Modified adeno-associated virus vectors and their use in treatment of central nervous system diseases

Abstract

A nucleic acid fragment comprising a nucleotide sequence encoding a human glucocerebrosidase (GCase) is provided. The invention also provides an expression vector containing the nucleic acid fragment, a recombinant adeno-associated virus (rAAV) containing the expression vector, a pharmaceutical composition containing the nucleic acid fragment, the expression vector or the rAAV, and application of the nucleic acid fragment, the expression vector or the rAAV in treatment of central nervous system diseases.

Description

Modified adeno-associated virus vector and its use in treating central nervous system diseases Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a nucleic acid fragment comprising a nucleotide sequence encoding human glucocerebrosidase (GCase), an expression vector and use thereof in treating central nervous system diseases. Background Art

[0002] Patients with mutations in one allele of GBA1 (glucosylceramidase beta 1, NCBI Gene ID: 2629) are at increased risk for Parkinson's disease (PD), while those with mutations in both alleles of the GBA1 gene are at increased risk for Gaucher disease (GD). Specifically, defects in the enzyme GBA1 encodes for glucocerebrosidase (GCase), leading to the accumulation of the GCase glycolipid substrates glucosylceramide and glucosylsphingosine, ultimately leading to toxicity and inflammation and negatively impacting many clinical aspects of Parkinson's disease and Gaucher disease, including disease risk, severity of manifestations, age of onset, and the likelihood of developing dementia.

[0003] Common symptoms of Parkinson's disease include gait difficulty, tremor at rest, stiffness and frequent depression, sleep difficulties and cognitive decline. Elevated alpha-synuclein levels are often observed in PD patients, and the severity of PD symptoms is related to the activity of lysosomes and the degree of reduction in the clearance rate of alpha-synuclein. Therefore, by exogenously expressing active GCase protein, restoring lysosomal activity and increasing the rate of clearance of alpha-synuclein from cells can treat or improve the cellular molecular changes in Parkinson's patients, which may play a positive role in the treatment of PD. Some existing studies (Neurobiol Dis, 2022, 166: 105663; Mol Neurodegener, 2019, 29; 14(1): 36; Mov Disord, 2023, 38(7): 1197-1208) suggest that re-establishing normal levels of GCase enzyme activity in patients carrying GBA1 mutations may slow the progression of Parkinson's disease.

[0004] GD, also known as glucocerebrosidosis, is also caused by mutations in the glucocerebrosidase gene and is closely related to lysosomal dysfunction. Existing studies (Cells, 2019, 19; 8(4): 364) suggest that exogenous expression of active GCase protein may be helpful in treating GD.

[0005] Adeno-associated virus (AAV) is a helper virus-dependent DNA parvovirus belonging to the genus Dependovirus. It has advantages such as long-term gene expression, inability to replicate automatically without a helper virus, ability to infect both dividing and non-dividing cells, and lack of pathogenicity caused by wild-type infection. It is an attractive gene therapy vector (Mol. Ther., 2010, 18(8): 1458-1461). However, existing AAV as a gene therapy vector has some disadvantages, such as: low efficiency of target gene expression; AAV capsid may cause immunogenicity depending on different serotypes, thereby causing safety issues; target genes may be overexpressed in non-target organs, thereby causing toxic side effects, etc. It can be seen that there is still a demand for the research and development of AAV in the field of gene therapy, and existing AAV vectors still need to be improved.

[0006] It should be noted that the approaches described in this section are not necessarily approaches that have been previously conceived or employed. Unless otherwise indicated, it should not be assumed that any approach described in this section is prior art simply because it is included in this section. Similarly, unless otherwise indicated, the issues mentioned in this section should not be considered to have been recognized in any prior art.

[0007] Summary of the Invention

[0008] To solve the above technical problems, the present application provides a nucleic acid fragment comprising a nucleotide sequence encoding GCase, wherein the nucleotide sequence encoding GCase comprises a nucleotide sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity with the nucleotide sequence shown in SEQ ID NO: 1 or 2. The present application effectively improves expression efficiency by codon-optimizing the nucleotide sequence encoding GCase.

[0009] According to one embodiment of the present application, an expression vector is also provided, comprising the nucleic acid fragment described herein. In addition to codon-optimizing the nucleotide sequence encoding GCase, the expression vector further incorporates high-expression efficiency promoter elements, intronic regulatory sequences, and enhancer elements to further increase GCase expression, thereby achieving an effective therapeutic effect.

[0010] In some embodiments, the expression vector further comprises a miRNA target sequence, which utilizes the ability of miRNA to inhibit gene expression in specific sites to reduce expression of the expression vector in non-target organs, thereby reducing the potential toxicity of gene therapy.

[0011] According to one embodiment of the present application, a recombinant adeno-associated virus (rAAV) is also provided, which comprises the expression vector described in the present application.

[0012] According to one embodiment of the present application, a pharmaceutical composition is also provided, which comprises the nucleic acid fragment described in the present application, the expression vector described in the present application, and / or the recombinant adeno-associated virus described in the present application, and pharmaceutically, physiologically or nutritionally acceptable excipients.

[0013] According to one embodiment of the present application, there is also provided a use of the nucleic acid fragment described in the present application, the expression vector described in the present application, the recombinant adeno-associated virus described in the present application, or the pharmaceutical composition described in the present application in the preparation of a drug for preventing, alleviating or treating central nervous system diseases.

[0014] According to one embodiment of the present application, a method for preventing, alleviating or treating central nervous system diseases is also provided, which comprises administering a therapeutically effective amount of the nucleic acid fragment described in the present application, the expression vector described in the present application, the recombinant adeno-associated virus described in the present application, or the pharmaceutical composition described in the present application to a subject in need.

[0015] It should be understood that the content described in this section is not intended to identify the key or important features of the embodiments of the present application, nor is it intended to limit the scope of the present application. Other features of the present application will become easily understood through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The accompanying drawings illustrate exemplary embodiments and constitute a part of the specification. Together with the description of the specification, they serve to explain exemplary implementation of the embodiments. The illustrated embodiments are for illustrative purposes only and do not limit the scope of the claims. Throughout the drawings, the same reference numerals designate similar, but not necessarily identical, elements.

[0017] FIG1 is a schematic diagram of the structure of the pGBA_v4 vector prepared in Example 1.

[0018] Figure 2 is a schematic diagram of the pGBA_v5 vector structure prepared in Example 1.

[0019] FIG3 is a schematic diagram of the structure of the pGBA_v6 vector prepared in Example 1.

[0020] FIG4 is a schematic diagram of the structure of the pGBA_v7 vector prepared in Example 1.

[0021] Figure 5 is a schematic diagram of the pGBA_v8 vector structure prepared in Example 1.

