Manipulated parking devices and their use

Abstract

Provided are parkin protein variants having activating mutations and / or fused to a mitochondrial targeting sequence. The engineered parkin can be a fusion protein comprising a mitochondrial targeting sequence (MTS), a transmembrane domain, and a parkin protein or a functional variant or fragment thereof, such as a parkin having an N-terminal deletion. The MTS can be the MTS of PINK1 or a functional variant thereof. Alternatively or additionally, the engineered parkin can have one or more activating mutations, such as a single amino acid substitution. The engineered parkin can be delivered by a vector, such as an adeno-associated virus (AAV) vector, and used to treat diseases or disorders, such as Parkinson's disease or any of a variety of neurodegenerative diseases. TIFF2023535121000081.tif28170

Description

【Technical Field】 【0001】 Cross - Reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 027,866, filed on May 20, 2020, and U.S. Provisional Patent Application No. 63 / 027,868, filed on May 20, 2020, the contents of which are hereby incorporated by reference in their entirety. 【0002】 Description of the Sequence Listing The sequence listing related to this application is provided in text format instead of a paper copy and is hereby incorporated by reference into this specification. The name of the text file containing the sequence listing is ROPA_016_01WO_ST25.txt. The text file is approximately 265 KB, was created on May 20, 2021, and was electronically submitted via EFS - Web. 【0003】 Field of the Invention The present invention generally relates to gene therapy for disorders associated with mitochondrial dysfunction, such as central nervous system (CNS) disorders, for example, Parkinson's disease. In particular, the present disclosure provides engineered Parkin protein variants having activating mutations and / or fused to mitochondrial targeting sequences. 【Background Art】 【0004】 Background PARK2, which codes for the protein parkin, is one of several genes involved in Parkinson's disease. Others include PARK1 (which codes for the protein α-synuclein), PARK6 (which codes for the protein PINK1), PARK7 (which codes for the protein DJ-1), and PARK8 (also known as dardarin, which codes for the protein LRRK2). (Creed et al. (2018) Mov Disord. 33:717-729 (Non-patent Literature 1), Blesa et al. (2014) Front. Neuroanat. 8:1-12 (Non-patent Literature 2), Alcalay et al. (2010) Arch Neurol. 67:1116-1122 (Non-patent Literature 3)). 【0005】 PINK1 and parkin work together to protect mitochondria from oxidative stress. PINK1 is translocated to mitochondria via the N-terminal mitochondrial signaling sequence (MTS). In the absence of mitochondrial stress, PINK1 is proteolytically cleaved within healthy mitochondria by mitochondrial processing peptidases (MMPs) and protease presenilin-associated rhomboid-like protein (PARL). When mitochondria are damaged, PINK1 does not completely translocate; instead, its transmembrane domain (TMD) is embedded in the membrane of the damaged mitochondria, protecting it from proteolysis by MMPs and PARL, and it accumulates on the mitochondrial surface. 【0006】 Uncleaved PINK1 then plays a role in activating parkin through a series of enzymatic steps. Various point mutations in parkin have been shown to artificially activate parkin without PINK1 activity, or to shift the equilibrium toward activation when PINK1 is activated. 【0007】 There has been a long-standing unmet need for gene therapy-based treatments for Parkinson's disease and other disorders associated with mitochondrial dysfunction. The gene therapies provided herein address this need. [Prior art documents] [Non-patent literature] 【0008】 [Non-Patent Document 1] Creed et al.(2018)Mov Disord.33:717-729 [Non-Patent Document 2] Blesa et al.(2014)Front. Neuroanat.8:1-12 [Non-Patent Document 3] Alcalay et al.(2010)Arch Neurol.67:1116-1122 [Overview of the project] 【0009】 overview In one embodiment, the disclosure provides a recombinant adeno-associated virus (rAAV) virion comprising a capsid and a vector genome, the vector genome comprising a polynucleotide sequence encoding an activated parkin protein operably ligated to a promoter. 【0010】 In another aspect, the Disclosure provides, for example, a method for increasing parkin activity in cells, the method comprising contacting the cells with the rAAV virion of the Disclosure. 【0011】 In another embodiment, the Disclosure provides, for example, a method for increasing parkin activity in cells, the method comprising administering the rAAV virion of the Disclosure to a target. 【0012】 In another embodiment, the Disclosure provides a method for promoting neuronal survival, the method comprising bringing the neuron into contact with the rAAV virion of the Disclosure. 【0013】 In another embodiment, the present disclosure provides a method for promoting neuronal survival, the method comprising administering the rAAV virion of the present disclosure to a target. 【0014】 In another embodiment, the Disclosure provides a method for treating a disease or disorder, the method comprising administering the rAAV virion of the Disclosure to a target. 【0015】 In another aspect, the disclosure provides a polynucleotide comprising a polynucleotide sequence encoding a fusion protein, comprising a mitochondrial targeting sequence (MTS), a transmembrane domain (TMD), and a parkin protein or a functional variant or fragment thereof. 【0016】 In another embodiment, the Disclosure provides a vector comprising the polynucleotides of the Disclosure. 【0017】 In another aspect, the Disclosure provides, for example, a method for increasing parkin activity in cells, the method comprising administering a polynucleotide or vector of the Disclosure to a target. 【0018】 In another embodiment, the Disclosure provides a method for promoting neuronal survival, the method comprising contacting a neuron with a polynucleotide or vector of the Disclosure. 【0019】 In another embodiment, the Disclosure provides a method for promoting neuronal survival, the method comprising administering the polynucleotide or vector of the Disclosure to a target. 【0020】 In another aspect, the Disclosure provides a method for treating a disease or disorder, the method comprising administering the polynucleotide or vector of the Disclosure to a target. 【0021】 In further embodiments, the Disclosure provides cells, proteins, pharmaceutical compositions, and kits comprising or encoded by the polynucleotides or vectors of the Disclosure. 【0022】 In a further aspect, the present disclosure provides a pharmaceutical composition and a kit comprising the rAAV virions of the present disclosure. 【0023】 In various embodiments, the present disclosure provides a polynucleotide comprising a polynucleotide sequence encoding a fusion protein comprising a mitochondrial targeting sequence (MTS), a transmembrane domain (TMD), and a parkin protein or a functional variant or fragment thereof. 【0024】 In some embodiments of the polynucleotide, the MTS is the MTS of PINK1 or a functional variant thereof. 【0025】 In some embodiments of the polynucleotide, the MTS comprises a mitochondrial processing peptidase (MPP) cleavage site. 【0026】 In some embodiments of the polynucleotide, the MTS is residues 1-34 of human PINK1: TIFF0007872737000001.tif3128 and comprises a polypeptide sequence that is at least 95% identical. 【0027】 In some embodiments of the polynucleotide, the MTS is residues 1-94 of human PINK1: TIFF0007872737000002.tif15128 and comprises a polypeptide sequence that is at least 95% identical. 【0028】 In some embodiments of the polynucleotide, the MTS is residues 1-94 of human PINK1: TIFF0007872737000003.tif15128 and comprises a polypeptide sequence that is identical. 【0029】 In some embodiments of the polynucleotide, the TMD is the TMD of PINK1 or a functional variant thereof. 【0030】 In some embodiments of polynucleotides, the TMD includes a PARL cleavage site. 【0031】 In some embodiments of polynucleotides, the TMD is derived from residues 95-110 of human PINK1: It contains a polypeptide sequence that is at least 95% identical to TIFF0007872737000004.tif3128. 【0032】 In some embodiments of polynucleotides, the TMD is derived from residues 95-110 of human PINK1: Contains a polypeptide sequence identical to TIFF0007872737000005.tif3128. 【0033】 In some embodiments of polynucleotides, the TMD is derived from residues 95-110 of human PINK1: Contains a polypeptide sequence identical to TIFF0007872737000006.tif3128. 【0034】 In some embodiments of the polynucleotide, the fusion protein comprises the MTS-TMD fragment of PINK1 or a functional variant thereof. 【0035】 In some embodiments of the polynucleotide, the MTS-TMD fragment is derived from residues 1-110 of human PINK1: It contains a polypeptide sequence that is at least 95% identical to TIFF0007872737000007.tif15128. 【0036】 In some embodiments of the polynucleotide, the MTS-TMD fragment is derived from residues 1-110 of human PINK1: Contains a polypeptide sequence identical to TIFF0007872737000008.tif15128. 【0037】 In some embodiments of the polynucleotide, the functional variant or fragment thereof is a Δ-Parkin protein comprising a deletion of the N-terminal ubiquitin-like (Ubl) domain and optionally a deletion of the Ubl-RING0 interdomain linker sequence. 【0038】 In some embodiments of the polynucleotide, the Δparkin protein is derived from residues 141-465 of human parkin F146A+W403A: It contains a polypeptide sequence that is at least 95% identical to TIFF0007872737000009.tif51128. 【0039】 In some embodiments of the polynucleotide, the Δparkin protein is derived from residues 141-465 of human parkin F146A+W403A: Contains a polypeptide sequence identical to TIFF0007872737000010.tif51128. 【0040】 In some embodiments of the polynucleotide, the ΔParkin protein is derived from residues 76-465 of human parkin F146A+W403A: It contains a polypeptide sequence that is at least 95% identical to TIFF0007872737000011.tif63128. 【0041】 In some embodiments of the polynucleotide, the ΔParkin protein is derived from residues 76-465 of human parkin F146A+W403A: Contains a polypeptide sequence identical to TIFF0007872737000012.tif63128. 【0042】 In some embodiments of the polynucleotide, the fusion protein contains an F146A substitution relative to the reference human parkin protein sequence of SEQ ID NO: 1. 【0043】 In some embodiments of the polynucleotide, the fusion protein contains a W403A substitution relative to the reference human parkin protein sequence of SEQ ID NO: 1. 【0044】 In some embodiments of the polynucleotide, the fusion protein contains the F463A substitution relative to the reference human parkin protein sequence of SEQ ID NO: 1. 【0045】 In some embodiments of the polynucleotide, the fusion protein contains a C457S substitution relative to the reference human parkin protein sequence of SEQ ID NO: 1. 【0046】 In some embodiments of the polynucleotide, the fusion protein includes both the F146A substitution and the W403A substitution with respect to the reference human parkin protein sequence of SEQ ID NO: 1. 【0047】 In some embodiments of the polynucleotide, the fusion protein contains the F104M substitution relative to the reference human PINK1 protein sequence of SEQ ID NO: 64. 【0048】 In some embodiments of the polynucleotide, the fusion protein includes both the F146A substitution and the W403A substitution with respect to the reference human parkin protein sequence of SEQ ID NO: 1, and the fusion protein includes the F104M substitution with respect to the reference human PINK1 protein sequence of SEQ ID NO: 64. 【0049】 In some embodiments of the polynucleotide, the fusion protein comprises a polypeptide sequence that is at least 95% identical to the sequence of SEQ ID NO: 97 or 98, and comprises two or more amino acid substitutions selected from F104M, W403A, and F463A. F104M is relative to the reference human PINK1 protein sequence of SEQ ID NO: 64, W403A is relative to the reference human parkin protein sequence of SEQ ID NO: 1, and F463A is relative to the reference human parkin protein sequence of SEQ ID NO: 1. 【0050】 In some embodiments of the polynucleotide, the fusion protein comprises a polypeptide sequence identical to either sequence 97 or 98, and comprises two or more amino acid substitutions selected from F104M, W403A, and F463A. F104M is relative to the reference human PINK1 protein sequence of sequence 64, W403A is relative to the reference human parkin protein sequence of sequence 1, and F463A is relative to the reference human parkin protein sequence of sequence 1. 【0051】 In various embodiments, this disclosure provides vectors comprising the polynucleotides of the embodiments. 【0052】 In some embodiments of the vector, the vector is an adeno-associated virus (AAV) vector. 【0053】 In some embodiments of the AAV vector, the vector includes an AAV9 capsid or a functional variant thereof. The AAV9 capsid may share at least 98%, 99%, or 100% identity with a reference AAV9 capsid. 【0054】 In various embodiments, the present disclosure provides a method for increasing parkin activity in cells, the method comprising contacting cells with a polynucleotide or vector described in any of the embodiments. 【0055】 In various embodiments, the Disclosure provides a method for increasing parkin activity in a subject, the method comprising administering to the subject one of the polynucleotides or vectors of the embodiments. 【0056】 In some embodiments of this method, the cells or subjects are deficient in parkin activity and / or contain loss-of-function mutations in parkin. 【0057】 In some embodiments of this method, parkin activity includes one or more of the following: co-localization of parkin with TOMM2 in response to neurotoxic treatment, ubiquitination of mitochondrial proteins in response to neurotoxic treatment, and an increase in parkin levels in the mitochondrial fraction in response to neurotoxic treatment. 【0058】 In various embodiments, the present disclosure provides a method for promoting neuronal survival, which involves contacting a neuron with a polynucleotide or vector of any of the embodiments. 【0059】 In various embodiments, the Disclosure provides a method for promoting neuronal survival in a subject, the method comprising administering to the subject one of the polynucleotides or vectors of the embodiments. 【0060】 In some embodiments of this method, the neurons are dopaminergic neurons. 【0061】 In various embodiments, the Disclosure provides a method for treating a disease or disorder in a subject requiring treatment, the method comprising administering a polynucleotide or vector of any embodiment to the subject. 【0062】 In some embodiments of this method, the subjects have a genetic deficiency in parkin expression or function. 【0063】 In some embodiments of this method, the subjects have a genetic deficiency in PINK1 expression or function. 【0064】 In some embodiments of this method, the disease or disorder is Parkinson's disease. 【0065】 In some embodiments of this method, Parkinson's disease is early-onset Parkinson's disease (EOPD). 【0066】 In some embodiments of this method, it alleviates one or more symptoms of Parkinson's disease. 【0067】 In some embodiments of this method, it reduces motor complications associated with neurodegeneration; reduces the need for antiparkinsonian drug therapy, optionally L-dopa and / or dopamine agonists; restores the function of degenerated neurons; and / or protects neurons from degeneration. 【0068】 In some embodiments of this method, the method enhances substantia nigra function, which is optionally evaluated by [18F]fluoro-L-dopa positron emission tomography (PET) or DaT-SPECT imaging. 【0069】 In some embodiments of this method, the method improves one or both of the target UPDRS or MDS-UPDRS. 【0070】 In various embodiments, this disclosure provides cells comprising polynucleotides of any embodiment. 【0071】 In various embodiments, this disclosure provides proteins encoded by polynucleotides of any embodiment. 【0072】 In various embodiments, the disclosure provides pharmaceutical compositions comprising a vector of any embodiment and one or more pharmaceutically acceptable carriers, diluents, or excipients. 【0073】 In various embodiments, this disclosure provides a kit including a vector of any embodiment and instructions for use. 【0074】 In various embodiments, the Disclosure provides a recombinant adeno-associated virus (rAAV) virion comprising a capsid and a vector genome, the vector genome comprising a polynucleotide sequence encoding an activated parkin protein operably linked to a promoter. 【0075】 In some embodiments of the rAAV virion, the activated parkin protein contains one or more amino acid substitutions at positions Phe-146, Trp-403, Cys-457, Phe-463, and Asn-273 relative to the reference parkin protein. 【0076】 In some embodiments of the rAAV virion, the activated parkin protein contains two or more amino acid substitutions at positions Phe-146, Trp-403, Cys-457, Phe-463, and Asn-273 relative to the reference parkin protein. 【0077】 In some embodiments of the rAAV virion, the activated parkin protein contains amino acid substitutions at positions Phe-146, Trp-403, Cys-457, Phe-463, and Asn-273 relative to the reference parkin protein. 【0078】 In some embodiments of the rAAV virion, the activated parkin protein comprises one or more amino acid substitutions selected from F146A, W403A, and / or N273K relative to the reference parkin protein. 【0079】 In some embodiments of the rAAV virion, the activated parkin protein contains amino acid substitutions F146A and W403A relative to the reference parkin protein. 【0080】 In some embodiments of the rAAV virion, the activated parkin protein contains amino acid substitutions F146A, N273K, and W403A relative to the reference parkin protein. 【0081】 In some embodiments of the rAAV virion, the activated parkin protein contains a polypeptide sequence that is at least 95% identical to human parkin N273K+W403A+F463A (SEQ ID NO: 93). 【0082】 In some embodiments of the rAAV virion, the activated parkin protein contains a polypeptide sequence identical to that of human parkin N273K+W403A+F463A (SEQ ID NO: 93). 【0083】 In some embodiments of the rAAV virion, the parkin protein is a Δ parkin protein containing a deletion of the ubiquitin-like (Ubl) domain. 【0084】 In some embodiments of the rAAV virion, the Δparkin protein is derived from residues 76-465 of human parkin F146A+W403A: It contains a polypeptide sequence that is at least 95% identical to TIFF0007872737000013.tif63128. 【0085】 In some embodiments of the rAAV virion, the Δparkin protein is derived from residues 76-465 of human parkin F146A+W403A: Contains a polypeptide sequence identical to TIFF0007872737000014.tif63128. 【0086】 In some embodiments of the rAAV virion, the activated parkin protein contains an amino acid substitution at position Cys-431 relative to the reference parkin protein. 【0087】 In some embodiments of the rAAV virion, the activated parkin protein contains a C431F amino acid substitution relative to the reference parkin protein. 【0088】 In some embodiments of the rAAV virion, the promoter is a constitutive promoter. 【0089】 In some embodiments of the rAAV virion, the promoter is a CAG promoter. 【0090】 In some embodiments of the rAAV virion, the promoter is a CMV promoter. 【0091】 In some embodiments of the rAAV virion, the promoter is a neuron-specific promoter. 【0092】 In some embodiments of the rAAV virion, the promoter is a SYN promoter. 【0093】 In some embodiments of the rAAV virion, the vector genome includes WPRE elements. 【0094】 In some embodiments of the rAAV virion, the vector genome includes an hGH polyadenylation site. 【0095】 In some embodiments of the rAAV billion, the capsid is the AAV9 capsid or a functional variant thereof. 【0096】 In some embodiments of the rAAV billion, the AAV9 capsid shares at least 98%, 99%, or 100% identity with the reference AAV9 capsid. 【0097】 In various embodiments, the present disclosure provides a method for increasing parkin activity in cells, the method comprising contacting the cells with rAAV virions of any embodiment. 【0098】 In various embodiments, the Disclosure provides a method for increasing parkin activity in a subject, the method comprising administering an effective amount of rAAV virion of any embodiment to the subject. 【0099】 In some embodiments of this method, the cells or subjects are deficient in parkin activity and / or contain loss-of-function mutations in parkin. 【0100】 In some embodiments of this method, parkin activity includes one or more of the following: co-localization of parkin with TOMM2 in response to neurotoxic treatment, ubiquitination of mitochondrial proteins in response to neurotoxic treatment, and an increase in parkin levels in the mitochondrial fraction in response to neurotoxic treatment. 【0101】 In various embodiments, the present disclosure provides a method for promoting neuronal survival, which includes bringing the neuron into contact with an rAAV virion of any embodiment. 【0102】 In various embodiments, the Disclosure provides a method for promoting neuronal survival in a subject, the method comprising administering an effective amount of rAAV virion of any embodiment to the subject. 【0103】 In some embodiments of this method, the neurons are dopaminergic neurons. 【0104】 In various embodiments, the Disclosure provides a method for treating a disease or disorder in a subject requiring treatment, the method comprising administering an effective amount of rAAV virion of any embodiment to the subject. 【0105】 In some embodiments of this method, the subjects have a genetic defect in Parkin. 【0106】 In some embodiments of this method, the subjects have a genetic deficiency in PINK1. 