Recombinant AAV vectors for cellular translation regulation and cellular reprogramming and methods of using the same

Abstract

The present inventive concept relates to expression cassettes, vector genomes, and recombinant viral vectors for reprogramming mRNA translation control, more specifically dual-therapeutic expression cassettes for reprogramming oligodendrocytes and / or oligodendrocyte precursor cells (OPCs) to neurons. The present inventive concept further relates to methods of treating central nervous system disorders and conditions using the same.

Description

Attorney Docket No. 5510-0002WGRECOMBINANT AAV VECTORS FOR CELLULAR TRANSLATION REGULATION AND CELLULAR REPROGRAMMING AND METHODS OF USING THE SAMECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of U.S. Provisional Patent Application No. 63 / 708,898, filed October 18, 2024, and U.S. Provisional Patent Application No. 63 / 849,456, filed July 23, 2025, the disclosures of each of which are hereby incorporated herein by reference in their entireties.STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING

[0002] A Sequence Listing XML file, submitted pursuant 37 C.F.R. §§ 1.831-835, entitled 55I0-0002WO_ST26.xml, 15,478 bytes in size, created on October 8, 2025, and filed electronically, is provided in lieu of a paper copy. The entire content of the Sequence Listing XML file is incorporated herein by reference in its entirety.FIELD

[0003] The present inventive concept is related to vectors for regulating cellular translation and cellular reprogramming, and methods of using vectors, such as recombinant AAV vectors in regulating cellular translation and cellular reprogramming in the treatment of disorders and disease.BACKGROUND

[0004] In vivo cell reprogramming of endogenous cell populations, such as glial cells, offers a promising possibility for neuron replacement in the adult mammalian nervous system. This approach may compensate for neuronal loss occurring in neurological disorders, but viable tools are needed to advance this strategy from bench to clinic. Recently, the successful neuronal conversion of glial cells through the repression of a single gene, polypyrimidine tract-binding protein 1 (Ptbpl which encodes a key RNA-binding protein, has been described. Newly converted neurons not only express correct markers but they also functionally integrate into endogenous brain circuits and modify disease symptoms in in vivo models of neurodegenerative diseases. However, doubts about the nature of "converted" neurons, in particular in vivo, haveAttorney Docket No. 5510-0002WO been raised, based on concerns about tracking reporter genes in converted cells. Accordingly, improved reagents for in vivo cell reprogramming, and its implementation, with approaches that can be translated into the clinic, e.g., with antisense oligonucleotides targeting a single gene, such as Ptbpl, and delivery systems for the same, such as, but not limited to, viral vectors and non-viral vectors, are needed.SUMMARY

[0005] Aspects of the present inventive concept include systems, such as recombinant adeno- associated virus (rAAV) vectors to drive reprogramming of cell populations, such as glial cells / oligodendrocytes into functional neurons. In vivo cell reprogramming by aspects of the inventive concept may include, e.g., vectors for the expression of 1) A factor, at least one factor, or more than one factors, that: promote cellular reprogramming, e.g., a factor that promotes PTBP1 knockdown, i.e., a factor that inhibits / attenuates PTBP1 activity, function, expression, and / or enhances / induces PTBP1 degradation and / or inactivation, and / or may include the expression of 2) A factor, at least one factor, or more than one factors, that: enhance tissue regeneration; enhance cell function; enhance cell survival and / or cell replenishment, e.g., enhance neuron survival and / or replenish neurons depleted as a result of neuron loss; promote / enhance cell development; promote / enhance cell structure development / regeneration, e.g., promoting / enhancing functional axon regeneration; and / or promote / enhance cell / cell structure stabilization, either alone or in combination, offer a promising way to bring beneficial effects in the treatment of, e.g., disorders / conditions related to neuron loss, neurodegeneration (including neuron degeneration), dysmyelination, and / or global central nervous system (CNS) injury, by generating new neurons, such as, but not limited to, by generating new neurons from oligodendrocytes and / or oligodendrocyte precursor cells (OPCs).

[0006] According to an aspect of the inventive concept, provided is a vector including: a polynucleotide encoding at least one factor for inducing cellular reprogramming; and a polynucleotide encoding at least one factor for enhancing tissue regeneration, for enhancing cell survival, for promoting / enhancing cell development, for promoting / enhancing cell structure development / regeneration, and / or for promoting / enhancing cell / cell structure stabilization. Also provided are recombinant virus particles, compositions, and pharmaceutical compositions including the vector described herein.Attorney Docket No. 5510-0002WG

[0007] According to another aspect of the inventive concept, provided is an expression cassette including: a polynucleotide encoding at least one factor for inducing cellular reprogramming; and a polynucleotide encoding at least one factor for enhancing tissue regeneration, for enhancing cell survival, for promoting / enhancing cell development, for promoting / enhancing cell structure development / regeneration, and / or for promoting / enhancing cell / cell structure stabilization. Also provided are vectors, recombinant virus particles, compositions, and pharmaceutical compositions including the expression cassette described herein.

[0008] According to another aspect of the inventive concept, provided is a method of inducing cellular reprogramming in a cell including exposing the cell to a vector, recombinant virus particle, composition, or pharmaceutical composition of the inventive concept described herein.

[0009] According to another aspect of the inventive concept, provided is a method of reprogramming an oligodendrocyte or an oligodendrocyte precursor cell (OPC) to a neuron including exposing the oligodendrocyte or OPC to a vector, recombinant virus particle, composition, or pharmaceutical composition of the inventive concept described herein, wherein the oligodendrocyte or OPC is reprogrammed into a neuron.

[0010] According to another aspect of the inventive concept, provided is a method of treating a central nervous system (CNS) disorder or condition responsive to an increase in the number of neurons in a subject in need thereof including delivering a vector, recombinant virus particle, composition, or pharmaceutical composition of the inventive concept described herein, wherein delivery of the vector or the recombinant virus particle treats the CNS disorder or condition.BRIEF DESCRIPTION OF THE DRAWINGS

[0011] FIG. 1 depicts the design of an exemplary recombinant AAV (rAAV)-based dual- therapeutic expression cassette(s) for cell reprogramming and mRNA translation control according to a first embodiment of the inventive concept.

[0012] FIG. 2 depicts the design of an exemplary rAAV-based dual-therapeutic expression cassette(s) for cell reprogramming and mRNA translation control according to a second embodiment of the inventive concept.Attorney Docket No. 5510-0002WG

[0013] FIG. 3 depicts the construction of dual-therapeutic expression cassette(s) for cell reprogramming and mRNA translation control according to the first embodiment of the inventive concept.

[0014] FIG. 4 depicts the construction of dual-therapeutic expression cassette(s) for cell reprogramming and mRNA translation control according to the second embodiment of the inventive concept.

[0015] FIG. 5 depicts anti-PTBPl approaches according to embodiments of the inventive concept, in addition to RNA-based (siRNA, shRNA, miRNA, and / or IncRNA) anti-PTBPl approaches.

[0016] Abbreviations used in the drawings. ITR: Inverted Terminal Repeat; scITR: self- complementary Inverted Terminal Repeat; PTBP1 : Polypyrimidine Tract Binding Protein 1 [also known as: PTB, PTB2, PTB3, PTB4, pPTB, HNRPI, PTB-1, PTB-T, HNRNPI, HNRNP-I]; TT: Polyadenylation, and / or Transcription Terminator; IS: Introns and Splicing Elements; MAG: Myelin Associated Glycoprotein; GDNF: glial cell line-derived neurotrophic factor; pro-GDNF: glial cell line-derived neurotrophic factor preproprotein.

[0017] FIG. 6 depicts DNA sequence similarity and homology analysis between (A) anti- PTBPl siRNA4 and PTBP1 of different species, and (B) sequence alignment of anti-PTBPl siRNA4 and humanized anti-PTBPl siRNA4.

[0018] FIG. 7 depicts the human PTBP1 knockdown by siRNA4 or humanized siRNA4 miRNA4_Hu) delivered as an artificial microRNA in a miR-155 backbone. (A) Representation of miRNA4 plasmid constructs with added termination signals (T8 for Pol III and SV40PolyA for Pol II). Indicated miRNAs were transcribed by RNA polymerase II (CMV) or III (Hl) promoters. (B) Knockdown analysis of Human PTBP1 co-transfected in Hek293 with miRNA4 or miRNA4_Hu. miRNA targeting Luciferase (miRNA_Luc) was used as a negative control. (C) Representative western blot after 24h post-co-transfection in Hek293 cells.

[0019] FIG. 8 depicts the secondary structures of mi miRNA4 and miRNA4_Hu in a in a miR-155 backbone (RNA4_miR155 and miRNA4_Hu_miR155, respectively). The sequence of miRNA4 and miRNA4_Hu include a rational deletion of the indicated nucleotides of miRNA4 and miRNA4_Hu, based on the secondary structure of native miR-155.

[0020] FIG. 9 depicts the secondary structures of miRNA4 and miRNA4_Hu in a miR-E backbone (miRNA4_miR-E and miRNA4_Hu_miR-E, respectively).Attorney Docket No. 5510-0002WG

[0021] FIG. 10 depicts (Panel A) Representation of miRNA4 and miRNA4_Hu plasmid constructs in either a miR-155 or miR-E backbone, with added termination signals (T8 for Pol III and SV40PolyA for Pol II). Indicated miRNAs were transcribed by RNA polymerase II (CMV) or III (Hl) promoters. (Panel B) Knockdown analysis of Human PTBP1 co-transfected in cells with miRNA4 or miRNA4_Hu. in either a miR-155 or miR-E backbone. A miRNA targeting luciferase (miRNA_Luc) was used as a negative control.

[0022] FIG. 11 depicts plasmid design and features of an expression cassette for both hGDNF and miRNA4_Hu (bottom right), an expression cassette for hGDNF and a negative miRNA control (miRNA_Scramble, top right), and an expression cassette hGDNF knockout (hGDNF. KO) construct that introduces several stop codons to prevent protein translation (middle right). (Panel A) RT-qPCR quantification of human PTBP1 (hPTBPl) knockdown under different plasmid design conditions after transfection in the HEK293 cell line. (Panel B) Quantification of secreted hGDNF by ELISA. * p < 0.05; ** p < 0.01.

[0023] FIG. 12 depicts human PTBP1 (hPTBPl) protein knockdown determined by Western blot after transfection in HEK293 cells for different plasmid designs and the features of the expression cassettes (hGDNF alone, and hGDNF with miRNA4_Hu) therein.

[0024] FIG. 13 (Panel A) Rat PTBPl(rPTBPl) protein knockdown as determined by Western blot in primary rat oligodendrocytes for different plasmid designs and features of the expression cassettes (hGDNF with miRNA_Scramble, top right, and hGDNF with miRNA4, bottom right) therein. (Panel B) Quantitation of secreted hGDNF by ELISA for the different plasmid designs. *p < 0.05; **p < 0.01.DETAILED DESCRIPTION

[0025] The foregoing and other aspects of the present inventive concept will now be described in more detail with respect to other embodiments described herein. It should be appreciated that the inventive concept can be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the inventive concept to those skilled in the art.

[0026] Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination. Moreover, the presentAttorney Docket No. 5510-0002WG invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A. B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.

[0027] Unless otherwise defined all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0028] Nucleotide sequences are presented herein by single strand only, in the 5' to 3' direction, from left to right, unless specifically indicated otherwise. Nucleotides and amino acids are represented herein in the manner recommended by the IUPAC-IUB Biochemical Nomenclature Commission, or (for amino acids) by either the one-letter code, or the three letter code, both in accordance with 37 C.F.R. § 1.822 and established usage.

[0029] Except as otherwise indicated, standard methods known to those skilled in the art may be used for production of recombinant and synthetic polypeptides, antibodies or antigen-binding fragments thereof, manipulation of nucleic acid sequences, production of transformed cells, the construction of, e.g., rAAV constructs, modified capsid proteins, packaging vectors expressing the AAV rep and / or cap sequences, and transiently and stably transfected packaging cells. Such techniques are known to those skilled in the art. See, e.g., SAMBROOK et al., MOLECULAR CLONING: A LABORATORY MANUAL 2nd Ed (Cold Spring Harbor, NY., 1989); F. M. AUSUBEL et al. CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Green Publishing Associates, Inc. and lohn Wiley & Sons, Inc., New York).

[0030] All publications, patent applications, patents, nucleotide sequences, amino acid sequences and other references mentioned herein are incorporated by reference in their entirety.

[0031] The terminology used in the description of the inventive concept herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the inventive concept. As used in the description of the inventive concept and the appended claims, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Additionally, as used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items and may be abbreviated as " / ".Attorney Docket No. 5510-0002WO

[0032] The term "comprise," as used herein, in addition to its regular meaning, may also include, and, in some embodiments, may specifically refer to the expressions "consist essentially of and / or "consist of." Thus, the expression "comprise" can also refer to, in some embodiments, the specifically listed elements of that which is claimed and does not include further elements, as well as embodiments in which the specifically listed elements of that which is claimed may and / or does encompass further elements, or embodiments in which the specifically listed elements of that which is claimed may encompass further elements that do not materially affect the basic and novel characteristic(s) of that which is claimed. For example, that which is claimed, such as a composition, formulation, method, system, etc. "comprising" listed elements also encompasses, for example, a composition, formulation, method, kit, etc. "consisting of," i.e., wherein that which is claimed does not include further elements, and a composition, formulation, method, kit, etc. "consisting essentially of," i.e., wherein that which is claimed may include further elements that do not materially affect the basic and novel characteristic(s) of that which is claimed. It is noted that aspects of the present inventive concept may comprise, consist essentially of, or consist of the various elements as described herein.

[0033] The term "about" is intended to qualify the numerical values which it modifies, denoting such a value as variable within a margin of error. When no particular margin of error, such as a standard deviation to a mean value given in a chart or table of data, is recited, the term "about" should be understood to mean that range which may encompass, for example, ± 20%, ± 15%, or ± 10%, and in some embodiments, ± 5%, ± 3%, or ± 2%, of the recited value and the range is included.

[0034] The term, "sequence identity" generally refers to the extent to which two optimally aligned polynucleotide or polypeptide sequences are invariant throughout a window of alignment of components, e.g., nucleotides or amino acids. "Identity" can be readily calculated by known methods including, but not limited to, those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton Press, New York (1991).Attorney Docket No. 5510-0002WG

[0035] The terms "substantially identical' or "corresponding to" generally means that two nucleic acid sequences or two amino acid sequences have at least about 80% sequence identity. In some embodiments, the two nucleic acid sequences or two amino acid sequences can have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0036] The term "adeno-associated virus" (AAV) in relation to the present inventive concept includes without limitation AAV type 1, AAV type 2, AAV type 3 (including types 3 A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8. AAV type 9, AAV type 10, AAV type 11, avian AAV, bovine AAV, canine AAV, equine AAV, and ovine AAV, etc., or any other AAV presently known or later discovered. See, e.g., BERNARD N. FIELDS et al., VIROLOGY, volume 2, chapter 69 (4th ed., Lippincott-Raven Publishers). A number of additional AAV serotypes and clades have been identified (see, e.g., Gao et al. (2004) J. Virol. 78, 6381-6388, and Table 1 of U.S. Patent Application Publication No. 2019 / 0224256, incorporated herein by reference), which are also encompassed by the term "AAV."

[0037] The terms "vector," "virus vector," "delivery vector" (and similar terms) generally refer to a virus particle that functions as a nucleic acid delivery vehicle, and which include the viral nucleic acid (i.e., the vector genome) packaged within the virion. Virus vectors according to the present inventive concept can package an AAV or rAAV genome, or any other nucleic acid including viral nucleic acids. Alternatively, in some contexts, the term "vector," "virus vector," "delivery vector" (and similar terms) may be used to refer to the vector genome (e.g., vDNA) in the absence of the virion and / or to a viral capsid that acts as a transporter to deliver molecules tethered to the capsid or packaged within the capsid. In some embodiments, the vector, virus vector, and / or delivery vector may be a recombinant vector, virus vector, and / or delivery vector, created by combining two or more nucleic acid fragments from different sources. Recombinant nucleic acid molecules, such as a vector, virus vector, and / or delivery vector, may be molecules formed by laboratory methods of genetic recombination, e.g., molecular cloning, that bring together genetic material from multiple sources that would not otherwise be found in nature. Recombinant nucleic acid molecules may include recombinant vector genomes, recombinant virus genomes, and / or recombinant genomes.

