Gene therapy vectors for use in Parkinson's disease

Abstract

Disclosed herein are recombinant gene therapy vectors comprising a gene encoding PTEN-induced kinase 1 (PINK1) operably linked to a promoter, and methods of using the recombinant therapy vectors to inhibit, reduce, or delay neuronal degeneration or death in a subject.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of U.S. Provisional Application No. 63 / 443,289, filed February 3, 2023, which is incorporated herein by reference in its entirety.

[0002] FIELD OF THE INVENTION The present invention relates to gene therapy for inhibiting, reducing, or slowing the degeneration or death of neurons in a subject. [Background technology]

[0003] Parkinson's disease (PD) is a progressive neurodegenerative disorder in which symptoms slowly progress from mild motor impairment to severe physical disability, and in some patients, cognitive decline or dementia is also present. Despite the urgent need, there are currently no clinically approved therapies to delay or cure PD. In most patients, the cause of the disease is unknown, but a combination of factors, including mitochondrial stress, likely contributes. Mitochondrial stress can lead to both neurodegeneration and neuroinflammation, and has been shown to induce numerous cellular dysregulations, particularly those associated with PD.

[0004] Mutations in the PTEN-induced kinase 1 (PINK1) and parkin RBR E3 ubiquitin-protein ligase (PARKIN) genes were the first to be associated with familial forms of PD. Homozygous and compound heterozygous loss-of-function mutations in the PINK1 gene cause early-onset autosomal recessive PD. PINK1 signaling is important for maintaining mitochondrial homeostasis due to its essential role in mitochondrial degradation, mobility, size, and network maintenance. Of these, the best-characterized function of PINK1 is PARKIN-dependent mitophagy. In healthy mitochondria, PINK1, located upstream of PARKIN, is constitutively imported into mitochondria, cleaved by proteases, and then further degraded by the ubiquitin / proteasome system. PARKIN remains inactive in the cytosol. Upon injury or mitochondrial dysfunction (most often due to mitochondrial depolarization), PINK1 accumulates at the outer mitochondrial membrane (OMM), leading to the recruitment of PARKIN from the cytosol to the OMM, where its E3 ligase activity ubiquitinates mitochondrial proteins, leading to mitochondrial degradation. Beyond its essential function in mitophagy, PINK1 is also involved in regulating mitochondrial bioenergetics (complex 1 activation), calcium homeostasis, and mitochondrial quality control (mitochondrial-derived vesicle pathway). Currently, there are few small molecule therapies targeting mitochondria in the pipeline, likely due to off-target systemic side effects associated with the regulation of ubiquitously present mitochondrial proteins. Furthermore, according to CiteLine, there are currently 3,153 ongoing or completed clinical trials for PD worldwide. However, none of these target PINK1.

[0005] There remains a need to develop therapeutic agents that can be delivered directly to the striatum or substantia nigra without systemic side effects and without side effects on non-targeted CNS resident cells. Summary of the Invention

[0006] Provided herein is a recombinant gene therapy vector comprising a polynucleotide operably linked to a promoter, wherein the vector is an adeno-associated virus (AAV), and the polynucleotide encodes PTEN-induced kinase 1 (PINK1) and has at least about 90% identity to SEQ ID NO:2 or SEQ ID NO:3.

[0007] In one embodiment of the recombinant gene therapy vector, the polynucleotide encoding PINK1 has at least 95% identity to SEQ ID NO:2 or SEQ ID NO:3.

[0008] In a further embodiment of the recombinant gene therapy vector, the polynucleotide encoding PINK1 has at least 98% identity to SEQ ID NO:2 or SEQ ID NO:3.

[0009] In still further embodiments of the recombinant gene therapy vector, the polynucleotide encoding PINK1 has at least 99% identity to SEQ ID NO:2 or SEQ ID NO:3.

[0010] In still further embodiments of the recombinant gene therapy vector, the polynucleotide encoding PINK1 has the sequence of SEQ ID NO:2 or SEQ ID NO:3.

[0011] In still further embodiments of the gene therapy vector, the vector is a serotype 1 adeno-associated virus (AAV1).

[0012] In still further embodiments of the gene therapy vector, the promoter is selected from the group consisting of hSYNI (human synapsin), INA (alpha-internexin), NES (nestin), hTH (human tyrosine hydroxylase), FOXA2 (forkhead box A2), CaMKII (calmodulin-dependent protein kinase II), NSE (neuron-specific enolase), CMV, CAG, UBC, PGK, EFl-alpha, GAPDH, SV40, HBV, chicken beta-actin, and human beta-actin promoters.

[0013] In yet further embodiments of the gene therapy vector, the vector further comprises one or more regulatory elements. In some embodiments, the one or more regulatory elements are selected from the group consisting of an enhancer, an intron, a polyA signal sequence, and a transcript stabilization element. In some embodiments, the enhancer is selected from the group consisting of a CMV enhancer, a GAPDH enhancer, a β-actin enhancer, and an EF1-α enhancer. In some embodiments, the transcript stabilization element is selected from the group consisting of a WPRE (woodchuck hepatitis virus posttranscriptional regulatory element) sequence, an HPRE (hepatitis posttranscriptional regulatory element) sequence, a scaffold binding region, a 3'UTR, and a 5'UTR.

[0014] In yet a further embodiment of the gene therapy vector, the vector comprises an expression cassette comprising, in 5' to 3' order: a) a CAG promoter and a polynucleotide encoding PINK1; b) a CAG promoter, a polynucleotide encoding PINK1, and a WPRE; c) an hTH promoter and a polynucleotide encoding PINK1; d) an EF1α promoter and a polynucleotide encoding PINK1; e) an EF1α promoter, a polynucleotide encoding PINK1, and a WPRE; f) a CBA promoter and a polynucleotide encoding PINK1; and g) a CBA promoter, a polynucleotide encoding PINK1, and a WPRE.

[0015] Further provided herein is a host cell comprising the gene therapy vector provided above.

[0016] In one embodiment of the host cell, the host cell is selected from the group consisting of HEK293, 293T, HeLa, Vero, and Sf9 cells.

[0017] Further provided herein is a method of increasing expression of PINK1 in a cell of a subject by contacting the cell with a recombinant gene therapy vector provided above.

[0018] In one embodiment of this method, the cell is a neuron, hi some embodiments, the neuron is a primary tyrosine hydroxylase-positive neuron.

[0019] In further embodiments of the method, the subject is a human subject. In some embodiments, the human subject comprises a mutation in the PINK1 gene. In some embodiments, the human subject has or is at risk of developing Parkinson's disease (PD). In some embodiments, the PD is early-onset PD. In some embodiments, the PD is early-onset autosomal recessive PD.

[0020] Further provided herein is a method for inhibiting, reducing, or delaying neuronal degeneration or death in a subject by contacting the neuron with a gene therapy vector according to any one of claims 1 to 12, thereby increasing the expression level of PINK1 in the neuron and inhibiting, reducing, or delaying neuronal dysfunction / degeneration or death. In one embodiment, the subject is a human subject. In one embodiment, the neuron is a primary tyrosine hydroxylase-positive neuron.

[0021] In one embodiment of the method, MPP+(1-methyl-4-phenylpyridinium)-induced neurodegeneration is attenuated in a subject.

[0022] In a further embodiment of the method, MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)-induced neuronal loss is prevented in the subject.

[0023] In yet a further embodiment of the method, the human subject comprises a mutation in the PINK1 gene.

[0024] In yet further embodiments of the method, the human subject has or is at risk of developing Parkinson's disease (PD). In some embodiments, the PD is early-onset PD. In some embodiments, the PD is early-onset autosomal recessive PD.

[0025] Further provided herein is a polynucleotide that encodes PINK1 and has at least about 90% identity to SEQ ID NO:2 or SEQ ID NO:3.

[0026] In one embodiment, the polynucleotide has at least 95% identity to SEQ ID NO:2 or SEQ ID NO:3.

[0027] In a further embodiment, the polynucleotide has at least 98% identity to SEQ ID NO:2 or SEQ ID NO:3.

[0028] In still further embodiments of the polynucleotide, the polynucleotide encoding PINK1 has at least 99% identity to SEQ ID NO:2 or SEQ ID NO:3.

[0029] In still further embodiments of the polynucleotide, the polynucleotide encoding PINK1 has the sequence of SEQ ID NO:2, or SEQ ID NO:3.

[0030] Further provided herein is a plasmid comprising the polynucleotide of any one of claims 32 to 36.

[0031] Further provided herein is a recombinant gene therapy vector comprising a mutant polynucleotide encoding PINK1, wherein cells transduced with the vector express PINK1 at a higher level compared to the level of PINK1 expressed in cells of the same type transduced with the same type of vector comprising a wild-type polynucleotide encoding PINK1.

