Gene therapy for CNS degeneration

Abstract

The present disclosure generally relates to compositions and methods for treating, preventing, inhibiting, or delaying central nervous system degeneration. The present disclosure relates to recombinant gene therapy vectors containing PARK2, PINK1, DJ-1, LRRK2, SCNA, c-Rel, ATG7, VMAT2, or GBA genes, or functional fragments or variants thereof. The present disclosure also relates to CRISPR / Cas-based gene editing systems for treating, preventing, inhibiting, or delaying central nervous system degeneration. [Selected Figure] Figure 1

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 664,006, filed on 27 April 2018, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0002] Incorporation of sequence lists The sequence listing relating to this application is provided in text format instead of as a hard copy and is incorporated herein by reference. The name of the text file containing the sequence listing is ROPA_009_01WO_ST25.txt. The text file is 254KB in size, was created on April 29, 2019, and submitted electronically via EFS-Web.

[0003] Areas of disclosure This disclosure relates, in general, to gene therapy and / or gene editing for the treatment of disorders associated with central nervous system degeneration, such as Parkinson's disease. More specifically, this disclosure provides compositions and methods for gene therapy or gene repair in neurons, both ex vivo and in vivo. [Background technology]

[0004] background Various genes are involved in central nervous system degeneration disorders such as Parkinson's disease (PD). These genes include PARK2, PINK1 (PARK6), DJ-1 (PARK7), LRRK2, alpha-synuclein, and DJ-1. (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)). PARK2, also known as PRKN, is involved in the morphology of PD, an autosomal recessive juvenile form of PD. Despite extensive attempts, there are few reports of successful gene therapy for central nervous system degeneration.

[0005] There is a great need in the art for new compositions and methods for treating and preventing central nervous system degeneration. [Prior art documents] [Non-patent literature]

[0006] [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]

[0007] Summary of Disclosure This disclosure provides, in part, compositions and methods for treating, preventing, inhibiting, or delaying central nervous system degeneration. More specifically, the inventors disclose various embodiments of recombinant gene therapy vectors and related methods comprising the Parkinson's protein 2 E3 ubiquitin protein ligase (PARK2) gene, the PTEN-induced putative kinase 1 (PINK1) gene, the protein deglycase DJ-1 (DJ-1) gene, the leucine-rich repeat kinase 2 (LRRK2) gene, the alpha-synuclein (SCNA) gene, the oncogene c-Rel (c-Rel) gene, the ubiquitin-like modifier-activating enzyme (ATG7) gene, the synaptic vesicle amine transporter (VMAT2) gene, or the glucocerebrosidase (GBA) gene, or functional fragments or variants thereof.

[0008] Further inventors disclose various embodiments and related methods of a gene editing system comprising Cas protein; guide RNA; the Parkinson's protein 2 E3 ubiquitin protein ligase (PARK 2) gene, the PTEN-induced putative kinase 1 (PINK 1) gene, the protein deglycase DJ-1 (DJ-1) gene, the leucine-rich repeat kinase 2 (LRRK 2) gene, the alpha-synuclein (SCNA) gene, the oncogene c-Rel (c-Rel) gene, the ubiquitin-like modifier-activating enzyme (ATG7) gene, the synaptic vesicle amine transporter (VMAT 2) gene, or the glucocerebrosidase (GBA) gene, or a repair template comprising a functional fragment or variant thereof.

[0009] In a first aspect, the present disclosure provides a method of inhibiting the degeneration or death of dopaminergic neurons that contain mutations in genes associated with Parkinson's disease (PD). The mutant gene can be the E3 ubiquitin protein ligase (PARK 2) gene of Parkinson protein 2, the PTEN-induced putative kinase 1 (PINK1) gene, the protein deglycase DJ-1 (DJ-1) gene, the leucine-rich repeat kinase 2 (LRRK2) gene, the alpha-synuclein (SCNA) gene, the proto-oncogene c-Rel (c-Rel) gene, the ubiquitin-like modifier activating enzyme (ATG7) gene, the synaptic vesicle amine transporter (VMAT2) gene, or the glucocerebrosidase (GBA) gene. In the method of this aspect, the method comprises contacting the neurons with a recombinant gene therapy vector comprising a polynucleotide encoding a wild-type protein expressed by the wild-type version of the mutant gene or a functional variant or fragment thereof. After contacting with the recombinant gene therapy vector, the neurons express the wild-type protein or a functional variant or fragment thereof.

[0010] In some embodiments, the PARK2, PINK1, LRRK2, SCNA, c-Rel, ATG7, VMAT2, or GBA proteins each contain the amino acid sequences shown in SEQ ID NOs. 1-9. In some embodiments, the gene is the PARK2 gene, and the wild-type PARK2 protein contains the amino acid sequence shown in any of SEQ ID NOs. 10-17. In some embodiments, the polynucleotides each contain sequences having at least 70%, 75%, 80%, 85%, 95%, or 99% identity with the PARK2, PINK1, LRRK2, SCNA, c-Rel, ATG7, VMAT2, or GBA polynucleotide sequences shown in SEQ ID NOs. 18-26. In some embodiments, the gene is the PARK2 gene, and the polynucleotides contain sequences having at least 70%, 75%, 80%, 85%, 95%, or 99% identity with the PARK2 isoform polynucleotide sequences shown in any of SEQ ID NOs. 27-34. In some embodiments, the polynucleotides are codon-optimized. In some embodiments, the polynucleotide contains less than 40, less than 30, less than 20, or 10 or fewer CpG islands. In some embodiments, the polynucleotide contains at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, or at least 10 CpG islands. In some embodiments, it contains 5 to 20 CpG islands.

[0011] A variety of viral or non-viral vectors can be used. In some embodiments, the recombinant gene therapy vector is a recombinant adeno-associated virus (AAV). Any of the known serotypes can be used. In some embodiments, the AAV has serotype AAV1, AAV2, AAV5, AAV8, AAV9, AAVrh10, or AAVrh74. In some embodiments, the recombinant gene therapy vector comprises self-complementary AAV. In some embodiments, the recombinant gene therapy vector comprises single-stranded AAV. In some embodiments, the AAV is wild-type AAV or modified AAV. In some embodiments, the AAV comprises a capsid protein having at least 95% identity to a wild-type VP1, VP2, or VP3 capsid protein.

[0012] The recombinant gene therapy vector may contain gene regulatory elements. In some embodiments, the recombinant gene therapy vector comprises a polynucleotide containing, in 5' to 3' order, a eukaryotically active promoter sequence and a sequence encoding a wild-type protein or a functional fragment or variant thereof. The sequence encoding a wild-type protein or a functional fragment or variant thereof is operably linked to the eukaryotically active promoter sequence.

[0013] Without being limited by the examples of this disclosure, in some embodiments, the recombinant gene therapy vector further comprises one or more of the following: a neuron-specific promoter optionally selected from the group consisting of 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) promoter; a ubiquitous promoter selected from the group consisting of CMV, CAG, UBC, PGK, EF1-alpha, GAPDH, SV40, HBV, and chicken beta-actin promoter; an enhancer; an intron; a poly(A) signal; a WPRE (woodchuck hepatitis virus post-transcriptional regulator); and an HPRE (hepatitis post-transcriptional regulator). The WPRE may be WPRE(r) or WPRE(x).

[0014] In various embodiments of the vectors and methods disclosed herein, the vector is HuBA promoter, transgene, WPRE(x), and pAglobin-Oc, CMV promoter, TPL-eMLP 5' enhancer, transgene, WPRE(r), and pAglobin-Oc, Syn promoter, transgene, WPRE(r), 3'UTR(globin), and pAGH-Bt CBA promoter, transgene, and pAGH-Bt, EF1a promoter, transgene, and pAglobin-Oc, HuBA promoter, transgene, R2V17, and pAGH-Bt, Syn promoter, transgene, WPRE(x), 3'UTR(globin), and pAGH-Hs, CaMKIIa promoter, transgene, WPRE(r), and pAGH-Hs, CMV and TPL promoters, transgenes, WPRE(r), and pAGH-Hs, HuBA promoter, transgene, and pAGH-Hs, CMV and TPL promoters, eMPL, transgenes, R2V17, 3'UTR (globin), and pAGH-Bt, EF1α promoter, transgene, WPRE(r), and pAGH-Bt, Syn promoter, transgene, R2V17, and pAglobin-Oc, CaMKIIa promoter, transgene, R2V17, and pAglobin-Oc, CBA promoter, transgene, WPRE(x), 3'UTR (globin), and pAGH-Hs, CBA promoter, transgene, 3'UTR (globin), and pA globin-Oc, CaMKIIa promoter, transgene, R2V17, and pAGH-Bt, EF1a promoter, transgene, R2V17, 3' globin, and pAGH-Hs, CMV promoter, transgene, R2V17, 3'UTR (globin), and pAGH-Hs, or CMV promoter, transgene, and pAGH-Hs It includes them in the order from 5' to 3', Optionally, the introduced gene encodes PARK2.

[0015] The methods of this disclosure may have a variety of effects. In some embodiments, neurons express a reduced amount of alpha-synuclein and / or contain a reduced amount of Lewy bodies after contact with a recombinant gene therapy vector. In some embodiments, neurons express a reduced amount of monoamine oxidase after contact with a recombinant gene therapy vector. In some embodiments, neurons produce and / or release an increased amount of dopamine after contact with a recombinant gene therapy vector. In some embodiments, neurons undergo increased mitophagy after contact with a recombinant gene therapy vector.

[0016] In some embodiments, neurons express a smaller amount of monoamine oxidase compared to the amount expressed in neurons not in contact with the recombinant gene therapy vector, optionally, the smaller amount is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% lower than the amount expressed in neurons not in contact with the recombinant gene therapy vector. In some embodiments, neurons produce and / or release an increased amount of dopamine compared to the amount produced and / or released by neurons not in contact with the recombinant gene therapy vector, optionally, the increased amount is at least 5%, 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 twice, at least three times, at least four times, at least five times, or at least ten times greater than the amount produced and / or released by neurons not in contact with the recombinant gene therapy vector. In some embodiments, neurons receive an increased amount of autophagy compared to the amount of autophagy received by neurons not in contact with the recombinant gene therapy vector, and optionally, the increased amount is at least 5%, 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 twice, at least three times, at least four times, at least five times, or at least ten times greater than the amount received by neurons not in contact with the recombinant gene therapy vector.

[0017] The neurons used in the methods of this disclosure may have a variety of characteristics. In some embodiments, the neurons are primary tyrosine hydroxylase-positive neurons. In some embodiments, the neurons are generated from induced pluripotent stem cells prepared from cells obtained from subjects diagnosed with Parkinson's disease.

[0018] In another aspect, the Disclosure provides a recombinant gene therapy vector comprising a polynucleotide encoding the wild-type Parkinson's protein 2 E3 ubiquitin protein ligase (PARK 2) gene, the PTEN-induced putative kinase 1 (PINK 1) gene, the protein deglycase DJ-1 (DJ-1) gene, the leucine-rich repeat kinase 2 (LRRK 2) gene, the alpha-synuclein (SCNA) gene, the oncogene c-Rel (c-Rel) gene, the ubiquitin-like modifier-activating enzyme (ATG7) gene, the synaptic vesicle amine transporter (VMAT 2) gene, or the glucocerebrosidase (GBA) gene, or a functional variant or fragment thereof, wherein the polynucleotide is operably linked to a promoter sequence active in eukaryotes, and neurons transduced with the recombinant gene therapy vector express the wild-type protein, or a functional variant or fragment thereof.

[0019] In some embodiments, the functional PARK2, PINK1, LRRK2, SCNA, c-Rel, ATG7, VMAT2, or GBA protein each contains the amino acid sequence shown in SEQ ID NOs. 1-9. In some embodiments, the gene is the PARK2 gene, and the wild-type PARK2 protein contains the amino acid sequence shown in any of SEQ ID NOs. 10-17. In some embodiments, the polynucleotide each contains a sequence having at least 70%, 75%, 80%, 85%, 95%, or 99% identity with the PARK2, PINK1, LRRK2, SCNA, c-Rel, ATG7, VMAT2, or GBA polynucleotide sequence shown in SEQ ID NOs. 18-26. In some embodiments, the gene is the PARK2 gene, and the polynucleotide contains a sequence having at least 70%, 75%, 80%, 85%, 95%, or 99% identity with the PARK2 isoform polynucleotide sequence shown in any of SEQ ID NOs. 27-34. In some embodiments, the vector is HuBA promoter, transgene, WPRE(x), and pAglobin-Oc, CMV promoter, TPL-eMLP 5' enhancer, transgene, WPRE(r), and pAglobin-Oc, Syn promoter, transgene, WPRE(r), 3'UTR(globin), and pAGH-Bt CBA promoter, transgene, and pAGH-Bt, EF1a promoter, transgene, and pAglobin-Oc, HuBA promoter, transgene, R2V17, and pAGH-Bt, Syn promoter, transgene, WPRE(x), 3'UTR(globin), and pAGH-Hs, CaMKIIa promoter, transgene, WPRE(r), and pAGH-Hs, CMV and TPL promoters, transgenes, WPRE(r), and pAGH-Hs, HuBA promoter, transgene, and pAGH-Hs, CMV and TPL promoters, eMPL, transgenes, R2V17, 3'UTR (globin), and pAGH-Bt, EF1a promoter, transgene, WPRE(r), and pAGH-Bt, Syn promoter, transgene, R2V17, and pAglobin-Oc, CaMKIIa promoter, transgene, R2V17, and pAglobin-Oc, CBA promoter, transgene, WPRE(x), 3'UTR (globin), and pAGH-Hs, CBA promoter, transgene, 3'UTR (globin), and pA globin-Oc, CaMKIIa promoter, transgene, R2V17, and pAGH-Bt, EF1a promoter, transgene, R2V17, 3'UTR (globin), and pAGH-Hs, CMV promoter, transgene, R2V17, 3'UTR (globin), and pAGH-Hs, or CMV promoter, transgene, and pAGH-Hs It includes an expression cassette containing the elements in the order from 5' to 3', Optionally, the introduced gene encodes PARK2.

[0020] In some embodiments, the polynucleotide is codon-optimized. In some embodiments, the polynucleotide comprises one of SEQ ID NOs: 35-38. In some embodiments, the recombinant gene therapy vector is recombinant adeno-associated virus (rAAV). In some embodiments, the rAAV has serotypes AAV1, AAV2, AAV5, AAV8, AAV9, AAVrh10, or AAVrh74. In some embodiments, the recombinant gene therapy vector comprises a self-complementary or single-stranded AAV genome. In some embodiments, the AAV is wild-type AAV or modified AAV. In some embodiments, the AAV comprises a capsid protein having at least 95% identity with wild-type VP1, VP2, or VP3 capsid protein.

[0021] In another aspect, the Disclosure provides a method for treating or inhibiting the onset of Parkinson's disease (PD) in a subject who has or is at risk of having PD, comprising administering a recombinant gene therapy vector to a subject containing a polynucleotide encoding wild-type Parkinson's protein 2 E3 ubiquitin protein ligase (PARK2), PTEN-induced putative kinase 1 (PINK1) gene, protein deglycase DJ-1 (DJ-1) gene, leucine-rich repeat kinase 2 (LRRK2) gene, alpha-synuclein (SCNA) gene, proto-oncogene c-Rel (c-Rel) gene, ubiquitin-like modifier-activating enzyme (ATG7) gene, synaptic vesicle amine transporter (VMAT2) gene, or glucocerebrosidase (GBA) gene, or a functional variant or fragment thereof, wherein the administration of the recombinant gene therapy vector treats or inhibits the onset of Parkinson's disease in the subject.

[0022] In some embodiments, PD is early-onset PD, optionally early-onset autosomal recessive PD. In some embodiments, the subject includes mutations in the PARK2 gene, PINK1 gene, LRRK2 gene, SCNA gene, c-Rel gene, ATG7 gene, VMAT2, or GBA gene. In some embodiments, the PARK2, PINK1, LRRK2, SCNA, c-Rel, ATG7, VMAT2, or GBA protein each contains the amino acid sequence shown in SEQ ID NOs. 1-9. In some embodiments, the gene is the PARK2 gene, and the wild-type PARK2 protein contains the amino acid sequence shown in any of SEQ ID NOs. 10-17. In some embodiments, the polynucleotide each contains a sequence having at least 70%, 75%, 80%, 85%, 95%, or 99% identity with the PARK2, PINK1, LRRK2, SCNA, c-Rel, ATG7, VMAT2, or GBA polynucleotide sequence shown in SEQ ID NOs. 18-26. In some embodiments, the gene is the PARK2 gene, and the polynucleotide includes a sequence having at least 70%, 75%, 80%, 85%, 95%, or 99% identity with the PARK2 isoform polynucleotide sequence shown in any of SEQ ID NOs. 27–34. In some embodiments, the polynucleotide is codon-optimized. In some embodiments, the polynucleotide includes a sequence having at least 70%, 75%, 80%, 85%, 95%, or 99% identity with the sequence shown in any of SEQ ID NOs. 35–38.

[0023] In some embodiments, the recombinant gene therapy vector is recombinant adeno-associated virus (AAV). In some embodiments, the AAV has serotype AAV1, AAV2, AAV5, AAV8, AAV9, AAVrh10, or AAVrh74. In some embodiments, the recombinant gene therapy vector includes a self-complementary AAV genome. In some embodiments, the recombinant gene therapy vector includes single-stranded AAV. In some embodiments, the AAV is wild-type AAV or modified AAV. In some embodiments, the AAV includes a capsid protein having at least 95% identity with wild-type VP1, VP2, or VP3 capsid protein.

[0024] In some embodiments, the recombinant gene therapy vector comprises a polynucleotide containing a eukaryotically active promoter sequence and a sequence encoding a wild-type protein or a functional fragment or variant thereof in a 5' to 3' order, wherein the sequence encoding the wild-type protein or a functional fragment or variant thereof is operably ligated to the eukaryotically active promoter sequence.

[0025] In some embodiments, the recombinant gene therapy vector further comprises one or more of the following: a neuron-specific promoter optionally selected from the group consisting of 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) promoter; a ubiquitous promoter selected from the group consisting of CMV, CAG, UBC, PGK, EF1-alpha, GAPDH, SV40, HBV, human beta-actin, and chicken beta-actin promoter; an enhancer; an intron; a poly(A) signaling molecule; a WPRE (woodchuck hepatitis virus post-transcriptional regulator); and an HPRE (hepatitis post-transcriptional regulator).

[0026] Recombinant gene therapy vectors or gene editing systems can be administered in a variety of ways. In some embodiments, the administration step includes systemic, parenteral, intravenous, intracerebral, cerebrospinal fluid, intrathecal, intracisional, intraputamen, intrahippocampal, striatal, or intracerebroventricular administration. In some embodiments, the administration step includes intravenous, intracerebral, cerebrospinal fluid, intrathecal, intracisional, intraputamen, intrahippocampal, striatal, or intracerebroventricular injection. In some embodiments, the administration step includes intrathecal injection with a Trendelenburg inclination. In some embodiments, the administration step includes direct injection into the compact portion of the substantia nigra of the brain. In some embodiments, the administration step includes introducing the recombinant gene therapy vector into the target brain or cerebrospinal fluid (CSF).

[0027] In some embodiments, 1 x 10 per kilogram of the subject's body weight 9 ~1x10 14 A gene therapy vector of vector genome (vg / kg) is administered to the subject. In some embodiments, 1 x 10⁶ per kilogram of the subject's body weight 9 ~1x10 14 In some embodiments, the gene therapy vector of vector genome (vg / kg) is administered to the target brain, with 1 x 10 per kilogram of the target's body weight. 9 ~1x10 14 The gene therapy vector, consisting of a vector genome (vg / kg), is administered to the target CSF. In some embodiments, 1 x 10⁶ per kilogram of the target's body weight. 7 ~1x10 9 The gene therapy vector (vg / kg) of the vector genome is administered to the target.

[0028] The methods disclosed herein relate to both adult and juvenile disorders. In some embodiments, the subjects are adults. In some embodiments, the subjects are children.

[0029] The methods of this disclosure may have various effects on subjects. In some embodiments, the number of dopaminergic neurons in a subject after the administration step is greater than the number of dopaminergic neurons in a subject before the administration step. In some embodiments, the dopamine level in a subject after the administration step is greater than the dopamine level in a subject before the administration step. In some embodiments, the number of dopaminergic neurons in a subject treated by the method increases compared to the number of dopaminergic neurons in a subject not treated in this way. In some embodiments, the dopamine level in a subject treated by the method increases compared to the dopamine level in a subject not treated in this way. In some embodiments, the dopamine level in the substantia nigra of a subject treated by the method increases compared to the dopamine level in the substantia nigra of a subject not treated in this way. In some embodiments, the PRKN level in the CSF of a subject after the administration step is greater than the PRKN level in the CSF of a subject before the administration step. In some embodiments, the Unified Parkinson's Disease Rating Scale (UPDRS) score of a subject before the administration step is improved compared to the UPDRS score of a subject before the administration step. In some embodiments, the level of PRKN in the CSF of the subject treated by this method is increased compared to the level of PRKN in the CSF of the subject not treated in this way. In some embodiments, the level of PRKN in the substantia nigra of the subject after the administration step is greater than the level of PRKN in the substantia nigra of the subject before the administration step. In some embodiments, the UPDRS score of the subject treated by this method is improved compared to the UPDRS score of the subject not treated in this way. In some embodiments, the neurons of the subject express a reduced amount of alpha-synuclein and / or contain a reduced amount of Lewy bodies after contact with the recombinant gene therapy vector.