[0038] A "recombinant AAV vector genome" or "rAAV genome" is an AAV genome (i.e., vDNA) that includes at least one inverted terminal repeat (ITR), e.g., one, two or three invertedAttorney Docket No. 5510-0002WO terminal repeats (TTRs), and one or more heterologous nucleotide sequences. rAAV vectors generally retain the terminal repeat(s) (TR(s)) in cis to generate virus; however, modified AAV TRs and non-AAV TRs including partially, or completely synthetic sequences can also serve this purpose. All other viral sequences are dispensable and may be supplied in trans (Muzyczka, (1992) Curr. Topics Microbiol. Immunol. 158, 97). The rAAV vector optionally includes two TRs (e.g., AAV TRs), which generally will be at the 5' and 3' ends of the heterologous nucleotide sequence(s), but do not need to be contiguous thereto. The TRs can be the same or different from each other. The vector genome can also contain a single ITR at its 3' or 5’ end.

[0039] The term "terminal repeat" or "TR" includes any viral terminal repeat or synthetic sequence that forms a hairpin structure and functions as an inverted terminal repeat (i.e., mediates the desired functions such as replication, virus packaging, integration and / or provirus rescue, and the like). The TR can be an AAV TR or a non-AAV TR. For example, a non-AAV TR sequence such as those of other parvoviruses (e g., canine parvovirus (CPV), mouse parvovirus (MVM), human parvovirus B-19) or the SV40 hairpin that serves as the origin of SV40 replication can be used as a TR, which can further be modified by truncation, substitution, deletion, insertion and / or addition. Further, the TR can be partially or completely synthetic, such as the "double-D sequence" as described in U.S. Pat. No. 5,478,745 to Samulski et al.

[0040] An "AAV inverted terminal repeat" or "AAV ITR" may be from any AAV, including but not limited to those from serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 or any other AAV now known or later discovered (see, e g., Table 1 of U.S. Patent Application Publication No. 2019 / 0224256, incorporated herein by reference). An AAV ITR need not have the native terminal repeat sequence (e.g., a native AAV ITR sequence may be altered by insertion, deletion, and / or truncation), as long as the terminal repeat mediates the desired functions, e.g., replication, virus packaging, integration, and / or provirus rescue, and the like.

[0041] In some embodiments, vectors of the inventive concept may include a mutant ITR (mITR), such as, but not limited to, an ITR in which the terminal resolution site (trs) has been abolished / deleted, such as the self-complementary ITR (scITR) included in e.g., self- complementary AAV (scAAV) vectors, e.g., as described in McCarty et al. (2003) Gene Ther. 10:2112-2118, Wang et al. (2003) Gene Ther. 2105-2111, and Wu et al. (2007) Hum. Gene Ther. 18: 171-182. In some embodiments, the vector may include replacing the mITR / scITR sequences present in traditional scAAV vectors with, e.g., sequences including a TelNAttorney Docket No. 5510-0002WO recognition site, to provide closed-end double-stranded AAV (cceAAV) vectors, e.g., as described in Zhang et al. (2024) Mol. Ther. Methods Clin. Dev. 32, 101206, incorporated herein by reference.

[0042] The terms "rAAV particle," rAAV virus particle," and "rAAV virion" may be used interchangeably. A "rAAV particle," "rAAV virus particle," or "rAAV virion" includes a rAAV vector genome packaged within an AAV capsid.

[0043] The AAV capsid structure is described in more detail, e.g., in BERNARD N. FIELDS et al., VIROLOGY, volume 2, chapters 69 and 70 (4th ed., Lippincott-Raven Publishers).

[0044] By "substantially retain" a property, it is meant that at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the property (e.g., activity, or other measurable characteristic) is retained.

[0045] The vectors of the inventive concept can further be "targeted" virus vectors (e.g., having a directed / preferential tropism), and / or a "hybrid" parvovirus (i.e., in which the viral ITRs and viral capsid are from different parvoviruses) as described in international patent publication WO 00 / 28004 and Chao et al., (2000) Mol. Therapy 2, 619. In some embodiments, the virus vectors of the inventive concept may include virus particles including capsid proteins that provide a tropism to the virus particle, for example, in some embodiments, viral vectors / virus particles having a tropism toward oligodendrocytes, oligodendrocyte precursor cells (OPCs), astrocytes, neurons, and / or in glial progenitor cells. The vectors of the inventive concept may also include "chimeric" virus vectors, wherein the capsid protein or capsid proteins of the virus particle may include amino acid sequences from more than one AAV serotype, such as, but not limited to, virus vectors and virus particles including capsid proteins having amino acid sequences from more than one AAV serotype and having a preferential oligodendrocyte tropism, such as those described in Powell et al. (2016) Gene Therapy 23, 807-814, incorporated herein by reference.

[0046] In some embodiments, the virus vectors of the inventive concept may include a capsid protein or capsid proteins that exert influence on transgene expression from promoters, such as constitutive promoters, included in the virus vector in, e.g., transgene expression in oligodendrocytes, astrocytes, neurons, and / or in glial progenitor cells, such as AAV9 capsids, and mutants thereof, and their influence on chicken P-actin promoter-driven gene expression, and gene expression from variants of the chicken P-actin promoter, such as described in Powell et al.Attorney Docket No. 5510-0002WO(2020) Mol. Ther. 28(5), 1373-1380), incorporated herein by reference, AAV9 capsids, and mutants thereof, and their influence the minimal synthetic Jetl promoter, such as described in Bohlen et al. (2020 Hum. Gene Ther. 31(21-22), 1155-1168, or AAV-LK03 capsids, and mutants thereof, on transgene expression across species, such as the capsids described in Gonzalez-Sandoval et al. (2023) Nat. Comm. 14, 2448, incorporated herein by reference, wherein capsid proteins affect the epigenetic status of the vector genome and histone H3 chemical modifications on the vector DNA.

[0047] The term "tropism" as used herein generally refers to preferential entry of the virus into certain cell or tissue type(s) and / or preferential interaction with the cell surface that facilitates entry into certain cell or tissue types, optionally and preferably followed by expression (e.g. transcription and, optionally, translation) of sequences carried by the viral genome in the cell, e.g., for a recombinant virus, expression of the heterologous nucleotide sequence(s). Those skilled in the art will appreciate that transcription of a heterologous nucleic acid sequence from the viral genome may not be initiated in the absence of trans-acting factors, e.g., for an inducible promoter or otherwise regulated nucleic acid sequence. In the case of a rAAV genome, gene expression from the viral genome may be from a stably integrated provirus and / or from a nonintegrated episome, as well as any other form which the virus nucleic acid may take within the cell.

[0048] The term "tropism profile" generally refers to the pattern of transduction of one or more target cells, tissues and / or organs. Representative examples of chimeric AAV capsids have a tropism profile characterized by efficient transduction of oligodendrocytes with only low transduction of neurons, astrocytes, and other CNS cells.

[0049] The terms "specific for oligodendrocytes and / or OPCs" and "has a tropism for oligodendrocytes and / or OPCs" as used herein generally refers to a viral vector that, when administered directly into the CNS, preferentially transduces oligodendrocytes and / or OPCs over neurons, astrocytes, and other CNS cell types. In some embodiments, at least about 80% of the transduced cells are oligodendrocytes and / or OPCs, e.g., at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more are oligodendrocytes and / or OPCs.

[0050] The term "central nervous system (CNS) disorder or condition responsive to an increase in the number of neurons" as used herein refers to a disease, disorder, condition, or injury in which CNS cells are damaged, lost, or function improperly and which show anAttorney Docket No. 5510-0002WO improvement in at least one symptom when the number of neurons in the CNS (e.g., at the site of tissue damage) is increased. The term includes diseases, disorders, conditions, and injuries in which CNS cells are directly affected as well as diseases, disorders, conditions, and injuries in which CNS cells become dysfunctional secondary to damage to other cells, tissues, or organs.

[0051] As used herein, "transduction" of a cell by a virus vector (e.g., an AAV vector) means entry of the vector into the cell and transfer of genetic material into the cell by the incorporation of nucleic acid into the virus vector and subsequent transfer into the cell via the virus vector.

[0052] Unless indicated otherwise, "efficient transduction" or "efficient tropism," or similar terms, can be determined by reference to a suitable positive or negative control (e.g., at least about 50%, 60%, 70%, 80%, 85%, 90%, 95% or more of the transduction or tropism, respectively, of a positive control or at least about 110%, 120%, 150%, 200%, 300%, 500%, 1000% or more of the transduction or tropism, respectively, of a negative control).

[0053] Similarly, it can be determined if a virus "does not efficiently transduce" or "does not have efficient tropism" for a target tissue, or similar terms, by reference to a suitable control. In some embodiments, the virus vector does not efficiently transduce (i.e., does not have efficient tropism for) liver, kidney, gonads and / or genu cells. In some embodiments, undesirable transduction of tissue(s) (e.g., liver) is 20% or less, 10% or less, 5% or less, 1% or less, 0.1% or less of the level of transduction of the desired target tissue(s) (e.g., oligodendrocytes and / or OPCs).

[0054] The terms "transdifferentiation," "transdifferentiating," and transdifferentiate" as used herein may refer to the conversion of one cell type to another cell type. The transdifferentiation can be confirmed by the loss of markers of the first cell type and gain of markers of the second cell type.

[0055] The term "differentiation factor" as used herein, generally refers to a compound, molecule, or polypeptide that promotes the differentiation of a cell from one cell type or stage to another.

[0056] The term "polypeptide" encompasses both peptides and proteins, unless indicated otherwise.

[0057] A "nucleic acid" or "nucleotide sequence" is a sequence of nucleotide bases, and may be RNA, DNA or DNA-RNA hybrid sequences (including both naturally occurring and nonAttorney Docket No. 5510-0002WO naturally occurring nucleotide), but is preferably either single or double stranded DNA sequences.

[0058] An "isolated" nucleic acid or nucleotide sequence (e.g., an "isolated DNA" or an "isolated RNA") describes a nucleic acid or nucleotide sequence separated or substantially free from at least some of the other components of the naturally occurring organism or virus, for example, the cell or viral structural components or other polypeptides or nucleic acids commonly found associated with the nucleic acid or nucleotide sequence.

[0059] Similarly, an "isolated" polypeptide describes a polypeptide that is separated or substantially free from at least some of the other components of the naturally occurring organism or virus, for example, the cell or viral structural components or other polypeptides or nucleic acids commonly found associated with the polypeptide.

[0060] A "heterologous nucleotide sequence" or "heterologous nucleic acid" is a sequence that is not naturally occurring in the virus. In some embodiments, the heterologous nucleic acid or nucleotide sequence includes an open reading frame that encodes a polypeptide and / or an untranslated RNA.

[0061] A "therapeutic polypeptide" can be a polypeptide that can alleviate or reduce symptoms that result from an absence or defect in a protein in a cell or subject. In addition, a "therapeutic polypeptide" can be a polypeptide that otherwise confers a benefit to a subject, e.g., anti-cancer effects or improvement in transplant survivability.

[0062] The term, "antisense nucleotide sequence" or "antisense oligonucleotide" generally refers to a nucleotide sequence that is complementary to a specified DNA or RNA sequence. Antisense oligonucleotides and nucleic acids that express the same can be made in accordance with conventional techniques. See, e.g., U.S. Pat. No. 5,023,243 to Tullis; U.S. Pat. No. 5,149,797 to Pederson et al. The antisense nucleotide sequence can be complementary to the entire nucleotide sequence encoding the polypeptide or a portion thereof of at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 300, or 500 contiguous bases or less than 500, 300, 200, 150, 100, 75, 50, 45, 40, 35, 30, 25, 20, 15, or 10 contiguous bases and will reduce the level of polypeptide production.

[0063] It will be appreciated that it is not necessary that an antisense nucleotide sequence be fully complementary its target sequence, so long as the degree of sequence similarity is sufficient for the antisense nucleotide sequence to hybridize to its target and reduce production of theAttorney Docket No. 5510-0002WO polypeptide. It will further be appreciated by one of skill in the art that a higher degree of sequence similarity is generally required for short antisense nucleotide sequences, whereas a greater degree of mismatched bases will be tolerated by longer antisense nucleotide sequences.

[0064] For example, hybridization of such nucleotide sequences can be carried out under conditions of reduced stringency, medium stringency or even stringent conditions (e.g., conditions represented by a wash stringency of 35-40% formamide with 5xDenhardt's solution, 0.5% SDS and 1XSSPE at 37°C.; conditions represented by a wash stringency of 40-45% formamide with 5><Denhardfs solution, 0.5% SDS, and 1XSSPE at 42°C.; and / or conditions represented by a wash stringency of 50% formamide with 5xDenhardt' s solution, 0.5% SDS and 1XSSPE at 42°C., respectively) to the nucleotide sequences specifically disclosed herein. See, e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual 2nd Ed (Cold Spring Harbor. NY, 1989).

[0065] In other embodiments, antisense nucleotide sequences of the inventive concept have at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence similarity with the complement of the coding sequences specifically disclosed herein and will reduce the level of polypeptide production.

[0066] The length of the antisense nucleotide sequence (i.e., the number of nucleotides therein) is not critical, so long as it binds selectively to the intended location and reduces transcription and / or translation of the target sequence, and the reduction can be determined in accordance with routine procedures. In general, the antisense nucleotide sequence will be from about 8, 10, 12, or 15 nucleotides in length, up to about 20, 30, 50, 75 or 100 nucleotides, or longer, in length.

[0067] An antisense nucleotide sequence can be constructed using chemical synthesis and enzymatic ligation reactions by procedures known in the art. For example, an antisense nucleotide sequence can be chemically synthesized using naturally occurring nucleotides or various modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleotide sequences, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. Examples of modified nucleotides which can be used to generate the antisense nucleotide sequence include 5 -fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine,Attorney Docket No. 5510-0002WO xanthine, 4-acetyl cytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2- thiouridine, 5-carboxymethylaminomethyl-halouracil, dihydrouracil, beta-D- galactosylqueuosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1 -methylinosine, 2,2- dimethylguanine, 2-methyladenine, 2-methylguanine, 3 -methylcytosine, 5-methylcytosine, N6- adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueuosine, 5 '-methoxy carboxymethyl-acid, 5-methoxyuracil, 2-methylthio-N6- isopentenyl adenine, uracil-5-oxyacetic acid, wybutoxosine, pseudouracil, queuosine, 2- thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5 -methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (cmo5U), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2- carboxypropyl) uracil, acp3U, and 2,6-diaminopurine. Alternatively, the antisense nucleotide sequence can be produced using an expression vector into which a nucleic acid has been cloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest).

[0068] Antisense nucleotide sequences may further include nucleotide sequences wherein at least one, some, or all, of the intemucleotide bridging phosphate residues are modified phosphates, such as methyl phosphonates, methyl phosphonothioates, phosphoromoipholidates, phosphoropiperazidates and phosphoramidates. For example, every other one of the internucleotide bridging phosphate residues can be modified as described. In another nonlimiting example, the antisense nucleotide sequence is a nucleotide sequence in which one, or all, of the nucleotides contain a 2' lower alkyl moiety (e g., C1-C4, linear or branched, saturated or unsaturated alkyl, such as methyl, ethyl, ethenyl, propyl, 1 -propenyl, 2-propenyl, and isopropyl). For example, every other one of the nucleotides can be modified as described. See also, Furdon et al. (1989) Nucleic Acids Res. 17, 9193; Agrawal at al. (1990) Proc. Natl. Acad. Sci. USA 87, 1401; Baker et al. (1990) Nucleic Acids Res. 18, 3537; Sproat et al. (1989) Nucleic Acids Res. 17, 3373; Walder and Walder (1988) Proc. Natl. Acad. Sci. USA 85, 5011; incorporated by reference herein in their entireties for their teaching of methods of making antisense molecules, including those containing modified nucleotide bases).

[0069] Antisense RNA nucleotide sequences may be classified as and are included among types of non-coding RNA (ncRNA). Typically, ncRNAs having nucleotide sequences of less than about 200 nucleotides in length may be classified as short ncRNAs, whereas ncRNAsAttorney Docket No. 5510-0002WO having nucleotide sequences of greater than about 200 nucleotides in length may be classified as long ncRNA (IncRNA) sequences.

[0070] The terms, "RNAi" or "RNA interference" generally refer to the process of sequencespecific post-transcriptional gene silencing, mediated by an "interfering RNA" which is a doublestranded RNA (dsRNA). The term, "dsRNA" refers to RNA that is partially or completely double stranded. Examples of double stranded RNA useful in RNA interference include small interfering RNA (siRNA), small interfering nucleic acid (siNA), short hairpin RNA (shRNA), microRNA (miRNA), cleavage-inducing tinyRNA (cityRNA), and the like. In the RNAi process, dsRNA, including a first (antisense) strand that is complementary to a portion of a target gene and a second (sense) strand that is fully or partially complementary to the first antisense strand, is introduced into a cell or organism.

