodiments, the age-related neurodegenerative disorders is Huntington's disease. In some embodiments, the age-related neurodegenerative disorders is amyotrophic lateral sclerosis. In some embodiments, the age-related neurodegenerative disorders is DS-AD.

[0033] "Astrocyte" can refer to characteristic star-shaped glial cells in the brain and spinal cord. As would be clear to one skilled in the art, astrocytes can be characterized in their star shape, expression of markers like glial fibrillary acidic protein (GFAP) and aldehyde dehydrogenase 1 family member Li (ALDH1L1), excitatory amino acid transporter 1 / glutamate aspartate transporter (EAATl/GLAST), glutamine synthetase, S100 beta, or excitatory amino acid transporter 1 / glutamate transporter 1 (EAAT2/GLT-1), participation of blood-brain barrier together with endothelial cells, transmitter uptake and release, regulation of ionic concentration in extracellular space, reaction to neuronal injury and participation in nervous system repair, and metabolic support of surrounding neurons. In certain cases of the present disclosure, an astrocyte can refer to a non-neuronal cell in a nervous system that expresses glial fibrillary acidic protein (GFAP), Aldehyde Dehydrogenase 1 Family Member Li (ALDH1L1), or both. In certain cases, an astrocyte can refer to a nonneuronal cell in a nervous system that expresses a glial fibrillary acidic protein (GFAP) promoter-driven transgene (e.g., red fluorescent protein (RFP), Cre recombinase).

[0034] "Cell lineage" or "lineage" can denote the developmental history of a tissue or organ from the fertilized embryo.

[0035] As used herein, the term "contacting" cells with a composition of the disclosure refers to placing the composition (e.g., compound, nucleic acid, viral vector etc.) in a location that will allow it to touch the cell in order to produce "contacted" cells. The contacting may be accomplished using any suitable method. For example, in one embodiment, contacting is by adding the compound to a culture of cells. Contacting may also be accomplished by injecting it or delivering the composition to a location within a body such that the composition "contacts" the cell type targeted. [0036] "Detecting the presence of" can include determining the amount of something present in addition to determining whether it is present or absent depending on the context.

[0037] The terms "determining," "measuring," "evaluating," "assessing," "assaying," and "analyzing" are often used interchangeably herein to refer to forms of measurement. The terms include determining if an element is present or not (for example, detection). These terms can include quantitative, qualitative, or quantitative and qualitative determinations. Assessing can be relative or absolute.

[0038] As used herein, the term "differentiation", of "differentiate" or "converting" of "inducing differentiation" are used interchangeably to refer to changing the default cell type (genotype and/or phenotype) to a non-default cell type (genotype and/or phenotype). Thus "inducing differentiation in an astrocyte cell" refers to inducing the cell to change its morphology from an astrocyte to a neuronal cell type (i.e., change in gene expression as determined by genetic analysis such as a microarray) and/or phenotype (i.e. change in expression of a protein).

[0039] The term "ex vivo" is used to describe an event that takes place outside of a subject's body. An ex vivo assay is not performed on a subject. Rather, it is performed upon a sample separate from a subject. An example of an ex vivo assay performed on a sample is an "in vitro" assay.

[0040] As used herein, the term "functional neuron" can refer to a neuron that is able to send or receive information through chemical or electrical signals. In some cases, a functional neuron exhibits one or more functional properties of a mature neuron that exists in a normal nervous system, including, but not limited to: excitability (e.g., ability to exhibit action potential, e.g., a rapid rise and subsequent fall in voltage or membrane potential across a cellular membrane), forming synaptic connections with other neurons, presynaptic neurotransmitter release, and post-synaptic response (e.g., excitatory postsynaptic current or inhibitory postsynaptic current). In some cases, a functional neuron is characterized in its expression of one or more markers of functional neurons, including, but not limited to, synapsin, synaptophysin, glutamate decarboxylase 67 (GAD67), glutamate decarboxylase 67 (GAD65), parvalbumin, dopamine- and cAMP-regulated neuronal phosphoprotein 32 (DARPP32), vesicular glutamate transporter 1 (vGLUTl), vesicular glutamate transporter 2 (vGLUT2), acetylcholine, tyrosine hydroxylase (TH), dopamine, vesicular GABA transporter (VGAT), and gamma-aminobutyric acid (GABA).

[0041] The term "glial cell" can generally refer to a type of supportive cell in the central nervous system (e.g., brain and spinal cord) and the peripheral nervous system. In some cases, unlike neurons, glial cells do not conduct electrical impulses or exhibit action potential. In some cases, glial cells do not transmit information with each other, or with neurons via synaptic connection or electrical signals. In a nervous system or in an in vitro culture system, glial cells can surround neurons and provide support for and insulation between neurons. Non-limiting examples of glial cells include oligodendrocytes, astrocytes, ependymal cells, Schwann cells, and satellite cells.

[0042] The term "in vivo" is used to describe an event that takes place in a subject's body.

[0043] The terms "iRNA", "RNAi agent," "iRNA agent,", "RNA interference agent" as used interchangeably herein, refer to an agent that contains RNA, and which mediates the targeted cleavage of an RNA transcript via an RNA-induced silencing complex (RISC) pathway. iRNA directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). For example, iRNA can modulate, e.g., inhibits, the expression of PTB in a cell, e.g., a cell within a subject, such as a mammalian subject. RNAi agents include, without limitation, "small interfering RNA (siRNA)", "endoribonuclease-prepared siRNA (e-siRNA)", "short hairpin RNA (shRNA)", and "small temporally regulated RNA (stRNA)"; "diced siRNA (d-siRNA)", and aptamers, oligonucleotides and other synthetic nucleic acids that comprise at least one uracil base. In some embodiments, such RNAi agents are delivered by a vector such as, but not limited to, a replication defective or replication competent viral vector (e.g., adenoviral vectors, lentiviral vectors, gamma- retroviral vectors etc.).

[0044] As used herein, the term "mature neuron" can refer to a differentiated neuron. In some cases, a neuron is to be a mature neuron if it expresses one or more markers of mature neurons, e.g., microtubule-associated protein 2 (MAP2) and Neuronal Nuclei (NeuN), neuron specific enolase (NSE), 160 kDa neurofilament medium, 200kDa neurofilament heavy, postsynaptic density protein 95(PDS-95), Synapsin I, Synaptophysin, glutamate decarboxylase 67 (GAD67), glutamate decarboxylase 67 (GAD65), parvalbumin, dopamine- and cAMP- regulated neuronal phosphoprotein 32 (DARPP32), vesicular glutamate transporter 1 (vGLUTl), vesicular glutamate transporter 2 (vGLUT2), acetylcholine, and/or tyrosine hydroxylase (TH).

[0045] A "microRNA" or "miRNA" refers to a non-coding nucleic acid (RNA) sequence that binds to at least partially complementary nucleic acid sequence (mRNAs) and negatively regulates the expression of the target mRNA at the post-transcriptional level. A microRNA is typically processed from a "precursor" miRNA having a double-stranded, hairpin loop structure to a "mature" form. Typically, a mature microRNA sequence is about 19-25 nucleotides in length.

[0046] "miR-9" is a short non-coding RNA gene involved in gene regulation and highly conserved from Drosophila and mouse to human. The mature ~21nt miRNAs are processed from hairpin precursor sequences by the Dicer enzyme. miR-9 can be one of the most highly expressed microRNAs in developing and adult vertebrate brain. Key transcriptional regulators such as FoxGl, Hesl or Tlx, can be direct targets of miR-9, placing it at the core of the gene network controlling the neuronal progenitor state. [0047] The sequence of miR-9 is reported at

[http://www.3mirbase.org. See also Yoo, A. S., Staahl, B. T., Chen, L., & Crabtree, G. R., MicroRNA-mediated switching of chromatin remodeling complexes in neural development. Nature 460 (7255), 642-

646 (2009). In one such embodiment, a third microRNA of interest is miR-124.

[0048] A miR-9 agent can comprise any agent that mimics or replicates the function of miR-9 within a cell. A miR-9 agent can be any agent that decreases the expression or activity of nPTB. In some cases, the miR-9 agent can be a small chemical molecule, interfering RNA, short hairpin RNA, microRNA, dominant negative PTB, sponge polynucleotide, ribozyme, antisense oligonucleotide, monoclonal antibody, or polyclonal antibody that is configured to suppress the expression or activity of nPTB.

[0049] In certain cases the miR-9 agent increases the expression of miR-9 within a cell. In certain cases, the miR-9 agent increases an amount of a nucleic acid encoding a miR-9 molecule. In one embodiment, the miR-9 agent is a miR-9 ribonucleic acid molecule. A miR-9 agent that increases the expression of miR-9 can be an expression plasmid, an expression vector, or an expression cassette containing a gene encoding miR-9. The expression plasmid, vector, or cassette once delivered to a non-neuronal cell can the express miR-9 from a miR-9 gene encoded in the plasmid.