[0032] Further provided herein is a method for increasing expression of PINK1 in a subject's cells, comprising contacting the cells with a recombinant gene therapy vector comprising a mutant polynucleotide encoding PINK1, wherein expression of PINK1 in the cells is increased compared to expression of PINK1 in the same type of cells contacted under the same conditions with the same amount of vector of the same type comprising a wild-type polynucleotide encoding PINK1.

[0033] Further provided herein is a method for inhibiting, reducing, or delaying neuronal dysfunction / degeneration or death in a subject, comprising contacting the neuron with a recombinant gene therapy vector comprising a mutant polynucleotide encoding PINK1, wherein expression of PINK1 in the neuron is increased compared to expression of PINK1 in the same type of neuron contacted under the same conditions with the same type and amount of vector comprising a wild-type polynucleotide encoding PINK1.

[0034] In one embodiment of the vector or method provided above, the variant polynucleotide is operably linked to a promoter.

[0035] In further embodiments of the vectors or methods provided above, the mutant polynucleotide and the wild-type polynucleotide are each operably linked to the same type of promoter.

[0036] In still further embodiments of the vector or method provided above, the vector is an adeno-associated virus (AAV).

[0037] In still further embodiments of the vectors or methods provided above, PINK1 is expressed at least about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2-fold more highly by cells or neurons contacted with or transduced with a vector comprising a mutant polynucleotide encoding PINK1 compared to cells or neurons contacted with or transduced with a vector comprising a wild-type polynucleotide encoding PINK1.

[0038] In still further embodiments of the vectors or methods provided above, the variant polynucleotide encoding PINK1 has at least about 70%, at least about 80%, at least about 85%, or at least about 90% identity to SEQ ID NO:2 or SEQ ID NO:3.

[0039] In still further embodiments of the vector or method provided above, the cell is a neuron.

[0040] In still further embodiments of the vectors or methods provided above, the neurons are selected from the group consisting of cortical neurons, ependymal cells, glutamatergic neurons, GABAergic neurons, dopaminergic neurons, oligodendrocytes, astrocytes, and microglial cells.

[0041] Further provided herein is a method for inhibiting, reducing, or slowing the degeneration or death of neurons in a subject, comprising contacting neurons with an amount of a recombinant gene therapy vector comprising a polynucleotide encoding PINK1, wherein the amount of vector is effective to inhibit, reduce, or slow the degeneration or death of neurons in the subject.

[0042] In one embodiment of this method, neurons are contacted with an amount of vector corresponding to a multiplicity of infection (MOI) of about 1 to about 10^5.

[0043] In a further embodiment of the method, the neurons are contacted with an amount of vector corresponding to an MOI of about 10 to about 10^4.

[0044] In still further embodiments of the method, the neurons are contacted with an amount of vector corresponding to an MOI of about 1, about 10, about 10^1, about 10^2, about 10^3, about 10^4, or about 10^5.

[0045] In yet a further embodiment of the method, the polynucleotide is operably linked to a promoter.

[0046] In yet a further embodiment of the method, the polynucleotide is a mutant polynucleotide.

[0047] In yet a further embodiment of the method, the vector is an adeno-associated virus (AAV).

[0048] In still further embodiments of the method, the variant polynucleotide encoding PINK1 has at least about 70%, at least about 80%, at least about 85%, or at least about 90% identity to SEQ ID NO:2 or SEQ ID NO:3.

[0049] In still further embodiments of the method, the neuron is selected from the group consisting of a cortical neuron, an ependymal cell, a glutamatergic neuron, a GABAergic neuron, a dopaminergic neuron, an oligodendrocyte, an astrocyte, and a microglial cell. [Brief explanation of the drawings]

[0050] [Figure 1A] 1 is a Western blot showing PINK1 mRNA expression levels in PINK1 KO HEK293T cells transduced with AAV1 CIS plasmids containing WT PINK1 (GT65) or PINK1 mutants (GT66, TG69, GT72, GT73, GT74, or GT75). [Figure 1B]1 is a graph showing relative PINK1 protein levels in PINK1 KO HEK293T cells transduced with AAV1 CIS plasmids containing WT PINK1 (GT65) or PINK1 mutants. [Figure 2A] Representative images from HEK293T wild-type and PINK1 knockout cells treated with DMSO vehicle or valinomycin drug before and after overexpression of AAV1-PINK1 are shown. Figure 2A shows immunocytochemistry of pS65-Ub in PINK1 WT and KO HEK293T cells treated with DMSO vehicle or valinomycin. [Figure 2B] Representative images are shown from HEK293T wild-type and PINK1 knockout cells treated with DMSO vehicle or valinomycin drug before and after overexpressing AAV1-PINK1. Figure 2B is a graph showing pS65-Ub readout in PINK1 WT and KO HEK293T cells overexpressing PINK1 WT or PINK1 mutants with or without valinomycin treatment. [Figure 3A] 3A is a graph showing the amount of LDH released in primary cortical neurons transduced with an AAV1 vector containing the PINK1 mutant GT67 (FIG. 3A). [Figure 3B] FIG. 3B is a graph showing the amount of LDH released in primary cortical neurons transduced with an AAV1 vector containing GT69 (FIG. 3B). [Figure 3C] FIG. 3C is a graph showing the amount of released LDH in primary cortical neurons transduced with an AAV1 vector containing GT74 (FIG. 3C). [Figure 3D] FIG. 3D is a graph showing the amount of LDH released in primary cortical neurons transduced with an AAV1 vector containing GT77 (FIG. 3D). [Figure 3E] FIG. 3C is a graph showing the amount of released LDH in primary cortical neurons transduced with an AAV1 vector containing GT79 (FIG. 3E). [Figure 4]1 is a graph showing the amount of LDH released in primary cortical neurons transduced with AAV1 vectors containing GT69 or GT74 at the indicated MOI compared to non-transduced neurons. [Figure 5] Figure 1 shows the in vivo expression of human PINK1 in rats transduced with AAV vectors containing PINK1 WT (Panel A), PINK1 mutant GT69 (Panel B), or PINK1 mutant GT74 (Panel C). Panel D shows human PINK1 mRNA levels in non-transduced rats. [Figure 6] Western blot and qPCR results from striatal samples are shown, respectively. [Figure 7] Western blot and qPCR results from striatal samples are shown, respectively. [Figure 8A] Dopamine (DA) levels in the striatum of wild-type and PINK1 KO rodents treated with various amounts of control and GT65 are shown. [Figure 8B] Dopamine (DA) levels in the striatum of wild-type and PINK1 KO rodents treated with various amounts of control and GT69 are shown. [Figure 9A] Figure 9A shows the levels of tyrosine hydroxylase (TH) in the midbrain of PINK1 KO rodents treated with various amounts of GT65 and GT69, as measured by mass spectrometry (Figure 9A) and Western blot (Figure 9B). [Figure 9B] Figure 9A shows the levels of tyrosine hydroxylase (TH) in the midbrain of PINK1 KO rodents treated with various amounts of GT65 and GT69, as measured by mass spectrometry (Figure 9A) and Western blot (Figure 9B). [Figure 10]Figures 10A-10D are pathway maps of WT rats, PINK1 KO rats, or PINK1 KO rats transduced with AAV containing the WT PINK1 gene (GT65) or mutant PINK1 gene (GT69). Figures 10E-10G are graphs showing changes in motor capacity for PINK1 KO rats after transduction with AAV containing the WT PINK1 gene (GT65) or mutant PINK1 gene (GT69). [Figure 11] In situ hybridization (ISH) and immunohistochemistry (IHC) of hPINK1 mRNA and protein, and canine tyrosine hydroxylase (TH) in dog brains following a single injection of an AAV1 vector containing GT69 into the substantia nigra are shown. DETAILED DESCRIPTION OF THE INVENTION

[0051] The present invention is described and claimed below, and it is well established that the subject matter described and claimed herein should be read and interpreted as of the time of filing of this patent application.

[0052] In the "Background" section and throughout this specification, various publications, articles, and patents are cited or described, and each of these references is incorporated herein by reference in its entirety. The discussion of documents, operations, materials, devices, articles and the like which is included in the specification is for the purpose of providing a context for the present invention. Such discussion is not an admission that any or all of these items constitute part of the prior art to any invention disclosed or claimed.

[0053] 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. Unless otherwise defined, certain terms used herein have the meanings described herein.

[0054] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0055] Unless otherwise specified, all numerical values, such as concentrations or concentration ranges, described herein should be understood in all instances to be modified by the term "about." Thus, numerical values ​​typically include ±10% of the stated value. For example, a concentration of 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Similarly, a concentration range of 1% to 10% (w / v) includes 0.9% (w / v) to 11% (w / v). As used herein, the use of numerical ranges explicitly includes all possible subranges, including integers and fractions of values ​​within that range, and all individual numerical values ​​within that range, unless the context clearly indicates otherwise.