[0071] In some embodiments, RNAi begins with the cleavage of longer dsRNAs into small interfering RNAs (siRNAs) by an RNaselll-like enzyme, dicer. SiRNAs are dsRNAs that are usually about 19 to about 28 nucleotides, or about 20 to about 25 nucleotides, or about 21 to about 22 nucleotides in length and often contain 2-nucleotide 3' overhangs, and 5' phosphate and 3 hydroxyl termini. One strand of the siRNA is incorporated into a ribonucleoprotein complex known as the RNA-induced silencing complex (RISC). RISC uses this siRNA strand to identify mRNA molecules that are at least partially complementary to the incorporated siRNA strand, then cleaves these target mRNAs or inhibits their translation. Therefore, the siRNA strand that is incorporated into RISC is known as the guide strand or the antisense strand. The other siRNA strand, known as the passenger strand or the sense strand, is eliminated from the siRNA and is at least partially homologous to the target mRNA. Those of skill in the art will recognize that, in principle, either strand of the siRNA can be incorporated into RISC and function as a guide strand. However, siRNA design (e.g., decreased siRNA duplex stability at the 5' end of the antisense strand) can favor incorporation of the antisense strand into RISC.

[0072] RISC-mediated cleavage of mRNAs having a sequence at least partially complementary to the guide strand leads to a decrease in the steady state level of that mRNA and of the corresponding protein encoded by this mRNA. Alternatively, RISC can also decrease expression of the corresponding protein via translational repression without cleavage of the target mRNA. Other RNA molecules and RNA-like molecules can also interact with RISC and silence gene expression. Examples of other RNA molecules that can interact with RISC includeAttorney Docket No. 5510-0002WO shRNAs, single-stranded siRNAs, miRNAs, cityRNAs, and dicer- substrate 27-mer duplexes. The term "interfering RNAs" may refer to a double-stranded interfering RNA unless otherwise noted. Examples of RNA and RNA-like molecules that can interact with RISC include RNA molecules containing one or more chemically modified nucleotides, one or more deoxyribonucleotides, and / or one or more non-phosphodiester linkages. All RNA or RNA-like molecules that can interact with the RISC, the RISC component protein Argonaute (AGO), and participate in RISC-mediated changes in gene expression may be referred to as "interfering RNAs." Accordingly, siRNAs, shRNAs, miRNAs, cityRNAs, and dicer-substrate 27-mer duplexes, including some of the embodiments of the inventive concept as described herein, may be considered as subsets of "interfering RNAs."

[0073] MicroRNAs (miRNAs) are non-protein coding RNAs, generally of between about 15 to about 25 nucleotides in length (commonly about 20-24 nucleotides in length in plants). These miRNAs direct cleavage in trans of target transcripts, negatively regulating the expression of genes involved in various regulation and development pathways (Bartel (2004) Cell 116, 281; Zhang et al. (2006) Dev. Biol. 289, 3).

[0074] CityRNAs are cleavage-inducing tinyRNAs (tyRNAs, typically < 17-nt guide RNAs that associate with AGOs). Certain 14-nt cityRNAs catalytically activate human Argonaute3 (AGO3) to direct cleavage of target transcripts, which AGOs utilize differently for target recognition than miRNAs, and which rely more heavily on target cleavage than miRNAs (Zhang et al. (2024) Cell Reports 43, 114806).

[0075] Genes encoding miRNAs yield primary miRNAs (termed a "pri-miRNA") of about 70 to 300 bp in length that can form imperfect stem-loop structures. A single pri-miRNA may contain from one to several miRNA precursors. In animals, pri-miRNAs may be processed in the nucleus into shorter hairpin RNAs of about 65 nt (pre-miRNAs) by the RNaselll enzyme Drosha and its cofactor DGCR8 / Pasha. The pre-miRNA may then exported to the cytoplasm, where it is further processed by another R-Naselll enzyme. Dicer, releasing a miRNA / miRNA* duplex of about 22 nt in size. Many reviews on microRNA biogenesis and function are available. For example, see, Bartel (2004) Cell 116, 281, Murchison et al. (2004) Curr. Opin. Cell Biol. 16, 223, Dugas et al. (2004) Curr. Opin. Plant Biol. 7, 512 and Kim (2005) Nature Rev. Mol. Cell Biol. 6, 376. Alternatively, it will be appreciated that miRNA biogenesis may include andAttorney Docket No. 5510-0002WO involve Dicer-independent pathways, such as the Dicer-independent pathway requiring Ago catalysis, e.g., as described by Cheloufi et al. (2010) Nature 465, 584.

[0076] It will be appreciated that an interfering RNA nucleotide sequence, such as the guide strand of an siRNA or miRNA, or a mature miRNA, may not necessarily be fully complementary to its target sequence / target mRNA sequence, so long as the degree of sequence similarity is sufficient for the interfering RNA nucleotide sequence to hybridize to its target and reduce production of the polypeptide. Moreover, it will be appreciated that it is not necessary that the guide strand of an siRNA or miRNA be fully complementary to its corresponding passenger strand, as well as other sequences that are part of / included in, e.g., pri-miRNAs, pre-miRNAs, and / or precursors to siRNA. It will further be appreciated by one of skill in the art that a higher degree of sequence similarity is generally required for short interfering RNA nucleotide sequences, whereas a greater degree of mismatched bases will be tolerated by longer interfering RNA nucleotide sequences.

[0077] In some embodiments, miRNA constructs of the inventive concept may include an interfering RNA nucleotide sequence and the complement of the interfering RNA nucleotide sequence, i.e., the construct includes both the interfering RNA nucleotide sequence and its complement. In some embodiments, miRNA constructs of the inventive concept may include an interfering RNA nucleotide sequence and a sequence that hybridizes to the interfering RNA nucleotide sequence, i.e., the sequence that hybridizes to the interfering RNA nucleotide sequence has a sufficient degree of sequence similarity to the interfering RNA sequence to permit hybridizing of the sequences to the interfering RNA nucleotide sequence. In some embodiments, the sequence that hybridizes to the interfering RNA nucleotide sequence may have about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence similarity / identity with the interfering RNA nucleotide sequence.

[0078] In some embodiments, miRNA constructs of the inventive concept may include, for example, an interfering RNA sequence incorporated / inserted into a miRNA backbone. The miRNA backbone that may be part of the vectors and constructs of the inventive concept is not particularly limited. In some embodiments, the miRNA backbone of the constructs of the inventive concept may be a miR-155 backbone. In some embodiments, the miRNA backbone of the constructs of the inventive concept may be a miR-E backbone. Nevertheless, although theseAttorney Docket No. 5510-0002WO miRNA backbones are presented herein as being exemplary, it should not be necessarily construed that the miRNA backbones are limited thereto, and the miRNA backbone may be any miRNA that may be appreciated by one of skill in the art. In some embodiments, the interfering RNA sequence incorporated / inserted into a miRNA backbone may include secondary structure similar to the secondary structure of the native miRNA backbone, e.g., include mismatches and / or deletions in the interfering RNA sequences that mimic / duplicate the secondary structure found in the native miRNA backbone.

[0079] As used herein, the terms "amount sufficient to inhibit expression" and "amount sufficient to attenuate expression" generally refer to a concentration or amount of the interfering RNA that is sufficient to reduce levels or stability of mRNA, e.g., as with siRNAs inducing mRNA degradation, or protein produced from a gene in a cell, e.g., as with miRNAs blocking mRNA translation. As used herein, "inhibiting expression" and "attenuating expression" generally refer to the absence or observable decrease in the level of protein and / or mRNA product from a gene.

[0080] As used herein, "complementary" polynucleotides are those that are capable of base pairing according to the standard Watson-Crick complementarity rules. Specifically, purines will base pair with pyrimidines to form a combination of guanine paired with cytosine (G:C) and adenine paired with either thymine (A:T) in the case of DNA, or adenine paired with uracil (A:U) in the case of RNA. For example, the sequence "A-G-T" binds to the complementary sequence "T-C-A." It is understood that two polynucleotides may hybridize to each other even if they are not completely complementary to each other, provided that each has at least one region that is substantially complementary to the other.

[0081] The terms "complementary" or “complementarity," may refer to the natural binding of polynucleotides under permissive salt and temperature conditions by base-pairing.Complementarity between two single- stranded molecules may be "partial," in which only some of the nucleotides bind, or it may be complete when total complementarity exists between the single stranded molecules. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands.

[0082] The terms "substantially complementary" or "partially complementary" can mean that two nucleic acid sequences are complementary at least at about e.g., 70%-80% of their nucleotides. In some embodiments, the two nucleic acid sequences can be complementary by atAttorney Docket No. 5510-0002WO least at about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more of their nucleotides. The terms "substantially complementary" and "partially complementary" can also mean that two nucleic acid sequences can hybridize under high stringency conditions and such conditions are well known in the art.

[0083] The terms "contacting," "introducing," "delivering," and "administering" are used interchangeably and may refer to a process by which antisense RNA or interfering RNA of the present inventive concept or a nucleic acid molecule encoding an antisense RNA or an interfering RNA of the inventive concept is delivered to a cell or a subject, in order to inhibit or alter or modify expression and / or biological activity of PTBP1 in the cell or subject. In some embodiments, the interfering RNA that modifies expression of PTBP 1 suppresses PTBP1 expression and / or activity, thereby promoting neuronal lineage conversion, e.g., conversion of non-neuronal cells, such as glial cells, astrocytes, oligodendrocytes, oligodendrocyte precursor cells, etc., into functional neurons. The antisense RNA or interfering RA may be administered in a number of ways, including, but not limited to, direct introduction into a cell (i.e., intracellularly) and / or extracellular introduction into a cavity, interstitial space, regional circulation feeding a particular organ or tissue, or into a tissue or structure (e.g., the striatum).

[0084] "Introducing" in the context of a cell or a subject can mean presenting the nucleic acid molecule to the cell or subject in such a manner that the nucleic acid molecule gains access to the interior of a cell. Where more than one nucleic acid molecule is to be introduced these nucleic acid molecules can be assembled as part of a single polynucleotide or nucleic acid construct, or as separate polynucleotide or nucleic acid constructs, and can be located on the same or different nucleic acid constructs. Accordingly, these polynucleotides can be introduced into cells in a single transformation event or in separate transformation events. Thus, the term "transformation" as used herein refers to the introduction of a heterologous nucleic acid into a cell. Transformation of a cell may be stable or transient.

[0085] Embodiments of the inventive concept are directed to expression cassettes designed to express the nucleic acids of the present inventive concept. "Expression cassette" means a nucleic acid molecule / polynucleotide sequence having at least a control sequence operably linked to a nucleotide sequence of interest. In this manner, e.g., promoters in operable interaction with the nucleotide sequences for a polynucleotide encoding a factor for inducing cellularAttorney Docket No. 5510-0002WO reprogramming, such as promoting neuronal lineage conversion, and a polynucleotide encoding a factor for enhancing tissue regeneration, for enhancing cell survival, promoting / enhancing cell development, for promoting / enhancing cell structure development / regeneration, and / or for promoting / enhancing cell / cell structure stabilization, which may include polynucleotides encoding for antisense RNAs and / or interfering RNAs, may be provided in expression cassettes for expression in a cell. In some embodiments, the cell is an oligodendrocyte, or an oligodendrocyte precursor cell (OPC). In some embodiments, the cell is a neuron, or a portion of a neuron, such as an axon. In some embodiments, the polynucleotide encoding a factor for inducing cellular reprogramming, and a polynucleotide encoding a factor for enhancing tissue regeneration, for enhancing cell survival, promoting / enhancing cell development, for promoting / enhancing cell structure development / regeneration, and / or for promoting / enhancing cell / cell structure stabilization are for promoting / enhancing axon regeneration, more particularly, for promoting / enhancing functional axon regeneration.

[0086] In some embodiments, the factor for inducing cellular reprogramming includes a sequence configured to inhibit / attenuate expression and / or biological activity of PTBP 1. In some embodiments, the factor for inducing cellular reprogramming suppresses PTBP1 expression and / or activity, thereby promoting neuronal lineage conversion. In some embodiments, the factor for inducing cellular reprogramming includes or expresses an inhibitory molecule, RNA interference construct, antisense sequence, or other agent that inhibits, silences, and / or attenuates expression and / or biological activity of PTBP 1, thereby facilitating neuronal reprogramming of non-neuronal cells (glial cells, astrocytes, oligodendrocytes, oligodendrocyte precursor cells, etc.) into functional neurons. In some embodiments, the factor for inducing cellular reprogramming includes or expresses an RNA aptamer sequence that blocks / inhibits PTBP1 biological activity. In some embodiments, the RNA aptamer sequence does not alter / significantly affect the expression / expression levels of PTBP1.

[0087] The term, "functional axon regeneration" refers to the regeneration of an axon that results in the restoration of lost axon function (e.g., movement, sensation, or autonomic control) and / or resolving / treating axon dysfunction resulting from, e.g., disease and / or injury. Functional axon regeneration may include complete and accurate rewiring of the axon to its appropriate target (e.g., muscle or sensory receptor) and re-formation of a functional synapse as part of the restoration of function. In some embodiments, functional axon regeneration further includesAttorney Docket No. 5510-0002WO restoration of normal electrophysiological properties, such as action potential propagation and synaptic transmission efficiency, confirming that regenerated axons conduct impulses properly. Functional axon regeneration / functional recovery may also include appropriate remyelination by oligodendrocytes or Schwann cells to re-establish conduction velocity and signal fidelity. Functional axon regeneration may include complete (100%) restoration of function, or may include partial (e.g., about 10% or greater, about 20% or greater, about 30% or greater, about 40% or greater, about 50% or greater, about 60% or greater, about 70% or greater, about 80% or greater, about 90% or greater, or about 95% or greater, up to about 99%) restoration of function, typically evaluated by behavioral, electrophysiological, or imaging assessments (e.g., motor coordination, evoked potentials, or connectivity mapping). In some embodiments, functional axon regeneration is mediated through activation of intrinsic neuronal growth programs (e.g., regeneration-associated genes) and / or modification of the extrinsic microenvironment (e.g., extracellular matrix remodeling or glial modulation) that promotes regrowth. In some embodiments, the factor for enhancing tissue regeneration, for enhancing cell survival, promoting / enhancing cell development, for promoting / enhancing cell structure development / regeneration, and / or for promoting / enhancing cell / cell structure stabilization may be for and / or facilitate generation / regeneration of neurons, and may include transdifferentiating non-neuronal cells, such as, but not limited to, OPCs and oligodendrocytes, into neurons as part of and / or along with functional axon regeneration. Proper axon regeneration further involves accurate and target-specific reconnection rather than maladaptive or aberrant sprouting, which may otherwise lead to spasticity, pain, or dysautonomia.

[0088] The term "promoter" refers to a region of a nucleotide sequence that incorporates the necessary signals for efficient expression of a nucleotide sequence, which may include expression of coding (e.g., mRNA for translation into a polypeptide / protein) and non-coding (e.g., non-coding RNAs) nucleotide sequences. This may include nucleotide sequences to which an RNA polymerase binds, but is not limited to such sequences, and can include regions to which other regulatory proteins bind together with regions involved in the control of protein translation, as well as include coding sequences.

[0089] Accordingly, a "promoter" of the inventive concept is capable of initiating transcription in a cell. Such promoters include those that drive expression of a nucleotide sequence constitutively, those that drive expression when induced, and those that driveAttorney Docket No. 5510-0002WO expression in a tissue- or a developmentally-specific manner, as these various types of promoters are known in the art.

[0090] For purposes of the inventive concept, regulatory regions (promoters, transcriptional regulatory regions, and translational termination regions) can be native / analogous to the cell and / or the regulator regions can be native / analogous to the other regulatory regions. Alternatively, the regulatory regions may be heterologous to the cell and / or to each other. Thus, for example, a promoter can be heterologous when it is operably linked to a polynucleotide from a species different from the species from which the polynucleotide was derived. Alternatively, a promoter can also be heterologous to a selected nucleotide sequence if the promoter is from the same / analogous species from which the polynucleotide is derived, but one or both (i.e., promoter and polynucleotide) are substantially modified from their original form and / or genomic locus, or the promoter is not the native promoter for the operably linked polynucleotide.