[0050] In some embodiments, the miR-9 agent is one that suppresses the expression or activity of nPTB, and can be any type of reagent that suppresses or eliminates the protein expression or protein activity of nPTB. In some cases, the miR-9 agent can be a small chemical molecule, interfering RNA, short hairpin RNA, microRNA, dominant negative nPTB, sponge polynucleotide, ribozyme, antisense oligonucleotide, monoclonal antibody, or polyclonal antibody that is configured to suppress the expression or activity of nPTB. In certain cases, the miR-9 agent inhibits the expression or activity of an nPTB molecule. In certain instances, the anti-nPTB inhibitor comprises an anti-nPTB shRNA, an anti-nPTB antisense oligonucleotide, an anti-nPTB antibody or fragment thereof, an anti- nPTB nanobody, an anti-nPTB affibody, an anti-nPTB polypeptide, an anti-nPTB small molecule, a dominant negative nPTB mutant, a nPTB miRNA, or a sponge polyribonucleotide containing polypyrimidine. In some instances, the composition may only comprise an anti-nPTB agent or miR-9 agent.

[0051] In certain instances, the miR-9 agent increases the expression or activity of miR-9 by at least about 2 fold, at least about 4 fold, at least about 6 fold, at least about 8 fold, at least about 10 fold, at least about 20 fold, at least about 50 fold, or at least about 100 fold.

[0052] In certain instances, the miR-9 agent suppresses or reduces the expression or activity of nPTB by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about

25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about

75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the endogenous or native level. In some cases, a miR-9 agent as provided herein directly suppress the expression level of nPTB, e.g., suppressing the transcription, translation, or protein stability of nPTB. In some cases, the miR-9 agent as provided herein directly suppresses the activity of nPTB, e.g., blocking the binding of nPTB to its target molecules. In some cases, the miR-9 agent as provided herein directly effects on the expression or activity of nPTB, without affecting other cellular signaling pathway.

[0053] The sequence miR-124 is reported at the website having an address of "mirbase.org" wherein www. is placed before the address. See also Yoo, A. S., Staahl, B. T., Chen, L., & Crabtree, G. R.,

MicroRNA-mediated switching of chromatin-remodelling complexes in neural development. Nature 460 (7255), 642-646 (2009).

[0054] miR-124 can optionally be provided in the same or a separate vector to promote neuronal induction by targeting PTB to reduce its expression. It is thought that when PTB inactivation is experimentally induced by shPTB, such negative REST/miR-124 loop is converted to a positive one to allow miR-124 to be more efficient in targeting REST and reduced REST further de-represses miR-124. Once this loop is activated, cells are progressively converted to functional neurons.

[0055] A miR-124 agent can comprise any agent that mimics or replicates the function of miR-124 within a cell. In some cases, the miR-124 agent can be a small chemical molecule, interfering RNA, short hairpin RNA, microRNA, dominant negative PTB, sponge polynucleotide, ribozyme, antisense oligonucleotide, monoclonal antibody, or polyclonal antibody that is configured to regulate PTB expression or activity and/or modulate REST.

[0056] The term "neurodegenerative disorder" is used in reference to diseases associated with a loss of functional neurons or an increase of neuronal dysfunction within a subject. Age-related neurodegenerative disorders can be associated and/or caused by the process of aging.

[0057] The term "neuron" or "neuronal cell" as used herein can have the ordinary meaning one skilled in the art would appreciate. In some cases, neuron can refer to an electrically excitable cell that can receive, process, and transmit information through electrical signals (e.g., membrane potential discharges) and chemical signals (e.g., synaptic transmission of neurotransmitters). As one skilled in the art would appreciate, the chemical signals (e.g., based on release and recognition of neurotransmitters) transduced between neurons can occur via specialized connections called synapses.

[0058] "Neuronal lineage" can refer to the developmental history from a neural stem cell to a mature neuron, including the various stages along this process (as known as neurogenesis), such as, but not limited to, neural stem cells (neuroepithelial cells, radial glial cells), neural progenitors (e.g., intermediate neuronal precursors), neurons, astrocytes, and oligodendrocytes.

[0059] The term "non-neuronal cell" can refer to any type of cell that is not a neuron. An exemplary nonneuronal cell is a cell that is of a cellular lineage other than a neuronal lineage (e.g., a hematopoietic lineage). In some embodiments, a non-neuronal cell is a cell of neuronal lineage but not a neuron, for example, a glial cell. In some embodiments, a non-neuronal cell is somatic cell that is not neuron, such as, but not limited to, glial cell, adult primary fibroblast, embryonic fibroblast, epithelial cell, melanocyte, keratinocyte, adipocyte, blood cell, bone marrow stromal cell, Langerhans cell, muscle cell, rectal cell, or chondrocyte. In some embodiments, a non-neuronal cell is from a non-neuronal cell line, such as, but not limited to, glioblastoma cell line, Hela cell line, NT2 cell line, ARPE19 cell line, or N2A cell line.

[0060] A "nucleic acid molecule" or "polynucleotide" can be composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for thymine when the polynucleotide is RNA. Thus, the term "polynucleotide sequence" is the alphabetical representation of a polynucleotide molecule. This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching. The terms "nucleic acid" and "polynucleotide" as used interchangeably herein can refer to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. The term can encompass nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, locked nucleic acids (LNAs), and peptide-nucleic acids (PNAs). As used in this disclosure, the term "polynucleotide" refers to a nucleic acid molecule that either is recombinant or has been isolated free of total genomic nucleic acid. Included within the term "polynucleotide" are oligonucleotides (nucleic acids 100 residues or less in length), recombinant vectors, including, for example, plasmids, cosmids, phage, viruses, and the like. Polynucleotides include, in certain aspects, regulatory sequences, isolated substantially away from their naturally occurring genes or protein encoding sequences. Polynucleotides may be RNA, DNA, analogs thereof, or a combination thereof. A nucleic acid encoding all or part of a RNA molecule, antisense oligonucleotide, polypeptide, vector, viral vector, gene therapy, or other biomolecules may contain a contiguous nucleic acid sequence encoding all or a portion of the following lengths: 10, 20, 30, 40,

50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180,

190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310,

320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440,

441, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560,

570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690,

700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820,

830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950,

960, 970, 980, 990, 1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, 1095, 1100, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 9000, 10000, or more nucleotides, nucleosides, or base pairs. It is also contemplated that a particular polypeptide from a given species may be encoded by nucleic acids containing natural variations that have slightly different nucleic acid sequences but, nonetheless, encode the same or substantially similar protein, polypeptide, or peptide. Any sequence provided herein includes both RNA ("T" replaced with "U") or DNA sequence ("U" replaced with "T") including full length sequences and fragments (e.g., domains of a polynucleotide).

[0061] "Oligodendrocyte" can refer to a type of glial call that can create myelin sheath that surrounds a neuronal axon to provide support and insulation to axons in the central nervous system. Oligodendrocyte can also be characterized in their expression of PDGF receptor alpha (PDGFR-a), SOX10, neural/glial antigen 2 (NG2),

Olig 1, 2, and 3, oligodendrocyte specific protein (OSP), Myelin basic protein (MBP), or myelin oligodendrocyte glycoprotein (MOG).

[0062] "Pluripotent" can refer to the ability of a cell to form all lineages of the body or soma (i.e., the embryo proper). Exemplary "pluripotent stem cells" can include embryonic stem cells and induced pluripotent stem cells. [0063] "Polypyrimidine tract binding protein" or "PTB" and its homolog neural PTB (nPTB) are both RNA-binding proteins with PTB being expressed ubiquitously in most cell types except neurons whereas nPTB being exclusively expressed in developing neurons. PTB can also be called polypyrimidine tract-binding protein 1, and in humans is encoded by the PTBP1 gene. The PTBP1 gene belongs to the subfamily of ubiquitously expressed heterogeneous nuclear ribonucleoproteins (hnRNPs). The hnRNPs are RNA-binding proteins and they complex with heterogeneous nuclear RNA (hnRNA). These proteins are associated with pre-mRNAs in the nucleus and appear to influence pre-mRNA processing and other aspects of mRNA metabolism and transport. PTB can have four repeats of quasi-RNA recognition motif (RRM) domains that bind RNAs. Consistent with its widespread expression, PTB can contribute to the repression of a large number of alternative splicing events. PTB can recognize short RNA motifs, such as UCUU and UCUCU, located within a pyrimidine-rich context and often associated with the polypyrimidine tract upstream of the 3' splice site of both constitutive and alternative exons. In some cases, binding site for PTB can also include exonic sequences, sequences in introns downstream of regulated exons, and sequences in 3' untranslated regions (3'UTRs). In most alternative splicing systems regulated by PTB, repression can be achieved through the interaction of PTB with multiple PTB binding sites surrounding the alternative exon. In some cases, repression can involve a single PTB binding site. Splicing repression by PTB can occur by a direct competition between PTB and U2AF65, which in turn can preclude the assembly of the U2 snRNP on the branch point. In some cases, splicing repression by PTB can involve PTB binding sites located on both sides of alternative exons, and can result from cooperative interactions between PTB molecules that would loop out the RNA, thereby making the splice sites inaccessible to the splicing machinery. Splicing repression by PTB can also involve multimerization of PTB from a high-affinity binding site that can create a repressive wave that covers the alternative exon and prevents its recognition. PTB can be widely expressed in non neuronal cells, while nPTB can be restricted to neurons. PTB and nPTB can undergo a programmed switch during neuronal differentiation. During neuronal differentiation, PTB is gradually down-regulated at the neuronal induction stage, coincidentally or consequentially, nPTB level is gradually upregulated to a peak level. Later, when the neuronal differentiation enters into neuronal maturation stage, nPTB level experiences reduction after its initial rise and then returns to a relatively low level as compared to the its peak level during neuronal differentiation, when the cell develops into a mature neuron. The sequences of PTB 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):el801. doi:10.1371/journal.pone .0001801; Makeyev et al., Mol. Cell (2007) August 3; 27(3):435-48); thus, one of skill in the art can design and construct antisense, miRNA, siRNA molecules and the like to modulate, e.g., to decrease or inhibit, the expression of PTB; to practice the methods of this disclosure.