【0107】 In some embodiments of this method, the subjects have a genetic defect in DJ-1. 【0108】 In some embodiments of this method, the disease or disorder is Parkinson's disease. 【0109】 In some embodiments of this method, Parkinson's disease is early-onset Parkinson's disease (EOPD). 【0110】 In some embodiments of this method, it alleviates one or more symptoms of Parkinson's disease. 【0111】 In some embodiments of this method, it reduces motor complications associated with neurodegeneration; reduces the need for antiparkinsonian drug therapy, optionally L-dopa and / or dopamine agonists; restores the function of degenerated neurons; and / or protects neurons from degeneration. 【0112】 In some embodiments of this method, the method enhances substantia nigra function, which is optionally evaluated by [18F]fluoro-L-dopa positron emission tomography (PET) or DaT-SPECT imaging. 【0113】 In some embodiments of this method, the method improves one or both of the target UPDRS or MDS-UPDRS. 【0114】 In various embodiments, the disclosure provides pharmaceutical compositions comprising an rAAV virion of any embodiment and one or more pharmaceutically acceptable carriers, diluents, or excipients. 【0115】 In various embodiments, this disclosure provides a kit comprising an rAAV vilion of any embodiment and instructions for use. 【0116】 In various embodiments, the present disclosure provides polynucleotides comprising a polynucleotide sequence encoding an activated parkin protein. 【0117】 In some embodiments of the polynucleotide, the activated parkin protein contains an amino acid substitution at position Cys-431 relative to the reference parkin protein. 【0118】 In some embodiments of the polynucleotide, the activated parkin protein contains a C431F amino acid substitution relative to the reference parkin protein. 【0119】 In some embodiments of the polynucleotide, the activated parkin protein contains one or more amino acid substitutions at positions Phe-146, Trp-403, Cys-457, Phe-463, and Asn-273 relative to the reference parkin protein. 【0120】 In some embodiments of the polynucleotide, the activated parkin protein contains two or more amino acid substitutions at positions Phe-146, Trp-403, Cys-457, Phe-463, and Asn-273 relative to the reference parkin protein. 【0121】 In some embodiments of the polynucleotide, the activated parkin protein contains amino acid substitutions at positions Phe-146, Trp-403, Cys-457, Phe-463, and Asn-273 relative to the reference parkin protein. 【0122】 In some embodiments of the polynucleotide, the activated parkin protein comprises one or more amino acid substitutions selected from F146A, W403A, and / or N273K relative to the reference parkin protein. 【0123】 In some embodiments of the polynucleotide, the activated parkin protein contains amino acid substitutions F146A and W403A relative to the reference parkin protein. 【0124】 In some embodiments of the polynucleotide, the activated parkin protein includes amino acid substitutions F146A, N273K, and W403A relative to the reference parkin protein. 【0125】 In some embodiments of the polynucleotide, the activated parkin protein contains a polypeptide sequence that is at least 95% identical to human parkin N273K+W403A+F463A (SEQ ID NO: 93). 【0126】 In some embodiments of the polynucleotide, the activated parkin protein contains a polypeptide sequence identical to human parkin N273K+W403A+F463A (SEQ ID NO: 93). 【0127】 In some embodiments of the polynucleotide, the parkin protein is a Δ parkin protein containing a deletion of the ubiquitin-like (Ubl) domain. 【0128】 In some embodiments of the polynucleotide, the ΔParkin protein is derived from residues 76-465 of human parkin F146A+W403A: Contains a polypeptide sequence that is at least 95% identical to TIFF0007872737000015.tif63128. 【0129】 In some embodiments of the polynucleotide, the ΔParkin protein is derived from residues 76-465 of human parkin F146A+W403A: Contains a polypeptide sequence identical to TIFF0007872737000016.tif63128. 【0130】 In some embodiments of the polynucleotide, the polynucleotide includes a promoter operably ligated to a polynucleotide sequence encoding an activated parkin protein. 【0131】 In some embodiments of polynucleotides, the promoter is a constitutive promoter. 【0132】 In some embodiments of polynucleotides, the promoter is a CAG promoter. 【0133】 In some embodiments of the polynucleotide, the promoter is the CMV promoter. 【0134】 In some embodiments of polynucleotides, the promoter is a neuron-specific promoter. 【0135】 In some embodiments of polynucleotides, the promoter is a SYN promoter. 【0136】 In some embodiments of polynucleotides, the vector genome includes WPRE elements. 【0137】 In some embodiments of polynucleotides, the vector genome includes hGH polyadenylation sites. 【0138】 In various embodiments, this disclosure provides vectors comprising polynucleotides of any embodiment. 【0139】 In some embodiments of the vector, the vector is an adeno-associated virus (AAV) vector. 【0140】 In some embodiments of the AAV vector, the vector includes an AAV9 capsid or a functional variant thereof. The AAV9 capsid may share at least 98%, 99%, or 100% identity with a reference AAV9 capsid. 【0141】 In various embodiments, the present disclosure provides a method for increasing parkin activity in cells, the method comprising contacting the cells with any one of the polynucleotides or vectors in the embodiments. 【0142】 In various embodiments, the Disclosure provides a method for increasing parkin activity in a subject, which includes administering one of the polynucleotides or vectors of the embodiments to the subject. 【0143】 In some embodiments of this method, the cells or subjects are deficient in parkin activity and / or contain loss-of-function mutations in parkin. 【0144】 In some embodiments of this method, parkin activity includes one or more of the following: co-localization of parkin with TOMM2 in response to neurotoxic treatment, ubiquitination of mitochondrial proteins in response to neurotoxic treatment, and an increase in parkin levels in the mitochondrial fraction in response to neurotoxic treatment. 【0145】 In various embodiments, the present disclosure provides a method for promoting neuronal survival, which includes contacting neurons with a polynucleotide or vector of any embodiment. 【0146】 In various embodiments, the present disclosure provides a method for promoting neuronal survival in a subject, the method comprising administering a polynucleotide or vector of any embodiment to the subject. 【0147】 In some embodiments of this method, the neurons are dopaminergic neurons. 【0148】 In various embodiments, the Disclosure provides a method for treating a disease or disorder in a subject requiring treatment, the method comprising administering a polynucleotide or vector of any embodiment to the subject. 【0149】 In some embodiments of this method, the subjects have a genetic deficiency in parkin expression or function. 【0150】 In some embodiments of this method, the subjects have a genetic deficiency in PINK1 expression or function. 【0151】 In some embodiments of this method, the disease or disorder is Parkinson's disease. 【0152】 In some embodiments of the method, the Parkinson's disease is early-onset Parkinson's disease (EOPD). 【0153】 In some embodiments of this method, it alleviates one or more symptoms of Parkinson's disease. 【0154】 In some embodiments of this method, it reduces motor complications associated with neurodegeneration; reduces the need for antiparkinsonian drug therapy, optionally L-dopa and / or dopamine agonists; restores the function of degenerated neurons; and / or protects neurons from degeneration. 【0155】 In some embodiments of this method, the method enhances substantia nigra function, which is optionally evaluated by [18F]fluoro-L-dopa positron emission tomography (PET) or DaT-SPECT imaging. 【0156】 In some embodiments of this method, the method improves one or both of the target UPDRS or MDS-UPDRS. 【0157】 In various embodiments, this disclosure provides cells comprising polynucleotides of any embodiment. 【0158】 In various embodiments, this disclosure provides proteins encoded by polynucleotides of any embodiment. 【0159】 In various embodiments, the Disclosure provides pharmaceutical compositions comprising a vector of any embodiment and one or more pharmaceutically acceptable carriers, diluents, or excipients. 【0160】 In various embodiments, this disclosure provides a kit including a vector of any embodiment and instructions for use. 【0161】 [Invention 1001] A fusion protein comprising a mitochondrial targeting sequence (MTS), a transmembrane domain (TMD), and the parkin protein or a functional variant or fragment thereof. A polynucleotide containing a polynucleotide sequence that codes for a specific type of polynucleotide. [Invention 1002] The polynucleotide of the present invention 1001, wherein the MTS is the MTS of PINK1 or a functional variant thereof. [Invention 1003] The MTS is a polynucleotide according to the present invention 1001 or 1002, wherein the MTS includes a mitochondrial processing peptidase (MPP) cleavage site. [Invention 1004] The aforementioned MTS is derived from residues 1-34 of human PINK1: TIFF0007872737000017.tif3128 A polynucleotide according to any one of the present invention 1001 to 1003, comprising a polypeptide sequence that is at least 95% identical to [another polynucleotide]. [Invention 1005] The aforementioned MTS is derived from residues 1-94 of human PINK1: TIFF0007872737000018.tif15128 The polynucleotide of the present invention 1004, comprising a polypeptide sequence that is at least 95% identical to [another polynucleotide]. [Invention 1006] The aforementioned MTS is derived from residues 1-94 of human PINK1: TIFF0007872737000019.tif15128 A polynucleotide of the present invention 1005, comprising a polypeptide sequence identical to that of the present invention. [Invention 1007] The polynucleotide according to any one of the invention 1001 to 1006, wherein the TMD is the TMD of PINK1 or a functional variant thereof. [Invention 1008] The TMD is a polynucleotide according to any one of the present invention 1001 to 1007, wherein the TMD includes a PARL cleavage site. [Invention 1009] The aforementioned TMD is human PINK1 residues 95-110: TIFF0007872737000020.tif3128 A polynucleotide of any of the present invention 1001 to 1008, comprising a polypeptide sequence that is at least 95% identical to [another polynucleotide]. [Invention 1010] The aforementioned TMD is human PINK1 residues 95-110: TIFF0007872737000021.tif3128 A polynucleotide of the present invention 1009, comprising a polypeptide sequence identical to that of the present invention. [Invention 1011] The aforementioned TMD is human PINK1 residues 95-110: TIFF0007872737000022.tif3128 A polynucleotide of the present invention 1009, comprising a polypeptide sequence identical to that of the present invention. [Invention 1012] The fusion protein comprises a polynucleotide according to any one of the inventions 1001 to 1011, wherein the fusion protein comprises a PINK1 MTS-TMD fragment or a functional variant thereof. [Invention 1013] The aforementioned MTS-TMD fragment corresponds to residues 1-110 of human PINK1: TIFF0007872737000023.tif15128 The polynucleotide of the present invention 1012, comprising a polypeptide sequence that is at least 95% identical to [another polynucleotide]. [Invention 1014] The aforementioned MTS-TMD fragment corresponds to residues 1-110 of human PINK1: TIFF0007872737000024.tif15128 A polynucleotide of the present invention 1012, comprising a polypeptide sequence identical to that of the present invention. [Invention 1015] The polynucleotide of any one of the invention 1001 to 1014, wherein the functional variant or fragment thereof is a Δ-Parkin protein comprising a deletion of the N-terminal ubiquitin-like (Ubl) domain and optionally a deletion of the Ubl-RING0 interdomain linker sequence. [Invention 1016] The aforementioned Δparkin protein is derived from residues 141-465 of human parkin F146A+W403A: TIFF0007872737000025.tif51128 A polynucleotide of the present invention 1015, comprising a polypeptide sequence that is at least 95% identical to [another polynucleotide]. [Invention 1017] The aforementioned Δparkin protein is derived from residues 141-465 of human parkin F146A+W403A: TIFF0007872737000026.tif51128 A polynucleotide of the present invention 1015, comprising a polypeptide sequence identical to that of the present invention. [Invention 1018] The aforementioned Δparkin protein is derived from residues 76-465 of human parkin F146A+W403A: TIFF0007872737000027.tif63128 A polynucleotide of the present invention 1015, comprising a polypeptide sequence that is at least 95% identical to [another polynucleotide]. [Invention 1019] The aforementioned Δparkin protein is derived from residues 76-465 of human parkin F146A+W403A: TIFF0007872737000028.tif63128 A polynucleotide of the present invention 1015, comprising a polypeptide sequence identical to that of the present invention. [Invention 1020] The aforementioned fusion protein has the following sequence: TIFF0007872737000029.tif75128 A polynucleotide of any of the present invention 1001 to 1019, comprising a polypeptide sequence that is at least 95% identical to [another polynucleotide]. [Invention 1021] The fusion protein is a polynucleotide of any of the present invention 1001 to 1020, comprising an F146A substitution with respect to the reference human parkin protein sequence of Sequence ID No. 1. [Invention 1022] The fusion protein is a polynucleotide of any of the present invention 1001 to 1021, comprising a W403A substitution with respect to the reference human parkin protein sequence of SEQ ID NO: 1. [Invention 1023] The fusion protein is a polynucleotide of any of the present invention 1001 to 1022, comprising the F463A substitution with respect to the reference human parkin protein sequence of Sequence ID No. 1. [Invention 1024] The fusion protein is a polynucleotide of any of the present invention 1001 to 1023, comprising a C457S substitution with respect to the reference human parkin protein sequence of SEQ ID NO: 1. [Invention 1025] The fusion protein is a polynucleotide of the present invention 1022, wherein the fusion protein contains both the F146A substitution and the W403A substitution with respect to the reference human parkin protein sequence of Sequence ID No. 1. [Invention 1026] The fusion protein is a polynucleotide of any of the present invention 1001 to 1025, comprising an F104M substitution with respect to the reference human PINK1 protein sequence of SEQ ID NO: 64. [Invention 1027] The polynucleotides of any of the present invention 1001 to 1026, wherein the fusion protein contains both the F146A substitution and the W403A substitution with respect to the reference human parkin protein sequence of SEQ ID NO: 1, and the fusion protein contains the F104M substitution with respect to the reference human PINK1 protein sequence of SEQ ID NO: 64. [Invention 1028] The fusion protein comprises a polypeptide sequence that is at least 95% identical to the sequence of SEQ ID NO: 97 or 98, and comprises two or more amino acid substitutions selected from F104M, W403A, and F463A. F104M corresponds to the reference human PINK1 protein sequence of SEQ ID NO: 64, W403A corresponds to the reference human parkin protein sequence of SEQ ID NO: 1, and F463A corresponds to the reference human parkin protein sequence of SEQ ID NO: 1. Polynucleotide according to the present invention 1001. [Invention 1029] The fusion protein comprises a polypeptide sequence identical to either sequence 97 or 98, and comprises two or more amino acid substitutions selected from F104M, W403A, and F463A. F104M corresponds to the reference human PINK1 protein sequence of SEQ ID NO: 64, W403A corresponds to the reference human parkin protein sequence of SEQ ID NO: 1, and F463A corresponds to the reference human parkin protein sequence of SEQ ID NO: 1. Polynucleotide according to the present invention 1001. [Invention 1030] A vector comprising any of the polynucleotides 1001 to 1029 of the present invention. [Invention 1031] The vector of the present invention 1030, which is an adeno-associated virus (AAV) vector. [Invention 1032] A vector according to the present invention 1031, comprising an AAV9 capsid or a functional variant thereof. [Invention 1033] The vector of the present invention 1032, wherein the AAV9 capsid shares at least 98%, 99%, or 100% identity with a reference AAV9 capsid. [Invention 1034] A method for increasing parkin activity in cells, comprising contacting the cells with any polynucleotide of any of Invention 1001 to 1029 or any vector of any of Invention 1030 to 1033. [Invention 1035] A method for increasing parkin activity in a subject, comprising administering to the subject any polynucleotide of any of Invention 1001 to 1029 or any vector of Invention 1030 to 1033. [Invention 1036] The method of the present invention 1034 or 1035, wherein the cells or subjects lack parkin activity and / or contain a loss-of-function mutation in parkin. [Invention 1037] Any method of the present invention 1034 to 1036, wherein parkin activity includes one or more of the following: co-localization of parkin with TOMM2 in response to neurotoxic treatment, ubiquitination of mitochondrial proteins in response to neurotoxic treatment, and an increase in parkin levels in the mitochondrial fraction in response to neurotoxic treatment. [Invention 1038] A method for promoting neuronal survival, comprising contacting the neuron with a polynucleotide of any of the inventions 1001 to 1029 or a vector of any of the inventions 1030 to 1033. [Invention 1039] A method for promoting neuronal survival in a subject, comprising administering to the subject a polynucleotide of any of Invention 1001 to 1029 or a vector of any of Invention 1030 to 1033. [Invention 1040] The method of the present invention 1038 or 1039, wherein the neuron is a dopaminergic neuron. [Invention 1041] A method for treating a disease or disorder in a subject requiring treatment, comprising administering to the subject a polynucleotide of any of Invention 1001 to 1029 or a vector of any of Invention 1030 to 1033. [Invention 1042] The method of the present invention 1041, wherein the subject has a genetic defect in parkin expression or function. [Invention 1043] The method of the present invention 1041 or 1042, wherein the subject has a genetic defect in PINK1 expression or function. [Invention 1044] A method according to any one of the present invention 1041 to 1043, wherein the disease or disorder is Parkinson's disease. [Invention 1045] The method of the present invention 1044, wherein the Parkinson's disease is early-onset Parkinson's disease (EOPD). [Invention 1046] A method according to any of invention 1041 to 1045 for alleviating one or more symptoms of Parkinson's disease. [Invention 1047] Any method of the present invention 1041 to 1046 for reducing motor complications associated with neurodegeneration; reducing the need for antiparkinsonian drug therapy, optionally L-dopa and / or dopamine agonists; restoring the function of degenerated neurons; and / or protecting neurons from degeneration. [Invention 1048] Nigestria function, optionally evaluated by [18F]fluoro-L-dopa positron emission tomography (PET) or DaT-SPECT imaging. A method of enhancing any of the present invention 1041 to 1047. [Invention 1049] A method of improving one or both of the aforementioned UPDRS or MDS-UPDRS, as described in item 1041 to 1048 of the present invention. [Invention 1050] A cell containing any of the polynucleotides 1001 to 1029 of the present invention. [Invention 1051] A protein encoded by any of the polynucleotides 1001 to 1029 of this invention. [Invention 1052] A pharmaceutical composition comprising any vector according to invention 1030 to 1033 and one or more pharmaceutically acceptable carriers, diluents, or excipients. [Invention 1053] A kit comprising any vector according to invention 1030 to 1033, and instructions for use. [Invention 1054] A recombinant adeno-associated virus (rAAV) virion comprising a capsid and a vector genome, wherein the vector genome comprises a polynucleotide sequence encoding an activated parkin protein operably linked to a promoter. [Invention 1055] The rAAV virion of the present invention 1055, wherein the activated parkin protein contains one or more amino acid substitutions at positions Phe-146, Trp-403, Cys-457, Phe-463, and Asn-273 relative to the reference parkin protein. [Invention 1056] The rAAV virion of the present invention 1055, wherein the activated parkin protein contains two or more amino acid substitutions at positions Phe-146, Trp-403, Cys-457, Phe-463, and Asn-273 relative to the reference parkin protein. [Invention 1057] The rAAV virion of the present invention 1056, wherein the activated parkin protein contains amino acid substitutions at positions Phe-146, Trp-403, Cys-457, Phe-463, and Asn-273 relative to the reference parkin protein. [Invention 1058] The rAAV virion according to any of Invention 1054 to 1057, wherein the activated parkin protein comprises one or more amino acid substitutions selected from F146A, W403A, and / or N273K relative to the reference parkin protein. [Invention 1059] The rAAV virion of the present invention 1058, wherein the activated parkin protein contains amino acid substitutions F146A and W403A relative to the reference parkin protein. [Invention 1060] The rAAV virion of the present invention 1059, wherein the activated parkin protein contains amino acid substitutions F146A, N273K, and W403A relative to the reference parkin protein. [Invention 1061] The rAAV virion of the present invention 1060, wherein the activated parkin protein comprises a polypeptide sequence that is