Modified adeno-associated viral vectors and their use in treatment of neurological diseases

Abstract

The present application provides a nucleic acid fragment comprising a modified nucleotide sequence encoding an aromatic L-amino acid decarboxylase (AADC). 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 neurological diseases.

Description

Modified adeno-associated virus vector and its use in treating neurological diseases Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to a nucleic acid fragment comprising a nucleotide sequence encoding an aromatic L-amino acid decarboxylase, an expression vector, and use thereof in treating neurological diseases. Background Art

[0002] Aromatic L-amino acid decarboxylase (AADC) is a homodimeric pyridoxal phosphate-dependent enzyme responsible for the synthesis of dopamine and serotonin. AADC catalyzes the decarboxylation of L-3,4-dihydroxyphenylalanine (L-DOPA or levodopa) to dopamine, the decarboxylation of L-5-hydroxytryptophan (L-5-HTP) to serotonin, and the decarboxylation of L-tryptophan to tryptamine.

[0003] Aromatic L-amino acid decarboxylase deficiency (AADCD) is an autosomal recessive neurometabolic disorder caused by a genetic defect that results in AADC deficiency, which in turn impairs the synthesis of dopamine and serotonin from L-DOPA and L-5-HTP, respectively. In the absence of neuronal dopamine, patients experience movement disorders, behavioral problems, autonomic dysfunction, and developmental delays. Traditional drug therapy for AADC deficiency involves a combination of vitamin B6, dopamine agonists, and monoamine oxidase inhibitors, but its efficacy is limited. A promising treatment for AADC deficiency is AADC gene therapy.

[0004] Parkinson's disease (PD) is a progressive neurodegenerative disease of the central nervous system characterized by the gradual loss of dopaminergic neurons, leading to dopamine deficiency. Exogenous L-DOPA supplementation is currently the most effective strategy for treating Parkinson's disease. However, as PD progresses, AADC levels continue to decline, requiring an increase in the dose of L-DOPA to maintain the desired clinical effect. In addition, the benefits of dopamine therapy become less significant over time, partly due to the progressive death of dopamine-producing cells and the corresponding loss of AADC activity. At the same time, systemic administration of high-dose dopamine is accompanied by side effects such as fluctuations in motor performance, movement disorders, and hallucinations caused by dopaminergic stimulation of the mesolimbic system. Therefore, one strategy to restore dopamine function and minimize side effects is to use gene therapy to deliver AADC directly to target areas of the CNS (EMBO Mol Med., 2021 Sep 7, 13(9): e14712; CN202011500265.X).

[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 AADC, wherein the nucleotide sequence encoding AADC 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: 3. The present application effectively improves expression efficiency by codon-optimizing the nucleotide sequence encoding AADC.

[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 AADC, the expression vector in the present application further incorporates high-expression efficiency promoters and enhancers, thereby further increasing the expression level of AADC and 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 neurological diseases.

[0014] According to one embodiment of the present application, a method for preventing, alleviating or treating neurological 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 pAADC_v0 vector prepared in Example 1.

[0018] FIG2 is a schematic diagram of the structure of the pAADC_v1 vector prepared in Example 1.

[0019] FIG3 is a schematic diagram of the structure of the pAADC_v2 vector prepared in Example 1.

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

[0021] FIG5 is a schematic diagram of the structure of the pAADC_vB vector prepared in Example 1.

[0022] FIG6 is a schematic diagram of the structure of the pAADC_vC vector prepared in Example 1.

[0023] FIG7 is a schematic diagram of the structure of the pAADC_vE vector prepared in Example 1.

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

[0025] FIG9 is a Western blot result diagram showing the effects of pAADC_v0, pAADC_v1, and pAADC_v2 on AADC expression in 293T cells in Example 2.

[0026] FIG10 is a Western blot result showing the effect of pAADC_vA and pAADC_vB on AADC expression in 293T cells in Example 3.

[0027] FIG11 is a Western blot result showing the effects of pAADC_v1 and pAADC_vC on AADC expression in 293T cells in Example 4.

[0028] Figure 12 is a graph showing the expression of target proteins in Neuro-2a cells after transfection of different vectors in Example 5. 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 sequence, 802 represents a vector containing miR-802 target sequence, 99a-5p represents a vector containing miR-99a-5p target sequence, 100-5p represents a vector containing miR-100-5p target sequence, and 1200 represents a vector containing miR-1200 target sequence.