[0064] The term "reprogramming" or "trans-differentiation" can refer to the generation of a cell of a certain lineage (e.g., a neuronal cell) from a different type of cell (e.g., a fibroblast cell) without an intermediate process of dedifferentiating the cell into a cell exhibiting pluripotent stem cell characteristics.

[0065] As used herein, "small molecules" can refer to small organic or inorganic molecules of molecular weight below about 3,000 Daltons. The small molecules can be natural products or synthetic products.

[0066] The terms "subject," "individual," or "patient" are often used interchangeably herein and refer to, except where indicated, mammals such as humans or non-human primates. In some embodiments, the subject is a human. In some embodiments, the subject is diagnosed with a neurodegenerative disorder. In some embodiments, the subject is suspected of or is at high risk for a disease.

[0067] As used herein, the terms "treatment" or "treating" are used in reference to a pharmaceutical composition or other intervention regimen for obtaining beneficial or desired results in the recipient. Beneficial or desired results include but are not limited to a therapeutic benefit and/or a prophylactic benefit. A therapeutic benefit may refer to eradication or amelioration of symptoms or of an underlying disorder being treated. Also, a therapeutic benefit can be achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder. A prophylactic effect includes delaying, preventing, or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof. For prophylactic benefit, a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease may undergo treatment, even though a diagnosis of this disease may not have been made.

[0068] A "vector" is a nucleic acid that can be capable of transporting another nucleic acid into a cell. A vector can be capable of directing expression of a protein or proteins encoded by one or more genes, or a microRNA encoded by a polynucleotide, carried by the vector when it is present in the appropriate environment. A vector can be a viral vector, plasmid, cosmid etc. and can be an RNA or a DNA vector.

[0069] A "viral vector" is a viral-derived nucleic acid that can be capable of transporting another nucleic acid into a cell. A viral vector can be capable of directing expression of a protein or proteins encoded by one or more genes, or a microRNA encoded by a polynucleotide, carried by the vector when it is present in the appropriate environment. In some embodiments, the nucleic acid is encapsidated in a virus. Examples of viral vectors include, but are not limited to, retroviral, adenoviral, lentiviral and adeno- associated viral vectors (AAV). An AAV vector can be an AAV2, AAV8, or AAV9 vector.

[0070] In order to develop regenerative neuro-medicine strategies that leverage trans-differentiation for broad therapeutic applications, there are multiple barriers to be overcome. It is well known in the field of neuroscience that astrocytes from older subjects are more difficult to convert than those from young subjects. Even with embryonic astrocytes, it becomes increasingly difficult to convert them after passaging in in vitro cell culture. Such properties can be related to the general problem known as age restriction. In order to develop approaches to treat neurodegenerative diseases in humans, the age restriction barrier needs to be both understood and overcome.

[0071] Disclosed herein, are compositions and methods that overcome age restriction for generating functional neurons within a subject and treating neurodegenerative disorders.

[0072] Disclosed herein are compositions and methods for reprogramming non-neuronal cells into functional neurons. In some cases, the non-neuronal cells exhibit an age-restricted phenotype.

An age-restricted phenotype can comprise any number of phenotypic changes resulting from the process of aging.

[0073] Disclosed herein are compositions for generating a functional neuron in vivo that comprise (a) a PTB inhibition agent that suppresses PTB expression or activity; (b) a miR-9 agent that increases miR-9 expression or activity and optionally (c) an miR- 124. In certain cases, the functional neuron is generated in a subject.

[0074] In certain cases, the PTB inhibition agent reduces or inhibits an activity of a PTB polypeptide. In certain cases, the PTB inhibition agent reduces an amount of a PTB polypeptide within a non-neuronal cell. In certain cases, the PTB inhibition agent is an anti-PTB inhibitor. In certain instances, the anti-PTB inhibitor comprises an antisense oligonucleotide (ASO), an anti-PTB shRNA, an anti-PTB siRNA, an anti-PTB miRNA, an anti-PTB antibody or fragment thereof, an anti-PTB nanobody, an anti-PTB affibody, an anti-PTB polypeptide, an anti-PTB small molecule, a dominant negative PTB mutant, or a sponge polyribonucleotide containing polypyrimidine. [0075] As provided herein, a PTB inhibition agent that suppresses the expression or activity of PTB can be any type of reagent that suppresses or eliminates the protein expression or protein activity of PTB. In some cases, the PTB inhibition agent can be a small chemical molecule, interfering RNA, short hairpin RNA, microRNA, dominant negative PTB, sponge polynucleotide, ribozyme, antisense oligonucleotide, monoclonal antibody, or polyclonal antibody that is configured to suppress the expression or activity of PTB.

[0076] A small chemical molecule inhibitor of PTB can be an organic or inorganic chemical compound. The small molecule inhibitor of PTB can have a structure that is based on an active fragment of PTB. For example, computer modeling methods known in the can be used to rationally design a molecule that has a structure similar to an active fragment of PTB, for example, the RNA-binding motifs (e.g.,

1, 2, 3, 4, or more different RNA-binding motifs).

[0077] RNA interference (RNAi) can be useful for reducing the expression level of target gene PTB. As provided herein, the methods can include use of small hairpin RNA (shRNA) for suppressing expression of PTB in a non-neuronal cell. shRNA molecules are believed to direct sequence-specific degradation of mRNA in cells of various types after first undergoing processing by an RNase III enzyme called DICER (Bernstein et al., Nature 409: 363, 2001) into smaller dsRNA molecules comprised of two 21 nt strands, each of which has a 5' phosphate group and a 3' hydroxyl, and includes a 19 nt region precisely complementary with the other strand, so that there is a 19 nt duplex region flanked by 2 nt-3' overhangs. RNAi can thus be mediated by short interfering RNAs (siRNA), which typically comprise a double-stranded region approximately 19 nucleotides in length with 1-2 nucleotide 3' overhangs on each strand, resulting in a total length of between approximately 21 and 23 nucleotides.

[0078] A short, interfering RNA (siRNA) that can be use in the methods provided herein can comprise an RNA duplex that can be approximately 19 base pairs long and optionally further comprise one or two single-stranded overhangs or loops, resulting in a total length of between approximately 21 and 23 nucleotides. A siRNA can comprise two RNA strands hybridized together, or can alternatively comprise a single RNA strand that includes a self-hybridizing portion. siRNAs can include one or more free strand ends, which can include phosphate and/or hydroxyl groups. siRNAs typically can include a portion that hybridizes under stringent conditions with a target transcript. One strand of the siRNA (or, the self-hybridizing portion of the siRNA) can be precisely complementary with a region of the target transcript (e.g., PTB mRNA transcript), meaning that the siRNA hybridizes to the target transcript without a single mismatch. In certain cases, perfect complementarity is not achieved. In some cases, the mismatches are located at or near the siRNA termini.

[0079] siRNAs can also include various RNA structures (e.g., short hairpin RNAs (shRNAs)) that can be processed in vivo to generate such molecules. shRNAs can include RNA strands containing two complementary elements that hybridize to one another to form a stem, a loop, and optionally an overhang, e.g., a 3' overhang. The stem can be approximately 19 bp long, the loop about 1-20, e.g., about 4-

10, and about 6-8 nt long, and/or the overhang about 1-20, e.g., about 2-15 nt long. In certain cases, the stem can be minimally 19 nucleotides in length and can be up to approximately 29 nucleotides in length. Classical siRNAs as provided herein can trigger degradation of mRNAs to which they are targeted (e.g., PTB mRNA transcript), thereby also reducing the rate of protein synthesis. In some cases, certain siRNAs (e.g., microRNAs) that bind to the 3' UTR of PTB mRNA transcript can inhibit expression of a protein encoded by the template transcript by a mechanism related to but distinct from classic RNA interference, e.g., by reducing translation of the transcript rather than decreasing its stability. MicroRNAs can be between approximately 20 and 26 nucleotides in length, e.g., 22 nt in length. MicroRNAs can be used to destabilize target transcripts and/or block their translation (e.g., PTB expression). Exemplary coding sequences for PTB-shRNA include SEQ ID NO:2 and 3. It should be noted that SEQ ID NO:2 and 3 are depicted as DNA, however, it should be noted that "T" can be replaced with "U" to arrive at an RNA sequence. Such DNA and RNA sequences can be part of vectors used in the methods and compositions of the disclosure.

