CN117018231A - Gene therapy for treatment of neuropathy and use thereof - Google Patents

Abstract

The invention belongs to the field of gene therapy, and specifically relates to gene therapy for treating neuropathy and its application. The invention provides a gene therapy, which involves a recombinant nucleic acid molecule encoding human glucocerebrosidase GCase and neurotrophic factors. The invention also provides a recombinant vector containing the recombinant nucleic acid molecule, a recombinant adenoid-related Viruses, host cells and pharmaceutical compositions, which are used to treat neuropathies involving reduced GCase activity, such as Parkinson's disease and type II and type III Gaucher's disease. They can be effectively administered by systemic injection or local injection. It can improve the GCase activity in the body of the subject and the anti-damage and anti-apoptosis ability of neurons.

Description

Gene therapy for treating neuropathy and its applications

Technical field

The invention belongs to the field of gene therapy, and specifically relates to gene therapy for treating neuropathy and its application.

Background technique

Parkinson's disease is the second most common neurodegenerative disorder in the world, with an estimated 5.8 million patients worldwide, and the number will increase further as the population ages. Studies have confirmed that the reduced GCase enzyme activity caused by GBA1 gene mutations will not only lead to Gaucher’s Disease (Gaucher’s Disease), but also increase the risk of early-onset Parkinson’s disease in carriers. Decreased GCase activity in nerve cells such as dopamine neurons will lead to the accumulation of misfolded α-synuclein (α-synuclein), forming Lewy Body, causing nerve cell damage and death, and eventually the carrier will develop Parkinson's disease. In addition to GBA1 gene mutations that lead to a decrease in GCase activity, aging can also lead to a decrease in GCase activity in the body, which is one of the reasons why aging leads to an increase in the incidence of Parkinson's disease.

Parkinson's disease is currently an incurable disease, and existing clinical interventions can only alleviate patients' symptoms. For patients carrying GBA1 mutations, there are currently three main treatment strategies. The first is small molecule chemical drugs. This type of therapeutic drug has two strategies. One is to act as a molecular chaperone for GCase, thereby increasing the activity of mutant GCase; the other is to act on the upstream of the metabolic pathway to reduce the accumulation of enzyme substrates. The second is enzyme replacement therapy, which uses genetic recombination technology to express GCase in vitro and then injects it into the body. The third method is gene therapy, which uses vector tools to deliver wild-type GBA1 into the body and achieve the required expression level under the action of cis-regulatory elements. The first two methods require lifelong continuous administration. Although the third method can achieve long-term effective effects with a single administration, only delivering the wild-type GBA1 gene cannot save the patient's damaged neurons.

Therefore, for neurological diseases caused by reduced GCase activity that currently have no effective cure, such as neurodegenerative diseases - Parkinson's disease and type II and III Gaucher's disease, a new treatment for neurological diseases has been explored and developed. Gene drugs are of great significance.

references:

Kelly E.Glajch,Tim E.Moors,Yi Chen,et al.Wild-type GBA1 increases theα-synuclein tetramer-monomer ratio,reduces lipid-rich aggregates,andattenuates 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.GlucosylsphingosinePromotesα-Synuclein Pathology in Mutant GBA-Associated Parkinson’sDisease.The Journal of Neuroscience,October 4,2017,37(40):9617-9631.

Contents of the invention

In view of the shortcomings of existing products and technologies, the present invention aims to provide gene therapy for treating neuropathy and its application. The present invention provides genetic medicines such as recombinant nucleic acid molecules, recombinant vectors, recombinant adeno-associated viruses, host cells, etc. for treating neuropathy. The genetic medicine of the present invention is a combination of GCase nucleic acid and neurotrophic factor nucleic acid. In addition to producing highly active GCase that can effectively clear Lewy Body and preventing the deposition of new pathological α-synuclein, it also produces neurotrophic factors. It can also reduce/rescue the damage caused to neurons by the stressful environment in the body and extend the life of neurons.

In order to achieve the above objects, the present invention adopts the following technical solutions:

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

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

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

MEFSSPSREECPKPLSRVSIMAGSLTGLLLLQAVSWASGARPCIPKSFGYSSVVCVCNATYCDSFDPPTFPALGTFSRYESTRSGRRMELSMGPIQANHTGTGLLLTLQPEQKFQKVKGFGGAMTDAAALNILALSPPAQNLLLKSYFSEEGIGYNIIRVPMASCDFSIRTYTYADTPDDFQLHNFSLPEEDTKLKIPLIHRALQLAQRPVSLLASPWT SPTWLKTNGAVNGKGSLKGQPGDIYHQTWARYFVKFLDAYAEHKLQFWAVTAENEPSAGLLSGYPFQCLGFTPEHQRDFIARDLGPTLANSTHHNVRLLMLDDQRLLLPHWAKVVLTDPEAAKYVHGIAVHWYLDFLAPAKATLGETHRLFPNTMLFASEACVGSKFWEQSVRLGSWDRGMQYSHSIITNLLYHVVGWTDWNLALNPEGGPNWVRNFV DSPIIVDITKDTFYKQPMFYHLGHFSKFIPEGSQRVGLVASQKNDLDAVALMHPDGSAVVVVLNRSSKDVPLTIKDPAVGFLETISPGYSIHTYLWRRQ*(SEQ ID NO.1);

MWCASPVAVVAFCAGLVSHPVLTQGQEAGGRPGADCEVCKEFLNRFYKSLIDRGVNFSLDTIEKELISFCLDTKGKENRLCYYLGATKDAATKILSEVTRPMSVHMPAMKICEKLKKLDSQICELKYEKTLDLASVDLRKMRVAELKQILHSWGEECRACAEKTDYVNLIQELAPKYAATHPKTEL*(SEQ ID NO.2);

MQRWKAAALASVLCSSVLSIWMCREGLLLSHRLGPALVPLHRLPRTLDARIARLAQYRALLQGAPDAMELRELTPWAGRPPGPRRRAGPRRRRARLGARPCGLRELEVRVSELGLGYASDETVLFRYCAGACEAAARVYDLGLRRLRQRRRLRRERVRAQPCCRPTAYEDEVSFLDAHSRYHTVHELSARECACV*(SEQ ID NO.3);

MKLWDVVAVCLVLLHTASAFPLPAGKRPPEAPAEDRSLGRRRAPFALSSDSNMPEDYPDQFDDVMDFIQATIKRLKRSPDKQMAVLPRRERNRQAAAANPENSRGKGRRGQRGKNRGCVLTAIHLNVTDLGLGYETKEELIFRYCSGSCDAAETTYDKILKNLSRNRRLVSDKVGQACCRPIAFDDDLSFLDDNLVYHILRKHSAKRCGCI*(SEQ ID NO.4);

MTILFLTMVISYFGCMKAHSDPARRGELSVCDSISEWVTAADKKTAVDMSGGTVTVLE KVPVSKGQLKQYFYETKCNPMGYTKEGCRGIDKRHWNSQCRTTQSYVRALTMDSKKRIG WRFIRIDTSCVCTLTIKRGR*(SEQID NO. 5).

Preferably, the gene linking element includes but is not limited to internal ribosome entry site sequence IRES, 2A peptide or 2A-like peptide; the IRES element includes but is not limited to encephalomyocarditis virus EMCV; the 2A peptide or 2A-like peptide , including but not limited to P2A originating from porcine Jieshen virus and T2A originating from P. lucidum virus.

The second object of the present invention is to provide a recombinant nucleic acid molecule comprising an operably linked promoter and the nucleic acid molecule of claim 1.

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

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

Preferably, the recombinant nucleic acid molecule further contains AAV inverted terminal repeat sequence ITR.

Preferably, the AAV inverted terminal repeat sequence ITR is AAV type 2.

Another object of the present invention is to provide a recombinant vector for gene therapy, which contains the above-mentioned recombinant nucleic acid molecule, and the vector is selected from plasmid vectors and viral vectors.

Preferably, the viral vector includes but is not limited to adeno-associated virus vector, adenovirus vector, lentiviral vector, hybrid virus vector and phage vector.

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

Another object of the present invention is to provide a recombinant adeno-associated virus for gene therapy. The recombinant adeno-associated virus contains an AAV capsid and a vector genome, and the vector genome contains the above-mentioned recombinant nucleic acid molecule.

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

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

Another object of the present invention is to provide a pharmaceutical composition for gene therapy, which includes the above-mentioned recombinant nucleic acid molecule, recombinant vector, recombinant adeno-associated virus, pharmaceutically acceptable carrier and/or other conventional pharmaceutical ingredients.

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

Preferably, the pharmaceutical composition acts on the patient's central nervous system (CNS) through systemic injection or local injection.

Preferably, the local injection includes one or more of intracerebral injection, intraparenchymal injection, intrathecal injection and intracisternal injection.

Preferably, the pharmaceutical composition enhances CED delivery by convection.

The present invention also provides the use of the above-mentioned recombinant nucleic acid molecule, recombinant vector and/or recombinant adeno-associated virus in the preparation of drugs for preventing or treating neuropathy with reduced GCase activity.

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

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

The present invention provides recombinant nucleic acid molecules, recombinant vectors, recombinant adeno-associated viruses, host cells and pharmaceutical compositions involved in gene therapy for treating neuropathy. It is a gene therapy that can increase GCase activity and rescue damaged neurons. It not only has The long-term effective effect of a single dose can also slow down or even reverse the course of the patient's disease.

Description of the drawings

Figure 1 is a schematic diagram of the isolated nucleic acid sequence structure;

Figure 2 is a schematic diagram of rAAV-EGFP plasmid;

Figure 3 is a schematic diagram of rAAV-GBA1-EGFP plasmid;

Figure 4 is a schematic diagram of rAAV-GBA1-CDNF plasmid;

Figure 5 is a schematic diagram of rAAV-GBA-NRTN plasmid;

Figure 6 is a schematic diagram of rAAV-GBA1-GDNF plasmid;

Figure 7 is a schematic diagram of rAAV-GBA1-BDNF plasmid;

Figure 8 shows the expression of GBA1 and CDNF in HEK293 cells in rAAV-GBA1-CDNF;

Figure 9 shows the expression of GBA1 and NRTN in HEK293 cells in rAAV-GBA1-NRTN;

Figure 10 shows the expression of GBA1 and GDNF in HEK293 cells in rAAV-GBA1-GDNF;

Figure 11 shows the expression of rAAV-GBA1-BDNF in GBA1 and BDNF in HEK293 cells;

Figure 12 shows the NRTN content in the culture supernatant after HEK293 cells were transfected with rAAV-GBA1-NRTN;

Figure 13 shows the GDNF content in the culture supernatant after HEK293 cells were transfected with rAAV-GBA1-GDNF;

Figure 14 shows the BDNF content in the culture supernatant after HEK293 cells were transfected with rAAV-GBA1-BDNF;

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

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

Figure 17 is a graph showing the GCase activity (n=3) of the GBA1 expression product in the cells after HEK293 cells were treated with 100uM CBE for 4 hours and transfected with the rAAV vector;

Figure 18 is a diagram showing the effect of NRTN in the rAAV-GBA1-NRTN vector on the activity of human neural cells such as SH-SY5Y;