[0091] The choice of promoters to be used depends upon several factors, including, but not limited to, cell- or tissue-specific expression, desired expression level, efficiency, inducibility and selectability. For example, where expression in a specific tissue or organ is desired, a tissuespecific or tumor-specific promoter can be used (e.g., a breast cancer specific promoter). In contrast, where expression in response to a stimulus is desired, an inducible promoter can be used. Where continuous expression is desired throughout the cells of a subject, a constitutive promoter can be used. It is a routine matter for one of skill in the art to modulate the expression of a nucleotide sequence by appropriately selecting and positioning promoters and other regulatory regions relative to that sequence.

[0092] "Subject" as used herein may be a patient. In some embodiments, the subject is a human; however, a subject of this disclosure can include an animal subject, including avian subjects, such as, but not limited to, chickens, ducks, geese, quail, turkeys, pheasant, parrots, parakeets, and mammalian subjects such as, but not limited to, canines, felines, bovines, caprines, equines, ovines, porcines, rodents e.g. rats and mice), lagomorphs, primates (including non-human primates), etc., including domesticated animals, companion animals and wild animals for veterinary medicine, treatment or pharmaceutical drug development purposes.

[0093] The human subjects relevant to this disclosure may be male or female and may be any species and of any race or ethnicity, including, but not limited to, Caucasian, African- American,Attorney Docket No. 5510-0002WOAfrican, Asian, Hispanic, Indian, etc., and combined backgrounds. The subjects may be of any age, including newborn, neonate, infant, child, adolescent, adult, and geriatric.

[0094] In some embodiments, the subject is in need of treatment. In further embodiments, the subject is at risk for or has been diagnosed with a central nervous system (CNS) disorder or condition. The disorder or condition is responsive to an increase in the number of neurons.

[0095] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this inventive concept belongs.

[0096] In some embodiments, the inventive concept provides a pharmaceutical composition including an expression vector, virus particle and / or composition as set forth herein in a pharmaceutically acceptable carrier and, optionally, other medicinal agents, pharmaceutical agents, stabilizing agents, buffers, carriers, adjuvants, diluents, etc. For injection, the carrier will typically be a liquid. For other methods of administration, the carrier may be either solid or liquid. For inhalation administration, the carrier will be respirable, and may be in solid or liquid particulate form. In some embodiments, the expression vector, virus particle and / or composition as set forth herein may be embedded in a thin film / fdm matrix suitable for buccal and / or sublingual administration, or reconstitution of the expression vector, virus particle and / or composition from the thin film / film matrix for administration.Viral vectors

[0097] Embodiments of the present inventive concept relate to recombinant viral vectors, such as recombinant adeno-associated (rAAV) vectors, for use in cellular reprogramming in a subject, such as in the reprogramming of astrocytes / OPCs / oligodendrocytes / glial cells into functional neurons. The recombinant viral vectors of the inventive concept may include, for example, a vector genome including an expression cassette or expression cassettes, i.e., a component of a recombinant vector or a recombinant vector genome including a gene and regulatory sequence, for 1) a factor, at least one factor, or more than one factors, for enhancing tissue regeneration, for enhancing cell survival and / or cell replenishment, e.g., enhancing neuron survival and / or replenish neurons depleted as a result of neuron loss, for promoting / enhancing cell development, for promoting / enhancing cell structure development / regeneration, e.g., for promoting / enhancing functional axon regeneration, or for promoting / enhancing cell / cellAttorney Docket No. 5510-0002WO structure stabilization, and / or 2) a factor, at least one factor, or more than one factors, for inducing cellular reprogramming, e.g., a factor for transdifferentiating oligodendrocytes and / or OPCs into neurons, either alone or in combination, to be expressed by a transduced cell, including an inverted terminal repeat (ITR), a self-complementary inverted terminal repeat (scITR), or a mutant inverted terminal repeat (mITR), e.g., as found in and for generating self- complementary AAV (scAAV) vectors, e.g., as described in McCarty et al. (2003) Gene Ther. 10:2112-2118, Wang et al. (2003) Gene Ther. 10:2105-2111, and Wu et al. (2007) Hum. Gene Ther. 18: 171-182, upstream and downstream (5' and 3') to the expression cassette(s) of the vector genome. In some embodiments, the recombinant vector genome may include sequences for generating covalently closed ends, e.g., a TelN recognition sequence, in place of the mITR typically found in scAAV vectors, to provide and for generating covalently closed-end double stranded AAV (cceAAV) vectors, e.g., as described in Zhang et al. (2024) Mol. Ther. Methods Clin. Dev. 32: 101206. In some embodiments, expression of the factor for enhancing tissue regeneration, for enhancing cell survival and / or replenishment, such as neuron survival and / or replenish neurons depleted as a result of neuron loss, for promoting / enhancing cell development, for promoting / enhancing cell structure development / regeneration, such as promoting / enhancing functional axon regeneration, and / or for promoting / enhancing cell / cell structure stabilization, and the factor for inducing cellular reprogramming, such as a factor that promotes PTBP1 knockdown, may be included within a single expression cassette / vector. In some embodiments, expression of the factor for enhancing tissue regeneration, for enhancing cell survival and / or replenishment, such as neuron survival and / or replenish neurons , for promoting / enhancing cell development, for promoting / enhancing cell structure development / regeneration, such as promoting / enhancing functional axon regeneration, and / or promoting / enhancing cell / cell structure stabilization, and the factor for inducing cellular reprogramming, such as a factor that promotes PTBP1 knockdown, may be included within separate expression cassette s / vectors. Expression cassettes may include a promoter sequence, and the open reading frame for the factor for enhancing tissue regeneration, for enhancing cell survival and / or replenishment, e.g., neuron survival and / or replenishment, and / or for promoting / enhancing cell development, for promoting / enhancing cell structure development / regeneration, such as promoting / enhancing functional axon regeneration, for promoting / enhancing cell / cell structure stabilization, and / or the factor for inducing cellular reprogramming. The expression cassette may further include a 3'Attorney Docket No. 5510-0002WO untranslated region that, in eukaryotes, typically contains a polyadenylation site and / or a transcription terminator. In some embodiments, the expression cassette or cassettes may further include introns and / or splicing elements (IS) between the promoter and the open reading frame and / or between the open reading frame and the 3' untranslated region. In some embodiments, the 3' UTR of the expression cassette may include sequences encoding for an interfering RNA (miRNA, siRNA, etc.) and / or sequences suitable for and take part in interfering RNA biogenesis. In some embodiments, wherein the factor for enhancing tissue regeneration, for enhancing cell survival, promoting / enhancing cell development, for promoting / enhancing cell structure development / regeneration, and / or for promoting / enhancing cell / structure stabilization, and the factor for inducing cellular reprogramming are included within separate expression cassettes / vectors, the separate expression cassettes / vectors, i.e., the expression cassette / vector including the factor for enhancing tissue regeneration, for enhancing cell survival and / or replenishment, promoting / enhancing cell development, for promoting / enhancing cell structure development / regeneration, and / or for promoting / enhancing cell / cell structure stabilization, and the expression cassette including the factor for inducing cellular reprogramming, may each be considered individually as embodiments of the inventive concept, that may be used independently, or in combination, in the methods of the inventive concept without departing from the scope of the inventive concept.

[0098] According to some embodiments, the promoter / promoter sequence of the expression cassette may be a natural promoter or a synthetically constructed promoter without departing from the scope of the inventive concept. The promoter may be ubiquitous, constitutive, cell typespecific, tissue-specific, and / or inducible. In some embodiments, the promoter / promoter sequence of the expression cassette may be a ubiquitous and / or a constitutive promoter, e.g., RNA polymerase-II (Pol-II) promoter leT or RNA polymerase-III (Pol-III) promoter 7SK, U6, or Hl. In some embodiments, the promoter / promoters may be an RNA Pol-II promoter, an RNA Pol-III promoter, or a combination of Pol-II and Pol-III promoters / promoter systems. In some embodiments, the promoter / promoter sequence supports expression in oligodendrocytes, astrocytes, neurons, and / or in glial progenitor cells. In some embodiments, the promoter / promoter sequence of the expression cassette may be a cell type-specific promoter, e.g., the myelin basic protein (MBP) promoter, or the myelin associated glycoprotein (MAG) promoter (see, e.g., Jonquieres et al. (2016) Front. Mol. Neurosci. 9, 13). In some embodiments,Attorney Docket No. 5510-0002WO the constitutive promoter is, e.g., the cytomegalovirus promoter and the SV40-derived initial promoter, and mammalian constitutive protein gene promoters such as the chicken 0-actin promoter, synthetic variants thereof, and truncated variants of the chicken -actin promoter (see, e.g., the CBA promoter, and the truncated constitutive chicken P-actin hybrid (CBh) promoter described by Gray et al. (2011) Hum. Gene Ther. 22, 1143 and Powell et al. (2020) Mol. Ther. (28(5), 1373-1380), or the synthetic Jetl promoter, the hybrid chicken P-actin (CAG) promoter, and the human synapsin (hSyn) promoter described by Bohlen et al. (2020) Hum. Gene Ther. 31(21-22), 1155-1168. In some embodiments, the promoter, and transgene expression therefrom, is influenced by capsid-promoter interactions (see, Powell et al. (2020) Mol. Ther. (28(5), 1373— 1380 and Bohlen et al. (2020 Hum. Gene Ther. 31(21-22), 1155-1168). In some embodiments, transgene expression may be influenced by capsid-mediated histone H3 chemical modifications related to active transcription and accumulation of epigenetic marks on vector chromatin, e.g., as described by Gonzalez-Sandoval et al. (2023) Nat. Comm. 14, 2448.

[0099] In some embodiments, tissue-specific or tumor-specific promoters can be used. Tissue-specific nucleic acids include, but are not limited to, B29 (B cells), CD14 (monocytic cells), CD43 (leukocytes and platelets), CD45 (hematopoietic cells), CD68 (macrophages), desmin (muscle), elastase-1 (pancreatic acinar cells), endoglin (endothelial cells), fibronectin (differentiating cells and healing tissues), FLT-1 (endothelial cells), GFAP (astrocytes), GPIIb (megakaryocytes), ICAM-2 (endothelial cells), INF-f (hematopoietic cells), Mb (muscle), NPHSI (podocytes), OG-2 (osteoblasts, SP-B (lungs), SYN1 (neurons), and WASP (hematopoietic cells). Some reported tumor-specific nucleic acids and promoters include, without limitation, AFP (hepatocellular carcinoma), CCKAR (pancreatic cancer), CEA (epithelial cancer), c-erbB2 (breast and pancreatic cancer). COX-2, CXCR4, E2F-1, HE4, LP, MUC1 (carcinoma), PRC1 (breast cancer), PSA (prostate cancer), RRM2 (breast cancer), survivin, TRPl (melanoma), and TYR (melanoma).

[0100] In some embodiments, inducible promoters can be used. Examples of inducible promoters include, but are not limited to, tetracycline repressor system promoters, Lac repressor system promoters, copper-inducible system promoters, salicylate-inducible system promoters (e.g., the PRI a system), glucocorticoid-inducible promoters, and ecdysone-inducible system promoters.Attorney Docket No. 5510-0002WO

[0101] As used herein, "regulatory sequences" may be nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences include, but are not limited to, enhancers, introns, translation leader sequences, non-translated leader sequences, and polyadenylation signal sequences.

[0102] The expression cassette also can optionally include a transcriptional and / or translational termination region (i.e., termination region) that is functional in the cell. A variety of transcriptional terminators are available for use in expression cassettes and are responsible for the termination of transcription beyond the transgene and correct mRNA polyadenylation. The termination region may be native to the transcriptional initiation region, may be native to the operably linked nucleotide sequence of interest, may be native to the host, or may be derived from another source (i.e., foreign or heterologous to the promoter, the nucleotide sequence of interest, the host, or any combination thereof). In addition, a native transcription terminator for a coding sequence can be used.

[0103] A signal sequence can be operably linked to nucleic acids of the present invention to direct the nucleotide sequence into a cellular compartment. In this manner, the expression cassette includes a nucleotide sequence encoding the interfering RNA operably linked to a nucleic acid sequence for the signal sequence. The signal sequence may be operably linked at the N- or C-terminus of the interfering RNA.

[0104] Regardless of the type of regulatory sequence(s) used, they can be operably linked to the nucleotide sequence of the antisense RNA or interfering RNA. "Operably linked" can mean that elements of a nucleic acid construct such as an expression cassette are configured to be able to perform their usual function. Thus, regulatory or control sequences (e.g., promoters) operably linked to a nucleotide sequence of interest are capable of effecting expression of the nucleotide sequence of interest. The control sequences need not be contiguous with the nucleotide sequence of interest, so long as they function to direct the expression thereof. Thus, for example, intervening untranslated, yet transcribed, sequences can be present between a promoter and a coding sequence, and the promoter sequence can still be considered "operably linked" to the coding sequence. A nucleotide sequence of the present invention (i.e., an antisense RNA orAttorney Docket No. 5510-0002WG interfering RNA) can be operably linked to a regulatory sequence, thereby allowing its expression in a cell and / or subject.

[0105] The expression cassette also can include a nucleotide sequence for a selectable marker, which can be used to select a transformed cell or subject. As used herein, "selectable marker" means a nucleic acid that when expressed imparts a distinct phenotype to the cell or subject expressing the marker and thus allows such transformed cells or subjects to be distinguished from those that do not have the marker. Such a nucleic acid may encode either a selectable or screenable marker, depending on whether the marker confers a trait that can be selected for by chemical means, such as by using a selective agent (e.g. an antibiotic or the like), or on whether the marker is simply a trait that one can identify through observation or testing, such as by screening. Of course, many examples of suitable selectable markers are known in the art and can be used in the expression cassettes described herein.

[0106] Examples of selectable markers include, but are not limited to, a nucleic acid encoding neo or nptll, which confers resistance to kanamycin, G418, and the like (Potrykus e al. (1985) Mol. Gen. Genet. 199, 183); a nucleic acid encoding a nitrilase such as bxn from Klebsiella ozcaenae that confers resistance to bromoxynil (Stalker et al. (1998) Science 242, 419); a nucleic acid encoding an altered acetolactate synthase (ALS) that confers resistance to imidazolinone, sulfonylurea or other ALS-inhibiting chemicals (EP Patent Application No. 154204); a nucleic acid encoding a methotrexate-resistant dihydrofolate reductase (DHFR) (Thillet et al. (1988) J. Biol. Chet. 263, 12500); a nucleic acid encoding a dalapon dehalogenase that confers resistance to dalapon; a nucleic acid encoding a mannose-6-phosphate isomerase (also referred to as phosphomannose isomerase (PMI) that confers an ability to metabolize mannose (U.S. Pat. Nos. 5,767,378 and 5,994,629); a nucleic acid encoding an altered anthranilate synthase that confers resistance to 5-methyl tryptophan; and / or a nucleic acid encoding hph that confers resistance to hygromycin. One of skill in the art is capable of choosing a suitable selectable marker for use in an expression cassette.

[0107] Additional selectable markers include, but are not limited to, a nucleic acid encoding P-glucuronidase or uidA (GUS) that encodes an enzyme for which various chromogenic substrates are known; a nucleic acid encoding P-lactamase, an enzyme for which various chromogenic substrates are known (e.g., PADAC, a chromogenic cephalosporin) (Sutcliffe (1978) Proc. Natl. Acad. Sci. USA 75, 3737); a nucleic acid encoding xylE that encodes aAttorney Docket No. 5510-0002WG catechol dioxygenase (Zukowsky et al. (1983) Proc. Natl. Acad. Sci. USA 80, 1101); a nucleic acid encoding tyrosinase, an enzyme capable of oxidizing tyrosine to DOPA and dopaquinone, which in turn condenses to form melanin (Katz et al. (1983) J. Gen. Microbiol. 729, 2703); a nucleic acid encoding i-galactosidase, an enzyme for which there are chromogenic substrates; a nucleic acid encoding luciferase (lux) that allows for bioluminescence detection (Ow et al. (1986) Science 234, 856); a nucleic acid encoding aequorin which may be employed in calciumsensitive bioluminescence detection (Prasher et al. (1985) Biochemn. Biophys. Res. Comm. 126, 1259); or a nucleic acid encoding green fluorescent protein (Niedz et al. (1995) Plant Cell Reports 14, 403). One of skill in the art is capable of choosing a suitable selectable marker for use in an expression cassette.