at least 95% identical to human parkin N273K+W403A+F463A (SEQ ID NO: 93). [Invention 1062] The rAAV virion of the present invention 1054, comprising a polypeptide sequence in which the activated parkin protein is identical to human parkin N273K+W403A+F463A (SEQ ID NO: 93). [Invention 1063] The rAAV virion according to any one of the present inventions 1054 to 1060, wherein the parkin protein is a Δ parkin protein containing a deletion of the ubiquitin-like (Ubl) domain. [Invention 1064] The aforementioned Δparkin protein is derived from residues 76-465 of human parkin F146A+W403A: TIFF0007872737000030.tif63128 The rAAV virion of the present invention 1063, comprising a polypeptide sequence that is at least 95% identical to [another name]. [Invention 1065] The aforementioned Δparkin protein is derived from residues 76-465 of human parkin F146A+W403A: TIFF0007872737000031.tif63128 The rAAV virion of the present invention 1063, comprising a polypeptide sequence identical to that of the present invention. [Invention 1066] The rAAV virion according to any of the inventions 1054 to 1065, wherein the activated parkin protein contains an amino acid substitution at position Cys-431 relative to the reference parkin protein. [Invention 1067] The rAAV virion of the present invention 1066, wherein the activated parkin protein contains a C431F amino acid substitution relative to the reference parkin protein. [Invention 1068] The rAAV virion according to any of the present invention 1054 to 1067, wherein the promoter is a constitutive promoter. [Invention 1069] The rAAV virion according to any of the inventions 1054 to 1068, wherein the promoter is a CAG promoter. [Invention 1070] The rAAV virion according to any of the inventions 1054 to 1068, wherein the promoter is a CMV promoter. [Invention 1071] The rAAV virion according to any of the present invention 1054 to 1067, wherein the promoter is a neuron-specific promoter. [Invention 1072] The rAAV virion according to any one of the inventions 1054-1067 or 1071, wherein the promoter is a SYN promoter. [Invention 1073] The vector genome is an rAAV virion according to any of the inventions 1054 to 1072, comprising a WPRE element. [Invention 1074] The vector genome is an rAAV virion according to any of the present invention 1054 to 1073, which contains an hGH polyadenylation site. [Invention 1075] An rAAV virion according to any of the present inventions 1054 to 1074, wherein the capsid is an AAV9 capsid or a functional variant thereof. [Invention 1076] The rAAV billion of the present invention 1075, wherein the AAV9 capsid shares at least 98%, 99%, or 100% identity with a reference AAV9 capsid. [Invention 1077] A method for increasing parkin activity in cells, comprising contacting the cells with any rAAV virions of the present invention 1054 to 1076. [Invention 1078] A method for increasing parkin activity in a subject, comprising administering an effective amount of any rAAV virion from invention 1054 to 1076 to the subject. [Invention 1079] The method of Invention 1077 or Invention 1078, wherein the cells or subjects lack parkin activity and / or contain a loss-of-function mutation in parkin. [Invention 1080] Any method of the present invention 1077 to 1079, wherein parkin activity comprises one or more of the following: co-localization of parkin with TOMM2 in response to neurotoxic treatment, ubiquitination of mitochondrial proteins in response to neurotoxic treatment, and an increase in parkin levels in the mitochondrial fraction in response to neurotoxic treatment. [Invention 1081] A method for promoting neuronal survival, comprising bringing the neuron into contact with an rAAV virion according to any of the present invention 1054 to 1076. [Invention 1082] A method for promoting neuronal survival in a subject, comprising administering an effective amount of any rAAV virion according to Invention 1054-1076 to the subject. [Invention 1083] The method of the present invention 1081 or 1082, wherein the neuron is a dopaminergic neuron. [Invention 1084] A method for treating a disease or disorder in a subject requiring treatment, comprising administering an effective amount of any rAAV virion from any of the present invention 1054 to 1076 to the subject. [Invention 1085] The method of the present invention 1084, wherein the subject has a Parkin gene deficiency. [Invention 1086] The method of the present invention 1084, wherein the subject has a PINK1 gene deficiency. [Invention 1087] The method of the present invention 1084, wherein the subject has a genetic defect in DJ-1. [Invention 1088] The method according to any one of the present invention 1084 to 1087, wherein the disease or disorder is Parkinson's disease. [Invention 1089] The method of the present invention 1088, wherein the Parkinson's disease is early-onset Parkinson's disease (EOPD). [Invention 1090] A method of alleviating one or more symptoms of Parkinson's disease, as described in any of invention 1084 to 1089. [Invention 1091] Any method of the present invention 1084 to 1090 for reducing motor complications associated with neurodegeneration; reducing the need for antiparkinsonian drug therapy, optionally L-dopa and / or dopamine agonists; restoring the function of degenerated neurons; and / or protecting neurons from degeneration. [Invention 1092] Nigestria function, optionally evaluated by [18F]fluoro-L-dopa positron emission tomography (PET) or DaT-SPECT imaging. A method of enhancing any of the invention 1084 to 1091. [Invention 1093] Any method 1084 to 1092 of the present invention for improving one or both of the aforementioned UPDRS or MDS-UPDRS. [Invention 1094] A pharmaceutical composition comprising any rAAV virion according to invention 1054 to 1076 and one or more pharmaceutically acceptable carriers, diluents, or excipients. [Invention 1095] A kit comprising an rAAV virion according to any of invention 1054 to 1076, and instructions for use. [Invention 1096] A polynucleotide containing a polynucleotide sequence that codes for the activated parkin protein. [Invention 1097] The activated parkin protein is a polynucleotide of the present invention 1096, comprising an amino acid substitution at position Cys-431 relative to the reference parkin protein. [Invention 1098] The activated parkin protein is a polynucleotide of the present invention 1096, wherein the activated parkin protein contains a C431F amino acid substitution relative to the reference parkin protein. [Invention 1099] The activated parkin protein is a polynucleotide according to any of the inventions 1096 to 1098, wherein the activated parkin protein contains one or more amino acid substitutions at positions Phe-146, Trp-403, Cys-457, Phe-463, and Asn-273 relative to the reference parkin protein. [Invention 1100] The polynucleotide of the present invention 1099, wherein the activated parkin protein comprises two or more amino acid substitutions at positions Phe-146, Trp-403, Cys-457, Phe-463, and Asn-273 relative to the reference parkin protein. [Invention 1101] The polynucleotide of the present invention 1100, wherein the activated parkin protein contains amino acid substitutions at positions Phe-146, Trp-403, Cys-457, Phe-463, and Asn-273 relative to the reference parkin protein. [Invention 1102] A polynucleotide according to any of Invention 1096 to 1101, wherein the activated parkin protein comprises one or more amino acid substitutions selected from F146A, W403A, and / or N273K relative to the reference parkin protein. [Invention 1103] The polynucleotide of the present invention 1102, wherein the activated parkin protein contains amino acid substitutions F146A and W403A relative to the reference parkin protein. [Invention 1104] The activated parkin protein comprises the amino acid substitutions F146A, N273K, and W403A relative to the reference parkin protein, according to the polynucleotide of the present invention 1103. [Invention 1105] The polynucleotide of the present invention 1096, wherein the activated parkin protein comprises a polypeptide sequence that is at least 95% identical to human parkin N273K+W403A+F463A (SEQ ID NO: 93). [Invention 1106] The polynucleotide of the present invention 1096, comprising a polypeptide sequence in which the activated parkin protein is identical to human parkin N273K+W403A+F463A (SEQ ID NO: 93). [Invention 1107] The polynucleotide according to any one of the present invention 1096 to 1104, wherein the parkin protein is a Δ parkin protein containing a deletion of the ubiquitin-like (Ubl) domain. [Invention 1108] The aforementioned Δparkin protein is derived from residues 76-465 of human parkin F146A+W403A: TIFF0007872737000032.tif63128 The polynucleotide of the present invention 1107, comprising a polypeptide sequence that is at least 95% identical to that of the present invention. [Invention 1109] The aforementioned Δparkin protein is derived from residues 76-465 of human parkin F146A+W403A: TIFF0007872737000033.tif63128 A polynucleotide of the present invention 1108, comprising the same polypeptide sequence as [another polynucleotide]. [Invention 1110] A polynucleotide according to any one of the invention 1096 to 1109, comprising a promoter operably ligated to the polynucleotide sequence encoding an activated parkin protein. [Invention 1111] The polynucleotide of the present invention 1110, wherein the promoter is a constitutive promoter. [Invention 1112] The polynucleotide of the present invention 1111, wherein the promoter is a CAG promoter or a CMV promoter. [Invention 1113] The polynucleotide of the present invention 1110, wherein the promoter is a neuron-specific promoter. [Invention 1114] The polynucleotide of the present invention 1110 or 1113, wherein the promoter is a SYN promoter. [Invention 1115] A polynucleotide according to any of invention 1096 to 1114, comprising a WPRE element. [Invention 1116] A polynucleotide according to any of the present invention 1096 to 1115, comprising an hGH polyadenylation site. [Invention 1117] A vector comprising any of the polynucleotides of Invention 1096 to 1116. [Invention 1118] The vector of the present invention 1117, which is an adeno-associated virus (AAV) vector. [Invention 1119] A vector according to the present invention 1118, comprising an AAV9 capsid or a functional variant thereof. [Invention 1120] The vector of the present invention 1119, wherein the AAV9 capsid shares at least 98%, 99%, or 100% identity with a reference AAV9 capsid. [Invention 1121] A method for increasing parkin activity in cells, comprising contacting the cells with a polynucleotide of any of Invention 1096 to 1116 or a vector of any of Invention 1117 to 1120. [Invention 1122] A method for increasing parkin activity in a subject, comprising administering to the subject a polynucleotide of any of Invention 1096 to 1116 or a vector of any of Invention 1117 to 1120. [Invention 1123] The method of the present invention 1121 or 1122, wherein the cells or subjects lack parkin activity and / or contain a loss-of-function mutation in parkin. [Invention 1124] Any method 1121 to 1123 of the present invention, wherein parkin activity comprises one or more of the following: co-localization of parkin with TOMM2 in response to neurotoxic treatment, ubiquitination of mitochondrial proteins in response to neurotoxic treatment, and an increase in parkin levels in the mitochondrial fraction in response to neurotoxic treatment. [Invention 1125] A method for promoting the survival of a neuron, comprising contacting the neuron with a polynucleotide of any of Invention 1096 to 1116 or a vector of any of Invention 1117 to 1120. [Invention 1126] A method for promoting neuronal survival in a subject, comprising administering to the subject a polynucleotide of any of Invention 1096 to 1116 or a vector of any of Invention 1117 to 1120. [Invention 1127] The method of the present invention 1125 or 1126, wherein the neuron is a dopaminergic neuron. [Invention 1128] A method for treating a disease or disorder in a subject requiring treatment, comprising administering to the subject a polynucleotide of any of Invention 1096 to 1116 or a vector of any of Invention 1117 to 1120. [Invention 1129] The method of the present invention 1128, wherein the subject has a genetic defect in parkin expression or function. [Invention 1130] The method of the present invention 1128 or 1129, wherein the subject has a genetic defect in PINK1 expression or function. [Invention 1131] A method according to any one of the present invention 1128 to 1130, wherein the disease or disorder is Parkinson's disease. [Invention 1132] The method of the present invention 1131, wherein the Parkinson's disease is early-onset Parkinson's disease (EOPD). [Invention 1133] A method of alleviating one or more symptoms of Parkinson's disease, as described in any of invention 1128 to 1132. [Invention 1134] Any method of the present invention 1128-1133 for reducing motor complications associated with neurodegeneration; reducing the need for antiparkinsonian drug therapy, optionally L-dopa and / or dopamine agonists; restoring the function of degenerated neurons; and / or protecting neurons from degeneration. [Invention 1135] Nigestria function, optionally evaluated by [18F]fluoro-L-dopa positron emission tomography (PET) or DaT-SPECT imaging. Any method of the present invention 1128 to 1134 to enhance the. [Invention 1136] Any method 1128 to 1135 of the present invention for improving one or both of the aforementioned UPDRS or MDS-UPDRS. [Invention 1137] A cell containing any of the polynucleotides of the present invention 1096 to 1116. [Invention 1138] A protein encoded by any of the polynucleotides of Invention 1096 to 1116. [Invention 1139] A pharmaceutical composition comprising any vector according to invention 1117 to 1120 and one or more pharmaceutically acceptable carriers, diluents, or excipients. [Invention 1140] A kit comprising a vector according to any of invention 1117 to 1120, and instructions for use. Further aspects and embodiments of the present invention will become apparent from the following detailed description. [Brief explanation of the drawing] 【0162】 [Figure 1] The diagram shows the domains of parkin with specific amino acid substitutions indicated by arrows. [Figure 2] A vector diagram of a non-restrictive example of a vector genome is shown. [Figure 3] A vector diagram of a non-restrictive example of a vector genome is shown. Amino acid substitutions at F146A, N273K, and W403A are indicated by arrows. [Figure 4] A vector diagram of a non-restrictive example of a vector genome is shown. Amino acid substitutions at F146A and W403A are indicated by arrows. [Figure 5] A vector diagram of a non-restrictive example of a vector genome is shown. Amino acid substitutions at F146A and W403A are indicated by arrows. [Figure 6] A vector diagram of a non-restrictive example of a vector genome is shown. [Figure 7] A vector diagram of a non-restrictive example of a vector genome is shown. Amino acid substitutions at F146A and W403A are indicated by arrows. [Figure 8]A vector diagram of a non-restrictive example of a vector genome is shown. Amino acid substitutions at F104M, F146A, and W403A are indicated by arrows. F104M is relative to the reference human PINK1 protein sequence of SEQ ID NO: 64, W403A is relative to the reference human parkin protein sequence of SEQ ID NO: 1, and F463A is relative to the reference human parkin protein sequence of SEQ ID NO: 1. [Figure 9] A vector diagram of a non-restrictive example of a vector genome is shown. Amino acid substitutions at C431F are indicated by arrows. [Figure 10] Figures 10A–10D show the testing of the bioactivity of the parkin construct in transfected N27A dopaminergic (DA) neurons. The luminescence units (LUs) represent neuronal proliferation and / or survival measured 3 days after treatment with μM of control (Figure 10A), 7.5 μM 6-hydroxydopamine (6-OHDA) (Figure 10B), 15 μM 6-OHDA (Figure 10C), or 30 μM 6-OHDA (Figure 10D). [Figure 11] Figures 11A–11D show the testing of the bioactivity of the parkin construct in transfected N27A dopaminergic (DA) neurons. Luminescent units (LUs) represent neuronal proliferation and / or survival measured 9 days after treatment with control (Figure 11A), 7.5 μM 6-OHDA (Figure 10B), 15 μM 6-OHDA (Figure 10C), or 30 μM 6-OHDA (Figure 10D). [Figure 12] This demonstrates the bioactivity of parkin constructs in transfected human PARK2- / -dopaminergic (DA) neurons. [Figure 13] Western blots of parkin protein expression after transduction of primary neurons using AAV vectors encoding parkin variants are shown. CON = control green fluorescent protein, ACT = activated parkin, DEL = Δ parkin, SUP1 = super parkin, SUP2 = super parkin V2, WT = wild-type parkin, C431F = C431F amino acid substitution. [Figure 14] A vector diagram of a non-restrictive example of a vector genome is shown. [Figure 15] A vector diagram of a non-restrictive example of a vector genome is shown. [Figure 16] A vector diagram of a non-restrictive example of a vector genome is shown. [Figure 17] A vector diagram of a non-restrictive example of a vector genome is shown. [Modes for carrying out the invention] 【0163】 Detailed description of the invention overview Adeno-associated virus vectors, such as the AAV2 vector, have been used to deliver potentially therapeutic transgenes to the brains of individuals with Parkinson's disease (PD), but with limited success. For example, a double-blind trial of AAV2-neuruturin delivery showed good tolerability but was not superior to sham surgery. Olanow et al. Ann Neurol. 78:248-57 (2015). Parkin expression from AAV vectors has been shown to have neuroprotective effects on substantia nigra dopamine neurons in preclinical models of neurodegeneration (Benskey et al., Neurotox, 2015; Paterna et al., Mol Ther, 2007; Yasuda et al., J Neuropath Exp Neurol, 2011; Klein et al. Neurosci Lett. 401:130-135 (2006)). AAV-mediated gene delivery of Nurr1 and Foxa2 in a PD mouse model significantly protected motor behavior associated with midbrain DA (mDA) neurons and substantia nigra striatal DA neurotransmission (Oh et al. EMBO Mol Med. 7:510-25 (2015)). 【0164】 The present invention generally relates to gene therapy for disorders associated with mitochondrial dysfunction, such as central nervous system (CNS) disorders like Parkinson's disease. In particular, this disclosure provides recombinant adeno-associated virus (rAAV) virions for the expression of activated parkin protein. 【0165】 In one embodiment, the disclosure provides a recombinant adeno-associated virus (rAAV) virion comprising a capsid and a vector genome, the vector genome comprising a polynucleotide sequence encoding an activated parkin protein operably linked to a promoter. 【0166】 In other embodiments, the Disclosure provides a method for promoting neuronal survival, which includes selectively exposing neurons to or administering an effective amount of the Disclosed rAAV virion to a subject. 【0167】 In another aspect, the disclosure provides a method for treating a disease or disorder, the method comprising administering an effective amount of the disclosed rAAV virion to a target. 【0168】 Furthermore, the Disclosure provides a polynucleotide sequence encoding a fusion protein in which a portion of the parkin protein is fused to a mitochondrial targeting sequence (MTS). A vector containing the polynucleotide of the Disclosure, for example, a recombinant adeno-associated virus (rAAV) vector, is further provided. 【0169】 In one embodiment, the disclosure provides a polynucleotide comprising a polynucleotide sequence encoding a fusion protein comprising a mitochondrial targeting sequence (MTS), a transmembrane domain (TMD), and a parkin protein or a functional variant thereof. 【0170】 In other embodiments, the Disclosure provides a vector comprising the polynucleotides of the Disclosure. 【0171】 In another aspect, the Disclosure provides a method for increasing parkin activity in cells, the method comprising contacting the cells with a polynucleotide or vector of the Disclosure. 【0172】 In another embodiment, the Disclosure provides a method for increasing parkin activity in a subject, the method comprising administering the polynucleotide or vector of the Disclosure to the subject. 【0173】 In another embodiment, the Disclosure provides a method for promoting neuronal survival, the method comprising contacting a neuron with a polynucleotide or vector of the Disclosure. 【0174】 In another aspect, the Disclosure provides a method for promoting neuronal survival in a subject, the method comprising administering the polynucleotide or vector of the Disclosure to the subject. 【0175】 In another embodiment, the Disclosure provides a method for treating a disease or disorder in a subject requiring treatment, the method comprising administering the polynucleotide or vector of the Disclosure to the subject. 【0176】 Various other aspects and embodiments are disclosed in the detailed description below. The present invention is limited only by the appended claims. 【0177】 definition Section headings are intended solely for organizational purposes and should not be construed as limiting the subject matter described to any particular aspect or embodiment. 【0178】 Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the present invention relates. Similar or equivalent methods and materials may be used in the practice of the present invention, but preferred methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety. In case of any conflict, this specification shall prevail, including definitions. Furthermore, the materials, methods, and examples described herein are illustrative and not intended to limit the scope of the invention. 