[0030] In another aspect, the disclosure relates to a method for inhibiting the degeneration or death of dopaminergic neurons having a mutant parkin (PRKN) gene, comprising: a Cas protein, or a polynucleotide encoding a Cas protein; guide RNA (gRNA); and the functional parkinson's protein 2 E3 ubiquitin protein ligase (PARK2) gene, PTEN-induced putative kinase 1 (PINK1) gene, protein deglycase DJ-1 (DJ-1) gene, leucine-rich repeat kinase 2 (LRRK2) gene, alpha-synucleus The present invention provides a method comprising contacting a neuron with a gene editing system comprising a repair template containing the rain (SCNA) gene, the oncogene c-Rel (c-Rel) gene, the ubiquitin-like modifier activator (ATG7) gene, the synaptic vesicle amine transporter (VMAT2) gene, or the glucocerebrosidase (GBA) gene, or a functional variant or fragment thereof, wherein the gene editing system is capable of repairing an endogenous gene within the neuron or inserting a functional gene into the neuron's genome.

[0031] In some embodiments, at least one component of the gene editing system is delivered by recombinant AAV.

[0032] In another aspect, the present disclosure provides a gene editing system for cells comprising a Cas protein, or a polynucleotide encoding a Cas protein; guide RNA (gRNA); and a repair template comprising the functional Parkinson's protein 2 E3 ubiquitin protein ligase (PARK2) gene, the PTEN-induced putative kinase 1 (PINK1) gene, the protein deglycase DJ-1 (DJ-1) gene, the leucine-rich repeat kinase 2 (LRRK2) gene, the alpha-synuclein (SCNA) gene, the oncogene c-Rel (c-Rel) gene, the ubiquitin-like modifier-activating enzyme (ATG7) gene, the synaptic vesicle amine transporter (VMAT2) gene, or the glucocerebrosidase (GBA) gene, or a functional variant or fragment thereof, wherein the gene editing system is capable of repairing an endogenous gene within a cell or inserting a functional gene into the cell's genome.

[0033] In some embodiments, at least one component of the gene editing system is delivered by recombinant AAV. In some embodiments, the gene editing system is delivered by recombinant AAV. In some embodiments, the cell is an ex vivo neuron. In some embodiments, the cell is a target cell.

[0034] In another aspect, the disclosure provides a recombinant gene therapy vector comprising a transgene polynucleotide encoding the E3 ubiquitin protein ligase (PARK2) gene, the transgene polynucleotide being operably linked to a promoter sequence active in eukaryotes.

[0035] In some embodiments, the transgene polynucleotide shares at least 95% identity with one of sequence numbers 35-38.

[0036] In some embodiments, the promoter sequence is selected from Table 5.

[0037] In some embodiments, the vector further includes a CMV enhancer.

[0038] In some embodiments, the vector further includes a 5' untranslated region (UTR) selected from Table 6.

[0039] In some embodiments, the vector further includes a 3' untranslated region selected from Table 7.

[0040] In some embodiments, the vector further comprises a polyadenylated sequence (Poly-A) selected from Table 8.

[0041] In some embodiments, polynucleotides are codon-optimized.

[0042] In some embodiments, the expression cassette shares at least 95% sequence identity with any one of sequence numbers 39-58.

[0043] In some embodiments, the vector is an adeno-associated virus (AAV) vector.

[0044] In some embodiments, the vector includes two AAV reverse terminal repeats (ITRs) adjacent to the expression cassette.

[0045] In some embodiments, the AAV has serotypes AAV1, AAV2, AAV5, AAV8, AAV9, AAVrh10, or AAVrh74.

[0046] In some embodiments, the recombinant gene therapy vector includes a self-complementary AAV.

[0047] In some embodiments, the recombinant gene therapy vector comprises a single-stranded AAV.

[0048] In some embodiments, AAV is either wild-type AAV or modified AAV.

[0049] In some embodiments, the AAV comprises a capsid protein having at least 95% identity with the wild-type VP1, VP2, or VP3 capsid protein.

[0050] In another embodiment, the disclosure provides a host cell containing one of the aforementioned recombinant gene therapy vectors.

[0051] In another aspect, the Disclosure provides a method for inhibiting the degeneration or death of dopaminergic neurons containing a mutation in a gene associated with Parkinson's disease (PD), wherein the mutated gene is the E3 ubiquitin protein ligase (PARK2) gene for Parkinson's protein 2, and the method comprises contacting the neuron with the recombinant gene therapy vector of the Disclosure, after which the neuron expresses wild-type protein.

[0052] In some embodiments, neurons express a reduced amount of alpha-synuclein and / or contain a reduced amount of Lewy bodies after contact with a recombinant gene therapy vector.

[0053] In some embodiments, neurons express reduced amounts of monoamine oxidase after contact with a recombinant gene therapy vector.

[0054] In some embodiments, neurons produce and / or release increased amounts of dopamine after contact with a recombinant gene therapy vector.

[0055] In some embodiments, neurons undergo increased mitophagy after contact with a recombinant gene therapy vector.

[0056] In some embodiments, neurons express a smaller amount of monoamine oxidase compared to the amount expressed in neurons not in contact with the recombinant gene therapy vector, and optionally, the smaller amount is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% lower than the amount expressed in neurons not in contact with the recombinant gene therapy vector.

[0057] In some embodiments, neurons produce and / or release an increased amount of dopamine compared to the amount of dopamine produced and / or released by neurons not in contact with the recombinant gene therapy vector, and optionally, the increased amount is at least 5%, 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 twice, at least three times, at least four times, at least five times, or at least ten times greater than the amount produced and / or released by neurons not in contact with the recombinant gene therapy vector.

[0058] In some embodiments, neurons receive an increased amount of autophagy compared to the amount of autophagy received by neurons not in contact with the recombinant gene therapy vector, and optionally, the increased amount is at least 5%, 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 twice, at least three times, at least four times, at least five times, or at least ten times greater than the amount received by neurons not in contact with the recombinant gene therapy vector.

[0059] In some embodiments, the neurons are primary tyrosine hydroxylase-positive neurons.

[0060] In some embodiments, neurons are generated from induced pluripotent stem cells prepared from cells obtained from subjects diagnosed with Parkinson's disease.

[0061] In another aspect, the Disclosure provides a method for treating or inhibiting the development of Parkinson's disease (PD) in a subject who has or is at risk of having PD, comprising administering the gene therapy vector of the Disclosure, wherein the administration of the recombinant gene therapy vector treats or inhibits the development of Parkinson's disease in the subject.

[0062] In some embodiments, PD is early-onset PD, and optionally, early-onset autosomal recessive PD.

[0063] In some embodiments, the subject has a mutation in the PARK2 gene.

[0064] In some embodiments, PARK2 includes the amino acid sequence shown in SEQ ID NO: 1.

[0065] In some embodiments, the administration step includes systemic, parenteral, intravenous, cerebral, cerebrospinal fluid, intrathecal, intracisional, intraputamen, intrahippocampal, intrastriatal, or intraventricular administration.

[0066] In some embodiments, the administration step includes intravenous, intracerebral, intraspinal, intrathecal, intracisional, intraputamental, intrahippocampal, intrastriatal, or intraventricular injection.

[0067] In some embodiments, the administration step includes intrathecal injection with a Trendelenburg incline.

[0068] In some embodiments, the administration step includes directly injecting the substance into the substantia nigra of the brain.

[0069] In some embodiments, the administration step includes introducing a recombinant gene therapy vector into the target brain or cerebrospinal fluid (CSF).

[0070] In some embodiments, a gene therapy vector of 1x10 9 ~1x10 14 vector genomes (vg / kg) per kilogram of the subject's body weight is administered to the subject.

[0071] In some embodiments, a gene therapy vector of 1x10 9 ~1x10 14 vector genomes (vg / kg) per kilogram of the subject's body weight is administered to the subject's brain.

[0072] In some embodiments, a gene therapy vector of 1x10 9 ~1x10 14 vector genomes (vg / kg) per kilogram of the subject's body weight is administered to the subject's CSF.

[0073] In some embodiments, a gene therapy vector of 1x10 7 ~1x10 9 vector genomes (vg / kg) per kilogram of the subject's body weight is administered to the subject.

[0074] In some embodiments, a total of 1×10 7 ~1×10 11 vector genomes is administered to the subject, for example, via direct injection into the putamen or substantia nigra.

[0075] In some embodiments, the subject is an adult.

[0076] In some embodiments, the subject is a child.

[0077] In some embodiments, the number of dopaminergic neurons in the subject after the administering step is greater than the number of dopaminergic neurons in the subject before the administering step.

[0078] In some embodiments, the dopamine level in the subject after the administration step is greater than the dopamine level in the subject before the administration step.

[0079] In some embodiments, the number of dopaminergic neurons in subjects treated by this method increases compared to the number of dopaminergic neurons in subjects not treated in this way.

[0080] In some embodiments, the dopamine levels in subjects treated by this method increase compared to the dopamine levels in subjects not treated in this manner.

[0081] In some embodiments, the level of dopamine in the substantia nigra of the subject treated by the method increases compared to the level of dopamine in the substantia nigra of the subject not treated in the same way.

[0082] In some embodiments, the level of PRKN in the target CSF after the administration step is greater than the level of PRKN in the target CSF before the administration step.

[0083] In some embodiments, the subject's Unified Parkinson's Disease Rating Scale (UPDRS) score prior to the administration step is improved compared to the subject's UPDRS score prior to the administration step.

[0084] In some embodiments, the level of PRKN in the CSF of subjects treated by this method is increased compared to the level of PRKN in the CSF of subjects not treated in this way.

[0085] In some embodiments, the UPDRS score of subjects treated by this method is improved compared to the UPDRS score of subjects not treated in this manner.

[0086] In some embodiments, the target neurons express a reduced amount of alpha-synuclein and / or contain a reduced amount of Lewy bodies after contact with the recombinant gene therapy vector.

