CN117018231B

CN117018231B - Gene therapy for treatment of neuropathy and use thereof

Info

Publication number: CN117018231B
Application number: CN202311039041.7A
Authority: CN
Inventors: 柳青慕; 牛德强; 朱振东; 熊燃
Original assignee: Kehui Zhiyao Shenzhen New Drug Research Center Co ltd
Current assignee: Kehui Zhiyao Shenzhen New Drug Research Center Co ltd
Priority date: 2023-08-16
Filing date: 2023-08-16
Publication date: 2024-05-10
Anticipated expiration: 2043-08-16
Also published as: CN117018231A

Abstract

The invention belongs to the field of gene therapy, and in particular relates to gene therapy for treating neuropathy and application thereof. The invention provides a gene therapy method, which relates to a recombinant nucleic acid molecule which codes for human glucocerebrosidase GCase and neurotrophic factors, and also provides a recombinant vector, a recombinant adeno-associated virus, a host cell and a pharmaceutical composition which comprise the recombinant nucleic acid molecule and are used for treating neuropathy caused by reduction of the activity of the GCase, such as Parkinson's disease, type II and type III Gaohu's disease, and the activity of the GCase and the anti-injury and anti-apoptosis capacity of neurons in a subject can be effectively improved by a systemic injection or local injection administration mode.

Description

Gene therapy for treatment of neuropathy and use thereof

Technical Field

The invention belongs to the field of gene therapy, and in particular relates to gene therapy for treating neuropathy and application thereof.

Background

Parkinson is the second largest neurodegenerative disease in the world, over 580 ten thousand patients worldwide are estimated, and as the population ages, the number of patients will increase further. Studies have shown that a decrease in GCase enzyme activity caused by GBA1 gene mutation not only results in Gaucher's Disease, but also increases the risk of early parkinson's Disease in the carrier. The decrease of GCase activity in nerve cells such as dopamine neurons can cause accumulation of misfolded alpha synuclein (alpha-synuclein) to form a Lewy Body, damage and death of nerve cells, and finally parkinsonism of a carrier occurs. In addition to the decrease in GCase activity caused by GBA1 gene mutation, aging also causes decrease in vivo GCase activity, which is one of the reasons for increased Parkinson's disease rate caused by aging.

Parkinson's disease is currently an incurable disease, and existing clinical interventions can only alleviate the symptoms of patients. There are currently three main therapeutic strategies for GBA1 mutant carrying patients. The first is a small molecule chemical. The therapeutic drugs have two strategies, namely, the therapeutic drugs are used as molecular chaperones of the GCase, so that the activity of the mutant GCase is improved; secondly, acts on the upstream of the metabolic pathway to reduce the accumulation of enzyme substrates. The second is enzyme replacement therapy, which uses gene recombination techniques to express GCase in vitro and then re-inject it into the body. The third is gene therapy, which uses vector means to deliver the wild-type GBA1 into the body and allow it to reach the desired expression level under the action of cis-regulatory elements. Both of the first two methods require continuous administration throughout the life, and the third, while achieving long-term efficacy with a single administration, delivers only the wild-type GBA1 gene and does not rescue neurons that have been damaged in the patient.

Therefore, aiming at the neurodegenerative diseases such as Parkinson's disease and type II and III Gaoshi's disease caused by the reduced GCase activity, which are not available in the prior art, a novel gene medicine for treating the neurodegenerative diseases is explored and researched, and has important significance.

Reference is made to:

Kelly E. Glajch, Tim E. Moors, Yi Chen, et al. Wild-type GBA1 increases the α-synuclein tetramer-monomer ratio, reduces lipid-rich aggregates, and attenuates motor and cognitive deficits in mice. PANS. 2021, 118（31）：e2103425118.

Sangjune Kin, Seung Pil Yun, Saebom Lee, et al. GBA1 deficiency negatively affects physiological α-synuclein tetramers and related multimers. PANS. January 23, 2018, 115 (4): 798-803.

Yumiko V. Taguchi, Jun Liu, Jiapeng Ruan, et al. Glucosylsphingosine Promotes α-Synuclein Pathology in Mutant GBa-associated Parkinson's Disease. The Journal of Neuroscience, October 4, 2017, 37(40): 9617-9631.

Disclosure of Invention

In view of the deficiencies of the existing products and techniques, the present invention is directed to providing gene therapy for the treatment of neuropathy and uses thereof. The present invention provides recombinant nucleic acid molecules, recombinant vectors, recombinant adeno-associated viruses, host cells, etc. for use in the treatment of neuropathy. The gene medicine is a composition of GCase nucleic acid and neurotrophic factor nucleic acid, not only can generate high-activity GCase capable of effectively clearing away Lewy Body and preventing new pathological alpha synuclein from depositing, but also can reduce/save damage to neurons caused by internal pressure environment and prolong the service life of the neurons.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

It is a first object of the present invention to provide a gene therapy involving a recombinant nucleic acid molecule encoding human glucocerebrosidase GCase and neurotrophic factor.

Preferably, the neurotrophic factor comprises human brain dopamine neurotrophic factor CDNF, human brain-derived neurotrophic factor BDNF, human glial cell-derived neurotrophic factor GDNF, and human nerve rank protein NRTN.

Preferably, the sequence encoding human glucocerebrosidase GCase is linked to the sequence encoding a neurotrophic factor by a gene linker element; the amino acid sequence of the coded human glucocerebrosidase GCase is SEQ ID NO.1; the amino acid sequence of the human brain dopamine neurotrophic factor CDNF is SEQ ID NO.2, the amino acid sequence of the human brain-derived neurotrophic factor BDNF is SEQ ID NO.3, the amino acid sequence of the human glial cell-derived neurotrophic factor GDNF is SEQ ID NO.4, and the amino acid sequence of the human nerve rank protein NRTN is SEQ ID NO.5.

MEFSSPSREECPKPLSRVSIMAGSLTGLLLLQAVSWASGARPCIPKSFGYSSVVCVCNATYCDSFDPPTFPALGTFSRYESTRSGRRMELSMGPIQANHTGTGLLLTLQPEQKFQKVKGFGGAMTDAAALNILALSPPAQNLLLKSYFSEEGIGYNIIRVPMASCDFSIRTYTYADTPDDFQLHNFSLPEEDTKLKIPLIHRALQLAQRPVSLLASPWTSPTWLKTNGAVNGKGSLKGQPGDIYHQTWARYFVKFLDAYAEHKLQFWAVTAENEPSAGLLSGYPFQCLGFTPEHQRDFIARDLGPTLANSTHHNVRLLMLDDQRLLLPHWAKVVLTDPEAAKYVHGIAVHWYLDFLAPAKATLGETHRLFPNTMLFASEACVGSKFWEQSVRLGSWDRGMQYSHSIITNLLYHVVGWTDWNLALNPEGGPNWVRNFVDSPIIVDITKDTFYKQPMFYHLGHFSKFIPEGSQRVGLVASQKNDLDAVALMHPDGSAVVVVLNRSSKDVPLTIKDPAVGFLETISPGYSIHTYLWRRQ*（SEQ ID NO.1）;

MWCASPVAVVAFCAGLLVSHPVLTQGQEAGGRPGADCEVCKEFLNRFYKSLIDRGVNFSLDTIEKELISFCLDTKGKENRLCYYLGATKDAATKILSEVTRPMSVHMPAMKICEKLKKLDSQICELKYEKTLDLASVDLRKMRVAELKQILHSWGEECRACAEKTDYVNLIQELAPKYAATHPKTEL*（SEQ ID NO.2）;

MQRWKAAALASVLCSSVLSIWMCREGLLLSHRLGPALVPLHRLPRTLDARIARLAQYRALLQGAPDAMELRELTPWAGRPPGPRRRAGPRRRRARARLGARPCGLRELEVRVSELGLGYASDETVLFRYCAGACEAAARVYDLGLRRLRQRRRLRRERVRAQPCCRPTAYEDEVSFLDAHSRYHTVHELSARECACV*（SEQ ID NO.3）;

MKLWDVVAVCLVLLHTASAFPLPAGKRPPEAPAEDRSLGRRRAPFALSSDSNMPEDYPDQFDDVMDFIQATIKRLKRSPDKQMAVLPRRERNRQAAAANPENSRGKGRRGQRGKNRGCVLTAIHLNVTDLGLGYETKEELIFRYCSGSCDAAETTYDKILKNLSRNRRLVSDKVGQACCRPIAFDDDLSFLDDNLVYHILRKHSAKRCGCI*（SEQ ID NO.4）;

MTILFLTMVISYFGCMKAHSDPARRGELSVCDSISEWVTAADKKTAVDMSGGTVTVLEKVPVSKGQLKQYFYETKCNPMGYTKEGCRGIDKRHWNSQCRTTQSYVRALTMDSKKRIGWRFIRIDTSCVCTLTIKRGR*（SEQ ID NO.5）.

Preferably, the gene-connecting element comprises, but is not limited to, an internal ribosome entry site sequence IRES, a 2A peptide or a class 2A; such IRES elements include, but are not limited to, encephalomyocarditis virus EMCV; the 2A peptide or the like 2A peptide comprises, but is not limited to, P2A derived from porcine teschovirus and T2A derived from Leptospira Ming virus.

It is a second object of the present invention to provide a recombinant nucleic acid molecule comprising an operably linked promoter.

Preferably, the promoter is the human cytomegalovirus enhancer and chicken β -actin promoter combined promoter CAG, the human cytomegalovirus strong promoter CMV, or the human synapsin I promoter hSyn.

Preferably, the recombinant nucleic acid molecule further comprises an intron such as the simian virus 40 intron SV40, a post-transcriptional regulatory element such as a nucleic acid sequence further comprises the woodchuck hepatitis virus post-transcriptional regulatory element WPRE and a polyadenylation such as SV40 PolyA.

Preferably, the recombinant nucleic acid molecule further comprises an AAV inverted terminal repeat ITR.

Preferably, the AAV inverted terminal repeat ITRs are AAV type 2.

It is another object of the present invention to provide a recombinant vector for gene therapy comprising the recombinant nucleic acid molecule described above, said vector being selected from the group consisting of a plasmid vector and a viral vector.

Preferably, the viral vectors include, but are not limited to, adeno-associated viral vectors, adenoviral vectors, lentiviral vectors, hybrid viral vectors, and phage vectors.