【0179】 All publications and patents described herein are incorporated herein by reference in whole, as if each individual publication or patent were specifically and individually indicated to be incorporated by reference. In the event of any conflict, this application shall prevail, including any definitions herein. However, any reference to any reference, article, publication, patent, patent publication, and patent application cited herein shall not be, and should not be construed as, an endorsement or proposal in any form of which that constitutes valid prior art or forms part of the common general knowledge in any country of the world. 【0180】 In this document, unless otherwise indicated, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer value within the enumerated range, and, where appropriate, fractions thereof (e.g., one-tenth and one-hundredth of an integer). The term “about” when preceding a number or figure means that the number or figure is within a range of plus or minus 10%. Where used herein, the terms “a” and “an” should be understood to refer to “one or more” of the enumerated components, unless otherwise indicated. The use of substitutes (e.g., “or”) should be understood to mean either one, both, or any combination thereof of the substitutes. The terms “and / or” should be understood to mean one or both of the options. Where used herein, the terms “include” and “comprise” are used synonymously. 【0181】 As used herein, the terms “identity” and “identical” refer to the percentage of residues that are perfectly identical in the alignment of a “query” sequence and a “target” sequence, such as the alignment generated by the BLAST algorithm, with respect to a polypeptide or polynucleotide sequence. Identity is calculated over the entire length of the target sequence unless otherwise specified. Thus, if, when the query sequence is aligned with the target sequence, at least x% (truncated) of residues in the target sequence are aligned to perfectly match the corresponding residues in the query sequence, then the query sequence “shares at least x% identity” with the target sequence. If the target sequence has variable positions (e.g., residues indicated by X), any alignment to any residue in the query sequence is counted as a match. Sequence comparisons for determining identity percentages can be achieved by many well-known methods, including the use of mathematical algorithms, such as those found in the BLAST suite of sequence analysis programs. Unless otherwise specified, the terms "identity" and "identical" in relation to a reference sequence refer to the full-length sequence identity of the reference sequence after aligning two sequences using the Blast-p program (for proteins) or Blast-n program (for polynucleotides) of the National Center for Biotechnology Information (NCBI) online alignment tool, version 2.11.0 (released October 19, 2020), available at blast.ncbi.nlm.nih.gov. See Altschul et al. J.Mol.Biol.215:403-410 (1990). 【0182】 As used herein, “AAV vector” or “rAAV vector” refers to a recombinant vector containing one or more target polynucleotides (or transgenes) flanked by an AAV terminal repeat sequence (ITR). Such an AAV vector may be replicated and 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, the AAV vector may be packaged into infectious particles using a host cell stably engineered to express the rep and cap genes. 【0183】 As used herein, “AAV virion,” “AAV virus particle,” or “AAV vector particle” refers to a virus particle comprising at least one AAV capsid protein and a capsidized polynucleotide AAV vector. As used herein, if a particle contains heterologous polynucleotides (i.e., polynucleotides other than the wild-type AAV genome, such as a transgene delivered to a mammalian cell), it is usually referred to as an “AAV vector particle” or simply an “AAV vector.” Thus, the production of an AAV vector particle necessarily involves the production of an AAV vector, since the vector is contained within the AAV vector particle. 【0184】 As used herein, “promoter” refers to a polynucleotide sequence that can promote the initiation of RNA transcription from a polynucleotide in eukaryotic cells. 【0185】 As used herein, “vector genome” refers to a polynucleotide sequence packaged by a vector (e.g., rAAV virion), including contiguous sequences (in AAV, inverted terminal sequences). The terms “expression cassette” and “polynucleotide cassette” refer to portions of the vector genome between contiguous sequences. “Expression cassette” means that the vector genome contains at least one gene encoding a gene product operably ligated to an element that drives expression (e.g., a promoter). 【0186】 As used herein, the terms “patient in need” or “subject in need” refer to a patient or subject at risk of or suffering from a disease, disorder, or condition suitable for treatment or improvement with the recombinant gene therapy vectors or gene editing systems disclosed herein. A patient or subject in need may, for example, be a patient or subject diagnosed with a disorder related to the degradation of the central nervous system. The subject may have mutations or dysfunction in PARK2, PARK6, PARK7, LRRK2, or α-synuclein, gene, or protein. “Subject” and “patient” are used interchangeably herein. A subject treated by the methods described herein may be an adult or a child. The subject may be within an age range. The subject may be a person identified as being at risk for Parkinson's disease, for example, early-onset Parkinson's disease. 【0187】 As used herein, “deficiency,” for example, “deficiency of parkin activity” in a cell or subject, refers to either a genetic deficiency due to partial or complete loss of function of the PARK2 gene, or a decrease in activity due to other causes such as lower-than-normal levels of protein (parkin) expression or reduced expression of factors affecting protein (parkin) activity. For example, since PINK1 activates parkin, cells expressing lower-than-normal levels of PINK1 may have reduced parkin activity. 【0188】 As used herein, “Parkin activity” refers to any enzymatic or cellular signaling activity of parkin. 【0189】 As used herein, “activated parkin” refers to a parkin protein variant that has increased intrinsic activity in one or more biochemical or cellular assays compared to a reference parkin protein (e.g., human parkin protein). 【0190】 As used herein, the terms “variant” or “functional variant” are interchangeable and refer to a protein having one or more amino acid substitutions, insertions, or deletions compared to the parent protein, which retains one or more desired activities of the parent protein. 【0191】 As used herein, “genetic defect” means a partial or complete loss of function in a gene. For example, a subject with a genetic defect in parkin function expression has one or more mutations in the PARK2 gene that reduce the expression of parkin protein or reduce its function in at least some of the subject’s cells (e.g., neurons). 【0192】 As used herein, “Parkinson’s disease” refers, for example, to “The Differential Diagnosis of Parkinson’s Disease.” Parkinson’s Disease: Pathogenesis and Clinical Aspects, Chapter 6. Codon Publications (2018), or Harrison’s Principles of Internal Medicine, 2018. th As defined in the ed., this name refers to any form of disease known in the art. 【0193】 As used herein, “treatment” means the suppression, reduction, or improvement of one or more symptoms of a disease or disorder, and / or the prevention of the progression of the disease or disorder. 【0194】 As used herein, the phrase “disorders associated with mitochondrial dysfunction” refers to any disease or disorder that develops or progresses in connection with mitochondrial dysfunction and can be prevented or reversed by parkin activity. 【0195】 Parkin protein This disclosure envisions compositions and methods of use related to various activated parkin proteins. An activated parkin protein is any parkin protein in which biochemical, cellular, or physiological activity is increased compared to a reference parkin protein (e.g., wild-type parkin proteins such as the parkin protein normally encoded by the human PRKN2 gene, i.e., H1 in Table 1). 【0196】 Furthermore, this disclosure envisions compositions and methods of use relating to various fusions of a portion of a parkin protein to a mitochondrial targeting sequence (MTS). The parkin protein may optionally be a Δ parkin protein, i.e., a parkin protein having one or more domain deletions compared to a reference parkin protein (e.g., a parkin protein normally encoded by the human PRKN2 gene, i.e., a wild-type parkin protein such as H1 in Table 1). 【0197】 Alternative splicing generates various alternative isoforms of human parkin, as shown in Table 1 (see Scuderi et al. BioMed Res.Int'l Vol.2014, Article 690796). 【0198】 (Table 1) Parkin isoforms TIFF0007872737000034.tif137163* The protein accession number does not exist in the database. 【0199】 The polypeptide sequence of a standard human parkine isoform (H1) is as follows: TIFF0007872737000035.tif66133 【0200】 The reference parkin protein may be Sequence ID No. 1. The activated parkin protein may also be another isoform of parkin, having amino acid substitutions at equivalent positions in the multiple sequence alignment of parkin protein isoforms, for example, prepared using the ClustalW or MUSCLE alignment algorithm. 【0201】 Further isoforms of perkin that may be used in the compositions and methods of this disclosure include the polypeptides of SEQ ID NOs: 2-8. 【0202】 In some embodiments, the polynucleotide encoding activated parkin comprises a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 9. 【0203】 The polynucleotide sequence encoding activating parkin may be codon-optimized. In some embodiments, the polynucleotide encoding activating parkin includes a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 10. 【0204】 In some embodiments, the activated parkin contains one or more amino acid substitutions selected from predicted residue mutations in the Ubl (S65D or S65E), linker (S131A), RING0 (Y143A, F146A), RING1 (N273K), REP (W403A), or RING2 (C457S or F463A) domains, numbered for SEQ ID NO: 1. That is, the activated parkin protein may contain one or more, two or more, three or more, or four or more amino acid substitutions selected from the group consisting of S65D or S65E, S131A, Y143A, F146A, N273K, W403A, C457S, and F463A. Alternative conserved or non-conserved mutations may be used in any of these sites, including, but not limited to, one, two or more, three or more, or four or more amino acid substitutions selected from the group consisting of S65X, S131X, Y143X, F146X, N273X, W403X, C457X, and F463X, where X represents any native or non-native amino acid other than those present in the reference parkin protein. 【0205】 Specific mutations intended by this disclosure include S65D, S65E, S65K, or S65R; S131A, S131L, or S131I; F146A, F146S, F146T, F146I, or F146L; N273K, N2773R, N273E, or N273Q; and F463A, F463S, F463T, F463I, or F463L. In some embodiments, the amino acid substitution disrupts intramolecular or intermolecular interfaces. In some embodiments, the amino acid substitution disrupts intramolecular or intermolecular interfaces while maintaining one or more properties of the residue, such as charge, size, and / or hydrophobicity. 【0206】 Activated parkin may contain one or more amino acid substitutions, insertions, or deletions (collective mutations) that reduce the binding of one structural domain of parkin to another structural domain, thereby reducing autoinhibition. For example, activated parkin may contain a mutation in Ubl that reduces binding to the RING1 domain, or a mutation in the RING1 domain that reduces binding to the Ubl domain (e.g., N273K). Activated parkin may contain a mutation in the REP domain that reduces binding to the RING1 domain (e.g., W403A), or a mutation in the RING1 domain that reduces binding to the REP domain. Activated parkin may contain a mutation in the RING0 domain that reduces binding to the RING2 domain (e.g., F146A), or a mutation in the RING2 domain that reduces binding to the RING0 domain (e.g., C457S and / or F463A). 【0207】 Alternatively, or in addition to the foregoing, the activated parkin may include mutations that protect against parkin degradation, mediated by kinase c-Abl (e.g., Y143A) or by kinase p38MAPK (e.g., S131A). 【0208】 In lieu of or in addition to the foregoing, activated parkin may also contain the amino acid substitution C431X, where X represents any native or non-native amino acid other than those present in the reference parkin protein. In some embodiments, activated parkin may also contain the amino acid substitution C431F. 【0209】 Various further embodiments of activated parkin are provided in Table 2A or Table 2B. 【0210】 (Table 2A) Exemplary combinations of amino acid substitutions TIFF0007872737000036.tif105160 【0211】 (Table 2B) Exemplary combinations of amino acid substitutions TIFF0007872737000037.tif128160 【0212】 In some embodiments, the activated parkin protein contains one or more amino acid substitutions at position Cys-431 relative to the reference parkin protein. 【0213】 In some embodiments, the activated parkin protein contains one or more amino acid substitutions at positions Phe-146, Trp-403, Cys-457, Phe-463, and Asn-273 relative to the reference parkin protein. 【0214】 In some embodiments, the activated parkin protein contains two or more amino acid substitutions at positions Phe-146, Trp-403, Cys-457, Phe-463, and Asn-273 relative to the reference parkin protein. 【0215】 In some embodiments, the activated parkin protein includes amino acid substitutions at positions Phe-146, Trp-403, Cys-457, Phe-463, and Asn-273 relative to the reference parkin protein. 【0216】 In some embodiments, the activated parkin protein comprises one or more amino acid substitutions selected from F146A, W403A, and / or N273K relative to the reference parkin protein. 【0217】 In some embodiments, the activated parkin protein contains amino acid substitutions F146A and W403A relative to the reference parkin protein. 【0218】 In some embodiments, the activated parkin protein includes amino acid substitutions F146A, N273K, and W403A relative to the reference parkin protein. 【0219】 In some embodiments, the activated parkin protein comprises a polypeptide sequence that is at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the human parkin isoforms listed in Table 1. In some embodiments, the activated parkin protein comprises a polypeptide sequence that is at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to human parkin of SEQ ID NO: 1. 【0220】 In some embodiments, the activated parkin protein is human parkin F146A+N273K+W403A: It contains a polypeptide sequence that is at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to TIFF0007872737000038.tif67133. 【0221】 In some embodiments, the activated parkin protein consists of the polypeptide sequence of human parkin F146A+N273K+W403A (SEQ ID NO: 11). 【0222】 In some embodiments, the activated parkin protein is identical to a portion of human parkin F146A+N273K+W403A (SEQ ID NO: 11) throughout the entire length of the polypeptide sequence, and the polypeptide sequence has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid C-terminal and / or N-terminal truncations relative to SEQ ID NO: 11. 【0223】 In some embodiments, the activated parkin protein is human parkin N273K+W403A+C457S: It contains a polypeptide sequence that is at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to TIFF0007872737000039.tif67133. 【0224】 In some embodiments, the activated parkin protein is human parkin N273K+W403A+F463A: It contains a polypeptide sequence that is at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to TIFF0007872737000040.tif67133. 【0225】 In some embodiments, the activated parkin protein is human parkin F146A+N273K+W403A+C457S: It contains a polypeptide sequence that is at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to TIFF0007872737000041.tif66133. 【0226】 In some embodiments, the activated parkin protein is human parkin F146A+N273K+W403A+C457S+F463A: It contains a polypeptide sequence that is at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to TIFF0007872737000042.tif67133. 【0227】 In some embodiments, the activated parkin protein is human parkin N273K+W403A+F463A: It contains a polypeptide sequence that is at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to TIFF0007872737000043.tif67133. 【0228】 In some embodiments, the activated parkin protein is human parkin C431F: It contains a polypeptide sequence that is at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to TIFF0007872737000044.tif66133. 【0229】 A fusion protein comprising the Parkin protein or a functional variant or fragment thereof. The Park protein may be SEQ ID NO: 1 or another isoform of Parkin, for example, having deletions and / or amino acid substitutions at equivalent positions in a multiple sequence alignment of the Parkin protein isoform, prepared using the ClustalW or MUSCLE alignment algorithm. 【0230】 Further isoforms of parkin available include the following, where the N-terminal portion in parentheses may be optionally removed: TIFF0007872737000045.tif227160TIFF0007872737000046.tif21159 【0231】 This disclosure provides a fusion protein comprising a mitochondrial targeting sequence (MTS), a transmembrane domain (TMD), and a parkin protein or a functional variant or fragment thereof. The MTS may be the MTS of PINK1 or a functional variant thereof. 【0232】 The MTS of PINK1 is post-translationally cleaved by mitochondrial processing peptidase (MPP) and presenilin-associated rhomboid-like (PARL) proteins. In some embodiments, the MTS, or another portion of the fusion protein, includes an MPP cleavage site. In some embodiments, the TMD includes a PARL cleavage site. The MPP and PARL cleavage sites, if present, are cleaved when mitochondria become polarized. We recognize that the inclusion of these cleavage sites in the fusion protein may specifically activate the fusion protein in damaged mitochondria. 【0233】 The fusion protein may optionally have amino acid substitutions that stabilize the PARL cleavage product. For example, the fusion protein may contain amino acid substitutions F104M, F104A, F104V, F104S, or F104G relative to the wild-type PINK1 sequence. An exemplary partial sequence of PINK1 is also shown below. TIFF0007872737000047.tif29128 【0234】 By functionally equivalent substitutions of F104M, F104A, F104V, F104S, F104G, or PINK1 at the same or different positions, the fusion protein can be cleaved in the MTS by MPP and PARL. As a result, parkin or parkin fragments of the fusion protein are released from the mitochondrial membrane in their active form. Advantageously, parkin fragments produced by cleavage by PARL (in undamaged mitochondria) can be released from the mitochondrial membrane into the cytoplasm in their active form. 【0235】 MTS is derived from residues 1-94 of human PINK1: TIFF0007872737000048.tif29128 may contain at least 95%, 96%, 97%, 98%, 99%, or 100% of the polypeptide sequence. 【0236】 MTS may be the smallest MTS. MTS is human PINK1 residues 1-34: It may contain polypeptide sequences that are at least 95%, 96%, 97%, 98%, 99%, or 100% identical to TIFF0007872737000049.tif15128. 【0237】 The fusion proteins of this disclosure may further have a transmembrane domain (TMD). A suitable transmembrane domain may include any TMD that can be cleaved by PARL. 【0238】 In some embodiments, the TMD is the TMD of PINK1 or a functional variant thereof. The TMD is derived from residues 95-110 of human PINK1: It may contain polypeptide sequences that are at least 95%, 96%, 97%, 98%, 99%, or 100% identical to TIFF0007872737000050.tif17128. 【0239】 In some embodiments, the TMD is the TMD of PINK1 or a functional variant thereof. The TMD is residues 95-110 of human PINK1 F104M: It may contain polypeptide sequences that are at least 95%, 96%, 97%, 98%, 99%, or 100% identical to TIFF0007872737000051.tif17128. 【0240】 In some embodiments, the TMD is the TMD of PINK1 or a functional variant thereof. The TMD is residues 95-110 of human PINK1 F104A: It may contain polypeptide sequences that are at least 95%, 96%, 97%, 98%, 99%, or 100% identical to TIFF0007872737000052.tif17128. 【0241】 In some embodiments, the fusion protein comprises the MTS of PINK1 and the TMD of PINK1-i, i.e., the MTS-TMD fragment of PINK1, or functional variants thereof. In some embodiments, the fusion protein comprises the MTS-TMD fragment of PINK1 or a functional variant thereof, optionally comprising residues 1-110 of human PINK1: It contains a polypeptide sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to TIFF0007872737000053.tif29128. 【0242】 The MTS-TMD fragment consists of residues 1-110 of human PINK1 F104M: It may contain a polypeptide sequence identical to TIFF0007872737000054.tif29128. 【0243】 The MTS-TMD fragment consists of residues 1-110 of human PINK1 F104A: It may contain a polypeptide sequence identical to TIFF0007872737000055.tif29128. 【0244】 In some cases, the parkin fragment is a fragment containing a deletion of the N-terminal ubiquitin-like (Ubl) domain and, optionally, a deletion of the Ubl-RING0 interdomain linker. This fragment is referred to herein as the "Δparkin protein." The "Δparkin protein" may optionally contain one or more activated amino acid substitutions, such as F146A and / or W403A and / or C457S and / or F463A. Thus, in some embodiments, the fusion protein is residues 141-465 of human parkin F146A+W403A: The ΔParkin protein contains a polypeptide sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to TIFF0007872737000056.tif65128. 【0245】 In some embodiments, the fusion protein is derived from residues 76-465 of human parkin F146A+W403A: The ΔParkin protein contains a polypeptide sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to TIFF0007872737000057.tif77128. 