[0080] In some embodiments, a plasmid containing a DNA sequence encoding a particular desired siRNA sequence is delivered into a target cell via transfection or virally-mediated infection. Once in the cell, the DNA sequence is continuously transcribed into RNA molecules that loop back on themselves and form hairpin structures through intramolecular base pairing. These hairpin structures, once processed by the cell, are equivalent to transfected siRNA molecules and are used by the cell to mediate RNAi of the desired protein. The use of shRNA has an advantage over siRNA transfection as the former can lead to stable, long-term inhibition of protein expression. Inhibition of protein expression by transfected siRNAs is a transient phenomenon that does not occur for times periods longer than several days. In some cases, this can be useful and desired. In cases where longer periods of protein inhibition are necessary, shRNA-mediated inhibition can be used. Short Hairpin RNAs (shRNA) can be comprised of stem-loop structures, which can be designed to contain a 5' flanking region, siRNA region segments, a loop region, a 3' siRNA region and a 3' flanking region. shRNAs can have effective knockdown of target sequences. In some embodiments the DNA sequence encoding shRNA against human PTB comprises the following sequence (SEQ ID NO:2):

5'-GCGTGAAGATCCTGTTCAATACrCGAGTATTGAACAGGATCTTCACGC-3'

The sense part, loop and antisense part are shown in bold, italics, and underline, respectively. In some embodiments, the DNA sequence encoding shRNA against mouse PTB (SEQ ID NO:3):

5'-GGGTGAAGATCCTGTTCAATACrCGAGTATTGAACAGGATCTTCACCC-3'

The sense part, loop and antisense part are shown in bold, italics, and underline, respectively.

[0081] Sponge polynucleotides that have a base sequence complementary to part or all of target RNA transcript (e.g., PTB mRNA transcript) can be used in the method provided herein as well. For instance, sponge polynucleotide can contain polypyrimidine tract. Sponge polynucleotides can be used to "trap" PTB mRNA transcripts, thereby blocking them from being normally spliced, translated, or transported, so that the expression level of PTB protein can be reduced.

[0082] In some cases, the PTB inhibition agent can be a dominant negative mutant that can inhibit an activity of PTB molecule. The dominant negative mutant can be a peptide or peptide mimetic that can inhibit an activity of PTB molecule, or a nucleic acid composition, in the form of a DNA vector or gene therapy vector, that expresses a dominant-negative polypeptide that can inhibit an activity of PTB. The dominant negative mutant can bind to a target RNA or ligand of PTB, affecting its target interaction. The dominant negative molecule can act, for example, by blocking protein-protein interactions or protein-RNA interactions.

[0083] Polypeptide mimetic compositions can contain any combination of non-natural structural components, which are typically from three structural groups: a) residue linkage groups other than the natural amide bond ("peptide bond") linkages; b) non-natural residues in place of naturally occurring amino acid residues; or c) residues which induce secondary structural mimicry, e.g., to induce or stabilize a secondary structure, e.g., a beta turn, gamma turn, beta sheet, alpha helix conformation, and the like. Individual peptidomimetic residues can be joined by peptide bonds, other chemical bonds or coupling means, such as, e.g., glutaraldehyde, N- hydroxysuccinimide esters, bifunctional maleimides, N,N'- dicyclohexylcarbodiimide (DCC) or N,N'- diisopropylcarbodiimide (DIC). Linking groups that can be an alternative to the traditional amide bond ("peptide bond") linkages include, e.g., ketomethylene (e.g., — C(=0)—CH2— for —C(=0)—NH— ), aminomethylene (CH2—NH), ethylene, olefin (CH=CH), ether (CH2—O), thioether (CH2—S), tetrazole (CN4— ), thiazole, retroamide, thioamide, or ester.

[0084] Another non-limiting example of a PTB inhibition agent can be anti-PTB antibody. An anti-PTB antibody can be a polyclonal antibody or a monoclonal antibody that specifically binds to PTB. An anti-PTB antibody as used herein can bind to PTB at its active fragment or inactive fragment. In some configurations, an anti-PTB antibody that binds to the active fragment of PTB can block PTB from interacting with its functional targets (e.g., target RNA transcript) or partners (e.g., protein ligands), thereby inhibiting activity of PTB. In other configurations, an anti-PTB antibody that binds to the inactive fragment of PTB can induce PTB aggregation in some cases, thereby immobilizing PTB inside the cell, preventing it from relocating to interact with its targets or partners. In some cases, an anti-PTB antibody can also induce protein degradation of PTB as being bound to an antibody.

[0085] Antisense nucleic acids (e.g., DNA, RNA, modified DNA, or modified RNA) are generally single-stranded nucleic acids complementary to a portion of a target nucleic acid (e.g., a PTB mRNA transcript) and therefore able to bind to the target to form a duplex. An anti-PTB antisense nucleotide can be configured to suppress the expression or activity of PTB, e.g., in a cell. Antisense oligonucleotides (ASOs) can pair with a target mRNA to render the RNA a substrate for cleavage by the intranuclear enzyme RNase H. In some cases, antisense oligonucleotide can mediate target mRNA degradation for ~3 months in the nervous system, e.g., the rodent and non-human primate nervous system after injection into the cerebral spinal fluid. As provided herein, antisense oligonucleotide that can be used in the methods provided herein are typically oligonucleotides that range from 15 to 35 nucleotides in length but can range from 10 up to approximately 50 nucleotides in length. Binding can reduce or inhibit the function of the target PTB nucleic acid. For example, antisense oligonucleotides can block transcription when bound to genomic DNA (e.g., PTB gene), inhibit translation when bound to mRNA (e.g., PTB mRNA transcript), and/or lead to degradation of the nucleic acid. Reduction in expression of PTB can be achieved by the administration of antisense nucleic acids or peptide nucleic acids comprising sequences complementary to those of the mRNA that encodes the polypeptide. Antisense technology and its applications are well known in the art and are described in (Phillips, M. I. (ed.) Antisense Technology, Methods Enzymol., 313, Bennett, C.F., Krainer, A.R. and Cleveland, D.W. (2019). Antisense oligonucleotide therapies for neurodegenerative diseases. Ann. Rev. Neurosci. 42, 385-406, and 314: 2000, and references mentioned therein. See also Crooke, S. "ANTISENSE DRUG TECHNOLOGY: PRINCIPLES, STRATEGIES, AND APPLICATIONS" (1st Edition) Marcel Dekker; and references cited therein. In some cases, antisense oligonucleotide as provided herein can comprise locked nucleic acids (LNAs). In some cases, LNAs refer to a modified RNA nucleotide, in which the ribose moiety is modified with an extra bridge connecting the 2' oxygen and 4' carbon and the bridge "locks" the ribose in the 3'- endo (North) conformation. In some cases, LNAs are be mixed with DNA or RNA residues in the oligonucleotide whenever desired and hybridize with DNA or RNA according to Watson-Crick base-pairing rules. The locked ribose conformation can enhance base stacking and backbone pre organization. Inclusion of LNAs in the oligonucleotide as provide herein, in some cases, increases the hybridization properties (melting temperature) of oligonucleotides. In some cases, inclusion of LNAs blocks translation of the target mRNA, but without inducing degradation of the target mRNA. Exemplary techniques and applications of using LNAs can be found in PCT/US2013/047157 and Campbell MA et al., Chem. Soc. Rev., 40(12), 5680-9, which are incorporated herein by reference in their entireties.

[0086] The PTB inhibition agent as provided herein can also include ribozymes or deoxyribozymes that can catalyze the sequence-specific cleavage of RNA molecules. The cleavage site is determined by complementary pairing of nucleotides in the RNA or DNA enzyme with nucleotides in the target RNA (e.g., PTB mRNA transcript). Thus, RNA and DNA enzymes can be designed to cleave PTB mRNA transcript, thereby increasing its rate of degradation.

[0087] In certain instances, the PTB inhibition agent suppresses or reduces the expression or activity of PTB by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about

40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about

90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the endogenous or native level. As provided herein, the PTB inhibition agent suppresses expression or activity of PTB by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% of the level without the PTB inhibition agent. In some cases, a PTB inhibition agent as provided herein directly suppress the expression level of PTB, e.g., suppressing the transcription, translation, or protein stability of PTB. In some cases, PTB inhibition agent as provided herein directly suppresses the activity of PTB, e.g., blocking the binding of PTB to its target molecules. In some cases, the PTB inhibition agent as provided herein directly effects on the expression or activity of PTB, without affecting other cellular signaling pathway.