[0087] [Invention 1001] The expression cassette includes an expression cassette containing a transgene encoding E3 ubiquitin protein ligase (PARK2), PTEN-induced putative kinase 1 (PINK1), protein deglycase DJ-1 (DJ-1), leucine-rich repeat kinase 2 (LRRK2), alpha-synuclein (SCNA), proto-oncogene c-Rel (c-Rel), ubiquitin-like modifier activator enzyme (ATG7), synaptic vesicle amine transporter (VMAT2), or glucocerebrosidase (GBA). The polynucleotides of the aforementioned transgene are operably linked to a promoter sequence that is active in eukaryotes. Recombinant gene therapy vector. [Invention 1002] The vector of the present invention 1001, wherein the introduced gene encodes PARK2, and the polynucleotide sequence of the introduced gene shares at least 95% identity with one of sequence numbers 35 to 38. [Invention 1003] A vector according to Invention 1001 or Invention 1002, wherein the promoter sequence is selected from Table 5. [Invention 1004] The vector according to any one of the invention 1001 to 1003, wherein the expression cassette further comprises a CMV enhancer. [Invention 1005] A vector according to any of invention 1001 to 1004, wherein the expression cassette further comprises a 5' untranslated region (UTR) selected from Table 6. [Invention 1006] A vector according to any one of the invention 1001 to 1006, wherein the expression cassette further comprises a 3' untranslated region selected from Table 7. [Invention 1007] A vector according to any of invention 1001 to 1006, wherein the expression cassette further comprises a polyadenylated sequence (PolyA) selected from Table 8. [Invention 1008] A vector according to any of the present invention 1001 to 1007, wherein the introduced gene is codon-optimized. [Invention 1009] A vector according to any of the present invention 1001 to 1008, wherein the expression cassette shares at least 95% sequence identity with any one of sequence numbers 39 to 58. [Invention 1010] The expression cassette, HuBA promoter, the aforementioned transgene, WPRE(x), and pAglobin-Oc; CMV promoter, TPL-eMLP enhancer, the aforementioned transgene, WPRE(r), and pAglobin-Oc; Syn promoter, the aforementioned transgene, WPRE(r), 3'UTR(globin), and pAGH-Bt; CBA promoter, the aforementioned transgene, and pAGH-Bt; EF1α promoter, the aforementioned transgene, and pA globin-Oc; HuBA promoter, the aforementioned transgene, R2V17, and pAGH-Bt; Syn promoter, the aforementioned transgene, WPRE(x), 3'UTR(globin), and pAGH-Hs; CaMKIIa promoter, the aforementioned transgene, WPRE(r), and pAGH-Hs; CMV promoter, TPL-eMLP5' enhancer, the aforementioned transgene, WPRE(r), and pAGH-Hs; HuBA promoter, the aforementioned transgene, and pAGH-Hs; CMV and TPL promoters, eMPL, the aforementioned transgenes, R2V17, 3'UTR (globin), and pAGH-Bt; EF1α promoter, the aforementioned transgene, WPRE(r), and pAGH-Bt; Syn promoter, the aforementioned transgene, R2V17, and pAglobin-Oc; CaMKIIa promoter, the aforementioned transgene, R2V17, and pAglobin-Oc; CBA promoter, the aforementioned transgene, WPRE(x), 3'UTR(globin), and pAGH-Hs; CBA promoter, the aforementioned transgene, 3'UTR(globin), and pAglobin-Oc; CaMKIIa promoter, the aforementioned transgene, R2V17, and pAGH-Bt; EF1α promoter, the aforementioned transgene, R2V17, 3'UTR (globin), and pAGH-Hs; CMV promoter, the aforementioned transgene, R2V17, 3'UTR (globin), and pAGH-Hs; or CMV promoter, the aforementioned transgene, and pAGH-Hs It includes them in the order from 5' to 3', Optionally, the introduced gene encodes PARK2. A vector according to any of invention 1001 to 1009. [Invention 1011] A vector according to any of the invention's 1001 to 1010, which is an adeno-associated virus (AAV) vector. [Invention 1012] A vector according to the present invention 1011, comprising two AAV reverse terminal repeats (ITRs) adjacent to the expression cassette. [Invention 1013] The vector of the present invention 1011 or 1012, wherein the AAV has serotype AAV1, AAV2, AAV5, AAV8, AAV9, AAVrh10, or AAVrh74. [Invention 1014] The recombinant gene therapy vector comprises a self-complementary AAV or a single-stranded AAV, as per any of invention 1011 to 1013. [Invention 1015] A vector according to any of invention 1011 to 1014, wherein the AAV is wild-type AAV or modified AAV. [Invention 1016] A vector according to any one of invention 1011 to 1015, wherein the AAV comprises a capsid protein having at least 95% identity with wild-type VP1, VP2, or VP3 capsid protein. [Invention 1017] A host cell containing any vector according to invention 1001 to 1016. [Invention 1018] A method for inhibiting the degeneration or death of dopaminergic neurons containing mutations in genes associated with Parkinson's disease (PD), This includes contacting the neuron with any recombinant gene therapy vector according to invention 1001 to 1016, After contact with the recombinant gene therapy vector, the neurons express PARK2, PINK1, DJ-1, LRRK2, SCNA, c-Rel, ATG7, VMAT2, or GBA. A method wherein the neuron contains a mutation in the PARK2 gene, and the expression cassette encodes PARK2. [Invention 1019] The method of the present invention 1018, wherein the neurons express a reduced amount of alpha-synuclein and / or contain a reduced amount of Lewy bodies after contact with the recombinant gene therapy vector. [Invention 1020] The method according to the present invention 1018 or 1019, wherein the neuron expresses a reduced amount of monoamine oxidase after contact with the recombinant gene therapy vector. [Invention 1021] Any method of the present invention 1018 to 1020, wherein the neuron produces and / or releases an increased amount of dopamine after contact with the recombinant gene therapy vector. [Invention 1022] A method according to any one of the present invention 1018 to 1021, wherein the neuron receives increased mitophagy after contact with the recombinant gene therapy vector. [Invention 1023] The neurons express a smaller amount of monoamine oxidase compared to the amount of monoamine oxidase expressed in neurons that have not been in contact with the recombinant gene therapy vector. Optionally, the lesser amount is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% lower than the amount expressed in the neurons that have not come into contact with the recombinant gene therapy vector. Any method described in 1018 to 1022 of this invention. [Invention 1024] The neurons produce and / or release an increased amount of dopamine compared to the amount of dopamine produced and / or released by neurons that are not in contact with the recombinant gene therapy vector. Optionally, the increased amount is at least 5%, 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 twice, at least three times, at least four times, at least five times, or at least ten times greater than the amount produced and / or released by the neurons not in contact with the recombinant gene therapy vector. Any method according to invention 1018 to 1023. [Invention 1025] The aforementioned neurons receive an increased amount of autophagy compared to the amount of autophagy received by neurons that are not in contact with the recombinant gene therapy vector. Optionally, the increased amount is at least 5%, 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 twice, at least three times, at least four times, at least five times, or at least ten times greater than the amount received by the neurons not in contact with the recombinant gene therapy vector. Any method according to invention 1018 to 1024. [Invention 1026] The method according to any one of items 1018 to 1025 of the present invention, wherein the neuron is a primary tyrosine hydroxylase-positive neuron. [Invention 1027] The method according to any one of items 1018 to 1026 of the present invention, wherein the neurons are generated from induced pluripotent stem cells prepared from cells obtained from a subject diagnosed with Parkinson's disease. [Invention 1028] A method for treating or inhibiting the onset of Parkinson's disease (PD) in a person who has or is at risk of having PD, This includes administering any of the vectors according to invention 1001 to 1016 to the subject, A method by which the administration of the recombinant gene therapy vector treats or inhibits the onset of Parkinson's disease in the subject. [Invention 1029] The method of the present invention 1028, wherein the PD is early-onset PD, and optionally, early-onset autosomal recessive PD. [Invention 1030] The method of the present invention 1028 or 1029, wherein the subject includes a mutation in the PARK2 gene. [Invention 1031] The method according to any one of the present invention 1028 to 1030, wherein PARK2 comprises the amino acid sequence shown in SEQ ID NO: 1. [Invention 1032] A method according to any one of the present invention 1028 to 1031, wherein the administration step includes systemic, parenteral, intravenous, cerebral, cerebrospinal fluid, intrathecal, intracisional, intraputamen, intrahippocampal, intrastriatal, or intraventricular administration. [Invention 1033] The method according to any one of items 1028 to 1031 of the present invention, wherein the administration step includes intravenous injection, injection into the cerebrum, cerebrospinal fluid, intrathecal cavity, intracisional cavity, intraputamen, intrahippocampus, intrastriatal cavity, or intraventricular cavity. [Invention 1034] The method of the present invention 1032, wherein the administration step includes intrathecal injection with a Trendelenburg incline. [Invention 1035] The method of the present invention 1032, wherein the administration step includes direct injection into the compact portion of the substantia nigra of the brain. [Invention 1036] Any method of the present invention 1028 to 1035, wherein the administration step includes introducing the recombinant gene therapy vector into the target brain or cerebrospinal fluid (CSF). [Invention 1037] 1 x 10 per kilogram of the aforementioned body weight 9 ~1x1014 A method according to any of items 1028 to 1036 of the present invention, wherein the gene therapy vector in the form of a vector genome (vg / kg) is administered to the subject. [Invention 1038] 1 x 10 per kilogram of the aforementioned body weight 9 ~1x10 14 A method according to any of items 1028 to 1036 of the present invention, wherein the gene therapy vector in the form of a vector genome (vg / kg) is administered to the brain of the subject. [Invention 1039] 1 x 10 per kilogram of the aforementioned body weight 9 ~1x10 14 A method according to any of items 1028 to 1036 of the present invention, wherein the gene therapy vector in the form of a vector genome (vg / kg) is administered to the target CSF. [Invention 1040] 1 x 10 per kilogram of the aforementioned body weight 7 ~1x10 9 A method according to any of items 1028 to 1036 of the present invention, wherein the gene therapy vector in the form of a vector genome (vg / kg) is administered to the subject. [Invention 1041] The method according to any of the present invention 1028 to 1040, wherein the subject is an adult. [Invention 1042] The method according to any of the present invention 1028 to 1040, wherein the subject is a child. [Invention 1043] The method according to any one of the present invention 1028 to 1042, wherein the number of dopaminergic neurons in the subject after the administration step is greater than the number of dopaminergic neurons in the subject before the administration step. [Invention 1044] A method according to any of the present invention 1028 to 1043, wherein the level of dopamine in the subject after the administration step is greater than the level of dopamine in the subject before the administration step. [Invention 1045] Any method of the present invention 1028 to 1044, wherein the number of dopaminergic neurons in a subject treated by the method is increased compared to the number of dopaminergic neurons in a subject not treated in the same manner. [Invention 1046] A method according to any one of the present invention 1028 to 1045, wherein the level of dopamine in a subject treated by the method is increased compared to the level of dopamine in a subject not treated in the same way. [Invention 1047] A method according to any one of the present invention 1028 to 1046, wherein the level of dopamine in the substantia nigra of a subject treated by the method is increased compared to the level of dopamine in the substantia nigra of a subject not treated in the same way. [Invention 1048] A method of the present invention, wherein the level of PRKN in the target CSF after the administration step is greater than the level of PRKN in the target CSF before the administration step. [Invention 1049] Any method 1028 to 1048 of the present invention, wherein the Unified Parkinson's Disease Rating Scale (UPDRS) score of the subject prior to the administration step is improved compared to the subject's UPDRS score prior to the administration step. [Invention 1050] Any method according to items 1028 to 1049 of the present invention, wherein the level of PRKN in the CSF of a subject treated by the method is increased compared to the level of PRKN in the CSF of a subject not treated in the same manner. [Invention 1051] Any method according to items 1028 to 1059 of the present invention, wherein the UPDRS score of a subject treated by the method is improved compared to the UPDRS score of a subject not treated in the same manner. [Invention 1052] Any method of the present invention 1028 to 1051, wherein the target neuron expresses a reduced amount of alpha-synuclein and / or contains a reduced amount of Lewy bodies after contact with the recombinant gene therapy vector. [Invention 1053] A method for inhibiting the degeneration or death of dopaminergic neurons containing mutations in genes associated with Parkinson's disease (PD), The aforementioned mutated genes are the Parkinson's protein 2 E3 ubiquitin protein ligase (PARK2) gene, the PTEN-induced putative kinase 1 (PINK1) gene, the protein deglycase DJ-1 (DJ-1) gene, the leucine-rich repeat kinase 2 (LRRK2) gene, the alpha-synuclein (SCNA) gene, the oncogene c-Rel (c-Rel) gene, the ubiquitin-like modifier activator enzyme (ATG7) gene, the synaptic vesicle amine transporter (VMAT2) gene, or the glucocerebrosidase (GBA) gene. This includes contacting the neuron with a recombinant gene therapy vector containing a polynucleotide encoding a wild-type protein or a functional variant or fragment thereof expressed by the wild-type version of the mutant gene, A method comprising the following steps: after contact with the recombinant gene therapy vector, the neuron expresses the wild-type protein or a functional variant or fragment thereof. [Invention 1054] The method of the present invention 1053, wherein the wild-type PARK2, PINK1, LRRK2, SCNA, c-Rel, ATG7, VMAT2, or GBA protein each contains the amino acid sequence shown in SEQ ID NOs: 1 to 9. [Invention 1055] The method of the present invention 1053, wherein the gene is the PARK2 gene, and the wild-type PARK2 protein contains the amino acid sequence shown in any of SEQ ID NOs: 10 to 17. [Invention 1056] The method of the present invention 1053, wherein the polynucleotides each include a sequence having at least 70%, 75%, 80%, 85%, 95%, or 99% identity with the PARK2, PINK1, LRRK2, SCNA, c-Rel, ATG7, VMAT2, or GBA polynucleotide sequences shown in SEQ ID NOs. 18-26. [Invention 1057] The method of the present invention 1053, wherein the gene is the PARK2 gene, and the polynucleotide comprises a sequence having at least 70%, 75%, 80%, 85%, 95%, or 99% identity with the PARK2 isoform polynucleotide sequence shown in any of SEQ ID NOs. 27 to 34. [Invention 1058] The method according to any one of the present invention 1053 to 1057, wherein the polynucleotide is codon-optimized. [Invention 1059] The method according to any one of the invention 1053 to 1058, wherein the recombinant gene therapy vector is recombinant adeno-associated virus (AAV). [Invention 1060] The method of the present invention 1059, wherein the AAV has serotype AAV1, AAV2, AAV5, AAV8, AAV9, AAVrh10, or AAVrh74. [Invention 1061] The method of the present invention 1059 or 1060, wherein the recombinant gene therapy vector comprises a self-complementary AAV. [Invention 1062] The method according to any one of the invention 1059 to 1061, wherein the recombinant gene therapy vector contains a single-stranded AAV. [Invention 1063] The method according to any one of the invention 1059 to 1062, wherein the AAV is wild-type AAV or modified AAV. [Invention 1064] The method according to any one of the invention 1059 to 1063, wherein the AAV comprises a capsid protein having at least 95% identity with wild-type VP1, VP2, or VP3 capsid protein. [Invention 1065] The recombinant gene therapy vector described above is as follows: a. Promoter sequences that are active in eukaryotes, b. The sequence encoding the wild-type protein or a functional fragment or variant thereof It contains a polynucleotide that has the elements in the order from 5' to 3', The sequence encoding the wild-type protein or a functional fragment or variant thereof is operably linked to the promoter sequence active in the eukaryote. Any method according to invention 1053 to 1064. [Invention 1066] The recombinant gene therapy vector, A neuron-specific promoter, optionally selected from the group consisting of 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) promoters. A ubiquitous promoter selected from the group consisting of CMV, CAG, UBC, PGK, EF1-alpha, GAPDH, SV40, HBV, and chicken beta-actin and human beta-actin promoters. enhancer, Intron, Poly-A signal, The sequence encoding the 2A peptide, and WPRE (Woodchuck hepatitis virus post-transcriptional regulatory element) Any method of the present invention 1053 to 1065, further comprising one or more of the above. [Invention 1067] Any method of the present invention 1053 to 1064, wherein the neurons express a reduced amount of alpha-synuclein and / or contain a reduced amount of Lewy bodies after contact with the recombinant gene therapy vector. [Invention 1068] A method according to any one of the present invention 1053 to 1067, wherein the neuron expresses a reduced amount of monoamine oxidase after contact with the recombinant gene therapy vector. [Invention 1069] Any method of the present invention 1053 to 1068, wherein the neuron produces and / or releases an increased amount of dopamine after contact with the recombinant gene therapy vector. [Invention 1070] A method according to any of the present invention 1053 to 1069, wherein the neuron receives increased mitophagy after contact with the recombinant gene therapy vector. [Invention 1071] The neurons express a smaller amount of monoamine oxidase compared to the amount of monoamine oxidase expressed in neurons that have not been in contact with the recombinant gene therapy vector. Optionally, the lesser amount is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% lower than the amount expressed in the neurons that have not come into contact with the recombinant gene therapy vector. Any method according to invention 1053 to 1070. [Invention 1072] The neurons produce and / or release an increased amount of dopamine compared to the amount of dopamine produced and / or released by neurons that are not in contact with the recombinant gene therapy vector. Optionally, the increased amount is at least 5%, 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 twice, at least three times, at least four times, at least five times, or at least ten times greater than the amount produced and / or released by the neurons not in contact with the recombinant gene therapy vector. Any method according to invention 1053 to 1071. [Invention 1073] The aforementioned neurons receive an increased amount of autophagy compared to the amount of autophagy received by neurons that are not in contact with the recombinant gene therapy vector. Optionally, the increased amount is at least 5%, 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 twice, at least three times, at least four times, at least five times, or at least ten times greater than the amount received by the neurons not in contact with the recombinant gene therapy vector. Any method according to invention 1053 to 1072. [Invention 1074] The method according to any one of the present invention 1053 to 1073, wherein the neuron is a primary tyrosine hydroxylase-positive neuron. [Invention 1075] The method according to any one of items 1053 to 1074 of the present invention, wherein the neurons are generated from induced pluripotent stem cells prepared from cells obtained from a subject diagnosed with Parkinson's disease. [Invention 1076] The polynucleotides include the wild-type Parkinson's protein 2 E3 ubiquitin protein ligase (PARK2) gene, the PTEN-induced putative kinase 1 (PINK1) gene, the protein deglycase DJ-1 (DJ-1) gene, the leucine-rich repeat kinase 2 (LRRK2) gene, the alpha-synuclein (SCNA) gene, the oncogene c-Rel (c-Rel) gene, the ubiquitin-like modifier-activating enzyme (ATG7) gene, the synaptic vesicle amine transporter (VMAT2) gene, or the glucocerebrosidase (GBA) gene, or a functional variant or fragment thereof. The polynucleotide is operably linked to an active promoter in eukaryotes, and neurons transduced by the recombinant gene therapy vector express the wild-type protein, or a functional variant or fragment thereof. Recombinant gene therapy vector. [Invention 1077] The recombinant gene therapy vector of the present invention 1076, wherein the wild-type PARK2, PINK1, LRRK2, SCNA, c-Rel, ATG7, VMAT2, or GBA protein each contains the amino acid sequence shown in SEQ ID NOs. 1 to 9. [Invention 1078] The recombinant gene therapy vector of the present invention 1076, wherein the gene is the PARK2 gene, and the wild-type PARK2 protein contains the amino acid sequence shown in any of SEQ ID NOs. 10 to 17. [Invention 1079] The recombinant gene therapy vector of the present invention 1076, wherein each of the polynucleotides contains a sequence having at least 70%, 75%, 80%, 85%, 95%, or 99% identity with the PARK2, PINK1, LRRK2, SCNA, c-Rel, ATG7, VMAT2, or GBA polynucleotide sequences shown in SEQ ID NOs. 18-26. [Invention 1080] The recombinant gene therapy vector of the present invention 1076, wherein the gene is the PARK2 gene, and the polynucleotide comprises a sequence having at least 70%, 75%, 80%, 85%, 95%, or 99% identity with the PARK2 isoform polynucleotide sequence shown in any of SEQ ID NOs. 27 to 34. [Invention 1081] The recombinant gene therapy vector of the present invention 1076, wherein the polynucleotide is codon-optimized. [Invention 1082] The recombinant gene therapy vector according to any of the inventions 1076 to 1081, wherein the recombinant gene therapy vector is recombinant adeno-associated virus (rAAV). [Invention 1083] The recombinant gene therapy vector of the present invention 1082, wherein the rAAV has serotype AAV1, AAV2, AAV5, AAV8, AAV9, AAVrh10, or AAVrh74. [Invention 1084] A recombinant gene therapy vector according to any of invention 1076 to 1083, comprising self-complementary AAV or single-stranded AAV. [Invention 1085] A recombinant gene therapy vector according to any of the invention items 1076 to 1083, wherein the AAV is wild-type AAV or modified AAV. [Invention 1086] A recombinant gene therapy vector according to any of invention 1076 to 1085, wherein the AAV comprises a capsid protein having at least 95% identity with wild-type VP1, VP2, or VP3 capsid protein. [Invention 1087] A method for treating or inhibiting the onset of Parkinson's disease (PD) in a person who has or is at risk of having PD, The method involves administering to the subject a recombinant gene therapy vector containing a polynucleotide sequence encoding wild-type Parkinson's protein 2 E3 ubiquitin protein ligase (PARK 2), PTEN-induced putative kinase 1 (PINK 1), protein deglycase DJ-1 (DJ-1), leucine-rich repeat kinase 2 (LRRK 2), alpha-synuclein (SCNA), proto-oncogene c-Rel (c-Rel), ubiquitin-like modifier activator enzyme (ATG7), synaptic vesicle amine transporter (VMAT 2), or glucocerebrosidase (GBA) protein or a functional variant or fragment thereof. A method by which the administration of the recombinant gene therapy vector treats or inhibits the onset of Parkinson's disease in the subject. [Invention 1088] The method of the present invention 1087, wherein the PD is early-onset PD, and optionally, early-onset autosomal recessive PD. [Invention 1089] The method of Invention 1087 or Invention 1088, wherein the subject includes a mutation in the PARK2 gene, PINK1 gene, LRRK2 gene, SCNA gene, c-Rel gene, ATG7 gene, VMAT2, or GBA gene. [Invention 1090] The method according to any one of the present invention 1087 to 1089, wherein the PARK2, PINK1, LRRK2, SCNA, c-Rel, ATG7, VMAT2, or GBA protein each contains the amino acid sequence shown in SEQ ID NOs. 1 to 9. [Invention 1091] The method according to any one of the present invention 1087 to 1089, wherein the sequence encodes the PARK2 gene, and the wild-type PARK2 protein contains the amino acid sequence shown in any of sequence numbers 10 to 17. [Invention 1092] The method according to any one of the invention 1087 to 1089, wherein the polynucleotide each comprises a sequence having at least 70%, 75%, 80%, 85%, 95%, or 99% identity with the PARK2, PINK1, LRRK2, SCNA, c-Rel, ATG7, VMAT2, or GBA polynucleotide sequences shown in SEQ ID NOs. 18 to 26. [Invention 1093] The method according to any one of the present invention 1087 to 1089, wherein the sequence encodes the PARK2 gene, and the polynucleotide comprises a sequence having at least 70%, 75%, 80%, 85%, 95%, or 99% identity with the PARK2 isoform polynucleotide sequence shown in any of SEQ ID NOs. 27 to 34. [Invention 1094] The method of any of the invention 1087 to 1093, wherein the sequence encoding the PARK2, PINK1, LRRK2, SCNA, c-Rel, ATG7, VMAT2, or GBA protein, or a functional variant or fragment thereof, is codon-optimized, and optionally the sequence includes any of SEQ ID NOs. 35 to 38. [Invention 1095] A recombinant adeno-associated virus (rAAV) is used in any of the methods described in 1087 to 1094 of this invention. [Invention 1096] The method of the present invention 1095, wherein the AAV has serotype AAV1, AAV2, AAV5, AAV8, AAV9, AAVrh10, or AAVrh74. [Invention 1097] The method of the present invention 1095 or 1096, wherein the recombinant gene therapy vector comprises a self-complementary AAV or a single-stranded AAV. [Invention 1098] The method according to any one of the invention 1095 to 1097, wherein the AAV is wild-type AAV or modified AAV. [Invention 1099] The method according to any one of the present invention 1095 to 1098, wherein the AAV comprises a capsid protein having at least 95% identity with wild-type VP1, VP2, or VP3 capsid protein. [Invention 1100] The recombinant gene therapy vector described above is as follows: a. Promoter sequences that are active in eukaryotes, b. The sequence encoding the wild-type protein or a functional fragment or variant thereof It contains a polynucleotide that has the elements in the order from 5' to 3', The sequence encoding the wild-type protein or a functional fragment or variant thereof is operably linked to the promoter sequence active in the eukaryote. Any method described in Invention 1095 to 1099. [Invention 1101] The recombinant gene therapy vector, A neuron-specific promoter, optionally selected from the group consisting of 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) promoters. A ubiquitous promoter selected from the group consisting of CMV, CAG, UBC, PGK, EF1-alpha, GAPDH, SV40, HBV, and chicken beta-actin and human beta-actin promoters. enhancer, Intron, Poly-A signal, The sequence encoding the 2A peptide, and The method of the present invention 1101, further comprising one or more WPREs (woodchuck hepatitis virus post-transcriptional regulatory elements). [Invention 1102] The recombinant gene therapy vector, HuBA promoter, the aforementioned transgene, WPRE(x), and pAglobin-Oc; CMV promoter, TPL-eMLP enhancer, the aforementioned transgene, WPRE(r), and pAglobin-Oc; Syn promoter, the aforementioned transgene, WPRE(r), 3'UTR(globin), and pAGH-Bt; CBA promoter, the aforementioned transgene, and pAGH-Bt; EF1α promoter, the aforementioned transgene, and pA globin-Oc; HuBA promoter, the aforementioned transgene, R2V17, and pAGH-Bt; Syn promoter, the aforementioned transgene, WPRE(x), 3'UTR(globin), and pAGH-Hs; CaMKIIa promoter, the aforementioned transgene, WPRE(r), and pAGH-Hs; CMV promoter, TPL-eMLP enhancer, the aforementioned transgene, WPRE(r), and pAGH-Hs; HuBA promoter, the aforementioned transgene, and pAGH-Hs; CMV and TPL promoters, eMPL, the aforementioned transgenes, R2V17, 3'UTR (globin), and pAGH-Bt; EF1α promoter, the aforementioned transgene, WPRE(r), and pAGH-Bt; Syn promoter, the aforementioned transgene, R2V17, and pAglobin-Oc; CaMKIIa promoter, the aforementioned transgene, R2V17, and pAglobin-Oc; CBA promoter, the aforementioned transgene, WPRE(x), 3'UTR(globin), and pAGH-Hs; CBA promoter, the aforementioned transgene, 3'UTR(globin), and pAglobin-Oc; CaMKIIa promoter, the aforementioned transgene, R2V17, and pAGH-Bt; EF1α promoter, the aforementioned transgene, R2V17, 3'UTR (globin), and pAGH-Hs; CMV promoter, the aforementioned transgene, R2V17, 3'UTR (globin), and pAGH-Hs; or CMV promoter, the aforementioned transgene, and pAGH-Hs It includes them in the order from 5' to 3', Optionally, the introduced gene encodes PARK2. The method of the present invention 1101. [Invention 1103] A method according to any of items 1087 to 1102 of the present invention, wherein the administration step includes systemic, parenteral, intravenous, cerebral, cerebrospinal fluid, intrathecal, intracisional, intraputamen, intrahippocampal, intrastriatal, or intraventricular administration. [Invention 1104] The method of the present invention 1103, wherein the administration step includes intravenous, intracerebral, intracerebrospinal, intramedullary, intracisional, intraputamental, intrahippocampal, intrastriatal, or intraventricular injection. [Invention 1105] The method of the present invention 1103, wherein the administration step includes intrathecal injection with a Trendelenburg incline. [Invention 1106] The method of the present invention 1103, wherein the administration step includes direct injection into the compact portion of the substantia nigra of the brain. [Invention 1107] Any method of the present invention 1087 to 1106, wherein the administration step includes introducing the recombinant gene therapy vector into the target brain or cerebrospinal fluid (CSF). [Invention 1108] 1 x 10 per kilogram of the aforementioned body weight 9 ~1x10 14 A method according to any of the present invention 1087 to 1107, wherein the gene therapy vector in the form of a vector genome (vg / kg) is administered to the subject. [Invention 1109] 1 x 10 per kilogram of the aforementioned body weight 9 ~1x10 14 A method according to any of the present invention 1087 to 1108, wherein the gene therapy vector in the form of a vector genome (vg / kg) is administered to the brain of the subject. [Invention 1110] 1 x 10 per kilogram of the aforementioned body weight 9 ~1x10 14 A method according to any of the present invention 1087 to 1108, wherein the gene therapy vector in the form of a vector genome (vg / kg) is administered to the target CSF. [Invention 1111] 1 x 10 per kilogram of the aforementioned body weight 7 ~1x10 9 A method according to any one of the present invention 1087 to 1110, wherein the gene therapy vector in the form of a vector genome (vg / kg) is administered to the subject. [Invention 1112] The method according to any of the present invention 1087 to 1111, wherein the subject is an adult. [Invention 1113] The method according to any of the invention 1087 to 1112, wherein the subject is a child. [Invention 1114] The method according to any one of the present invention 1087 to 1113, wherein the number of dopaminergic neurons in the subject after the administration step is greater than the number of dopaminergic neurons in the subject before the administration step. [Invention 1115] A method according to any of the present invention 1087 to 1114, wherein the level of dopamine in the subject after the administration step is greater than the level of dopamine in the subject before the administration step. [Invention 1116] Any method of the present invention 1087 to 1115, wherein the number of dopaminergic neurons in a subject treated by the method is increased compared to the number of dopaminergic neurons in a subject not treated in the same way. [Invention 1117] A method according to any one of the present invention 1087 to 1116, wherein the level of dopamine in a subject treated by the method is increased compared to the level of dopamine in a subject not treated in the same way. [Invention 1118] A method according to Invention 1087 or Invention 1117, wherein the level of dopamine in the substantia nigra of a subject treated by the method is increased compared to the level of dopamine in the substantia nigra of a subject not treated in the same way. [Invention 1119] A method according to any of the present invention 1087 to 1118, wherein the level of PRKN in the target CSF after the administration step is greater than the level of PRKN in the target CSF before the administration step. [Invention 1120] Any method 1087 to 1119 of the present invention, wherein the Unified Parkinson's Disease Rating Scale (UPDRS) score of the subject prior to the administration step is improved compared to the subject's UPDRS score prior to the administration step. [Invention 1121] Any method of the present invention 1087 to 1120, wherein the level of PRKN in the CSF of a subject treated by the method is increased compared to the level of PRKN in the CSF of a subject not treated in the same way. [Invention 1122] Any method 1087 to 1121 of the present invention, wherein the UPDRS score of a subject treated by the method is improved compared to the UPDRS score of a subject not treated in the same manner. [Invention 1123] Any method 1087 to 1112 of the present invention, wherein the target neuron expresses a reduced amount of alpha-synuclein and / or contains a reduced amount of Lewy bodies after contact with the recombinant gene therapy vector. [Invention 1124] A method for inhibiting the degeneration or death of dopaminergic neurons possessing a mutated parkin (PRKN) gene, a. Cas protein, or polynucleotide encoding a Cas protein; b. Guide RNA (gRNA); and c. Repair templates containing the functional Parkinson's protein 2 E3 ubiquitin protein ligase (PARK2) gene, PTEN-induced putative kinase 1 (PINK1) gene, protein deglycase DJ-1 (DJ-1) gene, leucine-rich repeat kinase 2 (LRRK2) gene, alpha-synuclein (SCNA) gene, proto-oncogene c-Rel (c-Rel) gene, ubiquitin-like modifier-activating enzyme (ATG7) gene, synaptic vesicle amine transporter (VMAT2) gene, or glucocerebrosidase (GBA) gene, or functional variants or fragments thereof. This includes bringing a gene editing system containing the neuron into contact with the neuron, A method comprising a gene editing system capable of repairing endogenous genes within a neuron or inserting functional genes into the genome of a neuron. [Invention 1125] The method of the present invention 1124, wherein at least one component of the gene editing system is delivered by recombinant AAV. [Invention 1126] The method of the present invention 1124, wherein the gene editing system is delivered by recombinant AAV. [Invention 1127] A gene editing system for cells, a. Cas protein, or polynucleotide encoding a Cas protein; b. Guide RNA (gRNA); and c. Repair templates containing the functional Parkinson's protein 2 E3 ubiquitin protein ligase (PARK2) gene, PTEN-induced putative kinase 1 (PINK1) gene, protein deglycase DJ-1 (DJ-1) gene, leucine-rich repeat kinase 2 (LRRK2) gene, alpha-synuclein (SCNA) gene, proto-oncogene c-Rel (c-Rel) gene, ubiquitin-like modifier-activating enzyme (ATG7) gene, synaptic vesicle amine transporter (VMAT2) gene, or glucocerebrosidase (GBA) gene, or functional variants or fragments thereof. Includes, The gene editing system is capable of repairing endogenous genes within the cell or inserting functional genes into the cell's genome. Gene editing system. [Invention 1128] The gene editing system of the present invention 1127, wherein at least one component of the gene editing system is delivered by recombinant AAV. [Invention 1129] A gene editing system according to the present invention 1127, delivered by recombinant AAV. [Invention 1130] The gene editing system according to any of the invention 1127 to 1129, wherein the aforementioned cells are ex vivo neurons. [Invention 1131] The aforementioned cells are the target cells, and the gene editing system is one of the inventions 1127 to 1130. Other features and advantages of the present invention are evident from and encompassed by the following detailed description and claims. [Brief explanation of the drawing]