【0246】 The complete fusion protein of this disclosure may, in some embodiments, include an MTS-TMD of PINK1 fused at the C-terminus to the ΔParkin protein. Therefore, in some embodiments, the fusion protein has the following sequence: It contains a polypeptide sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to TIFF0007872737000058.tif89128. 【0247】 In some embodiments, the fusion protein has the following sequence: It contains a polypeptide sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to TIFF0007872737000059.tif90128. Here, the sequence includes an F104M or F104A substitution relative to the reference human PINK1 protein sequence of SEQ ID NO: 64. 【0248】 In some embodiments, the fusion protein has the following sequence: It contains a polypeptide sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to TIFF0007872737000060.tif75128. 【0249】 In some embodiments, the fusion protein has the following sequence: It contains a polypeptide sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to TIFF0007872737000061.tif77128. Here, the sequence includes an F104M or F104A substitution relative to the reference human PINK1 protein sequence of SEQ ID NO: 64. 【0250】 The complete fusion protein of this disclosure may, in some embodiments, include an MTS-TMD of PINK1 fused at the C-terminus to the ΔParkin protein. Therefore, in some embodiments, the fusion protein has the following sequence: It contains a polypeptide sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to TIFF0007872737000062.tif88128. Here, the sequence includes the W403A and F463A substitutions. 【0251】 The complete fusion protein of this disclosure may, in some embodiments, include an MTS-TMD of PINK1 fused at the C-terminus to the ΔParkin protein. Therefore, in some embodiments, the fusion protein has the following sequence: It contains a polypeptide sequence that is at least 95%, 96%, 97%, 98%, 99%, or 100% identical to TIFF0007872737000063.tif89128. The sequence here includes the F104M, W403A, and F463A substitutions. F104M is for the reference human PINK1 protein sequence of SEQ ID NO: 64, W403A is for the reference human parkin protein sequence of SEQ ID NO: 1, and F463A is for the reference human parkin protein sequence of SEQ ID NO: 1. 【0252】 In some embodiments, the polynucleotide encoding the fusion protein includes a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 77. 【0253】 The polynucleotide sequence encoding the fusion protein may be codon-optimized. In some embodiments, the polynucleotide encoding the fusion protein includes a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 78. 【0254】 In some embodiments, the polynucleotide encoding the fusion protein includes a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 79. 【0255】 The polynucleotide sequence encoding the fusion protein may be codon-optimized. In some embodiments, the polynucleotide encoding the fusion protein includes a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 80. 【0256】 In some embodiments, the polynucleotide encoding the fusion protein includes a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 81. 【0257】 The polynucleotide sequence encoding the fusion protein may be codon-optimized. In some embodiments, the polynucleotide encoding the fusion protein includes a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 82. 【0258】 In some embodiments, the parkin protein comprises a polypeptide sequence or a functional fragment thereof that is at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the human parkin isoforms listed in Table 1, or the fragment comprises a deletion of the portion indicated in parentheses. In some embodiments, the parkin protein comprises a polypeptide sequence or a functional fragment thereof that is at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to human parkin of SEQ ID NO: 1. 【0259】 Parkin may include deletions of the ubiquitin-like (Ubl) domain of parkin, or deletions of a portion of the Ubl domain. Parkin having a deletion of the Ubl domain is referred to herein as "Δparkin". The boundary of the Ubl domain may vary depending on the sequence of the reference parkin. Generally, the Ubl domain of human parkin is considered to be the first 75 amino acid residues. Therefore, in some embodiments, the parkin protein is a Δparkin protein containing a deletion of the ubiquitin-like (Ubl) domain, for example, Δparkin may include deletions of residues 1-75, 5-75, 1-70, 5-75, etc. 【0260】 Activated parkin may further include deletions of the parkin linker domain (residues 76-140) or any part of the linker. 【0261】 In some embodiments, this Δ-Parkin protein is derived from residues 76-465 of human Parkin. It contains a polypeptide sequence that is at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to TIFF0007872737000064.tif66133. 【0262】 In some embodiments, the Δparkin protein is derived from human parkin residues 141-465: It contains a polypeptide sequence that is at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to TIFF0007872737000065.tif67133. 【0263】 In some embodiments, the Δparkin protein is derived from residues 76-465 of human parkin F146A+W403A: It contains a polypeptide sequence that is at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to TIFF0007872737000066.tif67133. 【0264】 In some embodiments, the Δparkin protein is derived from residues 141-465 of human parkin W403A+F463A: It contains a polypeptide sequence that is at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to TIFF0007872737000067.tif67133. 【0265】 In some embodiments, the activated Δ-Parkin protein consists of the polypeptide sequence of residues 76-465 of human parkin (SEQ ID NO: 16). In some embodiments, the activated Δ-Parkin protein consists of the polypeptide sequence of residues 76-465 of human parkin F146A+W403A (SEQ ID NO: 18). 【0266】 In some embodiments, the activated Δ-Parkin protein is identical to a portion of residues 76-465 of human parkin (SEQ ID NO: 16) throughout the entire length of the polypeptide sequence, and the polypeptide sequence has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid C-terminal and / or N-terminal truncations relative to SEQ ID NO: 16. 【0267】 In some embodiments, the activated Δ-Parkin protein is identical to a portion of residues 76-465 of human parkin F146A+W403A (SEQ ID NO: 18) throughout the entire length of the polypeptide sequence, and the polypeptide sequence has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid C-terminal and / or N-terminal truncations relative to SEQ ID NO: 18. 【0268】 In some embodiments, the activated parkin protein is derived from residues 76-465 (or residues 141-465) of human parkin N273K+W403A+C457S: It contains a polypeptide sequence that is at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to TIFF0007872737000068.tif52128. 【0269】 In some embodiments, the activated parkin protein is residues 76-465 (or residues 141-465) of human parkin N273K+W403A+F463A: It contains a polypeptide sequence that is at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to TIFF0007872737000069.tif62133. 【0270】 In some embodiments, the activated parkin protein is residues 76-465 (or residues 141-465) of human parkin F146A+N273K+W403A+C457S: It contains a polypeptide sequence that is at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to TIFF0007872737000070.tif62133. 【0271】 In some embodiments, the activated parkin protein is residues 76-465 (or residues 141-465) of human parkin F146A+N273K+W403A+C457S+F463A: It contains a polypeptide sequence that is at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to TIFF0007872737000071.tif62133. 【0272】 In some embodiments, the polynucleotide encoding Δ-Parkin includes a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 22. 【0273】 In some embodiments, the polynucleotide encoding Δ-Parkin includes a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 23. 【0274】 The polynucleotide encoding the Δ-Parkin protein may be codon-optimized. In some embodiments, the polynucleotide encoding the Δ-Parkin protein includes a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 24. 【0275】 In some embodiments, the polynucleotide encoding the ΔParkin protein comprises a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 25. 【0276】 Vector genome The AAV virion of this disclosure comprises a vector genome. The vector genome may comprise an expression cassette (or a polynucleotide cassette for gene editing applications that do not require the expression of a polynucleotide sequence). Any suitable inverted end sequence (ITR) may be used. The ITR may be derived from the same serotype as the capsid, or from a different serotype (for example, an AAV2 ITR may be used). 【0277】 In some embodiments, the 5'ITR includes a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 26. 【0278】 In some embodiments, the 5'ITR includes a polynucleotide sequence that is at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 27. 【0279】 In some embodiments, the vector genome includes, for example, one or more filler sequences that are at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to sequence number 28. 【0280】 promoter In some embodiments, a polynucleotide sequence encoding a parkin protein, such as activated parkin protein, or a functional variant or fragment thereof, is operably ligated to a promoter. 【0281】 This disclosure intends to utilize a variety of promoters. Promoters useful in embodiments of this disclosure include, but are not limited to, a cytomegalovirus (CMV) promoter, a phosphoglycerate kinase (PGK) promoter, or a promoter sequence comprising a CMV enhancer, a chicken beta-actin promoter, and a portion of the rabbit beta-globin gene (CAG). In some cases, the promoter may be a synthetic promoter. An example of a synthetic promoter is provided by Schlabach et al. PNAS USA. 107(6):2538-43 (2010). 【0282】 In some embodiments, a polynucleotide sequence encoding a parkin protein, or a functional variant or fragment thereof, is operably ligated to an inductive promoter. The inductive promoter may be configured to transcriptionally express or deexpress the polynucleotide sequence in response to the addition or accumulation of a drug, or in response to the removal, degradation, or dilution of a drug. The drug may be a pharmacologic agent. The drug may be, but is not limited to, a tetracycline or one of its derivatives, but includes doxycycline. In some cases, the inductive promoter is a tet-on promoter, a tet-off promoter, a chemically regulated promoter, or a physically regulated promoter (i.e., a promoter that responds to the presence or absence of light, or to low or high temperatures). Inductive promoters include heavy metal ion inductive promoters (such as mouse mammary tumor virus (mMTV) promoters or various growth hormone promoters), and T7 phage-derived promoters that are active in the presence of T7 RNA polymerase. This list of inductive promoters is non-exclusive. 【0283】 In some cases, the promoter is a tissue-specific promoter, such as a promoter that can drive expression in neurons rather than non-neuronal cells. In some embodiments, the tissue-specific promoter is selected from any variety of neuron-specific promoters, including but not limited to hSYN1 (human synapsin), INA (alpha internexin), NES (nestin), TH (tyrosine hydroxylase), FOXA2 (forkheadbox A2), CaMKII (calmodulin-dependent protein kinase II), and NSE (neuron-specific enolase). In some cases, the promoter is a ubiquitous promoter. "Ubiquitin promoter" refers to a promoter that is not tissue-specific under experimental or clinical conditions. In some cases, the ubiquitous promoter is one of the following: CMV, CAG, UBC, PGK, EF1-alpha, GAPDH, SV40, HBV, chicken beta-actin, and human beta-actin promoters. 【0284】 In some embodiments, the promoter sequence is selected from Table 3, and the sequence has at least 95%, at least 98%, or at least 99% identity thereto. 【0285】 (Table 3) TIFF0007872737000072.tif49128 【0286】 Further exemplary examples of promoters include the SV40 late promoter derived from Simian virus 40, baculovirus polyhedral enhancer / promoter elements, herpes simplex virus thymidine kinase (HSV tk), early promoters derived from cytomegalovirus (CMV), and various retroviral promoters including LTR elements. A wide variety of other promoters are known and commonly available in the art, and sequences of many such promoters are available in sequence databases such as the GenBank database. 【0287】 Other adjustment elements In some cases, the vectors of this disclosure further include one or more regulatory elements selected from the group consisting of enhancers, introns, polyA signals, 2A peptide coding sequences, WPREs (woodchuck hepatitis virus posttranscriptional regulatory elements), and HPREs (hepatitis B posttranscriptional regulatory elements). 【0288】 In some embodiments, the vector includes a CMV enhancer. 【0289】 In certain embodiments, the vector includes one or more enhancers. In certain embodiments, the enhancers are CMV enhancer sequences, GAPDH enhancer sequences, β-actin enhancer sequences, or EF1-α enhancer sequences. The aforementioned sequences are known in the art. For example, the sequence of the CMV earliest (IE) enhancer is sequence number 35. 【0290】 In certain embodiments, the vector includes one or more introns. In certain embodiments, the introns are rabbit globin intron sequences, chicken β-actin intron sequences, synthetic intron sequences, or EF1-α intron sequences. 【0291】 In certain embodiments, the vector includes a polyA sequence. In certain embodiments, the polyA sequence is a rabbit globin polyA sequence, a human growth hormone polyA sequence, a bovine growth hormone polyA sequence, a PGK polyA sequence, an SV40 polyA sequence, or a TK polyA sequence. In some embodiments, the polyA signal may be a bovine growth hormone polyadenylation signal (bGHpA). 【0292】 In certain embodiments, the vector includes one or more transcript stabilizing elements. In certain embodiments, the transcript stabilizing elements are a WPRE sequence, an HPRE sequence, a scaffold attachment region, a 3'UTR, or a 5'UTR. In certain embodiments, the vector includes both a 5'UTR and a 3'UTR. 【0293】 In some embodiments, the vector includes a 5' untranslated region (UTR) selected from Table 4. 【0294】 (Table 4) TIFF0007872737000073.tif56128 【0295】 In some embodiments, the vector includes a 3' untranslated region selected from Table 5. 【0296】 (Table 5) TIFF0007872737000074.tif56128 【0297】 In some embodiments, the vector includes a polyadenylated sequence (PolyA) selected from Table 6. 【0298】 (Table 6) TIFF0007872737000075.tif28128 【0299】 Exemplary vector genomes are shown in Figures 2-5, 6-8, and 14-17, provided as SEQ ID NOs. 53-58, 83-88, 91, 92, 94, 96, and 98. In some embodiments, the vector genome contains, is essentially, or consists of a polynucleotide sequence that shares at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any one of SEQ ID NOs. 53-58, 83-88, 91, 92, 94, 96, and 98. 【0300】 In one embodiment, the expression cassette comprises, in the order of 5' to 3', a HuBA promoter, a polynucleotide sequence encoding activating parkin, WPRE(x), and pAGlobin-Oc. 【0301】 In one embodiment, the expression cassette comprises, in 5' to 3' order, a CMV promoter, a TPL-eMLP enhancer, a polynucleotide sequence encoding activated parkin, WPRE(r), and pAGlobin-Oc. 【0302】 In one embodiment, the expression cassette includes, in the order of 5' to 3', a Syn promoter, a polynucleotide sequence encoding activated parkin, WPRE(r), 3'UTR(globin), and pAGH-Bt. 【0303】 In one embodiment, the expression cassette includes, in the order of 5' to 3', a CBA promoter, a polynucleotide sequence encoding activated parkin, and pAGH-Bt. 【0304】 In one embodiment, the expression cassette includes, in the order of 5' to 3', an EF1α promoter, a polynucleotide sequence encoding activated parkin, and pAGlobin-Oc. 【0305】 In one embodiment, the expression cassette includes, in the order of 5' to 3', a HuBA promoter, a polynucleotide sequence encoding activated parkin, R2V17, and pAGH-Bt. 【0306】 In one embodiment, the expression cassette includes, in 5' to 3' order, a Syn promoter, a polynucleotide sequence encoding activated parkin, WPRE(x), 3'UTR(globin), and pAGH-Hs. 【0307】 In one embodiment, the expression cassette includes, in the order of 5' to 3', a CaMKIIa promoter, a polynucleotide sequence encoding activated parkin, WPRE(r), and pAGH-Hs. 【0308】 In one embodiment, the expression cassette includes, in 5' to 3' order, a CMV promoter, a TPL-eMLP enhancer, a polynucleotide sequence encoding activated parkin, WPRE(r), and pAGH-Hs. 【0309】 In one embodiment, the expression cassette includes, in 5' to 3' order, a HuBA promoter, a polynucleotide sequence encoding activated parkin, and pAGH-Hs. 【0310】 In one embodiment, the expression cassette includes, in the order of 5' to 3', a CMV promoter, a TPL / eMLP enhancer, a polynucleotide sequence encoding activated parkin, R2V17, 3'UTR (globin), and pAGH-Bt. 【0311】 In one embodiment, the expression cassette includes, in the order of 5' to 3', the EF1α promoter, a polynucleotide sequence encoding activated parkin, WPRE(r), and pAGH-Bt. 【0312】 In one embodiment, the expression cassette includes, in the order of 5' to 3', a Syn promoter, a polynucleotide sequence encoding activating parkin, R2V17, and pAGlobin-Oc. 【0313】 In one embodiment, the expression cassette includes, in the order of 5' to 3', a CaMKIIa promoter, a polynucleotide sequence encoding activated parkin, R2V17, and pAGlobin-Oc. 【0314】 In one embodiment, the expression cassette includes, in 5' to 3' order, a CBA promoter, a polynucleotide sequence encoding activated parkin, WPRE(x), 3'UTR(globin), and pAGH-Hs. 【0315】 In one embodiment, the expression cassette includes, in the order of 5' to 3', a CBA promoter, a polynucleotide sequence encoding activated parkin, a 3'UTR (globin), and pAGlobin-Oc. 【0316】 In one embodiment, the expression cassette includes, in the order of 5' to 3', a CaMKIIa promoter, a polynucleotide sequence encoding activated parkin, R2V17, and pAGH-Bt. 【0317】 In one embodiment, the expression cassette includes, in the order of 5' to 3', the EF1α promoter, a polynucleotide sequence encoding activated parkin, R2V17, 3'UTR (globin), and pAGH-Hs. 【0318】 In one embodiment, the expression cassette includes, in 5' to 3' order, a CMV promoter, a polynucleotide sequence encoding activated parkin, R2V17, 3'UTR (globin), and pAGH-Hs. 【0319】 In one embodiment, the expression cassette includes, in the order of 5' to 3', a CMV promoter, a polynucleotide sequence encoding activated parkin, and pAGH-Hs. 【0320】 In the embodiments described above, the order of the 5' elements in the polynucleotide sequence encoding the activated parkin is reversed, resulting in either the promoter preceding the enhancer element or the enhancer element preceding the promoter element. 【0321】 Adeno-associated virus vector Adeno-associated virus (AAV) is a replication-deficient parvovirus with a single-stranded DNA genome approximately 4.7 kb long, containing two 145-nucleotide inverted end sequences (ITRs). AAV has several known variants, sometimes called serotypes, when classified by antigenic epitopes. The nucleotide sequences of the genomes of AAV serotypes are known. For example, the whole genome of AAV-1 is available under GenBank registry number NC_002077, the whole genome of AAV-2 is available under GenBank registry number NC_001401 and Srivastava et al., J. Virol., 45:555-564 (1983), the whole genome of AAV-3 is available under GenBank registry number NC_1829, the whole genome of AAV-4 is available under GenBank registry number NC_001829, the genome of AAV-5 is available under GenBank registry number AF085716, the whole genome of AAV-6 is available under GenBank registry number NC_001862, at least portions of the genomes of AAV-7 and AAV-8 are available under GenBank registry numbers AX753246 and AX753249, respectively, and the genome of AAV-9 is available under GenBank registry number Gao et al. The AAV-10 genome is provided in al., J. Virol., 78:6381-6388 (2004), the AAV-11 genome in Mol. Ther., 13(1):67-76 (2006), and the AAV-11 genome 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. The cis-acting sequences that lead to viral DNA replication (rep), capsid formation / packaging, and host cell chromosome integration are contained within the AAV ITR. Three AAV promoters (named p5, p19, and p40 from their relative map locations) drive the expression of two AAV internal open reading frames encoding the rep and cap genes.Two rep promoters (p5 and p19), linked to differential splicing of a single AAV intron (nucleotides 2107 and 2227), produce four rep proteins (rep78, rep68, rep52, and rep40) from the rep gene. The rep proteins possess multiple enzymatic properties that ultimately carry out viral genome replication. The cap gene, expressed from the p40 promoter, encodes three capsid proteins: VP1, VP2, and VP3. Alternative splicing and non-consensus translation initiation sites are involved in the production of the three related capsid proteins. A single consensus polyadenylation site is located at map position 95 of the AAV genome. The life cycle and genetics of AAV are outlined in Muzyczka, Current Topics in Microbiology and Immunology, 158:97-129 (1992). 