Preferably, the viral vector is selected from adeno-associated viral vectors.

It is another object of the present invention to provide a recombinant adeno-associated virus for use in gene therapy comprising an AAV capsid and a vector genome comprising the recombinant nucleic acid molecule described above.

Preferably, the capsid of the recombinant adeno-associated virus is an AAV2 or AAV9 capsid protein.

It is another object of the present invention to provide an isolated host cell for gene therapy comprising the recombinant nucleic acid molecule, recombinant vector or recombinant adeno-associated virus described above.

It is another object of the present invention to provide a pharmaceutical composition for gene therapy comprising the recombinant nucleic acid molecule, recombinant vector, recombinant adeno-associated virus as described above, and pharmaceutically acceptable carrier and/or other conventional pharmaceutical ingredients.

Preferably, the other conventional pharmaceutical ingredients include preservatives and/or stabilizers.

Preferably, the pharmaceutical composition is administered to the CNS of the patient by systemic injection or local injection.

Preferably, the local injection includes one or more of an intra-brain injection, an intra-parenchymal injection, an intrathecal injection, and an intracisternal injection.

Preferably, the pharmaceutical composition is delivered by convection enhanced CED.

The invention also provides application of the recombinant nucleic acid molecule, the recombinant vector and/or the recombinant adeno-associated virus in preparing medicines for preventing or treating the neuropathy with reduced GCase activity.

Preferably, the recombinant nucleic acid molecule, recombinant vector, recombinant adeno-associated virus, host cell and/or pharmaceutical composition may be administered with another therapy.

Compared with the prior art, the invention has the following beneficial effects:

The invention provides a recombinant nucleic acid molecule, a recombinant vector, a recombinant adeno-associated virus, a host cell and a pharmaceutical composition related to gene therapy for treating neuropathy, which are gene therapy capable of improving the activity of GCase and saving injured neurons, and have the long-term effective effect of single administration and can slow down or even reverse the course of the disease of patients.

Drawings

FIG. 1 is a schematic diagram of the structure of an isolated nucleic acid sequence;

FIG. 2 is a schematic drawing of a rAAV-EGFP plasmid;

FIG. 3 is a schematic representation of a rAAV-GBA1-EGFP plasmid;

FIG. 4 is a schematic representation of a rAAV-GBA1-CDNF plasmid;

FIG. 5 is a schematic drawing of a rAAV-GBA-NRTN plasmid;

FIG. 6 is a schematic representation of a rAAV-GBA1-GDNF plasmid;

FIG. 7 is a schematic representation of a rAAV-GBA1-BDNF plasmid;

FIG. 8 is a graph showing expression of GBA1 and CDNF in rAAV-GBA1-CDNF in HEK293 cells;

FIG. 9 is a graph showing expression of GBA1 and NRTN in rAAV-GBA1-NRTN in HEK293 cells;

FIG. 10 is a graph showing expression of GBA1 and GDNF in rAAV-GBA1-GDNF in HEK293 cells;

FIG. 11 is a graph showing expression of rAAV-GBA1-BDNF in GBA1 and BDNF in HEK293 cells;

FIG. 12 is a graph showing NRTN content in culture supernatant after transfection of rAAV-GBA1-NRTN by HEK293 cells;

FIG. 13 is a graph showing GDNF content in culture supernatants after HEK293 cells are transfected with rAAV-GBA 1-GDNF;

FIG. 14 is a graph showing BDNF content in culture supernatants after HEK293 cells are transfected with rAAV-GBA 1-BDNF;

FIG. 15 is a graph showing the expression level (n=3) of the GBA1 expression product GCase in HEK293 cells after transfection of the rAAV vector;

FIG. 16 is a graph showing the activity (n=3) of the GBA1 expression product GCase in HEK293 cells after transfection of the rAAV vector;

FIG. 17 is a graph showing the GCase activity (n=3) of the GBA1 expression product in cells after transfection of the rAAV vector by 100uM CBE-treated HEK293 cells 4 h;

FIG. 18 is a graph showing the effect of NRTN in a rAAV-GBA1-NRTN vector on the activity of human neural cells, such as SH-SY 5Y;

FIG. 19 is a graph showing the effect of GDNF on the viability of human neural cells, such as SH-SY5Y, in a rAAV-GBA1-GDNF vector;

FIG. 20 is a graph showing the effect of BDNF in a rAAV-GBA1-BDNF vector on the viability of human neural cells such as SH-SY 5Y;

FIG. 21 is a graph showing expression of GBA1 and CDNF after AAV2/2-GBA1-CDNF infection of human neural cells such as SH-SY 5Y;

FIG. 22 is a graph showing changes in expression levels of apoptosis markers (n=3) in human neural cells such as SH-SY5Y, 24h after AAV2/2-GBA1-CDNF infection with 5uM TG at 10 ⁵ vg/cell;

FIG. 23 is a graph showing changes in cell viability (n=3) of AAV2/2-GBA1-CDNF after infection of 5uM TG with 10 ⁶ vg/cell for 24h in human neural cells such as SH-SY 5Y;

FIG. 24 is a graph showing the inhibition of endogenous GCase activity (n=3) of human neuroblastoma SH-SY5Y cells at various concentrations of CBE;

FIG. 25 is a graph showing the expression of alpha-Syn protein and GCase in cells after the gene drugs prepared from rAAV vectors such as rAAV-GBA1-EGFP and rAAV-GBA1-CDNF are applied to a CBE-mediated SH-SY5Y cell model;

FIG. 26 is a graph showing the levels of GCase activity and the α -Syn tetramer to monomer ratios in cells after the gene drug prepared from the rAAV vector acts on a cell model for CBE-mediated inhibition of endogenous GCase activity in nerve cells, wherein FIG. 26 (A) is a graph showing the levels of GCase activity in cells after the gene drug acts on a CBE-mediated SH-SY5Y cell model; FIG. 26 (B) is a graph showing the change in the ratio of alpha-Syn protein tetramer to monomer in cells after the application of a genetic drug to a CBE-mediated cell model.

Detailed Description

The above-described aspects of the present invention will be described in further detail with reference to the following embodiments. It should not be construed that the scope of the above subject matter of the present invention is limited to the following examples.

EXAMPLE 1 construction of the nucleic acid sequences of the invention and expression vectors and recombinant adeno-associated viruses thereof

The gene therapy of the present invention relates to a recombinant nucleic acid molecule encoding human glucocerebrosidase GCase and neurotrophic factors. Wherein the neurotrophic factor comprises human brain dopamine neurotrophic factor CDNF, human brain-derived neurotrophic factor BDNF, human glial cell-derived neurotrophic factor GDNF and human nerve rank protein NRTN. The sequence encoding human glucocerebrosidase GCase is linked to the sequence encoding neurotrophic factor by a genetic connecting element. The amino acid sequences of the coding human glucocerebrosidase GCase, the coding human brain dopamine neurotrophic factor CDNF, the coding human brain-derived neurotrophic factor BDNF, the coding human glial cell-derived neurotrophic factor GDNF and the coding human nerve rank protein NRTN are shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO. 5. Wherein the gene-connecting element comprises, but is not limited to, an internal ribosome entry site sequence IRES, a 2A peptide or a class 2A; such IRES elements include, but are not limited to, EMCV derived from encephalomyocarditis virus; the 2A peptide or the like 2A peptide comprises, but is not limited to, P2A derived from porcine teschovirus and T2A derived from Leptospira Ming virus.

The recombinant nucleic acid molecules of the invention further comprise an operably linked promoter that is the human cytomegalovirus enhancer and chicken β -actin promoter combined promoter CAG, the human cytomegalovirus strong promoter CMV, or the human synapsin I promoter hSyn.

The recombinant nucleic acid molecules of the invention further comprise introns such as the Simian Virus 40 intron SV40, posttranscriptional regulatory elements such as nucleic acid sequences further comprise the woodchuck hepatitis Virus posttranscriptional regulatory element WPRE and polyadenylation such as SV40 PolyA.

The recombinant nucleic acid molecule of the invention further comprises an AAV inverted terminal repeat ITR, wherein the AAV inverted terminal repeat ITR is AAV type 2.

A recombinant vector comprising a recombinant nucleic acid molecule of the invention selected from the group consisting of plasmid vectors and viral vectors, including but not limited to adeno-associated viral vectors, adenoviral vectors, lentiviral vectors, hybrid viral vectors, and phage vectors; preferably selected from adeno-associated viral vectors.

(1) Vector construction

The gene therapy of the present invention relates to an isolated nucleic acid transfer whose sequence 5 'to 3' is in the order of 5'ITR, promoter, SV40 intron, GBA1, linker sequence, neurotrophic factor, WPRE, SV40 polyA and 3' ITR, and the schematic of the nucleic acid sequence of the nucleic acid transfer of the present invention is shown in FIG. 1. The construction of the vector is completed by Guangzhou Pi-type biological technology limited company, sequence information (the amino acid sequences are shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO. 5) for encoding human glucocerebrosidase GCase, encoding human brain dopamine neurotrophic factor CDNF, encoding human brain-derived neurotrophic factor BDNF, encoding human glial cell-derived neurotrophic factor GDNF and encoding human nerve rank protein NRTN, the nucleic acid sequence structure schematic diagram and the vector skeleton are required to be subjected to sequence synthesis and construction of a recombinant adeno-associated virus (rAAV) expression vector, and finally the constructed rAAV expression vectors rAAV-EGFP, rAAV-GBA1-CDNF, rAAV-GBA 1-NRNF, rAAV-GDA 1-GDNF and rAAV-GBA1-BDNF are provided to the inventor in the form of Stbl3 bacterial liquid and plasmid DNA, wherein the constructed vector sequence is shown as SEQ ID NO. 6-11. As shown in FIGS. 2-7, the plasmids are schematic for rAAV-EGFP, rAAV-GBA1-CDNF, rAAV-GBA1-NRTN, rAAV-GBA1-GDNF and rAAV-GBA 1-BDNF.