[0088] In certain cases, the PTB inhibition agent and the miR-9 agent are encoded by an expression vector. In some embodiments, the expression vector is a viral vector. In some embodiments, the viral vector is an adenoviral, a lentiviral or an adeno-associated viral (AAV) vector. In an instance, the expression vector is a viral vector wherein the viral vector is an adeno-associated viral (AAV) vector. In some embodiments, the AAV vector is AAV2, AAV8, or AAV9. An AAV vector can comprise one or more AAV inverted terminal repeats (ITRs) flanking a polynucleotide encoding a PTB inhibition agent and/or a polynucleotide encoding an miR-9 agent. The polynucleotide can be operatively linked to transcriptional control DNAs, specifically promoter DNA and polyadenylation signal sequence DNA that are functional in target cells to form an expression cassette. The expression cassette may also include intron sequences to facilitate processing of an RNA transcript when expressed in mammalian cells. In an instance, the AAV vector comprises an AAV2, AAV8, or AAV9 vector. In an instance, the viral vector comprises the PTB inhibition agent of SEQ ID NO:2. In an instance, the viral vector comprises the miR-9 agent of SEQ ID NO: 3. In certain instances, the viral vector comprises the PTB inhibition agent of SEQ ID NO:2 and/or 3 and miR-9 agent of SEQ ID NO:l from nucleotide 3409-3469.

[0089] The expression cassette may encode one or more anti-PTB shRNA ("shPTB") molecules and/or one or more miR-9 molecules. In some embodiments, the expression cassette contains a miR-124 sequence. In some embodiments, the expression cassette comprises a nucleotide sequence encoding a single shPTB molecule. In some embodiments, the expression cassette comprises a nucleotide sequence encoding a single miR-9 molecule. In some embodiments, the expression cassette comprises a nucleotide sequence encoding a single shPTB and a single miR-9. In some embodiments, the expression cassette comprises a nucleotide sequence encoding two or three shPTB molecules. In some embodiments, the expression cassette comprises a nucleotide sequence encoding two or three miR-9 molecules. In some embodiments, the expression cassette comprises a nucleotide sequence encoding one, two or three miR-124 molecules.

In some embodiments, the expression cassette comprises regulatory elements to differentially express the anti-PTB shRNA and the miR-9 and the optional miR-124. In some embodiments, the regulatory element is a polIII promoter. In one embodiment, the polIII promoter is an HI or U6 promoter.

[0090] In one embodiment a vector of the disclosure has the general structure of FIG. 2E or 5, wherein the vector comprises 1 or 2 shPTB coding sequences, one miR-9 coding sequence and an optional miR-124 coding sequence. In another embodiment, a vector of the disclosure comprises a sequence that is at least 85%-100% identical to SEQ ID NO:l and has the general format of FIG. 2E, "AAV-shPTB-124-9".

[0091] In one embodiment, the disclosure provides pharmaceutical compositions comprising a PTB inhibition agent and an miR-9 agent and an optional miR-124 molecule in an amount effective to reprogram an age restricted non-neuronal cell to a mature neuron by suppressing the expression or activity of PTB and increasing the miR-9 expression or activity in the non-neuronal cell. An exemplary pharmaceutical composition can further comprise a pharmaceutically acceptable carrier or excipient. As described above, a PTB inhibition agent and/or an miR-9 agent as provided herein can be a small chemical molecule, interfering RNA, short hairpin RNA, microRNA, dominant negative mutant, ribozyme, antisense oligonucleotide, protein inhibitor, monoclonal antibody, a polyclonal antibody, a peptide, or any form of modified nucleic acid.

[0092] A pharmaceutical composition provided herein can include one or more carriers and excipients (including but not limited to buffers, carbohydrates, 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, sesame oil and the like, saline solutions, aqueous dextrose and glycerol solutions, flavoring agents, coloring agents, detackifiers and other acceptable additives, or binders, other pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH buffering agents, tonicity adjusting agents, emulsifying agents, wetting agents and the like. Examples of excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. In another instance, the composition is substantially free of preservatives. In other embodiments, the composition contains at least one preservative. General methodology on pharmaceutical dosage forms can be found in Ansel et ah, Pharmaceutical Dosage Forms and Drug Delivery Systems (Lippencott Williams & Wilkins, Baltimore Md. (1999)). It will be recognized that, while any suitable carrier known to those of ordinary skill in the art can be employed to administer the pharmaceutical compositions described herein, the type of carrier can vary depending on the mode 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 by reference in their entirety herein. An exemplary pharmaceutical composition can be formulated for injection, parenteral administration, intravenous administration, or injection into the CSF. As one of ordinary skills in the art will appreciate, pharmaceutical compositions can comprise any appropriate carrier or excipient, depending on the type of PTB inhibition agent and miR-9 agent and the administration route the composition is designed for. For example, a composition comprising a PTB inhibition agent and miR-9 agent as provided herein can be formulated for parenteral administration and can be presented in unit dose form in ampoules, pre-filled syringes, small volume infusion or in multidose containers with an added preservative. The composition can take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, for example solutions in aqueous polyethylene glycol. For example, for injectable formulations, a vehicle can be chosen from those known in the art to be suitable, including aqueous solutions or oil suspensions, or emulsions, with sesame oil, corn oil, cottonseed oil, or peanut oil, as well as elixirs, mannitol, dextrose, or a sterile aqueous solution, and similar pharmaceutical vehicles. The formulation can also comprise polymer compositions which are biocompatible, biodegradable, such as poly(lactic-co- glycolic)acid. These materials can be made into micro or nanospheres, loaded with drug and further coated or derivatized to provide superior sustained release performance. Vehicles suitable for periocular or intraocular injection include, for example, suspensions of active agent in injection grade water, liposomes, and vehicles suitable for lipophilic substances and those known in the art. A composition as provided herein can further comprise additional agent besides a PTB inhibition agent and miR-9 agent and a pharmaceutically acceptable carrier or excipient. For example, additional agent can be provided for promoting neuronal survival purpose. Alternatively or additionally, additional agent can be provided for monitoring pharmacodynamics purpose. In some embodiments, a composition comprises additional agent as a penetration enhancer for delivery of constructs comprising, e.g.,

PTB inhibition agent and miR-9 agent and optional miR-124 molecules. [0093] Disclosed herein are methods for reprogramming a non neuronal cell into a neuron, wherein the method comprises contacting the non-neuronal cell with the composition comprising a PTB inhibition agent and an miR-9 agent thereby reprogramming the non neuronal cell into the neuron.

[0094] In some embodiments, a therapeutically effective amount of composition is administered to the non-neuronal cell. A "therapeutically effective amount" of a composition of the disclosure will vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the composition to elicit a desired response in the individual, the formulation or type of agent being delivered, etc. A therapeutically effective amount can also be one in which any toxic or detrimental effects of the composition are outweighed by the therapeutically beneficial effects. Without wishing to be bound by a particular theory, it is contemplated that, in some cases, a therapeutically effective amount of composition comprising a PTB inhibition agent and an miR-9 agent and an optional miR-124 molecule as provided herein can be an amount that converts a certain proportion of astrocytes in a brain region that experiences neuronal loss, conversion of such proportion of astrocytes to functional neurons in the brain region is sufficient to ameliorate or treating the disease or condition associated with the neuronal loss in the brain region, and meanwhile, such proportion of astrocytes does not exceed a threshold level that can lead to adverse effects that can outweigh the beneficial effects brought by the neuronal conversion, for instance, due to excessive reduction in the number of astrocytes in the brain region as a direct consequence of the neuronal conversion. In some embodiments, a therapeutically effective amount of the composition is an amount sufficient to inhibit PTB expression or activity and/or increase miR-9 expression or activity. A sufficient amount of composition can be determined empirically as one skilled in the art would readily appreciate. In some cases, the amount of composition can be determined by any type of assay that examines the activity of the composition in the non-neuronal cell. For example, when the composition is configured to suppress the expression of PTB and increase miR-9 expression in the non-neuronal cell, the sufficient amount of the composition can be determined by assessing the expression level of PTB and/or miR-9 in an exemplary non-neuronal cell after administration of the composition, e.g., by Western blot. In some cases, functional assays are utilized for assessing the activity of PTB and/or miR-9 after delivery of the composition to an exemplary non-neuronal cell. In some cases, other functional assays, such as, immunostaining for neuronal markers, electrical recording for neuronal functional properties, that examine downstream neuronal properties are used to determine a sufficient amount of composition. [0095] In certain cases, the non-neuronal cell is a glial cell. In certain case, the glial cell is an astrocyte. In an instance, the astrocyte expresses miR-9 at a reduced level as compared to an astrocyte of a younger age or as compared to a threshold level of expression. The term "a reduced level as compared to an astrocyte of a younger age" or "reduced level as compared to a threshold level of expression" can include instances wherein expression of miR-9 is reduced by about 20 % to about 100 %. In some instances, expression of miR-9 is reduced by about 20 % to about 25 %, about 20 % to about 30 %, about 20 % to about 40 %, about 20 % to about 50 %, about 20 % to about 60 %, about 20 % to about 70 %, about 20 % to about 75 %, about 20 % to about 80 %, about 20 % to about 90 %, about 20 % to about 95 %, about 20 % to about 100 %, about 25 % to about 30 %, about 25 % to about 40 %, about 25 % to about 50 %, about 25 % to about 60 %, about 25 % to about 70 %, about 25 % to about 75 %, about 25 % to about 80 %, about 25 % to about 90 %, about 25 % to about 95 %, about 25 % to about 100 %, about 30 % to about 40 %, about 30 % to about 50 %, about 30 % to about 60 %, about 30 % to about 70 %, about 30 % to about 75 %, about 30 % to about 80 %, about 30 % to about 90 %, about 30 % to about 95 %, about 30 % to about 100 %, about 40 % to about 50 %, about 40 % to about 60 %, about 40 % to about 70 %, about 40 % to about 75 %, about 40 % to about 80 %, about 40 % to about 90 %, about 40 % to about 95 %, about 40 % to about 100 %, about 50 % to about 60 %, about 50 % to about 70 %, about 50 % to about 75 %, about 50 % to about 80 %, about 50 % to about 90 %, about 50 % to about 95 %, about 50 % to about 100 %, about 60 % to about 70 %, about 60 % to about 75 %, about 60 % to about 80 %, about 60 % to about 90 %, about 60 % to about 95 %, about 60 % to about 100 %, about 70 % to about 75 %, about 70 % to about 80 %, about 70 % to about 90 %, about 70 % to about 95 %, about 70 % to about 100 %, about 75 % to about 80 %, about 75 % to about 90 %, about 75 % to about 95 %, about 75 % to about 100 %, about 80 % to about 90 %, about 80 % to about 95 %, about 80 % to about 100 %, about 90 % to about 95 %, about 90 % to about 100 %, or about 95 % to about 100 %. In some instances, expression of miR-9 is reduced by about 20 %, about 25 % about 30 %, about 40 %, about 50 %, about 60 %, about 70 %, about 75