[0056] Unless otherwise indicated, the term "at least" preceding a series of elements should be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0057] As used herein, the terms "comprises," "comprising," "includes," "including," "has," "having," "contains," or "containing," or any other variation thereof, are intended to include the stated element or elements, but not to exclude other elements or elements, and are understood to be non-exclusive or open-ended. For example, a composition, mixture, process, method, article, or device comprising a list of elements is not necessarily limited to only those elements, but may include other elements not expressly listed or inherent in such composition, mixture, process, method, article, or device. Further, unless expressly stated to the contrary, "or" refers to an inclusive "or," not an exclusive "or." For example, condition A or B is satisfied by one of the following: A is true (or exists) and B is false (or does not exist); A is false (or does not exist) and B is true (or exists); and both A and B are true (or exist).

[0058] As used herein, the connective term "and / or" between multiple listed elements is understood to encompass both individual and combined alternatives. For example, when two elements are connected by "and / or," the first alternative refers to the first element being applicable without the second element. The second alternative refers to the second element being applicable without the first element. The third alternative refers to the first element and the second element being applicable together. Any one of these alternatives is understood to be within the meaning and, therefore, meets the requirements of the term "and / or" as used herein. The simultaneous applicability of two or more of the alternatives is also understood to be within the meaning and, therefore, meets the requirements of the term "and / or."

[0059] As used herein, the term "consists of," or variations such as "consist of" or "consisting of," as used throughout the specification and claims, is inclusive of any listed element or elements, but indicates that no additional element or elements are added to the specified method, structure, or composition.

[0060] As used herein, the term "consists essentially of," or variations such as "consist essentially of" or "consisting essentially of," as used throughout the specification and claims, refers to the inclusion of any recited integer or group of integers, optionally including any recited integer or group of integers that does not materially alter the basic or novel characteristics of the specified method, structure, or composition of matter. See MPEP §2 111.03.

[0061] It should also be understood that terms such as "about," "approximately," "generally," and "substantially," as used herein when referring to dimensions or characteristics of preferred inventive components, indicate that the described dimensions / characteristics are not precise boundaries or parameters, but do not exclude minor variations therefrom that are functionally the same or similar, as would be understood by one of ordinary skill in the art. At a minimum, such references involving numerical parameters will include variations that do not change the least significant digit using mathematical and industrial principles accepted in the art (e.g., rounding, measurement or other systematic errors, manufacturing tolerances, etc.).

[0062] The term "identical" or percent "identity," in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that, when compared and aligned for maximum correspondence, are the same or have a specified percentage of the same amino acid residues or nucleotides, as determined using one of the following sequence comparison algorithms or by visual inspection. For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the designated program parameters.

[0063] Optimal alignment of sequences for comparison can be determined, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), the similarity search method of Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988), computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by visual inspection (see generally Current Protocols in Molecular Biology, FMAusubel et al., eds., Current Protocols, Greene Publishing Associates, Inc. and John This can be done through a collaboration with Wiley & Sons, Inc. (1995 Supplement) (see Ausubel).

[0064] Examples of suitable algorithms for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms described in Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match when aligned with words of the same length in database sequences or meet some positive threshold score T, referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased.

[0065] For nucleotide sequences, the cumulative score is calculated using the parameters M (reward score for a pair of matching residues, always >0) and N (penalty score for mismatching residues, always <0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of the word hits in each direction is stopped when the cumulative alignment score drops by an amount X from its maximum achieved value, when the accumulation of one or more negatively scoring residue alignments causes the cumulative score to fall below zero, or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, expectation (E) of 10, M of 5, N of -4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1992)).

[0066] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide sequences or two amino acid sequences will occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in the comparison of the test nucleic acid with the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

[0067] A further indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide; for example, the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions.

[0068] As used herein, the term "polynucleotide," also referred to interchangeably as "nucleic acid molecule," "nucleotide," or "nucleic acid," refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotide" includes, but is not limited to, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is a mixture of single- and double-stranded regions, and hybrid molecules containing DNA and RNA that may be single-stranded or, more typically, double-stranded, or a mixture of single- and double-stranded regions. In addition, "polynucleotide" refers to triple-stranded regions containing RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNA or RNA containing one or more modified bases and DNA or RNA with backbones modified for stability or other reasons. "Modified" bases include, for example, tritylated bases and unusual bases, such as inosine. Various modifications can be made to DNA and RNA. Thus, "polynucleotide" encompasses chemically, enzymatically, or metabolically modified forms of polynucleotides typically found in nature, as well as chemical forms characteristic of viral and cellular DNA and RNA. "Polynucleotide" also encompasses relatively short nucleic acid strands, often referred to as oligonucleotides.

[0069] As used herein, the terms "peptide," "polypeptide," or "protein" can refer to a molecule composed of amino acids and recognized as a protein by those skilled in the art. Conventional one-letter or three-letter codes for amino acid residues are used herein. The terms "peptide," "polypeptide," and "protein" can be used interchangeably herein to refer to polymers of amino acids of any length. The polymers may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. The term also encompasses amino acid polymers that are naturally modified or modified by intervention, e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, e.g., conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, e.g., unnatural amino acids), as well as other modifications known in the art.

[0070] The peptide sequences described herein are written according to the usual convention, with the N-terminal region of the peptide on the left and the C-terminal region on the right. Although isomeric forms of amino acids are known, unless expressly indicated otherwise, it is the L-form of the amino acid that is shown.

[0071] In one aspect, the present disclosure provides a recombinant gene therapy vector comprising an expression cassette for expressing the PTEN-induced putative kinase 1 (PINK1) protein (listed below, SEQ ID NO: 9), wherein the vector is an adeno-associated vector (AAV). In some embodiments, the expression cassette comprises a polynucleotide encoding PINK1 and is operably linked to a promoter.

[0072] A polynucleotide encoding PINK1 may comprise a sequence having at least about 70%, about 75%, 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO: 1 (corresponding to nucleotides 92-1834 of the human wild-type human PINK1 gene (NM_032409.3)). In some embodiments, a polynucleotide encoding PINK1 may also be a mutant polynucleotide derived from a modified wild-type human PINK1 gene. In some embodiments, a polynucleotide encoding PINK1 may comprise a sequence having at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to a sequence selected from SEQ ID NOs: 2-3. In some embodiments, a polynucleotide encoding PINK1 may comprise a sequence selected from SEQ ID NOs: 2-3. In some embodiments, a polynucleotide encoding PINK1 may comprise the sequence of SEQ ID NO: 2. In some embodiments, a polynucleotide encoding PINK1 may comprise the sequence of SEQ ID NO: 3.

[0073] As used herein, "mutant polynucleotide encoding PINK1," "mutant PINK1 gene," and "PINK1 mutant" are used interchangeably and refer to a polynucleotide sequence derived from an altered wild-type human PINK1 gene. In other words, a "mutant polynucleotide encoding PINK1," "mutant PINK1 gene," or "PINK1 mutant" contains at least one nucleotide change compared to the wild-type human PINK1 gene (SEQ ID NO: 1) and encodes a PINK1 protein (SEQ ID NO: 9).

[0074] SEQ ID NO: 1

[0075] SEQ ID NO: 2

[0076] SEQ ID NO: 3

[0077] SEQ ID NO:9 MAVRQALGRGLQLGRALLLRFTGKPGRAYGLGRPGPAAGCVRGERPGWAAGPGAEPRRVGLGLPNRLRFFRQSVAGLAARLQRQFVVRAWGCAGPCGRAVFLAFGLGLGLIEEKQAESRRAVSACQEIQAIFTQKSKPGPDPLDT RRLQGFRLEEYLIGQSIGKGCSAAVYEATMPTLPQNLEVTKSTGLLPGRGPGTSAPGEGQERAPGAPAFPLAIKMMWNISAGSSSEAILNTMSQELVPASRVALAGEYGAVTYRKSKRGPKQLAPHPNIIRVLRAFTSSVPLLPG ALVDYPDVLPSRLHPEGLGGHGRTLFLVMKNYPCTLRQYLCVNTPSPRLAAMMLLQLLEGVDHLVQQGIAHRDLKSDNILVELDPDGCPWLVIADFGCCLADESIGLQLPFSSWYVDRGGNGCLMAPEVSTARPGPRAVIDYSKAD AWAVGAIAYEIFGLVNPFYGQGKAHLESRSYQEAQLPALPESVPPDVRQLVRALLQREASKRPSARVAANVLHLSLWGEHILALKNLKLDKMVGWLLQQSAATLLANRLTEKCCVETKMKMLFLANLECETLCQAALLLCSWRAAL

[0078] Adeno-associated virus (AAV) is a single-stranded DNA parvovirus that grows only in cells in which certain functions are provided by a co-infecting helper virus. General information and reviews of AAV can be found, for example, in Carter, 1989, Handbook of Parvoviruses, Vol. 1, pp. 169-228, and Bems, 1990, Virology, pp. 1743-1764, Raven Press, New York. The same principles described in these reviews are fully expected to be applicable to additional AAV serotypes characterized after the publication date of these reviews, since the various serotypes are well known to be very closely related structurally, functionally, and at the genetic level (see, for example, Blacklowe, 1988, pp. 165-174 of Parvoviruses and Human Disease, J.R.P.Tattison, ed.; and Rose, Comprehensive Virology 3:1-61 (1974)). For example, all AAV serotypes clearly exhibit very similar replication properties mediated by homologous rep genes, and all possess three related capsid proteins, such as those expressed in AAV2. The extent of relatedness is further suggested by heteroduplex analysis, which reveals extensive cross-hybridization between serotypes along the length of the genome and the presence of similar self-annealing segments at the ends corresponding to the "inverted terminal repeats" (ITRs). Similar infectivity patterns also suggest that the replication functions in each serotype are under similar regulatory control.