Sequence information of vector rAAV-EGFP:

CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGCGGCCATTCGGTACAATTCACGCGTCGACATTGATTATTGACTAGCTCTGGTCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACTCGAGGCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGCGGGATCAGCCACCGCGGTGGCGGCCTAGAGTCGACGAGGAACTGAAAAACCAGAAAGTTAACTGGTAAGTTTAGTCTTTTTGTCTTTTATTTCAGGTCCCGGATCCGGTGGTGGTGCAAATCAAAGAACTGCTCCTCAGTGGATGTTGCCTTTACTTCTAGGCCTGTACGGAAGTGTTACTTCTGCTCTAAAAGCTGCGGAATTGTACCCGCGGCCGATCCACCGGTCGCCACCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAGAATTCCGCTCGAGATAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTAGTTCTTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATCTAGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGATGTGGGAGGTTTTTTAAAGCGGCCGCAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGGGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATACGTCAAAGCAACCATAGTACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTTGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGGCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGT（SEQ ID NO.6）;

sequence information of vector rAAV-GBA 1-EGFP:

CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGCGGCCATTCGGTACAATTCACGCGTCGACATTGATTATTGACTAGCTCTGGTCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACTCGAGGCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGCGGGATCAGCCACCGCGGTGGCGGCCTAGAGTCGACGAGGAACTGAAAAACCAGAAAGTTAACTGGTAAGTTTAGTCTTTTTGTCTTTTATTTCAGGTCCCGGATCCGGTGGTGGTGCAAATCAAAGAACTGCTCCTCAGTGGATGTTGCCTTTACTTCTAGGCCTGTACGGAAGTGTTACTTCTGCTCTAAAAGCTGCGGAATTGTACCCGCGGCCGATCCACCGGTCGCCACCATGGAGTTTTCAAGTCCTTCCAGAGAGGAATGTCCCAAGCCTTTGAGTAGGGTAAGCATCATGGCTGGCAGCCTCACAGGATTGCTTCTACTTCAGGCAGTGTCGTGGGCATCAGGTGCCCGCCCCTGCATCCCTAAAAGCTTCGGCTACAGCTCGGTGGTGTGTGTCTGCAATGCCACATACTGTGACTCCTTTGACCCCCCGACCTTTCCTGCCCTTGGTACCTTCAGCCGCTATGAGAGTACACGCAGTGGGCGACGGATGGAGCTGAGTATGGGGCCCATCCAGGCTAATCACACGGGCACAGGCCTGCTACTGACCCTGCAGCCAGAACAGAAGTTCCAGAAAGTGAAGGGATTTGGAGGGGCCATGACAGATGCTGCTGCTCTCAACATCCTTGCCCTGTCACCCCCTGCCCAAAATTTGCTACTTAAATCGTACTTCTCTGAAGAAGGAATCGGATATAACATCATCCGGGTACCCATGGCCAGCTGTGACTTCTCCATCCGCACCTACACCTATGCAGACACCCCTGATGATTTCCAGTTGCACAACTTCAGCCTCCCAGAGGAAGATACCAAGCTCAAGATACCCCTGATTCACCGAGCCCTGCAGTTGGCCCAGCGTCCCGTTTCACTCCTTGCCAGCCCCTGGACATCACCCACTTGGCTCAAGACCAATGGAGCGGTGAATGGGAAGGGGTCACTCAAGGGACAGCCCGGAGACATCTACCACCAGACCTGGGCCAGATACTTTGTGAAGTTCCTGGATGCCTATGCTGAGCACAAGTTACAGTTCTGGGCAGTGACAGCTGAAAATGAGCCTTCTGCTGGGCTGTTGAGTGGATACCCCTTCCAGTGCCTGGGCTTCACCCCTGAACATCAGCGAGACTTCATTGCCCGTGACCTAGGTCCTACCCTCGCCAACAGTACTCACCACAATGTCCGCCTACTCATGCTGGATGACCAACGCTTGCTGCTGCCCCACTGGGCAAAGGTGGTACTGACAGACCCAGAAGCAGCTAAATATGTTCATGGCATTGCTGTACATTGGTACCTGGACTTTCTGGCTCCAGCCAAAGCCACCCTAGGGGAGACACACCGCCTGTTCCCCAACACCATGCTCTTTGCCTCAGAGGCCTGTGTGGGCTCCAAGTTCTGGGAGCAGAGTGTGCGGCTAGGCTCCTGGGATCGAGGGATGCAGTACAGCCACAGCATCATCACGAACCTCCTGTACCATGTGGTCGGCTGGACCGACTGGAACCTTGCCCTGAACCCCGAAGGAGGACCCAATTGGGTGCGTAACTTTGTCGACAGTCCCATCATTGTAGACATCACCAAGGACACGTTTTACAAACAGCCCATGTTCTACCACCTTGGCCACTTCAGCAAGTTCATTCCTGAGGGCTCCCAGAGAGTGGGGCTGGTTGCCAGTCAGAAGAACGACCTGGACGCAGTGGCACTGATGCATCCCGATGGCTCTGCTGTTGTGGTCGTGCTAAACCGCTCCTCTAAGGATGTGCCTCTTACCATCAAGGATCCTGCTGTGGGCTTCCTGGAGACAATCTCACCTGGCTACTCCATTCACACCTACCTGTGGCGTCGCCAGGGAAGCGGAGCCACTAACTTCTCCCTGTTGAAACAAGCAGGGGATGTCGAAGAGAATCCCGGGCCAATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAGAATTCCGCTCGAGATAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTAGTTCTTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATCTAGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGATGTGGGAGGTTTTTTAAAGCGGCCGCAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGGGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATACGTCAAAGCAACCATAGTACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTTGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGGCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGT（SEQ ID NO. 7）

sequence information of vector rAAV-GBA 1-CDNF:

CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGCGGCCATTCGGTACAATTCACGCGTCGACATTGATTATTGACTAGCTCTGGTCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACTCGAGGCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGCGGGATCAGCCACCGCGGTGGCGGCCTAGAGTCGACGAGGAACTGAAAAACCAGAAAGTTAACTGGTAAGTTTAGTCTTTTTGTCTTTTATTTCAGGTCCCGGATCCGGTGGTGGTGCAAATCAAAGAACTGCTCCTCAGTGGATGTTGCCTTTACTTCTAGGCCTGTACGGAAGTGTTACTTCTGCTCTAAAAGCTGCGGAATTGTACCCGCGGCCGATCCACCGGTCGCCACCATGGAGTTTTCAAGTCCTTCCAGAGAGGAATGTCCCAAGCCTTTGAGTAGGGTAAGCATCATGGCTGGCAGCCTCACAGGATTGCTTCTACTTCAGGCAGTGTCGTGGGCATCAGGTGCCCGCCCCTGCATCCCTAAAAGCTTCGGCTACAGCTCGGTGGTGTGTGTCTGCAATGCCACATACTGTGACTCCTTTGACCCCCCGACCTTTCCTGCCCTTGGTACCTTCAGCCGCTATGAGAGTACACGCAGTGGGCGACGGATGGAGCTGAGTATGGGGCCCATCCAGGCTAATCACACGGGCACAGGCCTGCTACTGACCCTGCAGCCAGAACAGAAGTTCCAGAAAGTGAAGGGATTTGGAGGGGCCATGACAGATGCTGCTGCTCTCAACATCCTTGCCCTGTCACCCCCTGCCCAAAATTTGCTACTTAAATCGTACTTCTCTGAAGAAGGAATCGGATATAACATCATCCGGGTACCCATGGCCAGCTGTGACTTCTCCATCCGCACCTACACCTATGCAGACACCCCTGATGATTTCCAGTTGCACAACTTCAGCCTCCCAGAGGAAGATACCAAGCTCAAGATACCCCTGATTCACCGAGCCCTGCAGTTGGCCCAGCGTCCCGTTTCACTCCTTGCCAGCCCCTGGACATCACCCACTTGGCTCAAGACCAATGGAGCGGTGAATGGGAAGGGGTCACTCAAGGGACAGCCCGGAGACATCTACCACCAGACCTGGGCCAGATACTTTGTGAAGTTCCTGGATGCCTATGCTGAGCACAAGTTACAGTTCTGGGCAGTGACAGCTGAAAATGAGCCTTCTGCTGGGCTGTTGAGTGGATACCCCTTCCAGTGCCTGGGCTTCACCCCTGAACATCAGCGAGACTTCATTGCCCGTGACCTAGGTCCTACCCTCGCCAACAGTACTCACCACAATGTCCGCCTACTCATGCTGGATGACCAACGCTTGCTGCTGCCCCACTGGGCAAAGGTGGTACTGACAGACCCAGAAGCAGCTAAATATGTTCATGGCATTGCTGTACATTGGTACCTGGACTTTCTGGCTCCAGCCAAAGCCACCCTAGGGGAGACACACCGCCTGTTCCCCAACACCATGCTCTTTGCCTCAGAGGCCTGTGTGGGCTCCAAGTTCTGGGAGCAGAGTGTGCGGCTAGGCTCCTGGGATCGAGGGATGCAGTACAGCCACAGCATCATCACGAACCTCCTGTACCATGTGGTCGGCTGGACCGACTGGAACCTTGCCCTGAACCCCGAAGGAGGACCCAATTGGGTGCGTAACTTTGTCGACAGTCCCATCATTGTAGACATCACCAAGGACACGTTTTACAAACAGCCCATGTTCTACCACCTTGGCCACTTCAGCAAGTTCATTCCTGAGGGCTCCCAGAGAGTGGGGCTGGTTGCCAGTCAGAAGAACGACCTGGACGCAGTGGCACTGATGCATCCCGATGGCTCTGCTGTTGTGGTCGTGCTAAACCGCTCCTCTAAGGATGTGCCTCTTACCATCAAGGATCCTGCTGTGGGCTTCCTGGAGACAATCTCACCTGGCTACTCCATTCACACCTACCTGTGGCGTCGCCAGGGAAGCGGAGCCACTAACTTCTCCCTGTTGAAACAAGCAGGGGATGTCGAAGAGAATCCCGGGCCAATGTGGTGCGCGAGCCCAGTTGCTGTGGTGGCCTTTTGCGCCGGGCTTTTGGTCTCTCACCCGGTGCTGACGCAGGGCCAGGAGGCCGGGGGGCGGCCAGGGGCCGACTGTGAAGTATGTAAAGAATTCTTGAACCGATTCTACAAGTCACTGATAGACAGAGGAGTTAACTTTTCGCTGGACACTATAGAGAAAGAATTGATCAGTTTTTGCTTGGACACCAAAGGAAAAGAAAACCGCCTGTGCTATTATCTAGGAGCCACAAAAGACGCAGCCACAAAGATCCTAAGTGAAGTCACTCGCCCAATGAGTGTGCATATGCCTGCAATGAAGATTTGTGAGAAGCTGAAGAAGTTGGATAGCCAGATCTGTGAGCTGAAATATGAAAAAACACTGGACTTGGCATCAGTTGACCTGCGGAAGATGAGAGTGGCAGAGCTGAAGCAGATCCTGCATAGCTGGGGGGAGGAGTGCAGGGCCTGTGCAGAAAAAACTGACTATGTGAATCTCATTCAAGAGCTGGCCCCCAAGTATGCAGCGACACACCCCAAAACAGAGCTCTGAGAATTCCGCTCGAGATAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTAGTTCTTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATCTAGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGATGTGGGAGGTTTTTTAAAGCGGCCGCAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGGGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATACGTCAAAGCAACCATAGTACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTTGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGGCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGT（SEQ ID NO.8）;

sequence information of vector rAAV-GBA 1-NRTN:

CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGCGGCCATTCGGTACAATTCACGCGTCGACATTGATTATTGACTAGCTCTGGTCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACTCGAGGCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGCGGGATCAGCCACCGCGGTGGCGGCCTAGAGTCGACGAGGAACTGAAAAACCAGAAAGTTAACTGGTAAGTTTAGTCTTTTTGTCTTTTATTTCAGGTCCCGGATCCGGTGGTGGTGCAAATCAAAGAACTGCTCCTCAGTGGATGTTGCCTTTACTTCTAGGCCTGTACGGAAGTGTTACTTCTGCTCTAAAAGCTGCGGAATTGTACCCGCGGCCGATCCACCGGTCGCCACCATGGAGTTTTCAAGTCCTTCCAGAGAGGAATGTCCCAAGCCTTTGAGTAGGGTAAGCATCATGGCTGGCAGCCTCACAGGATTGCTTCTACTTCAGGCAGTGTCGTGGGCATCAGGTGCCCGCCCCTGCATCCCTAAAAGCTTCGGCTACAGCTCGGTGGTGTGTGTCTGCAATGCCACATACTGTGACTCCTTTGACCCCCCGACCTTTCCTGCCCTTGGTACCTTCAGCCGCTATGAGAGTACACGCAGTGGGCGACGGATGGAGCTGAGTATGGGGCCCATCCAGGCTAATCACACGGGCACAGGCCTGCTACTGACCCTGCAGCCAGAACAGAAGTTCCAGAAAGTGAAGGGATTTGGAGGGGCCATGACAGATGCTGCTGCTCTCAACATCCTTGCCCTGTCACCCCCTGCCCAAAATTTGCTACTTAAATCGTACTTCTCTGAAGAAGGAATCGGATATAACATCATCCGGGTACCCATGGCCAGCTGTGACTTCTCCATCCGCACCTACACCTATGCAGACACCCCTGATGATTTCCAGTTGCACAACTTCAGCCTCCCAGAGGAAGATACCAAGCTCAAGATACCCCTGATTCACCGAGCCCTGCAGTTGGCCCAGCGTCCCGTTTCACTCCTTGCCAGCCCCTGGACATCACCCACTTGGCTCAAGACCAATGGAGCGGTGAATGGGAAGGGGTCACTCAAGGGACAGCCCGGAGACATCTACCACCAGACCTGGGCCAGATACTTTGTGAAGTTCCTGGATGCCTATGCTGAGCACAAGTTACAGTTCTGGGCAGTGACAGCTGAAAATGAGCCTTCTGCTGGGCTGTTGAGTGGATACCCCTTCCAGTGCCTGGGCTTCACCCCTGAACATCAGCGAGACTTCATTGCCCGTGACCTAGGTCCTACCCTCGCCAACAGTACTCACCACAATGTCCGCCTACTCATGCTGGATGACCAACGCTTGCTGCTGCCCCACTGGGCAAAGGTGGTACTGACAGACCCAGAAGCAGCTAAATATGTTCATGGCATTGCTGTACATTGGTACCTGGACTTTCTGGCTCCAGCCAAAGCCACCCTAGGGGAGACACACCGCCTGTTCCCCAACACCATGCTCTTTGCCTCAGAGGCCTGTGTGGGCTCCAAGTTCTGGGAGCAGAGTGTGCGGCTAGGCTCCTGGGATCGAGGGATGCAGTACAGCCACAGCATCATCACGAACCTCCTGTACCATGTGGTCGGCTGGACCGACTGGAACCTTGCCCTGAACCCCGAAGGAGGACCCAATTGGGTGCGTAACTTTGTCGACAGTCCCATCATTGTAGACATCACCAAGGACACGTTTTACAAACAGCCCATGTTCTACCACCTTGGCCACTTCAGCAAGTTCATTCCTGAGGGCTCCCAGAGAGTGGGGCTGGTTGCCAGTCAGAAGAACGACCTGGACGCAGTGGCACTGATGCATCCCGATGGCTCTGCTGTTGTGGTCGTGCTAAACCGCTCCTCTAAGGATGTGCCTCTTACCATCAAGGATCCTGCTGTGGGCTTCCTGGAGACAATCTCACCTGGCTACTCCATTCACACCTACCTGTGGCGTCGCCAGGGAAGCGGAGCCACTAACTTCTCCCTGTTGAAACAAGCAGGGGATGTCGAAGAGAATCCCGGGCCAATGCAGCGCTGGAAGGCGGCGGCCTTGGCCTCAGTGCTCTGCAGCTCCGTGCTGTCCATCTGGATGTGTCGAGAGGGCCTGCTTCTCAGCCACCGCCTCGGACCTGCGCTGGTCCCCCTGCACCGCCTGCCTCGAACCCTGGACGCCCGGATTGCCCGCCTGGCCCAGTACCGTGCACTCCTGCAGGGGGCCCCGGATGCGATGGAGCTGCGCGAGCTGACGCCCTGGGCTGGGCGGCCCCCAGGTCCGCGCCGTCGGGCGGGGCCCCGGCGGCGGCGCGCGCGTGCGCGGTTGGGGGCGCGGCCTTGCGGGCTGCGCGAGCTGGAGGTGCGCGTGAGCGAGCTGGGCCTGGGCTACGCGTCCGACGAGACGGTGCTGTTCCGCTACTGCGCAGGCGCCTGCGAGGCTGCCGCGCGCGTCTACGACCTCGGGCTGCGACGACTGCGCCAGCGGCGGCGCCTGCGGCGGGAGCGGGTGCGCGCGCAGCCCTGCTGCCGCCCGACGGCCTACGAGGACGAGGTGTCCTTCCTGGACGCGCACAGCCGCTACCACACGGTGCACGAGCTGTCGGCGCGCGAGTGCGCCTGCGTGTGAGAATTCCGCTCGAGATAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTAGTTCTTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATCTAGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGATGTGGGAGGTTTTTTAAAGCGGCCGCAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGGGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATACGTCAAAGCAACCATAGTACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTTGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGGCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGT（SEQ ID NO.9）;

Sequence information of vector rAAV-GBA 1-GDNF:

CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGCGGCCATTCGGTACAATTCACGCGTCGACATTGATTATTGACTAGCTCTGGTCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACTCGAGGCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGCGGGATCAGCCACCGCGGTGGCGGCCTAGAGTCGACGAGGAACTGAAAAACCAGAAAGTTAACTGGTAAGTTTAGTCTTTTTGTCTTTTATTTCAGGTCCCGGATCCGGTGGTGGTGCAAATCAAAGAACTGCTCCTCAGTGGATGTTGCCTTTACTTCTAGGCCTGTACGGAAGTGTTACTTCTGCTCTAAAAGCTGCGGAATTGTACCCGCGGCCGATCCACCGGTCGCCACCATGGAGTTTTCAAGTCCTTCCAGAGAGGAATGTCCCAAGCCTTTGAGTAGGGTAAGCATCATGGCTGGCAGCCTCACAGGATTGCTTCTACTTCAGGCAGTGTCGTGGGCATCAGGTGCCCGCCCCTGCATCCCTAAAAGCTTCGGCTACAGCTCGGTGGTGTGTGTCTGCAATGCCACATACTGTGACTCCTTTGACCCCCCGACCTTTCCTGCCCTTGGTACCTTCAGCCGCTATGAGAGTACACGCAGTGGGCGACGGATGGAGCTGAGTATGGGGCCCATCCAGGCTAATCACACGGGCACAGGCCTGCTACTGACCCTGCAGCCAGAACAGAAGTTCCAGAAAGTGAAGGGATTTGGAGGGGCCATGACAGATGCTGCTGCTCTCAACATCCTTGCCCTGTCACCCCCTGCCCAAAATTTGCTACTTAAATCGTACTTCTCTGAAGAAGGAATCGGATATAACATCATCCGGGTACCCATGGCCAGCTGTGACTTCTCCATCCGCACCTACACCTATGCAGACACCCCTGATGATTTCCAGTTGCACAACTTCAGCCTCCCAGAGGAAGATACCAAGCTCAAGATACCCCTGATTCACCGAGCCCTGCAGTTGGCCCAGCGTCCCGTTTCACTCCTTGCCAGCCCCTGGACATCACCCACTTGGCTCAAGACCAATGGAGCGGTGAATGGGAAGGGGTCACTCAAGGGACAGCCCGGAGACATCTACCACCAGACCTGGGCCAGATACTTTGTGAAGTTCCTGGATGCCTATGCTGAGCACAAGTTACAGTTCTGGGCAGTGACAGCTGAAAATGAGCCTTCTGCTGGGCTGTTGAGTGGATACCCCTTCCAGTGCCTGGGCTTCACCCCTGAACATCAGCGAGACTTCATTGCCCGTGACCTAGGTCCTACCCTCGCCAACAGTACTCACCACAATGTCCGCCTACTCATGCTGGATGACCAACGCTTGCTGCTGCCCCACTGGGCAAAGGTGGTACTGACAGACCCAGAAGCAGCTAAATATGTTCATGGCATTGCTGTACATTGGTACCTGGACTTTCTGGCTCCAGCCAAAGCCACCCTAGGGGAGACACACCGCCTGTTCCCCAACACCATGCTCTTTGCCTCAGAGGCCTGTGTGGGCTCCAAGTTCTGGGAGCAGAGTGTGCGGCTAGGCTCCTGGGATCGAGGGATGCAGTACAGCCACAGCATCATCACGAACCTCCTGTACCATGTGGTCGGCTGGACCGACTGGAACCTTGCCCTGAACCCCGAAGGAGGACCCAATTGGGTGCGTAACTTTGTCGACAGTCCCATCATTGTAGACATCACCAAGGACACGTTTTACAAACAGCCCATGTTCTACCACCTTGGCCACTTCAGCAAGTTCATTCCTGAGGGCTCCCAGAGAGTGGGGCTGGTTGCCAGTCAGAAGAACGACCTGGACGCAGTGGCACTGATGCATCCCGATGGCTCTGCTGTTGTGGTCGTGCTAAACCGCTCCTCTAAGGATGTGCCTCTTACCATCAAGGATCCTGCTGTGGGCTTCCTGGAGACAATCTCACCTGGCTACTCCATTCACACCTACCTGTGGCGTCGCCAGGGAAGCGGAGCCACTAACTTCTCCCTGTTGAAACAAGCAGGGGATGTCGAAGAGAATCCCGGGCCAATGAAGTTATGGGATGTCGTGGCTGTCTGCCTGGTGCTGCTCCACACCGCGTCCGCCTTCCCGCTGCCCGCCGGTAAGAGGCCTCCCGAGGCGCCCGCCGAAGACCGCTCCCTCGGCCGCCGCCGCGCGCCCTTCGCGCTGAGCAGTGACTCAAATATGCCAGAGGATTATCCTGATCAGTTCGATGATGTCATGGATTTTATTCAAGCCACCATTAAAAGACTGAAAAGGTCACCAGATAAACAAATGGCAGTGCTTCCTAGAAGAGAGCGGAATCGGCAGGCTGCAGCTGCCAACCCAGAGAATTCCAGAGGAAAAGGTCGGAGAGGCCAGAGGGGCAAAAACCGGGGTTGTGTCTTAACTGCAATACATTTAAATGTCACTGACTTGGGTCTGGGCTATGAAACCAAGGAGGAACTGATTTTTAGGTACTGCAGCGGCTCTTGCGATGCAGCTGAGACAACGTACGACAAAATATTGAAAAACTTATCCAGAAATAGAAGGCTGGTGAGTGACAAAGTAGGGCAGGCATGTTGCAGACCCATCGCCTTTGATGATGACCTGTCGTTTTTAGATGATAACCTGGTTTACCATATTCTAAGAAAGCATTCCGCTAAAAGGTGTGGATGTATCTGAGAATTCCGCTCGAGATAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTAGTTCTTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATCTAGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGATGTGGGAGGTTTTTTAAAGCGGCCGCAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGGGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATACGTCAAAGCAACCATAGTACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTTGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGGCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGT（SEQ ID NO.10）;