%, about 80 %, about 90 %, about 95 %, or about 100 %. In some instances, expression of miR-9 is reduced by at least about 20 %, about 25 %, about 30 %, about 40 %, about 50 %, about 60 %, about 70

%, about 75 %, about 80 %, about 90 %, or about 95 %.

[0096] In an instance, the astrocyte expresses miR-9 at a reduced level as compared to a housekeeping or constitutively expressed gene. The term "housekeeping gene" can refer to genes involved in the maintenance of basal cellular functions that are essential for the existence of a cell. Non-limiting example of housekeeping genes can include ACTB, GAPDH, TBP, RRN18S, etc. The term "a reduced level as compared to a housekeeping or constitutively expressed gene" can include instances wherein expression of miR-9 is reduced by about 20 % to about 100 %. In some instances, expression of miR-9 is reduced by about 20 % to about 25 %, about 20 % to about 30 %, about 20 % to about 40 %, about 20 % to about 50 %, about 20 % to about 60 %, about 20 % to about 70 %, about 20 % to about 75 %, about 20 % to about 80 %, about 20 % to about 90 %, about 20 % to about 95 %, about 20 % to about 100 %, about 25 % to about 30 %, about 25 % to about 40 %, about 25 % to about 50 %, about 25 % to about 60 %, about 25 % to about 70 %, about 25 % to about 75 %, about 25 % to about 80 %, about 25 % to about 90 %, about 25 % to about 95 %, about 25 % to about 100 %, about 30 % to about 40 %, about 30 % to about 50 %, about 30 % to about 60 %, about 30 % to about 70 %, about 30 % to about 75 %, about 30 % to about 80 %, about 30 % to about 90 %, about 30 % to about 95 %, about 30 % to about 100 %, about 40 % to about 50 %, about 40 % to about 60 %, about 40 % to about 70 %, about 40 % to about 75 %, about 40 % to about 80 %, about 40 % to about 90 %, about 40 % to about 95 %, about 40 % to about 100 %, about 50 % to about 60 %, about 50 % to about 70 %, about 50 % to about 75 %, about 50 % to about 80 %, about 50 % to about 90 %, about 50 % to about 95 %, about 50 % to about 100 %, about 60 % to about 70 %, about 60 % to about 75 %, about 60 % to about 80 %, about 60 % to about 90 %, about 60 % to about 95 %, about 60 % to about 100 %, about 70 % to about 75 %, about 70 % to about 80 %, about 70 % to about 90 %, about 70 % to about 95 %, about 70 % to about 100 %, about 75 % to about 80 %, about 75 % to about 90 %, about 75 % to about 95 %, about 75 % to about 100 %, about 80 % to about 90 %, about 80 % to about 95 %, about 80 % to about 100 %, about 90 % to about 95 %, about 90 % to about 100 %, or about 95 % to about 100 %. In some instances, expression of miR-9 is reduced by about 20 %, about 25 %, about 30 %, about 40 %, about 50 %, about 60 %, about 70 %, about 75 %, about 80 %, about 90 %, about 95 %, or about 100 %. In some instances, expression of miR-9 is reduced by at least about 20 %, about 25 %, about 30 %, about 40 %, about 50 %, about 60 %, about 70 %, about 75 %, about 80 %, about 90 %, or about 95 %.

[0097] In certain cases, the non-neuronal cell and the neuron are located within a brain of a subject. In some embodiment, the non neuronal cell is in the substantia nigra. In some embodiments, the composition is administered to a brain region selected from the substantial nigra, the midbrain, the basal forebrain, and the ventral tegmental area. In some embodiments, the composition is administered into substantial nigra (SN). In some embodiments, the composition is administered to ventral tegmental area (VTA). In some embodiments, the composition is administered to a region defined by a local degeneration or loss of functional neurons resulting from a disorder or trauma.

[0098] In certain instances, the subject has a neurodegenerative disorder, such as an age-related neurodegenerative disorder. In certain cases, the functional neuron is generated in a subject having a neurodegenerative disorder. In an instance, the subject comprises a neurodegenerative disorder. In some embodiments, the neurodegenerative disorder is an age-related disorder. In certain instances, the neurodegenerative disorder can be Alzheimer's Disease, Parkinson's Disease, dementia, or a disease associated with a loss of functional neurons within the brain of a subject. In some embodiments, the neurodegenerative disorder is Alzheimer's Disease. In some embodiments, the neurodegenerative disorder is Parkinson's Disease. In some embodiments, the neurodegenerative disorder or trauma is stroke or cerebrovascular accident (CVA). [0099] Disclosed are methods for reprogramming a non-neuronal cell into a neuron, wherein the method comprises contacting the non neuronal cell with a PTB inhibition agent that suppresses PTB expression or activity and a miR-9 agent that increases miR-9 expression or activity in the non-neuronal cell, thereby reprogramming the non-neuronal cell into the neuron. In some cases, the non-neuronal cell is a glial cell that expresses reduced levels of miR-9. In certain cases, the non-neuronal cell is a glial cell.

In certain case, the glial cell is an astrocyte. In an instance, the astrocyte expresses miR-9 at a reduced level as compared to an astrocyte of a younger age.

[0100] In some embodiments, the glial cell is selected from the group consisting of an astrocyte, an oligodendrocyte, an ependymal cell, a Schwan cell, a NG2 cell, and a satellite cell. In some embodiments the glial cell is an astrocyte.

[0101] In some embodiments, the PTB inhibition agent reduces or inhibits an expression level of a nucleic acid encoding a PTB polypeptide within the non-neuronal cell. In some embodiments, the PTB inhibition agent reduces or inhibits an activity of a PTB polypeptide within the non-neuronal cell. In some embodiments, the PTB inhibition agent reduces an amount of a PTB polypeptide within the non-neuronal cell. In some cases, the PTB inhibition agent is an anti-PTB inhibitor. In certain instances, the anti-PTB inhibitor comprises an antisense oligonucleotide (ASO), an anti-PTB shRNA, an anti-PTB antibody or fragment thereof, an anti-PTB nanobody, an anti-PTB affibody, an anti-PTB polypeptide, an anti-PTB small molecule, a dominant negative PTB mutant, a PTB miRNA, or a sponge polyribonucleotide containing polypyrimidine.