[0079] As used herein, "AAV vector" or "rAAV vector" refers to a recombinant vector containing one or more polynucleotides of interest (or transgenes) flanked by AAV interterminal repeats (ITRs). Such AAV vectors can replicate and be packaged into infectious viral particles when present in a host cell transfected with a plasmid encoding and expressing the rep and cap gene products. Alternatively, AAV vectors can be packaged into infectious particles using host cells stably engineered to express the rep and cap genes.

[0080] As used herein, "AAV virion," or "AAV virus particle," or "AAV vector particle" refers to a viral particle composed of at least one AAV capsid protein and an encapsidated polynucleotide AAV vector. As used herein, when a particle contains a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, such as a transgene delivered to a mammalian cell), it is typically referred to as an "AAV vector particle," or simply an "AAV vector." Thus, the production of an AAV vector particle necessarily includes the production of an AAV vector, and such a vector is contained within the AAV vector particle.

[0081] AAV is a replication-deficient parvovirus, whose single-stranded DNA genome is approximately 4.7 kb in length, containing two 145-nucleotide inverted terminal repeats (ITRs). There are multiple known variants of AAV, which are sometimes called serotypes when classified by antigenic epitopes. The nucleotide sequences of the genomes of AAV serotypes are known. For example, the complete genome of AAV-1 is provided in GenBank accession number NC_002077; the complete genome of AAV-2 is provided in GenBank accession number NC_001401, and in Srivastava et al., J. Virol., 45:555-564 (1983); the complete genome of AAV-3 is provided in GenBank accession number NC_1829; the complete genome of AAV-4 is provided in GenBank accession number NC_001829; the AAV-5 genome is provided in GenBank accession number AF085716; the complete genome of AAV-6 is provided in GenBank accession number NC_001862; at least portions of the AAV-7 and AAV-8 genomes are provided in GenBank accession numbers AX753246 and AX753249, respectively; and the AAV-9 genome is provided in Gao et al. al., J. Virol., 78:6381-6388 (2004); the AAV-10 genome is provided in Mol. Ther., 13(1):67-76 (2006); and the AAV-11 genome is provided in Virology, 330(2):375-383 (2004). The sequence of the AAVrh.74 genome is provided in U.S. Patent No. 9,434,928, incorporated herein by reference. Cis-acting sequences that direct viral DNA replication (rep), encapsidation / packaging, and host cell chromosomal integration are contained within the AAV ITRs. Three AAV promoters (designated p5, p19, and p40 because of their relative map positions) drive expression of two AAV internal open reading frames encoding the rep and cap genes.Two rep promoters (p5 and p19), combined with differential splicing of a single AAV intron (nucleotides 2107 and 2227), result in the production of four rep proteins (rep78, rep68, rep52, and rep40) from the rep gene. The rep proteins have multiple enzymatic properties that are ultimately responsible for replicating the viral genome. The cap gene is expressed from the p40 promoter and encodes three capsid proteins, VP1, VP2, and VP3. Alternative splicing and non-consensus translation initiation sites are responsible for the production of the three related capsid proteins. A single consensus polyadenylation site is located at map position 95 of the AAV genome. The AAV life cycle and genetics are reviewed in Muzyczka, Current Topics in Microbiology and Immunology, 158:97-129 (1992).

[0082] AAV has unique features that make it attractive as a vector for delivering foreign DNA to cells, for example, in gene therapy. AAV infection of cells in culture is noncytopathic, and natural infection in humans and other animals is asymptomatic and asymptomatic. Furthermore, AAV infects many mammalian cell types, enabling the possibility of targeting many different tissues in vivo. Furthermore, AAV can slowly transduce dividing and non-dividing cells and persist essentially throughout the lifespan of these cells as transcriptionally active nuclear episomes (extrachromosomal elements). The AAV proviral genome is inserted as cloned DNA into plasmids, allowing the construction of recombinant genomes. Furthermore, because signals directing AAV replication and genome encapsidation are contained within the ITRs of the AAV genome, part or all of the internal approximately 4.3 kb region of the genome (encoding the replication and structural capsid protein, rep-cap) can be replaced with foreign DNA. To generate AAV vectors, the rep and cap proteins can be provided in trans. Another important feature of AAV is that it is an extremely stable and robust virus. AAV easily withstands the conditions used to inactivate adenovirus (56°C to 65°C for several hours). This reduces the importance of cryopreservation of AAV. AAV may also be lyophilized. Finally, AAV-infected cells are not resistant to superinfection.

[0083] The AAV DNA in the rAAV genome can be derived from any AAV variant or serotype from which a recombinant virus can be derived, including, but not limited to, AAV variants or serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, and AAVrhlO. The production of pseudotyped rAAV is disclosed, for example, in WO 01 / 83692. Other types of rAAV variants, such as rAAV with capsid mutations, are also contemplated. See, for example, Marsic et al., Molecular Therapy, 22(11):1900-1909 (2014). The nucleotide sequences of the genomes of various AAV serotypes are known in the art.

[0084] In some cases, rAAVs contain self-complementary genomes. As defined herein, rAAVs containing a "self-complementary" or "double-stranded" genome refer to rAAVs whose coding regions have been engineered to form an intramolecular double-stranded DNA template, as described in McCarty et al. "Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis," Gene Therapy, 8(16):1248-54 (2001). The present disclosure contemplates the use of rAAVs containing self-complementary genomes, in some cases, because upon infection (such transduction), rather than waiting for cell-mediated synthesis of the second strand of the rAAV genome, the two complementary halves of the scAAV assemble to form a single double-stranded DNA (dsDNA) unit that is ready for immediate replication and transcription. It will be appreciated that instead of the full coding capacity (4.7-6 kb) found in rAAV, rAAV containing self-complementary genomes may only retain about half that amount (=2.4 kb).

[0085] The full-length sequence of most known AAVs, as well as the sequence of the capsid gene, is provided in US Pat. No. 8,524,446, which is incorporated herein by reference in its entirety.

[0086] The AAV vector may comprise a wild-type AAV sequence or may comprise one or more modifications to the wild-type AAV sequence. In certain embodiments, the AAV vector comprises one or more amino acid modifications, e.g., substitutions, deletions, or insertions, in capsid proteins, e.g., VP1, VP2, and / or VP3. In certain embodiments, the modifications provide reduced immunogenicity when the AAV vector is administered to a subject.

[0087] According to the present disclosure, the AAV used herein is a wild-type AAV or a modified AAV. In some embodiments, the AAV used herein comprises a capsid protein having at least 95% identity to the wild-type VP1, VP2, or VP3 capsid protein. In some embodiments, the AAV used herein is AAV1.

[0088] According to the present disclosure, the polynucleotide encoding PINK1 is operably linked to a promoter. In some embodiments, the promoter is a eukaryotically active promoter capable of promoting the initiation of RNA transcription from the polynucleotide in eukaryotic cells. In some embodiments, the promoter is a tissue-specific promoter, such as a promoter capable of driving expression to a greater extent in neuronal cells than in non-neuronal cells. Suitable neuron-specific promoters include, but are not limited to, hSYNI (human synapsin), INA (α-internexin), NES (nestin), hTH (human tyrosine hydroxylase), FOXA2 (forkhead box A2), CaMKII (calmodulin-dependent protein kinase II), Prnp (prion protein promoter), Hb9, and NSE (neuron-specific enolase) promoters. In some cases, the promoter is a ubiquitous promoter. A "ubiquitous promoter" refers to a promoter that is not tissue-specific under experimental or clinical conditions. As used herein, suitable ubiquitous promoters include, but are not limited to, CMV, CAG, UBC, PGK, EFl-α, EFS (human eukaryotic translation elongation factor 1 α1 short form), SFFV (spleen focus forming virus promoter), GAPDH, SV40, HBV, chicken β-actin, and human β-actin promoters.