[0022] Figure 6 is a schematic diagram of the pGBA_v9 vector structure prepared in Example 1.

[0023] Figure 7 is a schematic diagram of the pGBA_v7_2 vector structure prepared in Example 1.

[0024] FIG8 is a schematic diagram of the structure of the pGBA_v7_EF vector prepared in Example 1.

[0025] Figure 9 is a schematic diagram of the pGBA_v7_WT vector structure prepared in Example 1.

[0026] FIG10 is a Western blot result diagram showing the effect of pGBA_v4 and pGBA_v5 on GCase expression in 293T and SH-SY5Y cells in Example 2.

[0027] Figure 11 is a graph showing the expression of target proteins in Neuro-2a cells after transfection of different vectors in Example 3. Different labels in the figure represent the use of different vectors for transfection: blank represents an untransfected blank group, PC represents a control vector without miR-183 and miR-122 target sequences, 14 represents a vector containing miR-183 and miR-122 target sequences, Let-7a-1 represents a vector containing let-7a-1 target sequences, 802 represents a vector containing miR-802 target sequences, 99a-5p represents a vector containing miR-99a-5p target sequences, 100-5p represents a vector containing miR-100-5p target sequences, and 1200 represents a vector containing miR-1200 target sequences.

[0028] FIG12 is a Western blot result showing the effects of pGBA_V7_WT, pGBA_v7, pGBA_v6, and pGBA_v7_EF on GCase expression in SH-SY5Y cells in Example 4.

[0029] FIG13 is a Western blot result diagram showing the effects of pGBA_v6, pGBA_v7, pGBA_v8, and pGBA_v9 on GCase expression in SH-SY5Y cells according to Example 5.

[0030] FIG14 is a Western blot result diagram showing the effect of pGBA_v6 and pGBA_v7 on GCase expression in 293T cells in Example 6.

[0031] Figure 15 is a Western blot result showing the effects of pGBA_v6 and pGBA_v7 on GCase expression in mouse brain tissue according to Example 7. The control group (Ctl group) shows the test results of three mice injected with empty virus shells; Group 1 shows the test results of three mice injected with AAV-GBA_v6; and Group 2 shows the test results of three mice injected with AAV-GBA_v7.

[0032] Figure 16 shows the Western blot results of Example 8, which demonstrate the effects of pGBA_v7 and pGBA_v7_2 on GCase expression in mouse brain and liver tissues. The control group (Ctl group) shows the test results for three mice injected with empty viral shells; Group 1 shows the test results for three mice injected with AAV-GBA_v7; and Group 2 shows the test results for three mice injected with AAV-GBA_v7_2.

[0033] Figure 17 is a graph showing the therapeutic effect of AAV-GBA_v7 on Parkinson's disease model mice A53T in Example 9. The control group (A53T-GBA + AAV empty shell control) shows the test results of three mice injected with empty virus shells; the experimental group (A53T-GBA + AAV-GBA_v7) shows the test results of three mice injected with AAV-GBA_v7. DETAILED DESCRIPTION

[0034] Unless otherwise indicated, all numbers used in this specification and claims to represent content, concentration, ratio, mass, volume, time, temperature, thickness, technical effect, etc. should be understood as being modified by the term "about" or "approximately" in any case. Therefore, unless otherwise indicated, the numerical parameters listed in the following specification and the appended claims are approximate values. For those skilled in the art, it can vary according to the desired properties and effects sought to be obtained through this disclosure, and each numerical parameter should be interpreted according to the number of significant digits and conventional rounding methods or in a manner understood by those skilled in the art.

[0035] Although the numerical ranges and parameters setting forth the broad scope of the present disclosure are approximations, the numerical values ​​set forth in the specific examples are provided as precisely as possible. However, any numerical value inherently contains certain errors necessarily resulting from the standard deviation found in its respective testing measurements. Every numerical range given herein will include every narrower numerical range that falls within that broader numerical range, as if each narrower numerical range were expressly written herein.

[0036] As used herein, the expression "A and / or B" includes three cases: (1) A; (2) B; and (3) A and B. The expression "A, B, and / or C" includes seven cases: (1) A; (2) B; (3) C; (4) A and B; (5) A and C; (6) B and C; and (7) A, B, and C. The meanings of similar expressions can be deduced analogously.

[0037] As used herein, "nucleic acid" and "polynucleotide" are used interchangeably to refer to a polymeric form of nucleotides of any length, including deoxyribonucleotides, ribonucleotides, combinations thereof, and analogs thereof.

[0038] As used herein, "polypeptide" and "peptide" are used interchangeably to refer to amino acid polymers of any length. Thus, polypeptides, oligopeptides, proteins, antibodies, and enzymes are all included within the definition of polypeptide.

[0039] It should be noted that in the context of this application, upstream refers to the 5' end of the gene or the N-terminus of the protein, and downstream refers to the 3' end of the gene or the C-terminus of the protein, and from upstream to downstream is from 5' end to 3' end or N-terminus to C-terminus.

[0040] As used herein, "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it has been linked. Examples of vectors include, but are not limited to, plasmids, viruses, bacteria, phages, and insertable DNA segments.

[0041] The "adeno-associated viral vector" described in this application is derived from an adeno-associated virus. An "adeno-associated viral vector" is derived from the wild-type genome of the virus by removing all or part of the wild-type genome from the adeno-associated viral genome using molecular methods and replacing it with a heterologous (non-natural) nucleic acid (e.g., a nucleic acid encoding a therapeutic protein or polynucleotide sequence). Typically, for an adeno-associated viral vector, one or two inverted terminal repeats (ITRs) in the adeno-associated viral genome are retained in the adeno-associated viral vector. Adeno-associated viral vectors can be used as gene therapy vectors because they can introduce nucleic acid / genetic material into cells so that the nucleic acid / genetic material can remain in the cells.

[0042] "Serotype" as described in this application refers to an adeno-associated virus with a capsid that is serologically different from other adeno-associated virus serotypes. Serological specificity is determined based on the lack of cross-reactivity between antibodies to an adeno-associated virus compared to another adeno-associated virus. Such cross-reactivity differences are usually due to differences in capsid protein sequences / antigenic determinants (e.g., due to differences in VP1, VP2, and / or VP3 sequences of adeno-associated virus serotypes). Under the traditional definition, a serotype means that the virus of interest has been tested for neutralizing activity against serum specific for all existing and characterized serotypes, and no antibodies to neutralize the virus of interest have been found. As more naturally occurring virus isolates are discovered and / or capsid mutants are generated, there may or may not be serological differences from any of the currently existing serotypes. Therefore, in the case where a new virus (e.g., an adeno-associated virus) does not have serological differences, this new virus (e.g., an adeno-associated virus) will be a subgroup or variant of the corresponding serotype. In many cases, serological testing for neutralizing activity has not been performed on mutant viruses with modified capsid sequences to determine whether they are of another serotype according to the traditional serotype definition. Therefore, for convenience and to avoid repetition, the term "serotype" is used broadly to refer to serologically distinct viruses (e.g., adeno-associated viruses) as well as viruses that are not serologically distinct (e.g., adeno-associated viruses) that may be within a subgroup or variant of a given serotype.