[0102] In some embodiments, the miR-9 agent increases an amount of a nucleic acid encoding for a miR-9 molecule within the non-neuronal cell. In some embodiments, the miR-9 agent is a miR-9 ribonucleic acid molecule within the non-neuronal cell. In some embodiments, wherein the miR-9 agent inhibits the expression or activity of an nPTB molecule within the non-neuronal cell. In some cases, the miR-9 agent is an anti-nPTB inhibitor. In certain embodiments, the anti- nPTB inhibitor comprises an anti-nPTB shRNA, an anti-nPTB antisense oligonucleotide, an anti-nPTB antibody or fragment thereof, an anti- nPTB nanobody, an anti-nPTB affibody, an anti-nPTB polypeptide, an anti-nPTB small molecule, a dominant negative nPTB mutant, a nPTB miRNA, or a sponge polyribonucleotide containing polypyrimidine. [0103] In some embodiments, a level of PTB expression or activity is reduced for an extended period of time. In certain instances, the level of PTB expression and/or activity is reduced for at least about 0.5 days to about 15 days. In certain embodiments, the level of PTB expression and/or activity is reduced for at least about 0.5 days to about 1 day, about 0.5 days to about 2 days, about 0.5 days to about 4 days, about 0.5 days to about 5 days, about 0.5 days to about 10 days, about 0.5 days to about 12 days, about 0.5 days to about 15 days, about 1 day to about 2 days, about 1 day to about 4 days, about 1 day to about 5 days, about 1 day to about 10 days, about 1 day to about 12 days, about 1 day to about 15 days, about 2 days to about 4 days, about 2 days to about 5 days, about 2 days to about 10 days, about 2 days to about 12 days, about 2 days to about

15 days, about 4 days to about 5 days, about 4 days to about 10 days, about 4 days to about 12 days, about 4 days to about 15 days, about 5 days to about 10 days, about 5 days to about 12 days, about

5 days to about 15 days, about 10 days to about 12 days, about 10 days to about 15 days, or about 12 days to about 15 days. . In certain embodiments, the level of PTB expression and/or activity is reduced for at least about 0.5 days, about 1 day, about 2 days, about 4 days, about 5 days, about 10 days, about 12 days, or about 15 days. In certain embodiments, the level of PTB expression and/or activity is reduced for at least at least about 0.5 days, about 1 day, about 2 days, about 4 days, about 5 days, about 10 days, or about 12 days. In certain instances, the level of PTB expression and/or activity is reduced for at least at most about 1 day, about 2 days, about 4 days, about 5 days, about 10 days, about 12 days, or about 15 days.

[0104] In some embodiments, the miR-9 agent increases a level of miR-9 expression or activity for an extended period of time. In certain embodiments, the level of miR-9 expression and/or activity is increased for at least about 0.5 days to about 15 days. In certain embodiments, the level of miR-9 expression and/or activity is increased for at least about 0.5 days to about 1 day, about 0.5 days to about 2 days, about 0.5 days to about 4 days, about 0.5 days to about 5 days, about 0.5 days to about 10 days, about 0.5 days to about 12 days, about 0.5 days to about 15 days, about 1 day to about 2 days, about 1 day to about 4 days, about 1 day to about 5 days, about 1 day to about 10 days, about 1 day to about 12 days, about 1 day to about 15 days, about 2 days to about 4 days, about 2 days to about 5 days, about 2 days to about 10 days, about 2 days to about 12 days, about 2 days to about 15 days, about 4 days to about 5 days, about 4 days to about 10 days, about 4 days to about 12 days, about 4 days to about 15 days, about 5 days to about 10 days, about

5 days to about 12 days, about 5 days to about 15 days, about 10 days to about 12 days, about 10 days to about 15 days, or about 12 days to about 15 days. In certain instances, the level of miR-9 expression and/or activity is increased for at least about 0.5 days, about 1 day, about 2 days, about 4 days, about 5 days, about 10 days, about 12 days, or about 15 days. In certain instances, the level of miR-9 expression and/or activity is increased for at least about 0.5 days, about 1 day, about 2 days, about 4 days, about 5 days, about 10 days, or about 12 days.

[0105] In some embodiments, the non-neuron cell is located within a brain region of a mammal. In an instance, the mammal is a human. In some embodiments, the subject comprises a phenotype wherein contacting the non-neuronal cell within the brain of the subject with the PTB inhibition agent alone does not reprogram the non neuronal cell into a functional neuron. In some embodiments, the subject is an elderly individual with a brain injury or an individual with an age-related neurodegenerative disorder. In some embodiments, the elderly individual is over 50 years of age, or over 55 years of age, or over 60 years of age, or over 65 years of age, or over 70 years of age, or over 75 years of age.

[0106] The disclosure further provides a method of generating a neuron within a subject, wherein the method comprises administering a PTB inhibition agent that suppresses PTB expression or activity and a miR-9 agent that increases miR-9 expression or activity in the non-neuronal cell and an optional miR-124 molecule to a brain region that comprises a non-neuronal cell that expresses miR-9 at a reduced level as compared to a non-neuronal cell from the same brain region of a younger subject, thereby reprogramming the non-neuronal cell into the neuron. In some embodiments, the neuron is a dopaminergic neuron or a cholinergic neuron. In some embodiments, the non neuronal cell is a glial cell that expresses miR-9. In some cases, the glial cell is selected from the group consisting of an astrocyte, an oligodendrocyte, an ependymal cell, a Schwan cell, NG2 cell, and a satellite cell. In certain instances, the glial cell is an astrocyte. In certain instances, the subject is a human and the younger subject can be a young mouse. In some cases, the PTB inhibition agent reduces or inhibits an expression level of a nucleic acid encoding a PTB polypeptide within the non-neuronal cell. In some cases, the PTB inhibition agent reduces or inhibits an activity of a PTB polypeptide within the non-neuronal cell. In some embodiments, the PTB inhibition agent reduces an amount of a PTB polypeptide within the non-neuronal cell. In some cases, the PTB inhibition agent is an anti-PTB inhibitor.

[0107] In some embodiment, the method comprises administering a construct of SEQ ID NO:l to a brain region of the subject to produce neuron(s) in the subject from non-neuronal cells. In one embodiment, the construct of SEQ ID NO:l is delivered by an AAV capsid.

[0108] In some embodiments, the subject is an elderly individual with a brain injury or an individual with an age-related neurodegenerative disorder.

[0109] In some embodiments, the PTB inhibition agent and miR-9 agent and an optional miR-124 molecule are contacted to a non neuronal cell by delivery in the form of a viral vector. A viral vector can comprise one or more copies of an expression construct encoding a PTB inhibition agent, miR-9 agent and optional miR-124 molecule, e.g., shRNA, ASO, microRNA, dominant negative mutant, or antibody, such as, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, or 100 copies. A viral vector can be titered to any appropriate amount for administration, as one skilled in the art will be able to determine. For example, the titer as determined by PCR, RT-PCR, or other methods can be at least about 10 ⁵ viral particles/mL, at least about 10 ⁶ particles/mL, at least about 10 ⁷ particles/mL, at least about 10 ⁸ particles/mL, at least about 10 ⁹ particles/mL, at least about 10 ¹⁰ particles/mL, at least about 10 ¹¹ particles/mL, at least about 10 ¹² particles/mL, at least about 10 ¹³ particles/mL, at least about 10 ¹⁴ particles/mL, or at least about 10 ¹⁵ particles/mL. In some cases, the titer of viral vector to be administered is at least about 10 ¹⁰ viral particles/mL. In one embodiment, the vector comprises the sequence of SEQ ID NO:l and is contained in an AAV capsid. In some cases, the composition is antisense oligonucleotide, and the antisense oligonucleotide can be delivered at any effective amount as one skilled in the art will appreciate. In certain cases, the antisense oligonucleotide can be administered at least about 0.05 pg, at least about 0.075 pg, at least about 0.1 pg, at least about 0.125 pg, at least about 0.15 pg, at least about 0.175 pg, at least about 0.2 pg, at least about 0.225 pg, at least about 0.25 pg, at least about 0.275 pg, at least about 0.3 pg, at least about 0.325 pg, at least about 0.35 pg, at least about 0.375 pg, at least about 0.4 pg, at least about 0.425 pg, at least about 0.045 pg, at least about 0.475 pg, at least about 0.5 pg, at least about 0.6 pg, at least about 0.7 pg, at least about 0.8 pg, at least about 0.9 pg, at least about 1.0 pg, at least about 1.2 pg, at least about 1.25 pg, at least about 1.3 pg, at least about 1.4 pg, at least about 1.5 pg, at least about

1.6 pg, at least about 1.7 pg, at least about 1.8 pg, at least about

1.9 pg, at least about 2.0 pg, at least about 2.1 pg, at least about

2.2 pg, at least about 2.3 pg, at least about 2.4 pg, at least about

2.5 pg, at least about 2.75 pg, at least about 3 pg, at least about 4 pg, at least about 5 pg, at least about 6 pg, at least about 7 pg, at least about 8 pg, at least about 9 pg, or at least about 10 pg.