[0088] A patent or application file must include at least one drawing, drawn in color. A copy of the published patent or patent application containing the color drawing(s) will be provided by the Office upon request and payment of the necessary fees. [Figure 1] One embodiment of the expression cassette of this disclosure is shown. [Figure 2-1] Representative micrographs of gene expression in untransfection-negative (Figure 2A) and transfection-positive (Figure 2B) SH-SY5Y cells are shown. Expression cassettes were tested in dual: WT (Figure 2C and Figure 2D), CO1 (Figure 2E and Figure 2F), CO2 (Figure 2G and Figure 2H), and CO3 (Figure 2I and Figure 2J). [Figure 2-2] Figure 2K shows the percentage of GFP+ cells. [Figure 2-3] Figure 2L shows the fluorescence intensity of GFP+ cells. [Figure 3] The results of an enzyme-linked immunosorbent assay (ELISA) of parkin performed on cell lysates from transfected cells are shown. [Figure 4-1] Representative micrographs of gene expression in untransfection-negative control (Figure 4A), WT (Figure 4B), CO1 (Figure 4C), or CO4 (Figure 4D) SH-SY5Y cells are shown. [Figure 4-2] Figure 4E shows the percentage of GFP+ cells. Figure 4F shows the fluorescence intensity of GFP+ cells. [Figure 5]The results of an enzyme-linked immunosorbent assay (ELISA) of parkin performed on cell lysates from transfected cells are shown. [Figure 6-1] Representative micrographs of gene expression in induced pluripotent stem cell (iPSC)-derived parkin knockout dopaminergic progenitor cells are shown, representing untransfection-negative controls (Figure 6A), wild-type cells (WT) (Figure 6B), CO1 (Figure 6C), or CO4 (Figure 6D). [Figure 6-2] Figure 6E shows the percentage of GFP+ cells and the fluorescence intensity of GFP+ cells. For WT, CO1, and CO$, the bar on the left shows the percentage change in GFP intensity relative to WT, and the bar on the right shows the percentage of GFP+ cells. [Figure 7] One embodiment of the AAV vector expression cassette of this disclosure is shown. [Figure 8] A diagram of the transgene cassette and its various components is shown. [Figure 9] Table 10 shows the parkin transgene expression in SH-SY5Y cells for each of the constructs listed. [Figure 10] Table 11 shows the expression of the parkin transgene in iPSC-derived parkin knockout dopaminergic progenitor cells for each of the constructs listed. [Figure 11A] Representative fluorescence micrographs of iPSC-derived parkin knockout dopaminergic progenitor cells transfected with each of the constructs listed in Table 11, imaged by immunofluorescence for bright-field microscopy (Figure 11A), parkin (Figure 11B), the neuron marker NeuN (Figure 11C), or the astrocyte marker GFAP (Figure 11D), are shown. [Figure 11B] See the explanation in Figure 11A. [Figure 11C] See the explanation in Figure 11A. [Figure 11D] See the explanation in Figure 11A. [Figure 12A] An enlarged image of Figure 11A is shown. [Figure 12B] An enlarged image of Figure 11B is shown. [Figure 12C] An enlarged image of Figure 11C is shown. [Figure 12D] An enlarged image of Figure 11D is shown. [Figure 13] An example diagram of a structure design is provided. [Modes for carrying out the invention]

[0089] Detailed explanation This disclosure provides, in part, compositions and methods for treating, preventing, inhibiting, or delaying central nervous system degeneration in the treatment of Parkinson's disease, for example. More specifically, the inventors disclose various embodiments of recombinant gene therapy vectors comprising the Parkinson's protein 2 E3 ubiquitin protein ligase (PARK 2) gene, the PTEN-induced putative kinase 1 (PINK 1) gene, the protein deglycase DJ-1 (DJ-1) gene, the leucine-rich repeat kinase 2 (LRRK 2) gene, the alpha-synuclein (SCNA) gene, the proto-oncogene c-Rel (c-Rel) gene, the ubiquitin-like modifier-activating enzyme (ATG7) gene, the synaptic vesicle amine transporter (VMAT 2) gene, or the glucocerebrosidase (GBA) gene, or functional fragments or variants thereof. As used herein, the terms “gene” and “transgene” are interchangeable and refer to polynucleotide sequences encoding polypeptides or proteins, for example, any of the proteins disclosed in Table 1.

[0090] This disclosure further includes various embodiments of a gene editing system comprising Cas protein; guide RNA; the Parkinson's protein 2 E3 ubiquitin protein ligase (PARK 2) gene, the PTEN-induced putative kinase 1 (PINK 1) gene, the protein deglycase DJ-1 (DJ-1) gene, the leucine-rich repeat kinase 2 (LRRK 2) gene, the alpha-synuclein (SCNA) gene, the oncogene c-Rel (c-Rel) gene, the ubiquitin-like modifier-activating enzyme (ATG7) gene, the synaptic vesicle amine transporter (VMAT 2) gene, or the glucocerebrosidase (GBA) gene, or a repair template comprising a functional fragment or variant thereof.

[0091] This disclosure further includes methods for inhibiting the degeneration or death of dopaminergic neurons having a mutated PARK2 gene, and methods for treating or inhibiting the onset or progression of Parkinson's disease in subjects who have or are at risk of having Parkinson's disease. In certain embodiments, Parkinson's disease is early-onset or juvenile Parkinson's disease. In certain embodiments, it is associated with or caused by, for example, an autosomal recessive mutation in the subject's PARK2, PINK 1, DJ-1, LRRK2, SCNA, c-Rel, ATG7, VMAT2, or GBA gene. In some embodiments, a viral vector, for example, adeno-associated virus (AAV), is used to deliver the recombinant gene therapy construct to the subject's body or more specifically, the brain. Recombinant gene therapy vectors or gene editing systems for PARK2, PINK 1, DJ-1, LRRK2, SCNA, c-Rel, ATG7, VMAT2, or GBA are also provided. Table 1 provides protein sequences. These genes are expressed as various isoforms. For example, without limiting this disclosure to PARK2, isoforms of PARK2 are provided in Table 2. Table 3 provides polynucleotide sequences. Table 4 provides polynucleotide sequences of isoforms of PARK2. This disclosure provides compositions and methods comprising or encoding either isoforms as proteins or polynucleotides, including codon-optimized polynucleotides and spliced ​​or unspliced ​​variants.

[0092] (Table 1) Non-limited examples of genes related to CNS degradation TIFF0007869633000001.tif246166TIFF0007869633000002.tif239166TIFF0007869633000003.tif238166 TIFF0007869633000004.tif239166TIFF0007869633000005.tif239166TIFF0007869633000006.tif224166

[0093] (Table 2) PARK2 isoforms TIFF0007869633000007.tif225166TIFF0007869633000008.tif232166TIFF0007869633000009.tif239166TIFF0007869633000010.tif37166

[0094] (Table 3) Polynucleotides of genes in non-specific examples related to CNS degradation TIFF0007869633000011.tif181166TIFF0007869633000012.tif239166TIFF0007869633000013.tif239166TIFF0007869633000014.tif239166TIFF000 7869633000015.tif238166TIFF0007869633000016.tif238166TIFF0007869633000017.tif238166TIFF0007869633000018.tif238166TIFF0007869633 000019.tif238166TIFF0007869633000020.tif239166TIFF0007869633000021.tif238166TIFF0007869633000022.tif239166TIFF0007869633000023. tif238166TIFF0007869633000024.tif239166TIFF0007869633000025.tif239166TIFF0007869633000026.tif238166TIFF0007869633000027.tif66166

[0095] (Table 4) Polynucleotides of PARK2 isoforms TIFF0007869633000028.tif160166TIFF0007869633000029.tif239166TIFF0007869633000030.tif239166TIFF0007869633000031.tif239166 TIFF0007869633000032.tif238166TIFF0007869633000033.tif239166TIFF0007869633000034.tif239166TIFF0007869633000035.tif225166

[0096] In certain embodiments, the compositions and methods disclosed herein intend to use functional variants and functional fragments of any of these proteins or polynucleotide sequences. The functional fragments and variants retain the biological properties or activity of the corresponding wild-type protein, but in certain cases, the properties or activity may be reduced to, for example, about 50%, about 60%, about 70%, or about 80% compared to the wild-type protein, or increased to, for example, 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 2 times, 3 times, 4 times, or 5 times compared to the wild-type protein. In certain embodiments, the functional fragment or variant of the protein has at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with the corresponding wild-type protein. In certain embodiments, the functional fragment of the protein comprises 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 95%, at least 98%, or at least 99% of the corresponding wild-type protein.

[0097] In another embodiment, the disclosure provides a recombinant gene therapy vector comprising a transgene polynucleotide encoding an E3 ubiquitin protein ligase (PARK2), the transgene polynucleotide being operably linked to a promoter sequence active in eukaryotes. In some embodiments, the transgene polynucleotide shares at least 95% identity with one of sequence numbers 35-38.

[0098] In some embodiments, this disclosure provides codon-optimized polynucleotides encoding PARK2. In some cases, the entire transgene sequence is codon-optimized for expression in mammalian cells. Codon optimization refers to the bias in the frequency of synonymous codons (i.e., codons encoding the same amino acid) in the encoding DNA across different species. Such codon denaturation allows the same polypeptide to be encoded by various nucleotide sequences. Various codon optimization methods are known in the art and include, for example, the methods disclosed in U.S. Patents No. 5,786,464 and No. 6,114,148.

[0099] In some embodiments, the codon-optimized polynucleotide encoding PARK2 contains fewer CpG islands than the natural human polynucleotide sequence encoding human PARK2. For example, in some embodiments, the natural human sequence contains 95 CpG islands, while the codon-optimized polynucleotide contains or does not contain 95, less than 90, less than 85, less than 80, less than 75, less than 70, less than 65, less than 60, less than 55, less than 50, less than 45, less than 40, less than 35, less than 30, less than 25, less than 20, less than 15, less than 10, or less than 5 CpG islands. In some embodiments, the codon-optimized polynucleotide sequence contains 2 to 20, 5 to 20, about 5, or about 10 CpG islands. In some embodiments, the codon-optimized polynucleotide sequence contains one or more CpG islands. In some embodiments, the expression cassette shares at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any one of sequence numbers 39–58. In some embodiments, the expression cassette contains, essentially consists of, or comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with any one of sequence numbers 39–58.

[0100] In some embodiments, the vector is an adeno-associated virus (AAV) vector. In some embodiments, the vector includes two AAV reverse terminal repeats (ITRs) adjacent to the expression cassette. In some embodiments, the AAV has serotype AAV1, AAV2, AAV5, AAV8, AAV9, AAVrh10, or AAVrh74. In some embodiments, the recombinant gene therapy vector includes a self-complementary AAV. In some embodiments, the recombinant gene therapy vector includes a single-stranded AAV. In some embodiments, the AAV is wild-type AAV or modified AAV. In some embodiments, the AAV includes a capsid protein having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with wild-type VP1, VP2, or VP3 capsid protein.

[0101] In another aspect, the disclosure provides host cells containing one of the aforementioned recombinant gene therapy vectors. Exemplary host cells include HEK293, 293T, HeLa, Vero, and Sf9 cells.

[0102] In another aspect, the Disclosure provides a method for inhibiting, reducing, or delaying the degeneration or death of dopaminergic neurons containing a mutation in a gene associated with Parkinson's disease (PD), wherein the mutated gene is the E3 ubiquitin protein ligase (PARK2) gene for Parkinson's protein 2, and the method comprises contacting the neuron with the recombinant gene therapy vector of the Disclosure, after which the neuron expresses wild-type protein. The method can be practiced in vitro or in vivo, for example, in subjects where it is needed.

[0103] In some embodiments, neurons express a reduced amount of alpha-synuclein and / or contain a reduced amount of Lewy bodies after contact with a recombinant gene therapy vector. In some embodiments, the reduction of alpha-synuclein and / or the reduced amount of Lewy bodies is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, or at least 40% or more.

[0104] In some embodiments, neurons express a reduced amount of monoamine oxidase after contact with a recombinant gene therapy vector. In some embodiments, the amount of monoamine oxidase is reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, or at least 40% or more.

[0105] In some embodiments, neurons produce and / or release increased amounts of dopamine after contact with a recombinant gene therapy vector. In some embodiments, the amount of dopamine produced and / or released increases by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, or at least 40% or more.

[0106] In some embodiments, neurons undergo increased mitophagy after contact with a recombinant gene therapy vector. In some embodiments, mitophagy is increased by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, or at least 40% or more.

[0107] In some embodiments, neurons express a smaller amount of monoamine oxidase compared to the amount expressed in neurons not in contact with the recombinant gene therapy vector, and optionally, the smaller amount is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% lower than the amount expressed in neurons not in contact with the recombinant gene therapy vector.

[0108] In some embodiments, neurons produce and / or release an increased amount of dopamine compared to the amount of dopamine produced and / or released by neurons not in contact with the recombinant gene therapy vector, and optionally, the increased amount is at least 5%, 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 twice, at least three times, at least four times, at least five times, or at least ten times greater than the amount produced and / or released by neurons not in contact with the recombinant gene therapy vector.

[0109] In some embodiments, neurons receive an increased amount of autophagy compared to the amount of autophagy received by neurons not in contact with the recombinant gene therapy vector, and optionally, the increased amount is at least 5%, 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 twice, at least three times, at least four times, at least five times, or at least ten times greater than the amount received by neurons not in contact with the recombinant gene therapy vector.

[0110] In some embodiments, the neurons are primary tyrosine hydroxylase-positive neurons. In some embodiments, the neurons are generated from induced pluripotent stem cells prepared from cells obtained from subjects diagnosed with Parkinson's disease.

[0111] In another aspect, the Disclosure provides a method for treating or inhibiting or delaying the onset or progression of Parkinson's disease (PD) in a subject who has or is at risk of having PD, comprising administering the gene therapy vector of the Disclosure to the subject, wherein the administration of the recombinant gene therapy vector treats or inhibits or delays the onset or progression of Parkinson's disease in the subject.

[0112] In some embodiments, PD is early-onset PD. In some embodiments, PD is early-onset autosomal recessive PD. In some embodiments, the subject has a mutation in the PARK2 gene.

[0113] In some embodiments, the gene therapy vector comprises an expression cassette containing a transgene encoding PARK2 or a functional fragment or variant thereof. In some embodiments, PARK2 comprises an amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto. In some embodiments, the transgene polynucleotide shares at least 95% identity with one of SEQ ID NOs: 35-38.

[0114] In some embodiments, the administration step includes systemic, parenteral, intravenous, cerebral, cerebrospinal fluid, intrathecal, intracisional, intraputamen, intrahippocampal, intrastriatal, or intraventricular administration.

[0115] In some embodiments, the administration step includes intravenous, intracerebral, intraspinal, intrathecal, intracisional, intraputamental, intrahippocampal, intrastriatal, or intraventricular injection.

[0116] In some embodiments, the administration step includes intrathecal injection with a Trendelenburg incline.

[0117] In some embodiments, the administration step includes directly injecting the substance into the substantia nigra of the brain.

[0118] In some embodiments, the administration step includes introducing a recombinant gene therapy vector into the target brain or cerebrospinal fluid (CSF).

[0119] In some embodiments, 1 x 10 per kilogram of the subject's body weight 9 ~1x10 14 The gene therapy vector (vg / kg) of the vector genome is administered to the target.

[0120] In some embodiments, 1 x 10 per kilogram of the subject's body weight 9 ~1x10 14 The gene therapy vector (vg / kg) of the vector genome is administered to the target brain.

[0121] In some embodiments, 1 x 10 per kilogram of the subject's body weight 9 ~1x10 14 The gene therapy vector (vg / kg) of the vector genome is administered to the target CSF.

[0122] In some embodiments, 1 x 10 per kilogram of the subject's body weight 7 ~1x10 9 The gene therapy vector (vg / kg) of the vector genome is administered to the target.

[0123] In some embodiments, the subjects are adults or children.

[0124] In some embodiments, the number of dopaminergic neurons in the subject after the administration step is greater than the number of dopaminergic neurons in the subject before the administration step.

[0125] In some embodiments, the dopamine level in the subject after the administration step is greater than the dopamine level in the subject before the administration step.

[0126] In some embodiments, the number of dopaminergic neurons in subjects treated by this method increases compared to the number of dopaminergic neurons in subjects not treated in this way.

[0127] In some embodiments, the dopamine levels in subjects treated by this method increase compared to the dopamine levels in subjects not treated in this manner.

[0128] In some embodiments, the level of dopamine in the substantia nigra of the subject treated by the method increases compared to the level of dopamine in the substantia nigra of the subject not treated in the same way.

[0129] In some embodiments, the level of PRKN in the target CSF after the administration step is greater than the level of PRKN in the target CSF before the administration step.

[0130] In some embodiments, the subject's Unified Parkinson's Disease Rating Scale (UPDRS) score prior to the administration step is improved compared to the subject's UPDRS score prior to the administration step.

[0131] In some embodiments, the level of PRKN in the CSF of subjects treated by this method is increased compared to the level of PRKN in the CSF of subjects not treated in this way.

[0132] In some embodiments, the UPDRS score of subjects treated by this method is improved compared to the UPDRS score of subjects not treated in this manner.

[0133] In some embodiments, the target neurons express a reduced amount of alpha-synuclein and / or contain a reduced amount of Lewy bodies after contact with the recombinant gene therapy vector.

[0134] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art in which the invention relates. Methods and materials similar to or equivalent to those described herein may be used in the practice of the invention, but suitable methods and materials are listed below. All publications, patent applications, patents, and other references made herein are expressly incorporated by reference in their entirety. In case of any conflict, this specification, including its definitions, shall prevail. Furthermore, the materials, methods, and examples described herein are illustrative and not intended to be limiting.

[0135] All publications and patents referenced herein are incorporated herein by reference in whole, as if each individual publication or patent were incorporated by reference in detail and individually. In case of any conflict, this application, including any definitions herein, shall prevail. However, any reference, article, publication, patent, patent publication, and patent application cited herein shall not be recognized, nor should be recognized, as constituting valid prior art or being part of common knowledge in any country of the world, or as any form of suggestion.

[0136] 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 (such as one-tenth and one-hundredth of an integer). The term “approximately” when preceding a number or numerical value means that the range of the number or numerical value is plus or minus 10%. The terms “a” and “an” as used herein should be understood to mean “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 term “and / or” should be understood to mean either one or both of the substitutes. As used herein, the terms “include” and “comprise” are used synonymously.

[0137] The abbreviations PRKN and PARK2 are used interchangeably in this specification.