[0108] According to some embodiments, the factor for enhancing tissue regeneration, for enhancing cell function, for enhancing cell survival, e.g., neuron survival, for promoting / enhancing cell structure development / regeneration, e.g., promoting / enhancing functional axon regeneration, and / or for promoting / enhancing cell / cell structure stabilization that is encoded for by polynucleotides that may be included as part of the vectors and expression cassettes of the inventive concept, may be a neurotrophic factor (NTF). NTFs include, for example: "classic" neurotrophins, e.g., nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4); glial-derived neurotrophic factor (GDNF) family neurotrophins, e.g., GDNF, neurturin (NRTN), artemin (ARTN), and persephin (PSPN); and neuroprotective cytokines, e.g. EGF, IGF-1, FGF, HGF, EPO, VEGF, CNTF, and TNF. Other NTFs include neurotrophin-6 and neurotrophin-7.

[0109] In some embodiments, the neurotrophic factor can promote survival and differentiation of neurons. In some embodiments, the neurotrophic factor can prevent apoptosis of neurons, for example, apoptosis of neurons induced by axotomy. In some embodiments, the neurotrophic factor can promote and support survival of both dopaminergic and motor neurons. In some embodiments, the neurotrophic factor can decrease the overall loss of neurons, including loss of neurons during development, can rescue cells from axotomy-induced cell death, and / or can prevent degeneration, including chronic degeneration, of neurons. In some embodiments, the neurotrophic factor is glial cell line-derived neurotrophic factor (GDNF), and the factor for enhancing tissue regeneration, for enhancing neuron survival, and / or for promoting / enhancing functional axon regeneration included as part of the vectors and expression cassettes of theAttorney Docket No. 5510-0002WO inventive concept may include an GDNF open reading frame (ORF) encoding a mature GDNF peptide / protein. In some embodiments, the factor for enhancing tissue regeneration, for enhancing neuron survival, and / or for promoting / enhancing functional axon regeneration. In some embodiments, the factor for enhancing tissue regeneration, for enhancing neuron survival, and / or for promoting / enhancing functional axon regeneration may be IGF-1. In some embodiments, the factor for enhancing tissue regeneration, for enhancing neuron survival, and / or for promoting / enhancing functional axon regeneration may be neuroprotective peptide, or a peptide that mimics natural regulatory peptides and hormones, e.g., but not limited to, neuroprotective peptides discussed and described by Dergunova et al. (2023) Genes (Basel) 14(5), 953, incorporated herein by reference.

[0110] An embodiment of the mature form of human GDNF is a 134 amino acid protein (e g., amino acids 78-211 ofNCBI Ref. Seq. NP_000505.1, UniProt No. P39905-1) that is expressed as and derived from a 211 amino acid precursor, pro-GDNF (e.g., NCBI Ref. Seq. NP_000505.1 / UniProt No. P39905-1). Accordingly, in some embodiments, the factor for enhancing tissue regeneration, for enhancing neuron survival, and / or for promoting / enhancing functional axon regeneration included as part of the vectors and expression cassettes of the inventive concept may include a pro-GDNF ORF. The precursor pro-GDNF is a preproprotein that includes signal / secretion pre-sequences (e.g., amino acids 1-19 of NCBI Ref. Seq. NP_000505.1 / UniProt No. P39905-1) for guiding secretion of GDNF, and pro-sequences (e.g., amino acid sequences 20-77 of NCBI Ref. Seq. NP_000505.1 / UniProt No. P39905-1) to ensure correct folding of GDNF, that are post-translationally removed after secretion / folding of the polypeptide to produce the mature form of GDNF.[OHl] It will be appreciated by one of skill in the art that engineered versions of pro-GDNF, wherein the wild-type signal / secretion pre-sequences (MKLWDVVAVC LVLLHTASA, SEQ ID NO: 1) may be replaced with a heterologous signal / secretion sequence, e.g., human IL-2 signal / secretion sequences (MYRMQLLSCI ALSLALVTNS, SEQ ID NO:2) or other secretion signals known in the art, e.g., a signal peptide / secretion signal as set forth at <www.signalpeptide.de>. Moreover, in some embodiments, FLAG (DYKDDDK, SEQ ID NO:3) or His (HHHHHH, SEQ ID NO:4) tags may be inserted / included between the C-terminal of the pro-sequences (F PLPAGKRPPE APAEDRSLGR RRAPFALSSD SNMPEDYPDQ FDDVMDFIQA TIKRLKR, SEQ ID NO:5) and the N-terminal of the mature GDNF wild-typeAttorney Docket No. 5510-0002WO protein sequences to produce FLAG- or His-tagged GDNF proteins that may be used in the alternative to the mature wild-type protein without departing from the scope of the inventive concept. In some embodiments, the mature GDNF protein may have at least about 80%, 85%, 90%, 95%, 96%, 97% 98%, 99% or 100% sequence identity to amino acids 78-211 of NCBI Ref. Seq. NP_000505.1 / UniProt No. P39905-1. In some embodiments, the mature GDNF protein may have 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids added to the N- terminal of the wild-type mature GDNF protein, e.g., FLAG or His tags as described above.

[0112] Although the exemplary GDNF peptide / protein sequences described herein relate to a human GDNF, other GDNF peptides / proteins, including, for example, other human GDNF peptide / protein variants, e.g., splice variants of human pro-GDNF, (e.g., UniProt Nos. P39905-2, P39905-3, P39905-4, and P39905-5), other mammalian GDNF / pro-GDNF peptides / proteins, and even other non-mammalian GDNF / pro-GDNF peptides / proteins, for use, for example, in other mammalian and other non-mammalian subjects, respectively, all of which may include modifications as described herein in relation to the exemplary human GDNF / pro-GDNF peptide / protein presented above are envisioned, and may be included in the vectors and expression cassettes of the inventive concept described herein, without departing from the scope of the inventive concept. In some embodiments, the pro-GDNF ORF of the vectors and expression cassettes of the inventive concept includes the sequence of UniProt No. P39905-2, including a pro-sequence of 32 amino acids (F PLPAANMPED YPDQFDDVMDFIQATIKRLK R, SEQ ID NO:6), rather than the 58 amino acid pro-sequence (SEQ ID NO:5) of UniProt No. P39905-1.

[0113] According to some embodiments, the factor for inducing cellular reprogramming, that is encoded for by polynucleotides that may be included as part of the vectors and expression cassettes of the inventive concept, may facilitate the cellular reprogramming / conversion of, for example, glial cells / oligodendrocytes, such as resident striatal oligodendrocytes, into functional neurons, such as, for example, expression inhibitors / attenuators of polypyrimidine tract-binding protein 1 (PTBP1, or PTB). Expression inhibitors / attenuators of PTB (anti-PTBPl) may include, in some embodiments, for example, antisense RNA, interfering RNA, and ncRNA(s) that target a polynucleotide / RNA encoding PTBP1. In some embodiments, the anti-PTBPl may include linear and / or circular RNAs, such as short hairpin RNA(s) (shRNA) against PTBP1, small interfering RNA(s) (siRNA) against PTBP1, microRNA(s) (miRNA) against PTBP1, and / or longAttorney Docket No. 5510-0002WO noncoding RNA(s) (IncRNA) against PTBP1 . Tn some embodiments, the anti-PTBPl may include pri-miRNA backbones to target PTBP1 RNA, such as a Dicer-dependent backbone, e.g., miR-155, or a Dicer-independent backbone e.g., miR-451. In some embodiments, the anti- PTBPl may be included in an expression cassette including a polynucleotide encoding an antisense RNA or an interfering RNA, or any RNA including RNAs as described above. In some embodiments, the anti-PTBPl may be targeted to a polynucleotide encoding a mammalian polypyrimidine tract binding protein 1 (PTBP1). Examples of mammalian PTBP1 include, but are not limited to rat, mouse, monkey, and human PTBP1. In some embodiments, the mammalian PTBP1 is a human PTBP1. In some embodiments, the anti-PTBPl may be a humanized siRNA or miRNA, i.e., the polynucleotide sequence of the anti-PTBPl RNA has been altered / modified / mutated to correspond to the human PTBP1 sequence. In some embodiments, the expression cassette of the inventive concept may be an AAV / recombinant AAV vector.

[0114] Accordingly, in some embodiments of the inventive concept, the expression cassette can include an expression control sequence operatively linked to a nucleotide sequence that is a template for one or both strands of an interfering RNA, e.g., an anti-PTBPl interfering RNA. The interfering RNA template may include (a) a first (antisense) stand having a sequence complementary to from about 15 to about 30 consecutive nucleotides of the nucleotide sequence of PTBP 1; and (b) a second (sense) strand having a nucleotide sequence fully complementary or substantially complementary to the first strand. In some embodiments, a promoter can flank either end of the template nucleotide sequence, wherein the promoters drive expression of each individual DNA strand, thereby generating two complementary (or substantially complementary) RNAs that hybridize and form the interfering RNA. Alternatively, the nucleotide sequence is transcribed into both strands of the interfering RNA on one transcription unit, wherein the sense strand is transcribed from the 5' end of the transcription unit and the antisense strand is transcribed from the 3' end, wherein the two strands are separated by about 3 to about 500 basepairs, and wherein after transcription, the RNA transcript folds on itself to form a shRNA molecule.

[0115] Examples of anti-PTBPl RNAs include, but are not limited to, the polynucleotide sequences of SEQ ID NOS: 1-4 in U.S. Patent Application Publication Nos. 2019 / 0224256 and 2022 / 0288141, incorporated herein by reference in their entireties, or a nucleotide sequence atAttorney Docket No. 5510-0002WO least 75%-80% identical thereto, e.g., at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto. In some embodiments, the sequence of an anti -PTBP 1 RNA may be, for example, CCAACACUAU GGUUAACUA (SEQ ID NO:7), or CCAACACCAU GGUGAACUA (SEQ ID NO: 8), anti -PTBP 1 siRNA4, and humanized anti-PTBPl siRNA4 (siRNA4_Hu), respectively, as set forth in FIG. 6, or an RNA sequence having at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or 100% sequence identity to CCAACACUAU GGUUAACUA (SEQ ID NO:7), or CCAACACCAU GGUGAACUA (SEQ ID NO: 8). Moreover, although the anti-PTBPl as described above for cellular reprogramming generally relate to anti-PTBPl targeting human PTBP1, other anti-PTBPl that target mammalian PTBP1, for example, for use in mammalian subjects, are envisioned, without departing from the scope of the inventive concept. In some embodiments, for example, a particular non-human anti- PTBPl will be selected for a particular non-human subject in which the compositions and methods of the inventive concept may be used.

[0116] In some embodiments, the polynucleotide or polynucleotides of the inventive concept is operably linked to a promoter. In some embodiments, the promoter is a constitutive promoter. A constitutive promoter is advantageous in the present inventive concept as it ensures that the antisense RNA or interfering RNA continues to be expressed as the oligodendrocyte or oligodendrocyte precursor cell transdifferentiates to a neuron. Alternatively, a cell-type specific promoter may be used, e.g., an oligodendrocyte-specific promoter.

[0117] Another aspect of the inventive concept relates to a virus particle including the expression cassette of the inventive concept. The virus particle packages (i.e., encapsidates) a vector genome, such as, but not limited to, an AAV vector genome. In some embodiments, the inventive concept provides an AAV particle including an AAV capsid, wherein the AAV capsid packages an AAV vector genome.

[0118] In some embodiments, the virus particle has a tropism for oligodendrocytes and / or OPCs. Examples of virus particles with this tropism are known in the art. In some embodiments, the virus particle with a tropism for oligodendrocytes and / or OPCs is an AAV particle, e.g., an AAV particle having a modified capsid protein that provides the tropism, e.g., is one described inAttorney Docket No. 5510-0002WOU.S. Publication No. 2015 / 0238550 or International Publication No. WO 2016 / 08181 1, incorporated by reference herein in their entirety.

[0119] In some embodiments, the factor for inducing cellular reprogramming may include inhibitors / attenuators of PTBP1 with an ability to inhibit the PTBP1 polypyrimidine tract targeting / binding process and / or formation of a complex between PTBP1 and its target. Inhibition of the targeting / binding process with its target RNA may include, for example: competition with target RNA for PTBP1 binding sites; binding induced PTBP1 protein conformational changes that inhibit binding or PTBP1 with its target RNA; and / or blocking of interactions of PTBP 1 with additional factors relevant / necessary for PTBP1 complex formation with its target RNA, stability, and / or function. Inhibitors / attenuators of PTBP 1 may include, for example: polypeptides (e.g., peptides, microproteins, and / or proteins) derived from, e.g., phage display, yeast display, RNA display, cell display, fragments of proteins that interact / bind to PTBP1, structural bioinformatics, docking algorithms, intrabodies (e.g., VHHs) from protein immunization in animals, and / or synthetic and / or semi-synthetic libraries, etc.; RNA aptamers e.g., expressed by pol III and / or pol II promoters in linear and / or circular configurations; and / or small molecules that inhibit PTBP1 binding with its target, etc., or any combination thereof (e.g., peptide + aptamer), and may include natural and / or non-natural amino acids, chemical modifications, natural and / or non-natural nucleotides, etc., or a multiple (more than one) of any one thereof (e.g., aptamer_l + aptamer_2).

[0120] In some embodiments, the factor for inducing cellular reprogramming may include inhibitors / attenuators of PTBP1 release from its complex with its target RNA / polypyrimidine tract targeting / binding sequence, wherein the target is trapped in the complex with PTBP1. These factors may include inhibitors / attenuators that inhibit conformational changes required for target RNA release, and / or block interactions with factors required for target RNA release. Inhibitors / attenuators of PTBP1 release of target RNAs may include, for example: polypeptides (e.g., peptides, microproteins, and / or proteins) derived from, e.g., phage display, yeast display, RNA display, cell display, fragments of proteins that interact / bind to PTBP, structural bioinformatics, docking algorithms, intrabodies (e.g., VHHs) from protein immunization in animals, and / or synthetic and / or semi-synthetic libraries, etc.; RNA aptamers e.g., expressed by pol III and / or pol II promoters in linear and / or circular configurations; and / or small molecules that inhibit release of target RNA from PTBP1, etc., or any combination thereof (e.g., peptide +Attorney Docket No. 5510-0002WO aptamer), and may include natural and / or non-natural amino acids, chemical modifications, natural and / or non-natural nucleotides, etc., or a multiple (more than one) of any one thereof (e.g., aptamer 1 + aptamer 2).

[0121] In some embodiments, the factor for inducing cellular reprogramming may include enhancers of PTBP1 degradation. For example, the factor may be an "anchor" with a signal(s) for rapid intracellular proteolysis (e.g., a PEST signal) and / or a signal(s) for binding-induced protein conformational changes for (fast) protein degradation. Enhancers of PTBP 1 degradation may include, for example: polypeptides (e.g., peptides, microproteins, and / or proteins) derived from, e.g., phage display, yeast display, RNA display, cell display, fragments of proteins that interact / bind to PTBP1, structural bioinformatics, docking algorithms, intrabodies (e.g., VHHs) from protein immunization in animals, and / or synthetic and / or semi-synthetic libraries, etc.; RNA aptamers e.g., expressed by pol III and / or pol II promoters in linear and / or circular configurations, hybrid / double-aptamer(s), e.g., anti-PTBPl + anti-Ub ligase; and / or small molecules that enhance PTBP1 degradation, e.g., PROTAC technology, etc., or any combination thereof (e.g., peptide + aptamer), and may include natural and / or non-natural amino acids, chemical modifications, natural and / or non-natural nucleotides, etc., or a multiple (more than one) of any one thereof (e.g., aptamer_l + aptamer_2).

[0122] In some embodiments, the factor for inducing cellular reprogramming may include enhancers / inducers of PTBP 1 posttranslational modifications (PTM), e.g., ubiquitination, that induce PTBP1 degradation and / or inactivation, enhancers of PTBP1 posttranslational modifications that induce PTBP1 degradation and / or inactivation may include, for example: polypeptides (e.g., peptides, microproteins, and / or proteins) derived from, e.g., phage display, yeast display, RNA display, cell display, fragments of proteins that interact / bind to PTBP, structural bioinformatics, docking algorithms, intrabodies (e.g., VHHs) from protein immunization in animals, and / or synthetic and / or semi-synthetic libraries, etc.; RNA aptamers e.g., expressed by pol III and / or pol II promoters in linear and / or circular configurations, hybrid / double-aptamer(s), e.g., anti-PTBPl + anti-Ub ligase; and / or small molecules that induce posttranslational modifications that enhance PTBP1 degradation, e.g., PROTAC technology, etc., or any combination thereof (e.g., peptide + aptamer), and may include natural and / or non-natural amino acids, chemical modifications, natural and / or non-natural nucleotides, etc., or a multiple (more than one) of any one thereof (e.g., aptamer l + aptamer_2). In some embodiments, theAttorney Docket No. 5510-0002WO enhancers of PTBP1 posttranslational modification that induce PTBP1 degradation and / or inactivation may include, e.g., a IncRNA, or an engineered artificial IncRNA (alncRNA), that may be involved in regulating protein stability / degradation via the ubiquintin-proteasome system, e.g., an engineered alncRNA that targets PTBP1 for ubiquintination and degradation via the ubiquintin-proteasome system. Engineering of alncRNAs for targeted protein degradation has been described, e.g., by Cao et al. Nat. Chem. Biol. (2024) https: / / doi.org / 10.1038 / s41589-024- 01719-w.