[0043] As used herein, "miRNA target sequence" and "miRNA binding site" are used interchangeably to refer to a nucleotide sequence that can bind to a specific miRNA. In some non-limiting embodiments of the present application, a miRNA target sequence is designed at the 3' end of the GCase gene, which can bind to a specific miRNA to inhibit the expression level of the GCase gene.

[0044] As used herein, "operably linked" refers to the connection of multiple nucleic acid segments in a functional relationship. A nucleic acid is "operably linked" when it forms a functional relationship with another nucleic acid sequence. For example, a promoter or other transcriptional regulatory sequence is operably linked to a coding sequence if it affects the transcription of the coding sequence (or gene), and the promoter is connected in such a way that it can induce transcription of the gene. Operably linked means that the nucleotide sequences being linked may be contiguous or discontinuous.

[0045] As used herein, the terms "relieve," "treat," and their synonyms refer to the improvement of a disease, disorder, and / or condition. "Relieve," "treat," can be an improvement in at least one measurable physical parameter, which is not necessarily recognizable by the patient. "Relieve," "treat," can also be the inhibition of the development of a disease, disorder, and / or condition physically (e.g., stabilizing recognizable symptoms), physiologically (e.g., stabilizing physical parameters), or both. "Relieve," "treat," can also be the slowing down of the development or reversal of a disease, disorder, and / or condition.

[0046] As used herein, the term "prevent," "prevent," and its synonyms refer to delaying the onset of or reducing the risk of acquiring a particular disease, disorder, and / or condition, or symptoms associated with such disease, disorder, and / or condition.

[0047] As used herein, a "pharmaceutically acceptable excipient" refers to a carrier, diluent, or adjuvant used in the formulation or administration of a drug, which is not itself an essential active ingredient and is not unduly toxic upon administration. Suitable pharmaceutically acceptable excipients are well known to those of ordinary skill in the art and include, but are not limited to, calcium bicarbonate, calcium phosphate, various sugars and starch types, cellulose derivatives, gelatin, vegetable oils, polyethylene glycol, and surfactants, including, for example, polysorbate 20.

[0048] The "physiologically acceptable excipient" and "nutritionally acceptable excipient" mentioned in the present application refer to carriers, diluents or adjuvants that do not cause significant irritation to an organism and do not eliminate the biological activity and properties of the administered polypeptide.

[0049] In order to make the above-mentioned objects, features and advantages of the present application more obvious and easy to understand, the specific implementation methods of the present application are described in detail below.

[0050] Nucleic acid fragments, expression vectors, recombinant adeno-associated viruses

[0051] According to one embodiment of the present application, a nucleic acid fragment is provided, comprising a nucleotide sequence encoding GCase, wherein the nucleotide sequence encoding GCase comprises a nucleotide sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the nucleotide sequence shown in SEQ ID NO: 1 or 2. In some embodiments, the nucleotide sequence encoding GCase comprises the nucleotide sequence shown in SEQ ID NO: 1 or 2. In some embodiments, the nucleotide sequence encoding GCase is the nucleotide sequence shown in SEQ ID NO: 1 or 2.

[0052] According to one embodiment of the present application, an expression vector is also provided, wherein the expression vector comprises the nucleic acid fragment described in the present application.

[0053] In some embodiments, the expression vector further comprises a miRNA target sequence. miRNA (microRNA) is a single-stranded non-coding RNA with a length of 18 to 25 nucleotides that is widely present in humans and animals. The RNA-induced silencing complex formed by miRNA and protein factors can recognize the target sequence in mRNA, reduce the expression level of mRNA by degrading mRNA molecules, promoting deadenylation of the 3' end of mRNA molecules and inhibiting translation, and regulate gene expression at the post-transcriptional level (Nat Rev Mol Cell Biol., 2005, 6(5):376-385). By using a miRNA that is highly expressed in a certain cell, inserting the target sequence of the miRNA into the 3'UTR of an exogenous gene can effectively inhibit the expression of the exogenous gene in the cell. In some preferred embodiments, the miRNA target sequence can reduce the expression efficiency of the nucleotide sequence encoding GCase in liver tissue. In some preferred embodiments, the miRNA target sequence can reduce the expression efficiency of the nucleotide sequence encoding GCase in dorsal root ganglion tissue. In some preferred embodiments, the miRNA target sequence is selected from at least one of miR-183, miR-182, miR-96, miR23b, miR-145, miR-148a, miR-22, miR-122, miR-143, miR-21, or miR-192. In some preferred embodiments, the miRNA target sequence comprises one or both of the miR-183 target sequence or the miR-122 target sequence. In some preferred embodiments, the miRNA target sequence comprises one or both of the nucleotide sequences set forth in SEQ ID NO: 6 or SEQ ID NO: 7. In some embodiments, the number of miRNA target sequences is 1, 2, 3, 4, 5, 6, 7, or 8. In some preferred embodiments, the number of miRNA target sequences is 4. In some preferred embodiments, the miRNA target sequence comprises two miR-183 target sequences and two miR-122 target sequences. The more than one miRNA target sequence can be linked together by various means known in the art. In some preferred embodiments, the more than one miRNA target sequence is operably linked by a spacer sequence, wherein the spacer sequence comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 17-19. In some preferred embodiments, the more than one miRNA target sequence is operably linked by being arranged in a staggered manner and spaced apart from each other.

[0054] In some embodiments, the expression vector further optionally comprises a regulatory sequence, comprising an SV40 intron regulatory sequence, a Chimeric chβ-Actini-MVMj regulatory sequence, and / or an hGlobin intro2 exon3 intron regulatory sequence. The addition of Chimeric chβ-Actini-MVMj can promote efficient expression of the target gene in neural cells (Hum Gene Ther., 2011, 22(9): 1143-53). The addition of hGlobin intro2 exon3 (US11027000B2) or SV40, which are also commonly used regulatory sequences (J Cell Mol Med., 2018, 22(4): 2231-2239), can also promote efficient expression of the target gene in cells. In some preferred embodiments, the SV40 intron regulatory sequence is shown as SEQ ID NO: 8, the Chimeric chβ-Actini-MVMj regulatory sequence is shown as SEQ ID NO: 9, and the hGlobin intro2 exon3 intron regulatory sequence is shown as SEQ ID NO: 10.