Figure 19 is a diagram showing the effect of GDNF in the rAAV-GBA1-GDNF vector on the activity of human neural cells such as SH-SY5Y;

Figure 20 is a diagram showing the effect of BDNF in the rAAV-GBA1-BDNF vector on the activity of human neural cells such as SH-SY5Y;

Figure 21 is a diagram showing the expression of GBA1 and CDNF after AAV2/2-GBA1-CDNF infects human neural cells such as SH-SY5Y;

Figure 22 shows the changes in the expression levels of apoptosis markers in the cells (n=3) after AAV2/2-GBA1-CDNF was infected with 10 ⁵ vg/cell of human neural cells such as SH-SY5Y and treated with 5uM TG for 24 hours;

Figure 23 shows the changes in cell viability (n=3) after AAV2/2-GBA1-CDNF was infected with 10 ⁶ vg/cell of human neural cells such as SH-SY5Y and treated with 5uM TG for 24 hours;

Figure 24 is a graph showing the inhibitory effect of different concentrations of CBE on the endogenous GCase activity of human neuroblastoma cells SH-SY5Y (n=3);

Figure 25 is a diagram showing the expression of α-Syn protein and GCase in the cells after genetic drugs prepared from rAAV vectors such as rAAV-GBA1-EGFP and rAAV-GBA1-CDNF act on the CBE-mediated SH-SY5Y cell model;

Figure 26 is a diagram of the GCase activity level and the ratio of α-Syn tetramers to monomers in cells after the gene drugs prepared by rAAV vector act on the cell model of CBE-mediated inhibition of endogenous GCase activity in nerve cells. Figure 26 ( A) is a graph showing the activity level of GCase in cells after genetic drugs act on CBE-mediated SH-SY5Y cell model; Figure 26(B) shows the tetramerization of α-Syn protein in cells after genetic drugs act on CBE-mediated cell model. Changes in the ratio of monomers to monomers.

Detailed ways

The above contents of the present invention will be further described in detail below through specific implementation methods in the form of examples. However, this should not be understood to mean 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 sequence of the present invention, its expression vector and recombinant adeno-associated virus

The gene therapy of the present invention involves a recombinant nucleic acid molecule encoding human glucocerebrosidase GCase and neurotrophic factors. Among them, neurotrophic factors include human brain dopamine neurotrophic factor CDNF, human brain-derived neurotrophic factor BDNF, human glial cell-derived neurotrophic factor GDNF and human neural rank protein NRTN. The sequence encoding human glucocerebrosidase GCase is connected to the sequence encoding neurotrophic factors through a genetic linking element. Amino acid sequences 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 neural rank protein NRTN As shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5. Wherein, the gene linking element includes but is not limited to the internal ribosome entry site sequence IRES, 2A peptide or 2A-like peptide; the IRES element includes but is not limited to the encephalomyocarditis virus EMCV; the 2A peptide or 2A-like peptide, Including, but not limited to, P2A derived from porcine Jieshen virus and T2A derived from T. lucidum virus.

The recombinant nucleic acid molecule of the present invention also contains an operably connected promoter, which is a combination promoter of human cytomegalovirus enhancer and chicken β-actin promoter CAG, a strong promoter of human cytomegalovirus CMV or Human synapsin I promoter hSyn.

The recombinant nucleic acid molecule of the present invention also includes introns such as simian virus 40 intron SV40, post-transcriptional regulatory elements such as nucleic acid sequences, and woodchuck hepatitis virus post-transcriptional regulatory element WPRE and polyadenylic acid such as SV40 PolyA.

The recombinant nucleic acid molecule of the present invention also includes an AAV inverted terminal repeat sequence ITR, and the AAV inverted terminal repeat sequence ITR is AAV type 2.

A recombinant vector containing the recombinant nucleic acid molecule of the present invention, which is selected from plasmid vectors and viral vectors, and the viral vectors include but are not limited to adeno-associated virus vectors, adenovirus vectors, lentiviral vectors, hybrid virus vectors and phage vectors; Preferred are selected from adeno-associated virus vectors.

(1)Vector construction

The gene therapy of the present invention involves an isolated nucleic acid transfer product, the sequence of the nucleic acid transfer product from 5' to 3' is 5'ITR, promoter, SV40 intron, GBA1, linker sequence, neurotrophic factor, WPRE , SV40 polyA and 3'ITR. Figure 1 is a schematic diagram of the nucleic acid sequence structure of the nucleic acid transfer product of the present invention. The construction of the vector of the present invention was entrusted to Guangzhou Paizhen Biotechnology Co., Ltd., based on the encoding of human glucocerebrosidase GCase, encoding of human brain dopamine neurotrophic factor CDNF, encoding of human brain-derived neurotrophic factor BDNF, and encoding provided by the inventor. Sequence information of human glial cell-derived neurotrophic factor GDNF and encoding human neural rank protein NRTN (amino acid sequences such as SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO .5), the schematic diagram of the nucleic acid sequence structure and the vector skeleton require sequence synthesis and construction of the recombinant adeno-associated virus (rAAV) expression vector. Finally, the constructed rAAV expression vectors rAAV-EGFP, rAAV-GBA1-EGFP, rAAV- GBA1-CDNF, rAAV-GBA1-NRTN, rAAV-GBA1-GDNF, and rAAV-GBA1-BDNF are provided to the inventor in the form of Stbl3 bacterial culture and plasmid DNA. Among them, the constructed vector sequence is as SEQ ID NO.6- Shown in 11. As shown in Figure 2-7, it is a schematic diagram of the plasmids of rAAV-EGFP, rAAV-GBA1-EGFP, rAAV-GBA1-CDNF, rAAV-GBA1-NRTN, rAAV-GBA1-GDNF and rAAV-GBA1-BDNF.

Sequence information of vector rAAV-EGFP:

CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGTGGCCAACTCCATCACTAGGGGTTCCTGCGGCCATTCGCGGTACAATTCACGCGTCGACATTGATTATTGACTAGCTCTGGTCGTTACATAACTTACGGTAAATGGCCCGCCTGCCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGA CGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACTCGAGGCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCTCCCCACCCCCAATTTTGTATTT ATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGCGGGATCAGCCACCGCGGTGGCGGCCTAGAGTCGACGAG GAACTGAAAAACCAGAAAGTTAACTGGTAAGTTTAGTCTTTTTGTCTTTTATTTCAGGTCCCGGATCCGGTGGTGGTGCAAATCAAAGAACTGTCCCTCAGTGGATGTTGCCTTTACTTCTAGGCCTGTACGGAAGTGTTACTTCTGCTCTAAAAGCTGCGGAATTGTACCCGCGGCCGATCCACCGGTCGCCACCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCG ACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCG CCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACC ACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAGAATTCCGCTCGAGATAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCT TTCATTTTCTCCTCCTTGTATAAATCCTGGTTAGTTCTTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTTTATTTGTGAAATTTGGTGATGCTATTGCTTTATTTGTAACCATCTAGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAA

GTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGATGTGGGAGGTT

TTTTAAAGCGGCCGCAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCT

CGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGG

CGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGGGCGCCTGATGCGGTATTTT

CTCCTTACGCATCTGTGCGGTATTTCACACCGCATACGTCAAAGCAACCATAGTACGCGC

CCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACA

CTTGCCAGCGCCCTAGCGCCCGCTCCTTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTC

GCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCT

TTACGGCACCTCGACCCCAAAAAACTTGATTTGGGTGATGGTTCACGTAGTGGGCCATC

GCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACT

CTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGGCTATTCTTTTGATTTATAAGG

GATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGC

GAATTTTAACAAAATATTAACGTTTACAATTTTATGGTGCACTCTCAGTACAATCTGCTCT

GATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACG

GGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCA

TGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATA

CGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTT

TTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTA

TCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTAT

GAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGGCATTTTGCCTTCCTGTTT

TTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACG

AGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCG

AAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCC

GTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTG

GTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATT

ATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGAT

CGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCC

TTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCAC

GATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCT

AGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTC

TGCGCTCGGCCCTTCCGGCTGGCTGGTTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTG

GGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTA

TCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATA

GGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGA

TTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCT

CATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAA

AGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAA

AAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTT

CCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCC

GTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAAT

CCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAA

GACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTTCGTGCACACA

GCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAG

AAAGCCGCCACGCTTCCCGAAGGGAGAAAGGCGGCAGGTATCCGGTAAGCGGCAGGG

TCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAG

TCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGG

GCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGT (SEQ ID NO. 6);

Sequence information of vector rAAV-GBA1-EGFP:

CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGTGGCCAACTCCATCACTAGGGGTTCCTGCGGCCATTCGCGGTACAATTCACGCGTCGACATTGATTATTGACTAGCTCTGGTCGTTACATAACTTACGGTAAATGGCCCGCCTGCCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGA CGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACTCGAGGCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCTCCCCACCCCCAATTTTGTATTT ATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGCGGGATCAGCCACCGCGGTGGCGGCCTAGAGTCGACGAG GAACTGAAAAACCAGAAAGTTAACTGGTAAGTTTAGTCTTTTTGTCTTTTATTTCAGGTCCCGGATCCGGTGGTGGTGCAAATCAAAGAACTGTCCCTCAGTGGATGTTGCCTTTACTTCTAGGCCTGTACGGAAGTGTTACTTCTGCTCTAAAAGCTGCGGAATTGTACCCGCGGCCGATCCACCGGTCGCCACCATGGAGTTTTCAAGTCCTTCCAGAGAGGAATGTCCCAAGCCTTTGAGTAGGGTAAGCATGGCT GGCAGCCTCACAGGATTGCTTCTACTTCAGGCAGTGTCGTGGGCATCAGGTGCCCGCCCCTGCATCCCTAAAAGCTTCGGCTACAGCTCGGTGTGTGTGTCTGCAATGCCACATACTGTGACTCCTTTGACCCCCCGACCTTTCCTGCCCTTGGTACCTTCAGCCGCTATGAGAGTACACGCAGTGGGCGACGGATGGAG