[0123] In some embodiments, the factor for inducing cellular reprogramming may include suppressors / repressors of PTBP 1 transcription, and / or chromatin condensers of the region of the PTBP1 gene. Transcriptional repressors may include TALE transcriptional repressors or other Zn finger transcriptional repressors (see, e.g., Kim and Kini (2017) Mol. Cells 40(8), 533-541, Moore et al. (2014) ACS Synth. Biol. 2(10), 708-716), any one of the CAS (e g., Cas9, Casl4, etc.) transcriptional repressors, synthetic transcription factor repressors, and / or small molecules that target specific DNA sequences (see, e.g., Nature (1997) 387(6629), 202-205). In some embodiments, the transcription repressor may be an endonuclease deficient Cas9 (dCas9) associated with / fused with a Kruppel associated box (KRAB) domain and / or a dCas9 associated with / fused with a KRAB-MeCP2 combo domain.

[0124] In some embodiments, the factor for inducing cellular reprogramming may include PTBP1 function inhibitors. Inhibitors of PTBP1 function / action, including inhibitors of function / action of PTBP 1 in association with its target RNA sequence(s) may include, for example: polypeptides (e.g., peptides, microproteins, and / or proteins) derived from, e.g., phage display, yeast display, RNA display, cell display, fragments of proteins that interact / bind to PTBP, structural bioinformatics, docking algorithms, intrabodies (e.g., VHHs) from protein immunization in animals, and / or synthetic and / or semi-synthetic libraries, etc.; RNA aptamers e.g., expressed by pol III and / or pol II promoters in linear and / or circular configurations; and / or small molecules that inhibit PTBP1 binding with its target, etc., or any combination thereof (e.g., peptide + aptamer), or a multiple (more than one) of any one thereof (e.g., aptamer_l + aptamer_2).

[0125] In some embodiments, the factor for inducing cellular reprogramming may include factors capable of targeting PTBP1 RNA(s) that: increase the degradation of PTBP 1 RNA(s) (e.g., by methylation); prevent PTBP1 mRNA translation (e.g., by condensation); and / or preventAttorney Docket No. 5510-0002WOPTBP1 mRNA biogenesis (Cap, Splicing, polyadenylation, etc.). In some embodiments, these factors include translation inhibitors, splicing modulators, protein-binding inhibitors, competitive inhibitors that inhibit mRNA translation, and factors that target PTBP1 RNA(s) for degradation. These factors may include, for example: polypeptides (e.g., peptides, microproteins, and / or proteins) derived from, e.g., phage display, yeast display, RNA display, cell display, fragments of proteins that interact / bind to PTBP1, structural bioinformatics, docking algorithms, intrabodies (e.g., VHHs) from protein immunization in animals, and / or synthetic and / or semisynthetic libraries, etc., including hybrid / double-target polypeptides / fusions; RNA aptamers e.g., expressed by pol III and / or pol II promoters in linear and / or circular configurations, including hybrid / double-aptamers (e.g., anti-PTBPl + anti-RNAse); and / or small molecules that target and increase degradation of PTBP 1 RNA(s), etc., including hybrid / double-target small molecules and / or a combination (e.g., small molecule + aptamer(s)), and may include natural and / or nonnatural amino acids, chemical modifications, natural and / or non-natural nucleotides, etc., or including any combination (polypeptide(s), RNA aptamer(s), small molecule(s)) thereof, or a multiple (more than one) of any one thereof (e.g., aptamer l + aptamer_2), etc.

[0126] It will be appreciated that peptides / proteins according to embodiments of the inventive concept described herein include, but are not limited to, natural amino acids, nonnatural amino acids, and / or chemically modified amino acids, etc. It will be also appreciated that nucleic acids / nucleotide sequences / oligonucleotide sequences according to embodiments of the inventive concept may include, but are not limited to, natural nucleobases, non-natural nucleobases, and / or modified nucleobases, etc. In some embodiments, the nucleic acid may be made up of deoxyribonucleic acids (DNA). In some embodiments, the nucleic acid is a DNA sequence. In some embodiments, the nucleic acid, or a part / portion thereof, includes modified DNA bases. Modification of DNA bases are known in the art and can include chemically modified bases including labels.

[0127] In some embodiments, the nucleic acid, or a part / portion thereof, includes ribonucleic acid (RNA) sequences or modified ribonucleotide bases. Modification of RNA bases are known in the art and can include chemically modified bases including labels. In still other embodiments, different portions of nucleic acid / oligonucleotide sequence can include DNA and RNA, modified bases / nucleobases, or modified nucleic acid base / nucleotide connections, such as, for example, phosphorothioate DNA, peptide nucleic acids (PNAs) and locked nucleic acids (LNAs). In someAttorney Docket No. 5510-0002WO embodiments, the nucleic acid / oligonucleotide includes phosphorothioate modifications, for example, such as phosphorothioate DNA, at some, most, or all nucleic acid base / nucleotide connections / linkages of the nucleic acid / oligonucleotide.

[0128] According to some embodiments, the 3' untranslated region (3' UTR) of the expression cassette may include a polyadenylation (poly-A) tail, such as a synthetic poly-A tail, an SV40 poly-A tail, or an hGH poly-A tail, and / or may include a transcription terminator, such as a pause site from the human a2 globin gene, or a poly-T (> 4T) sequence. In some embodiments, the 3' UTR of the expression cassette may include sequences encoding for an interfering RNA (miRNA, siRNA, etc.) and / or sequences suitable for interfering RNA biogenesis.

[0129] According to some embodiments, the ITR / scITR / mITR of the recombinant vectors of the inventive concept include AAV ITRs / scITRs / mITRs from / derived from any strain that would be appreciated by one of skill in the art, e.g., any strain / serotype of AAV as set forth in Table 1 of U.S. Patent Application Publication No. 2019 / 0224256, incorporated herein by reference in its entirety. It will be appreciated that the AAV ITRs / scITRs / mITRs of the recombinant vectors according to the inventive concept may be selected based on the ability to express the desired gene product over the course of initial transduction, as well as in the subsequently transdifferentiated / reprogrammed cell. In some embodiments, the rAAV vector, including the AAV ITRs / scITRs / mITRs thereof may be a hybrid AAV vector, having ITRs / scITRs / mITRs and capsids from different AAV serotypes. In some embodiments, the rAAV vector, including the AAV ITRs / scITRs / mITRs thereof may be a chimeric AAV vector, having capsids including sequences from more than one AAV serotype, for example, but not limited to, OligoOOl, as described in Powell et al. (2016) Gene Therapy 23, 807-814, incorporated herein by reference in its entirety.

[0130] Methods of preparing rAAV vectors and rAAV vector stocks of the inventive concept are not particularly limited and may be accomplished by any method that may be appreciated by one of skill in the art, for example, but not limited to, as described in Conway et al., (1999) Gene Therapy 6, 986 and WO 00 / 017377, U.S. Patent No. 5,658,785, Zolotukhin et al., (1999) Gene Therapy 6, 973, and U.S. Patent Application Publication No. 2019 / 0224256, the disclosures of which are incorporated herein by reference in their entireties.Attorney Docket No. 5510-0002WOAlternative vector systems

[0131] Although some embodiments of the inventive concept have been described herein in relation to reagents / vectors that are viral, e.g., AAV / rAAV in nature, it will be appreciated by one of skill in the art that vectors for delivering the factor(s) for enhancing tissue regeneration, enhancing neuron survival and / or for promoting / enhancing functional axon regeneration, and / or the factor(s) for inducing cellular reprogramming according to embodiments of the inventive concept, and / or nucleic acids encoding for these factors, including nucleic acids encoding for antisense and / or interfering RNAs, may include alternative viral and non-viral vectors as will be appreciated by one of skill in the art without departing from the scope of the inventive concept as envisioned herein. It will also be appreciated that more than one vector, including viral vectors and non-viral vectors, for example, a vector including / expressing the factor(s) for enhancing tissue regeneration, enhancing neuron survival and / or for promoting / enhancing functional axon regeneration, and a vector including / expressing the factor(s) for inducing cellular reprogramming, either vector of which may be viral or non-viral, fall within the scope of the inventive concept.

[0132] Suitable vectors include plasmid vectors, viral vectors (e.g., retrovirus, alphavirus; vaccinia virus; adenovirus, adeno-associated virus and other parvoviruses, lentivirus, poxvirus, or herpes simplex virus), and non-viral vectors (e.g., lipid vectors, such as lipid nanoparticles (LNPs), e.g., as described in Jeong et al. (2023) Adv. Drug Deliv. Rev. 200, 114990, poly-lysine vectors, synthetic polyamino polymer vectors, and the like), including / expressing the factor(s) for inducing cellular reprogramming, and / or including / expressing factor(s) for enhancing tissue regeneration, enhancing neuron survival and / or for promoting / enhancing functional axon regeneration, or non-viral vectors including / expressing the factors for inducing cellular reprogramming and for enhancing tissue regeneration, enhancing neuron survival and / or for promoting / enhancing functional axon regeneration, without departing from the scope of the inventive concept.

[0133] Moreover, in some embodiments, the vectors may include a combination of vectors, such as, a combination of different viral (e.g., but not limited to, AAV and non-AAV) vectors, a combination of different non-viral vectors (e.g., but not limited to, lipid and poly-lysine), or even a combination of viral and non-viral vectors (e.g., but not limited to, AAV and lipid) in any possible combination, without departing from the scope of the inventive concept.Attorney Docket No. 5510-0002WOMethods of Treatment

[0134] Another aspect of the inventive concept relates to a method of treating a central nervous system (CNS) disorder or condition responsive to an increase in the number of neurons in a mammalian subject in need thereof, the method may include, for example, delivering to the brain the vector, expression cassette, virus particle, and / or composition of the inventive concept, thereby treating the central nervous system disorder or condition. The subject may be any subject in need of treatment. In some embodiments, the mammalian subject is a laboratory animal or domesticated animal. In some embodiments, the mammalian subject is a human subject.

[0135] The term "treat," "treating," or "treatment of' (or grammatically equivalent terms) it is meant that the severity of the subject's condition is reduced or at least partially improved or ameliorated and / or that some alleviation, mitigation or decrease in at least one clinical symptom is achieved anchor there is a delay in the progression of the condition and / or prevention or delay of the onset of a disease or disorder. The term "treat," "treats," "treating" or "treatment of and the like also include prophylactic treatment of the subject (e.g., to prevent the onset of infection or cancer or a disorder). As used herein, the term "prevent," "prevents," or "prevention" (and grammatical equivalents thereof) are not meant to imply complete abolition of disease and encompasses any type of prophylactic treatment that reduces the incidence of the condition, delays the onset and / or progression of the condition, and / or reduces the symptoms associated with the condition. Thus, unless the context indicates otherwise, the term "treat," "treating," or "treatment of' (or grammatically equivalent terms) refer to both prophylactic and therapeutic regimens.

[0136] An "effective" or "therapeutically effective" amount as used herein is an amount that is sufficient to provide some improvement or benefit to the subject. Alternatively stated, an "effective" or "therapeutically effective" amount is an amount that will provide some alleviation, mitigation, or decrease in at least one clinical symptom in the subject. Those skilled in the art will appreciate that the therapeutic effects need not be complete or curative, as long as some benefit is provided to the subject.

[0137] The disorder or condition may be any one in which an increase and / or restoration in the number of neurons in the brain and / or central nervous system (CNS) would be beneficial, e g., increasing and / or restoring the number of neurons would improve at least one symptom ofAttorney Docket No. 5510-0002WO the disorder or condition. In some embodiments, the disorder or condition is a neurodegenerative disorder, e.g., Parkinson's disease, Alzheimer's disease and other memory disorders, Huntington’s disease, frontotemporal dementia (FTD), ataxia, motor neuron diseases, multiple system atrophy, progressive supranuclear palsy, or amyotrophic lateral sclerosis (ALS). In some embodiments, the disorder or condition is a traumatic brain and / or spinal cord injury, retinal injury, retinal dystrophies, Huntington's chorea, stroke (including both ischemic and nonischemic), aneurism, epilepsy, CNS tumors, hearing loss, and / or cerebral infarction. In some embodiments, the disorder or condition may include neurodevelopmental disorders, sensory neuron loss (beyond motor neurons), paralysis / paralyses, and / or movement disorders, such as but not limited to hereditary tremors or other tremors. In some embodiments, the disorder or condition is a natural condition such as aging.

[0138] In some embodiments, the methods of the inventive concept may further include administering an additional compound, molecule, or agent to enhance reprograming of the cell. In some embodiments, the methods further include delivering to the oligodendrocyte or OPC or the brain a differentiation factor that promotes reprogramming to neurons. The differentiation factor may be, but are not limited to, NeuroDl, Asci i, Brn2a, Mytl 1, SOX2, or any combination thereof.

[0139] In some embodiments, the methods further may include delivering to the oligodendrocyte or OPC or the brain an inhibitor of expression and / or activity of a factor, the inhibition of which results in reprogramming to neurons. In one embodiment, the methods further include delivering to the oligodendrocyte or oligodendrocyte precursor cell or the brain an inhibitor of expression of the RE1 silencing transcription factor complex. The inhibitor may be, for example, an antisense RNA or an interfering RNA targeted to one or more polynucleotides encoding proteins in the complex.

[0140] In some embodiments, the methods further include delivering to the oligodendrocyte or OPC or the brain a factor that promotes neuron growth, e.g., a growth factor or neurotrophic factor such as nerve growth factor, brain-derived neurotrophic factor, glial cell line-derived neurotrophic factor, neurotrophin-3, neurotrophin-4, and ciliary neurotrophic factor.

[0141] In some embodiments, the methods further include delivering to the oligodendrocyte or OPC or the brain an additional therapeutic agent for the disorder or condition be treated. Any suitable therapeutic agent known in the art to be useful for treating the particular disorder orAttorney Docket No. 5510-0002WO condition may be used. Examples of therapeutic agents for neurological disorders include, without limitation, for Alzheimer's disease: caprylidene, donepezil, galantamine, tacrine, vitamin E, ergoloid mesylates, rivastigmine; for Parkinson's disease: nadolol, zonisamide, amantadine, apomorphine, belladonna, benztropine, biperiden, bromocriptine, carbidopa, entacapone, levodopa, pergolide mesylate, pramipexole, procyclidine, rasagiline, ropinirole, rotiotine, scopolamine, tolcapone, trihexylphenidyl, rivastigmine, seleginline; for Huntington's disease: baclofen, pregabalin, tetrabenazine, methylprednisolone, desvenlafaxine, nortriptyline; and for dementia: haloperidol and ergoloid mesylates; for amyotrophic lateral sclerosis: riluzole, edaravone; for traumatic brain or spinal cord injury: diuretics (e.g., mannitol, furosemide, glycerol, urea), anti-seizure drugs, coma-inducing drugs; for stroke: anticoagulants / antiplatelets (e.g., aspirin, warfarin), antihypertensives, tissue plasminogen activator.

[0142] In these embodiments, the additional compound, molecule, or agent may be delivered by contacting the oligodendrocyte or OPC or the brain with the additional compound, molecule, or agent itself or with an expression vector encoding the additional compound, molecule, or agent.

[0143] The rAAV vectors according to the present inventive concept may be used in veterinary and / or medical applications. Optionally, the subject is "in need of' the methods of the present inventive concept, e.g., because the subject has or is believed at risk for a disorder including those described herein or that would benefit from the delivery of a nucleic acid including those described herein. For example, in particular embodiments, the subject has (or has had) or is at risk for a neurodegenerative disorder or a spinal cord or brain injury. As a further option, the subject can be a laboratory animal and / or an animal model of disease.