[0055] In some embodiments, the expression vector further comprises a promoter. The promoter can be any suitable promoter sequence, i.e., a nucleotide sequence that can be recognized by the host cell expressing the nucleic acid sequence. The promoter sequence contains a transcriptional regulatory sequence that mediates protein or polypeptide expression. The promoter can be any nucleotide sequence with transcriptional activity in the selected host cell, including mutant, truncated and hybrid promoters, and can be derived from genes encoding extracellular or intracellular proteins or polypeptides that are homologous or heterologous to the host cell. In some embodiments, the promoter is selected from the promoter of chicken beta actin (CBA) promoter, cytomegalovirus (CMV) promoter, mini-CMV promoter and human SYN1 gene. In some preferred embodiments, the promoter is selected from CBA promoter, CMV promoter or mini-CMV promoter.

[0056] In some embodiments, the expression vector optionally further comprises an enhancer. In some embodiments, the enhancer comprises a CMV enhancer or a CBh enhancer. In some preferred embodiments, the enhancer is a CMV enhancer.

[0057] In some embodiments, the expression vector further comprises a tail signal. In some embodiments, the tail signal is a bovine growth hormone (bGH) poly A signal tail or a simian vacuolating virus 40 (SV40) poly A signal tail. Transcription termination sequences known in the art can be used in this application.

[0058] In some embodiments, the expression vector comprises, from the 5' end to the 3' end, the following: an adeno-associated virus ITR sequence at the 5' end; a promoter; the nucleic acid fragment described in the present application; a tail signal; and an adeno-associated virus ITR sequence at the 3' end.

[0059] In some embodiments, the expression vector includes, from the 5' end to the 3' end, the following: an adeno-associated virus ITR sequence at the 5' end; a promoter; the nucleic acid fragment described in this application; a miRNA target sequence; a tail signal; and an adeno-associated virus ITR sequence at the 3' end.

[0060] In some embodiments, the expression vector includes, from the 5' end to the 3' end, the following: an adeno-associated virus ITR sequence at the 5' end; a promoter; optionally, an enhancer; optionally, a regulatory sequence; the nucleic acid fragment described in the present application; a miRNA target sequence; a tail signal; and an adeno-associated virus ITR sequence at the 3' end.

[0061] In some preferred embodiments, the expression vector includes, from the 5' end to the 3' end, the following sequence: an adeno-associated virus ITR sequence at the 5' end; a promoter; an enhancer; a regulatory sequence; the nucleic acid fragment described in this application; a miRNA target sequence; a tail signal; and an adeno-associated virus ITR sequence at the 3' end.

[0062] Any suitable vector can be used to deliver the expression vector. In some embodiments, the expression vector is a plasmid vector. In some embodiments, the expression vector is a viral vector. Suitable plasmid vectors and viral vectors are well known in the art.

[0063] Virion particles containing the gene expression cassettes described herein are typically produced in packaging cells that are capable of replicating the viral genome, expressing viral proteins, and assembling virion particles. Techniques for producing AAV vector particles in packaging cells are well known in the art. In some non-limiting embodiments, packaging cells can be produced by simply transforming suitable cells with one or more plasmids encoding the AAV genome, AAV proteins, and any desired helper virus functions, a so-called "triple transfection" approach: using three plasmids, each of which carries a set of such genes. See Grieger et al., Nature Protocols, 2006, 1(3): 1412-28.

[0064] In some preferred embodiments, the expression vector is an AAV vector. In some embodiments, the expression vector further comprises an adeno-associated virus inverted terminal repeat (ITR) sequence at the 5' end and an adeno-associated virus ITR sequence at the 3' end. In some preferred embodiments, the adeno-associated virus ITR sequence at the 5' end is a nucleotide sequence shown in SEQ ID NO: 3 or SEQ ID NO: 5, and wherein the adeno-associated virus ITR sequence at the 3' end is a nucleotide sequence shown in SEQ ID NO: 4 or SEQ ID NO: 38. Wherein, compared with the wild-type adeno-associated virus ITR sequence at the 5' end (SEQ ID NO: 5), the mutant adeno-associated virus ITR sequence at the 5' end (SEQ ID NO: 3) can further enhance the expression efficiency of the target gene.

[0065] According to one embodiment of the present application, a recombinant adeno-associated virus (rAAV) is also provided, which comprises the expression vector described in the present application. In some embodiments, the serotype of the rAAV includes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9 (hu14), AAV10, AAV11, AAV12, AAV13, AAVrh8, AAVrh10, AAV-DJ, or AAV-DJ8. In some preferred embodiments, the serotype of the rAAV is AAV9. The nucleotide sequences of the genomes of different AAV serotypes are known in the art.

[0066] Pharmaceutical composition

[0067] According to one embodiment of the present application, a pharmaceutical composition is also provided, which comprises the nucleic acid fragment described in the present application, the expression vector described in the present application, and / or the recombinant adeno-associated virus described in the present application, and pharmaceutically, physiologically or nutritionally acceptable excipients.

[0068] In some embodiments, the pharmaceutical composition of the present application, in addition to the nucleic acid fragments, expression vectors and / or recombinant adeno-associated viruses provided herein, may also contain a pharmaceutically, nutritionally or physiologically acceptable carrier, such as a liquid, gel or solid carrier, an aqueous vehicle, a non-aqueous vehicle, an antimicrobial agent, an isotonic agent, a buffer, an antioxidant, a suspending agent / dispersing agent, a chelating agent, a diluent, an adjuvant, an excipient or a non-toxic auxiliary substance, other components known in the art, or various combinations thereof.

[0069] In some embodiments, the pharmaceutical composition is administered by intravenous injection or suboccipital injection.

[0070] use

[0071] According to one embodiment of the present application, there is also provided a use of the nucleic acid fragment, the expression vector, the recombinant adeno-associated virus, or the pharmaceutical composition described herein in the preparation of a medicament for preventing, alleviating, or treating a central nervous system disease. In some embodiments, the disease is a GCase-related disease. In some embodiments, the GCase-related disease includes: a disease associated with insufficient GCase protein expression, a disease associated with GCase protein deficiency, a disease associated with GCase gene deletion, and a disease associated with GCase gene mutation. In some embodiments, the central nervous system disease includes Parkinson's disease or Gaucher disease.

[0072] Disease treatment methods

[0073] According to one embodiment of the present application, a method for preventing, alleviating, or treating a central nervous system disease is also provided, comprising administering to a subject in need thereof a therapeutically effective amount of the nucleic acid fragment, expression vector, recombinant adeno-associated virus, or pharmaceutical composition described herein. In some embodiments, the central nervous system disease comprises Parkinson's disease or Gaucher disease.