sequence information of vector rAAV-GBA 1-BDNF:

CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGCGGCCATTCGGTACAATTCACGCGTCGACATTGATTATTGACTAGCTCTGGTCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACTCGAGGCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGCGGGATCAGCCACCGCGGTGGCGGCCTAGAGTCGACGAGGAACTGAAAAACCAGAAAGTTAACTGGTAAGTTTAGTCTTTTTGTCTTTTATTTCAGGTCCCGGATCCGGTGGTGGTGCAAATCAAAGAACTGCTCCTCAGTGGATGTTGCCTTTACTTCTAGGCCTGTACGGAAGTGTTACTTCTGCTCTAAAAGCTGCGGAATTGTACCCGCGGCCGATCCACCGGTCGCCACCATGGAGTTTTCAAGTCCTTCCAGAGAGGAATGTCCCAAGCCTTTGAGTAGGGTAAGCATCATGGCTGGCAGCCTCACAGGATTGCTTCTACTTCAGGCAGTGTCGTGGGCATCAGGTGCCCGCCCCTGCATCCCTAAAAGCTTCGGCTACAGCTCGGTGGTGTGTGTCTGCAATGCCACATACTGTGACTCCTTTGACCCCCCGACCTTTCCTGCCCTTGGTACCTTCAGCCGCTATGAGAGTACACGCAGTGGGCGACGGATGGAGCTGAGTATGGGGCCCATCCAGGCTAATCACACGGGCACAGGCCTGCTACTGACCCTGCAGCCAGAACAGAAGTTCCAGAAAGTGAAGGGATTTGGAGGGGCCATGACAGATGCTGCTGCTCTCAACATCCTTGCCCTGTCACCCCCTGCCCAAAATTTGCTACTTAAATCGTACTTCTCTGAAGAAGGAATCGGATATAACATCATCCGGGTACCCATGGCCAGCTGTGACTTCTCCATCCGCACCTACACCTATGCAGACACCCCTGATGATTTCCAGTTGCACAACTTCAGCCTCCCAGAGGAAGATACCAAGCTCAAGATACCCCTGATTCACCGAGCCCTGCAGTTGGCCCAGCGTCCCGTTTCACTCCTTGCCAGCCCCTGGACATCACCCACTTGGCTCAAGACCAATGGAGCGGTGAATGGGAAGGGGTCACTCAAGGGACAGCCCGGAGACATCTACCACCAGACCTGGGCCAGATACTTTGTGAAGTTCCTGGATGCCTATGCTGAGCACAAGTTACAGTTCTGGGCAGTGACAGCTGAAAATGAGCCTTCTGCTGGGCTGTTGAGTGGATACCCCTTCCAGTGCCTGGGCTTCACCCCTGAACATCAGCGAGACTTCATTGCCCGTGACCTAGGTCCTACCCTCGCCAACAGTACTCACCACAATGTCCGCCTACTCATGCTGGATGACCAACGCTTGCTGCTGCCCCACTGGGCAAAGGTGGTACTGACAGACCCAGAAGCAGCTAAATATGTTCATGGCATTGCTGTACATTGGTACCTGGACTTTCTGGCTCCAGCCAAAGCCACCCTAGGGGAGACACACCGCCTGTTCCCCAACACCATGCTCTTTGCCTCAGAGGCCTGTGTGGGCTCCAAGTTCTGGGAGCAGAGTGTGCGGCTAGGCTCCTGGGATCGAGGGATGCAGTACAGCCACAGCATCATCACGAACCTCCTGTACCATGTGGTCGGCTGGACCGACTGGAACCTTGCCCTGAACCCCGAAGGAGGACCCAATTGGGTGCGTAACTTTGTCGACAGTCCCATCATTGTAGACATCACCAAGGACACGTTTTACAAACAGCCCATGTTCTACCACCTTGGCCACTTCAGCAAGTTCATTCCTGAGGGCTCCCAGAGAGTGGGGCTGGTTGCCAGTCAGAAGAACGACCTGGACGCAGTGGCACTGATGCATCCCGATGGCTCTGCTGTTGTGGTCGTGCTAAACCGCTCCTCTAAGGATGTGCCTCTTACCATCAAGGATCCTGCTGTGGGCTTCCTGGAGACAATCTCACCTGGCTACTCCATTCACACCTACCTGTGGCGTCGCCAGGGAAGCGGAGCCACTAACTTCTCCCTGTTGAAACAAGCAGGGGATGTCGAAGAGAATCCCGGGCCAATGACCATCCTTTTCCTTACTATGGTTATTTCATACTTTGGTTGCATGAAGGCTCACTCTGACCCTGCCCGCCGAGGGGAGCTGAGCGTGTGTGACAGTATTAGTGAGTGGGTAACGGCGGCAGACAAAAAGACTGCAGTGGACATGTCGGGCGGGACGGTCACAGTCCTTGAAAAGGTCCCTGTATCAAAAGGCCAACTGAAGCAATACTTCTACGAGACCAAGTGCAATCCCATGGGTTACACAAAAGAAGGCTGCAGGGGCATAGACAAAAGGCATTGGAACTCCCAGTGCCGAACTACCCAGTCGTACGTGCGGGCCCTTACCATGGATAGCAAAAAGAGAATTGGCTGGCGATTCATAAGGATAGACACTTCTTGTGTATGTACATTGACCATTAAAAGGGGAAGATAGGAATTCCGCTCGAGATAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTAGTTCTTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATCTAGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGATGTGGGAGGTTTTTTAAAGCGGCCGCAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGGGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATACGTCAAAGCAACCATAGTACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTTGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGGCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGT（SEQ ID NO.11）.

(2) Recombinant adeno-associated virus production

HEK293 cells are inoculated into 20 cell culture dishes of 15cm, and rAAV expression vectors, capsid protein plasmid AAV2 (or AAV 9) and auxiliary packaging plasmid pHelper are mixed according to a mole ratio of 1:1:1 cotransfection under PEI mediation. Capsid protein plasmids and helper packaging plasmids were purchased from zhili middlet biotechnology limited. The mass ratio of plasmid to PEI is 1:3, mixing, standing at room temperature for 5min, dropwise adding into a culture dish, culturing at 37deg.C in a 5% CO ₂ incubator for 72h, collecting cells and culture supernatant, centrifuging at 3000g for 5min, separating cells and culture supernatant, adding PEG 8000 and 0.5M sodium chloride with final solubility of 8% into the collected culture supernatant, standing on ice for 3h, centrifuging at 4deg.C for 3000×g for 30min, removing supernatant, mixing the precipitated and collected cells, transferring into a 50mL centrifuge tube, adding 15mL lysate (50 mM Tris-HCl, 100mM NaCl, 0.2M MgCl ₂, pH8.0), repeatedly freezing and thawing in dry ice/ethanol water bath at 37deg.C for three times, adding Benzonase (Sigma, E1014-5 KU) with final solubility of 50U/mL and RNase (Sigma, 101109142001) with final concentration of 10U/mL, centrifuging at 37deg.C in water bath for 30min, adding 0.1% sodium deoxidize for 30min, centrifuging at 2500×g for 10min, and collecting supernatant.