[0144] Delivery of the rAAV vectors is not particularly limited and may be accomplished by any effective method and in any effective dosages for the particular method that may be appreciated by one of skill in the art, e.g., methods described in U.S. Patent Application Publication No. 2019 / 0224256.EXAMPLES

[0145] The following examples, which highlight certain features and properties of the exemplary embodiments of the inventive concept described herein are provided for purpose of illustration.Attorney Docket No. 5510-0002WO

[0146] FIG. 1 illustrates a design of a novel dual -therapeutic expression cassettes included in and rAAV vector for reprogramming and mRNA translation control according to a first embodiment of the inventive concept. The vector includes independent transcriptional units for anti -PTBP 1 and glial cell line-derived neurotrophic factor (GDNF), or the precursor of GDNF, pro-GDNF. In addition, the expression cassettes may optionally include IS sequences between the anti -PTBP 1 promoter sequence and the expressed anti -PTBP 1 sequences, between the expressed anti -PTBP 1 sequences and the anti-PTBPl polyadenylation and / or transcription termination sequences, and / or between the GDNF or pro-GDNF promoter sequences and the expressed GDNF or pro-GDNF sequences.

[0147] FIG. 2 illustrates a design of a novel dual-therapeutic expression cassette included in an rAAV vector for reprogramming and mRNA translation control according to a second embodiment of the inventive concept. The vector includes an expression cassette under which both the expressed anti-PTBPl sequences and GDNF or pro-GDNF sequences are part of the same transcriptional unit. Either the expressed anti-PTBPl sequences (top) or the expressed GDNF or pro-GDNF sequences (bottom) may be immediately 3' of the promoter. In addition, the expression cassette may optionally include IS sequences between the promoter sequence and the expressed anti-PTBPl sequences, and / or between the expressed anti-PTBPl sequences and the expressed GDNF or pro-GDNF sequences (top), or optionally include IS sequences between the promoter sequences and the expressed GDNF or pro-GDNF sequences, and / or between the expressed GDNF or pro-GDNF sequences and the expressed anti-PTBPl sequences (bottom).

[0148] FIG. 3 further details the components of the novel dual-therapeutic expression cassettes included in the rAAV vector for reprogramming and mRNA translation control shown in FIG. 1, wherein expression of anti-PTBPl and GDNF / pro-GDNF includes independent transcriptional control units. The IS sequences depicted in FIG. 3 are optionally present.

[0149] FIG. 4 further details the components of the novel dual-therapeutic expression cassette included in an rAAV vector for reprogramming and mRNA translation control shown in FIG. 2, wherein expression of anti-PTBPl and GDNF / pro-GDNF is part of the same transcription al unit. The IS sequences depicted in FIG. 4 are optionally present.

[0150] FIG. 5 broadly illustrates how factors for inducing cellular reprogramming (anti- PTBPl) that may be included in the novel dual therapeutic expression cassettes for inhibiting and / or interfering with PTBP1 expression and / or function, including its isoforms, including, butAttorney Docket No. 5510-0002WO not limited to, use of siRNA, shRNA, microRNA, and / or IncRNA, either configured as linear and / or circular RNAs. These solutions may include, e.g.: 1) factors that inhibit targeting / binding of PTBP 1 to its target RNA sequence(s); 2) factors that inhibit PTBP1 release of its target RNA sequence(s); 3) factors that enhance PTBP1 degradation when associated with PTBP1, e.g., enhance proteasome-mediated PTBP1 degradation; 4) factors that enhance posttranslational modifications of PTBP 1 which induce protein degradation and / or inactivity, e.g., ubiquintination; 5) factors which suppress / attenuate transcription of PTBP 1 mRNA; 6) factors which inhibit function of PTBP 1, e.g., interfere with PTBP1 function related to its binding / interacting with its target RNA sequences; and 7) factors, such as small molecules that bind to / interact with PTBP1 target RNA, and thus interfere with its binding to / interacting with PTBP1, and such small molecules in combination with aptamers.

[0151] FIG. 6 illustrates a DNA sequence similarity and homology analysis of anti-PTBPl siRNA4 with the corresponding sequences of PTBP 1 from various species. Panel A shows a multiple sequence alignment of the anti-PTBPl siRNA4, with the rat, mouse, human, and monkey PTBP1. Target-mismatches between the anti-PTBPl siRNA4, rat and mouse PTBP1, and human and monkey PTBP1 are highlighted. Panel B shows the sequence alignment of the anti-PTBPl siRNA4, and a humanized anti-PTBPl siRNA4, wherein the species-specific mismatches of the anti-PTBPl siRNA4 and human PTBP1 have been changed to correspond to the sequence of human PTBP1.

[0152] FIG. 7 illustrates human PTBP1 knockdown by siRNA4 or humanized siRNA4 (miRNA4_hu) delivered as an artificial microRNA (miRNA) in a miR-155 backbone. Panel A is a representation of the miRNA4 plasmid constructs for expressing the artificial miRNA, with added termination signals (T8 for Pol III and SV40PolyA for Pol II). The indicated artificial miRNAs were transcribed by RNA polymerase II (CMV) or III (Hl) promoters. Panel B shows the results of a knockdown analysis of human PTBP1 co-transfected in HEK293 cells with miRNA4 or miRNA4_hu. A miRNA targeting luciferase (miRNA_Luc) was used as a negative control. These results show that the artificial humanized miRNA4, miRNA4_hu, was effective at knocking down PTBP1 expression from either Hl and CMV promoters when compared to miRNA4 or the negative control. Panel C depicts a representative western blot after 24h post-co- transfection in HEK293 cells. The results of the western blot analysis mirror the results from the knockdown analysis.Attorney Docket No. 5510-0002WG

[0153] FIG. 8 illustrates the secondary structure and sequence of miRNA4 and miRNA4_Hu in a miR-155 backbone (miRNA4_miR155 (SEQ ID NO:9) and miRNA4_Hu_miR155 (SEQ ID NO: 10), respectively). Based on the secondary structure of native / wild-type miR-155, a deletion of CU and CC from siRNA4 and siRNA4_Hu, respectively, that duplicates the secondary structure found in native miR-155, was made as indicated in the constructs within the miR-155 backbone. The resulting siRNA4 sequence with CU deletion, CCAACAAUGG UUAACUA (SEQ ID NO: 11), is located at nucleotides 71-87 of SEQ ID NO:9 and hybridizes with UAGUUAACCA UAGUGUUGG (SEQ ID NO: 12), corresponding to nucleotides 29-47 of SEQ ID NO:9 shown in FIG. 8. The resulting siRNA4_Hu sequence with CC deletion, CCAACAAUGG UGAACUA (SEQ ID NO: 13), is located at nucleotides 71-87 of SEQ ID NO: 10 and hybridizes with UAGUUCACCA UGGUGUUGG (SEQ ID NO: 14), corresponding to nucleotides 29-47 of SEQ ID NO: 10 shown in FIG. 8.

[0154] FIG. 9 illustrates the secondary structure and sequence of miRNA4 and miRNA4_Hu in a miR-E backbone (miRNA4_miR-E (SEQ ID NO: 15) and miRNA4_Hu_miR-E (SEQ ID NO:16), respectively). In contrast to the miR-155 construct, no deletion of nucleotides in siRNA4 and siRNA4_Hu was made in the generation of the miR-E constructs. The siRNA4 sequence (SEQ ID NO:7) of miRNA4_miR-E is at nucleotides 59-87 of SEQ ID NO: 15 and hybridizes to UAGUUAACCA UAGUGUUGG (SEQ ID NO: 12), corresponding to nucleotides 97-115 of SEQ ID NO: 15 shown in FIG. 9. The siRNA4_Hu sequence (SEQ ID NO:8) of miRNA4_Hu_miR-E is at nucleotides 59-87 of SEQ ID NO: 16 and hybridizes to UAGUUCACCA UGGUGUUGG (SEQ ID NO: 14), corresponding to nucleotides 97-115 of SEQ ID NO: 16 shown in FIG. 9.

[0155] FIG. 10 illustrates PTBP1 knockdown by siRNA4 or siRNA4_Hu delivered as an artificial miRNA in a second miRNA backbone, miR-E, a miR-30 variant, and compared with siRNA4 or siRNA4_Hu delivered as an artificial miRNA in miR-155. Panel A is a representation of the miRNA4 plasmid constructs for expressing the artificial miRNA in a miR-155 or miR-E backbone, with added termination signals (T8 for Pol III and SV40PolyA for Pol II). The indicated artificial miRNAs were transcribed by RNA polymerase II (CMV) or III (Hl) promoters. Panel B shows the results of a knockdown analysis of human PTBP1 co-transfected in cells with miRNA4 or miRNA4_hu. A miRNA targeting luciferase (miRNA_Luc) was used as a negative control. These results show that the artificial humanized miRNA4, miRNA4_Hu, inAttorney Docket No. 5510-0002WG either a miR-155 or a miR-E backbone with an Hl promoter was effective at knocking down PTBP1 expression when compared to miRNA4 or the negative control. Panel C depicts the quantitation if the results shown in Panel B. These results show that miRNA4 generated in an miR-155 backbone with either an Hl or CMV promoter can downregulate human PTBP1 expression in HEK293 cells, similar to the results shown in FIG. 7, and to a lesser extent with miRNA4 generated in an mIR-E backbone and an Hl promoter. However, significant increases are exhibited for the downregulation of human PTBP1 expression, between 5.07-8.09-fold for miRNA4_Hu in an mIR-E backbone with an Hl promoter, between 13.12-15.94-fold for miRNA4_Hu in an miR-155 backbone with an Hl promoter, and between about 8.42-14.22-fold for miRNA4_Hu in an miR-155 backbone with a CMV promoter. These results show that while miRNA4 exhibits some downregulation of human PTBP1 in human cells, the humanized construct, miRNA4_Hu shows marked improvement in downregulation of human PTBP1 compared to miRNA4.

[0156] FIG. 11 illustrates PTBP1 mRNA knockdown by miRNA4_Hu in an expression cassette for both hGDNF and miRNA4_Hu (Panel A) and quantitation of secreted hGDNF by ELISA (Panel B). Quantitation of PTBP 1 mRNA and secreted hGDNF from constructs including a negative miRNA control (miRNA Scramble) and a negative hGDNF knockout control demonstrate that non-specific miRNA does not reduce PTBP1 mRNA levels, and that PTBP1 mRNA knockdown occurs independently of hGDNF expression.

[0157] FIG. 12 illustrates human PTBP1 (hPTBPl) protein knockdown by Western blot analysis for an expression cassette including only hGDNF and an expression cassette including both hGDNF and miRNA4 Hu after transfection of HEK293 cells. These results show that miRNA4_Hu is responsible for hPTBPl protein knockdown and is not affected by hGDNF expression.

[0158] FIG. 13 illustrates rat PTBP1 (rPTBPl) protein knockdown in primary rat oligodendrocytes by Western blot analysis for an expression cassette including hGDNF and a negative miRNA control (miRNA_Scramble), and an expression cassette including hGDNF and miRNA4. (Panel A) Western blot analysis of the two constructs shows that the non-specific miRNA Scramble-containing construct does not result in a reduction of rPTBPl protein levels. (Panel B) Quantitation of secreted hGDNF by ELISA. These results show that there is noAttorney Docket No. 5510-0002WO significant effect of the presence of miRNA in the construct on the production / secretion of hGDNF.

[0159] Further embodiments of the inventive concept are described in the following clauses.

[0160] 1. An expression cassette including: a polynucleotide encoding at least one factor for inducing cellular reprogramming; and a polynucleotide encoding at least one factor for enhancing tissue regeneration, for enhancing cell survival, for promoting / enhancing cell development, for promoting / enhancing cell structure development / regeneration, and / or for promoting / enhancing cell / cell structure stabilization.

[0161] 2 The expression cassette of Clause 1, wherein the at least one factor for enhancing tissue regeneration, for enhancing cell survival, for promoting / enhancing cell development, for promoting / enhancing cell structure development / regeneration, and / or for promoting / enhancing cell / cell structure stabilization is a factor for enhancing tissue regeneration, for enhancing neuron survival, and / or for promoting / enhancing functional axon regeneration.

[0162] 3. The expression cassette of Clause 1 or 2, wherein the polynucleotide encoding at least one factor for inducing cellular reprogramming and / or the polynucleotide encoding at least one factor for enhancing tissue regeneration, for enhancing cell survival, for promoting / enhancing cell development, for promoting / enhancing cell structure development / regeneration, and / or for promoting / enhancing cell / cell structure stabilization is operably linked to a promoter.

[0163] 4. The expression cassette of Clause 3, wherein the promoter is a cell-type-specific, tissue-specific, ubiquitous, inducible, and / or constitutive promoter.

[0164] 5. The expression cassette of Clause 3 or 4, wherein the promoter is an RNA polymerase-II (Pol-II) promoter, RNA polymerase-III (Pol-III) promoter, or a combination of RNA Pol II and RNA Pol III promoters / promoter systems.

[0165] 6. The expression cassette of any one of Clauses 3-5, wherein the promoter supports expression in oligodendrocytes, oligodendrocyte precursor cells (OPCs), astrocytes, neurons, and / or in glial progenitor cells.

[0166] 7 The expression cassette of any one of Clauses 3-6, wherein the promoter supports expression in oligodendrocytes and / or OPCs.

[0167] 8. The expression cassette of Clause 4, wherein the promoter is a constitutive promoter.Attorney Docket No. 5510-0002WO

[0168] 9 The expression cassette of any one of Clauses 1—7, wherein the expression cassette includes an adeno-associated virus (AAV) or recombinant AAV (rAAV) genome.

[0169] 10. The expression cassette of any one of Clauses 1-8, wherein the expression cassette is included in a lipid vector, poly-lysine vector, or synthetic polyamino polymer vector.

[0170] 11. The expression cassette of any one of Clauses 1-10, wherein the at least one factor for inducing cellular reprogramming inhibits / attenuates expression and / or biological activity of polypyrimidine tract binding protein 1 (PTBP1).

[0171] 12. The expression cassette of Clause 11, wherein the polynucleotide encoding at least one factor for inducing cellular reprogramming encodes for an antisense or an interfering RNA targeted to a polynucleotide encoding a human PTBP1.

[0172] 13. The expression cassette of Clause 11 or 12, wherein the interfering RNA is a cityRNA, a shRNA, a siRNA, and / or a miRNA.

[0173] 14. The expression cassette of Clause 13, wherein the interfering RNA is a miRNA and / or a siRNA.

[0174] 15. The expression cassette of Clause 14, wherein the interfering RNA encoded for includes a sequence having at least 80% sequence identity to SEQ ID NO: 8, and a sequence that hybridizes to SEQ ID NO:8.

[0175] 16. The expression cassette of Clause 15, wherein the interfering RNA encoded for includes the sequence of SEQ ID NO: 8.

[0176] 17. The expression cassette of Clause 15 or 16, wherein the sequence that hybridizes to the sequence having at least 80% sequence identity to SEQ ID NO: 8 includes a sequence having at least 80% identity to SEQ ID NO: 14.

[0177] 18. The expression cassette of Clause 17, wherein the sequence that hybridizes to the sequence having at least 80% sequence identity to SEQ ID NO: 8 includes the sequence of SEQ ID NO: 14.

[0178] 19. The expression cassette of any one of Clauses 15-18, wherein the interferingRNA encoded for includes a sequence having at least 80% sequence identity to SEQ ID NO: 10.

[0179] 20. The expression cassette of Clause 19, wherein the interfering RNA encoded for includes the sequence of SEQ ID NO: 10.

[0180] 21. The expression cassette of any one of Clauses 15-18, wherein the interferingRNA encoded for includes a sequence having at least 80% sequence identity to SEQ ID NO: 16.Attorney Docket No. 5510-0002WO

[0181] 22. The expression cassette of Clause 21, wherein the interfering RNA encoded for includes the sequence of SEQ ID NO: 16.

[0182] 23. The expression cassette of Clause 14, wherein the interfering RNA encoded for includes a sequence having at least 80% sequence identity to SEQ ID NO: 13, and a sequence that hybridizes to SEQ ID NO: 13.

[0183] 24. The expression cassette of Clause 23, wherein the interfering RNA encoded for includes the sequence of SEQ ID NO: 13.

[0184] 25. The expression cassette of Clause 23 or 24, wherein the sequence that hybridizes to the sequence having at least 80% sequence identity to SEQ ID NO: 13 includes a sequence having at least 80% identity to SEQ ID NO: 14.

[0185] 26. The expression cassette of Clause 25, wherein the sequence that hybridizes to the sequence having at least 80% sequence identity to SEQ ID NO: 13 includes the sequence of SEQ ID NO: 14.

[0186] 27. The expression cassette of any one of Clauses 23-26, wherein the interferingRNA encoded for includes a sequence having at least 80% sequence identity to SEQ ID NOTO.

[0187] 28. The expression cassette of Clause 19, wherein the interfering RNA encoded for includes the sequence of SEQ ID NOTO.

[0188] 29. The expression cassette of Clause 11, wherein the at least one factor for inducing cellular reprogramming comprises an RNA aptamer sequence configured to block and / or inhibit PTBP1 biological activity.