[0074] The specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including the activity of the particular expression vector employed, the age, weight, general health, sex, diet, time of administration, rate of excretion, drug combination, the severity and course of the disease, condition or symptom, the patient's predisposition to the disease, condition or symptom, and the judgment of the treating physician.

[0075] An effective amount can be administered in one or more administrations, applications, or dosages. The expression vector or pharmaceutical composition can be administered once or more daily to once or more weekly, including once every other day. It will be appreciated by those skilled in the art that certain factors may influence the dosage and timing required to effectively treat a subject, including, but not limited to, the severity of the disease or condition, previous treatments, the subject's overall health and / or age, and the presence of other diseases. Furthermore, treating a subject with a therapeutically effective amount of a therapeutic compound described herein can include a single treatment or a series of treatments. For example, an effective amount can be administered at least once.

[0076] Unless otherwise specified or contradicted by the context, the terms or expressions used in this article should be read in conjunction with the entire content of this article and as understood by one of ordinary skill in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0077] The various embodiments and preferences disclosed above can be combined with each other (as long as they are not inherently contradictory to each other), and the various embodiments formed by such combination are all considered to be part of the disclosure of this application.

[0078] The following description will be made of exemplary embodiments of the present application in conjunction with the accompanying drawings, including various details of the embodiments of the present application to facilitate understanding. It should be understood that they are considered to be merely exemplary and are in no way intended to limit the scope of protection of the present application. The scope of protection of the present application is defined solely by the claims. Therefore, it should be appreciated by those skilled in the art that various changes and modifications may be made to the embodiments described herein without departing from the scope of the present application. Similarly, for the sake of clarity and conciseness, descriptions of well-known functions and structures are omitted in the following description.

[0079] Example

[0080] The examples described below are exemplary and are intended only to explain the present invention and are not to be construed as limiting the present invention. Where specific techniques or conditions are not specified in the examples, the methods or conditions described in the literature within the art or in the product specifications were used. Where the manufacturer of the reagents or instruments is not specified, all are commercially available conventional products.

[0081] Example 1: Construction of expression vector

[0082] 1.1 Nucleotide sequence encoding GCase

[0083] The amino acid sequence of human GCase protein (SEQ ID NO: 15; GenBank: NP_000148.2) was searched through the NCBI GenBank database (https: / / www.ncbi.nlm.nih.gov / gene), and the GCase protein coding region sequence was codon optimized to obtain the optimized sequence GZ (SEQ ID NO: 1) and the optimized sequence EF (SEQ ID NO: 2).

[0084] 1.2 Construction of adeno-associated virus vector

[0085] The adeno-associated virus vector constructed in this example includes, from the 5' end to the 3' end, the following: 5' ITR; promoter; enhancer; regulatory sequence; nucleotide sequence encoding human GCase; miRNA target sequence; tail signal; 3' adeno-associated virus ITR sequence; and backbone sequence.

[0086] Table 1 Sequence information of expression vector

[0087] According to the sequence information in Table 1, the designed gene sequence was synthesized by gene synthesis services (GeneScript, Qingke, etc.) to construct the following vectors:

[0088] The structure of pGBA_v4 is shown in Figure 1, which contains: an adeno-associated virus ITR sequence with a mutated 5' end, a mini-CMV promoter, an SV40 intron regulatory sequence, a GBA1 optimized sequence EF, two mir-183 and two mir-122 sequences (the four miRNA target sequences are connected in sequence using spacer sequences as shown in SEQ ID NO: 17-19), a simian vacuolating virus 40 signal tail, an adeno-associated virus ITR sequence with a mutated 3' end, and a backbone sequence.

[0089] The structure of pGBA_v5 is shown in Figure 2. It contains: an adeno-associated virus (AAV) ITR sequence with a mutated 5' end, a mini-CMV promoter, an SV40 intronic regulatory sequence, an optimized GBA1 EF sequence, a simian vacuolating virus 40 signal tail, an AAV ITR sequence with a mutated 3' end, and a backbone sequence. It does not contain the mir-183 and mir-122 sequences.

[0090] The structure of pGBA_v6 is shown in Figure 3, which contains: adeno-associated virus ITR sequence at the 5' end, CMV enhancer, CBA promoter, Chimeric chβ-Actini-MVMj regulatory sequence, GBA1 optimized sequence GZ, 2 each of mir-183 and mir-122 sequences (the four miRNA target sequences are connected in sequence using spacer sequences as shown in SEQ ID NO: 17-19), bovine growth hormone poly A signal tail, adeno-associated virus ITR sequence at the 3' end, and backbone sequence.

[0091] The structure of pGBA_v7 is shown in Figure 4, which contains: adeno-associated virus ITR sequence at the 5' end, CBA promoter, CMV enhancer, SV40 intron regulatory sequence, GBA1 optimized sequence GZ, 2 mir-183 and mir-122 sequences each (the four miRNA target sequences are connected in sequence using spacer sequences shown in SEQ ID NO: 17-19), bovine growth hormone poly A signal tail, adeno-associated virus ITR sequence at the 3' end, and backbone sequence.

[0092] The structure of pGBA_v8 is shown in Figure 5, which contains: adeno-associated virus ITR sequence at the 5' end, CMV promoter, CMV enhancer, hGlobin intro2 exon3 intron regulatory sequence, GBA1 optimized sequence GZ, 2 mir-183 and mir-122 sequences each (the four miRNA target sequences are connected in sequence using spacer sequences shown in SEQ ID NO: 17-19), bovine growth hormone poly A signal tail, adeno-associated virus ITR sequence at the 3' end, and backbone sequence.

[0093] The structure of pGBA_v9 is shown in Figure 6, which contains: adeno-associated virus ITR sequence at the 5' end, CMV promoter, CMV enhancer, hGlobin intro2 exon3 intron regulatory sequence, GBA1 optimized sequence EF, 2 mir-183 and mir-122 sequences each (the four miRNA target sequences are connected in sequence using spacer sequences shown in SEQ ID NO: 17-19), bovine growth hormone poly A signal tail, adeno-associated virus ITR sequence at the 3' end, and backbone sequence.

[0094] The structure of pGBA_v7_2 is shown in Figure 7. It contains: an adeno-associated virus (AAV) ITR sequence at the 5' end, a CBA promoter, a CMV enhancer, an SV40 intronic regulatory sequence, an optimized GBA1 sequence GZ, a bovine growth hormone (BGH) poly A signal tail, an AAV ITR sequence at the 3' end, and a backbone sequence. It does not contain the mir-183 and mir-122 sequences.

[0095] The structure of pGBA_v7_EF is basically the same as that of pGBA_v7, except that the optimized sequence GZ in pGBA_v7 is replaced by the optimized sequence EF. Its structure is shown in Figure 8.