CTGAGTATGGGGCCCATCCAGGCTAATCACACGGGCACAGGCCTGCTACTGACCCTGCA

GCCAGAACAGAAGTTCCAGAAAGTGAAGGGATTTGGAGGGGCCATGACAGATGCTGCT

GCTCTCAACATCCTTGCCCTGTCACCCCCTGCCCAAAATTTGCTACTTAAATCGTACTTCT

CTGAAGAAGGAATCGGATATAACATCATCCGGGTACCCATGGCCAGCTGTGACTTCTCCA

TCCGCACCTACACCTATGCAGACACCCTGATGATTTCCAGTTGCACAACTTCAGCCTCC

CAGAGGAAGATACCAAGCTCAAGATACCCCTGATTCACCGAGCCCTGCAGTTGGCCCAG

CGTCCCGTTTCACTCCTTGCCAGCCCCTGGACATCACCCACTTGGCTCAAGACCAATGG

AGCGGTGAATGGGAAGGGGTCACTCAAGGGACAGCCCGGAGACATCTACCACCAGACC

TGGGCCAGATACTTTGTGAAGTTCCTGGATGCCTATGCTGAGCACAAGTTACAGTTCTGG

GCAGTGACAGCTGAAAATGAGCCTTCTGCTGGGCTGTTGAGTGGATACCCCTTCCAGTG

CCTGGGCTTCACCCCTGAACATCAGCGAGACTTCATTGCCCGTGACCTAGGTCCTACCCT

CGCCAACAGTACTCACCACAATGTCCGCCTACTCATGCTGGATGACCAACGCTTGCTGC

TGCCCCACTGGGCAAAGGTGGTACTGACAGACCCAGAAGCAGCTAAATATGTTCATGGC

ATTGCTGTACATTGGTACCTGGACTTTCTGGCTCCAGCCAAAGCCACCCTAGGGGAGAC

ACACCGCCTGTTCCCCAACACCATGCTCTTTGCCTCAGAGGCCTGTGTGGGCTCCAAGT

TCTGGGAGCAGAGTGTGCGGCTAGGCTCCTGGGATCGAGGGATGCAGTACAGCCACAG

CATCATCACGAACCTCCTGTACCATGTGGTCGGCTGGACCGACTGGAACCTTGCCCTGA

ACCCCGAAGGAGGACCCAATTGGGTGCGTAACTTTGTCGACAGTCCCATCATTGTAGAC

ATCACCAAGGACACGTTTTTACAAACAGCCCATGTTCTACCACCTTGGCCACTTCAGCAA

GTTATTCCTGAGGGCTCCCAGAGAGTGGGGCTGGTTGCCAGTCAGAAGAACGACCTG

GACGCAGTGGCACTGATGCATCCCGATGGCTCTGCTGTTGTGGTCGTGCTAAACCGCTC

CTCTAAGGATGTGCCTCTTACCATCAAGGATCCTGCTGTGGGCTTCCTGGAGACAATCTC

ACCTGGCTACTCCATTCACACCTACCTGTGGCGTCGCCAGGGAAGCGGAGCCACTAACT

TCTCCCTGTTGAAACAAGCAGGGGATGTCGAAGAGAATCCCGGGCCAATGGTGAGCAA

GGGCGAGGAGCTGTTCACCGGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTA

AACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGC

TGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTG

ACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCGACCACATGAAGCAGCA

CGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCA

AGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGG

TGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCA

CAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAG

AACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACCGGCAGCGTGCAGC

TCGCCGACCACTACCAGCAGAACACCCCATCGGCGACGGCCCCGTGCTGCTGCCCGA

CAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGAT

CACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCT

GTACAAGTAAGAATTCCGCTCGAGATAATCAACCTCTGGATTACAAAATTTGTGAAAGAT

TGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCT

TTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTT

AGTTCTTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTC

GGCTGTTGGGCACTGACAATTCCGTGGTGTTTATTTGTGAAATTTGTGATGCTATTGCTTT

ATTTGTAACCATCTAGCTTTATTTGTGAAATTTGGTGATGCTATTGCTTTATTTGTAACCATT

ATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAG

GGGGAGATGTGGGAGGTTTTTTAAAGCGGCCGCAGGAACCCCTAGTGATGGAGTTGGC

CACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGA

CGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG

GGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATACGTCA

AAGCAACCATAGTACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTAC

GCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTTCGCTTTCTTCCC

TTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGTCCCCTTT

AGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTTGGGTGATG

GTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCA

CGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGGCT

ATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGGCTGAT

TTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTTATGGTGCACT

CTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACC

CGCTGACGCGCCCTGACGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGA

CCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGA

CGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTT

AGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCT

AAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATA

TTGAAAAAGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGC

GGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTG

AAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATC

CTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTA

TGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACA

CTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGG

CATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCA

ACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATG

GGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAA

CGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAA

CTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATA

AAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTTATTGCTGATAAAT

CTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAG

CCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAAT

AGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGT

TTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGA

AGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAG

CGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAA

TCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAA

GAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATAC

TGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTAC

ATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCT

TACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACG

GGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACC

TACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGCGGGACAGGTA

TCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAA

CGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTG

TGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGT(SEQ ID NO.7)

Sequence information of vector rAAV-GBA1-CDNF:

CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGTGGCCAACTCCATCACTAGGGGTTCCTGCGGCCATTCGCGGTACAATTCACGCGTCGACATTGATTATTGACTAGCTCTGGTCGTTACATAACTTACGGTAAATGGCCCGCCTGCCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGA CGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACTCGAGGCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCTCCCCACCCCCAATTTTGTATTT ATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGCGGGATCAGCCACCGCGGTGGCGGCCTAGAGTCGACGAG GAACTGAAAAACCAGAAAGTTAACTGGTAAGTTTAGTTCTTTTTGTC

TTTTATTTCAGGTCCCGGATCCCGGTGGTGGTGCAAATCAAAGAACTGCTCCTCAGTGGAT

GTTGCCTTTACTTCTAGGCCTGTACGGAAGTGTTACTTCTGCTCTAAAAGCTGCGGAATT

GTACCCGCGGCCGATCCACCGGTCGCCACCATGGAGTTTTCAAGTCCTTCCAGAGAGGA

ATGTCCCAAGCCTTTGAGTAGGGTAAGCATCATGGCTGGCAGCCTCACAGGATTGCTTCT

ACTTCAGGCAGTGTCGTGGGCATCAGGTGCCCGCCCCTGCATCCCTAAAAGCTTCGGCT

ACAGCTCCGGTGGTGTGTGTCTGCAATGCCACATACTGTGACTCCTTTGACCCCCGACCT

TTCCTGCCCTTGGTACCTTCAGCCGCTATGAGAGTACACGCAGTGGGCGACGGATGGAG

CTGAGTATGGGGCCCATCCAGGCTAATCACACGGGCACAGGCCTGCTACTGACCCTGCA

GCCAGAACAGAAGTTCCAGAAAGTGAAGGGATTTGGAGGGGCCATGACAGATGCTGCT

GCTCTCAACATCCTTGCCCTGTCACCCCCTGCCCAAAATTTGCTACTTAAATCGTACTTCT

CTGAAGAAGGAATCGGATATAACATCATCCGGGTACCCATGGCCAGCTGTGACTTCTCCA

TCCGCACCTACACCTATGCAGACACCCTGATGATTTCCAGTTGCACAACTTCAGCCTCC

CAGAGGAAGATACCAAGCTCAAGATACCCCTGATTCACCGAGCCCTGCAGTTGGCCCAG

CGTCCCGTTTCACTCCTTGCCAGCCCCTGGACATCACCCACTTGGCTCAAGACCAATGG

AGCGGTGAATGGGAAGGGGTCACTCAAGGGACAGCCCGGAGACATCTACCACCAGACC

TGGGCCAGATACTTTGTGAAGTTCCTGGATGCCTATGCTGAGCACAAGTTACAGTTCTGG

GCAGTGACAGCTGAAAATGAGCCTTCTGCTGGGCTGTTGAGTGGATACCCCTTCCAGTG

CCTGGGCTTCACCCCTGAACATCAGCGAGACTTCATTGCCCGTGACCTAGGTCCTACCCT

CGCCAACAGTACTCACCACAATGTCCGCCTACTCATGCTGGATGACCAACGCTTGCTGC

TGCCCCACTGGGCAAAGGTGGTACTGACAGACCCAGAAGCAGCTAAATATGTTCATGGC

ATTGCTGTACATTGGTACCTGGACTTTCTGGCTCCAGCCAAAGCCACCCTAGGGGAGAC

ACACCGCCTGTTCCCCAACACCATGCTCTTTGCCTCAGAGGCCTGTGTGGGCTCCAAGT

TCTGGGAGCAGAGTGTGCGGCTAGGCTCCTGGGATCGAGGGATGCAGTACAGCCACAG

CATCATCACGAACCTCCTGTACCATGTGGTCGGCTGGACCGACTGGAACCTTGCCCTGA

ACCCCGAAGGAGGACCCAATTGGGTGCGTAACTTTGTCGACAGTCCCATCATTGTAGAC

ATCACCAAGGACACGTTTTTACAAACAGCCCATGTTCTACCACCTTGGCCACTTCAGCAA

GTTATTCCTGAGGGCTCCCAGAGAGTGGGGCTGGTTGCCAGTCAGAAGAACGACCTG

GACGCAGTGGCACTGATGCATCCCGATGGCTCTGCTGTTGTGGTCGTGCTAAACCGCTC

CTCTAAGGATGTGCCTCTTACCATCAAGGATCCTGCTGTGGGCTTCCTGGAGACAATCTC

ACCTGGCTACTCCATTCACACCTACCTGTGGCGTCGCCAGGGAAGCGGAGCCACTAACT

TCTCCCTGTTGAAACAAGCAGGGGATGTCGAAGAGAATCCCGGGCCAATGTGGTGCGC

GAGCCCAGTTGCTGTGGTGGCCTTTTGCGCCGGGCTTTTGGTCTTCCACCCGGTGCTGA

CGCAGGGCCAGGAGGCCGGGGGGCGGCCAGGGGCCGACTGTGAAGTATGTAAAGAATT

CTTGAACCGATTCTACAAGTCACTGATAGACAGAGGAGTTAACTTTTCGCTGGACACTAT

AGAGAAAGAATTGATCAGTTTTTGCTTGGACACCAAAGGAAAAGAAAACCGCCTGTGC

TATTATCTAGGAGCCACAAAAGACGCAGCCACAAAGATCCTAAGTGAAGTCACTCGCCC

AATGAGTGTGCATATGCCTGCAATGAAGATTTGTGAGAAGCTGAAGAAGTTGGATAGCC

AGATCTGTGAGCTGAAATATGAAAAAACACTGGACTTGGCATCAGTTGACCTGCGGAAG

ATGAGAGTGGCAGAGCTGAAGCAGATCCTGCATAGCTGGGGGGAGGAGTGCAGGGCCT

GTGCAGAAAAAAACTGACTATGTGAATTCTCATTCAAGAGCTGGCCCCCAAGTATGCAGCG

ACACACCCAAAACAGAGCTCTGAGAATTCCGCTCGAGATAATCAACCTCTGGATTACA

AAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATA

CGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATTGGCTTTCATTTTCTCCTCCT

TGTATAAATCCTGGTTAGTTCTTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCT

GCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTTTATTTGTGAAATTT

GTGATGCTATTGCTTTATTTGTAACCATCTAGCTTTATTTGTGAAATTTGTGATGCTATTGC

TTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTT

ATGTTTCAGGTTCAGGGGGAGATGTGGGAGGTTTTTTAAAGCGGCCGCAGGAACCCTA

GTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACC

AAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGC

AGCTGCCTGCAGGGGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCA

CACCGCATACGTCAAAGCAACCATAGTACGCGCCCTGTAGCGGCGCATTAAGCGCGGCG

GGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCC

TTTCGCTTTCTTCCCTTCCTTTCTCCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAAT

CGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACT

TGATTTGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTT

GACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCA

ACCCTATCTCGGGCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTA

AAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACA

ATTTTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGA

CACCCGCCAACACCCGCTGACGCGCCCTGACGGCTTGTCTGCTCCCGGCATCCGCTTA

CAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCAC

CGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGA

TAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTA

TTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAA

ATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTA

TTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAG

TAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAAC

AGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTT

AAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGT

CGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCA

TCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAA

CACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTT

TGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAA

GCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGC

GCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGA

TGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTT

ATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGG

GCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTAT

GGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAAC

TGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAA

AGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTT

TCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTT

TTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTT

GTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCG

CAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCT

GTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGC

GATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCG

GTCGGGCTGAACGGGGGGTTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACC

GAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAA

AGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGC

TTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTG

AGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGT (SEQ ID NO. 8);

Sequence information of vector rAAV-GBA1-NRTN:

CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGTGGCCAACTCCATCACTAGGGGTTCCTGCGGCCATTCGCGGTACAATTCACGCGTCGACATTGATTATTGACTAGCTCTGGTCGTTACATAACTTACGGTAAATGGCCCGCCTGCCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGA CGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACTCGAGGCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCTCCCCACCCCCAATTTTGTATTT ATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGCGCGCGCCAGGCGG

GGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCA

ATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCC

CTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGCGGGATCAGCCACCGCGGTGGCGGCC

TAGAGTCGACGAGGAACTGAAAAACCAGAAAGTTAACTGGTAAGTTTAGTCTTTTTGTC

TTTTATTTCAGGTCCCGGATCCCGGTGGTGGTGCAAATCAAAGAACTGCTCCTCAGTGGAT

GTTGCCTTTACTTCTAGGCCTGTACGGAAGTGTTACTTCTGCTCTAAAAGCTGCGGAATT

GTACCCGCGGCCGATCCACCGGTCGCCACCATGGAGTTTTCAAGTCCTTCCAGAGAGGA

ATGTCCCAAGCCTTTGAGTAGGGTAAGCATCATGGCTGGCAGCCTCACAGGATTGCTTCT

ACTTCAGGCAGTGTCGTGGGCATCAGGTGCCCGCCCCTGCATCCCTAAAAGCTTCGGCT

ACAGCTCCGGTGGTGTGTGTCTGCAATGCCACATACTGTGACTCCTTTGACCCCCGACCT

TTCCTGCCCTTGGTACCTTCAGCCGCTATGAGAGTACACGCAGTGGGCGACGGATGGAG

CTGAGTATGGGGCCCATCCAGGCTAATCACACGGGCACAGGCCTGCTACTGACCCTGCA

GCCAGAACAGAAGTTCCAGAAAGTGAAGGGATTTGGAGGGGCCATGACAGATGCTGCT

GCTCTCAACATCCTTGCCCTGTCACCCCCTGCCCAAAATTTGCTACTTAAATCGTACTTCT

CTGAAGAAGGAATCGGATATAACATCATCCGGGTACCCATGGCCAGCTGTGACTTCTCCA

TCCGCACCTACACCTATGCAGACACCCTGATGATTTCCAGTTGCACAACTTCAGCCTCC

CAGAGGAAGATACCAAGCTCAAGATACCCCTGATTCACCGAGCCCTGCAGTTGGCCCAG

CGTCCCGTTTCACTCCTTGCCAGCCCCTGGACATCACCCACTTGGCTCAAGACCAATGG

AGCGGTGAATGGGAAGGGGTCACTCAAGGGACAGCCCGGAGACATCTACCACCAGACC

TGGGCCAGATACTTTGTGAAGTTCCTGGATGCCTATGCTGAGCACAAGTTACAGTTCTGG

GCAGTGACAGCTGAAAATGAGCCTTCTGCTGGGCTGTTGAGTGGATACCCCTTCCAGTG

CCTGGGCTTCACCCCTGAACATCAGCGAGACTTCATTGCCCGTGACCTAGGTCCTACCCT

CGCCAACAGTACTCACCACAATGTCCGCCTACTCATGCTGGATGACCAACGCTTGCTGC

TGCCCCACTGGGCAAAGGTGGTACTGACAGACCCAGAAGCAGCTAAATATGTTCATGGC

ATTGCTGTACATTGGTACCTGGACTTTCTGGCTCCAGCCAAAGCCACCCTAGGGGAGAC

ACACCGCCTGTTCCCCAACACCATGCTCTTTGCCTCAGAGGCCTGTGTGGGCTCCAAGT

TCTGGGAGCAGAGTGTGCGGCTAGGCTCCTGGGATCGAGGGATGCAGTACAGCCACAG

CATCATCACGAACCTCCTGTACCATGTGGTCGGCTGGACCGACTGGAACCTTGCCCTGA

ACCCCGAAGGAGGACCCAATTGGGTGCGTAACTTTGTCGACAGTCCCATCATTGTAGAC

ATCACCAAGGACACGTTTTTACAAACAGCCCATGTTCTACCACCTTGGCCACTTCAGCAA

GTTATTCCTGAGGGCTCCCAGAGAGTGGGGCTGGTTGCCAGTCAGAAGAACGACCTG

GACGCAGTGGCACTGATGCATCCCGATGGCTCTGCTGTTGTGGTCGTGCTAAACCGCTC

CTCTAAGGATGTGCCTCTTACCATCAAGGATCCTGCTGTGGGCTTCCTGGAGACAATCTC

ACCTGGCTACTCCATTCACACCTACCTGTGGCGTCGCCAGGGAAGCGGAGCCACTAACT

TCTCCCTGTTGAAACAAGCAGGGGATGTCGAAGAGAATCCCGGGCCAATGCAGCGCTG

GAAGGCGGCGGCCTTGGCCTCAGTGCTCTGCAGCTCCGTGCTGTCCATCTGGATGTGTC

GAGAGGGCCTGCTTCTCAGCCACCGCCTCGGACCTGCGCTGGTCCCCCTGCACCGCCTG

CCTCGAACCCTGGACGCCCGGATTGCCCGCCTGGCCCAGTACCGTGCACTCCTGCAGG

GGCCCCGGATGCGATGGAGCTGCGCGAGCTGACGCCCTGGGCTGGGCGGCCCCCAGGT

CCGCGCCGTCGGGCGGGGCCCCGGCGGCGGCGCGCGCGTGCGCGGTTGGGGGCGCGG

CCTTGCGGGCTGCGCGAGCTGGAGGTGCGCGTGAGCGAGCTGGGCCTGGGCTACGCGT

CCGACGAGACGGTGCTGTTCCGCTACTGCGCAGGCGCCTGCGAGGCTGCCGCGCGCGT

CTACGACCTCGGGCTGCGACGACTGCGCCAGCGGCGGCGCCTGCGGCGGGAGCGGGTG

CGCGCGCAGCCCTGCTGCCGCCCGACGGCCTACGAGGACGAGGTGTCCTTCCTGGACG

CGCACAGCCGCTACCACACGGTGCACGAGCTGTCGGCGCGCGAGTGCGCCTGCGTGTG

AGAATTCCGCTCGAGATAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTA

TTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCAT

GCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTAGTTCTTGC

CACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGG

GCACTGACAATTCCGTGGTGTTTTATTTGTGAAATTTGTGATGCTATTGCTTTTATTTGTAAC

CATCTAGCTTTATTTGTGAAATTTGGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGC

AATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGGAGATG

TGGGAGGTTTTTTAAAGCGGCCGCAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCT

CTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCT

TTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGGGCGCCTGAT

GCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATACGTCAAAGCAACCAT

AGTACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTG

ACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTTCGCTTTCTTCCCTTCCTTTCTC

GCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGA

TTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTTGGGTGATGGTTCACGTAGT

GGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAAT

AGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGGCTATTCTTTTGAT

TTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAAT

TTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTTATGGTGCACTCTCAGTACAAT

CTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGC

CCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGG

AGCTGCATGTGTCAGAGGTTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCC

TCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTTAGACGTCAGGT

GGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAA

ATATTGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAA

GAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTT

CCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGG

TGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTC

GCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTAT

TATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATG

ACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGA

GAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACA

ACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAAC

TCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGAC

ACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACT

TACTCTAGCTTCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGAC

CACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTTATTGCTGATAAATCTGGAGCCGGTG

AGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATC

GTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGC

TGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATA

CTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTG

ATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCG

TAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGC

AAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAAC

TCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGT

GTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCT

GCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGA

CTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGC

ACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGC

TATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGCAGGTATCCGGTAAGCGG

CAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTT

TATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCA

GGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGT (SEQ ID NO. 9);

Sequence information of vector rAAV-GBA1-GDNF:

CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGTGGCCAACTCCATCACTAGGGGTTCCTGCGGCCATTCGCGGTACAATTCACGCGTCGACATTGATTATTGACTAGCTCTGGTCGTTACATAACTTACGGTAAATGGCCCGCCTGCCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGA CGTATGTTCCCATAGTAACGCCAATAGGGACTTTC

CATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTG

TATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATT

ATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACTCGAGGCCAC

GTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTT

TTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCCAGGCGG

GGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCA

ATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCC

CTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGCGGGATCAGCCACCGCGGTGGCGGCC