[0029] Figure 13 is a Western blot result diagram showing the effects of AAV-AADC_v1, AAV-AADC_vE, and AAV-AADC_vF on AADC expression in 293T cells in Example 6.

[0030] Figure 14 is a Western blot result diagram showing the effects of AAV9-AADC_vA, AAV9-AADC_v1, AAV1-AADC_v1, and AAV2-AADC_v1 on AADC expression in mouse brain tissue in Example 7.

[0031] FIG15 is a graph showing the immunofluorescence staining results of the effect of AAV9-AADC_vA on AADC expression in mouse brain tissue in Example 7.

[0032] Figure 16A shows the effect of AAV2-hAADC (an AADC expression vector in the prior art) on AADC expression in mouse brain tissue in Example 8. Figure 16B shows the DAB staining results of the effect of AAV9-AADC_vA on AADC expression in mouse brain tissue in Example 8.

[0033] 17 is a Western blot result graph and statistical graph characterizing the effects of AAV9-AADC_vE and AAV9-AADC_vA on AADC expression in mouse brain tissue and liver tissue in Example 9.

[0034] FIG18 is a graph showing the behavioral test results of the Parkinson's disease rat model before drug treatment in Example 10. ns represents no statistically significant difference.

[0035] Figure 19 shows the statistical results of the effects of AAV9-AADC_vA on the behavioral characteristics of the Parkinson's disease rat model described in Example 10. Group 1 shows the test results for rats injected with empty viral shells; Group 2 shows the test results for rats injected with AAV9-AADC_vA. * indicates statistically significant difference; ns indicates no statistical difference.

[0036] Figure 20 shows the statistical results of the effects of AAV9-AADC_vA on the behavioral characteristics of the Parkinson's disease rat model in Example 10. Group 1 shows the test results for rats injected with empty viral shells; Group 2 shows the test results for rats injected with AAV9-AADC_vA. * and ** indicate statistically significant differences.

[0037] Figure 21 shows the results of the AAV9-AADC_vA assay for striatal dopamine levels in a Parkinson's disease rat model in Example 11. Group 1 shows the results for rats injected with empty viral shells; Group 2 shows the results for rats injected with AAV9-AADC_vA. ** indicates statistically significant differences. DETAILED DESCRIPTION

[0038] 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" under any circumstances. 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 by this application, 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.

[0039] Although the numerical ranges and parameters setting forth the broad scope of this application 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 such broader numerical range, as if such narrower numerical ranges were expressly written herein.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] "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.

[0048] 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 AADC gene, which can bind to a specific miRNA to inhibit the expression level of the AADC gene.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] Nucleic acid fragments, expression vectors, recombinant adeno-associated viruses

[0056] According to one embodiment of the present application, a nucleic acid fragment is provided, comprising a nucleotide sequence encoding AADC, wherein the nucleotide sequence encoding AADC has a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to the nucleotide sequence shown in SEQ ID NO: 3. In some embodiments, the nucleotide sequence encoding AADC comprises the nucleotide sequence shown in SEQ ID NO: 3. In some embodiments, the nucleotide sequence encoding AADC is the nucleotide sequence shown in SEQ ID NO: 3.

[0057] 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.

[0058] 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 the 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 AADC in liver tissue. In some preferred embodiments, the miRNA target sequence can reduce the expression efficiency of the nucleotide sequence encoding AADC in dorsal root ganglion tissue. In some preferred embodiments, the miRNA target sequence is selected from at least one or more 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 sequence comprises one or both of the nucleotide sequences set forth in SEQ ID NO:5 or SEQ ID NO:6. 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 the nucleotide sequence set forth in any one of SEQ ID NOs: 16-18. 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.

[0059] In some embodiments, the expression vector further comprises a promoter. The promoter can be any suitable promoter sequence, i.e., a nucleic acid 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 nucleic acid sequence that has transcriptional activity in the selected host cell, including mutant, truncated, and hybrid promoters, and can be derived from a gene encoding an extracellular or intracellular protein or polypeptide that is homologous or heterologous to the host cell. In some embodiments, the promoter includes the chicken beta actin (CBA) promoter, the cytomegalovirus (CMV) promoter, and the promoter of the human SYN1 gene.

[0060] In some embodiments, the expression vector further optionally comprises an enhancer. The enhancer can be any suitable enhancer sequence. In some embodiments, the enhancer is a CMV enhancer.