[0096] pGBA_v7_WT is basically the same as pGBA_v7, except that the optimized sequence GZ in pGBA_v7 is replaced by the wild-type nucleic acid sequence encoding human GCase protein (SEQ ID NO: 39, GenBank: NP_000148.2). Its structure is shown in Figure 9.

[0097] Example 2:

[0098] Using the plasmids pGBA_v4 and pGBA_v5 constructed in Example 1, (Polyplus) was used to transfect 293T cells and SH-SY5Y cells (purchased from ATCC) according to the instructions of the reagent. 1 μg of plasmid was transfected using 3 μL Two days after transfection, the expression level of GBA gene expression protein GCase was detected by immunoblotting (GCase antibody purchased from Sigma-Aldrich, catalog number G4171-25UL). The results are shown in FIG10 .

[0099] pGBA_v4 is a plasmid with mir-183 and mir-122 target sequences, and pGBA_v5 does not have mir-183 and mir-122 sequences. The results of Figure 10 show that in 293T cells, the addition of mir-183 and mir-122 target sequences can reduce the expression of GCase, indicating that the addition of miRNA target sequences to the vector can reduce the expression of GCase in non-target cells; in the neural cell SH-SY5Y, the GCase expression level did not change much after the addition of mir-183 and mir-122 target sequences, indicating that the addition of miRNA target sequences to the vector does not affect the expression of GCase in target cells (neuronal cells). The above results show that miRNA target sequences can regulate the expression of GCase in different cells. When it is desired to specifically express GCase in neural cells, miRNA target sequences (such as mir-183 and mir-122 target sequences) can be added to the vector, which can ensure the normal expression of GCase in neural cells and reduce the expression of GCase in non-target cells.

[0100] Example 3:

[0101] A control scAAV vector containing a control miRNA target sequence was constructed using the same method as in Example 1. The construction method is briefly described as follows:

[0102] Amplification primers for control miRNAs (hsa-let-7a-1, hsa-miR-99a-5p, hsa-miR-100-5p, hsa-miR-802, and hsa-miR-1200) were synthesized in the form of long primers. The primer sequences are shown in Table 1. Circular PCR was performed on the vector scAAV-MeP426-Mecp2-pA1-pA2 (SEQ ID NO: 20) to introduce the miRNA target sequence. The amplified fragments were circularized by seamless cloning, and the ligated products were transformed into recombinase-deficient Escherichia coli stabl3 (full gold, CD521-01) for amplification to obtain a variety of control scAAV vectors containing control miRNA target sequences.

[0103] Table 2 PCR primer sequences for control miRNA

[0104] After the construction was completed, the control scAAV vector containing the control miRNA target sequence was transfected into Neuro-2a cells according to the method in Example 2, and the expression level of the target protein was then detected by Western blot. The results are shown in Figure 11. The different labels in the figure represent the use of different vectors for transfection:

[0105] Blank represents the untransfected blank group.

[0106] PC represents a control vector (scAAV-MeP426-Mecp2-pA1-pA2, the sequence of the exogenous nucleic acid fragment between its ITR sequences is shown in SEQ ID NO: 31) that does not contain miR-183 and miR-122 target sequences.

[0107] 14 represents a vector containing miR-183 and miR-122 target sequences (scAAV-MeP426-Mecp2-pA1-pA2-183-483-122-130, the sequence of the exogenous nucleic acid fragment between its ITR sequences is shown in SEQ ID NO: 32),

[0108] Let-7a-1 represents a vector containing the let-7a-1 target sequence (scAAV-MeP426-Mecp2-pA1-pA2-let-7a-1, the sequence of the exogenous nucleic acid fragment between its ITR sequences is shown in SEQ ID NO: 33),

[0109] 802 represents a vector containing the miR-802 target sequence (scAAV-MeP426-Mecp2-pA1-pA2-802, the sequence of the exogenous nucleic acid fragment between its ITR sequences is shown in SEQ ID NO: 36),

[0110] 99a-5p represents a vector containing the miR-99a-5p target sequence (scAAV-MeP426-Mecp2-pA1-pA2-99a-5p, the sequence of the exogenous nucleic acid fragment between its ITR sequences is shown in SEQ ID NO: 34),

[0111] 100-5p represents a vector containing the miR-100-5p target sequence (scAAV-MeP426-Mecp2-pA1-pA2-100-5p, the sequence of the exogenous nucleic acid fragment between its ITR sequences is shown in SEQ ID NO: 35),

[0112] 1200 represents a vector containing the miR-1200 target sequence (scAAV-MeP426-Mecp2-pA1-pA2-1200, the sequence of the exogenous nucleic acid fragment between its ITR sequences is shown in SEQ ID NO: 37).

[0113] The results in Figure 11 show that different miRNA target sequence combinations have different effects on reducing the expression of the target gene.

[0114] Example 4:

[0115] Using the plasmids pGBA_v6, pGBA_v7, pGBA_v7_EF and pGBA_V7_WT constructed in Example 1, (Polyplus) was used to transfect the neural cell line SH-SY5Y (purchased from ATCC) according to the reagent instructions. 1 μg of plasmid was transfected using 3 μL Two days after transfection, the expression level of GCase was detected by immunoblotting (GCase antibody purchased from Sigma-Aldrich, catalog number G4171-25UL). The results are shown in FIG12 .

[0116] The results in Figure 12 show that compared to the unoptimized wild-type vector pGBA_V7_WT, the expression levels of GCase in pGBA_v7, pGBA_v6, and pGBA_v7_EF were significantly increased, with pGBA_v6 and pGBA_v7 being preferred. The results in Figure 12 demonstrate that the codon-optimized sequences GZ and EF significantly increased the expression efficiency of the GCase protein.

[0117] Example 5:

[0118] Using the plasmids pGBA_v6, pGBA_v7, pGBA_v8 and pGBA_v9 constructed in Example 1, (Polyplus) transfected SH-SY5Y cell line (purchased from ATCC) according to the reagent instructions, using 3 μL of 1 μg plasmid. Two days after transfection, the expression level of GCase was detected by immunoblotting (GCase antibody purchased from Sigma-Aldrich, catalog number G4171-25UL). The results are shown in FIG13 .

[0119] The results in FIG13 show that the expression efficiency of GCase is in the order of pGBA_v6≥pGBA_v7>pGBA_v8>pGBA_v9 from high to low, among which pGBA_v6 and pGBA_v7 have the highest expression efficiency.

[0120] The combination strategy of promoter, enhancer and regulatory sequence of pGBA_v8 and pGBA_v9 is derived from US10898585B2 (other partial elements such as optimized sequence GZ and EF are derived from the present application). By contrasting the expression efficiency of pGBA_v6, pGBA_v7, pGBA_v8 and pGBA_v9, it can be seen that the element combination strategy such as promoter, enhancer and regulatory sequence selected in the present application can further improve the expression efficiency of optimized sequence GZ and EF, which is significantly better than the element combination strategy in prior art US10898585B2. The above results show that, although the element combination strategy in US10898585B2 can play the role of enhancing the expression efficiency of target gene in the vector disclosed in the patent, it can not play a role in the optimized sequence GZ and EF provided in the present application.