【0322】 AAV possesses unique characteristics 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 non-cellular, and natural infection in humans and other animals is asymptomatic. Furthermore, AAV infects many mammalian cells, allowing for the potential to target many different tissues in vivo. Additionally, AAV transduces both slowly dividing and non-dividing cells and can essentially persist for the lifetime of these cells as a transcriptionally active nuclear episome (extrachromosomal element). The AAV proviral genome is inserted as cloned DNA within a plasmid, enabling the construction of a recombinant genome. Furthermore, since the signals directing AAV replication and genome capsidation are contained within the ITR of the AAV genome, some or all of the approximately 4.3kb inside the genome (encoding the replication and structural capsid protein, rep-cap) may be replaced with foreign DNA. To generate an AAV vector, the rep and cap proteins may be provided in trans. Another important characteristic of AAV is that it is a very stable and potent virus. AAV readily tolerates the conditions used to inactivate adenoviruses (56°C to 65°C for several hours), reducing the importance of low-temperature storage of AAV. AAV can also be freeze-dried. Finally, AAV-infected cells are not resistant to co-infection. 【0323】 The AAV DNA in the rAAV genome may be derived from any AAV variant or serotype from which recombinant virus can be induced, including, but not limited to, 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 AAVrh10. 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 being considered. See, for example, Marsic et al., Molecular Therapy, 22(11):1900-1909 (2014). Nucleotide sequences of various AAV serotype genomes are publicly known in the art. 【0324】 In some cases, rAAVs contain a self-complementary genome. As defined herein, rAAVs containing a “self-complementary” or “double-stranded” genome refer to rAAVs that have been manipulated so that the coding region of the rAAV forms 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). This disclosure intends to use rAAVs containing a self-complementary genome in some cases, so that, at the time of infection (such as transduction), the two complementary halves of the scAAV immediately form a single double-stranded DNA (dsDNA) unit that can replicate and transcribe, rather than waiting for cell-mediated synthesis of the second strand of the rAAV genome. It should be understood that, instead of the full coding capacity (4.7-6kb) seen in rAAVs, rAAVs containing a self-complementary genome can only hold about half that amount (≒2.4kb). 【0325】 In other cases, rAAV vectors contain a single-stranded genome. As defined herein, a “single standard” genome refers to a genome that is not self-complementary. In most cases, non-recombinant AAVs have a single-stranded DNA genome. There have been some indications that rAAVs should be scAAVs to achieve efficient transduction of cells. However, this disclosure intends for rAAV vectors that may have a single-stranded genome rather than a self-complementary genome, based on the understanding that other genetic modifications of rAAV vectors may be beneficial in obtaining optimal gene transcription in target cells. In some cases, this disclosure relates to a single-stranded rAAV vector that can achieve efficient gene transfer to the anterior segment of the mouse eye. See Wang et al. Single stranded adeno-associated virus achieves efficient gene transfer to anterior segment in the mouse eye. PLoS ONE 12(8):e0182473(2017). 【0326】 In some cases, the rAAV vector is a vector for serotype AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh10, or AAVrh74. 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 intended. See, for example, Marsic et al., Molecular Therapy, 22(11):1900-1909 (2014). In some cases, the rAAV vector is a vector for serotype AAV9. In some embodiments, the rAAV vector is a vector for serotype AAV9 and contains a single-stranded genome. In some embodiments, the rAAV vector is a vector for serotype AAV9 and contains a self-complementary genome. In some embodiments, the rAAV vector contains the inverted end sequence (ITR) of AAV2. In some embodiments, the rAAV vector contains the AAV2 genome, and as a result, the rAAV vector is an AAV-2 / 9 vector, an AAV-2 / 6 vector, or an AAV-2 / 8 vector. 【0327】 The most well-known full-length sequence and capsid gene sequence for AAV are provided in U.S. Patent No. 8,524,446, which is incorporated herein by reference in its entirety. 【0328】 The AAV vector may contain a wild-type AAV sequence or one or more modifications to the wild-type AAV sequence. In certain embodiments, the AAV vector contains one or more amino acid modifications, such as substitutions, deletions, or insertions, within the capsid protein, e.g., VP1, VP2, and / or VP3. In certain embodiments, the modifications result in reduced immunogenicity when the AAV vector is provided to a subject. 【0329】 The capsid protein of rAAV may be modified so that rAAV targets specific target tissues, such as neurons, or more specifically, dopaminergic neurons. See, for example, 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. US20120082650A1. These disclosures are incorporated herein by reference. In some embodiments, rAAV is injected directly into the substantia nigra of the target. 【0330】 In some embodiments, the rAAV virion is an AAV2 rAAV virion. Many of the capsids are AAV2 capsids or functional variants thereof. In some embodiments, the AAV2 capsid shares at least 98%, 99%, or 100% identity with a reference AAV2 capsid, such as sequence number 59. 【0331】 In some embodiments, the rAAV virion is an AAV9 rAAV virion. Many of the capsids are AAV9 capsids or functional variants thereof. In some embodiments, the AAV9 capsid shares at least 98%, 99%, or 100% identity with a reference AAV9 capsid, such as sequence number 60. 【0332】 In some embodiments, the rAAV virion is the AAV-PHP.B rAAV virion or its neutrophil variant, such as those disclosed in International Patent Publications WO2015 / 038958A1 and WO2017 / 100671A1, but is not limited thereto. For example, the AAV capsid may comprise at least four consecutive amino acids from the sequence TLAVPFK (SEQ ID NO: 62) or KFPVALT (SEQ ID NO: 63), which are inserted, for example, between the sequences encoding amino acids 588 and 589 of AAV9. 【0333】 Many capsids are AAV-PHP.B capsids or functional variants thereof. In some embodiments, AAV-PHP.B capsids share at least 98%, 99%, or 100% identity with reference AAV-PHP.B capsids, such as sequence number 61. 【0334】 Further AAV capsids used in the rAAV vilion of this disclosure include those disclosed in International Patent Publications WO2009 / 012176A2 and WO2015 / 168666A2. 【0335】 In certain embodiments, the Disclosure provides an rAAV viron, such as an AAV2 rAAV viron or an AAV9 rAAV viron, which includes an expression cassette disclosed herein. 【0336】 In certain embodiments of rAAV virons, such as AAV2 rAAV viron or AAV9 rAAV viron, the expression cassette comprises, in 5' to 3' order, a HuBA promoter, a polynucleotide sequence encoding activated parkin, WPRE(x), and pAGlobin-Oc. 【0337】 In certain embodiments of rAAV virons, such as AAV2 rAAV viron or AAV9 rAAV viron, the expression cassette comprises, in 5' to 3' order, a CMV promoter, a TPL-eMLP enhancer, a polynucleotide sequence encoding activated parkin, WPRE(r), and pAGlobin-Oc. 【0338】 In certain embodiments of rAAV virons, such as AAV2 rAAV viron or AAV9 rAAV viron, the expression cassette comprises, in 5' to 3' order, a Syn promoter, a polynucleotide sequence encoding activated parkin, WPRE(r), 3'UTR(globin), and pAGH-Bt. 【0339】 In certain embodiments of rAAV virons, such as AAV2 rAAV viron or AAV9 rAAV viron, the expression cassette comprises, in 5' to 3' order, a CBA promoter, a polynucleotide sequence encoding activated parkin, and pAGH-Bt. 【0340】 In certain embodiments of rAAV virons, such as AAV2 rAAV viron or AAV9 rAAV viron, the expression cassette comprises, in 5' to 3' order, an EF1α promoter, a polynucleotide sequence encoding activated parkin, and pAGlobin-Oc. 【0341】 In certain embodiments of rAAV virons, such as AAV2 rAAV viron or AAV9 rAAV viron, the expression cassette comprises, in 5' to 3' order, a HuBA promoter, a polynucleotide sequence encoding activated parkin, R2V17, and pAGH-Bt. 【0342】 In certain embodiments of rAAV virons, such as AAV2 rAAV viron or AAV9 rAAV viron, the expression cassette comprises, in 5' to 3' order, a Syn promoter, a polynucleotide sequence encoding activated parkin, WPRE(x), 3'UTR(globin), and pAGH-Hs. 【0343】 In certain embodiments of rAAV virons, such as AAV2 rAAV viron or AAV9 rAAV viron, the expression cassette comprises, in 5' to 3' order, a CaMKIIa promoter, a polynucleotide sequence encoding activated parkin, WPRE(r), and pAGH-Hs. 【0344】 In certain embodiments of rAAV virons, such as AAV2 rAAV viron or AAV9 rAAV viron, the expression cassette comprises, in 5' to 3' order, a CMV promoter, a TPL-eMLP enhancer, a polynucleotide sequence encoding activated parkin, WPRE(r), and pAGH-Hs. 【0345】 In certain embodiments of rAAV virons, such as AAV2 rAAV viron or AAV9 rAAV viron, the expression cassette comprises, in 5' to 3' order, a HuBA promoter, a polynucleotide sequence encoding activated parkin, and pAGH-Hs. 【0346】 In certain embodiments of rAAV virons, such as AAV2 rAAV viron or AAV9 rAAV viron, the expression cassette includes, in 5' to 3' order, a CMV promoter, a TPL / eMLP enhancer, a polynucleotide sequence encoding activated parkin, R2V17, 3'UTR (globin), and pAGH-Bt. 【0347】 In certain embodiments of rAAV virons, such as AAV2 rAAV viron or AAV9 rAAV viron, the expression cassette comprises, in 5' to 3' order, the EF1α promoter, a polynucleotide sequence encoding activated parkin, WPRE(r), and pAGH-Bt. 【0348】 In certain embodiments of rAAV virons, such as AAV2 rAAV viron or AAV9 rAAV viron, the expression cassette comprises, in 5' to 3' order, a Syn promoter, a polynucleotide sequence encoding activating parkin, R2V17, and pAGlobin-Oc. 【0349】 In certain embodiments of rAAV virons, such as AAV2 rAAV viron or AAV9 rAAV viron, the expression cassette comprises, in 5' to 3' order, a CaMKIIa promoter, a polynucleotide sequence encoding activating parkin, R2V17, and pAGlobin-Oc. 【0350】 In certain embodiments of rAAV virons, such as AAV2 rAAV viron or AAV9 rAAV viron, the expression cassette includes, in 5' to 3' order, a CBA promoter, a polynucleotide sequence encoding activated parkin, WPRE(x), 3'UTR(globin), and pAGH-Hs. 【0351】 In certain embodiments of rAAV virons, such as AAV2 rAAV viron or AAV9 rAAV viron, the expression cassette comprises, in 5' to 3' order, a CBA promoter, a polynucleotide sequence encoding activated parkin, a 3'UTR (globin), and pAGlobin-Oc. 【0352】 In certain embodiments of rAAV virons, such as AAV2 rAAV viron or AAV9 rAAV viron, the expression cassette comprises, in 5' to 3' order, a CaMKIIa promoter, a polynucleotide sequence encoding activated parkin, R2V17, and pAGH-Bt. 【0353】 In certain embodiments of rAAV virons, such as AAV2 rAAV viron or AAV9 rAAV viron, the expression cassette comprises, in 5' to 3' order, an EF1α promoter, a polynucleotide sequence encoding activated parkin, R2V17, 3'UTR (globin), and pAGH-Hs. 【0354】 In certain embodiments of rAAV virons, such as AAV2 rAAV viron or AAV9 rAAV viron, the expression cassette comprises, in 5' to 3' order, a CMV promoter, a polynucleotide sequence encoding activated parkin, R2V17, 3'UTR (globin), and pAGH-Hs. 【0355】 In certain embodiments of rAAV virons, such as AAV2 rAAV viron or AAV9 rAAV viron, the expression cassette comprises, in 5' to 3' order, a CMV promoter, a polynucleotide sequence encoding activated parkin, and pAGH-Hs. 【0356】 In certain embodiments of the aforementioned rAAV virons, such as the AAV2 rAAV viron or the AAV9 rAAV viron, the order of the 5' elements relative to the polynucleotide sequence encoding the activated parkin is reversed, resulting in either the promoter preceding the enhancer element or the enhancer element preceding the promoter element. 【0357】 Pharmaceutical compositions and kits In one embodiment, the Disclosure provides a pharmaceutical composition comprising the rAAV virion of the Disclosure and one or more pharmaceutically acceptable carriers, diluents, or excipients. 【0358】 For example, various solutions, such as sterile aqueous solutions, can be used for administration by injection. Such aqueous solutions may be buffered if desired, and the liquid diluent can be initially isotonic with physiological saline or glucose. Solutions of rAAV as a free acid (DNA contains acidic phosphate groups) or a pharmacokinetically acceptable salt can be prepared in water suitably mixed with 0.001% or 0.01% of a surfactant, such as Pluronic® F-68. Dispersions of rAAV may be prepared in glycerol, liquid polyethylene glycol and mixtures thereof, and oil. Under normal storage and use conditions, these preparations contain preservatives to prevent microbial growth. In this regard, all sterile aqueous media used are readily available by standard techniques well known to those skilled in the art. 【0359】 Suitable pharmaceutical forms for injectable use include, but are not limited to, sterile aqueous solutions or dispersions, and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and fluid enough to be readily injectable (syringability). It must be stable under manufacturing and storage conditions and protected against contamination by microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Adequate fluidity can be maintained, for example, by the use of coatings such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action can be provided by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it is preferable to include isotonic agents, such as sugars or sodium chloride. Long-term absorption of injectable compositions can be achieved by using absorption-delaying agents, such as aluminum monostearate and gelatin. 【0360】 Sterile injectable solutions can be prepared by incorporating the required amount of rAAV in a suitable solvent, along with various other components listed above as needed, and then sterilizing by filtration. Generally, dispersants are prepared by incorporating the sterile active ingredient into a sterile vehicle containing a basic dispersion medium and other components required from those listed above. For sterile powders for the preparation of sterile injectable solutions, preferred preparation methods are vacuum drying and freeze-drying techniques, which yield powders of the active ingredient plus any additional desired components from a pre-sterilized filtered solution. 【0361】 In another embodiment, the Disclosure includes a kit, which includes the rAAV Virion of the Disclosure and instructions for use. 【0362】 How to use In one embodiment, the Disclosure provides a method for increasing parkin activity in cells, the method comprising contacting cells with rAAV of the Disclosure. In another embodiment, the Disclosure provides a method for increasing parkin activity in a subject, the method comprising administering rAAV of the Disclosure to the subject. In some embodiments, cells and / or subjects are deficient in parkin activity and / or contain loss-of-function mutations in parkin. The cells may be neurons, for example, dopaminergic neurons. In some embodiments, cells and / or subjects are deficient in PINK1 activity and / or contain loss-of-function mutations in PINK1. In various embodiments, activated parkin is expressed in cells or subjects. 【0363】 In certain embodiments, an rAAV viron, such as an AAV2 rAAV viron or an AAV9 rAAV viron, containing an expression cassette disclosed herein, is brought into contact with cells or administered to them. 【0364】 In certain embodiments, an rAAV viron, such as an AAV2 rAAV viron or an AAV9 rAAV viron, comprising an expression cassette containing a HuBA promoter, a polynucleotide sequence encoding activated parkin, WPRE(x), and pAGlobin-Oc in the order of 5' to 3', is brought into contact with cells or administered to a target. 【0365】 In certain embodiments, cells are contacted with or administered an expression cassette comprising, in the order of 5' to 3', a CMV promoter, a TPL-eMLP enhancer, a polynucleotide sequence encoding activated parkin, WPRE(r), and pAGlobin-Oc, an rAAV viron, such as an AAV2 rAAV viron or an AAV9 rAAV viron. 【0366】 In certain embodiments, an rAAV viron, such as AAV2 rAAV viron or AAV9 rAAV viron, comprising an expression cassette containing a Syn promoter, a polynucleotide sequence encoding activated parkin, WPRE(r), 3'UTR(globin), and pAGH-Bt in the order of 5' to 3', is brought into contact with cells or administered to the target. 【0367】 In certain embodiments, an rAAV viron, such as an AAV2 rAAV viron or an AAV9 rAAV viron, comprising an expression cassette containing a CBA promoter, a polynucleotide sequence encoding activated parkin, and pAGH-Bt in the order of 5' to 3', is brought into contact with cells or administered to a target. 【0368】 In certain embodiments, an rAAV viron, such as AAV2 rAAV viron or AAV9 rAAV viron, comprising an expression cassette containing an EF1α promoter, a polynucleotide sequence encoding activated parkin, and pAGlobin-Oc in the order of 5' to 3', is brought into contact with cells or administered to the target. 【0369】 In certain embodiments, an rAAV viron, such as an AAV2 rAAV viron or an AAV9 rAAV viron, comprising an expression cassette containing a HuBA promoter, a polynucleotide sequence encoding activated parkin, R2V17, and pAGH-Bt in the order of 5' to 3', is brought into contact with cells or administered to a target. 【0370】 In certain embodiments, an rAAV viron, such as AAV2 rAAV viron or AAV9 rAAV viron, comprising an expression cassette containing a Syn promoter, a polynucleotide sequence encoding activated parkin, WPRE(x), 3'UTR(globin), and pAGH-Hs in the order of 5' to 3', is brought into contact with cells or administered to the target. 【0371】 In certain embodiments, an rAAV viron, such as an AAV2 rAAV viron or an AAV9 rAAV viron, comprising an expression cassette containing a CaMKIIa promoter, a polynucleotide sequence encoding activated parkin, WPRE(r), and pAGH-Hs in the order of 5' to 3', is brought into contact with cells or administered to a target. 【0372】 In certain embodiments, cells are contacted with or administered an expression cassette comprising, in the order of 5' to 3', a CMV promoter, a TPL-eMLP enhancer, a polynucleotide sequence encoding activated parkin, WPRE(r), and pAGH-Hs, an rAAV viron, such as an AAV2 rAAV viron or an AAV9 rAAV viron. 【0373】 In certain embodiments, an rAAV viron, such as an AAV2 rAAV viron or an AAV9 rAAV viron, comprising an expression cassette containing a HuBA promoter, a polynucleotide sequence encoding activated parkin, and pAGH-Hs in the order of 5' to 3', is brought into contact with cells or administered to a target. 【0374】 In certain embodiments, cells are brought into contact with or administered an expression cassette comprising a CMV promoter, a TPL / eMLP enhancer, a polynucleotide sequence encoding activated parkin, R2V17, 3'UTR (globin), and pAGH-Bt in the order of 5' to 3', such as an AAV2 rAAV viron or an AAV9 rAAV viron. 【0375】 In certain embodiments, an rAAV viron, such as AAV2 rAAV viron or AAV9 rAAV viron, comprising an expression cassette containing an EF1α promoter, a polynucleotide sequence encoding activated parkin, WPRE(r), and pAGH-Bt in the order of 5' to 3', is brought into contact with cells or administered to a target. 【0376】 In certain embodiments, cells are contacted with or administered an expression cassette comprising a Syn promoter, a polynucleotide sequence encoding activated parkin, R2V17, and pAGlobin-Oc in the order of 5' to 3', such as an AAV2 rAAV viron or an AAV9 rAAV viron. 【0377】 In certain embodiments, cells are contacted with or administered an expression cassette comprising, in the order of 5' to 3', a CaMKIIa promoter, a polynucleotide sequence encoding activated parkin, R2V17, and pAGlobin-Oc, an rAAV viron, such as an AAV2 rAAV viron or an AAV9 rAAV viron. 【0378】 In certain embodiments, an rAAV viron, such as an AAV2 rAAV viron or an AAV9 rAAV viron, comprising an expression cassette containing a CBA promoter, a polynucleotide sequence encoding activated parkin, WPRE(x), 3'UTR(globin), and pAGH-Hs in the order of 5' to 3', is brought into contact with cells or administered to a target. 【0379】 In certain embodiments, an rAAV viron, such as AAV2 rAAV viron or AAV9 rAAV viron, comprising an expression cassette containing a CBA promoter, a polynucleotide sequence encoding activated parkin, a 3'UTR (globin), and pAGlobin-Oc in the order of 5' to 3', is brought into contact with cells or administered to the target. 【0380】 In certain embodiments, cells are contacted with or administered an expression cassette comprising, in the order of 5' to 3', a CaMKIIa promoter, a polynucleotide sequence encoding activated parkin, R2V17, and pAGH-Bt, an rAAV viron, such as an AAV2 rAAV viron or an AAV9 rAAV viron. 【0381】 In certain embodiments, cells are brought into contact with or administered an expression cassette comprising an EF1α promoter, a polynucleotide sequence encoding activated parkin, R2V17, 3'UTR (globin), and pAGH-Hs in the order of 5' to 3', an rAAV viron, such as an AAV2 rAAV viron or an AAV9 rAAV viron. 【0382】 In a particular embodiment, cells are brought into contact with or administered an expression cassette comprising a CMV promoter, a polynucleotide sequence encoding activated parkin, R2V17, 3'UTR (globin), and pAGH-Hs in the order of 5' to 3', such as an AAV2 rAAV viron or an AAV9 rAAV viron. 【0383】 In certain embodiments, an rAAV viron, such as AAV2 rAAV viron or AAV9 rAAV viron, comprising an expression cassette containing a CMV promoter, a polynucleotide sequence encoding activated parkin, and pAGH-Hs in the order of 5' to 3', is brought into contact with cells or administered to the target. 