(3) Virus purification

In the present invention, iodixanol (Sigma, D1556) ultracentrifugation (Beckman ultracentrifuge) was used for virus purification. Specifically, from top to bottom, a cell lysate, a 15% iodixanol solution, a 25% iodixanol solution, a 40% iodixanol solution, and a 60% iodixanol solution were sequentially added into an ultracentrifuge tube, and centrifuged at 10 ℃, 350,000Xg for 90min after heat sealing. After centrifugation, the needle was used to withdraw 40% iodixanol layer, and the layer was added to a 50KD ultrafiltration tube (Sigma, UFC 905008), concentrated by ultrafiltration and the virus was finally stored at-80℃in a 0.001% Pluronic F-68 (Bio, A600749)/PBS solution.

(4) Virus titer assay

The virus titer was determined by fluorescent quantitative PCR. The sequence information of the fluorescent quantitative PCR primers is shown in SEQ ID NO.12 and SEQ ID NO. 13. The specific operation is as follows:

Taking 10ug of the rAAV expression plasmid for packaging, carrying out enzyme digestion on HindIII (NEB, # 3104V) overnight, cutting the circular plasmid into linearity, separating by 1% agarose, recovering the target fragment by using a DNA gel recovery kit (Biyun Tian, D0056), and calculating the molar mass of the digested rAAV plasmid according to the formula of molar mass = plasmid base pair number multiplied by 662g/mol. The number of molecules per microliter of post-restriction linear rAAV plasmid was counted according to 1M = 6.02 x10 ²³ molecules. Then, the linear rAAV vector was prepared as 100ul of 1X10 ¹⁰ concentration standard, and then diluted to 10 ⁹、10⁸、10⁷、10⁶、10⁵、10⁴、10³ and 10 ² concentration standard in sequence. The SYBR Green method (Tiangen, FP 215) performs fluorescent quantitative PCR with the following PCR system:

table 1 PCR System

The PCR reaction procedure was:

Table 2 PCR reaction procedure

Forward primer sequence: 5'-CTGACCGCGTTAATCCCACA-3' (SEQ ID NO. 12);

reverse primer sequence: 5'-ACAAGCCGTGATTAAACCAAGA-3' (SEQ ID NO. 13).

5Ul of purified virus was prepared into 33ul of water, 5ul of 10 XDnase buffer, 2ul of DNaseI (Invitrogen, AM 2222), 5ul of 1% Fluronic F-68 reaction system, and incubated at 37℃for 60min. Then 10ul of 10 Xproteinase K buffer, 5ul of 1% Fluronic F-68, 1ul proteinase K (Invitrogen) and 34ul of water were added and incubated for 30min at 55℃and inactivated for 15min at 95 ℃. The virus samples after treatment were diluted 10×, 100×and1000×, and fluorescent quantitative PCR was performed using the same system as the standard, and the titer of the virus samples was calculated from the Ct values obtained. The titer of the viruses used in the invention is more than or equal to 10 ¹³ vg/mL (vg: virus genome). The logarithm of the molecular number of the standard substance is the ordinate, and the Ct value is the abscissa, so that a standard curve is drawn.

Example 2 rAAV efficient expression of vectors in mammalian cells such as HEK293

PEI mediates transfection of HEK293 cells by the rAAV vectors rAAV-GBA1-CDNF, rAAV-GBA1-NRTN, rAAV-GBA1-GDNF and rAAV-GBA1-BDNF, and only PEI and the HEK293 cells transfected with rAAV-EGFP and rAAV-GBA1-EGFP are added as a control group. The method comprises the following steps: HEK293 cells are inoculated into a 6-hole cell culture plate, and when the fusion degree reaches about 70%, the complete culture medium is replaced by 1mL of serum-free culture medium; PEI and rAAV carrier according to mass ratio of 3:1 is added into 500ul serum-free culture medium, vortex mixing is carried out, after standing for 15min at 37 ℃, dropwise added into cells, and gentle shaking mixing is carried out; culturing in a 5% CO ₂ incubator at 37 ℃ for 4 hours; the medium was replaced with 3mL of complete medium; culturing in a 5% CO ₂ incubator at 37 ℃ for 48 hours; cells and supernatant were collected.

(1) Expression of rAAV vectors in HEK293 cells

Extracting total protein in the collected HEK293 cells, and measuring the protein concentration by a BCA method (Thermo: 23227); the expression of the target protein is detected by Western blot, and the specific steps comprise: loading 30ug of the sample into each hole, carrying out electrophoresis separation on 12% SDS-PAGE gel, and transferring the sample; 5% skim milk powder was blocked for 1h and primary antibody （GBA1,Abcam：ab55080;CDNF,Abcam：ab205551;NRNT,Abcam：ab274417;GDNF,Affinity：DF7727;BDNF,Affinity：DF6387;β-actin,Abcam：ab8227）4℃ was incubated overnight; TBST is washed three times, secondary antibody is incubated for 1h at room temperature, TBST is washed three times, and the substrate is added for development.

The experimental results of GBA1 and neurotrophic factor expression (n=3) in the rAAV vector after PEI-mediated transfection of HEK293 cells are shown in fig. 8-11. The results show that: after the rAAV vectors rAAV-GBA1-CDNF, rAAV-GBA1-NRTN, rAAV-GBA1-BDNF and rAAV-GBA1-GDNF are transfected by the PEI, compared with a control group transfected with rAAV-EGFP, the expression of the GBA1 gene expression product GCase is improved by more than 5 times. From FIGS. 8-11, it can also be seen that the endogenous neurotrophic factor CDNF was not substantially expressed in HEK293 cells, and that the CDNF expression level was significantly increased after transfection of the vector rAAV-GBA 1-CDNF. The transfected HEK293 cells and untransfected cells were slightly less expressed after transfection of neurotrophins NRTN, GDNF and BDNF, probably because all three neurotrophins were secreted, and were secreted outside after intracellular processing maturation.

(2) Analysis of NRTN, GDNF and BDNF content in HEK293 cell culture supernatants transfected with rAAV-GBA1-NRTN, rAAV-GBA1-GDNF and rAAV-GBA1-BDNF

The culture supernatants of HEK293 cells of the collected PEI-mediated transfection vectors rAAV-GBA1-NRTN, rAAV-GBA1-GDNF and rAAV-GBA1-BDNF were centrifuged at 12000rpm and 4℃for 10min, and the supernatants were collected and analyzed for the contents of NRTN, GDNF and BDNF by using a commercial ELISA kit (NRTN: enzyme-free Cat No. MM-61013H; GDNF: boshide Cat No. EK0362; BDNF: boshide Cat No. EK0307), and the culture supernatants of HEK293 cells of PEI, transfected rAAV-EGFP and rAAV-GBA1-EGFP were used as control groups, and the specific operation method was as described in the kit.

Following transfection of HEK293 cells with the rAAV vector, the neurotrophic factor content (n=3) in the culture supernatants is shown in fig. 12-14. The results show that the concentration of the neurotrophic factor in the cell culture supernatant transfected with the neurotrophic factor is far higher than that of a control group, namely the concentration of the neurotrophic factor is 2403.55 +/-209.65 pg/mL, the concentration of the GDNF is 2235.6 +/-144.1 pg/mL, the concentration of the BDNF is 2166.05 +/-127.25 pg/mL, and the concentration of the BDNF with higher endogenous expression level in the control group is only 232.72 +/-31.20 pg/mL. This indicates that in the intracellular protein expression analysis described above, the reason why the difference in expression of neurotrophins is not obvious when rAAV-GBA1-NRTN, rAAV-GBA1-GDNF and rAAV-GBA1-BDNF cells are transfected as compared with the control group is that the expressed neurotrophins NRTN, GDNF and BDNF are secreted outside the cells after the processing is mature.

The results show that the rAAV vector can be efficiently expressed in mammalian cells, and the secreted neurotrophic factor can still be normally expressed and secreted outside the cells.

EXAMPLE 3 GCase expressed in mammalian cells by GBA1 in the construction product has an activity

PEI mediates rAAV vector rAAV-GBA1-CDNF, rAAV-GBA1-NRTN, rAAV-GBA1-GDNF and rAAV-GBA1-BDNF to transfect HEK293 cells, and only HEK293 cells transfected with PEI and HEK293 cells transfected with rAAV-EGFP are used as controls. Inoculating the culture medium into a 6-hole cell culture plate, and replacing the complete culture medium with 1mL of serum-free culture medium when the fusion degree reaches about 70%; PEI and rAAV carrier according to mass ratio of 3:1 is added into 500ul serum-free culture medium, vortex mixing is carried out, after standing for 15min at 37 ℃, dropwise added into cells, and gentle shaking mixing is carried out; culturing in a 5% CO ₂ incubator at 37 ℃ for 4 hours; the medium was replaced with 3mL of complete medium; culturing in a 5% CO ₂ incubator at 37 ℃ for 48 hours; cells were collected into 1.5mL centrifuge tubes.

Centrifuging the collected cells at 12000rpm for 1min, and removing the supernatant; adding 1mL PBS, centrifuging at 12000rpm for 1min, and removing supernatant; adding lysate (pH 5.4 citric acid-phosphate buffer, 0.25% Triton X-100, 1mM EDTA, protease-phosphatase inhibitor), stirring, mixing, ice-bath for 30min,12000rpm, and centrifuging for 10 min; the BCA method measures total protein concentration, adds samples to 96 Kong Baiban ug/well and supplements the volume to 50 ul/well; 50ul of substrate 4-methylumbelliferyl beta-D-glucoside working solution (pH 5.4 citric acid-phosphate buffer, 0.25% Triton X-100, 1mM EDTA, 1% BSA, 5mM 4-methylumbelliferyl beta-D-glucoside) was added, and the mixture was incubated at 37℃for 15min; adding an equal volume of stop solution (pH 12.5,1M glycine solution); biotek SynergyH1 detection by a full-function microplate reader (fluorescence: ex360, em 460), only lysate wells were added as blank.

After the HEK293 cells are transfected with the rAAV vector, the expression quantity and the activity experimental result of the GBA1 expression product GCase in the cells are shown in figures 15-16. The result shows that after the rAAV vector mediated by PEI transfects HEK293 cells, the intracellular GCase enzyme activity is increased by more than 3 times (GBA 1-CDNF 5.5 times, GBA1-NRNT 3.6.6 times, GBA1-GDNF 5.9 times and GBA-BDNF 4.6 times) compared with a control group, which is consistent with the trend of increasing the expression level of GCase protein.