[0061] In some embodiments, the expression vector further comprises a tail signal. Transcription termination sequences known in the art can be used in the present application. 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.

[0062] 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.

[0063] 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.

[0064] 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; an enhancer; a promoter; the nucleic acid fragment described in this application; a tail signal; and an adeno-associated virus ITR sequence at the 3' end.

[0065] In some preferred 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; an enhancer; 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.

[0066] 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.

[0067] 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.

[0068] 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: 12 or SEQ ID NO: 14, and wherein the adeno-associated virus ITR sequence at the 3' end is a nucleotide sequence shown in SEQ ID NO: 13 or SEQ ID NO: 39. Wherein, compared with the wild-type adeno-associated virus ITR sequence at the 5' end (SEQ ID NO: 14), the mutant adeno-associated virus ITR sequence at the 5' end (SEQ ID NO: 12) can further enhance the expression efficiency of the target gene.

[0069] 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 includes AAV1, AAV2, AAV9. 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.

[0070] Pharmaceutical composition

[0071] 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.

[0072] 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.

[0073] In some embodiments, the pharmaceutical composition is administered by intravenous injection or suboccipital injection.

[0074] use

[0075] 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 neurological disease. In some embodiments, the neurological disease is a neurological disease caused by a deficiency or decreased activity of an aromatic L-amino acid decarboxylase. In some embodiments, the neurological disease includes Parkinson's disease and aromatic L-amino acid decarboxylase deficiency.

[0076] Disease treatment methods

[0077] According to one embodiment of the present application, a method for preventing, alleviating or treating a neurological disease is also provided, comprising administering to a subject in need thereof a therapeutically effective amount of the nucleic acid fragment described herein, the expression vector described herein, the recombinant adeno-associated virus described herein, or the pharmaceutical composition described herein. In some embodiments, the neurological disease is a neurological disease caused by a deficiency or decreased activity of an aromatic L-amino acid decarboxylase. In some embodiments, the neurological disease includes Parkinson's disease and aromatic L-amino acid decarboxylase deficiency.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] Example

[0082] The following describes 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 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.

[0083] Unless otherwise stated, all reagents and instruments used in the following examples are commercially available conventional products. Unless otherwise stated, experiments were performed under conventional conditions or conditions recommended by the manufacturer.

[0084] Example 1: Construction of expression vector

[0085] 1.1 Nucleotide sequence encoding AADC

[0086] The amino acid sequence of human AADC protein (SEQ ID NO: 2; GenBank: NP_001076440.2) and the nucleotide sequence encoding human AADC (SEQ ID NO: 1; GenBank: NM_001082971.2) were searched through the NCBI GenBank database (https: / / www.ncbi.nlm.nih.gov / gene). The AADC protein coding region sequence was codon optimized to obtain the AADC optimized sequence AZ3 (SEQ ID NO: 3) and the AADC optimized sequence EF (SEQ ID NO: 4).

[0087] 1.2 Construction of adeno-associated virus vector

[0088] The adeno-associated virus vector constructed in this example includes, from the 5' end to the 3' end, the following: 5' ITR; enhancer; promoter; regulatory sequence; nucleotide sequence encoding human AADC; miRNA target sequence; tail signal; 3' adeno-associated virus ITR sequence; and backbone sequence.

[0089] Table 1 Sequence information of expression vector

[0090] 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:

[0091] The structure of pAADC_v0 is shown in Figure 1 , which contains: an adeno-associated virus ITR sequence with a mutated 5' end, a CMV enhancer, a CBA promoter, a wild-type sequence encoding AADC, mir-183 and mir-122 target sequences, a simian vacuolating virus 40 signal tail, an adeno-associated virus ITR sequence with a mutated 3' end, and a backbone sequence.

[0092] The structure of pAADC_v1 is shown in Figure 2 , which contains: an adeno-associated virus ITR sequence with a mutated 5' end, a CMV enhancer, a CBA promoter, an optimized sequence AZ3, mir-183 and mir-122 target sequences, a simian vacuolating virus 40 signal tail, an adeno-associated virus ITR sequence with a mutated 3' end, and a backbone sequence.

[0093] The structure of pAADC_v2 is shown in Figure 3 , which contains: an adeno-associated virus ITR sequence with a mutated 5' end, a CMV enhancer, a CBA promoter, an optimized EF sequence, mir-183 and mir-122 target sequences, a simian vacuolating virus 40 signal tail, an adeno-associated virus ITR sequence with a mutated 3' end, and a backbone sequence.