[0089] In some embodiments, the polynucleotide encoding PINK1 is operably linked to the hSYNI promoter (SEQ ID NO: 4).

[0090] SEQ ID NO:4 CTGCAGAGGGCCCTGCGTATGAGTGCAAGTGGGTTTTAGGACCAGGATGAGGCGGGGTGGGGGTGCCTACCTGACGACCGACCCCGACCCACTGGACAAGCACCCAACCCCCATTCCCCAAATTGCGCATCCCCTATCAGAGAGGGGGAGGGGAAACAGGATGCGGCGAGGCGCGTGCGCACTGCCAGCTTCAGCACCGCGGACAGTGCCTTCGCCCCCGCCTGGCGGCGCGCG CCACCGCCGCTCAGCACTGAAGGCGCCTGACGTCACTCGCCGGTCCCCGCAAACTCCCCTTCCCGGCCACCTTGGTCGCGTCCGCGCCGCCGCCGGCCCAGCCGGACCGCACCACGCGAGGCGCGAGATAGGGGGGCACGGGCGCGACCATCTGCGCTGGCGGCGCCGGCGACTCAGCGCTGCCTCAGTCTGCGGTGGGCAGCGGAGGAGTCGTGTCGTGCCTGAGAGCGCAG

[0091] In some embodiments, the polynucleotide encoding PINK1 is operably linked to a CAG promoter (SEQ ID NO: 5).

[0092] SEQ ID NO:5

[0093] The recombinant gene therapy vectors disclosed herein may further comprise one or more regulatory elements, such as an enhancer, an intron, a polyA signal sequence, and a transcript stabilization element.

[0094] In some embodiments, the transcript stabilization element can be a WPRE (woodchuck hepatitis virus post-transcriptional regulatory element) sequence, an HPRE (hepatitis post-transcriptional regulatory element) sequence, a scaffold binding region, a 3'UTR, or a 5'UTR. In some embodiments, the recombinant gene therapy vectors disclosed herein comprise both a 5'UTR and a 3'UTR.

[0095] Suitable enhancers for use herein include, but are not limited to, a CMV enhancer, a GAPDH enhancer, a β-actin enhancer, or an EF1-α enhancer.

[0096] In some embodiments, the recombinant gene therapy vectors disclosed herein comprise one or more intron sequences. For example, the intron sequence can be selected from a rabbit globin intron sequence, a chicken β-actin intron sequence, a synthetic intron sequence, or an EF1-α intron sequence.

[0097] In some embodiments, the recombinant gene therapy vectors disclosed herein comprise a polyA signal sequence. For example, the polyA signal sequence is a rabbit globin polyA signal sequence, a human growth hormone polyA signal sequence, a bovine growth hormone polyA signal sequence, a PGK polyA signal sequence, an SV40 polyA signal sequence, or a TK polyA signal sequence. In some embodiments, the polyA signal sequence can be a bovine growth hormone polyA signal sequence.

[0098] In some embodiments of the recombinant gene therapy vectors disclosed herein, the expression cassette may include an additional polynucleotide encoding a protein other than PINK1, and the polynucleotide encoding PINK1 and the additional polynucleotide are linked by a linker. Suitable linkers include, but are not limited to, an IRES (internal ribosome entry site) and a 2A peptide coding sequence.

[0099] In various embodiments of the recombinant gene therapy vectors disclosed herein, the expression cassette comprises, in 5' to 3' order: a CAG promoter, and a polynucleotide encoding PINK1; CAG promoter, a polynucleotide encoding PINK1, and WPRE; an hTH promoter, and a polynucleotide encoding PINK1; an EF1α promoter, and a polynucleotide encoding PINK1; EF1α promoter, a polynucleotide encoding PINK1, and WPRE; a CBA promoter and a polynucleotide encoding PINK1; or It comprises a CBA promoter, a polynucleotide encoding PINK1, and a WPRE.

[0100] Also within the scope of the present disclosure, recombinant gene therapy vectors include mutant polynucleotides encoding PINK1 and exhibit higher levels of PINK1 expression, i.e., cells transduced with the vector express PINK1 at a higher level compared to the level of PINK1 expressed in cells of the same type transduced with the same type of vector containing a wild-type polynucleotide encoding PINK1.

[0101] In a further aspect, the present disclosure provides a host cell comprising any of the recombinant gene therapy vectors provided above. Exemplary host cells include, but are not limited to, HEK293, 293T, HeLa, Vero, and Sf9 cells.

[0102] In still a further aspect, the disclosure provides a variant polynucleotide that encodes PINK1 and has the sequence of SEQ ID NO:2 or 3, or has at least about 70%, about 75%, 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% identity to SEQ ID NO:2 or 3. In some embodiments, the variant polynucleotide has at least 90% identity to SEQ ID NO:2 or 3. In some embodiments, the variant polynucleotide has at least 95% identity to SEQ ID NO:2 or 3. In some embodiments, the variant polynucleotide has at least 98% identity to SEQ ID NO:2 or 3. In some embodiments, the variant polynucleotide has at least 99% identity to SEQ ID NO:2 or 3.

[0103] In a still further aspect, the disclosure provides a plasmid comprising a mutant polynucleotide encoding PINK1, as described above.

[0104] In still a further aspect, the present disclosure provides methods for increasing PINK1 expression in a cell (e.g., a neuron) in a subject by contacting the cell with a recombinant gene therapy vector as provided above. The method can be performed in vitro or in vivo, e.g., in a subject in need thereof. In some embodiments, the subject is a human subject. In some embodiments, the human subject has or is at risk of developing PD. In some embodiments, the PD is early-onset PD. In some embodiments, the PD is early-onset autosomal recessive PD. In some embodiments, the human subject comprises a mutation in the PINK1 gene. In some embodiments, the neuron is a primary tyrosine hydroxylase-positive neuron. As shown in Example 2, when a gene therapy vector containing the PINK1 gene (wild-type or mutant) was transduced into PINK1 knockout (KO) HEK 293T cells, both PINK1 protein and mRNA expression were detected. Furthermore, compared with cells transduced with a vector containing the WT PINK1 gene (SEQ ID NO: 1), cells transduced with a vector containing the PINK1 mutant GT69 (SEQ ID NO: 2) or GT74 (SEQ ID NO: 3) exhibit higher protein and mRNA expression levels of PINK1. Furthermore, in Example 3, elevated PINK1 function was detected in PINK1 KO HEK 293T cells transduced with a vector containing the PINK1 mutant GT69 or GT74.

[0105] In a still further aspect, the disclosure provides a method for increasing expression of PINK1 in a cell in a subject, the method comprising contacting the cell with a recombinant gene therapy vector comprising a mutant polynucleotide encoding PINK1, as described above, wherein expression of PINK1 in the cell is increased compared to expression of PINK1 in a cell of the same type contacted with the same amount of the same type of vector comprising a wild-type polynucleotide encoding PINK1 under the same conditions.

[0106] In still a further aspect, the present disclosure provides methods of inhibiting, reducing, or delaying neuronal degeneration or death by contacting a neuron with a recombinant gene therapy vector as provided above. The methods can be performed in vitro or in vivo, e.g., in a subject in need thereof. In some embodiments, the subject is a human subject. In some embodiments, the human subject has or is at risk of developing PD. In some embodiments, the PD is early-onset PD. In some embodiments, the PD is early-onset autosomal recessive PD. In some embodiments, the human subject comprises a mutation in the PINK1 gene. In some embodiments, the neuron is a primary tyrosine hydroxylase-positive neuron. In some embodiments, MPP+(1-methyl-4-phenylpyridinium)-induced neurodegeneration is attenuated. In some embodiments, MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)-induced neuronal loss is prevented. As shown in Examples 4-5, cortical neurons transduced with vectors containing PINK1 mutants (GT69 or GT74) exhibit reduced levels of LDH release compared to untransduced cells.

[0107] Administration of the recombinant gene therapy vector or a composition comprising the recombinant gene therapy vector can be by routes standard in the art, including, but not limited to, systemic administration, local administration, direct injection, parenteral administration, intravenous administration, brain administration, cerebrospinal administration, intrathecal administration, intracisternal administration, intraputaminal administration, intrahippocampal administration, intranigra administration, intrastriatal administration, or intraventricular administration. In some cases, administration includes intravenous, cerebral, cerebrospinal, intrathecal, intracisternal, intraputaminal, intrahippocampal, intrastriatal, or intraventricular injection.

[0108] In some embodiments, the present disclosure provides for local and systemic administration of a therapeutically effective amount of a recombinant gene therapy vector disclosed herein. For example, systemic administration can be administration into the circulatory system so that the entire body is affected. Systemic administration includes enteral administration, such as absorption through the digestive tract, and parenteral administration via injection, infusion, or implantation.