In summary, the expression product GCase of GBA1 in the rAAV vector in mammalian cells such as HEK293 has enzymatic activity.

Example 4 rAAV expression of GBA1 product in the vector is still active in mammalian cells with reduced endogenous GCase activity, such as HEK293

CBE (conduritol-beta-epoxy) is a small molecule inhibitor of GCase, can inhibit the activity of enzyme on protein level, and is often used for constructing Gauss disease model. In the study, HEK293 cells are inoculated into a 6-hole cell culture plate, 100uM PBS-CBE solution (MedBio, MED 17946) with the final concentration is added when the cell fusion degree reaches 70%, and the mixture is incubated for 4 hours at 37 ℃ with 5% CO ₂; 2% FBS-PBS was washed twice; serum-free medium was added, and then PEI-mediated transfection of rAAV vectors rAAV-GBA1-CDNF, rAAV-GBA1-NRTN, rAAV-GBA1-GDNF and rAAV-GBA1-BDNF, with PEI alone and rAAV-EGFP transfected HEK293 cells as controls. The mass ratio of PEI to rAAV carrier is 3:1 is added into 500ul serum-free culture medium, vortex mixing is carried out, after standing for 15min at 37 ℃, dropwise added into cells, and gentle shaking mixing is carried out; culturing in a 5% CO ₂ incubator at 37 ℃ for 4 hours; the medium was replaced with 3mL of complete medium; culturing in a 5% CO ₂ incubator at 37 ℃ for 48 hours; collect cells into 1.5mL centrifuge tube

Centrifuging the collected cells at 12000rpm for 1min, and removing the supernatant; adding 1mL PBS, centrifuging at 12000rpm for 1min, and removing supernatant; adding lysate (pH 5.4 citric acid-phosphate buffer, 0.25% Triton X-100, 1mM EDTA, protease-phosphatase inhibitor), stirring, mixing, ice-bath for 30min,12000rpm, and centrifuging for 10 min; the BCA method measures total protein concentration, adds samples to a 96-white plate at 5 ug/well and supplements the volume to 50 ul/well; 50ul of substrate 4-methylumbelliferyl beta-D-glucoside working solution (pH 5.4 citric acid-phosphate buffer, 0.25% Triton X-100, 1mM EDTA, 1% BSA, 5mM 4-methylumbelliferyl beta-D-glucoside) was added, and the mixture was incubated at 37℃for 15min; adding an equal volume of stop solution (pH 12.5,1M glycine solution); biotek SynergyH1 detection by using a full-functional microplate enzyme-labeled instrument (fluorescence: ex360, em 460), only adding lysate wells as blank control

The experimental results of GCase activity (n=3) of GBA1 expression product in the cells after transfection of the rAAV vector by 100uM CBE treatment HEK293 cells for 4h are shown in fig. 17. As a result, compared with the control group, the activity of GCase was increased by more than 3 times (GBA 1-CDNF 4.9 times, GBA1-NRTN 3 times, GBA1-GDNF 4.7 times, GBA-BDNF 3.9 times) in the experimental group transfected with the rAAV vector. After CBE treatment, the GCase activity in cells transfected with either the transfection reagent (CBE group) or EGFP (CBE/EGFP group) was only about 50% of that in the control group.

In conclusion, the GBA1 expression product in the rAAV vector still has activity in mammalian cells with reduced endogenous GCase activity, such as HEK293, which also shows that the gene medicine can be used for treating Gaofu disease.

Example 5 neurotrophic factor NRTN, GDNF, BDNF and CDNF in rAAV vector have neuroprotective effects

(1) RAAV-GBA1-NRTN, rAAV-GBA1-GDNF and rAAV-GBA1-BDNF have neuroprotective effect

PEI mediates transfection of rAAV vectors rAAV-GBA1-NRTN, rAAV-GBA1-GDNF and rAAV-GBA1-BDNF in HEK293 cells, wherein rAAV-EGFP is used as a control group, and the mass ratio of PEI to rAAV vectors is 3:1 is added into 500ul serum-free culture medium, vortex mixing is carried out, after standing for 15min at 37 ℃, dropwise added into cells, and gentle shaking mixing is carried out; culturing in a 5% CO ₂ incubator at 37 ℃ for 4 hours; the medium was replaced with 3mL of complete medium; culturing in a 5% CO ₂ incubator at 37deg.C for 48 hr, collecting supernatant, filtering with 0.22uM microporous membrane, and storing at-20deg.C.

SH-SY5Y cells were inoculated into a flat bottom 96-well plate at a rate of 2.5X10 ⁴ cells/well, cultured in 10% FBS DMEM for 12 hours, and then replaced with 2% FBS DMEM for further culturing for 48 hours; after the medium was removed, 50ul of the above supernatant was added every 24 hours, and after 72 hours, the cell viability was measured by CTG method (Promega: G8641), specifically by adding an equal volume of CTG reagent to the cells, shaking 10min with a horizontal shaker at room temperature, and reading the luminescence value with an ELISA reader.

ELISA assay results in example 2 showed that the concentrations of NRTN, GDNF and BDNF in the above supernatants were 2403.55.+ -. 209.65pg/mL, 2235.6.+ -. 144.1pg/mL and 2166.05.+ -. 127.25pg/mL, respectively.

The experimental results of the effect of neurotrophic factors NRTN, GDNF, and BDNF on the viability of neural cells, such as SH-SY5Y, in rAAV vectors are shown in FIGS. 18-20. Cell viability results showed that the SH-SY5Y viability of the NRTN-added supernatant was 3.3 times that of the control group (as shown in FIG. 18), that of the GDNF group was 2.7 times that of the control group (as shown in FIG. 19), and that of the BDNF-added group was 14.6 times that of the control group (as shown in FIG. 20). This shows that the neurotrophic factors NRTN, GDNF and BDNF expressed by the rAAV vector can play an extremely remarkable role in protecting human nerve cells such as SH-SY5Y when the cells are subjected to 'starvation' treatment in the invention under the condition of environmental stress.

(2) AAV2/2-GBA1-CDNF has neuroprotective effect.

SH-SY5Y cells were inoculated into 6-well cell culture plates at 3X 10 ⁵ cells/well, cultured for 12h, AAV2/2-EGFP, AAV2/2-GBA1-EGFP and AAV2/2-GBA1-CDNF were infected with cells at a viral load of 10 ⁵ vg/cell for 72h (vg/cell), cells were collected, AAV2/2-EGFP and AAV2/2-GBA1-EGFP were used as control groups, and AAV2/2-GBA1-CDNF was used as experimental groups for analyzing the expression of GBA1 and CDNF in cells. Specifically, extracting total protein in the cells, and measuring the protein concentration by a BCA method; heating at 100deg.C for 5min for protein denaturation; loading 30ug of the sample into each hole, carrying out electrophoresis separation on 12% SDS-PAGE gel, and transferring the sample; 5% nonfat dry milk was blocked for 1h and incubated overnight at 4deg.C with primary antibody (GBA 1, abcam: ab55080; CDNF, abcam: ab205551; beta-actin, abcam: ab 8227); TBST was washed three times, secondary antibody incubated for 1h at room temperature, TBST was washed three times, and developed.

SH-SY5Y cells were seeded at 2.5X10 ⁴ cells/well in 96-well cell culture plates, cultured for 12h, added with TG (Thapsigargin) at a final concentration of 5uM, and the medium was aspirated. Complete medium (10% FBS, DMEM) containing the above viruses was added to the cells at 10 ⁵ vg/cell and 10 ⁶ vg/cell, respectively, and cultured in a 5% CO ₂ incubator at 37 ℃.

10 After culturing ⁵ vg/cell for 72h, removing the culture medium, adding PBS, washing twice, adding RNA extraction reagent (full gold: ET 101-01) according to 150 ul/hole to extract total RNA of cells, and specific operation is shown in the product specification. The total RNA extracted was reverse transcribed into cDNA (Takara: RR 047A). GAPDH was used as an internal control, and the relative expression levels of apoptosis markers CHOP and Capase were measured (Vazyme: Q111-02). 10 After culturing for 72 hours in ⁶ vg/cell experimental group, the cell viability was measured by CTG method (Promega: G8641).

The experimental results of the influence of the rAAV vector such as rAAV-GBA1-CDNF on apoptosis markers and the activation rate after the rAAV-GBA1-CDNF is prepared into the gene drug AAV2/2-GBA1-CDNF, after the nerve cells such as SH-SY5Y are infected, the expression conditions of GBA1 and CDNF in the cells and after the effect of 5 uM TG are performed are shown in figures 21-23.

The results of the protein expression analysis showed that the amount of the GBA1 expression product GCase was significantly increased in AAV2/2-GBA1-EGFP and AAV2/2-GBA 1-CDNF-infected cells, and that only AAV2/2-GBA 1-CDNF-infected cells had significant CDNF protein expression, as shown in FIG. 21, as compared to AAV 2/2-EGFP-infected SH-SY5Y cells. The rAAV vector can infect mammalian nerve cells such as SH-SY5Y and can be normally expressed in the cells after being prepared into gene therapy medicine adeno-associated virus.

In the result of the cell viability measured by the CTG method, in the experimental group infected by the viral load of 10 ⁵ vg/cell, the difference of the cell viability between the CDNF virus-infected group and the non-virus-infected group was not obvious (P > 0.05). Further analysis of expression of apoptosis marker CHOP and Caspase3 showed that apoptosis marker CHOP expression was significantly lower in CDNF virus infected groups than in non-viral infected groups (P < 0.01) and Caspase3 significantly lower than in non-CDNF virus groups (P < 0.05), as shown in fig. 22.

In the experimental group with 10 ⁶ vg/cell virus infection, the cell viability of the CDNF virus-infected group was significantly higher than that of the non-virus-infected group (P < 0.05), as shown in FIG. 23. After the constructed product is prepared into the gene medicine, the CDNF can save damage of mammalian nerve cells such as SH-SY5Y caused by TG, and improve the survival rate of the nerve cells.

In conclusion, after the rAAV vector is prepared into a gene therapy drug, namely adeno-associated virus, the rAAV vector can be normally expressed in mammalian nerve cells such as SH-SY5Y and can also perform normal physiological functions, such as the 'rescue' function of CDNF.