[0094] The structure of pAADC_vA is shown in Figure 4 , which contains: an adeno-associated virus ITR sequence with a mutated 5' end, a human SYN1 promoter, an optimized sequence AZ3, mir-183 and mir-122 target sequences, a simian vacuolating virus 40 signal tail, an adeno-associated virus ITR sequence with a mutated 3' end, and a backbone sequence.

[0095] The structure of pAADC_vB is shown in Figure 5 , which contains: an adeno-associated virus ITR sequence with a mutated 5' end, a human SYN1 promoter, a wild-type sequence encoding AADC, mir-183 and mir-122 target sequences, a simian vacuolating virus 40 signal tail, an adeno-associated virus ITR sequence with a mutated 3' end, and a backbone sequence.

[0096] The structure of pAADC_vC is basically the same as that of pAADC_v1, except that the pAADC_vC vector does not contain the mir-183 and mir-122 target sequences. Its structure is shown in Figure 6.

[0097] The structure of pAADC_vE is shown in Figure 7, which contains: adeno-associated virus ITR sequence at the 5' end, CMV promoter, CMV enhancer, hGlobin intro2 exon3 intron regulatory sequence, optimized sequence AZ3 encoding AADC, mir-183 and mir-122 target sequences, bovine growth hormone (bGH) poly A signal tail, adeno-associated virus ITR sequence at the 3' end, and backbone sequence.

[0098] The structure of pAADC_vF is shown in Figure 8, which contains: adeno-associated virus ITR sequence at the 5' end, CMV enhancer, CBA promoter, Chimeric chβ-Actini-MVMj regulatory sequence, optimized sequence AZ3 encoding AADC, mir-183 and mir-122 target sequences, bovine growth hormone (bGH) poly A signal tail, adeno-associated virus ITR sequence at the 3' end, and backbone sequence.

[0099] Example 2:

[0100] Using the plasmids pAADC_v0, pAADC_v1 and pAADC_v2 constructed in Example 1, (Polyplus) transfected 293T cells (purchased from ATCC) according to the reagent instructions, using 3 μL for 1 μg of plasmid. Two days after transfection, the expression level of AADC was detected by immunoblotting (AADC antibody was purchased from abcam, catalog number ab3905). The results are shown in FIG9 .

[0101] The results showed that the pAADC_v0 (carrying the wild-type sequence encoding AADC), pAADC_v1 (carrying the optimized sequence AZ3), and pAADC_v2 (carrying the optimized sequence EF) vectors all expressed AADC protein in cells. Compared to pAADC_v0, the expression efficiency of pAADC_v1 was significantly increased; the expression efficiency of pAADC_v2 was not significantly increased, but rather decreased to a certain extent. These results indicate that the codon-optimized AZ3 sequence significantly improved the expression efficiency of the AADC protein, but the codon-optimized EF sequence did not achieve an increase in expression efficiency. Furthermore, in addition to EF, the applicants discovered a large number of optimized sequences that did not improve expression efficiency during the screening process, confirming that the strategy of improving expression efficiency through codon optimization is affected by multiple factors such as protein type, protein length, secondary structure, and species type, and is unpredictable. Therefore, the preferred sequence for efficient expression of AADC in the vectors provided in this application is AZ3.

[0102] Example 3:

[0103] Using the plasmids pAADC_vA and pAADC_vB constructed in Example 1, (Polyplus) transfected 293T cell lines (purchased from ATCC) according to the reagent instructions, using 3 μL of 1 μg of plasmid. Two days after transfection, the expression level of AADC was detected by immunoblotting (AADC antibody was purchased from Abcom, catalog number 3905). The results are shown in FIG10 .

[0104] The results showed that both vectors effectively expressed AADC protein in cells, and the expression level of pAADC_vA (carrying the optimized sequence AZ3) was significantly higher than that of pAADC_vB (carrying the wild-type sequence encoding AADC). These results indicate that the AZ3 sequence obtained by optimizing the AADC coding sequence in this application can significantly improve the expression efficiency of AADC.

[0105] Example 4:

[0106] The plasmids pAADC_v1 and pAADC_vC constructed in Example 1 were used to (Polyplus) transfected 293T cell lines (purchased from ATCC) according to the reagent instructions, using 3 μL of 1 μg of plasmid. Two days after transfection, the expression level of AADC was detected by immunoblotting (AADC antibody was purchased from Abcam, catalog number 3905). The results are shown in FIG11 .