[0138] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0139] Adeno-associated virus (AAV) As used herein, the term "AAV" is a standard abbreviation for adeno-associated virus or its recombinant vector. Adeno-associated viruses are single-stranded DNA parvoviruses that replicate only in cells where specific functions are provided by co-infecting helper viruses. General information and reviews of AAVs can be found, for example, in Carter, 1989, Handbook of Parvoviruses, Vol. 1, pp. 169–228, and Berns, 1990, Virology, pp. 1743–1764, Raven Press, (New York). It is fully expected that the same principles described in these reviews will apply to additional AAV serotypes characterized after the publication date of the reviews, as it is well known that various serotypes are quite closely related structurally and functionally, even at the genetic level. (See, for example, Blacklowe, 1988, pp. 165-174 Parvoviruses and Human Disease, JR Pattison, ed., and Rose, Comprehensive Virology 3:1-61 (1974)). For example, all AAV serotypes clearly exhibit very similar replication characteristics mediated by homologous rep genes, and all possess three related capsid proteins, such as the one expressed in AAV2. The degree of relatedness is further suggested by heteroduplex analysis revealing extensive cross-hybridization between serotypes along genome length and the presence of similar self-annealing segments at the ends corresponding to “reverse terminal repeat sequences” (ITRs). Similar infectivity patterns also suggest that replication function in each serotype is under similar control.

[0140] As used herein, “AAV vector” or “rAAV vector” refers to a recombinant vector containing one or more polynucleotides (or transgenes) of interest flanked by an AAV terminal repeat sequence (ITR). Such an AAV vector can replicate and package into infectious viral particles when present in host cells transfected with plasmids encoding and expressing rep and cap gene products. Alternatively, an AAV vector can be packaged into infectious particles using host cells that have been stably engineered to express rep and cap genes.

[0141] As used herein, “AAV virion,” “AAV virus particle,” or “AAV vector particle” refers to a virus particle consisting of 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 typically referred to as an “AAV vector particle,” or simply an “AAV vector.” Thus, since such a vector is contained within the AAV vector particle, the production of an AAV vector particle necessarily includes the production of an AAV vector.

[0142] Adeno-associated virus (AAV) is a replication-deficient parvovirus whose single-stranded DNA genome is approximately 4.7 kb long and contains two 145-nucleotide reverse-end repeats (ITRs). There are several known variants of AAV, also called serotypes, when classified by antigenic epitopes. The nucleotide sequences of the genomes of AAV serotypes are known. For example, the complete genome of AAV-1 is provided to GenBank accession number NC_002077, the complete genome of AAV-2 is provided to GenBank accession number NC_001401 and Srivastava et al., J. Virol., 45:555-564 (1983), the complete genome of AAV-3 is provided to GenBank accession number NC_1829, the complete genome of AAV-4 is provided to GenBank accession number NC_001829, the genome of AAV-5 is provided to GenBank accession number AF085716, the complete genome of AAV-6 is provided to GenBank accession number NC_001862, at least portions of the genomes of AAV-7 and AAV-8 are provided to GenBank accession numbers AX753246 and AX753249 respectively, and the genome of AAV-9 is provided to Gao et al. The AAV-10 genome is provided in al., J. Virol., 78:6381-6388 (2004), the AAV-11 genome is provided in Mol. Ther., 13(1):67-76 (2006), and the AAV-11 genome is provided in Virology, 330(2):375-383 (2004). The sequence of the AAVrh.74 genome is provided in U.S. Patent No. 9,434,928, incorporated herein by reference. Cis-acting sequences directing viral DNA replication (rep), inclusion / packaging, and host cell chromosome integration are contained within the AAV ITR. Three AAV promoters (named p5, p19, and p40 with respect to 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) combine with differential splicing of a single AAV intron (at nucleotides 2107 and 2227) to produce four rep proteins (rep78, rep68, rep52, and rep40) from the rep gene. The rep proteins possess multiple enzymatic properties that ultimately contribute to the replication of the viral genome. The cap gene is expressed from the p40 promoter and encodes three capsid proteins: VP1, VP2, and VP3. Alternative splicing and non-consensus translation initiation sites are 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).

[0143] AAV possesses unique and attractive properties as a vector for delivering foreign DNA to cells, such as in gene therapy. AAV infection of cells in culture is non-cytotoxic, and natural infection in humans and other animals is silent and asymptomatic. Furthermore, AAV infects many mammalian cells, offering the potential to target many different tissues in vivo. Additionally, AAV can transduce slowly dividing and non-dividing cells and persist essentially throughout the lifespan of these cells as a transcriptionally active nuclear episome (extrachromosomal element). The AAV proviral genome can be inserted as cloned DNA into plasmids, making the construction of recombinant genomes feasible. Moreover, because signals directing AAV replication and genomic capsid formation 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 proteins, rep-cap) can be replaced with foreign DNA. To generate AAV vectors, the rep and cap proteins can be supplied in trans. Another significant characteristic of AAV is its extremely stable and vigorous nature. AAV readily withstands the conditions used to inactivate adenoviruses (56°C to 65°C for several hours), reducing the risks of refrigerated storage of AAV. AAV may also be freeze-dried. Finally, AAV-infected cells are not resistant to co-infection.

[0144] The AAV DNA in the rAAV genome may originate from any AAV variant or serotype from which recombinant viruses can be induced, including, but not limited to, AAV variants or serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, and AAVrh10. The production of pseudotyped rAAV is disclosed, for example, in WO 01 / 83692. Other types of rAAV variants, e.g., rAAV with capsid mutations, are also considered. See, for example, Marsic et al., Molecular Therapy, 22(11):1900-1909 (2014). Nucleotide sequences of genomes of various AAV serotypes are known in the art. AAV6, AAV8, or AAV9 may be used to promote eye-specific expression.

[0145] 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 is configured to form an intramolecular double-stranded DNA template, as described in McCarty et al. Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis. Gene Therapy. 8(16):1248-54 (2001). This disclosure intends to use rAAVs containing a self-complementary genome in which, in some cases, at the time of infection (such transduction), the two complementary halves of the scAAV combine to form a single double-stranded DNA (dsDNA) unit that is immediately ready for replication and transcription, rather than waiting for cell-mediated synthesis of the second strand of the rAAV genome. It's important to understand that, unlike rAAVs (4.7-6kb) which possess full coding capabilities, rAAVs containing a self-complementary genome can only hold about half that amount (approximately 2.4kb).

[0146] In other cases, rAAV vectors contain a single-stranded genome. Where 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 are several indicators that rAAVs should be scAAVs to achieve efficient transduction of cells such as ocular cells. However, this disclosure intends for rAAV vectors that may have a single-stranded genome rather than a self-complementary genome, with 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).

[0147] In some cases, the rAAV vector is of serotype AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, or AAVrh10. The production of pseudotype 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 of serotype AAV9. In some embodiments, the rAAV vector is of serotype AAV9 and contains a single-stranded genome. In some embodiments, the rAAV vector is of serotype AAV9 and contains a self-complementary genome. In some embodiments, the rAAV vector contains an AAV2 reverse terminal repeat (ITR) sequence. In some embodiments, the rAAV vector contains an AAV2 genome such that the rAAV vector is an AAV-2 / 9 vector, an AAV-2 / 6 vector, or an AAV-2 / 8 vector.

[0148] The full-length and sequenced versions of the most well-known AAV capsid gene are provided in U.S. Patent No. 8,524,446, which are incorporated herein by reference in their entirety.

[0149] AAV vectors may contain a wild-type AAV sequence, or they may contain one or more modifications to the wild-type AAV sequence. In certain embodiments, the AAV vector includes one or more amino acid modifications, e.g., substitutions, deletions, or insertions, within the capsid protein, e.g., VP1, VP2, and / or VP3. In certain embodiments, the modifications provide reduced immunogenicity when the AAV vector is provided to a subject.

[0150] promoter In some embodiments, polynucleotide sequences encoding wild-type PARK2, PINK1, LRRK2, SCNA, c-Rel, ATG 7, VMAT2, or GBA proteins or their functional variants or fragments are operably ligated to the CMV promoter. This disclosure further intends to explore the use of other promoter sequences. Useful promoters in embodiments of this disclosure include, but are not limited to, cytomegalovirus (CMV) promoters, phosphoglycate kinase (PGK) promoters, or CMV enhancers, as well as promoter sequences consisting of chicken beta-actin promoters and portions of the rabbit beta-globin gene (CAG). In some cases, the promoter may be a synthetic promoter. Exemplary synthetic promoters are provided in Schlabach et al. Synthetic design of strong promoters. Proc Natl Acad Sci US A. 2010 Feb 9;107(6):2538-2543.

[0151] In some cases, a polynucleotide sequence encoding a therapeutic protein or a functional variant or fragment of the wild-type PARK2, PINK1, LRRK2, SCNA, c-Rel, ATG7, VMAT2, or GBA protein is operably ligated to an inductive promoter. The polynucleotide sequence operably ligated to the inductive promoter may be configured to transcribe or de-transcribe in response to the addition or accumulation of a drug, or to the removal, degradation, or dilution of a drug. The drug may be a pharmacologic agent. The drug may be one of tetracyclines or their derivatives, including but not limited to doxycyclines. In some cases, the inductive promoter is a tet-on promoter, a tet-off promoter, a chemically modulated promoter, or a physically modulated promoter (i.e., a promoter that responds to the presence or absence of light, or to low or high temperatures). This list of inductive promoters is non-exclusive.

[0152] As used herein, “eukaryotically active promoter” or “promoter” is used interchangeably and refers to a promoter capable of promoting the initiation of RNA transcription from polynucleotides within eukaryotic cells. In some cases, the promoter is a tissue-specific promoter, such as a promoter capable of driving expression in neurons over a wider range than in non-neuronal cells. In some embodiments, the tissue-specific promoter is selected from a list of neuron-specific promoters consisting of 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. “Ubiquitous promoter” refers to a promoter that is not tissue-specific under experimental or clinical conditions. In some cases, the ubiquitous promoter is selected from the group consisting of CMV, CAG, UBC, PGK, EF1-alpha, GAPDH, SV40, HBV, chicken beta-actin, and human beta-actin.

[0153] In some embodiments, the promoter sequence is selected from Table 5 and has at least 95%, at least 98%, or at least 99% identity with it.

[0154] (Table 5) TIFF0007869633000036.tif218166TIFF0007869633000037.tif232166

[0155] 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 containing LTR elements. Inducible promoters include heavy metal ion-inducible promoters (such as mouse mammary tumor virus (mMTV) promoters or various growth hormone promoters) and promoters derived from T7 phage that are active in the presence of T7 RNA polymerase. Examples of tissue-specific promoters include various surfactant promoters (for expression in the lungs), myosin promoters (for expression in muscles), and albumin promoters (for expression in the liver). A wide variety of other promoters are known and generally available in the art, and sequences of many such promoters are available in sequence databases such as the GenBank database.

[0156] In some embodiments, the vector further includes a CMV enhancer.

[0157] Other controllable elements In some cases, the vectors of the present disclosure further include one or more regulatory elements selected from the group consisting of enhancers, introns, polyA signals, 2A peptide encoding sequences, WPREs (woodchuck hepatitis virus posttranscriptional regulators), and HPREs (hepatitis B posttranscriptional regulators).

[0158] 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 as follows: TIFF0007869633000038.tif49145

[0159] 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.

[0160] 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).

[0161] 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 scaffolding-binding region, a 3'UTR, or a 5'UTR. In certain embodiments, the vector includes both a 5'UTR and a 3'UTR.

[0162] In some embodiments, the vector includes a 5' untranslated region (UTR) selected from Table 6.

[0163] (Table 6) TIFF0007869633000039.tif124166TIFF0007869633000040.tif239166TIFF0007869633000041.tif239166TIFF0007869633000042.tif239166

[0164] In some embodiments, the vector includes a 3' untranslated region selected from Table 7.

[0165] (Table 7) TIFF0007869633000043.tif218166TIFF0007869633000044.tif239166TIFF0007869633000045.tif37166

[0166] In some embodiments, the vector includes a polyadenylated sequence (PolyA) selected from Table 8.

[0167] (Table 8) TIFF0007869633000046.tif210166

[0168] Exemplary expression cassettes are shown in Figure 13 and provided as sequence numbers 39–58 listed in Table 10. In some embodiments, the expression cassette includes, essentially consists of, or comprises a polynucleotide sequence that shares at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any one of sequence numbers 39–58. In some embodiments, the expression cassette includes, essentially consists of, or comprises a polynucleotide sequence that shares at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with any one of sequence numbers 39–58, excluding the sequence encoding the therapeutic gene product. In some embodiments, the sequence encoding the therapeutic gene product si is replaced by a sequence encoding a different therapeutic gene product.

[0169] In one embodiment, the expression cassette includes the HuBA promoter, the transgene, WPRE(x), and pAglobin-Oc in 5' to 3' order. In a particular embodiment, the transgene encodes PARK2, PINK1 (PARK6), DJ-1 (PARK7), LRRK2, α-synuclein, and DJ-1. In a particular embodiment, it encodes PARK2 and, in a particular embodiment, includes the sequence shown in SEQ ID NOs. 27 or 35-38.

[0170] In one embodiment, the expression cassette includes a CMV promoter, a TPL-eMLP enhancer, a transgene, WPRE(r), and pAglobin-Oc in 5' to 3' order. In a particular embodiment, the transgene encodes PARK2, PINK1 (PARK6), DJ-1 (PARK7), LRRK2, α-synuclein, and DJ-1. In a particular embodiment, it encodes PARK2 and, in a particular embodiment, includes the sequence shown in SEQ ID NOs. 27 or 35-38.

[0171] In one embodiment, the expression cassette includes a Syn promoter, a transgene, WPRE(r), 3'UTR(globin), and pAGH-Bt in 5' to 3' order. In a particular embodiment, the transgene encodes PARK2, PINK1 (PARK6), DJ-1 (PARK7), LRRK2, α-synuclein, and DJ-1. In a particular embodiment, it encodes PARK2 and, in a particular embodiment, includes the sequence shown in SEQ ID NOs. 27 or 35-38.

[0172] In one embodiment, the expression cassette includes a CBA promoter, a transgene, and pAGH-Bt in 5' to 3' order. In a particular embodiment, the transgene encodes PARK2, PINK1 (PARK6), DJ-1 (PARK7), LRRK2, α-synuclein, and DJ-1. In a particular embodiment, it encodes PARK2 and, in a particular embodiment, includes the sequence shown in SEQ ID NOs. 27 or 35-38.

[0173] In one embodiment, the expression cassette includes the EF1α promoter, the transgene, and pAglobin-Oc in 5' to 3' order. In a particular embodiment, the transgene encodes PARK2, PINK1 (PARK6), DJ-1 (PARK7), LRRK2, α-synuclein, and DJ-1. In a particular embodiment, it encodes PARK2 and, in a particular embodiment, includes the sequence shown in SEQ ID NOs. 27 or 35-38.

[0174] In one embodiment, the expression cassette includes the HuBA promoter, the transgene, R2V17, and pAGH-Bt in 5' to 3' order. In a particular embodiment, the transgene encodes PARK2, PINK1 (PARK6), DJ-1 (PARK7), LRRK2, α-synuclein, and DJ-1. In a particular embodiment, it encodes PARK2 and, in a particular embodiment, includes the sequence shown in SEQ ID NOs. 27 or 35-38.

[0175] In one embodiment, the expression cassette includes a Syn promoter, a transgene, WPRE(x), 3'UTR(globin), and pAGH-Hs in 5' to 3' order. In a particular embodiment, the transgene encodes PARK2, PINK1 (PARK6), DJ-1 (PARK7), LRRK2, α-synuclein, and DJ-1. In a particular embodiment, it encodes PARK2 and, in a particular embodiment, includes the sequence shown in SEQ ID NOs. 27 or 35-38.

[0176] In one embodiment, the expression cassette includes the CaMKIIa promoter, the transgene, WPRE(r), and pAGH-Hs in 5' to 3' order. In a particular embodiment, the transgene encodes PARK2, PINK1 (PARK6), DJ-1 (PARK7), LRRK2, α-synuclein, and DJ-1. In a particular embodiment, it encodes PARK2 and, in a particular embodiment, includes the sequence shown in SEQ ID NOs. 27 or 35-38.

[0177] In one embodiment, the expression cassette includes a CMV promoter, a TPL-eMLP enhancer, a transgene, WPRE(r), and pAGH-Hs in 5' to 3' order. In a particular embodiment, the transgene encodes PARK2, PINK1 (PARK6), DJ-1 (PARK7), LRRK2, α-synuclein, and DJ-1. In a particular embodiment, it encodes PARK2 and, in a particular embodiment, includes the sequence shown in SEQ ID NOs. 27 or 35-38.

[0178] In one embodiment, the expression cassette includes a HuBA promoter, a transgene, and pAGH-Hs in 5' to 3' order. In a particular embodiment, the transgene encodes PARK2, PINK1 (PARK6), DJ-1 (PARK7), LRRK2, α-synuclein, and DJ-1. In a particular embodiment, it encodes PARK2 and, in a particular embodiment, includes the sequence shown in SEQ ID NOs. 27 or 35-38.

[0179] In one embodiment, the expression cassette includes a CMV promoter, a TPL / eMLP enhancer, a transgene, R2V17, 3'UTR (globin), and pAGH-Bt in 5' to 3' order. In a particular embodiment, the transgene encodes PARK2, PINK1 (PARK6), DJ-1 (PARK7), LRRK2, α-synuclein, and DJ-1. In a particular embodiment, it encodes PARK2 and, in a particular embodiment, includes the sequence shown in SEQ ID NOs. 27 or 35-38.

[0180] In one embodiment, the expression cassette includes the EF1α promoter, the transgene, WPRE(r), and pAGH-Bt in 5' to 3' order. In a particular embodiment, the transgene encodes PARK2, PINK1 (PARK6), DJ-1 (PARK7), LRRK2, α-synuclein, and DJ-1. In a particular embodiment, it encodes PARK2 and, in a particular embodiment, includes the sequence shown in SEQ ID NOs. 27 or 35-38.

[0181] In one embodiment, the expression cassette includes the Syn promoter, the transgene, R2V17, and pAglobin-Oc in 5' to 3' order. In a particular embodiment, the transgene encodes PARK2, PINK1 (PARK6), DJ-1 (PARK7), LRRK2, α-synuclein, and DJ-1. In a particular embodiment, it encodes PARK2 and, in a particular embodiment, includes the sequence shown in SEQ ID NOs. 27 or 35-38.

[0182] In one embodiment, the expression cassette includes the CaMKIIa promoter, the transgene, R2V17, and pAglobin-Oc in 5' to 3' order. In a particular embodiment, the transgene encodes PARK2, PINK1 (PARK6), DJ-1 (PARK7), LRRK2, α-synuclein, and DJ-1. In a particular embodiment, it encodes PARK2 and, in a particular embodiment, includes the sequence shown in SEQ ID NOs. 27 or 35-38.

[0183] In one embodiment, the expression cassette includes a CBA promoter, a transgene, WPRE(x), 3'UTR(globin), and pAGH-Hs in 5' to 3' order. In a particular embodiment, the transgene encodes PARK2, PINK1 (PARK6), DJ-1 (PARK7), LRRK2, α-synuclein, and DJ-1. In a particular embodiment, it encodes PARK2 and, in a particular embodiment, includes the sequence shown in SEQ ID NOs. 27 or 35-38.

[0184] In one embodiment, the expression cassette includes a CBA promoter, a transgene, 3'UTR (globin), and pAglobin-Oc in 5' to 3' order. In a particular embodiment, the transgene encodes PARK2, PINK1 (PARK6), DJ-1 (PARK7), LRRK2, α-synuclein, and DJ-1. In a particular embodiment, it encodes PARK2 and, in a particular embodiment, includes the sequence shown in SEQ ID NOs. 27 or 35-38.

[0185] In one embodiment, the expression cassette includes the CaMKIIa promoter, the transgene, R2V17, and pAGH-Bt in 5' to 3' order. In a particular embodiment, the transgene encodes PARK2, PINK1 (PARK6), DJ-1 (PARK7), LRRK2, α-synuclein, and DJ-1. In a particular embodiment, it encodes PARK2 and, in a particular embodiment, includes the sequence shown in SEQ ID NOs. 27 or 35-38.

[0186] In one embodiment, the expression cassette includes the EF1α promoter, the transgene, R2V17, 3'UTR (globin), and pAGH-Hs in the order of 5' to 3'. In a particular embodiment, the transgene encodes PARK2, PINK1 (PARK6), DJ-1 (PARK7), LRRK2, α-synuclein, and DJ-1. In a particular embodiment, it encodes PARK2 and, in a particular embodiment, includes the sequence shown in SEQ ID NOs. 27 or 35-38.

[0187] In one embodiment, the expression cassette includes a CMV promoter, a transgene, R2V17, 3'UTR (globin), and pAGH-Hs in 5' to 3' order. In a particular embodiment, the transgene encodes PARK2, PINK1 (PARK6), DJ-1 (PARK7), LRRK2, α-synuclein, and DJ-1. In a particular embodiment, it encodes PARK2 and, in a particular embodiment, includes the sequence shown in SEQ ID NOs. 27 or 35-38.