TAGAGTCGACGAGGAACTGAAAAACCAGAAAGTTAACTGGTAAGTTTAGTCTTTTTGTC

TTTTATTTCAGGTCCCGGATCCCGGTGGTGGTGCAAATCAAAGAACTGCTCCTCAGTGGAT

GTTGCCTTTACTTCTAGGCCTGTACGGAAGTGTTACTTCTGCTCTAAAAGCTGCGGAATT

GTACCCGCGGCCGATCCACCGGTCGCCACCATGGAGTTTTCAAGTCCTTCCAGAGAGGA

ATGTCCCAAGCCTTTGAGTAGGGTAAGCATCATGGCTGGCAGCCTCACAGGATTGCTTCT

ACTTCAGGCAGTGTCGTGGGCATCAGGTGCCCGCCCCTGCATCCCTAAAAGCTTCGGCT

ACAGCTCCGGTGGTGTGTGTCTGCAATGCCACATACTGTGACTCCTTTGACCCCCGACCT

TTCCTGCCCTTGGTACCTTCAGCCGCTATGAGAGTACACGCAGTGGGCGACGGATGGAG

CTGAGTATGGGGCCCATCCAGGCTAATCACACGGGCACAGGCCTGCTACTGACCCTGCA

GCCAGAACAGAAGTTCCAGAAAGTGAAGGGATTTGGAGGGGCCATGACAGATGCTGCT

GCTCTCAACATCCTTGCCCTGTCACCCCCTGCCCAAAATTTGCTACTTAAATCGTACTTCT

CTGAAGAAGGAATCGGATATAACATCATCCGGGTACCCATGGCCAGCTGTGACTTCTCCA

TCCGCACCTACACCTATGCAGACACCCTGATGATTTCCAGTTGCACAACTTCAGCCTCC

CAGAGGAAGATACCAAGCTCAAGATACCCCTGATTCACCGAGCCCTGCAGTTGGCCCAG

CGTCCCGTTTCACTCCTTGCCAGCCCCTGGACATCACCCACTTGGCTCAAGACCAATGG

AGCGGTGAATGGGAAGGGGTCACTCAAGGGACAGCCCGGAGACATCTACCACCAGACC

TGGGCCAGATACTTTGTGAAGTTCCTGGATGCCTATGCTGAGCACAAGTTACAGTTCTGG

GCAGTGACAGCTGAAAATGAGCCTTCTGCTGGGCTGTTGAGTGGATACCCCTTCCAGTG

CCTGGGCTTCACCCCTGAACATCAGCGAGACTTCATTGCCCGTGACCTAGGTCCTACCCT

CGCCAACAGTACTCACCACAATGTCCGCCTACTCATGCTGGATGACCAACGCTTGCTGC

TGCCCCACTGGGCAAAGGTGGTACTGACAGACCCAGAAGCAGCTAAATATGTTCATGGC

ATTGCTGTACATTGGTACCTGGACTTTCTGGCTCCAGCCAAAGCCACCCTAGGGGAGAC

ACACCGCCTGTTCCCCAACACCATGCTCTTTGCCTCAGAGGCCTGTGTGGGCTCCAAGT

TCTGGGAGCAGAGTGTGCGGCTAGGCTCCTGGGATCGAGGGATGCAGTACAGCCACAG

CATCATCACGAACCTCCTGTACCATGTGGTCGGCTGGACCGACTGGAACCTTGCCCTGA

ACCCCGAAGGAGGACCCAATTGGGTGCGTAACTTTGTCGACAGTCCCATCATTGTAGAC

ATCACCAAGGACACGTTTTTACAAACAGCCCATGTTCTACCACCTTGGCCACTTCAGCAA

GTTATTCCTGAGGGCTCCCAGAGAGTGGGGCTGGTTGCCAGTCAGAAGAACGACCTG

GACGCAGTGGCACTGATGCATCCCGATGGCTCTGCTGTTGTGGTCGTGCTAAACCGCTC

CTCTAAGGATGTGCCTCTTACCATCAAGGATCCTGCTGTGGGCTTCCTGGAGACAATCTC

ACCTGGCTACTCCATTCACACCTACCTGTGGCGTCGCCAGGGAAGCGGAGCCACTAACT

TCTCCCTGTTGAAACAAGCAGGGGATGTCGAAGAGAATCCCGGGCCAATGAAGTTATGG

GATGTCGTGGCTGTCTGCCTGGTGCTGCTCCACACCGCGTCCCGCCTTCCCGCTGCCCGC

CGGTAAGAGGCCTCCCGAGGCGCCCGCCGAAGACCGCTCCCTCGGCCGCCGCCGCGCG

CCCTTCGCGCTGAGCAGTGACTCAAATATGCCAGAGGATTATCCTGATCAGTTCGATGAT

GTCATGGATTTTATTCAAGCCACCATTAAAAGACTGAAAAGGTCACCAGATAAACAAAT

GGCAGTGCTTCCTAGAAGAGAGCGGAATCGGCAGGCTGCAGCTGCCAACCCAGAGAAT

TCCAGAGGAAAAGGTCGGAGAGGCCAGAGGGGCAAAAACCGGGGTTGTGTCTTAACT

GCAATACATTTAAATGTCACTGACTTGGGTCTGGGCTATGAAACCAAGGAGGAACTGAT

TTTTAGGTACTGCAGCGGCTCTTGCGATGCAGCTGAGACAACGTACGACAAAATATTGA

AAAACTTATCCAGAAATAGAAGGCTGGTGAGTGACAAAGTAGGGCAGGCATGTTGCAG

ACCCATCGCCTTTGATGATGACCTGTCGTTTTTAGATGATAACCTGGTTTACCATATTCTA

AGAAAGCATTCCGCTAAAAGGTGTGGATGTATCTGAGAATTCCGCTCGAGATAATCAAC

CTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTAC

GCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCA

TTTTTCCTCCTTGTATAAATCCTGGTTAGTTCTTGCCACGGCGGAACTCATCGCCGCCTG

CCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTTA

TTTGTGAAATTTGGTGATGCTATTGCTTTATTTGTAACCATCTAGCTTTATTTGTGAAATTTG

TGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAAT

TGCATTCATTTTATGTTTCAGGTTCAGGGGGAGATGTGGGAGGTTTTTTAAAGCGGCCGC

AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAG

GCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCG

AGCGAGCGCGCAGCTGCCTGCAGGGGCGCCTGATGCGGTATTTTCTCCTTACGCATCTG

TGCGGTATTTCACACCGCATACGTCAAAGCAACCATAGTACGCGCCCTGTAGCGGCGCAT

TAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTA

GCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCGTC

AAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACC

CCAAAAAACTTGATTTGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTT

TTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGA

ACAACACTCAACCCTATCTCGGGCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGG

CCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATT

AACGTTTACAATTTTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGC

CAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGG

CATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCA

CCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTT

AATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGC

GGAACCCCTATTTGTTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATA

ACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCG

TGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACG

CTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACT

GGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGA

TGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAG

AGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCA

CAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACC

ATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCT

AACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGG

AGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGC

AACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATT

AATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGG

CTGGCTGGTTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTG

CAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGT

CAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAA

GCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCAT

TTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTT

AACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCGTAGAAAAGATCAAAGGATCTTCT

TGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCA

GCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTT

CAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTT

CAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGC

TGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATA

AGGCTGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACAGCCCAGCTTGGAGCGAA

CGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCC

GAAGGGAGAAAGCGGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGC

ACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCA

CCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAA

ACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGT

(SEQ ID NO.10);

Sequence information of vector rAAV-GBA1-BDNF:

CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGTGGCCAACTCCATCACTAGGGGTTCCTGCGGCCATTCGCGGTACAATTCACGCGTCGACATTGATTATTGACTAGCTCTGGTCGTTACATAACTTACGGTAAATGGCCCGCCTGCCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGA CGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACTCGAGGCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCTCCCCACCCCCAATTTTGTATTT ATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGCGGGATCAGCCACCGCGGTGGCGGCCTAGAGTCGACGAG GAACTGAAAAACCAGAAAGTTAACTGGTAAGTTTAGTCTTTTTGTCTTTTATTTCAGGTCCCGGATCCGGTGGTGGTGCAAATCAAAGAACTGTCCCTCAGTGGATGTTGCCTTTACTTCTAGGCCTGTACGGAAGTGTTACTTCTGCTCTAAAAGCTGCGGAATTGTACCCGCGGCCGATCCACCGGTCGCCACCATGGAGTTTTCAAGTCCTTCCAGAGAGGAATGTCCCAAGCCTTTGAGTAGGGTAAGCATGGCT GGCAGCCTCACAGGATTGCTTCTACTTCAGGCAGTGTCGTGGGCATCAGGTGCCCGCCCCTGCATCCCTAAAAGCTTCGGCTACAGCTCGGTGTGTGTGTCTGCAATGCCACATACTGTGACTCCTTTGACCCCCCGACCTTTCCTGCCCTTGGTACCTTCAGCCGCTATGAGAGTACACGCAGTGGGCGACGGATGGAGCTGAGTATGGGGCCCATCCAGGCTAATCACACGGGCACAGGCCTGCTACTGACCCTGCA GCCAGAACAGAAGTTCCAGAAAGTGAAGGGATTTGGAGGGGCCATGACAGATGCTGCTGCTCTCAACATCCTTGCCCTGTCACCCCCTGCCCAAAATTTGCTACTTAAATCGTACTTCTCTGAAGAAGGAATCGGATAACATCATCCGGGTACCCATGGCCAGCTGTGACTTCTCCATCCGCACCTACACCTATGCAGACACCCCTGATGATTTCCAGTTGCACAACTTCAGCCTCCCAGAGGAAGATACCAAGCTCAAGATACCC CTGATTCACCGAGCCCTGCAGTGGCCCAGCGTCCCGTTTCACTCCTTGCCAGCCCCTGGACATCACCCACTTGGCTCAAGACCAATGGAGCGGTGAATGGGAAGGGGTCACTCAAGGGACAGCCCGGAGACATCTACCACCAGACCTGGGCCAGATACTTTGTGAAGTTCCTGGATGCCTATGCTGAGCACAAGTTACAGTTCTGGGCAGTGACAGCTGAAAATGAGCCTTCTGCTGGGCTGTTGAGTGGATACCCCTTC CAGTGCCTGGGCTTCACCCCTGAACATCAGCGAGACTTCATTGCCCGTGACCTAGGTCCTACCCTCGCCAACAGTACTCACCACAATGTCCGCCTACTCATGCTGGATGACCAACGCTTGCTGCCCCACTGGGCAAAGGTGGTACTGACAGACCCAGAAGCAGCTAAATATGTTCATGGC

ATTGCTGTACATTGGTACCTGGACTTTCTGGCTCCAGCCAAAGCCACCCTAGGGGAGAC

ACACCGCCTGTTCCCCAACACCATGCTCTTTGCCTCAGAGGCCTGTGTGGGCTCCAAGT

TCTGGGAGCAGAGTGTGCGGCTAGGCTCCTGGGATCGAGGGATGCAGTACAGCCACAG

CATCATCACGAACCTCCTGTACCATGTGGTCGGCTGGACCGACTGGAACCTTGCCCTGA

ACCCCGAAGGAGGACCCAATTGGGTGCGTAACTTTGTCGACAGTCCCATCATTGTAGAC

ATCACCAAGGACACGTTTTTACAAACAGCCCATGTTCTACCACCTTGGCCACTTCAGCAA

GTTATTCCTGAGGGCTCCCAGAGAGTGGGGCTGGTTGCCAGTCAGAAGAACGACCTG

GACGCAGTGGCACTGATGCATCCCGATGGCTCTGCTGTTGTGGTCGTGCTAAACCGCTC

CTCTAAGGATGTGCCTCTTACCATCAAGGATCCTGCTGTGGGCTTCCTGGAGACAATCTC

ACCTGGCTACTCCATTCACACCTACCTGTGGCGTCGCCAGGGAAGCGGAGCCACTAACT

TCTCCCTGTTGAAACAAGCAGGGGATGTCGAAGAGAATCCCGGGCCAATGACCATCCTT

TTCCTTACTATGGTTATTTCATACTTTGGTTGCATGAAGGCTCACTCTGACCCTGCCCGCC

GAGGGGAGCTGAGCGTGTGTGACAGTATTAGTGAGTGGGTAACGGCGGCAGACAAAAA

GACTGCAGTGGACATGTCGGGCGGGACGGTCACAGTCCTTGAAAAGGTCCCTGTATCA

AAAGGCCAACTGAAGCAATACTTCTACGAGACCAAGTGCAATCCCATGGGTTACACAAA

AGAAGGCTGCAGGGGCATAGACAAAAGGCATTGGAACTCCCAGTGCCGAACTACCCAG

TCGTACGTGCGGGCCCTTACCATGGATAGCAAAAAGAGAATTGGCTGGCGATTCATAAG

GATAGACACTTCTTGTGTATGTACATTGACCATTAAAAGGGGAAGATAGGAATTCCGCTC

GAGATAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGT

TGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCC

GTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTAGTTCTTGCCACGGCGGAACT

CATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATT

CCGTGGTGTTTATTTGTGAAATTTGGTGATGCTATTGCTTTATTTGTAACCATCTAGCTTTAT

TTGTGAAATTTGGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTT

AACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGATGTGGGAGGTTTTT

TAAAGCGGCCGCAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGC

TCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGG

CCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGGGCGCCTGATGCGGTATTTTCTC

CTTACGCATCTGTGCCGGTATTTCACACCGCATACGTCAAAGCAACCATAGTACGCGCCCT

GTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTT

GCCAGCGCCCTAGCGCCCGCTCCTTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCC

GGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTA

CGGCACCTCGACCCCAAAAAACTTGATTTGGGTGATGGTTCACGTAGTGGGCCATCGCC

CTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTT

GTTCCAAACTGGAACAACACTCAACCCTATCTCGGGCTATTCTTTTGATTTATAAGGGAT

TTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAA

TTTTAACAAAATATTAACGTTTACAATTTTATGGTGCACTCTCAGTACAATCTGCTCTGAT

GCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGG

CTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATG

TGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATAC

GCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTT

CGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATC

CGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGA

GTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGGCATTTTGCCTTCCTGTTTTT

GCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAG

TGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAA

GAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTA

TTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTT

GAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATG

CAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCG

GAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTT

GATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGA

TGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAG

CTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTG

CGCTCGGCCCTTCCGGCTGGCTGGTTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGG

GTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTAT

CTACACGAGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAG

GTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGAT

TGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTC

ATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCGTAGAAAA

GATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAA

AAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTC

CGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCG

TAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATC

CTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAG

ACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTTCGTGCACACAG

CCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGA

AACGCCACGCTTCCCGAAGGGAGAAAGGCGGCAGGTATCCGGTAAGCGGCAGGGTC

GGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTC

CTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGG

CGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGT (SEQ ID NO. 11).