[0107] pAADC_v1 is a plasmid containing the mir-183 and mir-122 target sequences, while pAADC_vC does not. The results showed that the addition of the mir-183 and mir-122 target sequences could downregulate AADC expression, indicating that the addition of miRNA target sequences to the vector can reduce the expression of the target gene.

[0108] Example 5:

[0109] 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:

[0110] 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: 21) 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.

[0111] Table 2 PCR primer sequences for control miRNA

[0112] 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 12. The different labels in the figure represent the use of different vectors for transfection:

[0113] Blank represents the untransfected blank group.

[0114] 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: 32) that does not contain miR-183 and miR-122 target sequences.

[0115] 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: 33),

[0116] 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: 34),

[0117] 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: 37),

[0118] 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: 35),

[0119] 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: 36),

[0120] 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: 38).

[0121] The results in Figure 12 show that different miRNA target sequence combinations have different effects on reducing the expression of the target gene.

[0122] Example 6:

[0123] In the plasmids pAADC_v1, pAADC_vE, and pAADC_vF constructed in Example 1, AAV-AADC_v1, AAV-AADC_vE, and AAV-AADC_vF were packaged using AAV9. 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 plasmids used in each experiment (such as pAADC_v1, pAADC_vE, or pAADC_vF). 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-AADC_vE, AAV-AADC_vF, and AAV-AADC_v1 obtained by the above packaging were used to infect the human 293T cell line. Two days later, the expression level of AADC was detected by immunoblotting (AADC antibody was purchased from abcam, catalog number ab3905). The results are shown in Figure 13.

[0127] The combination strategy of promoter, enhancer, and regulatory sequence elements in pAADC_vE is derived from US10898585B2 (other elements, such as the optimized sequence AZ3, are derived from this application). By comparing the expression efficiency of pAADC_vE and pAADC_v1, it can be seen that the combination strategy of promoter, enhancer, regulatory sequence, and other elements selected in this application can further improve the expression efficiency of the optimized sequence AZ3, significantly outperforming the element combination strategy in the prior art US10898585B2. These results indicate that while the element combination strategy in US10898585B2 can enhance the expression efficiency of the target gene in the vector disclosed in that patent, it does not work for the optimized sequence AZ3 provided in this application.

[0128] Example 7:

[0129] Of the plasmids pAADC_vA and pAADC_v1 constructed in Example 1, pAADC_vA was packaged using AAV9 to obtain AAV9-AADC_vA, and pAADC_v1 was packaged using AAV1, AAV2, and AAV9 to obtain AAV1-AADC_v1, AAV2-AADC_v1, and AAV9-AADC_v1. The packaging methods for AAV1, AAV2, and AAV9 are commonly used in the art (see Neurosci Res, 2015, 93:144-57. doi:10.1016 / j.neures.2014.09.002). C57BL / 6 mice were infected at an infection titer of 5E+10 vg / mouse. After 21 days, mouse brains were harvested and immunoblotted for AADC expression (AADC antibody purchased from Abcam, Cat. No. ab3905). The results are shown in Figure 14.

[0130] The results showed that both AAV9-AADC_vA and AAV9-AADC_v1 effectively expressed AADC protein in the mouse brain, with AAV9-AADC_vA having higher expression efficiency. Since the expression activities of AAV-AADC_vF and AAV-AADC_v1 in cells in Example 5 were comparable, and the expression activity of AAV9-AADC_vA was much higher than that of AAV-AADC_v1, AAV9-AADC_vA was selected as the optimal strategy.

[0131] Simultaneously, frozen sections of the left brain of the mice were immunostained, and the results are shown in Figure 15. The striatum is enclosed within the border. The results indicate that AAV9-AADC_vA is well expressed in the striatum, the target brain region for AADC treatment.

[0132] Example 8:

[0133] The AAV9-AADC_vA obtained in Example 7 was microinjected into the right striatum of SD rats at a dose of 1.5E+11 vg per animal. Two to four weeks later, the rat brains were harvested, fixed with 4% PFA, and frozen sections were prepared. The sections were then stained using DBA staining (AADC antibody purchased from abcam, Catalog No. ab3905; DAB colorimetric kit, Beyotime, Catalog No. P0202). Microscopic images were taken and assembled to produce a map of AADC distribution in the brain, as shown in Figure 16B.