[0188] In one embodiment, the expression cassette includes a CMV promoter, a transgene, and pAGH-Hs in 5' to 3' order. In a particular embodiment, the transgene encodes PARK2, PINK1 (PARK6), DJ-1 (PARK7), LRRK2, α-synuclein, and DJ-1. In a particular embodiment, it encodes PARK2 and, in a particular embodiment, includes the sequence shown in SEQ ID NOs. 27 or 35-38.

[0189] In the embodiments described above, the promoter precedes the enhancer element, or the order of the 5' elements relative to the transgene is reversed so that the enhancer element precedes the promoter element.

[0190] Therapeutic compositions and methods Where used herein, the terms “requiring patient” or “requiring subject” refer to a patient or subject who is 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 requiring patient or subject may, for example, be a patient or subject diagnosed with a disorder related to central nervous system degradation. A subject may have mutations or dysfunctions in the 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. A subject may have an age range. A subject may be a person identified as being at risk for Parkinson's disease, for example, early-onset Parkinson's disease.

[0191] Combination therapy is also intended by the present invention. Combinations as used herein include concurrent or sequential treatments. Combinations of the methods of the present invention with standard medical treatments (e.g., corticosteroids or topical decompressants) are particularly intended, as are combinations with novel therapies. In some cases, subjects may be treated with steroids to prevent or reduce the immune response to the administration of rAAV as described herein. In certain cases, subjects may receive topical decompressants before, during, or after the administration of rAAV as described herein.

[0192] A therapeutically effective dose of rAAV vector 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 The dosages of rAAVs are in the range of 1e9vg / kg to about 5e9vg / kg, or about 1e10vg / kg to about 5e10vg / 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.

[0193] For example, in certain embodiments, therapeutically effective doses of rAAV vector are approximately 1 e10 vg / kg, 2 e10 vg / kg, 3 e10 vg / kg, 4 e10 vg / kg, 5 e10 vg / kg, 6 e10 vg / kg, 7 e10 vg / kg, 8 e10 vg / kg, 9 e10 vg / kg, 1 e12 vg / kg, 2 e12 vg / kg, 3 e12 vg / kg, 4 e12 vg / kg, and 5 e12 vg / kg. The present invention also includes compositions comprising these doses of rAAV vector.

[0194] In some embodiments, for example, when injected directly into the substantia nigra, a therapeutically effective dose of rAAV vector is 1e7vg to 1e11vg, or a dose in the range of approximately 1e7vg, approximately 1e8vg, approximately 1e9vg, approximately 1e10vg, or approximately 1e11vg.

[0195] In some embodiments, for example, when injected directly into the putamen, a therapeutically effective dose of rAAV vector is 1e7vg to 1e11vg, or a dose in the range of approximately 1e7vg, approximately 1e8vg, approximately 1e9vg, approximately 1e10vg, or approximately 1e11vg.

[0196] In some cases, the therapeutic composition contains more than approximately 1e9, 1e10, or 1e11 of rAAV vector genomes per volume of the therapeutic composition injected. In some cases, the therapeutic composition contains more than approximately 1e9, 1e10, or 1e11 of rAAV vector genomes per volume of the therapeutic composition injected. 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.

[0197] Administration of composition The effective dose of the composition may be administered by standard routes in the art, including, but not limited to, systemic, topical, direct injection, parenteral, intravenous, cerebral, cerebrospinal fluid, intrathecal, intracisional, intraputamen, intrahippocampal, striatal, or intracerebroventricular administration. In some cases, administration may include intravenous, cerebral, cerebrospinal fluid, intrathecal, intracisional, intracisional, intraputamen, intrahippocampal, striatal, or intracerebroventricular injection. Administration may be performed by intrathecal injection with a Trendelenburg inclination. The administration routes of the rAAV (in particular AAV ITR and capsid protein) and serotypes(s) of the AAV components of the present invention may be selected and / or adapted by those skilled in the art, taking into account the disorder being treated and the target cells / tissues expressing the repaired and / or exogenously provided genes.

[0198] In certain embodiments, this disclosure provides topical and systemic administration of effective doses of rAAV and compositions of the present invention. For example, systemic administration may be administration to the circulatory system in which the whole body is affected. Systemic administration includes enteral administration, such as absorption through the gastrointestinal tract, and parenteral administration via infusion, infusion, or transplantation.

[0199] In detail, the actual administration of rAAV according to the present invention can be achieved by using any physical method to deliver the rAAV recombinant vector to the target tissue of an animal. Administration according to the present invention includes, but is not limited to, injection into the central nervous system (CNS) or cerebrospinal fluid (CSF), and / or direct injection into the brain.

[0200] The capsid protein of rAAV can be modified so that rAAV targets specific target tissues of the subject, 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, and also see U.S. Patent No. 6,180,613 and U.S. Patent Publication No. US2012 / 0082650A1, both of which are incorporated herein by reference. In some embodiments, rAAV is injected directly into the substantia nigra of the subject.

[0201] For example, various solutions, such as sterile aqueous solutions, can be used for administration by injection. Such aqueous solutions can be buffered as needed, and the liquid diluent is first 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 a surfactant such as hydroxypropylcellulose. Dispersions of rAAV can also be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof, as well as in 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 known to those skilled in the art.

[0202] Suitable pharmaceutical forms for injection applications include, but are not limited to, sterile aqueous solutions, or dispersions and sterile powders for the immediate preparation of sterile injection solutions or dispersions. In all cases, the form must be sterile and fluid to the extent that easy injection is possible. It must be stable under manufacturing and storage conditions and protected from microbial contamination 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 activity can be achieved by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. Often, it is preferable to include isotonic agents, such as sugars or sodium chloride. Sustained absorption of the injectionable composition can be achieved by the use of absorption-delaying agents, such as aluminum monostearate and gelatin.

[0203] Sterile injectable solutions can be prepared by incorporating the required amount of rAAV into a suitable solvent containing, if necessary, various other components listed above, and then sterilizing by filtration. Generally, dispersions 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 preparing sterile injectable solutions, preferred preparation methods are vacuum drying and freeze-drying techniques, which yield powders of the active ingredient and any additional desired components from the pre-sterilized filtered solution.

[0204] Transduction by rAAV can also be performed in vitro. In one embodiment, desired target cells are removed from the subject, transduced with rAAV, and then reintroduced into the subject.

[0205] Suitable methods for transduction and retransduction of transduced cells into a target are known in the art. In one embodiment, cells can be transduced in vitro, for example, by combining rAAV with the cells in a suitable culture medium and screening cells retaining the DNA of interest using conventional techniques such as Southern blotting and / or PCR, or by using a selectable marker. The transduced cells can then be formulated into a pharmaceutical composition, which is introduced into the target by various techniques such as systemic, topical, direct injection, parenteral, intravenous, intracerebral, intracerebrospinal, intrathecal, intracisional, intrahippocampal, striatal, or intraventricular administration. In some cases, administration includes intravenous, intracerebral, intracerebrospinal, intrathecal, intracisional, intracisional, intrahippocampal, striatal, or intraventricular injection. Administration may be performed by intrathecal injection with a Trendelenburg inclination.

[0206] Transduction of cells by rAAV according to the present invention results in sustained expression of a target gene such as PARK2, PARK6, PARK7, LRRK2, or α-synuclein. Accordingly, the present invention provides methods for administering or delivering recombinant gene therapy vectors (e.g., rAAV vectors) that express genes associated with CNS degeneration in mammalian subjects, preferably humans. These methods involve transducing tissue (including, but not limited to, brain tissue) with one or more rAAVs of the present invention. Transduction may be carried out with a gene cassette containing tissue-specific regulatory elements. For example, one embodiment of the present invention provides a method for transducing neuronal cells and brain tissue directed by neuron-specific regulatory elements, including, but not limited to, those derived from neuron enrichment promoters and other regulatory elements.

[0207] Gene editing systems As used herein, a gene editing system is a system comprising one or more proteins or polynucleotides capable of sequence-specifically editing an endogenous target gene or locus. In some embodiments, the gene editing system is a protein-based gene regulatory system comprising a protein comprising one or more zinc finger-binding domains and an enzyme domain. In some embodiments, the protein-based gene regulatory system comprises a protein comprising a transcription activator-like effector nuclease (TALEN) domain and an enzyme domain. Such embodiments are referred to herein as "TALEN".

[0208] 1. Zinc finger system The zinc finger system comprises a fusion protein containing two protein domains: a zinc finger DNA-binding domain and an enzyme domain. A "zinc finger DNA-binding domain," "zinc finger protein," or "ZFP" is a domain within a protein or larger protein that binds to DNA in a sequence-specific manner via one or more zinc fingers, and is a region of amino acid sequence within the binding domain whose structure is stabilized by the coordination of zinc ions. The zinc finger domain, by binding to a target DNA sequence, directs the activity of the enzyme domain near the sequence, and thus induces modification of an endogenous target gene near the target sequence. Zinc finger domains can be designed to bind to substantially any desired sequence. Therefore, after identifying a target locus containing a target DNA sequence to be cleaved or recombined (e.g., a target locus within a target gene referenced in Table 1), one or more zinc finger-binding domains can be manipulated to bind to one or more target DNA sequences within the target locus. Expression of a fusion protein containing a zinc finger-binding domain and an enzyme domain in cells results in modification of the target locus.

[0209] In some embodiments, a zinc finger binding domain contains one or more zinc fingers. (Miller et al. (1985) EMBOJ.4:16010-1714; Rhodes (1993) Scientific American Febuary:56-65, U.S. Patent No. 6,453,242) Typically, a single zinc finger domain is about 30 amino acids long. Each zinc finger binds to a 3-nucleotide (i.e., triplet) sequence (or a 4-nucleotide sequence that can overlap by 1 nucleotide with the 4-nucleotide binding site of an adjacent zinc finger). Therefore, the length of the sequence to which the zinc finger binding domain is manipulated to bind (e.g., the target sequence) determines the number of zinc fingers in the manipulated zinc finger binding domain. For example, for a ZFP that does not bind to a subsite where the finger motif overlaps, a 6-nucleotide target sequence is bound by two finger binding domains, a 9-nucleotide target sequence is bound by three finger binding domains, and so on. The binding sites of individual zinc fingers (i.e., subsites) at the target site do not need to be consecutive, but can be separated by one or more nucleotides depending on the length and nature of the amino acid sequence between zinc fingers (i.e., inter-finger linkers) within the multi-finger binding domain. In some embodiments, the DNA-binding domain of an individual ZFN contains 3 to 6 zinc finger repeats, each capable of recognizing 9 to 18 base pairs.

[0210] Zinc finger binding domains can be designed to bind to selected sequences. See, for example, Beerli et al. (2002) Nature Biotechnol. 20:135-141, Pabo et al. (2001) Ann. Rev. Biochem. 70:313-340, Isalan et al. (2001) Nature Biotechnol. 19:656-660, Segal et al. (2001) Curr. Opin. Biotechnol. 12:632-637, and Choo et al. (2000) Curr. Opin. Struct. Biol. 10:411-416. Manipulated zinc finger binding domains can exhibit novel binding specificity compared to naturally occurring zinc finger proteins. Manipulation methods include, but are not limited to, rational design and various types of selection.

[0211] The selection of target DNA sequences for binding by zinc finger domains can be achieved, for example, by the method disclosed in U.S. Patent No. 6,453,242. It will be apparent to those skilled in the art that a simple visual inspection of nucleotide sequences can also be used for the selection of target DNA sequences. Therefore, any means for the selection of target DNA sequences can be used in the methods described herein. Target sites generally have a length of at least 9 nucleotides and are therefore bound by zinc finger binding domains containing at least 3 zinc fingers. However, for example, 4 finger binding domains with a 12-nucleotide target site, 5 finger binding domains with a 15-nucleotide target site, or 6 finger binding domains with an 18-nucleotide target site are also possible. As will be apparent, binding of larger binding domains (e.g., 7, 8, 9 or more fingers) with longer target sites is also possible.

[0212] In some embodiments, the zinc finger binding domain binds to a target DNA sequence that is at least 90% identical to the target DNA sequence within the target locus of a target gene selected from those listed in Table 1. In some embodiments, the zinc finger binding domain binds to a target DNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the target DNA sequence within the target locus of a target gene selected from those listed in Table 1. In some embodiments, the zinc finger binding domain binds to a target DNA sequence that is 100% identical to the target DNA sequence within the target locus of a target gene selected from those listed in Table 1.

[0213] The enzyme domain portion of a zinc finger fusion protein can be obtained from any endonuclease or exonuclease. Exemplary endonucleases from which the enzyme domain may originate include, but are not limited to, restriction endonucleases and homing endonucleases. See, for example, 2002-2003 Catalogue, New England Biolabs, Beverly, Mass, and Belfort et al. (1997) Nucleic Acids Res. 25:3379-3388. Additional enzymes that cleave DNA are known (see, for example, 51 nuclease, manguinea nuclease, pancreatic DNase I, micrococcal nuclease, yeast HO endonuclease, and Linn et al. (eds.) Nucleases, Cold Spring Harbor Laboratory Press, 1993). One or more of these enzymes (or functional fragments thereof) can be used as a source for the cleavage domain.

[0214] Exemplary restriction endonucleases (restriction enzymes) suitable for use as the enzyme domains of ZFPs described herein are present in many species and can sequence-specifically bind to DNA (at the recognition site) and cleave the DNA at or near the binding site. Certain restriction enzymes (e.g., IIS type) cleave the DNA at the site removed from the recognition site and have separable binding and cleavage domains. For example, the IIS type enzyme Fok I catalyzes double-strand breaks of DNA, catalyzing 9 nucleotides from its recognition site on one strand and 13 nucleotides from its recognition site on the other strand. See, for example, U.S. Patent Nos. 5,356,802, 5,436,150, and 5,487,994, as well as Li et al. (1992) Proc. Natl. Acad. Sci. USA 89:4275-4279, Li et al. (1993) Proc. Natl. Acad. Sci. USA 90:2764-2768, Kim et al. (1994a) Proc. Natl. Acad. Sci. USA 91:883-887, and Kim et al. (1994b) J. Biol. Chem. 269:31,978-31,982. Thus, in one embodiment, the fusion protein comprises an enzyme domain from at least one IIS-type restriction enzyme and one or more zinc finger-binding domains.

[0215] An exemplary IIS-type restriction enzyme in which the cleavage domain is separable from the binding domain is Fok I. This particular enzyme is active as a dimer. Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA 95:10,570-10,575. Therefore, for targeted double-strand DNA cleavage using zinc finger-Fok I fusion, the catalytically active cleavage domain can be reconstituted using two fusion proteins, each containing a Fok I enzyme domain. Alternatively, a single polypeptide molecule containing a zinc finger binding domain and two Fok I enzyme domains can also be used. An exemplary ZFP containing Fok I enzyme domains is described in U.S. Patent No. 9,782,437.

[0216] 2. TALEN systems The TALEN system includes proteins containing TAL effector DNA-binding domains and enzyme domains. These are constructed by fusing the TAL effector DNA-binding domain to a DNA-cleaving domain (a nuclease that cleaves DNA strands). The FokI restriction enzyme mentioned above is an exemplary enzyme domain suitable for use in TALEN gene regulatory systems.

[0217] TAL effectors are proteins secreted by Xanthomonas fungi via their type III secretory system when infecting plants. The DNA-binding domain contains a repeating, highly conserved sequence of 33-34 amino acids, with the 12th and 13th amino acids differing. These two positions, referred to as repeating variable duos (RVDs), are highly variable and strongly correlate with specific nucleotide recognition. Therefore, the TAL effector domain can be manipulated to bind to specific target DNA sequences by selecting combinations of repeating segments containing appropriate RVDs. The nucleic acid specificity for RVD combinations is as follows: HD targets cytosine, NI targets adenine, NG targets thymine, and NN targets guanine (although in some embodiments, NN can also bind to adenine with lower specificity).

[0218] In some embodiments, the TAL effector domain binds to a target DNA sequence that is at least 90% identical to the target DNA sequence within the target locus of a target gene selected from those listed in Table 1. In some embodiments, the TAL effector domain binds to a target DNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the target DNA sequence within the target locus of a target gene selected from those listed in Table 1. In some embodiments, the TAL effector domain binds to a target DNA sequence that is 100% identical to the target DNA sequence within the target locus of a target gene selected from those listed in Table 1.

[0219] Methods and compositions for constructing TAL effector repeats are known in the art. See, for example, Cermak et al, Nucleic Acids Research, 39:12, 2011, e82. Plasmids for constructing TAL effector repeats are commercially available from Addgene.

[0220] In some embodiments, the gene editing system is a combination gene control system comprising a site-directed modification polypeptide and a nucleic acid guide molecule. In this specification, “site-directed modification polypeptide” refers to a polypeptide that binds to a nucleic acid guide molecule and, by the nucleic acid guide molecule it binds to, targets a target nucleic acid sequence, such as a DNA sequence, and modifies the target DNA sequence (e.g., cleavage, mutation, or methylation of the target DNA). The site-directed modification polypeptide comprises two parts: a part that binds to the nucleic acid guide and an active part. In some embodiments, the site-directed modification polypeptide includes an active part that exhibits site-directed enzymatic activity (e.g., DNA methylation, DNA cleavage, histone acetylation, histone methylation, etc.), and the enzymatic active site is determined by the guide nucleic acid.

[0221] The nucleic acid guide comprises two parts: a first part that is complementary to and can bind to an endogenous target DNA sequence (referred to herein as the “DNA-binding segment”), and a second part that can interact with a site-directed modified polypeptide (referred to herein as the “protein-binding segment”). In some embodiments, the DNA-binding segment and the protein-binding segment of the nucleic acid guide are contained within a single polynucleotide molecule. In some embodiments, the DNA-binding segment and the protein-binding segment of the nucleic acid guide are each contained within separate polynucleotide molecules, and as a result, the nucleic acid guide comprises two polynucleotide molecules that associate with each other to form a functional guide.

[0222] Nucleic acid guides mediate the target specificity of a combined protein / nuclear gene regulatory system by specifically hybridizing with target DNA sequences contained within the DNA sequence of a target gene. References to target genes herein encompass the full-length DNA sequence of that particular gene, and the full-length DNA sequence of a particular target gene includes multiple target loci pointing to a portion of a particular target gene sequence (e.g., an exon or intron). Within each target locus, there exists a shorter stretch of the DNA sequence referred to herein as the “target DNA sequence” or “target sequence,” which can be modified by the gene regulatory system described herein. Furthermore, each target locus includes a “target modification site” pointing to the precise location of a modification induced by the gene regulatory system (e.g., a site of an insertion, deletion, or mutation, a site of a DNA break, or a site of an epigenetic modification). The gene regulatory systems described herein may include a single nucleic acid guide or multiple nucleic acid guides (e.g., two, three, four, five, six, seven, eight, nine, ten, or more nucleic acid guides).

[0223] The CRISPR / Cas systems described below are exemplary embodiments of combined protein / nucleic acid systems.

[0224] 3. CRISPR / Cas gene regulatory system In some embodiments, the gene editing systems described herein are CRISPR (clustered, regularly spaced, short palindromic repetitions) / Cas (CRISPR-related) nuclease systems. In such embodiments, the site-directed modification polypeptide is a CRISPR-related endonuclease ("Cas" endonuclease), and the nucleic acid guide molecule is a guide RNA (gRNA).

[0225] Cas polypeptides are polypeptides that can interact with gRNA molecules and cooperate with gRNA molecules to home to or localize to target DNA sequences, and include naturally occurring Cas proteins and Cas proteins that have been manipulated, modified, or otherwise modified in different ways by a naturally occurring Cas sequence and one or more amino acid residues.

[0226] In some embodiments, the Cas protein is the Cas9 protein. Cas9 is a multi-domain enzyme that uses an HNH nuclease domain to cleave the target strand of DNA and a RuvC-like domain to cleave the non-target strand. In some embodiments, Cas9 can be produced by selective domain inactivation, which allows conversion of WT Cas9 to an enzymatically inactive variant (e.g., dCas9) that is unable to cleave DNA, or the nickase variant can produce single-strand DNA breaks by cleaving either the target or non-target strand, or the other.

[0227] Guide RNA (gRNA) comprises two segments: a DNA-binding segment and a protein-binding segment. In some embodiments, the protein-binding segment of the gRNA is contained within one RNA molecule, and the DNA-binding segment is contained within another separate RNA molecule. Such embodiments are referred to herein as “dual-molecule gRNA” or “two-molecule gRNA” or “dual gRNA.” In some embodiments, the gRNA is a single RNA molecule and is referred herein as “single-guide RNA” or “single-guide RNA.” The terms “guide RNA” or “gRNA” are inclusive and refer to both two-molecule guide RNA and sgRNA.

[0228] The protein-binding segment of gRNA partially contains two complementary nucleotide stretches that hybridize with each other to form a double-stranded RNA (dsRNA) double, which facilitates binding to the Cas protein.