[0109] In some embodiments, the actual administration of the recombinant gene therapy vectors disclosed herein can be achieved by using any physical method that delivers the vector to the target tissue of the subject. Suitable routes of administration include, but are not limited to, injection into the central nervous system (CNS) or cerebrospinal fluid (CSF), and / or direct injection into the brain.

[0110] The capsid protein of a recombinant gene therapy vector can be modified to target the vector to a specific target tissue of interest, such as neurons, or more specifically, dopaminergic neurons. See, e.g., Albert et al. AAV Vector-Mediated Gene Delivery to Substantia Nigra Dopamine Neurons: Implications for Gene Therapy and Disease Models. Genes. 2017 Feb. 8; see also U.S. Patent No. 6,180,613 and U.S. Patent Publication No. US20120082650(A1), which are incorporated by reference in their entireties. In some embodiments, the recombinant gene therapy vector is injected directly into the substantia nigra of a subject.

[0111] For example, for purposes of administration by injection, various solutions can be used, such as sterile aqueous solutions. Such aqueous solutions can be buffered, if desired, and the liquid diluent is first made isotonic with physiological saline or glucose. Solutions of the vector as a free acid (DNA contains acidic phosphate groups) or pharmacologically acceptable salt can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions of the vector can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, as well as in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. In this regard, the sterile aqueous media employed are all readily available by standard techniques well known to those skilled in the art.

[0112] Pharmaceutical forms suitable for injectable use include, for example, sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the composition must be sterile and must be fluid to the extent that easy syringability exists. The composition must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0113] Sterile injectable solutions can be prepared by incorporating the required amount of recombinant gene therapy vector into an appropriate solvent with various other ingredients as enumerated above, as needed, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the sterilized active ingredient into a sterile vehicle containing a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for preparing sterile injectable solutions, certain preferred preparation methods are vacuum drying and freeze-drying techniques, which yield a powder of the active ingredient and any additional desired ingredients from a previously sterile-filtered solution thereof.

[0114] Transduction with a recombinant gene therapy vector can also be performed in vitro. In one embodiment, the desired target cells are removed from a subject, transduced with a recombinant gene therapy vector, and reintroduced into the subject.

[0115] Suitable methods for transduction and reintroduction of transduced cells into a subject are known in the art. In one embodiment, cells can be transduced in vitro, for example, by combining a recombinant gene therapy vector with the cells in a suitable medium and screening for cells harboring the DNA of interest using conventional techniques such as Southern blot and / or PCR, or by using a selectable marker. The transduced cells can then be formulated into a pharmaceutical composition, and the composition can be introduced into a subject by various techniques (e.g., systemic administration, local administration, direct injection, parenteral administration, intravenous administration, intracerebral administration, cerebrospinal administration, intrathecal administration, intracisternal administration, intraputaminar administration, intrahippocampal administration, intrastriatal administration, or intracerebroventricular administration). In some cases, administration includes intravenous, cerebral, cerebrospinal, intrathecal, intracisternal, intraputaminar, intrahippocampal, intrastriatal, or intracerebroventricular injection. Administration can be performed by intrathecal injection on the Trendelenburg slope.

[0116] Transduction of cells with the recombinant gene therapy vectors disclosed herein results in sustained or increased expression of PINK1. Accordingly, provided herein are methods of administering or delivering a recombinant gene therapy vector expressing PINK1 to a mammalian subject, preferably a human. These methods include transducing a tissue (including, but not limited to, brain tissue) with the recombinant gene therapy vectors disclosed herein. Transduction can be carried out using a gene expression cassette containing tissue-specific control elements. For example, in one embodiment, transduction of neuronal cells and brain tissue is directed by neuron-specific control elements (including, but not limited to, those derived from neuron-enriched promoters and other control elements).

[0117] In still a further aspect, the present disclosure provides a recombinant gene therapy vector comprising a mutant polynucleotide encoding PINK1, wherein cells transduced with the vector express PINK1 at a higher level compared to the level of PINK1 expressed in cells of the same type transduced with the same type vector comprising a wild-type polynucleotide encoding PINK1.

[0118] In some embodiments, cells are contacted with a recombinant gene therapy vector comprising a mutant polynucleotide encoding PINK1, thereby increasing the expression of PINK1 in the cells compared to the expression of PINK1 in cells of the same type contacted with the same amount of the same type of vector comprising a wild-type polynucleotide encoding PINK1 under the same conditions.

[0119] In some embodiments, neurons of a subject are contacted with a recombinant gene therapy vector comprising a mutant polynucleotide encoding PINK1, thereby increasing the expression of PINK1 in the neurons compared to the expression of PINK1 in neurons of the same type contacted with the same amount of a vector of the same type comprising a wild-type polynucleotide encoding PINK1 under the same conditions, thereby inhibiting, reducing, or delaying neuronal dysfunction / degeneration or death in the subject.

[0120] In some embodiments, the mutant polynucleotide and / or the wild-type polynucleotide are operably linked to a promoter.

[0121] In some embodiments, the vector is an adeno-associated virus (AAV).

[0122] In some embodiments, PINK1 is expressed at least about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2-fold more highly by cells or neurons contacted with or transduced with a vector comprising a mutant polynucleotide encoding PINK1 compared to cells or neurons contacted with or transduced with a vector comprising a wild-type polynucleotide encoding PINK1.

[0123] In some embodiments, the variant polynucleotide encoding PINK1 has at least about 70%, at least about 80%, at least about 85%, or at least about 90% identity to SEQ ID NO:2 or SEQ ID NO:3.

[0124] In some embodiments, the cell is a neuron and may be selected from a cortical neuron, an ependymal cell, a glutamatergic neuron, a GABAergic neuron, a dopaminergic neuron, an oligodendrocyte, an astrocyte, and a microglial cell.

[0125] In still a further aspect, the disclosure provides a method for inhibiting, reducing, or delaying degeneration or death of a neuron in a subject, the method comprising contacting the neuron with an amount of a recombinant gene therapy vector comprising a polynucleotide encoding PINK1, wherein the amount of vector is effective to inhibit, reduce, or delay degeneration or death of the neuron in the subject.

[0126] In some embodiments, neurons are contacted with an amount of vector corresponding to a multiplicity of infection (MOI) of about 1 to about 10^5, or about 10 to about 10^4, or about 1, or about 10, or about 10^1, or about 10^2, or about 10^3, or about 10^4, or about 10^5.

[0127] In some embodiments, the polynucleotide is operably linked to a promoter.

[0128] In some embodiments, the polynucleotide is a variant polynucleotide encoding PINK1, and the variant polynucleotide encoding PINK1 has at least about 70%, at least about 80%, at least about 85%, or at least about 90% identity to SEQ ID NO:2 or SEQ ID NO:3.

[0129] In some embodiments, the vector is an adeno-associated virus (AAV).

[0130] In some embodiments, the cell is a neuron, and the neuron may be selected from a cortical neuron, an ependymal cell, a glutamatergic neuron, a GABAergic neuron, a dopaminergic neuron, an oligodendrocyte, an astrocyte, and a microglial cell. [Example]

[0131] Those skilled in the art will appreciate that changes can be made to the embodiments described above without departing from the broad inventive concept. It is therefore understood that the invention is not limited to the particular embodiments disclosed, but is intended to encompass modifications within the spirit and scope of the invention as defined by this specification. It is also well established that the subject matter described and claimed herein should be viewed and interpreted at the time of the filing of this patent application.

[0132] Example 1: Design of human PINK1 gene mutants Sixteen human PINK1 gene variants were designed using various design algorithms (see Table 1) and cloned into an AAV1 vector (GT0065 is wild-type PINK1 (WT PINK1)).

[0133] [Table 1]

[0134] SEQ ID NO:6

[0135] SEQ ID NO:7

[0136] SEQ ID NO:8

[0137] Example 2: Protein expression screening AAV1 CIS plasmids for WT PINK1 (GT65) and 16 PINK1 mutants (GT66–GT81) were generated. The plasmids were then tested for protein and mRNA expression in PINK1 knockout (KO) human embryonic kidney (HEK) 293T cells. Figure 1A shows the relative PINK1 protein levels of six representative PINK1 mutant constructs in PINK1 KO HEK293T cells. PINK1 KO HEK293T cells were cultured at the same cell density for 24 hours. The following day, these cells were treated with plasmid DNA containing the six different PINK1 mutants at concentrations of 0.3 micrograms and 1 microgram. The treated cells were incubated for 48 hours and then harvested for protein estimation using Western blot. Specifically, cells were lysed, and the lysates were subjected to electrophoresis to separate proteins by size and charge. The separated proteins were then queried with an antibody specific for PINK1 protein and detected by autoradiography. A housekeeping protein, actin, was used as an internal control to normalize protein levels across samples. PINK1 and actin protein bands were measured for intensity using imaging software and plotted as shown in Figure 1B. Figure 1B shows a representative Western blot analysis of 16 PINK1 mutant constructs overexpressed in PINK1 KO HEK293T cells. The graph represents the relative enrichment of PINK1 protein compared to GT65 (WT PINK1), plotted in ascending order of expression. Similar experiments were repeated three times. Statistical analysis was performed using GraphPad Prism software (one-way ANOVA, p<0.0001).