[0134] The expression of other AADC expression vectors AAV2-hAADC in the prior art (see PLoS One, 2015, 10 (4): e0122708. doi: 10.1371 / journal.pone.0122708) is more limited to the injection site (see Fig. 3A in PLoS One, 2015, 10 (4): e0122708. and Fig. 16A in this application). Compared with AAV2-hAADC, the vector AAV9-AADC_vA provided in this application is expressed more widely in the same part of rats and can diffuse out of the injection area and be diffusely distributed throughout the striatum (Fig. 16B). As shown in the figure, the white arrow in Fig. 16A indicates the area where AAV2-AADC does not express AADC in the literature, but the AAV9-AADC_vA provided in this application is expressed in this area. Since the volume of human striatum is much larger than that of rat, the better diffusion of AAV9-AADC_vA indicates that it has better therapeutic effect.

[0135] Example 9:

[0136] The AAV9-AADC_vE obtained in Example 6 or the AAV9-AADC_vA obtained in Example 7 was microinjected into the right striatum of C57BL / 6 mice at a dose of 3.4E+10 vg per animal. 21 days later, the brain and liver of the mice were harvested and immunoblotted (AADC antibody purchased from abcam, catalog number ab3905) to detect the expression of AADC. The results are shown in Figure 17. Group 1 shows the test results of 3 mice injected with empty virus shells (the virus does not carry the AADC gene); Group 2 shows the test results of 3 mice injected with the virus AAV9-AADC_vE; and Group 3 shows the test results of 3 mice injected with the virus AAV9-AADC_vA. At the same time, the grayscale values ​​of the immunoblot bands were read using Clinx Image Analysis software, and statistical analysis was performed using GraphPad Prism. The t-test was used to analyze the significance of the differences between the two groups of data marked in the figure, where * and ** represent statistical differences, and ns represents no statistical difference.

[0137] The results in Figure 17 show that animals injected with AAV9-AADC_vE and AAV9-AADC_vA were found to have increased expression in brain tissue, proving that AADC can be expressed in mouse brain tissue; at the same time, the AADC expression level of AAV9-AADC_vA was significantly higher than that of the control vector AAV9-AADC_vE. This demonstrates that the expression efficiency of AAV9-AADC_vA in the brain is better than that of AAV9-AADC_vE. The above results once again verify the results of Example 6, confirming that although the element combination strategy in US10898585B2 can enhance the expression efficiency of the target gene in the vector disclosed in the patent, it cannot work on the optimized sequence AZ3 provided in this application.

[0138] The results of Figure 17 also show that the expression of AADC in the liver of animals injected with AAV9-AADC_vE and AAV9-AADC_vA did not increase, indicating that the vectors to which mir-183 and mir-122 target sequences were added did not result in increased expression of AADC in the liver, and thus had less liver toxicity. 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 the dorsal root ganglion toxicity caused by AAV administration. The above results confirm that the vector in this application can solve the toxicity problem caused by traditional AAV vectors and is a more preferred treatment option.

[0139] Example 10:

[0140] After SD rats were injected unilaterally with 6-OHDA to establish a Parkinson's disease model, L-dopamine was injected intraperitoneally, and rotation-positive animals were selected for testing. The positive animals were randomly divided into two groups and behavioral tests were performed: different doses of L-dopamine (13 mpk, 62 mpk) were injected intraperitoneally daily, and the number of rotations of the animals from 15 minutes to 45 minutes after injection was counted. The results are shown in Figure 18. The two groups of data marked in the figure were statistically analyzed using t-test, and ns represents no statistical difference. The results in Figure 17 show that there was no statistical difference in behavior between the two groups of animals before treatment with AAV drugs.

[0141] Subsequently, the AAV9-AADC_vA obtained in Example 7 was microinjected into the right striatum of the two groups of animals at doses of 1.5E+11 vg, 2.76E+10 vg, 9.21E+9 vg, and 3.07E+9 vg per animal. Behavioral testing began in the third week after the injection: the animals were intraperitoneally injected with different doses of levodopamine (3.25 mpk, 6.5 mpk, 13 mpk, 26 mpk, and 62 mpk) daily, and the number of rotations of the animals from 15 minutes to 45 minutes after injection was counted. The results are shown in Figure 19. Group 1 shows the test results of rats injected with empty virus shells (the virus does not carry the AADC gene); Group 2 shows the test results of rats injected with virus AAV9-AADC_vA. The two groups of data marked in the figure were statistically analyzed using t-test. * and ** represent statistically significant differences, and ns represents no statistical difference.