[0121] In addition, by comparing the expression efficiency of pGBA_v8 and pGBA_v9, it was found that the expression efficiency of GBA1 optimized sequence GZ was better than that of GBA1 optimized sequence EF, that is, GBA1 optimized sequence GZ was a more preferred optimized sequence.

[0122] Example 6:

[0123] In the plasmids pGBA_v6 and pGBA_v7 constructed in Example 1, AAV9 was used to package the above plasmids to obtain AAV-GBA_v6 and AAV-GBA_v7. The plasmids used in the packaging step include: an AAV9 expression capsid plasmid (addgeen 37825-AAV9.T), a Helper plasmid (genemedi P-HP01), and the target gene plasmid used in each experiment (such as GBA_v6 or GBA_v7). The three plasmids were transfected in 293T cells (purchased from ATCC) at a molar ratio of 1:1:1, and a cell fluid containing the virus was obtained. The specific transfection method is as follows:

[0124] 1) Prepare the complex: Prepare the DNA-PEI nucleic acid-transfection reagent complex according to the following system: For each well of cells, dilute 2 μg of target plasmid with 100 μL serum-free medium and mix thoroughly to make DNA dilution solution. Immediately add 6 μL of Add transfection reagent (Polyplus), mix gently, and incubate at room temperature for 10-20 minutes to form a DNA-PEI nucleic acid transfection reagent complex.

[0125] 2) Transfection: Directly add 100 μL of the DNA-PEI nucleic acid-PEI complex to the 293T cell culture medium. Shake the culture plate and gently mix. Incubate at 37°C in a 5% CO2 incubator. Expression of the transfected gene can be detected as early as 48 hours after transfection.

[0126] The AAV-GBA_v6 and AAV-GBA_v7 packaged above were used to infect human 293T cells at three infection titers (MOI: 300K, 150K, and 75K, respectively). The high dose of the virus infection group was the same, as were the medium and low doses. Two days later, GCase expression was detected by immunoblotting (GCase antibody purchased from Sigma-Aldrich, Catalog No. G4171-25UL). The results are shown in Figure 14.

[0127] The results in Figure 14 show that compared to the control group, pGBA_v6 and pGBA_v7 only expressed lower levels of GCase protein in 293T cells, with pGBA_v6 expressing even less GCase protein. This indicates that the vectors incorporating miRNA target sequences in this application can express less GCase protein in non-target cells. These results confirm that the vectors in this application can address the poor targeting issues associated with traditional AAV vectors, thereby reducing the toxicity of expression vectors to non-target organs, representing a significant technological advancement.

[0128] Example 7:

[0129] The viral vectors AAV-GBA_v6 and AAV-GBA_v7 constructed in Example 6 were used to infect C57bl / 6 mice at an infection titer of 5E+10 vg / mouse. 21 days later, brain tissues of the mice were collected and immunoblotted for GCase expression (GCase antibody purchased from Sigma-Aldrich, catalog number G4171-25UL). The results are shown in Figure 15. The control group (Ctl group) is the test result of 3 mice injected with empty virus shells (the virus does not carry the GCase gene); Group 1 is the test result of 3 mice injected with the virus AAV-GBA_v6; Group 2 is the test result of 3 mice injected with the virus AAV-GBA_v7.

[0130] The results in Figure 15 show that both pGBA_v6 and pGBA_v7 can effectively express GCase protein in the mouse brain, and the expression efficiency of pGBA_v6 is higher, which reflects the high efficiency of the expression vector provided in this application in expressing GCase in the animal brain.

[0131] Example 8:

[0132] Plasmids pGBA_v7 and pGBA_v7_2 constructed in Example 1 were packaged using AAV9 to obtain AAV-GBA_v7 and AAV-GBA_v7_2, which were then infected into C57BL / 6 mice at an infection titer of 5E+10 vg / mouse. 21 days later, brain tissues were collected from the mice and immunoblotted for GCase expression (GCase antibody purchased from Sigma-Aldrich, catalog number G4171-25UL). The results are shown in Figure 16. The control group (Ctl group) shows the test results for three mice injected with empty virus shells (the virus did not carry the GCase gene); Group 1 shows the test results for three mice injected with the AAV-GBA_v7 virus; and Group 2 shows the test results for three mice injected with the AAV-GBA_v7 virus.

[0133] The left figure in Figure 16 shows the expression of GCase in brain tissue. Compared with the control group, both AAV-GBA_v7 and AAV-GBA_v7_2 can effectively express GCase protein in the mouse brain.

[0134] The right panel of FIG16 shows the expression of GCase in liver tissue. Compared with AAV-GBA_v7_2 without miRNA target sequence, AAV-GBA_v7 containing miRNA target sequence expressed lower GCase in liver tissue.

[0135] The above results confirm that the addition of miRNA target sequences (such as mir-183 and mir-122 target sequences) to the expression vector can effectively reduce the nonspecific expression of GCase protein in animal livers, thereby achieving the effect of reducing liver toxicity. In addition, all vectors provided in this application did not cause obvious dorsal root ganglion lesions (results not shown), confirming that the addition of miRNA target sequences (such as mir-183 and mir-122 target sequences) to the vector can also effectively reduce dorsal root ganglion toxicity caused by AAV administration.

[0136] Example 9:

[0137] The Parkinson's model mouse A53T (Jicui Yaokang T054329) was hybridized with a mouse carrying a GBA mutation (Nanjing Model Organisms NM-KI-18060) to obtain the pathological model mouse A53T-GBA.

[0138] A53T-GBA mice were infected using the viral vector AAV-GBA_v7 constructed in Example 7 with an infection titer of 5E+10 vg / mouse. 15 weeks after infection, the mice were subjected to a limb grip strength test, and the results were shown in Figure 17. The method for the limb grip strength test is as follows: Use a mouse grip strength meter (Yuyan, instrument model: 47200) for testing, place the mouse on the grid, make the trunk parallel to the grid, and allow both the forepaws and hind paws to attach to the grid, and then measure. Gently pull the tail of the mouse to ensure that the trunk is parallel to the grid, and record the maximum grip strength value of the mouse displayed on the screen. Repeat this process twice to obtain 3 forelimb / hindlimb grip strength measurements, take the average value, and perform statistical analysis using the student-T test. * indicates a significant difference. Among them, the control group (A53T-GBA+AAV empty shell control) is the test results of 3 mice injected with virus empty shells (the virus does not carry the GCase gene); the experimental group (A53T-GBA+AAV-GBA_v7) is the test results of 3 mice injected with the virus AAV-GBA_v7.