In some embodiments, the antisense oligonucleotide is administered about 0.05 pg, about 0.075 pg, about 0.1 pg, about 0.125 pg, about 0.15 pg, about 0.175 pg, about 0.2 pg, about 0.225 pg, about 0.25 pg, about 0.275 pg, about 0.3 pg, about 0.325 pg, about 0.35 pg, about 0.375 pg, about 0.4 pg, about 0.425 pg, about 0.045 pg, about 0.475 pg, about 0.5 pg, about 0.6 pg, about 0.7 pg, about 0.8 pg, about 0.9 pg, about 1.0 pg, about 1.2 pg, about 1.25 pg, about 1.3 pg, about 1.4 pg, about 1.5 pg, about 1.6 pg, about 1.7 pg, about 1.8 pg, about 1.9 pg, about 2.0 pg, about 2.1 pg, about 2.2 pg, about 2.3 pg, about 2.4 pg, about 2.5 pg, about 2.75 pg, about 3 pg, about 4 pg, about 5 pg, about 6 pg, about 7 pg, about 8 pg, about 9 pg, or about 10 pg. In some cases, the antisense oligonucleotide is administered in vitro at about 0.075 to about 0.325 pg, about 0.1 to about 0.3 pg, or about 0.125 to about 0.25 pg. In some cases, the antisense oligonucleotide is administered in vivo at about 1 to about 10 pg, at about 1 to about 5 pg, at about 1 to about 3 pg, about 1.5 to about 2.5 pg, or about 1.75 to about 2.25 pg.

[0110] A PTB inhibition agent, a miR-9 agent and an optional miR- 124 molecule can be delivered directly to the brain or by systemic injection into a subject. In some embodiments, a composition comprising the PTB inhibition agent and the miR-9 agent with or without a miR-124 molecule is administered or delivered to the central nervous system. Methods of therapeutic delivery to the central nervous system and the brain is appreciated and understood to one skilled in the art. In another embodiment, a composition as provided herein is delivered systemically to a subject or to a region in nervous system, e.g., brain or spinal cord, of a subject, e.g., delivered to cerebrospinal fluid or cerebral ventricles. In some embodiments, the administration is to the brain or a region of the brain. In some embodiments, the administration is to the substantial nigra. In some embodiments, the administration is to the midbrain. In some embodiments, the administration is to the basal forebrain. In some embodiments, the composition is administered to the cerebrospinal fluid (CSF). In some embodiments, the administration is via intracranial injection, intracerebroventricular injection, intracisternal injection, or intrathecal injection. In some embodiments, administration is via intravenous injection. In some embodiments, administration is via retro-orbital injection.

[0111] Also disclosed herein are methods for generating function- specific neurons within the brain of a subject. A function-specific neuron can be a specific type of neuron (e.g., dopaminergic, cholinergic, etc.) or a function-specific neuron can be a neuron having a specific, intended function (e.g., release of a specific neurotransmitter, neurons comprising a specific phenotype, etc.). FIG. 3 demonstrates the molecular basis for regional specificity in non-neuronal cells. Shown are examples of region-specific transcription profiles in astrocytes from the midbrain and cortical region of the brain. The differential, region-specific transcription program phenotypes can potentiate the ability of astrocytes to be reprogrammed into specific types of neurons and/or function-specific neurons. FIG. 4 demonstrates an example targeting the regional specificity of non-neuronal cells to generate a function-specific neuron, specifically, astrocytes within the basal forebrain can be reprogrammed into cholinergic neurons, which originate from the basal forebrain region.

[0112] Disclosed are methods of generating a neuron within a brain of a subject, wherein the method comprises contacting a non-neuronal cell that is located within a region of the brain that the functional neuron originates from with a PTB inhibition agent that suppresses PTB expression or activity, thereby reprogramming the non-neuronal cell into the neuron. In some cases, the method further comprises contacting a non-neuronal cell that is located within a region of the brain that the functional neuron originates with a miR-9 agent that increases miR-9 expression or activity in the non neuronal cell. In some cases, the functional neuron comprises a transcriptional phenotype similar to the non-neuronal cell that is located within a region of the brain. In certain instances, the functional neuron is a dopaminergic neuron and the non-neuronal cell that is located within a mesencephalon region of the brain. In certain instances, the functional neuron is a cholinergic neuron and the non-neuronal cell that is located within a basal forebrain region of the brain. In some cases, the non-neuronal cell is a glial cell. In certain instances, the non-neuronal cell is an astrocyte. [0113] Disclosed is a method of generating a functional neuron within a brain of a subject with Alzheimer's Disease, wherein the method comprises contacting a non-neuronal cell with a PTB inhibition agent that suppresses PTB expression or activity and a miR-9 agent that increases miR-9 expression or activity and optionally a miR-124 agent, thereby reprogramming the non-neuronal cell into a functional neuron, wherein the non-neuronal cell is within a region of the brain from which the functional neuron originates.

[0114] Disclosed is a method of generating a functional neuron within the brain of a subject with Parkinson's Disease, wherein the method comprises contacting a non-neuronal cell with a PTB inhibition agent that suppresses PTB expression or activity and a miR-9 agent that increases miR-9 expression or activity and optionally a miR-124 agent, thereby reprogramming the non-neuronal cell into a functional neuron wherein the non-neuronal cell is within a region of the brain from which the functional neuron originates.

[0115] Disclosed herein are kits for generating a functional neuron within a brain of a subject comprising a PTB inhibition agent that suppresses PTB expression or activity, a miR-9 agent that increases miR-9 expression or activity, and a set of instructions for administering the PTB inhibition agent and the miR-9 agent to the brain of the subject.

EXAMPLES

[0116] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

Example 1: Strategy to overcome age restriction to neuronal reprogramming

[0117] Two regulatory loops were elucidated, one for neuronal conversion and the other for neuronal maturation. As provided herein expression of components in both loops were examined to determine which factors were linked to aging. Quantitative PCR analysis of key microRNAs was conducted in astrocytes derived from mice of different ages. Young astrocytes were isolated from the brain of postnatal day 5 mice. Old astrocytes were isolated from the brain of postnatal day 180 mice. miR-124 and miR-9 expression levels are shown. Interestingly, the results suggested that two microRNAs, miR-124 and miR-9, were both down-regulated during aging (FIG. 1), and suggested that the levels of these microRNAs may present barriers to neuronal reprogramming.

[0118] Because miR-124 is involved in the first loop for neuronal induction, which is suppressed by the transcription repressor REST, knockdown of PTB was expected to induce the expression of this microRNA. However, miR-9 is part of the second regulatory loop for neuronal maturation. One of the key problems in neuronal conversion experiments is cellular death during forced expression of neuronal reprogramming agents. Little is known about how to manage this problem. It was examined whether miR-9 was important in this process, and if so, if co-expression of miR-9 along PTB knockdown might help prevent cell death, thus leading to improved neuronal reprogramming efficiency. To test this hypothesis, miR-9 along with shPTB (example construct, FIG. 5) was co-expressed in astrocytes isolated from postnatal day 180, which is "old" compared to astrocytes from young mice (postnatal day 60). Interestingly, co expression of miR-9 is able to dramatically increase the neuronal conversion efficiency in those old astrocytes (FIG. 2A).

Quantitative analysis showed that both cell survival (FIG. 2B, left) and conversion efficiency (FIG. 2B, right) were significantly elevated with shPTB plus miR-9 compared to shPTB alone. Western blotting analysis showed that the two key neuronal markers Tau and Tuj1 were both greatly induced in response to PTB depletion in combination with miR-9 overexpression (FIG. 2C). These data suggested a means to overcome age-restriction to neuronal reprogramming from astrocytes.

Example 2: Molecular basis for regional specificity in astrocytes

[0119] Astrocytes from different parts of the brain appear to have intrinsic ability to develop into different neuronal subtypes once being converted. For example, astrocytes from the substantia nigra regions in midbrain are able to generate TH-positive dopaminergic neurons both in vitro and in the brain. To explore the molecular basis of this apparent regional specificity, several key transcription factors (TFs) that are uniquely expressed in dopaminergic neurons were examined, such as FOXA2, LMX1A, LMX1B, and EN2. By quantitative PCR, it was found that these TFs were essentially absent in cortical astrocytes, but readily detectable in midbrain astrocytes (FIG. 3). This finding indicated that midbrain astrocytes can inherit a key transcription program related to dopaminergic neurons because astrocytes and neurons are known to both originate from their common ancestors called radial glial cells during neurogenesis. Thus, the existing epigenetic landscape that permits detectable expression of neuronal-specific TFs may potentiate those astrocytes to develop into specific neuronal subtypes once being converted.

Example 3: The ability to convert astrocytes to cholinergic neurons in basal forebrain

[0120] The basal forebrain is a critical region associated with Alzheimer's disease, which has been attributed to the loss of cholinergic neurons. AAV2-shPTB vector was injected into the basal forebrain region of mice. Six weeks after AAV delivery, brain slices were stained with the cholinergic marker choline acetyltransferase (ChAT). Interestingly, detection of many RFP-marked cells was observed (because the AAV vector expresses RFP to allow lineage tracing) with a fraction of which stained positively for ChAT in the substantia innominate, which is a part of basal forebrain (FIG. 4). This observation established a foundation for region-specific targeting in generating specific types of neurons.

[0121] While certain embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

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