Example 6 GCase expressed by GBA1 in rAAV vector increases the ratio of alpha-synuclein (alpha-Syn) tetramer to monomer in endogenous GCase function deficient mammalian neural cells such as SH-SY5Y

Related studies have found that reduced activity of the GCase enzyme can reduce the tetramer to monomer ratio of the alpha-Syn protein in mammalian nerve cells, which in turn leads to the formation of pathological aggregates of alpha-Syn monomers, resulting in nerve cell death (Sangjune Kim et al.,2017; yumiko V. Taguchi et al.,2017; kelly E. Glajch et al., 2021).

In the invention, an endogenous GCase enzyme function defect cell model is constructed by applying CBE to SH-SY5Y, then gene medicine prepared by the rAAV vector is applied to cells, and the improvement of the GCase activity is researched on alpha-Syn protein tetramer in the model cells: the ratio of monomers affects.

(1) And constructing an endogenous GCase enzyme function defect model of SH-SY5Y cells based on CBE.

SH-SY5Y cells were inoculated into 48-well cell culture plates at 10 ⁵ cells/well, cultured for 24 hours, PBS-CBE solution was added to the cells at final solubilities of 0.01uM, 0.1uM, 1uM, 10uM, 100uM and 1000uM, incubated for 24 hours, and the cells were collected and assayed for GCase enzyme activity. Based on the change of the GCase enzyme activity under the action of the CBE, as shown in figure 24, the invention adopts 100uM final concentration to construct an endogenous GCase enzyme function defective cell model, and when the ratio of the alpha-Syn protein tetramer to the monomer in the model cell is obviously reduced compared with that of a normal cell (P < 0.05), the model construction is successful.

(2) The GBA1 expression product GCase in the rAAV vector has influence on the ratio of alpha-Syn protein tetramer to monomer in a CBE mediated endogenous GCase function defective nerve cell model.

Three replicates were set up and the above CBE-mediated SH-SY5Y model cells were seeded at 3X 10 ⁵ cells/well in 6-well plates and cultured in 100uM CBE-containing complete medium for 12h with normal SH-SY5Y cells as control. After 12h, adeno-associated virus AAV2/2-EGFP (5X 10 ⁵ vg/cell), AAV2/2-GBA1-EGFP (5X 10 ⁵ vg/cell) and AAV2/2-GBA1-CDNF (10 ⁵ vg/cell) were added, and the two groups of CBE-mediated model cells served as control groups; after 24h, the CBE-complete medium of the virus-infected group and one of the model control groups CBE (r) was replaced with complete medium without CBE, and the culture was continued for 48h, and the cells were collected.

Each treatment in the above collection of cells separated 3X 10 ⁴ cells, assayed by GCase activity. After mixing the three repeated groups into one group, extracting total cell proteins, and determining protein concentration by a BCA method (Thermo: 23227); protein denaturation; 15ug of each well is loaded, and 12% SDS-PAGE gel is used for electrophoretic separation and transfer; fixing 4% paraformaldehyde for 10min, washing with double distilled water for 3 times, sealing with 5% skimmed milk powder for 1h, and incubating primary antibody (GBA 1, abcam: ab55080; alpha-Syn, abcam: ab138501; beta-actin, abcam: ab 8227) at 4deg.C overnight; TBST was washed three times, secondary antibody incubated for 1h at room temperature, TBST was washed three times, developed, and analyzed for alpha-Syn protein tetramer and monomer expression.

After the gene medicine prepared by the rAAV vector acts on a cell model of CBE mediated inhibition of the endogenous GCase activity of nerve cells, the experimental results of the GCase activity level and the alpha-Syn tetramer to monomer ratio in the cells are shown in FIG. 25 and FIG. 26. As a result, it was found that when the drugs CBE (r) and AAV 2/2-EGFP) inhibiting the GCase activity were removed, the ratio of the tetramer to the monomer of the alpha-Syn protein increased as the GCase activity in the cells was increased as compared with the CBE group. The enzyme activity measurement result shows that the expression product GCase of the rAAV vector has enzyme activity after the GBA1 is input into cells through adeno-associated virus, and the ratio of alpha-Syn protein tetramer to monomer of AAV2/2-GBA1-EGFP and AAV2/2-GBA1-CDNF groups is obviously higher than that of AAV2/2-EGFP groups, which indicates that the GCase expressed by the rAAV vector can perform normal physiological functions as endogenous GCase. GCase is an enzyme in lysosomes that has phagocytic activity, and thus, adeno-associated viral infection may stimulate the expression of endogenous GCase, resulting in slightly higher enzyme activity in the AAV2/2-EGFP group than in the CBE (r) group, but the α -Syn protein tetramer to monomer ratio in the cell will increase whenever the activity of GCase is increased, either by exogenous introduction (GBA 1 in rAAV vector described in the present invention) or by induction of increased endogenous expression.

In summary, the GCase activity in mammalian nerve cells such as SH-SY5Y is positively correlated with the tetramer to monomer ratio of the α -Syn protein, i.e., after the GCase activity in the cells is increased, the α -Syn protein can exist in more stable tetramer form, reducing the probability of formation of insoluble fiber form by the unstable monomers, and further prolonging the lifetime of the nerve cells. The constructed product can express GCase with normal physiological functions after being input into nerve cells, which shows that the gene medicine prepared by the rAAV vector has the functions of reducing alpha-Syn protein fibrosis and prolonging the service life of the nerve cells. Because Lewy Body finally formed by alpha-Syn protein fibrosis is a parkinsonism marker, the construction product and the gene medicine prepared by the construction product have the function of treating the parkinsonism of the neurodegenerative disease.

The above embodiments are merely illustrative of the principles of the present invention and its effectiveness, and are not intended to limit the invention. Modifications and variations may be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the invention. Accordingly, it is intended that all equivalent modifications and variations of the invention be covered by the claims, which are within the ordinary skill of the art, be within the spirit and scope of the present disclosure.

Claims

1. A recombinant nucleic acid molecule encoding human glucocerebrosidase GCase and a neurotrophic factor;

the neurotrophic factors comprise human brain dopamine neurotrophic factor CDNF, human brain-derived neurotrophic factor BDNF, human glial cell-derived neurotrophic factor GDNF and human nerve rank protein NRTN;

The sequence encoding human glucocerebrosidase GCase is linked to the sequence encoding neurotrophic factor by a gene linker element; the amino acid sequence of the coded human glucocerebrosidase GCase is SEQ ID NO.1; the amino acid sequence of the human brain dopamine neurotrophic factor CDNF is SEQ ID NO.2, the amino acid sequence of the human brain-derived neurotrophic factor BDNF is SEQ ID NO.3, the amino acid sequence of the human glial cell-derived neurotrophic factor GDNF is SEQ ID NO.4, and the amino acid sequence of the human nerve rank protein NRTN is SEQ ID NO.5;

The sequence information of the recombinant nucleic acid molecules rAAV-GBA1-CDNF, rAAV-GBA1-NRTN, rAAV-GBA1-GDNF and rAAV-GBA1-BDNF is shown as SEQ ID NO. 8-11;

The gene connecting element comprises an internal ribosome entry site sequence IRES, a 2A peptide or a class 2A; the IRES element comprises an encephalomyocarditis virus EMCV; the 2A peptide or the similar 2A peptide comprises P2A derived from porcine teschovirus and T2A derived from Leptospira Minus virus.

2. The recombinant nucleic acid molecule of claim 1, comprising an operably linked promoter.

3. The recombinant nucleic acid molecule of claim 2, wherein the promoter is the human cytomegalovirus enhancer and chicken β -actin promoter combined promoter CAG, the human cytomegalovirus strong promoter CMV, or the human synapsin I promoter hSyn.

4. A recombinant nucleic acid molecule according to any one of claims 1 to 3, further comprising the intron simian virus 40 intron SV40, the posttranscriptional regulatory element nucleic acid sequence, further comprising the woodchuck hepatitis virus posttranscriptional regulatory element WPRE and polyadenylation such as SV40 PolyA.

5. The recombinant nucleic acid molecule of claim 4, further comprising an AAV inverted terminal repeat ITR.

6. The recombinant nucleic acid molecule of claim 5, wherein the AAV inverted terminal repeat ITR is AAV type 2.

7. A recombinant vector for use in gene therapy, characterized in that it comprises a recombinant nucleic acid molecule according to any one of claims 1-6, said vector being selected from viral vectors.

8. The recombinant vector according to claim 7, wherein the viral vector is selected from adeno-associated viral vectors.

9. A recombinant adeno-associated virus for use in gene therapy, comprising an AAV capsid and a vector genome comprising the recombinant nucleic acid molecule of any one of claims 1-6.

10. The recombinant adeno-associated virus of claim 9, wherein the capsid of the recombinant adeno-associated virus is an AAV2 or AAV9 capsid protein.

11. An isolated host cell for use in gene therapy comprising the recombinant nucleic acid molecule of any one of claims 1-6, the recombinant vector of any one of claims 7-8, or the recombinant adeno-associated virus of any one of claims 9-10.

12. A pharmaceutical composition for gene therapy, characterized in that it comprises a recombinant nucleic acid molecule according to any one of claims 1-6, a recombinant vector according to any one of claims 7-8, a recombinant adeno-associated virus according to any one of claims 9-10, and a pharmaceutically acceptable carrier and/or other conventional pharmaceutical ingredients.

13. The pharmaceutical composition according to claim 12, wherein the other conventional pharmaceutical ingredients comprise a preservative and/or a stabilizer.

14. The pharmaceutical composition according to any one of claims 12-13, wherein the pharmaceutical composition is administered to the CNS of a patient by systemic injection or local injection.

15. The pharmaceutical composition of claim 14, wherein the local injection comprises one or more of an intra-brain injection, an intra-parenchymal injection, an intrathecal injection, and an intracisternal injection.

16. The pharmaceutical composition of claim 12, wherein the pharmaceutical composition is delivered by convection enhanced CED.

17. Use of a recombinant nucleic acid molecule according to any one of claims 1 to 6, a recombinant vector according to any one of claims 7 to 8 and/or a recombinant adeno-associated virus according to any one of claims 9 to 10 for the preparation of a medicament for the treatment of parkinson's disease and type II, III gaucher's disease.

18. The use according to claim 17, wherein the recombinant nucleic acid molecule, recombinant vector, recombinant adeno-associated virus, host cell and/or pharmaceutical composition is administered with another therapy.