[0139] The results in Figure 17 show that the grip strength of model mice injected with AAV-GBA_v7 was improved, demonstrating that the expression vector provided in this application can significantly improve the symptoms of Parkinson's disease in animal models, confirming that it is an effective treatment strategy for Parkinson's disease. In addition, since expressing active GCase in vivo is helpful in treating Gaucher disease (Cells, 2019, 19; 8(4): 364.), the expression vector provided in this application is also an effective treatment strategy for Gaucher disease.

[0140] It should be noted that the above are only preferred embodiments of the present application and are not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Although specific implementation methods have been described, for the applicant or other skilled in the art, there may be or are currently no alternatives, modifications, changes, improvements and substantial equivalents of the above implementation methods. Therefore, the attached claims submitted and the claims that may be amended are intended to cover all such alternatives, modifications, changes, improvements and substantial equivalents. It is important to note that as technology evolves, many of the elements described herein may be replaced by equivalent elements that appear after the present application.

Claims

1. A nucleic acid fragment, characterized in that The nucleic acid fragment comprises a nucleotide sequence encoding glucocerebrosidase (GCase), wherein the nucleotide sequence encoding GCase comprises a nucleotide sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity with the nucleotide sequence shown in SEQ ID NO: 1 or 2. 2 . The nucleic acid fragment according to claim 1 , wherein the nucleotide sequence encoding GCase comprises the nucleotide sequence shown in SEQ ID NO: 1 or 2. .

3. An expression vector, characterized in that: The expression vector comprises the nucleic acid fragment according to claim 1 or 2. The expression vector according to claim 3 , which is a plasmid vector or a viral vector.

5. The expression vector of claim 4, wherein the viral vector comprises an adeno-associated virus (AAV) vector. 6 . The expression vector according to claim 5 , further comprising an adeno-associated virus inverted terminal repeat (ITR) sequence at the 5′ end and an adeno-associated virus ITR sequence at the 3′ end.

7. The expression vector according to claim 6, wherein The adeno-associated virus ITR sequence at the 5' end includes the nucleotide sequence shown in SEQ ID NO:3 or SEQ ID NO:5, and the adeno-associated virus ITR sequence at the 3' end includes the nucleotide sequence shown in SEQ ID NO:4 or SEQ ID NO:

38.

8. The expression vector according to any one of claims 3 to 7, further comprising a miRNA target sequence, wherein: The miRNA target sequence can reduce the expression efficiency of the nucleotide sequence encoding GCase in liver tissue; and / or The miRNA target sequence can reduce the expression efficiency of the nucleotide sequence encoding GCase in dorsal root ganglion tissue.

9. The expression vector according to claim 8, wherein the miRNA target sequence comprises one or both of the miR-183 target sequence or the miR-122 target sequence, preferably, the miRNA target sequence comprises one or both of the nucleotide sequences shown in SEQ ID NO:6 or SEQ ID NO:

7.

10. The expression vector according to claim 9, wherein the number of the miRNA target sequences is 1, 2, 3, 4, 5, 6, 7, or 8, preferably, the number of the miRNA target sequences is 4.

11. The expression vector according to claim 9, wherein the miRNA target sequence comprises 2 miR-183 target sequences and 2 miR-122 target sequences.

12. The expression vector according to any one of claims 3 to 10, further optionally comprising a regulatory sequence, wherein the regulatory sequence includes at least one of an SV40 intron regulatory sequence, a Chimeric chβ-Actini-MVMj regulatory sequence, or an hGlobin intro2 exon3 intron regulatory sequence.

13. The expression vector according to claim 12, wherein: The SV40 intron regulatory sequence is shown in SEQ ID NO:8, the Chimeric chβ-Actini-MVMj regulatory sequence is shown in SEQ ID NO:9, and the hGlobin intro2 exon3 intron regulatory sequence is shown in SEQ ID NO:

10.

14. The expression vector according to any one of claims 3 to 13, further comprising a promoter, and optionally, further comprising an enhancer. 15 . The expression vector according to claim 14 , wherein the promoter comprises a chicken beta actin (CBA) promoter, a cytomegalovirus (CMV) promoter, or a mini-CMV promoter, and the enhancer comprises a CMV enhancer or a CBh enhancer.

16. The expression vector of any one of claims 3 to 15, further comprising a tail signal, wherein the tail signal comprises a poly A signal tail of bovine growth hormone (Bgh) or a poly A signal tail of simian virus 40 (SV40).

17. The expression vector according to any one of claims 3 to 16, comprising, from the 5' end to the 3' end: a) Adeno-associated virus ITR sequence at the 5' end; b) promoter; c) optionally, further comprising an enhancer; d) optionally, further comprising regulatory sequences; e) The nucleic acid fragment according to claim 1 or 2; f) miRNA target sequence; g) tail signal; h) Adeno-associated virus ITR sequence at the 3' end.

18. A recombinant adeno-associated virus (rAAV), characterized in that It comprises the expression vector according to any one of claims 3 to 17.

19. The recombinant adeno-associated virus of claim 18, wherein the serotype of the rAAV comprises AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hu14), AAV10, AAV11, AAV12, AAV13, AAVrh8, AAVrh10, AAV-DJ, or AAV-DJ8.

20. The recombinant adeno-associated virus according to claim 19, wherein the serotype of the rAAV is AAV9.

21. A pharmaceutical composition, characterized in that It comprises the nucleic acid fragment according to claim 1 or 2, the expression vector according to any one of claims 3 to 17, and / or the recombinant adeno-associated virus according to any one of claims 18 to 20, and pharmaceutically, physiologically or nutritionally acceptable excipients.

22. The pharmaceutical composition according to claim 21, which is administered by intravenous injection or suboccipital injection.

23. Use of the nucleic acid fragment of claim 1 or 2, the expression vector of any one of claims 3-17, the recombinant adeno-associated virus of any one of claims 18-20, or the pharmaceutical composition of claim 21 or 22 in the preparation of a medicament for preventing, alleviating or treating a central nervous system disease.

24. The use according to claim 23, wherein the central nervous system disease comprises Parkinson's disease or Gaucher's disease.

25. A method for preventing, alleviating or treating a central nervous system disease, characterized in that: It comprises administering to a subject in need thereof a therapeutically effective amount of the nucleic acid fragment of claim 1 or 2, the expression vector of any one of claims 3-17, the recombinant adeno-associated virus of any one of claims 18-20, or the pharmaceutical composition of claim 21 or 22.

26. The method of claim 25, wherein the central nervous system disease comprises Parkinson's disease or Gaucher's disease.

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