[0229] The DNA-binding segment (or "DNA-binding sequence") of a gRNA contains a nucleotide sequence that is complementary to and capable of binding to a specific target DNA sequence. The protein-binding segment of the gRNA interacts with a Cas polypeptide, and the interaction between the gRNA molecule and the site-directed modification polypeptide results in Cas binding to endogenous DNA, causing one or more modifications within or around the target DNA sequence. The precise location of the target modification site is determined by both (i) the base-pair complementarity between the gRNA and the target DNA sequence, and (ii) the location of a short motif called a protospacer-adjacent motif (PAM) in the target DNA sequence. The PAM sequence is required for Cas binding to the target DNA sequence. Various PAM sequences are known in the art and are known to be suitable for use with specific Cas endonucleases (e.g., Cas9 endonuclease) (see, e.g., Nat Methods. 2013 Nov;10(11):1116-1121 and Sci Rep. 2014;4:5405). In some embodiments, the PAM sequence is located within 50 base pairs of the target modification site. In some embodiments, the PAM sequence is located within 10 base pairs of the target modification site. The DNA sequences that can be targeted by this method are limited only by the relative distance from the PAM sequence to the target modification site and the presence of a specific 20-base pair sequence for mediating sequence-specific gRNA-mediated Cas binding. In some embodiments, the target modification site is located at the 5' end of the target locus. In some embodiments, the target modification site is located at the 3' end of the target locus. In some embodiments, the target modification site is located within an intron or exon of the target locus.

[0230] In some embodiments, the disclosure provides polynucleotides encoding gRNA. In some embodiments, the gRNA-encoding nucleic acid is contained in an expression vector, for example, a recombinant expression vector. In some embodiments, the disclosure provides polynucleotides encoding site-specific modified polypeptides. In some embodiments, the polynucleotides encoding site-specific modified polypeptides are contained in an expression vector, for example, a recombinant expression vector.

[0231] a. Cas protein In some embodiments, the site-directed modified polypeptide is a Cas protein. Various species of Cas molecules can be used in the methods and compositions described herein, including S. pyogenes, S. aureus, N. meningitidis, S. thermophiles, Acidovorax avenae, Actinobacillus pleuropneumoniae, Actinobacillus succinogenes, Actinobacillus suis, Actinomyces sp., Cycliphilus denitrificans, Aminomonas paucivorans, and Bacillus cereus. Bacillus cereus, Bacillus smithii, Bacillus thuringiensis, Bacteroides sp., Blastopirellula marina, Bradyrhizobium sp., Brevibacillus laterospoxus, Campylobacter coli, Campylobacter jejuni, Campylobacter lari, Candidatus puniceispirillum, Clostridium cellulolyticum, Clostridium perfringens Corynebacterium perfringens, Corynebacterium accolens, Corynebacterium diphtheriaediphtheria), Corynebacterium matruchotii, Dinoroseobacter shibae, Eubacterium dolichum, Gammaproteobacterium, Gluconacetobacter diazotrophicus, Haemophilus parainfluenzae, Haemophilus sputomm, Helicobacter canadensis, Helicobacter cinaedi, Helicobacter mustelae, Ilyobacter polytropus Polytropus), Kingella kingae, Lactobacillus crispatus, Listeria ivanovii, Listeria monocytogenes, Listeriaceae bacterium, Methylocystis sp., Methylosinus trichosporium, Mobiluncus mulieris, Neisseria bacilliformis, Neisseria cinerea, Neisseria flavescens, Neisseria lactamica, Neisseria meningitidis, Neisseria species (Neisseria sp.), Neisseria wadsworthii, Nitrosomonas speciessp.), Parvibaculum lavamentivorans, Pasteurella multocida, Phascolarctobacterium succinatutens, Ralstonia syzygii, Rhodopseudomonas palustris, Rhodovulum sp., Simonsiella muelleri, Sphingomonas sp., Sporolactobacillus vineae, Staphylococcus aureus, Staphylococcus rugdunensis It contains Cas molecules derived from *Streptococcus lugdunensis*, *Streptococcus sp.*, *Subdoligranulum sp.*, *Tistrella mobilis*, *Treponema sp.*, or *Verminephrobacter eiseniae*.

[0232] In some embodiments, the Cas protein is a Cas9 protein or Cas9 orthologue, selected from the group consisting of SpCas9, SpCas9-HF1, SpCas9-HF2, SpCas9-HF3, SpCas9-HF4, SaCas9, FnCpf, FnCas9, eSpCas9, and NmeCas9. In some embodiments, the endonuclease is C2C1, C2C3, Cpf1 (also called Cas12a), Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also called Csnl and Csx12), Cas10, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, The group is selected from Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, Csx10, Csx16, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, and Csf4. Additional Cas9 orthologs are described in International PCT Publication WO2015 / 071474.

[0233] In some embodiments, the Cas9 protein is a naturally occurring Cas9 protein. Exemplary naturally occurring Cas9 molecules are described in Chylinski et al., RNA Biology 2013 10:5, 727-737. Such Cas9 molecules include the cluster 1 bacterial family, cluster 2 bacterial family, cluster 3 bacterial family, cluster 4 bacterial family, cluster 5 bacterial family, cluster 6 bacterial family, cluster 7 bacterial family, cluster 8 bacterial family, cluster 9 bacterial family, cluster 10 bacterial family, cluster 11 bacterial family, cluster 12 bacterial family, cluster 13 bacterial family, cluster 14 bacterial family, cluster 15 bacterial family, cluster 16 bacterial family, cluster 17 bacterial family, cluster 18 bacterial family, cluster 19 bacterial family, cluster 20 bacterial family, cluster 21 bacterial family, cluster 22 bacterial family, cluster 23 bacterial family, cluster 24 bacterial family, cluster 25 bacterial family, cluster 26 bacterial family, cluster 27 bacterial family, cluster 28 bacterial family, and cluster 29 bacterial family. - Cluster 30 bacterial family, Cluster 31 bacterial family, Cluster 32 bacterial family, Cluster 33 bacterial family, Cluster 34 bacterial family, Cluster 35 bacterial family, Cluster 36 bacterial family, Cluster 37 bacterial family, Cluster 38 bacterial family, Cluster 39 bacterial family, Cluster 40 bacterial family, Cluster 41 bacterial family, Cluster 42 bacterial family, Cluster 43 bacterial family, Cluster 44 bacterial family, Cluster 45 bacterial family, Cluster 46 bacterial family, Cluster 47 bacterial family, Cluster 48 bacterial family, Cluster 49 bacterial family, Cluster 50 bacterial family, Cluster 51 bacterial family, Cluster 52 bacterial family, Cluster 53 bacterial family, Cluster 54 bacterial family, Cluster 55 bacterial family, Cluster 56 bacterial family, Cluster 57 bacterial family, Cluster 58 bacterial family,This includes Cas9 molecules from the following bacterial families: Cluster 59, Cluster 60, Cluster 61, Cluster 62, Cluster 63, Cluster 64, Cluster 65, Cluster 66, Cluster 67, Cluster 68, Cluster 69, Cluster 70, Cluster 71, Cluster 72, Cluster 73, Cluster 74, Cluster 75, Cluster 76, Cluster 77, or Cluster 78.

[0234] In some embodiments, the Cas9 protein includes an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the Cas9 amino acid sequence described in Chylinski et al., RNA Biology 2013 10:5, 727-737, Hou et al., PNAS Early Edition 2013, 1-6).

[0235] In some embodiments, the Cas polypeptide comprises one or more of the following activities: a) Nickase activity, that is, the ability to cleave a single strand of nucleic acid molecule, e.g., the non-complementary or complementary strand. b) Double-stranded nuclease activity, i.e., the ability to cleave both strands of a double-stranded nucleic acid and, in one embodiment, create a double-stranded break, which is the presence of two nickase activities. c) Endonuclease activity, d) Exonuclease activity, and / or e) Helicase activity, that is, the ability to unwind the helical structure of double-stranded nucleic acids.

[0236] In some embodiments, Cas9 is either a wild-type (WT) Cas9 protein or an ortholog. WT Cas9 contains two catalytic domains (HNH and RuvC). Binding of WT Cas9 to DNA based on gRNA specificity results in a double-strand DNA break that can be repaired by non-homologous end joining (NHEJ) or homology-directed repair (HDR). In some embodiments, Cas9 fuses with heterologous proteins that recruit DNA damage signaling proteins, exonucleases, or phosphatases to further increase the likelihood or rate of repair of the target sequence by one or another repair mechanism. In some embodiments, WT Cas9 is co-expressed with a nucleic acid repair template to facilitate the incorporation of exogenous nucleic acid sequences by homology-directed repair.

[0237] In some embodiments, different Cas9 proteins (i.e., Cas9 proteins from different species) may be advantageous to be used in various provided ways to take advantage of the diverse enzymatic properties of different Cas9 proteins (e.g., to preferentialize different PAM sequences, to increase or decrease enzymatic activity, to increase or decrease levels of cytotoxicity, to alter the balance between NHEJ, homology-directed repair, single-strand breaks, double-strand breaks, etc.).

[0238] In some embodiments, the Cas protein is a Cas9 protein derived from S. pyogenes that recognizes the PAM sequence motifs NGG, NAG, and NGA (Mali et al, Science 2013;339(6121):823-826). In some embodiments, the Cas protein is a Cas9 protein derived from S. thermophilus that recognizes the PAM sequence motifs NGGNG and / or NNAGAAW (W=A or T) (see, for example, Horvath et al, Science, 2010;327(5962):167-170 and Deveau et al, J BACTERIOL 2008;190(4):1390-1400). In some embodiments, the Cas protein is a Cas9 protein derived from S. mutans and recognizes the PAM sequence motif NGG and / or NAAR (R=A or G) (see, e.g., Deveau et al, J BACTERIOL 2008;190 (4):1390-1400). In some embodiments, the Cas protein is a Cas9 protein derived from S. aureus and recognizes the PAM sequence motif NNGRR (R=A or G). In some embodiments, the Cas protein is a Cas9 protein derived from S. aureus and recognizes the PAM sequence motif N GRRT (R=A or G). In some embodiments, the Cas protein is a Cas9 protein derived from S. aureus and recognizes the PAM sequence motif N GRRV (R=A or G). In some embodiments, the Cas protein is a Cas9 protein derived from N. meningitidis that recognizes the PAM sequence motif N GATT or N GCTT (R=A or G, V=A, G, or C) (see, e.g., Hou et ah, PNAS 2013, 1-6). In the embodiments described above, N may be any nucleotide residue, e.g., A, G, C, or T.

[0239] In some embodiments, polynucleotides encoding Cas proteins are provided. In some embodiments, the polynucleotides encode Cas proteins that are at least 90% identical to the Cas proteins described in International PCT Publication WO2015 / 071474 or Chylinski et al., RNA Biology 2013 10:5,727-737. In some embodiments, the polynucleotides encode Cas proteins that are at least 95%, 96%, 97%, 98%, or 99% identical to the Cas proteins described in International PCT Publication WO2015 / 071474 or Chylinski et al., RNA Biology 2013 10:5,727-737. In some embodiments, the polynucleotide encodes a Cas protein that is 100% identical to the Cas protein described in International PCT Publication No. WO2015 / 071474, or Chylinski et al., RNA Biology 2013 10:5, 727-737.

[0240] i.Cas variant In some embodiments, a Cas polypeptide is manipulated to alter one or more properties of the Cas polypeptide. For example, in some embodiments, the Cas polypeptide may have altered enzymatic properties, such as altered nuclease activity (compared to naturally occurring or other reference Cas molecules) or altered helicase activity. In some embodiments, the manipulated Cas polypeptide may have changes that alter its size, such as deletions of amino acid sequences that reduce its size without significantly affecting another property of the Cas polypeptide. In some embodiments, the manipulated Cas polypeptide may have changes that affect PAM recognition. For example, the manipulated Cas polypeptide may be modified to recognize PAM sequences other than those recognized by the corresponding wild-type Cas protein.

[0241] Cas polypeptides possessing desired properties can be prepared in several ways, including modifications to naturally occurring Cas polypeptides or parent Cas polypeptides, to provide mutant or modified Cas polypeptides with the desired properties. For example, one or more mutations may be introduced into the sequence of a parent Cas polypeptide (e.g., a naturally occurring or engineered Cas polypeptide). Such mutations and differences may include substitutions (e.g., conservative or substitution of non-essential amino acids), insertions, or deletions. In some embodiments, the mutant Cas polypeptide contains one or more mutations (e.g., at least 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, or 50 mutations) relative to the parent Cas polypeptide.

[0242] In one embodiment, the mutant Cas polypeptide has different cleavage properties than the naturally occurring Cas polypeptide. In some embodiments, Cas is a Cas nickase mutant. The Cas nickase mutant contains only one catalytically active domain (either an HNH domain or a RuvC domain). The Cas nickase mutant retains DNA binding based on gRNA specificity but can cleave only one strand of DNA, resulting in a single-strand break (e.g., a "nick"). In some embodiments, two complementary Cas nickase mutants (e.g., one Cas nickase mutant with an inactive RuvC domain and one Cas nickase mutant with an inactive HNH domain) are expressed in the same cell having two gRNAs corresponding to two respective target sequences, one target sequence on the sense DNA strand and one target sequence on the antisense DNA strand. This dual nickase system can shift double-strand breaks and enhance target specificity because it is unlikely that two off-target nicks will be generated close enough to produce a double-strand break. In some embodiments, Cas nickase mutations are co-expressed with nucleic acid repair templates to promote the uptake of exogenous nucleic acid sequences by homology-directed repair.

[0243] In some embodiments, the Cas polypeptides described herein may be manipulated to alter the PAM specificity of the Cas polypeptide. In some embodiments, the mutant Cas polypeptide has a different PAM specificity than the parent Cas polypeptide. For example, naturally occurring Cas proteins can be modified to alter the PAM sequence recognized by the mutant Cas polypeptide to reduce nonspecific sites, improve specificity, or eliminate PAM recognition requirements. In some embodiments, the Cas protein may be modified to increase the length of the PAM recognition sequence. In some embodiments, the length of the PAM recognition sequence is at least 4, 5, 6, 7, 8, 9, 10, or 15 amino acids. Cas polypeptides that recognize different PAM sequences and / or have reduced off-target activity can be generated using directed evolution. Exemplary methods and systems that can be used for directed evolution of Cas polypeptides are described, for example, in Esvelt et al. Nature 2011, 472(7344):499-503.

[0244] An exemplary Cas mutation is described in International PCT Publication WO2015 / 161276, which is incorporated herein by reference in its entirety.

[0245] 2. gRNA This disclosure provides guide RNA (gRNA) for guiding site-directed modified polypeptides to specific target DNA sequences. The gRNA comprises a DNA targeting segment and a protein-binding segment. The DNA targeting segment of the gRNA contains a nucleotide sequence complementary to the sequence in the target DNA sequence. Thus, the DNA targeting segment of the gRNA interacts with the target DNA in a sequence-specific manner via hybridization (i.e., base pairing), and the nucleotide sequence of the DNA targeting segment determines the location within the target DNA to which the gRNA will bind. The DNA targeting segment of the gRNA can be modified (e.g., by genetic engineering) to hybridize with any desired sequence within the target DNA sequence.

[0246] The protein-binding segment of the guide RNA interacts with a site-specific modified polypeptide (e.g., the Cas9 protein) to form a complex. The guide RNA, via the DNA-targeting segment described above, guides the bound polypeptide to a specific nucleotide sequence within the target DNA. The protein-binding segments of the guide RNA are complementary and contain two nucleotide stretches that form a double-stranded RNA double helix.

[0247] In some embodiments, the gRNA comprises two distinct RNA molecules. In such embodiments, each of the two RNA molecules comprises stretches of nucleotides that are complementary to each other so that the complementary nucleotides of the two RNA molecules hybridize to form a double-stranded RNA double helix of a protein-binding segment. In some embodiments, the gRNA comprises a single RNA molecule (sgRNA).

[0248] The specificity of the gRNA to a target locus is mediated by the sequence of a DNA-binding segment containing approximately 20 nucleotides complementary to the target DNA sequence within the target locus. In some embodiments, the corresponding target DNA sequence is approximately 20 nucleotides long. In some embodiments, the DNA-binding segment of the gRNA sequence of the present invention is at least 90% complementary to the target DNA sequence within the target locus. In some embodiments, the DNA-binding segment of the gRNA sequence of the present invention is at least 95%, 96%, 97%, 98%, or 99% complementary to the target DNA sequence within the target locus. In some embodiments, the DNA-binding segment of the gRNA sequence of the present invention is 100% complementary to the target DNA sequence within the target locus.

[0249] In some embodiments, the DNA-binding segment of the gRNA sequence binds to a target DNA sequence that is at least 90% identical to the target DNA sequence within the target locus of a target gene selected from those listed in Table 1. In some embodiments, the DNA-binding segment of the gRNA sequence binds to a target DNA sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the target DNA sequence within the target locus of a target gene selected from those listed in Table 1. In some embodiments, the DNA-binding segment of the gRNA sequence binds to a target DNA sequence that is 100% identical to the target DNA sequence within the target locus of a target gene selected from those listed in Table 1.

[0250] In some embodiments, the DNA-binding segments of the gRNA sequences described herein are designed to minimize off-target binding using algorithms known in the art (e.g., Cas-OFF finders) to identify target sequences that are specific to a particular target locus or target gene.

[0251] In some embodiments, the gRNAs described herein may contain one or more modified nucleosides or nucleotides that introduce stability to nucleases. In such embodiments, these modified gRNAs may induce a reduced innate immune response compared to unmodified gRNAs. The term “innate immune response” generally includes cellular responses to exogenous nucleic acids, including single-stranded nucleic acids of viral or bacterial origin, which involve cytokine expression and release, particularly the induction of interferons, as well as cell death.

[0252] In some embodiments, the gRNAs described herein are modified at or near their 5' end (e.g., within 1-10 nucleotides, 1-5 nucleotides, or 1-2 nucleotides of their 5' end). In some embodiments, the 5' end of the gRNA is modified by including a eukaryotic mRNA cap structure or cap analogue (e.g., G(5')ppp(5')G cap analogue, m7G(5')ppp(5')G cap analogue, or 3'-O-Me-m7G(5')ppp(5')G anti-reversal cap analogue (ARCA)). In some embodiments, the in vitro transcribed gRNA is modified by treatment with a phosphatase (e.g., alkaline phosphatase from calf intestine) to remove the 5' triphosphate group. In some embodiments, the gRNA is modified at or near its 3' end (e.g., within 1-10 nucleotides, 1-5 nucleotides, or 1-2 nucleotides of its 3' end). For example, in some embodiments, the 3' end of the gRNA is modified by adding one or more (e.g., 25-200) adenine (A) residues.

[0253] In some embodiments, modified nucleosides and modified nucleotides can be present in gRNA, but they can also be present in other gene regulatory systems, such as mRNA, RNAi, or siRNA systems. In some embodiments, modified nucleosides and nucleotides may include one or more of the following: a) Modification, e.g., substitution of one or both of the unbound phosphate oxygen atoms and / or one or more bound phosphate oxygen atoms in the phosphate diester skeleton bond, b) Modifications, e.g., components of ribose sugar, e.g., substitution of 2'-hydroxyl groups on ribose sugar, c) Large-scale substitution of the phosphate group with a "dephospho" linker. d) Modification or substitution of naturally occurring nucleic acid bases, e) Substitution or modification of the ribose-phosphate skeleton, f) Modification of the 3' or 5' end of oligonucleotides, e.g., removal, modification, or substitution of terminal phosphate groups, or partial conjugation, g) Sugar modification.

[0254] In some embodiments, the modifications listed above can be combined to provide modified nucleosides and nucleotides having two, three, four, or more modifications. For example, in some embodiments, the modified nucleosides or nucleotides may have modified sugars and modified nucleic acid bases. In some embodiments, all bases of the gRNA are modified. In some embodiments, each of the phosphate groups of the gRNA molecule is replaced with a phosphorothioate group.

[0255] In some embodiments, a software tool can be used to optimize the selection of gRNAs within a user's target sequence, for example, minimizing overall off-target activity across the genome. Off-target activity may be other than cleavage. For example, for each possible gRNA selection using S. pyogenes Cas9, the software tool can identify all potential off-target sequences (before either NAG or NGG PAM) across the genome containing up to a certain number (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) mismatched base pairs. The cleavage efficiency at each off-target sequence can be predicted, for example, using an experimentally derived weighting scheme. Each possible gRNA can then be ranked according to its total predicted off-target cleavage, with higher-ranked gRNAs representing those likely to have the largest on-target and smallest off-target cleavage. Other functions, such as automated reagent design for gRNA vector construction, primer design for on-target measurement assays, and primer design for high-throughput detection and quantification of off-target cleavage via next-generation sequencing, may also be included in the tool.