[0138] Example 3: Functional Assay of Selected PINK1 Mutants. This dataset demonstrates that the AAV1 CIS plasmids selected from the expression data above are functional in the cells expressing them. Figure 2A shows representative images from HEK293T wild-type and PINK1 knockout cells treated with DMSO vehicle or the drug valinomycin before and after overexpressing AAV1-PINK1. Valinomycin is a mitochondrial stressor that stabilizes the PINK1 protein on the surface of mitochondria and induces a PINK1-specific modification on another protein called ubiquitin. This modification, called pS65-Ub, can be detected by immunocytochemistry (Figure 2A) and mesoscale discovery (MSD, Figure 2B). Assaying pS65-Ub is a direct readout of PINK1 function. Both HEK293T wild-type and KO cells overexpressing AAV1-PINK1 show very strong pS65-Ub signals after valinomycin treatment (last panel) compared to DMSO (first and third panels) or background levels (second panel). Overexpression of PINK1 mutants was performed as previously described.

[0139] The five selected PINK1 mutants (GT67, GT69, GT74, GT77, and GT79), along with WT PINK1 (GT65), were further tested in a PINK1-specific functional assay measuring PINK1 phosphorylation of ubiquitin at serine-65 (p-S65-Ub) after induction of mitophagic stress with valinomycin. Figure 2B shows representative data from pS65-Ub MSD assays in PINK1 WT and KO HEK293T cells overexpressing the five PINK1 mutants (GT67, GT69, GT74, GT77, and GT79) compared to WT PINK1 (GT0065). Blue bars represent pS65-Ub levels under normal, or unstressed, conditions, while red bars indicate stable pS65-Ub levels in response to PINK1 overexpression and function.

[0140] Example 4: Neuronal survival assessment Neurotoxin rescue experiments were performed in primary neuronal cultures. LDH release assays measure cytotoxicity in cortical neurons transduced with AAV1-PINK1 vectors. Shown here are the amounts of LDH released into the medium for vectors containing one of five selected PINK1 mutants compared with the WT PINK1 vector ("GT65") after neuronal cultures were challenged with MPP+ (500 μM) at the indicated MOI on day 7 posttransduction. Embryonic day 18 (E18) rat cortices were dissociated using standard protocols, plated on PDL-coated 96-well plates, and incubated at 37°C for 7 days. E18 neurons were then transduced with AAV1 vectors containing one of the five PINK1 mutants or WT PINK1 at multiplicities of infection (MOI) ranging from 10 to 10^4 viral genomes per cell. Cells were incubated for an additional 7 days, and aliquots of medium were collected from all wells as pretreatment controls. The neurotoxin MPP+ was added at a concentration of 500 μM, and cells were incubated with the toxin for 24 hours. Medium containing LDH (released due to cell death) was collected after MPP+ treatment, and LDH levels were measured for pre- and post-treatment conditions using a commercially available colorimetric assay. Graphs represent the amount of LDH released from the various treatment conditions, as identified on the x-axis for GT67 (Figure 3A), GT69 (Figure 3B), GT74 (Figure 3C), GT77 (Figure 3D), and GT79 (Figure 3E). AAV1.rPINK1 (rPINK1 = rat PINK1; MOI = 10^4) is a positive control that demonstrates cell death rescue, i.e., less LDH release compared to the control (first two bars). In Figures 3A and 3B, the control was rat cortical neurons transduced with an empty AAV1 vector, whereas in Figures 3C, 3D, and 3E, the control was untransduced cortical neurons. Only GT69 (Figure 3B) and GT74 (Figure 3C) showed effects similar to those of the positive control at lower MOIs.

[0141] Example 5: Batch-to-batch reproducibility of the effects of GT69 and GT74 on neuronal survival AAV1 vectors containing GT69 and GT74 performed well in transducing neurons and attenuating cell death after toxin challenge, suggesting reproducibility across production sites with these sequences. Figure 4 shows an LDH release assay to measure cytotoxicity as described above in primary cortical neurons transduced with GT69 or GT74 at the indicated MOI. Statistical results from one-way ANOVA are presented. * Shown in.

[0142] Example 6: In vivo PINK1 expression in rats Robust in vivo PINK1 expression is achieved after direct injection of AAVs encoding all three constructs into the WT rat brain (Figure 5). Adult WT rats were stereotactically injected directly into their midbrains with 6e9 GCs, GT65 (A), GT69 (B), or GT74 (C), at a fixed volume of 4 μL per injection (n=4 per vector). Three weeks later, brains were collected and processed for in situ hybridization (ISH) to determine human PINK1 mRNA levels (top image) and immunohistochemistry (bottom image). A representative image of a whole coronal brain slice from an injected hemisphere is shown on the left (scale bar = 1 mm), and the image of the red-outlined box on the right is enlarged (scale bar = 100 μM). (D) This IHC method was optimized to detect only human PINK1; therefore, no signal is detected in uninjected WT rats. Furthermore, Figures 6 and 7 show Western blot and qPCR results, respectively, from striatal samples, which demonstrate that GT69 and GT74 are as effective as or better than GT65 in inducing human PINK1 protein expression in the striatum of WT rats.

[0143] At 12 months of age, Pink1 KO mice are known to have increased dopamine (DA) levels in the striatum compared with WT controls (compare the checkerboard pattern vs. the solid gray line in Figures 8A and 8B). This is thought to be the result of presynaptic compensation resulting from nigrostriatal injury. Treatment with GT69 at all tested doses resulted in dopamine levels that were significantly different from PINK1 KO controls without any AAV treatment at 6 months postinjection (Figure 8B). Meanwhile, the AAV1.wtPINK1 vector (GT65) at the matched dose level did not significantly reduce the increased striatal DA levels to near WT control levels (Figure 8A), although there was a non-significant trend toward correction at the highest dose tested. These results demonstrate that GT69 normalizes dopamine levels in striatal samples from PINK1 KO rats.

[0144] Furthermore, GT69 rescued TH loss at lower doses than GT65 in midbrain samples from PINK1 KO rats. Tyrosine hydroxylase (TH) is a marker of dopaminergic neurons. In 12-month-old Pink1 KO rats, the minimum effective dose that significantly increased midbrain TH levels was 5 e9 vg for GT65, whereas GT69 rescued TH loss at a dose of 2 e9. Similar results were obtained by mass spectrometry (Figure 9A) and Western blot (Figure 9B).

[0145] Example 7: Motor Skills Figures 10A-10D show pathway maps of WT rats, PINK1 KO rats, or PINK1 KO rats transduced with AAV containing the WT PINK1 gene (GT65) or mutant PINK1 gene (GT69). Figures 10E-10G show graphs showing changes in motor capacity for PINK1 KO rats after transduction with AAV containing the WT PINK1 gene (GT65) or mutant PINK1 gene (GT69).

[0146] Example 8: In vivo PINK1 expression in the dog brain In this example, a single injection of an AAV1 vector containing GT69 was given into the substantia nigra of the dog brain, resulting in robust hPINK1 transgene expression in that region. As shown in Figure 11, in situ hybridization (ISH) and immunohistochemistry (IHC) for hPINK1 mRNA and protein, and canine tyrosine hydroxylase (TH), confirmed the desired transduction pattern in the dog brain. These images are representative images of dog midbrain sections (rep. 1 / 6) one week after injection of GT69 (1e10 vg in 20 μL).

Claims

1. A recombinant gene therapy vector comprising a polynucleotide operably linked to a promoter, wherein the vector is an adeno-associated virus (AAV), and the polynucleotide encodes PTEN-induced kinase 1 (PINK1) and has at least about 90% identity to SEQ ID NO:2 or SEQ ID NO:

3.

2. The recombinant gene therapy vector of claim 1 , wherein the polynucleotide encoding PINK1 has at least 95% identity to SEQ ID NO: 2 or SEQ ID NO:

3.

3. The recombinant gene therapy vector of claim 1 , wherein the polynucleotide encoding PINK1 has at least 98% identity to SEQ ID NO: 2 or SEQ ID NO:

3.

4. The recombinant gene therapy vector of claim 1 , wherein the polynucleotide encoding PINK1 has at least 99% identity to SEQ ID NO: 2 or SEQ ID NO:

3.

5. The recombinant gene therapy vector of claim 1 , wherein the polynucleotide encoding PINK1 has the sequence of SEQ ID NO: 2 or SEQ ID NO:

3.

6. The gene therapy vector of any one of claims 1 to 5, wherein the vector is a serotype 1 adeno-associated virus (AAV1).