(2) Preparation of recombinant adeno-associated virus

HEK293 cells were seeded into 20 15cm cell culture dishes. When the confluence reached 80%, the rAAV expression vector, capsid protein plasmid AAV2 (or AAV9) and auxiliary packaging plasmid pHelper were mixed in a PEI-mediated solution at a molar ratio of 1:1:1. co-transfection. Capsid protein particles and auxiliary packaging plasmids were purchased from Zhilizhongte (Wuhan) Biotechnology Co., Ltd. The mass ratio of plasmid to PEI is 1:3. After mixing, let it stand at room temperature for 5 minutes, add it drop by drop to the culture dish, and culture it in a 37°C, 5% _CO2 incubator for 72 hours. At the same time, collect the cells and culture supernatant, and centrifuge at 3000g. Separate the cells and culture supernatant for 5 minutes. Add PEG 8000 with a final concentration of 8% and 0.5M sodium chloride to the collected culture supernatant. Place it on ice for 3 hours, then centrifuge at 4°C and 3000 × g for 30 minutes. Remove the supernatant. Mix the precipitated and collected cells and transfer them to a 50mL centrifuge tube, add 15mL lysis solution (50mM Tris-HCl, 100mM NaCl, 0.2M MgCl ₂ , pH8.0), dry ice/ethanol and freeze and thaw in a 37°C water bath three times. , add Benzonase (Sigma, E1014-5KU) with a final concentration of 50U/mL and RNase (Sigma, 101109142001) with a final concentration of 10U/mL into the lysis solution, bathe in a 37°C water bath for 30min, add 0.1% sodium deoxycholate and continue the action for 30min. Centrifuge at 2500×g for 10 min to collect the supernatant.

(3) Virus purification

In the present invention, virus purification adopts iodixanol (Sigma, D1556) ultracentrifugation method (Beckman ultracentrifuge). Specifically, from top to bottom, add the cell lysate, 15% iodixanol solution, 25% iodixanol solution, 40% iodixanol solution and 60% iodixanol solution to the ultracentrifuge tube. After heat sealing, centrifuge at 10°C and 350,000 × g for 90 minutes. After centrifugation, the needle extracts the 40% iodixanol layer, adds it to a 50KD ultrafiltration tube (Sigma, UFC905008), and concentrates it by ultrafiltration. The virus is finally stored at -80°C in 0.001% Pluronic F-68 (Sangon, A600749)/ in PBS solution.

(4) Virus titer determination

Fluorescence quantitative PCR method was used to determine the virus titer. The fluorescent quantitative PCR primer sequence information is shown in SEQ ID NO. 12 and SEQ ID NO. 13 below. The specific operations are as follows:

Take 10ug of the rAAV expression plasmid used for packaging, digest it with HindIII (NEB, #3104V) overnight, cut the circular plasmid into linear lines, and separate it with 1% agarose. Use a DNA gel recovery kit (Beyotime, D0056) to recover the target fragment. Calculate the molar mass of rAAV plasmid digested by the enzyme according to the formula molar mass = number of plasmid base pairs × 662g/mol.bp. According to the number of molecules of 1M=6.02×10 ²³ , count the number of molecules of linear rAAV plasmid per microliter after digestion. Then, prepare the linear rAAV vector into a 100ul1×10 ¹⁰ concentration standard, and then dilute it into a 10 ⁹ , 10 ⁸ , 10 ⁷ , 10 ⁶ , 10 ⁵ , 10 ⁴ , 10 ³ and 10 ² concentration standard. SYBR Green method (Tiangen, FP215) was used to perform fluorescence quantitative PCR. The PCR system was:

Table 1 PCR system

The PCR reaction procedure is:

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).

Take 5ul of the purified virus and prepare a reaction system of 33ul water, 5ul 10×DNase buffer, 2ul DNaseI (Invitrogen, AM2222), and 5ul 1% Fluronic F-68, and incubate at 37°C for 60 minutes. Then add 10ul of 10× Proteinase K buffer, 5ul of 1% Fluronic F-68, 1ul of Proteinase K (Invitrogen) and 34ul of water, incubate at 55°C for 30 minutes, and inactivate at 95°C for 15 minutes. The treated virus samples were diluted at 10×, 100× and 1000×, and fluorescent quantitative PCR was performed in the same system as the standard. Based on the obtained Ct value, the titer of the virus sample was calculated. The titers of the viruses used in the present invention are all ≥10 ¹³ vg/mL (vg: virus genome). Among them, the logarithm of the number of molecules of the standard substance is the ordinate, and the Ct value is the abscissa to draw the standard curve.

Example 2 High-efficiency expression of rAAV vector in mammalian cells such as HEK293

PEI mediates the rAAV vector rAAV-GBA1-CDNF, rAAV-GBA1-NRTN, rAAV-GBA1-GDNF and rAAV-GBA1-BDNF to transfect HEK293 cells, only adding PEI and transfecting rAAV-EGFP and rAAV-GBA1-EGFP HEK293 cells served as control group. Specifically: HEK293 cells were seeded into a 6-well cell culture plate. When the confluence reached about 70%, the complete medium was replaced with 1 mL of serum-free medium; PEI and rAAV vector were added to 500 ul of serum-free culture in a mass ratio of 3:1. medium, vortex to mix, let stand at 37°C for 15 minutes, add dropwise to the cells, and gently shake to mix; culture in a 37°C, 5% CO ₂ incubator for 4 hours; replace the medium with 3 mL of complete medium ;Culture in 37℃, 5% _CO2 incubator for 48h; collect cells and supernatant.

(1) Expression of rAAV vector in HEK293 cells

Extract the total protein in the HEK293 cells collected above, and use the BCA method to determine the protein concentration (Thermo: 23227); Western blot detects the expression of the target protein. The specific steps include: loading 30ug per well, and performing electrophoretic separation on 12% SDS-PAGE gel. , transferred to membrane; blocked with 5% skimmed milk powder for 1h, primary antibodies (GBA1, Abcam: ab55080; CDNF, Abcam: ab205551; NRNT, Abcam: ab274417; GDNF, Affinity: DF7727; BDNF, Affinity: DF6387; β-actin, Abcam: ab8227) was incubated at 4°C overnight; washed three times with TBST, incubated with the secondary antibody at room temperature for 1 hour, washed three times with TBST, and developed with substrate.

After PEI-mediated transfection of HEK293 cells, the experimental results of the expression of GBA1 and neurotrophic factors in the rAAV vector (n=3) are shown in Figures 8-11. The results show that after PEI-mediated transfection of rAAV vectors rAAV-GBA1-CDNF, rAAV-GBA1-NRTN, rAAV-GBA1-BDNF and rAAV-GBA1-GDNF, compared with the control group transfected with rAAV-EGFP, GBA1 gene expression The expression of the product GCase increased by more than 5 times. It can also be seen from Figures 8-11 that in HEK293 cells, the endogenous neurotrophic factor CDNF is basically not expressed. After transfection with the vector rAAV-GBA1-CDNF, the expression of CDNF is significantly increased. After transfection of neurotrophic factors NRTN, GDNF and BDNF, the expression difference between transfected HEK293 cells and untransfected cells was slightly smaller. This may be because the above three neurotrophic factors are secreted and mature after intracellular processing. That is, it is secreted outside the cell.

(2) Analysis of NRTN, GDNF and BDNF contents in the culture supernatant of HEK293 cells transfected with rAAV-GBA1-NRTN, rAAV-GBA1-GDNF and rAAV-GBA1-BDNF

Centrifuge the culture supernatant of HEK293 cells collected above with PEI-mediated transfection vectors rAAV-GBA1-NRTN, rAAV-GBA1-GDNF and rAAV-GBA1-BDNF at 12000 rpm and 4°C for 10 min, collect the supernatant, and use commercial ELISA The kit (NRTN: enzyme immunoassay Cat No.MM-61013H; GDNF: Boster Cat No.EK0362; BDNF: Boster Cat No.EK0307) is used to analyze the content of NRTN, GDNF and BDNF. Only PEI is added, rAAV-EGFP and rAAV are transfected. The HEK293 cell culture supernatant of rAAV-GBA1-EGFP was used as the control group. Please refer to the kit instruction manual for specific operation methods.

After HEK293 cells were transfected with the rAAV vector, the neurotrophic factor content (n=3) in the culture supernatant is shown in Figures 12-14. The results showed that the concentration of neurotrophic factors in the culture supernatant of cells transfected with neurotrophic factors was much higher than that in the control group, which were NRTN concentration 2403.55±209.65pg/mL, GDNF concentration 2235.6±144.1pg/mL, and BDNF concentration 2166.05±127.25 pg/mL, while the concentration of BDNF with higher endogenous expression level in the control group was only 232.72±31.20pg/mL. This shows that in the above intracellular protein expression analysis, the reason why the expression of neurotrophic factors is not significantly different in cells transfected with rAAV-GBA1-NRTN, rAAV-GBA1-GDNF and rAAV-GBA1-BDNF compared with the control group is that the expressed neuronal The nutritional factors NRTN, GDNF and BDNF are secreted out of the cell after being processed and matured.

The above results show that the rAAV vector described in the present invention can be expressed efficiently in mammalian cells, and the secreted neurotrophic factors can still be expressed normally and secreted out of the cells.