[0256] The present invention is further described in the following embodiments, which do not limit the scope of the invention as described in the claims. [Examples]

[0257] Example 1 Evaluation of Parkin transgene variants A series of plasmid vectors were generated to evaluate the expression of Parkin transgene variants. The expression cassette (Figure 1) contained the CMV early-stage (IE) enhancer / promoter and 5'UTR, the Parkin transgene 2A and 3'UTR linked to enhanced green fluorescent protein (eGFP), and the rabbit globin polyadenylation sequence (PolyA) in 5'-3' order. For the Parkin transgene, either the wild-type human PRKN(WT) sequence or one of four codon-optimized variants (CO1-CO4) was tested. Sequences of the wild-type and codon-optimized Parkin transgenes are provided in SEQ ID NOs. 27, 35, 36, 37, and 38.

[0258] One consideration in the design of codon-optimized CO1-CO4 is the number of CpG sites. A CpG island is a DNA region in which a cytosine nucleotide is followed by a guanine nucleotide in a linear sequence of bases along its 5'→3' direction. It is well known that CpG islands (typically defined as a polynucleotide sequence of at least 200 bp, a GC percentage greater than 50%, and an observed-to-expected CpG ratio greater than 60%) are associated with vector immunogenicity. Therefore, it was expected that reducing the number of CpG sites would improve vector performance by reducing immunogenicity. The number of CpG sites (5'-CG-3') in each Parkin codon variant is shown in Table 9.

[0259] (Table 9) CpG sites in exemplary parkin codone variants TIFF0007869633000047.tif54128

[0260] SH-SY5Y cells. Gene expression in nervous system cells was evaluated using the human neuroblastoma cell line, SH-SY5Y. SH-SY5Y cells were cultured in 96-well plates at a seeding density of 10,000 cells per well. After 24 hours, cells were transfected with 0.10 μg of WT, CO1, CO2, or CO3 plasmids conjugated with 4 μL of Fugene HD per 1 μg plasmid. Cells were cultured for a further 48 hours and then assayed for eGFP expression by fluorescence microscopy.

[0261] Representative micrographs are shown in Figures 2A–2J. Controls included untransfected cells as a negative control (Figure 2A) and a known plasmid as a positive control (Figure 2B). Expression cassettes were tested in duplication: WT (Figures 2C–2D), CO1 (Figures 2E–2F), CO2 (Figures 2G–2H), and CO3 (Figures 2I–2J). The percentage of GFP+ cells and the fluorescence intensity of GFP+ cells were plotted in Figures 2H and 2K, respectively. Cell lysates were collected 7 days after transfection and assayed for parkin by enzyme-linked immunosorbent assay (ELISA), as shown in Figure 3. These experiments showed that CO1–CO3 did not increase parkin expression, but rather decreased the percentage of cells that actually expressed parkin and the overall level of parkin expression.

[0262] Next, the codon variant CO4 was tested against WT and CO1. Fluorescence micrographs of SH-SY5Y cells for untransfection-negative control (Figure 4A), WT (Figure 4B), CO1 (Figure 4C), or CO4 (Figure 4D) are shown, and the results were quantified in terms of GFP+ percentage (Figure 4E) and intensity (Figure 4F). Cell lysates were assayed for parkin expression by ELISA (Figure 5). In SH-SY5Y cells, CO4 expression was similar to that of WT and CO1.

[0263] iPSC-derived Parkin knockout dopaminergic progenitor cells. Human induced pluripotent stem cell (iPSC)-derived Parkin knockout dopaminergic progenitor cells were cultured in 96-well plates at a seeding density of 10,000 cells per well. After 7 days, cells were transfected with 0.15 μg of WT, CO1, and CO4 plasmids (conjugated with 4 μL of ViaFect per 1 μg plasmid). Transgene expression was evaluated 7 days post-transfection in untransfected wells (Figure 6A), WT (Figure 6B), CO1 (Figure 6C), or CO4 (Figure 6D) wells. As shown in Figure 6E, the three transgenes (WT, CO1, and CO4) resulted in similar percentages of GFP+ cells, but surprisingly, CO4 showed a 20% increase in GFP intensity compared to WT.

[0264] Example 2 Selection of a cassette for CO4 parkin transgene expression To evaluate other regulatory elements, various AAV expression cassettes were constructed in the form of transplasms for use in a helper-free AAV packaging system. The AAV expression cassette (Figure 7) contains the 5' ITR of AAV2, with or without a CMV enhancer (Enh), a promoter selected from Table 5, the 5' untranslated region (UTR) selected from Table 6, the Parkin transgene variant CO4 (SEQ ID NO: 38), the 3' untranslated region selected from Table 7, a polyadenylated sequence (PolyA) selected from Table 8, and the 3' ITR of AAV2 in 5' to 3' order. Figures of the cassettes and various elements are provided in Figure 8. For detection of transgene expression in in vitro studies, a polynucleotide sequence (SEQ ID NO: 80) encoding an N-terminal flag / HA tag (SEQ ID NO: 81) was inserted after the start codon on each Parkin transgene sequence listed in Table 10.

[0265] SH-SY5Y cells. SH-SY5Y cells were cultured in 24-well plates at a seeding density of 50,000 cells per well. After 24 hours, the cells were transfected with a 0.75 μg plasmid conjugated with 4 μL of Fugene6 per 1 μg plasmid. The cells were cultured for a further 48 hours, and Parkin expression was assayed by ELISA on cell lysates using an anti-Parkin primary antibody. The results for each construct are shown in Table 10 and graphed in Figure 9. Notably, protein expression was lower with nerve-specific promoters (Syn and CaMKIIa).

[0266] (Table 10) Parkin expression in SH-SY5Y cells (average of replication) TIFF0007869633000048.tif173170

[0267] iPSC-derived Parkin knockout dopaminergic progenitor cells. iPSC-derived Parkin knockout dopaminergic progenitor cells were cultured in 96-well plates at a seeding density of 10,000 cells per well. After 15 days, the cells were transfected with a 0.15 μg plasmid conjugated with 4 μL of ViaFect per 1 μg plasmid. Parkin expression was assayed at 2 days later by ELISA on cell lysates using an anti-Parkin primary antibody. The results for each construct are shown in Table 11 and graphed in Figure 10 (three copies are shown for each construct).

[0268] (Table 11) Parkin expression (average of replication) in iPSC-derived cells TIFF0007869633000049.tif113160

[0269] In fluorescence microscopy, transfected cells were fixed and stained 8 days after transfection and imaged using bright-field imaging (Figures 11A and 12A), or immunofluorescence for Parkin (Figures 11B and 12B), the neuron marker NeuN (Figures 11C and 12C), or the astrocyte marker GFAP (Figures 11D and 12D). Figures 12A-12D show magnified images of Figures 11A-11D.

[0270] Example 3 In vivo and clinical trials of ParkinAAV gene therapy AAV vectors were generated by packaging AAV vector genomes from constructs 1–20 of Example 2, particularly constructs 1, 7, 11, and 15 in Table 11. Tests were conducted in rats, and one or more constructs were selected for testing in non-human primates (NHPs). Dose-finding studies were performed, and the starting dose for clinical trials was determined by observing protein expression and measuring toxicity.

[0271] Clinical trials were conducted in subjects identified as having recessive mutations in the PRKN gene (also known as PRK2). The optimal dose was determined using protein expression and observed toxicity. Efficacy was evaluated using improvements in the Unified Parkinson's Disease Rating Scale (UPDRS).

[0272] Example 4 In vitro testing Recombinant cassettes were tested in human neuroblastoma cell line (SH-SY5Y). (See Jiang et al. Extracellular dopamine induces the oxidative toxicity of SH-SY5Y cells. Synapse. 2008 Nov;62(11):797-803. doi:10.1002 / syn.20554). The target genes tested were the Parkinson's protein 2 E3 ubiquitin protein ligase (PARK 2) gene, the PTEN-induced putative kinase 1 (PINK1) gene, the protein deglycase DJ-1 (DJ-1) gene, the leucine-rich repeat kinase 2 (LRRK2) gene, the alpha-synuclein (SCNA) gene, the oncogene c-Rel (c-Rel) gene, the ubiquitin-like modifier-activating enzyme (ATG7) gene, the synaptic vesicle amine transporter (VMAT2) gene, or the glucocerebrosidase (GBA) gene.

[0273] Each cell line was treated with a recombinant gene therapy vector for each target gene. Expression of the target gene increased. Function of the target gene improved. In some cases, improved mitophagy, reduced cytotoxicity, and / or reduced oxidative stress were observed.

[0274] Example 5 In vivo testing Rodents or non-human primates with one or more known mutation combinations in the target gene were treated with recombinant gene therapy vectors for the target gene. The target genes tested were the Parkinson's protein 2 E3 ubiquitin protein ligase (PARK 2) gene, the PTEN-induced putative kinase 1 (PINK 1) gene, the protein deglycase DJ-1 (DJ-1) gene, the leucine-rich repeat kinase 2 (LRRK 2) gene, the alpha-synuclein (SCNA) gene, the oncogene c-Rel (c-Rel) gene, the ubiquitin-like modifier-activating enzyme (ATG7) gene, the synaptic vesicle amine transporter (VMAT 2) gene, or the glucocerebrosidase (GBA) gene.

[0275] The study subjects were those with one or more of the following characteristics: dopamine (DA) loss, loss of 20% or more substantia nigra cells, dyskinesia, Lewy bodies, mitochondrial dysfunction, ROS, inflammation, other motor behavior disorders, and neurodegenerative symptoms.

[0276] The animals in which the methods of this disclosure are tested include non-disease and diseased animals, as well as mutant and non-mutant animals. Animal models in which the methods of this disclosure are tested and exemplary motor behavior readings are provided in Table 12.

[0277] (Table 12) TIFF0007869633000050.tif189160

[0278] The subjects in the study were to show improvement in any of the motor behavior readings or one or more of the following characteristics: increased DA in the striatum, increased substantia nigra cells, decreased Lewy bodies, improved behavior or other motor dysfunction, improved mitochondrial function, decreased inflammatory markers, or improved lifespan.

[0279] Example 6 Clinical trials The diagnosis of the parkin type in early-onset Parkinson's disease is primarily considered in individuals with early-onset Parkinson's disease (under 40 years of age), especially when autosomal recessive inheritance is suspected. PRKN (formerly known as PARK2), the gene encoding the parkin protein, is the main gene known to cause the parkin type in early-onset Parkinson's disease when pathogenic variants are identified. The diagnosis of the parkin type in early-onset Parkinson's disease can only be confirmed if pathogenic variants are identified on both alleles of PRKN (i.e., the individual is either homozygous for the same pathogenic allele or heterozygous for two different pathogenic alleles). The frequency of variant detection varies depending on family history and age of onset.

[0280] Table 13 provides other mutations associated with Parkinson's disease.

[0281] (Table 13) TIFF0007869633000051.tif200160

[0282] Human subjects with one or more mutations associated with Parkinson's disease are treated with recombinant gene therapy vectors encoding the wild-type or functional variant of the mutated gene. Subjects show improvement in any of the motor behavior readings listed in Table 12, or in any of the following features: increased dopamine in the brain, particularly in the substantia nigra; increased number of dopaminergic neurons; increased expression of the gene of interest, including PRKN; or improved Unified Parkinson's Disease Rating Scale (UPDRS).

[0283] All of the above-mentioned U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications referenced herein and / or listed in the application data sheets are incorporated herein by reference in their entirety.

[0284] The present invention is broadly described herein and may be embodied in other specific forms without departing from its structure, method, or other essential features as claimed below. The embodiments described are to be considered illustrative and non-limiting in all aspects. Accordingly, the scope of the invention is indicated by the appended claims rather than by the foregoing description. All modifications within the meaning and scope of equivalence of the claims are to be accepted within their respective scopes.

Claims

1. It includes an expression cassette containing a transgene encoding E3 ubiquitin protein ligase (PARK2), The aforementioned transgene is operably linked to a promoter sequence that is active in eukaryotes, and The aforementioned introduced gene includes sequence number 38, Recombinant gene therapy vector.

2. The vector according to claim 1, wherein the promoter sequence is selected from the human synapsin 1 (Syn), human beta-actin (HuBa), chicken beta-actin (CBA), cytomegalovirus (CMV), human EF1-alpha (EF1-α), and human CamKIIa (CaMKIIa) promoter sequences, and optionally the promoter sequence is selected from sequence numbers 63, 59-62, and 64 or sequences having at least 95% identity thereto.

3. The vector according to claim 1 or claim 2, wherein the expression cassette further comprises a CMV enhancer.

4. The vector according to any one of claims 1 to 3, wherein the expression cassette further comprises human beta-actin exon / intron, chicken beta-actin exon / intron + rabbit globin intron, synapsin-1 exon, CMV IE exon, TPL-eMLP (adenovirus-derived enhancer element), human EF1a intron / exon, and a 5' untranslated region (UTR) selected from 5' UTR human CamKIIa, and optionally the 5' untranslated region (UTR) has a sequence selected from SEQ ID NOs.65 to 71.

5. The vector according to any one of claims 1 to 4, wherein the expression cassette further comprises a 3' untranslated region selected from WPRE(x) (mutant woodchuck hepatitis regulatory element), R2V17 (HepB-derived enhancer element), 3'UTR (globin), and WPRE(r), and optionally the 3' untranslated region has a sequence selected from SEQ ID NOs. 72 to 75.

6. The vector according to any one of claims 1 to 5, wherein the expression cassette further comprises a polyadenylated sequence (PolyA) selected from human growth hormone (pAGH-Hs), rabbit globin (pAGlobin-Oc), and bovine growth hormone (pAGH-Bt) polyadenylated sequences, and optionally the polyadenylated sequence is selected from sequence numbers 78, 76, and 77.

7. The vector according to any one of claims 1 to 6, wherein the expression cassette shares at least 95% sequence identity with any one of sequence numbers 45, 39-44, and 46-58.

8. The expression cassette, Syn promoter, the transgene, WPRE(x), 3'UTR (globin), and pAGH-Hs; HuBA promoter, the transgene, WPRE(x), and pAglobin-Oc; CMV promoter, TPL-eMLP enhancer, the transgene, WPRE(r), and pAglobin-Oc; Syn promoter, the transgene, WPRE(r), 3'UTR (globin), and pAGH-Bt; CBA promoter, the transgene, and pAGH-Bt; EF1α promoter, the aforementioned transgene, and pAglobin-Oc; HuBA promoter, the transgene, R2V17, and pAGH-Bt; CaMKIIa promoter, the transgene, WPRE(r), and pAGH-Hs; CMV promoter, TPL-eMLP5' enhancer, the transgene, WPRE(r), and pAGH-Hs; HuBA promoter, the transgene, and pAGH-Hs; CMV and TPL promoter, eMPL, the transgene, R2V17, 3'UTR (globin), and pAGH-Bt; EF1α promoter, the transgene, WPRE(r), and pAGH-Bt; Syn promoter, the transgene, R2V17, and pAglobin-Oc; CaMKIIa promoter, the transgene, R2V17, and pAglobin-Oc; CBA promoter, the transgene, WPRE(x), 3'UTR (globin), and pAGH-Hs; CBA promoter, the transgene, 3'UTR (globin), and pAglobin-Oc; CaMKIIa promoter, the transgene, R2V17, and pAGH-Bt; EF1α promoter, the transgene, R2V17, 3'UTR (globin), and pAGH-Hs; CMV promoter, the transgene, R2V17, 3'UTR (globin), and pAGH-Hs; or CMV promoter, the transgene, and pAGH-Hs It includes in the order from 5' to 3', The vector according to any one of claims 1 to 7.

9. It is an adeno-associated virus (AAV) vector, and optionally: (a) The vector includes two AAV reverse terminal repeats (ITRs) adjacent to the expression cassette; and / or (b) The AAV has serotype AAV9, AAV1, AAV2, AAV5, AAV8, AAVrh10, or AAVrh74; and / or (c) The vector includes a self-complementary AAV or a single-stranded AAV; and / or (d) The AAV is wild-type AAV or modified AAV; and / or (e) The AAV comprises a capsid protein having at least 95% identity with wild-type VP1, VP2, or VP3 capsid protein. The vector according to any one of claims 1 to 8.

10. A host cell comprising the vector according to any one of claims 1 to 9.

11. A method for in vitro inhibiting the degeneration or death of dopaminergic neurons containing mutations in genes associated with Parkinson's disease (PD), This includes contacting the neuron with the recombinant gene therapy vector described in any one of claims 1 to 9, After contact with the recombinant gene therapy vector, the neurons express PARK2, Optionally, the neuron contains a mutation in the PARK2 gene; and / or Optionally, the neurons express a reduced amount of alpha-synuclein and / or contain a reduced amount of Lewy bodies after contact with the recombinant gene therapy vector; and / or Optionally, the neurons express a reduced amount of monoamine oxidase after contact with the recombinant gene therapy vector; and / or Optionally, the neuron produces and / or releases an increased amount of dopamine after contact with the recombinant gene therapy vector; and / or Optionally, the neurons receive increased mitophagy after contact with the recombinant gene therapy vector; and / or Optionally, the neuron expresses a smaller amount of monoamine oxidase compared to the amount expressed in a neuron not in contact with the recombinant gene therapy vector, and optionally, the smaller amount is at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% lower than the amount expressed in the neuron not in contact with the recombinant gene therapy vector; and / or Optionally, the neuron produces and / or releases an increased amount of dopamine compared to the amount of dopamine produced and / or released by a neuron not in contact with the recombinant gene therapy vector, and optionally, the increased amount is at least 5%, 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 twice, at least three times, at least four times, at least five times, or at least ten times greater than the amount produced and / or released by the neuron not in contact with the recombinant gene therapy vector; and / or Optionally, the neuron receives an increased amount of autophagy compared to the amount of autophagy received by a neuron not in contact with the recombinant gene therapy vector, and optionally, the increased amount is at least 5%, 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 twice, at least three times, at least four times, at least five times, or at least ten times greater than the amount received by the neuron not in contact with the recombinant gene therapy vector. method.

12. A pharmaceutical agent for treating or inhibiting the onset of Parkinson's disease (PD) in a subject who has or is at risk of having said PD, comprising a recombinant gene therapy vector according to any one of claims 1 to 9.

13. The pharmaceutical product according to claim 12, wherein the PD is early-onset PD, and optionally, early-onset autosomal recessive PD.

14. The pharmaceutical product according to claim 12 or 13, wherein the subject contains a mutation in the PARK2 gene, and optionally, the PARK2 contains the amino acid sequence shown in SEQ ID NO:

1.

15. The subject may be introduced into the subject by systemic, parenteral, intravenous, cerebral, cerebrospinal fluid, intrathecal cavity, cisterna magna, putamen, hippocampus, striatum, or intracerebroventricular administration, optionally. By intrathecal injection in the Trendelenburg incline, or By directly injecting it into the compact region of the substantia nigra of the brain, or By introducing the recombinant gene therapy vector into the brain or cerebrospinal fluid (CSF) of the subject, The above-mentioned target will be introduced, The pharmaceutical product according to any one of claims 12 to 14.

16. 1 x 10 per kilogram of the aforementioned body weight ９ ~1x10 １４ The gene therapy vector in the vector genome (vg / kg) is administered to the subject, or 1 x 10 per kilogram of the aforementioned body weight ７ ~1x10 ９ The gene therapy vector in the form of a vector genome (vg / kg) is administered to the subject. A pharmaceutical product according to any one of claims 12 to 15, characterized in that

17. (a) The number of dopaminergic neurons in the subject after administration of the drug is greater than the number of dopaminergic neurons in the subject before administration of the drug; (b) The level of dopamine in the subject after administration of the drug is greater than the level of dopamine in the subject before administration of the drug; (c) The number of dopaminergic neurons in the subject treated by the drug increases compared to the number of dopaminergic neurons in the subject not treated in the same way; (d) The level of dopamine in the subject treated by the drug increases compared to the level of dopamine in the subject not treated in the same way; (e) The level of dopamine in the substantia nigra of the subject treated by the drug increases compared to the level of dopamine in the substantia nigra of the subject not treated in the same way; (f) The level of PRKN in the subject's CSF after administration of the drug is greater than the level of PRKN in the subject's CSF before administration of the drug; (g) The Unified Parkinson's Disease Rating Scale (UPDRS) score of the subject before administration of the drug is improved compared to the subject's UPDRS score before administration of the drug; (h) The level of PRKN in the CSF of the subject treated with the drug increases compared to the level of PRKN in the CSF of the subject not treated in the same way; (i) The UPDRS score of the subject treated by the drug is improved compared to the UPDRS score of the subject not treated in the same way; and / or (j) The target neuron expresses a reduced amount of alpha-synuclein and / or contains a reduced amount of Lewy bodies after contact with the recombinant gene therapy vector. A pharmaceutical product according to any one of claims 12 to 16.

18. A pharmaceutical agent for inhibiting the degeneration or death of dopaminergic neurons containing a mutation in a gene associated with Parkinson's disease (PD), comprising a recombinant gene therapy vector according to any one of claims 1 to 9, characterized in that the neurons express PARK2 after contact with the recombinant gene therapy vector, and optionally the neurons contain a mutation in the PARK2 gene.

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