7. 7. The gene therapy vector of any one of claims 1 to 6, wherein the promoter is selected from the group consisting of hSYNI (human synapsin), INA (α-internexin), NES (nestin), hTH (human tyrosine hydroxylase), FOXA2 (forkhead box A2), CaMKII (calmodulin-dependent protein kinase II), NSE (neuron-specific enolase), CMV, CAG, UBC, PGK, EFl-α, GAPDH, SV40, HBV, chicken β-actin, and human β-actin promoters.

8. The gene therapy vector of any one of claims 1 to 7, further comprising one or more regulatory elements.

9. 9. The gene therapy vector of claim 8, wherein the one or more regulatory elements are selected from the group consisting of enhancers, introns, polyA signal sequences, and transcript stabilization elements.

10. The gene therapy vector of claim 9, wherein the enhancer is selected from the group consisting of a CMV enhancer, a GAPDH enhancer, a β-actin enhancer, and an EF1-α enhancer.

11. The gene therapy vector of claim 9, wherein the transcript stabilization element is selected from the group consisting of a WPRE (woodchuck hepatitis virus post-transcriptional regulatory element) sequence, a HPRE (hepatitis post-transcriptional regulatory element) sequence, a scaffold binding region, a 3'UTR, and a 5'UTR.

12. 12. The gene therapy vector of any one of claims 1 to 11, comprising an expression cassette comprising, in 5' to 3' order: a) a CAG promoter and a polynucleotide encoding PINK1; b) a CAG promoter, a polynucleotide encoding PINK1, and WPRE; c) an hTH promoter and a polynucleotide encoding PINK1; d) an EF1α promoter and a polynucleotide encoding PINK1; e) an EF1α promoter, a polynucleotide encoding PINK1, and WPRE; f) a CBA promoter and a polynucleotide encoding PINK1; and g) a CBA promoter, a polynucleotide encoding PINK1, and WPRE.

13. A host cell comprising a gene therapy vector according to any one of claims 1 to 12.

14. 14. The host cell of claim 13, selected from the group consisting of HEK293, 293T, HeLa, Vero, and Sf9 cells.

15. A method of increasing expression of PINK1 in a cell in a subject by contacting the cell with a recombinant gene therapy vector according to any one of claims 1 to 12.

16. 16. The method of claim 15, wherein the cell is a neuron.

17. 17. The method of claim 16, wherein the neuron is a primary tyrosine hydroxylase-positive neuron.

18. The method of any one of claims 15 to 17, wherein the subject is a human subject.

19. 19. The method of claim 18, wherein the human subject comprises a mutation in the PINK1 gene.

20. 20. The method of claim 18 or 19, wherein the human subject has or is at risk of developing Parkinson's disease (PD).

21. 21. The method of claim 20, wherein the PD is early-onset PD.

22. 21. The method of claim 20, wherein the PD is early-onset autosomal recessive PD.

23. 13. A method of inhibiting, reducing, or delaying degeneration or death of neurons in a subject by contacting the neurons with a gene therapy vector of any one of claims 1 to 12, thereby increasing the expression level of PINK1 in the neurons and inhibiting, reducing, or delaying neuronal dysfunction / degeneration or death.

24. 24. The method of claim 23, wherein the subject is a human subject.

25. 25. The method of claim 23 or 24, wherein the neuron is a primary tyrosine hydroxylase positive neuron.

26. 26. The method of any one of claims 23 to 25, wherein MPP+(1-methyl-4-phenylpyridinium)-induced neurodegeneration is attenuated in the subject.

27. 26. The method of any one of claims 23 to 25, wherein MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) induced neuronal loss is prevented in the subject.

28. 28. The method of any one of claims 24 to 27, wherein the human subject comprises a mutation in the PINK1 gene.

29. 29. The method of any one of claims 24 to 28, wherein the human subject has or is at risk of developing Parkinson's disease (PD).

30. 30. The method of claim 29, wherein the PD is early-onset PD.

31. 30. The method of claim 29, wherein the PD is early-onset autosomal recessive PD.

32. A polynucleotide encoding PINK1 and having at least about 90% identity to SEQ ID NO:2 or SEQ ID NO:

3.

33. 33. The polynucleotide of claim 32, wherein the polynucleotide has at least 95% identity to SEQ ID NO:2 or SEQ ID NO:

3.

34. 33. The polynucleotide of claim 32, wherein the polynucleotide has at least 98% identity to SEQ ID NO:2 or SEQ ID NO:

3.

35. The polynucleotide of claim 32, wherein the polynucleotide encoding PINK1 has at least 99% identity to SEQ ID NO: 2 or SEQ ID NO:

3.

36. The polynucleotide of claim 32, wherein the polynucleotide encoding PINK1 has the sequence of SEQ ID NO: 2 or SEQ ID NO:

3.

37. A plasmid comprising the polynucleotide of any one of claims 32 to 36.

38. A recombinant gene therapy vector comprising a mutant polynucleotide encoding PINK1, wherein cells transduced with said vector express PINK1 at a higher level compared to the level of PINK1 expressed in cells of the same type transduced with the same type of vector comprising a wild-type polynucleotide encoding PINK1.

39. A method for increasing the expression of PINK1 in a subject's cells, said method comprising contacting the cells with a recombinant gene therapy vector comprising a mutant polynucleotide encoding PINK1, wherein the expression of PINK1 in the cells is increased compared to the expression of PINK1 in the same type of cells contacted with the same amount of the same type of vector comprising a wild-type polynucleotide encoding PINK1 under the same conditions.

40. 1. A method for inhibiting, reducing, or delaying neuronal dysfunction / degeneration or death in a subject, said method comprising contacting a neuron with a recombinant gene therapy vector comprising a mutant polynucleotide encoding PINK1, wherein expression of PINK1 in said neuron is increased compared to expression of PINK1 in the same type of neuron contacted with the same amount of the same type of vector comprising a wild-type polynucleotide encoding PINK1 under the same conditions.

41. 41. The vector or method of any one of claims 38 to 40, wherein the mutant polynucleotide is operably linked to a promoter.

42. 41. The vector or method of any one of claims 38 to 40, wherein the mutant polynucleotide and the wild-type polynucleotide are each operably linked to the same type of promoter.

43. 43. The vector or method of any one of claims 38 to 42, wherein the vector is an adeno-associated virus (AAV).

44. 44. The vector or method of any one of claims 38-43, wherein PINK1 is expressed at least about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2-fold more highly by cells or neurons contacted with or transduced with a vector comprising a mutant polynucleotide encoding PINK1 compared to cells or neurons contacted with or transduced with a vector comprising a wild-type polynucleotide encoding PINK1.

45. 45. The vector or method of any one of claims 38 to 44, wherein the variant polynucleotide encoding PINK1 has at least about 70%, at least about 80%, at least about 85%, or at least about 90% identity to SEQ ID NO:2 or SEQ ID NO:

3.

46. 46. ​​The vector or method of any one of claims 38 to 45, wherein the cell is a neuron.

47. 47. The vector or method of claim 46, wherein the neuron is selected from the group consisting of a cortical neuron, an ependymal cell, a glutamatergic neuron, a GABAergic neuron, a dopaminergic neuron, an oligodendrocyte, an astrocyte, and a microglial cell.

48. 1. A method for inhibiting, reducing, or slowing the degeneration or death of neurons in a subject, the method comprising contacting neurons with an amount of a recombinant gene therapy vector comprising a polynucleotide encoding PINK1, wherein the amount of vector is effective to inhibit, reduce, or slow the degeneration or death of neurons in the subject.

49. 49. The method of claim 48, wherein the neurons are contacted with an amount of vector corresponding to a multiplicity of infection (MOI) of about 1 to about 10^5.

50. 49. The method of claim 48, wherein the neuron is contacted with an amount of the vector corresponding to an MOI of about 10 to about 10^4.

51. 49. The method of claim 48, wherein the neuron is contacted with an amount of vector corresponding to an MOI of about 1, about 10, about 10^1, about 10^2, about 10^3, about 10^4, or about 10^5.

52. 52. The method of any one of claims 48 to 51, wherein the polynucleotide is operably linked to a promoter.

53. 52. The method of any one of claims 48 to 51, wherein the polynucleotide is a mutant polynucleotide.

54. 54. The method of any one of claims 48 to 53, wherein the vector is an adeno-associated virus (AAV).

55. 55. The method of any one of claims 48 to 54, wherein the variant polynucleotide encoding PINK1 has at least about 70%, at least about 80%, at least about 85%, or at least about 90% identity to SEQ ID NO:2 or SEQ ID NO:

3.

56. 57. The method of claim 56, wherein the neuron is selected from the group consisting of a cortical neuron, an ependymal cell, a glutamatergic neuron, a GABAergic neuron, a dopaminergic neuron, an oligodendrocyte, an astrocyte, and a microglial cell.

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