【0384】 In certain embodiments of the aforementioned rAAV virions, such as the AAV2 rAAV viron or the AAV9 rAAV viron, the order of the 5' elements relative to the polynucleotide sequence encoding the activated parkin is reversed, resulting in either the promoter preceding the enhancer element or the enhancer element preceding the promoter element. 【0385】 The efficacy of activated parkin can be determined as an increase compared to untreated cells / control or compared to treatment with a reference parkin protein in one or more non-limiting assays, for example, as shown below: (1) expression of activated parkin protein, (2) increased ubiquitination of mitochondrial proteins, (3) improved mitophagy, (4) reduced cytotoxicity, (5) reduced oxidative stress, and / or (6) increased survival of neurons, e.g., dopaminergic neurons. In certain embodiments, the increase is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2x, at least 3x, at least 4x, at least 5x, at least 10x, at least 20x, at least 50x, or at least 100x. The aforementioned parameters and other parameters can be measured by methods well known in the art, including but not limited to those described in Examples 4-5. 【0386】 In some embodiments, the method promotes neuronal survival in cell culture and / or in vivo. The neurons may be dopaminergic neurons. Survival can be measured using one or more assays, such as those described in the following examples. In certain embodiments, survival is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least twofold, at least threefold, at least fourfold, at least fivefold, at least tenfold, at least 20fold, at least 50fold, or at least 100fold. 【0387】 Treatment method In another embodiment, the Disclosure provides a method for treating a subject in need of treatment for a disease or disorder, the method comprising administering an effective amount of the rAAV virion of the Disclosure to the subject. In some embodiments, the subject has a genetic deficit in the expression or function of parkin. The subject may have a genetic deficit (i.e., diagnosed or undiagnosed) in PRKN (i.e., PARK2, AR-DJ, ubiquitin E3 ligase), PARK7 (i.e., DJ-1), PINK1 (i.e., PARK6, PTEN-induced putative kinase 1, BRPK), LRRK2, SNCA (i.e., PARK1, PARK4, alpha-synuclein). In some embodiments, the subject has a genetic deficit in the expression or function of PINK1. In some embodiments, the subject has a genetic deficit in the expression or function of DJ-1. 【0388】 In certain embodiments, the subject is administered an rAAV viron, such as an AAV2 rAAV viron or an AAV9 rAAV viron, which includes an expression cassette disclosed herein. 【0389】 In a particular embodiment, the subject is administered an rAAV viron, such as an AAV2 rAAV viron or an AAV9 rAAV viron, which comprises an expression cassette containing a HuBA promoter, a polynucleotide sequence encoding activated parkin, WPRE(x), and pAGlobin-Oc in the order of 5' to 3'. 【0390】 In a particular embodiment, the subject is administered an rAAV viron, such as AAV2 rAAV viron or AAV9 rAAV viron, which comprises an expression cassette containing a CMV promoter, a TPL-eMLP enhancer, a polynucleotide sequence encoding activated parkin, WPRE(r), and pAGlobin-Oc in the order of 5' to 3'. 【0391】 In a particular embodiment, the subject is administered an rAAV viron, such as AAV2 rAAV viron or AAV9 rAAV viron, which comprises an expression cassette containing a Syn promoter, a polynucleotide sequence encoding activated parkin, WPRE(r), 3'UTR(globin), and pAGH-Bt in the order of 5' to 3'. 【0392】 In a particular embodiment, the subject is administered an rAAV viron, such as AAV2 rAAV viron or AAV9 rAAV viron, which comprises an expression cassette containing a CBA promoter, a polynucleotide sequence encoding activated parkin, and pAGH-Bt in the order of 5' to 3'. 【0393】 In a particular embodiment, the subject is administered an rAAV viron, such as AAV2 rAAV viron or AAV9 rAAV viron, which includes an expression cassette containing an EF1α promoter, a polynucleotide sequence encoding activated parkin, and pAGlobin-Oc in the order of 5' to 3'. 【0394】 In a particular embodiment, the subject is administered an rAAV viron, such as AAV2 rAAV viron or AAV9 rAAV viron, which includes an expression cassette containing a HuBA promoter, a polynucleotide sequence encoding activated parkin, R2V17, and pAGH-Bt in the order of 5' to 3'. 【0395】 In a particular embodiment, the subject is administered an rAAV viron, such as AAV2 rAAV viron or AAV9 rAAV viron, which includes an expression cassette containing a Syn promoter, a polynucleotide sequence encoding activated parkin, WPRE(x), 3'UTR(globin), and pAGH-Hs in the order of 5' to 3'. 【0396】 In a particular embodiment, the subject is administered an rAAV viron, such as AAV2 rAAV viron or AAV9 rAAV viron, which comprises an expression cassette containing a CaMKIIa promoter, a polynucleotide sequence encoding activated parkin, WPRE(r), and pAGH-Hs in the order of 5' to 3'. 【0397】 In a particular embodiment, the subject is administered an rAAV viron, such as AAV2 rAAV viron or AAV9 rAAV viron, which comprises an expression cassette containing a CMV promoter, a TPL-eMLP enhancer, a polynucleotide sequence encoding activated parkin, WPRE(r), and pAGH-Hs in the order of 5' to 3'. 【0398】 In a particular embodiment, the subject is administered an rAAV viron, such as AAV2 rAAV viron or AAV9 rAAV viron, which comprises an expression cassette containing a HuBA promoter, a polynucleotide sequence encoding activated parkin, and pAGH-Hs in the order of 5' to 3'. 【0399】 In a particular embodiment, the subject is administered an rAAV viron, such as AAV2 rAAV viron or AAV9 rAAV viron, which comprises an expression cassette containing, in the order of 5' to 3', a CMV promoter, a TPL / eMLP enhancer, a polynucleotide sequence encoding activated parkin, R2V17, 3'UTR (globin), and pAGH-Bt. 【0400】 In a particular embodiment, the subject is administered an rAAV viron, such as AAV2 rAAV viron or AAV9 rAAV viron, which includes an expression cassette containing the EF1α promoter, a polynucleotide sequence encoding activated parkin, WPRE(r), and pAGH-Bt in the order of 5' to 3'. 【0401】 In a particular embodiment, the subject is administered an rAAV viron, such as AAV2 rAAV viron or AAV9 rAAV viron, which includes an expression cassette containing a Syn promoter, a polynucleotide sequence encoding activated parkin, R2V17, and pAGlobin-Oc in the order of 5' to 3'. 【0402】 In a particular embodiment, the subject is administered an rAAV viron, such as AAV2 rAAV viron or AAV9 rAAV viron, which comprises an expression cassette containing, in the order of 5' to 3', a CaMKIIa promoter, a polynucleotide sequence encoding activated parkin, R2V17, and pAGlobin-Oc. 【0403】 In a particular embodiment, the subject is administered an rAAV viron, such as AAV2 rAAV viron or AAV9 rAAV viron, which comprises an expression cassette containing a CBA promoter, a polynucleotide sequence encoding activated parkin, WPRE(x), 3'UTR(globin), and pAGH-Hs in the order of 5' to 3'. 【0404】 In a particular embodiment, the subject is administered an rAAV viron, such as AAV2 rAAV viron or AAV9 rAAV viron, which includes an expression cassette containing a CBA promoter, a polynucleotide sequence encoding activated parkin, a 3'UTR (globin), and pAGlobin-Oc in the order of 5' to 3'. 【0405】 In a particular embodiment, the subject is administered an rAAV viron, such as AAV2 rAAV viron or AAV9 rAAV viron, which includes an expression cassette containing, in the order of 5' to 3', a CaMKIIa promoter, a polynucleotide sequence encoding activated parkin, R2V17, and pAGH-Bt. 【0406】 In a particular embodiment, the subject is administered an rAAV viron, such as AAV2 rAAV viron or AAV9 rAAV viron, which includes an expression cassette containing, in the order of 5' to 3', an EF1α promoter, a polynucleotide sequence encoding activated parkin, R2V17, 3'UTR (globin), and pAGH-Hs. 【0407】 In a particular embodiment, the subject is administered an rAAV viron, such as AAV2 rAAV viron or AAV9 rAAV viron, which includes an expression cassette containing a CMV promoter, a polynucleotide sequence encoding activated parkin, R2V17, 3'UTR (globin), and pAGH-Hs in the order of 5' to 3'. 【0408】 In a particular embodiment, the subject is administered an rAAV viron, such as AAV2 rAAV viron or AAV9 rAAV viron, which comprises an expression cassette containing a CMV promoter, a polynucleotide sequence encoding activated parkin, and pAGH-Hs in the order of 5' to 3'. 【0409】 In certain embodiments of the aforementioned rAAV virons, such as the AAV2 rAAV viron or the AAV9 rAAV viron, the order of the 5' elements relative to the polynucleotide sequence encoding the activated parkin is reversed, resulting in either the promoter preceding the enhancer element or the enhancer element preceding the promoter element. 【0410】 In some embodiments, the disease or disorder is Parkinson's disease. The disclosure provides treatments for any of the various neurodegenerative diseases. For example, the rAAV virion of the disclosure treats early-onset Parkinson's disease (EOPD) or juvenile PD, also known as juvenile-onset, early-onset, and autosomal recessive early-onset Parkinson's disease. 【0411】 The rAAV virion of this disclosure further treats idiopathic PD, nigrostriatal degeneration, dopamine deficiency due to primary dopamine neuron loss, sporadic PD, PD of unknown etiology, neurodegenerative diseases associated with loss of function, and / or obvious neurodegeneration of dopaminergic neurons in the midbrain (including the substantia nigra and / or ventral tegmental area) associated with neurodegenerative diseases of unknown etiology, idiopathic, and sporadic onset. 【0412】 The methods of this disclosure can prevent the loss of dopaminergic neurons in the substantia nigra in a variety of disorders, including but not limited to those associated with age and / or genetic causes and / or Parkinson's disease of unknown cause (i.e., idiopathic PD). They can treat a variety of neurodegenerative conditions associated with primary neuronal loss in the substantia nigra, whether of unknown or known etiology. 【0413】 In some embodiments, the compositions of the present disclosure may act as therapeutic agents having neuroprotective and neuroregenerative properties to halt and / or prevent further loss of dopaminergic neurons in the substantia nigra due to the absence or mutation of the PARK2 gene or the PINK1 gene. The compositions of the present disclosure may be administered as neuroprotective therapy and may mitigate substantia nigra-striatal degeneration and loss of dopaminergic neurons located in the substantia nigra region of the midbrain in patients with early-onset Parkinson's disease as a result of mutations or deletions in the PARK2 gene and / or the PINK1 gene. 【0414】 AAV-mediated delivery of activated parkin protein to the CNS may improve anatomical, neurochemical, and behavioral measures indicating neuroprotection and / or neurological recovery of the dopaminergic nigrostriatal system. 【0415】 Combination therapy is also envisioned by the present invention. Combination therapy may include the administration of rAAV virion as disclosed herein, as well as either or both l-3,4-dihydroxyphenylalanine (L-dopa) and / or dopamine agonists. In some embodiments, administration of rAAV virion reduces the need for administration of L-dopa and / or DA. As used herein, combinations include concurrent or sequential treatments. Combinations of the methods of the present invention with standard medical treatments (e.g., corticosteroids or topical decompressants) are specifically envisioned, as are combinations with novel therapies. In some cases, the subject may be treated with steroids to prevent or reduce the immune response to the administration of rAAV as described herein. 【0416】 For example, a therapeutically effective dose of rAAV vector for intravenous injection is approximately 1e7vg / kg to 5e15vg / kg, or approximately 1e7vg / kg to 1e14vg / kg, or approximately 1e8vg / kg to 1e14vg / kg, or approximately 1e9vg / kg to 1e13vg / kg, or approximately 1e9vg / kg to 1e12vg / kg, or approximately 1e7vg / kg to 5e7vg / kg, or approximately 1e8vg / kg to 5e8vg / kg, or approximately 1e9vg / kg to 5e9vg / kg, or approximately 1e10vg / kg to 5e10 The doses of rAAV are in the range of vg / kg, or about 1e11vg / kg to about 5e11vg / kg, or about 1e12vg / kg to about 5e12vg / kg, or about 1e13vg / kg to about 5e13vg / kg, or about 1e14vg / kg to about 5e14vg / kg, or about 1e15vg / kg to about 5e15vg / kg. The present invention also includes compositions comprising rAAV vectors in these ranges. 【0417】 For example, in certain embodiments, therapeutically effective doses of rAAV vector are doses of about 1 e10 vg / kg, about 2 e10 vg / kg, about 3 e10 vg / kg, about 4 e10 vg / kg, about 5 e10 vg / kg, about 6 e10 vg / kg, about 7 e10 vg / kg, about 8 e10 vg / kg, about 9 e10 vg / kg, about 1 e12 vg / kg, about 2 e12 vg / kg, about 3 e12 vg / kg, about 4 e12 vg / kg, and 5 e12 vg / kg. The present invention also includes compositions comprising these doses of rAAV vector. 【0418】 In some embodiments, for example, when direct injection into the substantia nigra is performed, the therapeutically effective dose of the rAAV vector is in the range of 1e7 / hemisphere vg to 1e11vg / hemisphere, or approximately 1e7vg / hemisphere, approximately 1e8vg / hemisphere, approximately 1e9vg / hemisphere, approximately 1e10vg / hemisphere, or approximately 1e11vg / hemisphere. 【0419】 In some embodiments, for example, when direct injection into the putamen (intraputamen) is performed, the therapeutically effective dose of the rAAV vector is in the range of 1e9vg / hemisphere to 6e11vg / hemisphere, or approximately 1e9vg / hemisphere, approximately 1e10vg / hemisphere, approximately 1e11vg / hemisphere, approximately 2e11vg / hemisphere, approximately 3e11vg / hemisphere, or approximately 6e11vg / hemisphere. 【0420】 In some cases, the therapeutic composition contains more than approximately 1e9, 1e10, or 1e11 of rAAV vector genomes per volume of the injected therapeutic composition. In some cases, the therapeutic composition contains more than approximately 1e9, 1e10, or 1e11 of rAAV vector genomes per volume of the injected therapeutic composition. In some cases, the therapeutic composition contains more than approximately 1e10, 1e11, 1e12, or 1e13 of rAAV vector genomes per mL. In certain embodiments, the therapeutic composition contains less than approximately 1e14, 1e13, or 1e12 of rAAV vector genomes per mL. 【0421】 In some embodiments, the Disclosure provides a method for treating and / or preventing Parkinson's disease, the method comprising optionally administering the vector of the Disclosure before, during, or after the onset of the disease. Parkinson's disease may be early-onset Parkinson's disease (EOPD). In some embodiments, the Method reduces one or more symptoms of Parkinson's disease, such as EOPD. It can reduce motor complications associated with neurodegeneration, nigrostriatal degeneration, and / or ataxia, reduce the need for antiparkinsonian drug therapy (including, but not limited to, L-DOPA and dopamine agonists), restore function of degenerated neurons, and / or protect neurons from degeneration. 【0422】 Evidence of functional improvement, clinical benefit, or efficacy in patients may be assessed by surrogate markers of enhanced substantia nigra-striatal function, such as [18F]fluoro-L-dopa positron emission tomography (PET) uptake in the midbrain regions of the putamen and substantia nigra, or by analysis of markers of presynaptic dopamine terminal activity, such as dopamine transporters (DaT) by DaT-SPECT imaging of the putamen. Evidence of symptomatic or clinical benefit may also be determined using standard Parkinson's disease rating scales, such as the Unified Parkinson's Disease Rating Scale (UPDRS) or the Movement Disorder Society-sponsored version of the UPDRS (MDS-UPDRS), which are assessed with or without concomitant antiparkinsonian medications. These or similar scales, as well as patient-reported outcomes regarding quality of life, can demonstrate improvements in both the motor and non-motor components of the disease. Further methods for evaluating therapeutic effects are known in the art, including, but not limited to, the methods used in Example 6. 【0423】 Administration of composition The effective dose of the composition may be administered by standard routes in the art, but are not limited to systemic, topical, direct injection, intravenous, intracerebral, intracerebrospinal, subarachnoid, intracisor, intraputamen, intrahippocampal, intramacula, intrastriatal (putamen and / or caudate nucleus), or intracerebroventricular injection. In some cases, administration may include intravenous, intracerebral, intracerebrospinal, subarachnoid, intracisor, intraputamen, intrahippocampal, intrastriatal (putamen and / or caudate nucleus), or intracerebroventricular injection. Administration may be performed by intraarachnoid injection with or without Trendelenburg tilt. 【0424】 In some embodiments, this disclosure provides topical and systemic administration of effective doses of the rAAV and compositions of the present invention. For example, systemic administration may be administered into the circulatory system so that the whole body is affected. Systemic administration includes parental administration by injection, infusion, or implantation. 【0425】 In particular, the administration of rAAV according to the present invention can be achieved by using any physical method for delivering the rAAV recombinant vector to the target tissue of an animal. Administration includes, but is not limited to, injection into the central nervous system (CNS) or cerebrospinal fluid (CSF), and / or direct injection into the brain. 【0426】 In some embodiments, the methods of the present disclosure include direct intraparenchymal delivery to a region of the midbrain (or just above the midbrain), including, for example, a region of the substantia nigra (and surrounding regions) by neurosurgery. Infusion may be performed using an infusion pump, a dedicated cannula, catheter, or syringe / needle. Optionally, targeting of the injection site may be achieved using MRI-guided imaging. Administration may include delivery to the CNS of an effective amount of rAAV virion, or a pharmaceutical composition containing rAAV virion. These may be achieved, for example, via intracisternal magna infusion with the Trendelenburg tilt procedure, or intracisternal magna infusion without the Trendelenburg tilt procedure, intraarachnoid infusion with the Trendelenburg tilt procedure, or intraarachnoid infusion without the Trendelenburg tilt procedure. The compositions of the present disclosure may further be administered intravenously. 【0427】 Direct delivery to the central nervous system (CNS) may involve targeting a specific neuronal region or a more general brain region containing a specific neuronal target. The selection of individual patient brain regions and / or neuronal targets, as well as subsequent intraoperative delivery of AAVs, can be achieved by employing any number of software planning programs (e.g., Stealth System, Clearpoint Neuronavigation System, Brainlab, Neuroinspire, etc.) using a variety of imaging techniques (MRI, CT, CT combined with MRI merging). Brain region targeting and delivery can involve standard stereotactic frames (Leksell, CRW) or, with or without intraoperative MRI, a frameless approach. Actual delivery of AAVs may be by injection through a needle or cannula with or without a material-coated inner lumen to prevent adsorption of the AAV vector (e.g., Smartflow cannula, MRI Interventions cannula). The delivery device interfaces the syringe with an automated or microinjection pump having pre-programmed injection rates and volumes. A syringe / needle combination, or a needle alone, may be directly interfaced with a stereotactic frame. Infusion may involve a constant flow rate or a variable rate due to convective-enhanced delivery. [Examples] 【0428】 Example 1: Bioactivity in vitro Plasmid vectors containing AAV expression cassettes encoding each of the following Parkin variants are constructed using conventional cloning methods. TIFF0007872737000076.tif105160TIFF0007872737000077.tif203150 【0429】 The constructs are screened for parkin expression by Western blotting, ELISA, and / or immunolabeling after in vitro transfection of HEK293, HeLa cells, rat primary neurons, and / or ChoLec2 cells. 【0430】 Selected constructs exhibiting Parkin expression are transfected to or converted to AAV virions using a helper-free packaging system and are also used to transduce ChoLec2 and / or SH-SY5Y cells. Cells are treated with uncoupling agents (carbonyl cyanide 3-chlorophenylhydrazone [CCCP] or carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone [FCCP]) and mitochondrial damage assays. The localization of Parkin variants to mitochondria is measured using fluorescence microscopy. Cells are also tested for damaged mitochondrial clearance by measuring the co-localization of exogenous Parkin and translocase complex subunit 20 (TOMM20) of the mitochondrial outer membrane, and by Western blotting of the mitochondrial membrane fraction. Levels of autophagosome (e.g., LC3) markers are also measured. 【0431】 Parkin mutants are further assayed, such as by the MitoSOX assay, to assess their ability to enhance cell survival and normalize mitochondrial morphology and function, for example, by reducing reactive oxygen species. 【0432】 To further demonstrate the physiological activity of parkin constructs, modifications of the parkin substrate, such as ubiquitination or total expression levels of AIMP2, CISD1, Miro, STEP-61, RTP-801, porin, mitofusion, PARIS, and PGC-1α, are measured compared to appropriate controls (endogenous proteins, e.g., β-actin). 