[0189] 30. The expression cassette of Clause 29, wherein the RNA aptamer sequence does not alter PTBP1 expression levels.

[0190] 31 The expression cassette of Clause any one of Clauses 1-30, wherein the at least one factor for enhancing tissue regeneration, for enhancing cell survival, for promoting / enhancing cell development, for promoting / enhancing cell structure development / regeneration, and / or for promoting / enhancing cell / cell structure stabilization is a neurotrophic factor (NTF).

[0191] 32. The expression cassette of Clause 31, wherein the NTF is GDNF.

[0192] 33. The expression cassette of Clause 31, wherein the NTF is expressed as a protein precursor of the NTF.

[0193] 34. The expression cassette of Clause 33, wherein the protein precursor is pro-GDNF.Attorney Docket No. 5510-0002WO

[0194] 35. A vector comprising the expression cassette of any one of Clauses 1-34.

[0195] 36. A recombinant virus particle including the expression cassette or vector of any one of Clauses 1-33.

[0196] 37. The recombinant virus particle of Clause 36, wherein the recombinant virus particle has a tropism for oligodendrocytes, OPCs, astrocytes, neurons, and / or glial progenitor cells.

[0197] 38. The recombinant virus particle of Clause 36 or 37, wherein the recombinant virus particle has a tropism for oligodendrocytes and / or OPCs.

[0198] 39. The recombinant virus particle of any one of Clauses 36-38, wherein the recombinant virus particle includes a capsid protein providing a tropism for the virus particle.

[0199] 40. The recombinant virus particle of Clause 39, wherein the tropism is for oligodendrocytes, OPCs, astrocytes, neurons, and / or glial progenitor cells.

[0200] 41. The recombinant virus particle of Clause 39 or 40, wherein the tropism is for oligodendrocytes and / or OPCs.

[0201] 42. The recombinant virus particle of Clause 39, wherein the capsid protein modulates expression of the at least one factor for inducing cellular reprogramming and / or the polynucleotide encoding at least one factor for enhancing tissue regeneration, for enhancing cell survival, for promoting / enhancing cell structure development / regeneration, and / or for promote / enhance cell / cell structure stabilization.

[0202] 43. The recombinant virus particle of Clause 36, wherein the expression of the at least one factor for inducing cellular reprogramming and / or the polynucleotide encoding a factor for enhancing tissue regeneration, for enhancing cell survival, for promoting / enhancing cell structure development / regeneration, and / or for promote / enhance cell / cell structure stabilization is influenced by epigenetic marking and / or epigenetic status of the expression cassette or vector.

[0203] 44. The recombinant virus particle of any one of Clauses 39-43, wherein the tropism is for oligodendrocytes, OPCs, astrocytes, neurons, and / or glial progenitor cells.

[0204] 45. The recombinant virus particle of any one of Clauses 39-44, wherein the tropism is for oligodendrocytes and / or OPCs.

[0205] 46. A composition including the expression cassette or the recombinant virus particle of any one of Clauses 1-45.Attorney Docket No. 5510-0002WO

[0206] 47. A pharmaceutical composition including the expression cassette or recombinant virus particle of any one of Clauses 1-45 and a pharmaceutically acceptable carrier.

[0207] 48. A method of inducing cellular reprogramming in a cell including exposing the cell with the expression cassette, the recombinant virus particle, the composition, or the pharmaceutical composition of any one of Clauses 1-47.

[0208] 49. The method of Clause 48, wherein inducing cellular reprogramming includes inhibiting / attenuating PTBP1 expression.

[0209] 50. The method of Clause 48 or 49, wherein cellular reprogramming further includes enhancing tissue regeneration, enhancing cell survival, promoting / enhancing cell development, promoting / enhancing cell structure development / regeneration, and / or promoting / enhancing cell / cell structure stabilization.

[0210] 51 A method of reprogramming an oligodendrocyte or an oligodendrocyte precursor cell (OPC) to a neuron including exposing the oligodendrocyte or OPC to the vector, the recombinant virus particle, the composition, or the pharmaceutical composition of any one of Clauses 1-47, wherein the oligodendrocyte or OPC is reprogrammed into a neuron.

[0211] 52. The method of Clause 51, wherein the oligodendrocyte or OPC is in the brain of a mammalian subject.

[0212] 53. A method of treating a central nervous system (CNS) disorder or condition responsive to an increase in the number of neurons in a subject in need thereof including delivering the expression cassette, the recombinant virus particle, the composition, or the pharmaceutical composition of any one of Clauses 1-47 to the brain of the subject, wherein delivery of the vector or the recombinant virus particle treats the CNS disorder or condition.

[0213] 54. The method of Clause 53, wherein the CNS disorder or condition is a neurodegenerative disorder.

[0214] 55. The method of Clause 54, wherein the neurodegenerative disorder is Parkinson's disease, Alzheimer's disease, Huntington's disease, frontotemporal dementia (FTD), ataxia, motor neuron diseases, multiple system atrophy, progressive supranuclear palsy, or amyotrophic lateral sclerosis (ALS).

[0215] 56. The method of Clause 53, wherein the CNS disorder or condition is a traumatic brain and / or spinal cord injury, retinal injury, retinal dystrophies, Huntington's chorea, stroke,Attorney Docket No. 5510-0002WO aneurism, epilepsy, CNS tumors, hearing loss, and / or cerebral infarction, a neurodevelopmental disorder, sensory neuron loss (beyond motor neurons), paralysis / paralyses, or tremors.

[0216] 57. The method of Clause 53, wherein the CNS disorder or condition is aging.

[0217] The foregoing is illustrative of embodiments of the inventive concept and is not to be construed as limiting thereof. Although some embodiments have been described, those skilled in the art will readily appreciate that many modifications are possible in embodiments without materially departing from the novel teachings and advantages of the inventive concept.Accordingly, all such modifications are intended to be included within the scope of the inventive concept and as defined in the claims that follow.

Claims

Attorney Docket No. 5510-0002WOTHAT WHICH IS CLAIMED:

1. A vector compri sing : a polynucleotide encoding at least one factor for inducing cellular reprogramming; and a polynucleotide encoding at least one factor for enhancing tissue regeneration, for enhancing cell survival, for promoting / enhancing cell development, for promoting / enhancing cell structure development / regeneration, and / or for promoting / enhancing cell / cell structure stabilization.

2. The vector of Claim 1, wherein the at least one factor for enhancing tissue regeneration, for enhancing cell survival, for promoting / enhancing cell development, for promoting / enhancing cell structure development / regeneration, and / or for promoting / enhancing cell / cell structure stabilization is a factor for enhancing tissue regeneration, for enhancing neuron survival, and / or for promoting / enhancing functional axon regeneration.

3. The vector of Claim 1 or 2, wherein the polynucleotide encoding at least one factor for inducing cellular reprogramming and / or the polynucleotide encoding at least one factor for enhancing tissue regeneration, for enhancing cell survival, for promoting / enhancing cell development, for promoting / enhancing cell structure development / regeneration, and / or for promoting / enhancing cell / cell structure stabilization is operably linked to a promoter.

4. The vector of Claim 3, wherein the promoter is a cell-type-specific, tissuespecific, ubiquitous, inducible, and / or constitutive promoter.

5. The vector of Claim 3 or 4, wherein the promoter is an RNA polymerase-II (Pol- Il) promoter, RNA polymerase-III (Pol -III) promoter, or a combination of RNA Pol II and RNA Pol III promoters / promoter systems.

6. The vector of any one of Claims 3-5, wherein the promoter supports expression in oligodendrocytes, oligodendrocyte precursor cells (OPCs), astrocytes, neurons, and / or in glial progenitor cells.Attorney Docket No. 5510-0002WO7. The vector of any one of Claims 3-6, wherein the promoter supports expression in oligodendrocytes and / or OPCs.

8. The vector of Claim 4, wherein the promoter is a constitutive promoter.

9. The vector of any one of Claims 1-7, wherein the vector comprises an adeno- associated virus (AAV) or recombinant AAV (rAAV) vector genome.

10. The vector of any one of Claims 1-8, wherein the vector is a lipid vector, polylysine vector, or synthetic polyamino polymer vector.

11. The vector of any one of Claims 1-10, wherein the at least one factor for inducing cellular reprogramming inhibits / attenuates expression and / or biological activity of polypyrimidine tract binding protein 1 (PTBP1).

12. The vector of Claim 11, wherein the polynucleotide encoding at least one factor for inducing cellular reprogramming encodes for an antisense or an interfering RNA targeted to a polynucleotide encoding a human PTBP1.

13. The vector of Claim 11 or 12, wherein the interfering RNA is a cityRNA, a shRNA, a siRNA, and / or a miRNA.

14. The vector of Claim 13, wherein the interfering RNA is a miRNA and / or a siRNA.

15. The vector of Claim 14, wherein the interfering RNA comprises a sequence having at least 80% sequence identity to SEQ ID NO: 8, and a sequence that hybridizes to SEQ ID N0:8.Attorney Docket No. 5510-0002WO16. The vector of Claim 15, wherein the interfering RNA comprises the sequence of SEQ ID N0:8.

17. The vector of Claim 15 or 16, wherein the sequence that hybridizes to the sequence having at least 80% sequence identity to SEQ ID NO:8 comprises a sequence having at least 80% identity to SEQ ID NO: 14.

18. The vector of Claim 17, wherein the sequence that hybridizes to the sequence having at least 80% sequence identity to SEQ ID NO: 8 comprises the sequence of SEQ ID NO:14.

19. The vector of any one of Claims 15-18, wherein the interfering RNA comprises a sequence having at least 80% sequence identity to SEQ ID NO: 10.

20. The vector of Claim 19, wherein the interfering RNA encoded for comprises the sequence of SEQ ID NO: 10.

21. The vector of any one of Claims 15-18, wherein the interfering RNA encoded for comprises a sequence having at least 80% sequence identity to SEQ ID NO: 16.

22. The vector of Claim 21, wherein the interfering RNA encoded for comprises the sequence of SEQ ID NO: 16.

23. The vector of Claim 14, wherein the interfering RNA comprises a sequence having at least 80% sequence identity to SEQ ID NO: 13, and a sequence that hybridizes to SEQ ID NO: 13.

24. The vector of Claim 23, wherein the interfering RNA comprises the sequence of SEQ ID NO: 13.Attorney Docket No. 5510-0002WO25. The vector of Claim 23 or 24, wherein the sequence that hybridizes to the sequence having at least 80% sequence identity to SEQ ID NO: 13 comprises a sequence having at least 80% identity to SEQ ID NO: 14.

26. The vector of Claim 25, wherein the sequence that hybridizes to the sequence having at least 80% sequence identity to SEQ ID NO: 13 comprises the sequence of SEQ ID NO:14.

27. The vector of any one of Claims 23-26, wherein the interfering RNA comprises a sequence having at least 80% sequence identity to SEQ ID NO: 10.

28. The vector of Claim 19, wherein the interfering RNA comprises the sequence of SEQ ID NO: 10.

29. The vector of Claim 11, wherein the at least one factor for inducing cellular reprogramming comprises an RNA aptamer sequence configured to block and / or inhibit PTBP1 biological activity.

30. The vector of Claim 29, wherein the RNA aptamer sequence does not alter PTBP1 expression levels.

31. The vector of Claim any one of Claims 1-30, wherein the at least one factor for enhancing tissue regeneration, for enhancing cell survival, for promoting / enhancing cell development, for promoting / enhancing cell structure development / regeneration, and / or for promoting / enhancing cell / cell structure stabilization is a neurotrophic factor (NTF).

32. The vector of Claim 31, wherein the NTF is GDNF.

33. The vector of Claim 31, wherein the NTF is expressed as a protein precursor of the NTF.Attorney Docket No. 5510-0002WO34. The vector of Claim 33, wherein the protein precursor is pro-GDNF.

35. A recombinant virus particle comprising the vector of any one of Claims 1-34.

36. The recombinant virus particle of Claim 35, wherein the recombinant virus particle has a tropism for oligodendrocytes, OPCs, astrocytes, neurons, and / or glial progenitor cells.

37. The recombinant virus particle of Claim 35 or 36, wherein the recombinant virus particle has a tropism for oligodendrocytes and / or OPCs.

38. The recombinant virus particle of any one of Claims 35-37, wherein the recombinant virus particle comprises a capsid protein providing a tropism for the virus particle.

39. The recombinant virus particle of Claim 38, wherein the tropism is for oligodendrocytes, OPCs, astrocytes, neurons, and / or glial progenitor cells.

40. The recombinant virus particle of Claim 38 or 39, wherein the tropism is for oligodendrocytes and / or OPCs.

41. The recombinant virus particle of Claim 38, wherein the capsid protein modulates expression of the at least one factor for inducing cellular reprogramming and / or the polynucleotide encoding at least one factor for enhancing tissue regeneration, for enhancing cell survival, for promoting / enhancing cell structure development / regeneration, and / or for promote / enhance cell / cell structure stabilization.

42. The recombinant virus particle of Claim 35, wherein the expression of the at least one factor for inducing cellular reprogramming and / or the polynucleotide encoding a factor for enhancing tissue regeneration, for enhancing cell survival, for promoting / enhancing cell structure development / regeneration, and / or for promote / enhance cell / cell structure stabilization is influenced by epigenetic marking and / or epigenetic status of the vector.Attorney Docket No. 5510-0002WO43. The recombinant virus particle of any one of Claims 38-42, wherein the tropism is for oligodendrocytes, OPCs, astrocytes, neurons, and / or glial progenitor cells.

44. The recombinant virus particle of any one of Claims 38-43, wherein the tropism is for oligodendrocytes and / or OPCs.

45. A composition comprising the vector or the recombinant virus particle of any one of Claims 1-44.

46. A pharmaceutical composition comprising the vector or the recombinant virus particle of any one of Claims 1-44 and a pharmaceutically acceptable carrier.

47. A method of inducing cellular reprogramming in a cell comprising exposing the cell with the vector, the recombinant virus particle, the composition, or the pharmaceutical composition of any one of Claims 1-46.

48. The method of Claim 47, wherein inducing cellular reprogramming comprises inhibiting / attenuating PTBP1 expression.

49. The method of Claim 47 or 48, wherein cellular reprogramming further comprises enhancing tissue regeneration, enhancing cell survival, promoting / enhancing cell development, promoting / enhancing cell structure development / regeneration, and / or promoting / enhancing cell / cell structure stabilization.

50. A method of reprogramming an oligodendrocyte or an oligodendrocyte precursor cell (OPC) to a neuron comprising exposing the oligodendrocyte or OPC to the vector, the recombinant virus particle, the composition, or the pharmaceutical composition of any one of Claims 1-46, wherein the oligodendrocyte or OPC is reprogrammed into a neuron.Attorney Docket No. 5510-0002WO51 . The method of Claim 50, wherein the oligodendrocyte or OPC is in the brain of a mammalian subject.

52. A method of treating a central nervous system (CNS) disorder or condition responsive to an increase in the number of neurons in a subject in need thereof comprising delivering the vector, the recombinant virus particle, the composition, or the pharmaceutical composition of any one of Claims 1-46 to the brain of the subject, wherein delivery of the vector or the recombinant virus particle treats the CNS disorder or condition.

53. The method of Claim 52, wherein the CNS disorder or condition is a neurodegenerative disorder.

54. The method of Claim 53, wherein the neurodegenerative disorder is Parkinson's disease, Alzheimer's disease, Huntington's disease, frontotemporal dementia (FTD), ataxia, motor neuron diseases, multiple system atrophy, progressive supranuclear palsy, or amyotrophic lateral sclerosis (ALS).

55. The method of claim 52, wherein the CNS disorder or condition is a traumatic brain and / or spinal cord injury, retinal injury, retinal dystrophies, Huntington's chorea, stroke, aneurism, epilepsy, CNS tumors, hearing loss, and / or cerebral infarction, a neurodevel opmental disorder, sensory neuron loss (beyond motor neurons), paralysis / paralyses, or tremors.

56. The method of Claim 52, wherein the CNS disorder or condition is aging.

57. An expression cassette comprising: a polynucleotide encoding at least one factor for inducing cellular reprogramming; and a polynucleotide encoding at least one factor for enhancing tissue regeneration, for enhancing cell survival, for promoting / enhancing cell development, for promoting / enhancing cell structure development / regeneration, and / or for promoting / enhancing cell / cell structure stabilization.Attorney Docket No. 5510-0002WO58. A vector comprising the expression cassette of Claim 57.

59. A recombinant virus particle comprising the expression cassette of Claim 57 or vector of Claim 58.

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