Example 3: GBA1 in the constructed product is active in GCase expressed in mammalian cells

PEI mediates rAAV vectors rAAV-GBA1-CDNF, rAAV-GBA1-NRTN, rAAV-GBA1-GDNF and rAAV-GBA1-BDNF to transfect HEK293 cells. HEK293 cells with PEI alone and HEK293 cells transfected with rAAV-EGFP were used as controls. Specifically, it is inoculated into a 6-well cell culture plate. When the confluence reaches about 70%, the complete medium is replaced with 1 mL of serum-free medium; PEI and rAAV vector are added to 500 ul of serum-free medium at a mass ratio of 3:1. Vortex to mix, let stand at 37°C for 15 minutes, add dropwise to the cells, and gently shake to mix; culture in a 37°C, 5% CO ₂ incubator for 4 hours; replace the culture medium with 3 mL of complete culture medium; 37°C , cultured in a 5% CO ₂ incubator for 48 h; collect cells into a 1.5 mL centrifuge tube.

The cells collected above were centrifuged at 12000 rpm and 1 min to remove the supernatant; add 1 mL of PBS, centrifuge at 12000 rpm and 1 min to remove the supernatant; add lysis solution (pH 5.4 citric acid-phosphate buffer, 0.25% Triton X-100, 1mM EDTA, Protease-phosphatase inhibitor), mix by pipetting, keep in ice bath for 30 minutes, 12000 rpm, centrifuge for 10 minutes; measure the total protein concentration by BCA method, add the sample to a 96-well white plate at 5ug/well, and add the volume to 50ul/well; add 50ul substrate 4-methylumbelliferone β-D-glucoside working solution (pH 5.4 citric acid-phosphate buffer, 0.25% Triton X-100, 1mM EDTA, 1% BSA, 5mM 4-methylumbelliferone Keto β-D-glucoside), incubate at 37°C for 15 minutes; add an equal volume of stop solution (pH 12.5, 1M glycine solution); detect with Biotek SynergyH1 full-function microplate reader (fluorescence: Ex360, Em460), Only the lysate well was added as a blank control.

After HEK293 cells were transfected with the rAAV vector, the experimental results of the expression amount and activity of GBA1 expression product GCase in the cells are shown in Figures 15-16. The results showed that after the PEI-mediated rAAV vector transfected HEK293 cells, compared with the control group, the intracellular GCase enzyme activity increased more than 3 times (GBA1-CDNF 5.5 times, GBA1-NRNT 3.6 times, GBA1-GDNF5. 9 times, GBA-BDNF 4.6 times), which is consistent with the trend of increasing GCase protein expression.

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

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

CBE (conduritol-β-epoxide) is a small molecule inhibitor of GCase that can inhibit enzyme activity at the protein level and is often used in the construction of Gaucher's disease models. In this study, HEK293 cells were seeded into a 6-well cell culture plate. When the cell confluence reached 70%, PBS-CBE solution (MedBio, MED17946) with a final concentration of 100uM was added and incubated at 37°C and 5% CO ₂ for 4 hours; Wash twice with 2% FBS-PBS; add serum-free medium, then PEI-mediated transfection of rAAV vectors rAAV-GBA1-CDNF, rAAV-GBA1-NRTN, rAAV-GBA1-GDNF and rAAV-GBA1-BDNF, add only PEI HEK293 and HEK293 cells transfected with rAAV-EGFP were used as controls. Specifically, PEI and rAAV vector were added to 500ul of serum-free culture medium at a mass ratio of 3:1, vortexed to mix, and after standing at 37°C for 15 minutes, they were added dropwise to the cells, and gently shaken to mix; 37°C, Culture in a 5% CO ₂ incubator for 4 hours; replace the culture medium with 3 mL of complete culture medium; culture in a 5% CO ₂ incubator at 37°C for 48 hours; collect cells into a 1.5 mL centrifuge tube

The cells collected above were centrifuged at 12000 rpm and 1 min to remove the supernatant; add 1 mL of PBS, centrifuge at 12000 rpm and 1 min to remove the supernatant; add lysis solution (pH 5.4 citric acid-phosphate buffer, 0.25% Triton X-100, 1mM EDTA, Protease-phosphatase inhibitor), mix by pipetting, ice bath for 30 minutes, 12000 rpm, centrifuge for 10 minutes; measure the total protein concentration by BCA method, add the sample to the 96 white plate at 5ug/well, and add the volume to 50ul/well; add 50ul Substrate 4-methylumbelliferone β-D-glucoside working solution (pH 5.4 citric acid-phosphate buffer, 0.25% Triton X-100, 1mM EDTA, 1% BSA, 5mM 4-methylumbelliferyl Keto β-D-glucoside), incubate at 37°C for 15 minutes; add equal volume of stop solution (pH12.5, 1M glycine solution); Biotek SynergyH1 full-function microplate reader (fluorescence: Ex360, Em460), only Add lysate well as blank control

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

In summary, the GBA1 expression product in the rAAV vector is still active in mammalian cells with reduced endogenous GCase activity, such as HEK293, which also shows that the genetic medicine of the present invention can be used to treat Gaucher's disease.

Example 5 Neurotrophic factors NRTN, GDNF, BDNF and CDNF in rAAV vector have neuroprotective effects

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

PEI mediates the transfection of rAAV vectors rAAV-GBA1-NRTN, rAAV-GBA1-GDNF and rAAV-GBA1-BDNF into HEK293 cells. rAAV-EGFP is used as the control group. Specifically, PEI and rAAV vector are added to 500ul at a mass ratio of 3:1. Serum-free medium, vortex to mix, let stand at 37°C for 15 minutes, add dropwise to the cells, and gently shake to mix; culture in a 37°C, 5% CO ₂ incubator for 4 hours; replace the medium with 3 mL Complete culture medium; incubate for 48 hours in a 37°C, 5% _CO2 incubator. Collect the supernatant, filter it with a 0.22uM microporous filter membrane and store it at -20°C for later use.

SH-SY5Y cells were seeded into a flat-bottomed 96-well plate at 2.5×10 ⁴ cells/well. After culturing for 12 hours in 10% FBSDMEM, they were replaced with 2% FBSDMEM and continued to culture for 48 hours. After the culture medium was removed, 50ul of the above-mentioned medium was added every 24 hours. After 72 hours, the cell viability was measured by the CTG method (Promega: G8641). Specifically, an equal volume of CTG reagent was added to the cells, shaken at room temperature for 10 minutes on a horizontal shaker, and the luminescence value was read with a microplate reader.

The ELISA measurement results in Example 2 show that the concentrations of NRTN, GDNF and BDNF in the above supernatant are 2403.55±209.65pg/mL, 2235.6±144.1pg/mL and 2166.05±127.25pg/mL respectively.

The experimental results of the effects of neurotrophic factors NRTN, GDNF and BDNF on the activity of neural cells such as SH-SY5Y in rAAV vector are shown in Figures 18-20. The cell viability results showed that the viability of SH-SY5Y added with NRTN supernatant was 3.3 times that of the control group (as shown in Figure 18), the viability of the GDNF group was 2.7 times that of the control group (as shown in Figure 19), and The activity rate of SH-SY5Y in the group adding BDNF was 14.6 times that of the control group (as shown in Figure 20). This shows that the neurotrophic factors NRTN, GDNF and BDNF expressed by the rAAV vector can play an extremely significant protective role on human neural cells such as SH-SY5Y under environmental stress (the present invention adopts cell "starvation" treatment).

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

SH-SY5Y cells were seeded into a 6-well cell culture plate at 3 × 10 ⁵ cells/well, and cultured for ¹² hours. Cells were infected with the virus amount for 72h (vg/cell, virus genome/cell). The cells were collected. AAV2/2-EGFP and AAV2/2-GBA1-EGFP were used as the control group, and AAV2/2-GBA1-CDNF was used as the experimental group to analyze the virus. The expression of GBA1 and CDNF in cells. Specifically, the total protein in the above cells was extracted, and the protein concentration was determined by the BCA method; the protein was denatured by heating at 100°C for 5 minutes; 30ug was loaded into each well, electrophoresed with 12% SDS-PAGE gel, and transferred to a membrane; blocked with 5% skimmed milk powder for 1 hour. The primary antibody (GBA1, Abcam: ab55080; CDNF, Abcam: ab205551; β-actin, Abcam: ab8227) was incubated overnight at 4°C; washed three times with TBST, incubated with the secondary antibody for 1 hour at room temperature, washed three times with TBST, and developed.

SH-SY5Y cells were seeded in a 96-well cell culture plate at 2.5×10 ⁴ cells/well and cultured for 12 hours. TG (Thapsigargin) with a final concentration of 5uM was added and cultured for 24 hours, and the culture medium was aspirated. Complete medium (10% FBS, DMEM) containing the above virus was added to the cells at 10 ⁵ vg/cell and 10 ⁶ vg/cell respectively, and cultured in a 37°C, 5% CO ₂ incubator.

After culturing for 72 hours in the experimental group of 10 ⁵ vg/cell, remove the culture medium, add PBS and wash twice. Add RNA extraction reagent (full gold: ET101-01) at 150ul/well to extract total cell RNA. For specific operations, see the product instructions. . The extracted total RNA was reverse transcribed into cDNA (Takara: RR047A). GAPDH was used as an internal reference to determine the relative expression of apoptosis markers CHOP and Capase3 (Vazyme: Q111-02). After culturing the 10 ⁶ vg/cell experimental group for 72 hours, the cell viability was measured by CTG method (Promega: G8641).

After the rAAV vector of the present invention, such as rAAV-GBA1-CDNF, is prepared into the genetic drug AAV2/2-GBA1-CDNF, and after infecting neural cells such as SH-SY5Y, the expression of GBA1 and CDNF in the cells, and after the action of 5uM TG, The experimental results of the effect of CDNF on cell apoptosis markers and viability are shown in Figures 21-23.

Protein expression analysis results showed that compared with AAV2/2-EGFP-infected SH-SY5Y cells, the amount of GBA1 expression product GCase in AAV2/2-GBA1-EGFP and AAV2/2-GBA1-CDNF-infected cells was significantly increased, and only There was obvious CDNF protein expression in cells infected with AAV2/2-GBA1-CDNF, as shown in Figure 21. It shows that after the rAAV vector is prepared into a gene therapy drug adeno-associated virus, it can infect mammalian neural cells such as SH-SY5Y, and can be expressed normally in the cells.

In the cell viability results measured by the CTG method, in the experimental group infected with a virus dose of 10 ⁵ vg/cell, the difference in cell viability between the CDNF virus-infected group and the non-virus-infected group was not significant (P>0.05). Further analysis of the expression of apoptosis markers CHOP and Caspase3 showed that the expression of apoptosis marker CHOP in the CDNF virus infection group was extremely significantly lower than that in the non-virus infection group (P<0.01), and that of Caspase3 was significantly lower than that in the non-CDNF virus infection group (P<0.05 ), as shown in Figure 22.

In the 10 ⁶ vg/cell virus-infected experimental group, the cell viability rate of the CDNF virus-infected group was significantly higher than that of the non-virus-infected group (P<0.05), as shown in Figure 23. It shows that after the constructed product is prepared into a genetic drug, CDNF can rescue mammalian nerve cells such as SH-SY5Y caused by TG and improve the survival rate of nerve cells.

In summary, after the rAAV vector in the present invention is prepared into a gene therapy drug - adeno-associated virus, it can be expressed normally in mammalian neural cells such as SH-SY5Y, and can also perform normal physiological functions, such as the above-mentioned CDNF The "rescue" effect.