【0433】 The selected AAV virions are further evaluated in primary neurons from rodents lacking the normal PARK2 or PARK6 gene, and in human patient-derived cells lacking the normal PARK2 or PARK6 gene. Neurons may differentiate into dopaminergic neurons before, during, or after contact with the AAV virion. The bioactivity assays described above are repeated in primary neuron assays or patient-derived cell assays. 【0434】 Example 2: In vivo efficacy Therapies using AAV virions encoding selected parkin constructs will be tested in animal models of the disease. Specifically, mice, rats, or non-human primates (NHPs) will be treated with dopaminergic neurotoxins to induce neurological disorders. The neurotoxins used in the experiments include 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 6-hydroxydopamine. 【0435】 Treatment with AAV virions encoding selected parkin constructs will also be tested in mouse or rat models with loss-of-function (e.g., null) mutations in the PARK2 or PARK6 gene. The neuroprotective and neuroregenerative effects of the treatment will be measured. 【0436】 The evaluation includes tests for preventing the loss of dopaminergic neurons in the substantia nigra and / or ventral tegmental area, or for rescuing them from further degeneration. Neuroprotective / neuroregenerative effects on the substantia nigra-striatal system are measured using techniques such as quantification of the number of neuronal cell bodies, general morphology of neuronal cell bodies and their axons (e.g., size, shape), and integrity of axonal projections in pathways to other brain regions (e.g., striatum), as disclosed, for example, in Kirik et al., Eur J Neurosci, 2000. Characterization of dopaminergic neurons (quantification of neuronal number) and fiber density (optical density measurement) is achieved using immunolabeling of tyrosine hydroxylase and / or vesicular monoamine transporters. Neurochemical levels of dopamine and / or its metabolites (e.g., 3,4-dihydroxyphenylacetic acid [DOPAC], homovanillic acid [HVA]) in the striatum and / or substantia nigra are also quantified. 【0437】 Characterization of the functional / behavioral outcomes of treatment with AAV virions encoding Parkinson's constructs is achieved by examining motor behavior using known test paradigms for characterizing nigrostriatal function in rodents (Bjorklund et al., Br Res, 2000; Kirik et al., Nat Neurosci, 2004), but is not limited to these, and includes, amphetamine-induced turning, spontaneous turning, forelimb use preference, cylinder test, modified stepping task, general spontaneous motor behavior in open fields, and rotorod. NHP is assessed by behavioral tests using NHP equivalent to the Unified Parkinson's Disease Rating Scale (Kordower et al., Ann Neurol, 2006). 【0438】 Example 4: Prevention of neuronal loss: A 6-OHDA model of Parkinson's disease The parkin variant was tested in assays known in the art as a 6-OHDA toxicity model, such as those described in Simola et al. Neurotox Res. 2007 Apr;11(3-4):151-67 (2007) and Hanrott et al. J. Biol. Chem. 281:5373-82 (2006), as a model of neuronal damage caused by Parkinson's disease. This model produces potent dopaminergic neuronal oxidative stress and neuronal loss, which are characteristic of the disease pathology of patients with Parkinson's disease. 【0439】 Table 8 summarizes the specific AAV structures evaluated in these experiments. 【0440】 (Table 8) Tested structures TIFF0007872737000078.tif91160 【0441】 C431F has been documented in the literature as a catalytic center mutation. See Fiesel et al. Hum Mutat. 36:774-786 (2015). It was intended as a negative control for parkin activation. 【0442】 The mitigation of the neurotoxic effects of the dopaminergic (DA) toxin 6-OHDA (6-hydroxydopamine) was evaluated in a rat dopaminergic neuronal cell line (N27-A; END Millipore, Millipore, Temecula, CA). Cells were seeded in 96-well plates and transfected with plasmid DNA encoding each of the engineered parkin variants, a fluorescent reporter control, or a mock transfection. After 24 hours of culture, cells were exposed to 7.5 μM, 15 μM, or 30 μM 6-OHDA (6-hydroxydopamine hydrobromide, Sigma-Aldrich, cat.162957). The cell viability of all neurons under each condition was measured by a luminescence-based assay 3 days (Figures 10A-10D; RealTime-Glo™ MT Cell Viability Assay, Promega cat. G9712) or 9 days (Figures 11A-11D; Cell Titer-Glo 2.0 Assay, Promega cat. G9241) after the addition of 6-OHDA. Non-parameter analyses (Kruskal-Wallis) were performed to evaluate the overall effect of the transfection status, and Dunn's multiple comparison post-hoc analyses differences were performed where appropriate. 【0443】 In both experiments, the "activated" and "super" parkin constructs prevented a dose-dependent decrease in neuronal cell count compared to the control (CON GFP) experiment. Surprisingly, C431F parkin was more effective than wild-type parkin (WT). Furthermore, in both of these 6-OHDA experiments, "activated" parkin, Δ parkin, and "super parkin V2" were superior to wild-type parkin, respectively. 【0444】 In summary, the manipulated parkin constructs tested in this embodiment are superior to wild-type parkin in preventing neuronal damage in an in vitro model of recognized Parkinson's disease. 【0445】 Example 4: Human IPSC-derived PARK2 - / - Increased cell number and preservation of mitochondrial membrane potential in dopaminergic neurons Another acceptable in vitro model for Parkinson's disease is an assay to prevent the harmful cellular effects of oxidative stress promoters in dopaminergic neurons. This involves treating Parkinnull (i.e., PARK2) with hydrogen peroxide (H2O2). - / - This includes preventing the dissipation of mitochondrial membrane potential in dopaminergic neurons. (Ferrari et al. J. Neuroscience Methods 340:108741 (2020); Avazzadeh et al. Blain Sci. 11:373 (2021)) This model uses human cells. Since substantia nigra degeneration is observed in subjects with Parkinson's disease, therapeutic approaches that can mitigate the loss of dopaminergic neurons in the model system are considered to predict therapeutic effects in Parkinson's disease. 【0446】 iPS-derived human PARK2 - / - dopaminergic neurons (Applied StemCell®, Milpitas, CA) were seeded in 384-well plates and cultured for 7 days. They were then transfected with plasmid DNA encoding each parkin variant using Viafect (Promega® #E4981). As a control, cells were treated with 0.1% DMSO medium and hydrogen peroxide (150 μM H2O2) on day 10 (6 wells / condition). After treating cells with H2O2 for 24 and 48 hours, mitochondrial membrane potential was assessed using a red fluorescent dye that stains mitochondria in viable cells. Its accumulation was membrane potential dependent (MitoTracker®, ThermoFisher® Cat. M7512). Immediately before fixation, cells were stained with 250 nM MitoTracker™ Red CMXRos for 30 minutes according to a standard kit protocol. After fixation, permeabilization, and blocking, the cells were subsequently stained with Hoechst nuclear dye. Quantification was achieved by imaging the plate at 20x magnification, capturing data from 9 fields / well using a ThermoFisher® CX7 LED high-content microscope. Automated quantitative image analysis was performed using the ThermoFisher® CellInsight® software package. 【0447】 Under untransfected control and mock control (CON-mock) conditions, cell numbers were significantly reduced after exposure to 150 μM H2O2 (Figure 12, left). Superparkin, Superparkin V2, and the mutant C431F construct were found to prevent the loss of H2O2-mediated neuronal numbers (Figure 12, left) and to prevent the dissipation of mitochondrial membrane potential (Figure 12, right). 【0448】 The Δ-Parkin, SuperParkin, SuperParkin V2, and C431F-Parkin constructs resulted in an increase in cell number after H2O2 treatment. SuperParkin, SuperParkin V2, and C431F-Parkin constructs prevented an increase in mitochondrial membrane trackers. 【0449】 Example 5: Expression of a manipulated parkin variant from an AAV vector This example demonstrates the expression of an engineered Parkin construct from an adeno-associated virus (AAV) vector in primary cortical neurons specific to physiologically relevant primary cells. 【0450】 Primary cortical neuron cultures were prepared using embryonic fetuses (approximately E18) from pregnant Wistar rats, as described by Banker and Goslin (1998). Briefly, after brain isolation, the hippocampus and cortex were dissected, washed with Hanks equilibrium salt solution (HBSS), and incubated with trypsin (0.25%) for 15 minutes. Cells were then dissociated by very gentle pipetting several times with a microglass pipette. The isolated cells were then placed in approximately 0.5 × 10⁶ cells in Neurobasal medium (Gibco®) containing B27 (2%), GlutaMax® (2nM) Penn / strep (1%), and glucose (6.5%), v / v on a tissue culture plate i treated with poly-L-lysine. 6 Cells were seeded at a cell / well density. The cells were grown in a humidified incubator (37°C, 5% CO2) for approximately 3 weeks, with approximately 1 ml of culture medium replenished weekly. Neurons were used for AAV gene transduction on day 14. 【0451】 Using the AAV9 vector for each manipulated parkin structure, 3 × 10 5 Primary neuron cultures were transduced based on the multiplicity of infection (MOI). 【0452】 Cell lysates were collected after 7 days. Total protein was measured using a BCA kit (Thermo® catalog number 23225), and 10 mg of protein was loaded onto a 4-12% Bis-Tris gel. The protein was transferred to a PVDF membrane, blocked in TBS-T + 10% powdered milk, and incubated overnight with anti-Parkin (CST #2132) antibody. Anti-GAPDH (Abcam ab8245) was also used experimentally as a loading control. 【0453】 Western blotting analysis of parkin protein expression revealed potent protein expression after transduction in primary neurons. Lanes 5 and 6 show the endogenous levels of WT parkin detected in neurons. Lane 1 reveals activated parkin-mediated overexpression of full-length human parkin protein of approximately 52 kDa size. The upper band in lane 2 represents the endogenous level of human parkin, while the lower band (approximately 36 kDa, arrow) reflects the Δ parkin form of the protein. Lanes 3 and 4 show superparkin-mediated overexpression of human parkin in both full-length (approximately 54 kDa) and its cleaved form (approximately 43 kDa). The cleavage band of the superparkin V2 vector is stronger than that of superparkin V1, consistent with V2 being more resistant to ubiquitination and subsequent degradation. 【0454】 Example 6: In vivo study in an MPTP mouse model of nigrostriatal degeneration This embodiment demonstrates the treatment of Parkinson's disease with an adeno-associated virus (AAV) vector expressing the engineered parkin variant disclosed herein. 【0455】 The animal model used is a substantia nigra-striatal degeneration model in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mice. In this model, repeated intraperitoneal injections of the neurotoxin MPTP induce bilateral loss of dopamine-producing neurons in the substantia nigra and simultaneous depletion of dopamine levels in the striatum. 【0456】 Unilateral injection into the substantia nigra of an AAV9 vector encoding each of the four parkin variants is performed four weeks prior to MPTP administration. As a positive pharmacological control, the mouse group receives chronic injections of nilotinib, a tyrosine kinase inhibitor with several neuroprotective properties, prior to MPTP administration. A summary of the treatment groups and the overall study design can be found in Table 9. 【0457】 (Table 9) TIFF0007872737000079.tif46128FB=Formulation buffer control, Sal=Physiological saline, ACT=Activated parkin, DEL=Δparkin, SUP1=Superparkin, SUP2=Superparkin V2, MPTP=1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine 【0458】 A fresh brain sample is collected six days after the initial MPTP injection. Neurochemical analysis includes quantifying dopamine and its metabolite levels in the striatum using high-performance liquid chromatography (HPLC). Anatomical analysis includes quantifying the number of tyrosine hydroxylase (TH)-positive cells in the substantia nigra (SN) (region of compacta; SNc). These data may provide evidence for the potential treatment of dopaminergic neuron loss in Parkinson's disease using the engineered parkin variants disclosed herein.

Claims

[Claim 1] (a) Mitochondrial targeting sequence (MTS), (b) Transmembrane domain (TMD), and (c) (i) Deletion of the N-terminal ubiquitin-like (Ubl) domain, (ii) Deletion of the N-terminal ubiquitin-like (Ubl) domain and deletion of the Ubl-RING0 interdomain linker sequence, (iii) A polypeptide sequence that is at least 95% identical to sequence number 73 or sequence number 74, and / or (iv) polypeptide sequence of sequence number 73 or sequence number 74 Parkin protein, which includes A fusion protein containing A polynucleotide comprising a polynucleotide sequence encoding a polynucleotide wherein the MTS and TMD are derived from PINK1, respectively. [Claim 2] (i) The MTS includes a mitochondrial processing peptidase (MPP) cleavage site, and / or (ii) The MTS comprises the polypeptide sequence of sequence number 65 or 66, The polynucleotide according to claim 1. [Claim 3] (i) The TMD includes a PARL cutting site and / or (ii) The TMD comprises the polypeptide sequence of SEQ ID NO: 67 or SEQ ID NO: 68, The polynucleotide according to claim 1 or 2. [Claim 4] (i) The fusion protein comprises an MTS-TMD polypeptide, (ii) The fusion protein comprises an MTS-TMD polypeptide having an amino acid sequence that is at least 95% identical to SEQ ID NO: 70, and / or (iii) The fusion protein comprises an MTS-TMD polypeptide containing the amino acid sequence of SEQ ID NO: 70, The polynucleotide according to any one of claims 1 to 3. [Claim 5] The polynucleotide according to any one of claims 1 to 4, wherein the fusion protein comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO:

75. [Claim 6] The polynucleotide according to any one of claims 1 to 5, wherein the fusion protein comprises one or more substitutions to the reference human parkin protein sequence of SEQ ID NO: 1, and the one or more substitutions are selected from F146A, W403A, F463A, C457S, and W403A. [Claim 7] (i) The fusion protein contains an F104M substitution to the reference human PINK1 protein sequence of SEQ ID NO: 64, or (ii) The fusion protein contains both the F146A substitution and the W403A substitution with respect to the reference human parkin protein sequence of SEQ ID NO: 1, and contains the F104M substitution with respect to the reference human PINK1 protein sequence of SEQ ID NO:

64. The polynucleotide according to any one of claims 1 to 6. [Claim 8] The polynucleotide according to claim 1, wherein the fusion protein comprises a polypeptide sequence that is at least 95% identical to the sequence of SEQ ID NO: 97 or 99, or the fusion protein comprises the polypeptide sequence of SEQ ID NO: 97 or 99, and the polypeptide sequence comprises two or more amino acid substitutions selected from F104M, W403A, and F463A, wherein F104M corresponds to the reference human PINK1 protein sequence of SEQ ID NO: 64, W403A corresponds to the reference human parkin protein sequence of SEQ ID NO: 1, and F463A corresponds to the reference human parkin protein sequence of SEQ ID NO:

1. [Claim 9] The polynucleotide according to claim 1, wherein the parkin protein is an activated parkin protein. [Claim 10] (i) The activated parkin protein contains an amino acid substitution at position Cys-431 relative to the reference human parkin protein sequence of Sequence ID No. 1, or (ii) The activated parkin protein contains a C431F amino acid substitution relative to the reference human parkin protein sequence of Sequence ID No. 1, The polynucleotide according to claim 9. [Claim 11] (i) The activated parkin protein contains one or more amino acid substitutions at positions Phe-146, Trp-403, Cys-457, Phe-463, and Asn-273 relative to the reference human parkin protein sequence of Sequence ID No. 1, (ii) The activated parkin protein contains two or more amino acid substitutions at positions Phe-146, Trp-403, Cys-457, Phe-463, and Asn-273 relative to the reference human parkin protein sequence of Sequence ID No. 1, or (iii) The activated parkin protein contains amino acid substitutions at positions Phe-146, Trp-403, Cys-457, Phe-463, and Asn-273 relative to the reference human parkin protein sequence of Sequence ID No. 1, The polynucleotide according to claim 9 or 10. [Claim 12] (i) The activated parkin protein comprises one or more amino acid substitutions selected from F146A, W403A, and / or N273K relative to the reference human parkin protein sequence of Sequence ID No. 1, (ii) The activated parkin protein contains amino acid substitutions F146A and W403A relative to the reference human parkin protein sequence of Sequence ID No. 1, or (iii) The activated parkin protein contains amino acid substitutions F146A, N273K, and W403A relative to the reference human parkin protein sequence of Sequence ID No. 1, The polynucleotide according to any one of claims 9 to 11. [Claim 13] The polynucleotide according to claim 9, wherein the activated parkin protein comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO: 93, or the activated parkin protein comprises the polypeptide sequence of SEQ ID NO:

93. [Claim 14] The polynucleotide according to any one of claims 9 to 12, wherein the parkin protein is a Δ parkin protein containing a deletion of a ubiquitin-like (Ubl) domain, the Δ parkin protein contains a polypeptide sequence that is at least 95% identical to SEQ ID NO: 18, and / or the Δ parkin protein contains the polypeptide sequence of SEQ ID NO:

18. [Claim 15] The polynucleotide according to any one of claims 1 to 14, comprising a promoter operably ligated to the polynucleotide sequence. [Claim 16] The polynucleotide according to claim 15, wherein the promoter is (i) a constitutive promoter, a CAG promoter, or a CMV promoter, or (ii) a neuron-specific promoter or a SYN promoter. [Claim 17] (i) comprising a WPRE element, (ii) comprising an hGH polyadenylation site, or (iii) comprising both (i) and (iii), according to any one of claims 1 to 16. [Claim 18] A recombinant adeno-associated virus (rAAV) virion comprising a capsid and a vector genome, wherein the vector genome comprises a polynucleotide as described in any one of claims 1 to 17. [Claim 19] The rAAV virion according to claim 18, wherein the capsid is an AAV9 capsid, and the AAV9 capsid shares at least 98%, 99%, or 100% identity with sequence number 60. [Claim 20] A composition for use in increasing Parkin activity in cells by contacting the composition with cells, comprising a polynucleotide according to any one of claims 1 to 17 or an rAAV virion according to claim 18 or 19. [Claim 21] A composition for use in increasing parkin activity in a subject by administering the composition to the subject, comprising a polynucleotide according to any one of claims 1 to 17 or rAAV virion according to claim 18 or 19. [Claim 22] The aforementioned cells or subjects lack parkin activity and / or contain loss-of-function mutations in parkin and / or Parkin activity includes one or more of the following in response to neurotoxic treatment: co-localization of parkin with TOMM2, ubiquitination of mitochondrial proteins in response to neurotoxic treatment, and increased parkin levels in the mitochondrial fraction in response to neurotoxic treatment. The composition according to claim 20 or claim 21. [Claim 23] A composition for use in promoting neuronal survival by bringing a neuron into contact with the composition, comprising a polynucleotide according to any one of claims 1 to 17 or an rAAV virion according to claim 18 or 19. [Claim 24] A composition for use in promoting neuronal survival in a subject by administering the composition to the subject, comprising a polynucleotide according to any one of claims 1 to 17 or rAAV virion according to claim 18 or 19. [Claim 25] The composition according to claim 23 or claim 24, wherein the neuron is a dopaminergic neuron. [Claim 26] A composition for use in the treatment of a disease or disorder in a subject requiring treatment of a disease or disorder by administering the composition to a subject, comprising a polynucleotide according to any one of claims 1 to 17 or rAAV virion according to claim 18 or 19. [Claim 27] The composition according to claim 26, wherein the disease or disorder is (i) related to a genetic defect in parkin expression or function, (ii) related to a genetic defect in PINK1 expression or function, or (iii) Parkinson's disease and / or early-onset Parkinson's disease (EOPD). [Claim 28] The composition according to claim 26 or 27, which is effective in (i) alleviating one or more symptoms of Parkinson's disease, (ii) reducing motor complications associated with neurodegeneration, (iii) reducing the need for antiparkinsonian drug therapy, L-dopa, or dopamine agonists, (iv) restoring the function of degenerated neurons, (v) protecting neurons from degeneration, (vi) enhancing substantia nigra-striatal function as assessed by [18F]fluoro-L-dopa positron emission tomography (PET) or DaT-SPECT imaging, (vii) improving one or both of the above-mentioned UPDRS or MDS-UPDRS, or (viiii) any combination of (i) to (vii). [Claim 29] A cell comprising the polynucleotide described in any one of claims 1 to 17. [Claim 30] A protein encoded by a polynucleotide according to any one of claims 1 to 17. [Claim 31] A pharmaceutical composition comprising the rAAV virion described in claim 18 or 19 and one or more pharmaceutically acceptable carriers, diluents, or excipients.

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