[0142] The results in Figure 19 show that rats injected with AAV9-AADC_vA can induce obvious rotation at lower levodopa doses (e.g., 6.5 mpk and 13 mpk), proving that AAV9-AADC_vA can reduce the dosage of levodopa while achieving the effect of high-dose levodopa.

[0143] At the same time, the animals were intraperitoneally injected with the same dose of levodopa (62 mpk), and the number of rotations after injection was counted. The results are shown in Figure 20. Group 1 shows the test results for rats injected with empty virus shells (the virus does not carry the AADC gene); Group 2 shows the test results for rats injected with the virus AAV9-AADC_vA. Fisher's LSD test was used to statistically analyze the two groups of data marked in the figure. * and ** indicate statistically significant differences. The results in Figure 20 confirm that the rotation time of animals injected with AAV9-AADC_vA was significantly longer.

[0144] The results in Figures 18-20 above indicate that treatment with AAV9-AADC_vA in Parkinson's model animals can significantly reduce the dosage of levodopa and prolong the duration of action of the drug levodopa.

[0145] Example 11:

[0146] The rats injected with the empty virus shells (Group 1) and the rats injected with the AAV9-AADC_vA virus (Group 2) in Example 10 were sacrificed 1 hour after intraperitoneal injection of levodopa, and the striatum on the side injected with 6-OHDA was immediately removed. The striatal dopamine content was detected and statistically analyzed using the ELISA method (Elabscience, Catalog No. E-EL-0046). The results are shown in Figure 21. The two groups of data marked in the figure were tested using t-test, and ** indicates statistically significant differences.

[0147] The results in Figure 21 show that rat brain tissue injected with AAV9-AADC_vA contains more dopamine, indicating that AAV9-AADC_vA can improve the efficiency of converting levodopa to dopamine at the injection site. Therefore, expressing AADC in the striatum to increase the conversion of levodopa to dopamine is the mechanism of action of the AAV vector provided in this application for treating Parkinson's disease.

[0148] 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 an aromatic L-amino acid decarboxylase (AADC), wherein the nucleotide sequence encoding AADC 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:

3.

2. The nucleic acid fragment according to claim 1, wherein the nucleotide sequence encoding AADC comprises the nucleotide sequence shown in SEQ ID NO:

3.

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:12 or SEQ ID NO:14, and the adeno-associated virus ITR sequence at the 3' end includes the nucleotide sequence shown in SEQ ID NO:13 or SEQ ID NO:

39.

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 AADC in liver tissue; and / or The miRNA target sequence can reduce the expression efficiency of the nucleotide sequence encoding AADC 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:5 or SEQ ID NO:

6.

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 11, further comprising a promoter. 13 . The expression vector according to claim 12 , wherein the promoter comprises a chicken beta actin (CBA) promoter, a cytomegalovirus (CMV) promoter, and a promoter of a human SYN1 gene.

14. The expression vector according to any one of claims 3 to 13, further optionally comprising an enhancer, wherein the enhancer comprises a CMV enhancer.

15. The expression vector according to any one of claims 3 to 14, further comprising a tail signal.

16. The expression vector according to claim 15, wherein the tail signal comprises a simian vacuolating virus 40 (SV40) poly A signal tail, or a bovine growth hormone (bGH) poly A signal tail.

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) optionally, further comprising an enhancer; c) promoter; d) the nucleic acid fragment according to claim 1 or 2; e) miRNA target sequence; f) tail signal; g) 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 includes AAV1, AAV2, and AAV9, and preferably, 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 to 17, the recombinant adeno-associated virus of any one of claims 18 to 20, or the pharmaceutical composition of claim 21 or 22 in the preparation of a drug for preventing, alleviating or treating neurological diseases.

24. The use according to claim 23, wherein the neurological disease is a neurological disease caused by a deficiency or reduced activity of aromatic L-amino acid decarboxylase.

25. The use according to claim 24, wherein the neurological disease comprises Parkinson's disease, aromatic L-amino acid decarboxylase deficiency.

26. A method for preventing, alleviating or treating neurological diseases, 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.

27. The method according to claim 26, wherein the neurological disease is a neurological disease caused by a deficiency or reduced activity of aromatic L-amino acid decarboxylase.

28. The method of claim 27, wherein the neurological disease comprises Parkinson's disease, aromatic L-amino acid decarboxylase deficiency.

Images