Example 6 GCase expressed by GBA1 in rAAV vector can improve the functional defect of endogenous GCase enzyme - the ratio of α-synuclein (α-Syn) tetramers to monomers in mammalian neural cells such as SH-SY5Y

Relevant studies have found that reduced GCase enzyme activity can reduce the ratio of α-Syn protein tetramers to monomers in mammalian nerve cells, leading to the formation of pathological aggregation of α-Syn monomers and ultimately the death of nerve cells (SangjuneKim et al., 2017; Yumiko V. Taguchi et al., 2017; Kelly E. Glajch et al., 2021).

In the present invention, CBE is used to act on SH-SY5Y to construct a cell model with endogenous GCase enzyme function deficiency, and then the genetic medicine prepared by the rAAV vector is used to act on the cells to study the effect of increased GCase activity on α-Syn protein tetramerization in the above model cells. Body: The ratio effect of the single body.

(1) Based on CBE, a functional defect model of endogenous GCase enzyme in SH-SY5Y cells was constructed.

SH-SY5Y cells were seeded into a 48-well cell culture plate at 10 ⁵ cells/well and cultured for 24 hours. PBS-CBE solution was added to the cells at a final concentration of 0.01uM, 0.1uM, 1uM, 10uM, 100uM and 1000uM, and incubated. 24 h, cells were collected, and GCase activity in the cells was measured. Based on the above-mentioned changes in GCase enzyme activity under the action of CBE, as shown in Figure 24, the present invention uses a final concentration of 100uM to construct a cell model with endogenous GCase enzyme function deficiency. When the ratio of α-Syn protein tetramers to monomers in the model cells is the same as that of normal A significant decrease (P<0.05) compared to the number of cells indicates that the model is successfully constructed.

(2) The effect of GCase, the GBA1 expression product in the rAAV vector, on the ratio of α-Syn protein tetramers to monomers in CBE-mediated neuronal cell models with endogenous GCase function deficiency.

Three replicate groups were set up. The above-mentioned CBE-mediated SH-SY5Y model cells were seeded in a 6-well plate at 3×10 ⁵ cells/well, and cultured in complete medium containing 100uM CBE for 12 hours. Normal SH-SY5Y cells were used as the control group. After 12 hours, adeno-associated virus AAV2/2-EGFP (5×10 ⁵ vg/cell), AAV2/2-GBA1-EGFP (5×10 ⁵ vg/cell) and AAV2/2-GBA1-CDNF (10 ⁵ vg /cell), two groups of CBE-mediated model cells served as the control group; 24 hours later, the CBE-complete medium in the virus-infected group and one of the model control groups, the CBE(r) group, was replaced with complete medium without CBE, and continued After culturing for 48 h, cells were collected.

3 × 10 ⁴ cells were separated from each treatment in the above collected cells, and GCase activity was measured. After the remaining cells were mixed into three replicate groups, total cell protein was extracted, and the protein concentration was measured by BCA method (Thermo: 23227); the protein was denatured; 15ug was loaded into each well, and 12% SDS-PAGE gel was used for electrophoresis separation and transferred to membrane. ; Fixed with 4% paraformaldehyde for 10 minutes, washed with double-distilled water 3 times, blocked with 5% skimmed milk powder for 1 hour, primary antibody (GBA1, Abcam: ab55080; α-Syn, Abcam: ab138501; β-actin, Abcam: ab8227) at 4°C Incubate overnight; wash three times with TBST, incubate with secondary antibody for 1 hour at room temperature, wash three times with TBST, develop, and analyze the expression of α-Syn protein tetramers and monomers.

After the gene drug prepared by rAAV vector acts on the cell model of CBE-mediated inhibition of endogenous GCase activity in nerve cells, the experimental results of the GCase activity level and α-Syn tetramer to monomer ratio in the cells are shown in Figure 25 and Figure 26 . The results showed that when the drug CBE that inhibits GCase activity was removed (CBE(r) and AAV 2/2-EGFP), compared with the CBE group, as the GCase activity in the cells increased, α-Syn protein tetramers and monomers The ratio also increases. The results of the enzyme activity assay showed that the expression product GCase after the GBA1 of the rAAV vector was imported into the cells through adeno-associated virus had enzymatic activity, and the α-Syn protein of the AAV2/2-GBA1-EGFP and AAV2/2-GBA1-CDNF groups The ratios of tetramers and monomers were significantly higher than those in the AAV2/2-EGFP group, indicating that the GCase expressed by the rAAV vector can perform normal physiological functions like endogenous GCase. GCase is an enzyme in lysosomes with phagocytosis. Therefore, adeno-associated virus infection may stimulate the expression of endogenous GCase, resulting in the enzyme activity of the AAV2/2-EGFP group being slightly higher than that of the CBE(r) group, but Whether through exogenous introduction (GBA1 in the rAAV vector described in the present invention) or induction of endogenous expression, as long as the activity of GCase increases, the ratio of α-Syn protein tetramers to monomers in cells will also increase accordingly.

To sum up, the GCase activity in mammalian neural cells such as SH-SY5Y is positively correlated with the ratio of α-Syn protein tetramers to monomers. That is, when the GCase activity in cells increases, more α-Syn can be synthesized. The protein exists in the form of a stable tetramer, which reduces the chance of unstable monomers forming insoluble fibrils, thereby extending the lifespan of nerve cells. The constructed product of the present invention can express GCase with normal physiological functions after being input into nerve cells, indicating that the genetic medicine prepared by the rAAV vector involved in the present invention has the effect of reducing α-Syn protein fibrosis and extending the lifespan of nerve cells. . Because the Lewy Body eventually formed by α-Syn protein fibrosis is a marker of Parkinson's disease, it is further shown that the constructed product of the present invention and the gene drug prepared therefrom have the effect of treating the neurodegenerative disease Parkinson's disease.

The above embodiments only illustrate the principles and effects of the present invention, but are not intended to limit the present invention. Anyone familiar with this technology can modify or change the above embodiments without departing from the spirit and scope of the invention. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the technical field without departing from the spirit and technical ideas disclosed in the present invention shall still be covered by the claims of the present invention.

Claims (22)

1. A gene therapy involving a recombinant nucleic acid molecule, characterized in that it encodes human glucocerebrosidase GCase and neurotrophic factors. 2. The recombinant nucleic acid molecule according to claim 1, wherein the neurotrophic factors include human brain dopamine neurotrophic factor CDNF, human brain-derived neurotrophic factor BDNF, and human glial cell-derived neurotrophic factor GDNF. and human neural rank protein NRTN. 3. The recombinant nucleic acid molecule according to claim 2, wherein the sequence encoding human glucocerebrosidase GCase is connected to the sequence encoding neurotrophic factor through a gene link element; encoding the human glucocerebrosidase The amino acid sequence of the enzyme GCase is SEQ ID NO.1; the amino acid sequence encoding the human brain dopamine neurotrophic factor CDNF is SEQ ID NO.2; the amino acid sequence encoding the human brain-derived neurotrophic factor BDNF is SEQ ID NO. 3. The amino acid sequence encoding the human glial cell-derived neurotrophic factor GDNF is SEQ ID NO.4, and the amino acid sequence encoding the human neural rank protein NRTN is SEQ ID NO.5. 4. The recombinant nucleic acid molecule according to claim 1, wherein the gene linking element includes but is not limited to internal ribosome entry site sequence IRES, 2A peptide or class 2A; the IRES element includes but is not limited to Encephalomyocarditis virus EMCV; the 2A peptide or 2A-like peptide includes, but is not limited to, P2A derived from porcine Jieshen virus and T2A derived from Aphrodisiac virus. 5. A recombinant nucleic acid molecule, characterized in that it contains an operably linked promoter and the nucleic acid molecule of claim 1. 6. The recombinant nucleic acid molecule according to claim 5, characterized in that the promoter is a combination promoter of human cytomegalovirus enhancer and chicken β-actin promoter CAG, and a strong promoter of human cytomegalovirus CMV. or the human synapsin I promoter hSyn. 7. The recombinant nucleic acid molecule according to any one of claims 1 to 6, characterized in that the recombinant nucleic acid molecule further comprises an intron such as simian virus 40 intron SV40, a post-transcriptional regulatory element such as a nucleic acid sequence further comprising Woodchuck hepatitis virus post-transcriptional regulatory element WPRE and polyadenylates such as SV40 PolyA. 8. The recombinant nucleic acid molecule according to claim 7, wherein the recombinant nucleic acid molecule further comprises an AAV inverted terminal repeat sequence ITR. 9. The recombinant nucleic acid molecule according to claim 8, wherein the AAV inverted terminal repeat ITR is AAV type 2. 10. A recombinant vector for gene therapy, characterized in that it contains the recombinant nucleic acid molecule according to any one of claims 1 to 9, and the vector is selected from the group consisting of plasmid vectors and viral vectors. 11. The recombinant vector according to claim 10, wherein the viral vector includes but is not limited to adeno-associated virus vector, adenovirus vector, lentiviral vector, hybrid virus vector and phage vector. 12. The recombinant vector according to claim 10, characterized in that the viral vector is selected from adeno-associated virus vectors. 13. A recombinant adeno-associated virus for gene therapy, characterized in that the recombinant adeno-associated virus contains an AAV capsid and a vector genome, and the vector genome contains the recombinant nucleic acid according to any one of claims 1-9 molecular. 14. The recombinant adeno-associated virus according to claim 13, wherein the capsid of the recombinant adeno-associated virus is AAV2 or AAV9 capsid protein. 15. An isolated host cell for gene therapy, characterized by comprising the recombinant nucleic acid molecule according to any one of claims 1-9, the recombinant vector according to any one of claims 10-12, or the recombinant nucleic acid molecule according to any one of claims 10-12. The recombinant adeno-associated virus according to any one of 13-14. 16. A pharmaceutical composition for gene therapy, characterized in that it contains the recombinant nucleic acid molecule according to any one of claims 1-9, the recombinant vector according to any one of claims 10-12, and the recombinant vector according to any one of claims 10-12. The recombinant adeno-associated virus described in any one of 13-14 and pharmaceutically acceptable carriers and/or other conventional pharmaceutical ingredients. 17. The pharmaceutical composition according to claim 16, wherein the other conventional pharmaceutical ingredients include preservatives and/or stabilizers. 18. The pharmaceutical composition according to any one of claims 16 to 17, characterized in that the pharmaceutical composition acts on the patient's central nervous system (CNS) through systemic injection or local injection. 19. The pharmaceutical composition according to claim 18, wherein the local injection includes one or more of intracerebral injection, intraparenchymal injection, intrathecal injection and intracisternal injection. 20. The pharmaceutical composition of any one of claims 18, wherein the pharmaceutical composition enhances CED delivery by convection. 21. The recombinant nucleic acid molecule described in any one of claims 1-9, the recombinant vector described in any one of claims 10-12 and/or the recombinant adeno-associated virus described in any one of claims 13-14 is prepared in Use in drugs for preventing or treating neuropathy with reduced GCase activity. 22. Use according to claim 21, characterized in that the recombinant nucleic acid molecule, recombinant vector, recombinant adeno-associated virus, host cell and/or pharmaceutical composition can be administered with another therapy.